Professional Documents
Culture Documents
Volume 4
Series Editors
A. Kamkin
Department of Fundamental and Applied Physiology, Russian State Medical
University, Ostrovitjanova Str. 1, 117997 Moscow, Russia
I. Kiseleva
Department of Fundamental and Applied Physiology, Russian State Medical
University, Ostrovitjanova Str.1, 117997 Moscow, Russia
Mechanosensitivity and
Mechanotransduction
123
Editors
Prof. Andre Kamkin Prof. Irina Kiseleva
Department of Fundamental and Applied Department of Fundamental
Physiology and Applied Physiology
Russian State Medical University Russian State Medical University
Ostrovitjanova Str. 1 Ostrovitjanova Str. 1
117997 Moscow 117997 Moscow
Russia Russia
Kamkin.A@g23.relcom.ru Kamkin.A@g23.relcom.ru
Editorial Assistant
Dr. Natalia E. Lapina
Division of Neurosurgical Research
Medical Faculty Mannheim
Ruprecht-Karls-University Heidelberg
Theodor-Kutzer-Ufer 1-3
D-68167 Mannheim
Germany
Natalia.Lapina@medma.uni-heidelberg.de
v
vi Foreword
Mechanical stretch resulting from pulsatile blood flow can modulate vascular cell
differentiation and function. Proteoglycans have a crucial role in mechanobiological
responses of the arterial system. The issue of mechanosensing and mechanotrans-
duction in vascular cells is discussed in Chapters 8 and 9. Emphasis is put on the
importance of haemodynamic forces for the maintenance of homeostasis in the
cardiovascular system. It is pointed out that a detailed knowledge of the molecu-
lar mechanisms underlying mechanosensing and mechanotransduction in vascular
cells is essential to develop novel concepts for the prevention and therapy of
cardiovascular diseases.
Mechanotransduction in the lung, the topic highlighted in part IV, is estab-
lished through the transient receptor potential vanilloid-4 (TRPV4) non-selective
cation channel. Activation of TRPV4 initiates increased vascular permeability in
response to high airway pressure or vascular pressure in the lung. Understanding
the molecular regulation of this ion channel may therefore provide novel insights
into various pulmonary disease processes including ventilator induced lung injury
and pulmonary oedema.
Primary cilia are organelles that have recently been recognized as mechanosen-
sors. Cilia can sense the movement of body fluids in various tissues including
endolymph in the inner ear, urine in renal tubules, bile in the hepatic biliary sys-
tem, and digestive fluid in the pancreatic duct. Mechanosensing by primary cilia is
discussed in Chapter 14. As outlined here genetic defects of the molecular compo-
nents of primary cilia, i.e. polycystins and other interacting proteins, can give rise
to various disorders such as polycystic kidney disease.
The final chapter addresses the role of mechanosensing and mechanotransduc-
tion in blood cells. Blood cells encounter substantial mechanical stimuli due to
variable haemodynamics. Large-conductance background K+ channels (LKbg ) have
been identified as mechanosensors in mouse B lymphocytes and their molecular
regulation by phospholipase C-dependent signalling pathways is discussed.
In summary, Mechanosensitivity and Mechanotransduction compiles in-depth
reviews of the current knowledge of mechanosensing and mechanical signal trans-
duction in various tissues. The readers attention is attracted by the profound and
clearly written contributions. Articles cover the many facets of cellular mechanosen-
sation ranging from the molecular gating of mechanosensitive ion channels to
perspectives for the future treatment of diseases.
vii
viii Editorial
and PKC (Lal et al., 2010). For example, in fibroblasts (Kumar et al., 2006; Zhao
et al., 2007; Zhang X et al., 2007), endothelial (Mathur et al., 2007), or vascular
smooth muscle cells (Na et al., 2007) stress-stiffening has been attributed to an
integrin-Rho-dependent activation of stress fibers. Stretch-induced conformational
changes in the ECM may alter integrin structure, resulting in activation of liganded
integrin receptors and focal contact-associated secondary messenger pathways in
the cell, such as FAK, Src family kinases, Abl and integrin-linked kinase ILK (Li
et al., 1999; Liu et al., 2000). Many studies have demonstrated that the physical net-
work involving the extracellular matrix, integrins and the cytoskeleton is critical for
the response of cells to mechanical forces (see for review Carver and Fuseler, 2010).
Thus, during the deformation of the whole cell the possibility of the transmission of
membrane forces into the cell is realized from the extracellular matrix through inte-
grin receptors to the cytoskeleton and then to channel proteins (see for review Boriek
and Kumar, 2008). It is beyond any doubt that integrins are major mechanosensors
or mechanotransducers in many different cell types because of their unique abil-
ity to transduce extracellular mechanical signals exerted by extracellular matrix into
intracellular signals. Integrins and cytoskeletal proteins act together as mechanosen-
sors (see for review Thampatty and Wang, 2008). Despite of the achieved progress
in addressing a number of questions, in many respects the mechanisms by which
different cells sense, transduce and convert mechanical stimuli into cellular signals
remain to be completely revealed.
For this reason the first part of this volume is devoted to a network involving
the extracellular matrix, integrins and the cytoskeleton. The first part of the volume
is beginning with an article devoted to the role of integrin-mediated mechanotrans-
duction by Yip et al. (2011). Vascular smooth muscle cells are used as a model for
a discussion of the main issues concerned with integrins: Integrins and their asso-
ciated kinases, integrins as mechanotransducers, mechanisms of coupling integrin
activation to myogenic constriction. Depending on the types of integrin involved
and the specific vascular beds, integrin dependent myogenic vasoconstriction can
be mediated through Ca2+ dependent mechanisms of L-type Ca2+ channel poten-
tiation, activation of global and local Ca2+ release from ryanodine sensitive Ca2+
stores; as well as Ca2+ independent mechanisms of FAK, c-Src, MAPK, and ILK
activation and reactive oxygen species release (Yip et al., 2011).
The second article is devoted to the role of the actin cytoskeleton in mechanosen-
sation (Luo and Robinson, 2011). The actin cytoskeleton composed of actin fila-
ments, myosin motors, and actin crosslinking proteins plays a critical role in force
propagation and in response to deformation. The chapter discusses the microstruc-
tures and deformations of the actin cytoskeleton, functions of the actin cytoskeleton
in mechanosensation. There are numerous overlaps between the mechanotrans-
duction and traditional chemical signal transduction pathways. Because of the
overlap between these pathways, mechanical-chemical coupling and feedback loops
are a natural consequence of this system integration. Because the actin cytoskele-
ton is structurally integrated with nearly every aspect of the cell, mechanical
inputs can be transmitted quickly throughout the cell. Challenges for understanding
mechanosensation through the actin cytoskeleton include revealing how proteins
Editorial ix
The second chapter also demonstrates that joint tissues, including cartilage,
bone, meniscus, tendon, ligament and synovial membrane, are exposed to high
levels of mechanical stimulation. This review is devoted to the mechanosensitiv-
ity of cells in joint tissues. Under discussion is the role of the mechanosensitivity
in the pathogenesis of joint diseases (Sanchez et al., 2011). The chapter reviews
the issues of mechanical stimuli and cartilage matrix remodeling and chondrocyte
mechanotransduction. The discussion covers mechanical stimuli and subchondral
bone and other joint tissues mechanosensitivity.
The following Part presents the review devoted to primary cilia as mechanosen-
sory organelles in vestibular tissues (Nauli, 2011). In this chapter the author
describes cilia as newly recognized mechanosensory organelles. The author dis-
cusses ciliary classification, function and disease relevance in mammals. Besides,
structural and functional ciliary proteins based on their ciliary domains are pre-
sented. The importance of sensory cilia in other organ systems is yet to be
discovered, and many more cilia-related diseases are still to be identified.
The final Part of the Volume presents the review Mechanosensitive K+ channels
in mouse B lymphocytes: PLC-mediated release of TREK-2 from Inhibition by PIP2
(Kim and Nam, 2011). There is a limited amount of data regarding mechanosensi-
tivity and mechanotransduction in blood cells, therefore this chapter is of special
interest to our readers. Blood cells can encounter significant mechanical stim-
uli in variable flow. In mouse B lymphocytes and their cell line WEHI-231, the
authors found large-conductance background K+ channels (LKbg ) that show sig-
nificant mechanosensitivity. The biophysical characteristics of LKbg were similar
to those of TREK-2. The activity of TREK-2 is under tonic inhibition by PIP2 ,
and a relatively mild membrane stretch relieves this inhibition via activation of PLC
hydrolysing PIP2 . Apart from the mechano-biochemical signaling mechanism, more
direct regulation by stronger membrane stretch is also suggested.
Thus, this book is a unique collection of reviews outlining current knowledge and
future developments in this rapidly growing field. Currently, investigations of the
molecular mechanisms of mechanosensitivity and mechanotransduction are focused
on several issues. The majority of studies investigate intracellular signaling path-
ways. Knowledge of the mechanisms which underlie these processes is necessary
for the understanding of the normal functioning of different living organs and tissues
and allows to predict changes, which arise due to alterations of their environment,
and possibly will allow to develop new methods of artificial intervention. The book
brings up the problem closer to the experts in related medical and biological sci-
ences as well as practicing doctors besides just presenting the latest achievements
in the field.
References
Baker AB (2011) Role of proteoglycans in vascular mechanotransduction. In: Kamkin A,
Kiseleva I (eds) Mechanosensitivity in cells and tissues 4. Mechanosensitivity and mechan-
otransduction. Springer, pp 219236
xiv References
Young SRL and Pavalko FM (2011) Cellular mechanisms of mechanotransduction in bone. In:
Kamkin A, Kiseleva I (eds) Mechanosensitivity in cells and tissues 4. Mechanosensitivity and
mechanotransduction. Springer, pp 277296
Zhang X, Stewart JA Jr, Kane ID, Massey EP, Cashatt DO, Carver WE (2007) Effects of elevated
glucose levels on interactions of cardiac fibroblasts with the extracellular matrix in vitro. Cell
Dev Biol Anim 43:297305
Zhao XH, Laschinger C, Arora P, Szarszi K, Kapus A, McCulloch CA (2007) Force activates
smooth muscle alpha-actin promoter activity through the Rho signaling pathway. Cell Sci
120:18011809
Contents
xvii
xviii Contents
xxi
xxii Contributors
Mary I. Townsley Department of Physiology and Center for Lung Biology, MSB
3074, University of South Alabama, Mobile, AL, 36688, USA,
mtownsley@usouthal.edu
Yajuan Xu Department of Prosthodontics, Guanghua School & Hospital of
Stomatology, Sun Yat-Sen University, Guangzhou 510055, Guangdong, PR China
Kay-Pong Yip Department of Molecular Pharmacology and Physiology,
University of South Florida, Tampa, FL 33612, USA, dyip@health.usf.edu
Suzanne R.L. Young Department of Cellular and Integrative Physiology, Indiana
University School of Medicine, 635 Barnhill Drive, MS 346A, Indianapolis, IN
46202, USA
Yiping Zhang Department of Prosthodontics, Guanghua School & Hospital of
Stomatology, Sun Yat-Sen University, Guangzhou 510055, Guangdong, PR China
Part I
The Role of Cytoskeleton
in Mechanosensitivity
and Mechanotransduction
Chapter 1
Integrin-Mediated Mechanotransduction
in Vascular Smooth Muscle Cells
1.1 Introduction
the major mechanisms preventing the conversion of pressure fluctuations into fluc-
tuations in blood flow is the myogenic response of vascular smooth muscle cells
(VSMCs). Myogenic response is an intrinsic mechanism of VSMCs by which the
vasculature constricts on elevation and dilates on reduction of perfusion pressure
(Bayliss, 1902). It is necessary for maintaining constant blood flow and capillary
hydrostatic pressure (Davis and Hill, 1999). End organ damage in hypertension
has been attributed to the transmission of increased blood pressure into the kid-
ney (Bidani et al., 1987; Karlsen et al., 1997; Abu-Amarah et al., 2005). Impaired
myogenic response in fawn-hooded hypertensive rats showed glomerular damage
(Simons et al., 1993; van Dokkum et al., 1999) highlighting the importance of nor-
mal myogenic response in maintaining a constant GFR and hence renal integrity.
However, the mechanisms of how VSMCs sense the changes in the perfusion
pressure are not well defined (Davis and Hill, 1999).
Mechanotransduction of VSMCs is not only involved in acute adaptation of
vascular resistance in myogenic response, but also in chronic adaptation such
as vascular remodeling in hypertension. Vascular remodeling is closely related
the VSMCs proliferation and extracellular matrix (ECM) accumulation. Genetic
hypertension and angiotensin II induced hypertension are associated with changes
in integrins and ECM expression in resistance arterioles (Intengan et al., 1999;
Intengan and Schiffrin, 2000; Mori and Cowley, 2004; Seubert et al., 2005).
(Wellner and Isenberg, 1994; Wellner and Isenberg, 1995). The stretch-induced
depolarization could be explained by the activation of mechanosensitive ion chan-
nels promoting Na+ or Ca2+ influx, Cl efflux, or inhibiting K+ efflux (Davis and
Hill, 1999). Ion transport pumps in the plasma membrane have also been suggested
as a plausible sensor. Stretching the VSMCs showed an increase in the expression
and translocation of the -subunit of the Na+ , K+ ATPase to the plasma membrane
(Sevieux et al., 2003).
Another group of NSCCs that has been implicated in myogenic response is the
transient receptor potential (TRP) superfamily of channels (Inoue et al., 2006). Most
TRPC channels can be activated by PLC pathway, which can likely be triggered
by the increase in intravascular pressure. In frog oocytes, TRPC1 forms stretch
sensitive cation channel (Maroto et al., 2005), and TRPC6 has been shown to
regulate myogenic response by their involvement in pressure-induced membrane
depolarization in rat cerebral arteries (Welsh et al., 2002; Spassova et al., 2006).
Increasing evidence suggests that TRPM4 channels play a major role in myogenic
response. Suppression of TRPM4 expression using antisense oligodeoxynucleotide
decreased pressure-induced depolarization and myogenic response in vitro, and
altered autoregulation in vivo in rat cerebral arteries (Earley et al., 2004; Reading
and Brayden, 2007). Another study proposed that TRPM4 may actually affect myo-
genic response by stretch-induced Ca2+ release via ryanodine receptors (Morita
et al., 2007). Besides cerebral arteries, TRPC3 and TRPC6 are highly expressed
in rat renal resistance vessels (Facemire et al., 2004; Inoue et al., 2006); TRPC1,
TRPC6, TRPM4, TRPM8, TRPV2, and TRPV4 are also abundant in pulmonary
arterial smooth muscle cells (Lin et al., 2004; Yang et al., 2006). However, their
roles in myogenic response in these vascular beds are unclear. Moreover, TRPV1
and TRPV2 have been implicated as mechanosensitive channels in aortic smooth
muscle cells; but their involvement in myogenic response is debatable (Roman,
2002; Muraki et al., 2003). It is noteworthy that even though stretch-sensitive
membrane bound channels may operate as mechanosensors accounting for the ini-
tiation of myogenic vasoconstriction, they could not provide an error signal to
sustain myogenic constriction because of the abrogation of stretch after myogenic
constriction.
The other plausible sensors for the myogenic phenomenon are integrins, the het-
erodimeric receptors of ECM. They are considered as stress sensing and transducing
elements in many cell types. Of the 24 known integrins, 16 have been reported in
various aspects in vascular biology. Among them, 1 1 , 2 1 , 3 1 , 4 1 , 5 1 ,
6 1 , 7 1 , 8 1 , 9 1 , v 1 , v 3 , v 5 , and 6 4 are expressed in VSMCs
(Glukhova et al., 1994; Moiseeva, 2001). They connect the ECM to the internal
cytoskeleton (CSK) via the short cytoplasmic tail of the -subunit and trans-
duce both outside-in (modification of cellular events on integrin ligation) and
inside-out signals (modification of the ECM in response to integrin ligation). An
6 K.-P. Yip et al.
increase in perfusion pressure might provide the mechanical signal to activate the
VSMC integrins via attachment between native ECM and integrin during passive
dilation. Stretch of ECM may induce conformational changes in integrin resulting
in the activation of focal adhesion associated secondary messenger pathways, such
as focal adhesion kinase (FAK), Src family kinases, and integrin-linked kinase (ILK)
(Lal et al., 2009).
FAK is a cytosolic soluble tyrosine kinase, which plays a major role in integrin-
mediated signaling. It consists of an N-terminal functional domain that interacts
with -integrin and growth factor receptors, a catalytic domain for tyrosine kinase
activity, and a focal-adhesion targeting domain that localizes FAK to focal adhe-
sions and binding sites for associated signaling molecules (Schaller, 2001). Integrins
induce auto-phosphorylation of FAK Tyr397 creating binding sites for cellular-
sarcoma (c-Src) family of tyrosine kinase. These FAK/c-Src complexes trigger
other downstream signals including stimulating mitogen-activated protein kinases
(MAPKs). Activation of MAPKs like extracellular signal-regulated kinase 1/2 (ERK
1/2) lead to cell proliferation and/or trigger other vascular remodeling signaling
in response to mechanical stimulus. ERK1/2 has been shown to be activated by
mechanical stretch in a time and strength-dependent manner in the VSMCs isolated
from rat aorta (Hu et al., 1998). Moreover phosphorylation of ERK 1/2 is essen-
tial for integrin-mediated phenotypic change of cultured aortic smooth muscle cells
(Roy et al., 2001).
ILK is a serine/threonine protein kinase that binds to the cytoplasmic tail of
the integrin subunit. It plays a central role in translating the external mechani-
cal stimulus in to intracellular biochemical signals. ILK has been shown to involve
in Ca2+ -independent phosphorylation of smooth muscle myosin light chain, hence
it may provide an alternative pathway through which integrin activation regulates
vascular resistance independent of Ca2+ (Deng et al., 2001). It has been shown as
an essential component of mechanical stretch sensor by controlling the contractility
in the zebrafish heart (Bendig et al., 2006); and deletion of ILK in zebra fish using
antisense morpholino oligonucleotides results in marked patterning abnormalities
of the vasculature and is lethal (Friedrich et al., 2004). Furthermore, inhibiting ILK
using siRNA was shown to inhibit vascular oxidative stress mediated by hyper-
tension (Vecchione et al., 2009). In addition, integrins is known to mediate the
activation of the serine/threonine protein kinase Akt through FAK-dependent and
independent mechanisms (Chen et al., 1996; Velling et al., 2004), and members
of Rho GTPases, including RhoA and Rac1, which interact with a wide-variety of
downstream effectors to engage in specific vascular responses.
Jones, 1999; Davis et al., 2001). Extracellular mechanical force can be transmit-
ted across the plasma membrane via integrins to initiate intracellular signaling in
non-muscle cells (Ingber, 1990; Wang et al., 1993; Martinez-Lemus et al., 2003;
Katsumi et al., 2004). It is known that an intact ECM- integrin-CSK axis is essential
for mechanosensing and mechanotransduction in the endothelial cells and fibrob-
lasts. The tensegrity model proposed by Ingber suggests that cellular tension or
prestress is balanced and stabilized by the forces within the intracellular acto-
myosin cytoskeleton network (Ingber, 1997, 2000, 2003a, b; Wang et al., 2001).
Disturbances in external mechanical stress can restructure the microtubules in the
cytoskeleton to offset the tension in adherent cells. This transduced force and
restructuring of microtubules can then initiate secondary messengers and signal-
ing cascades including an increase in the [Ca2+ ]i causing contraction. Accordingly,
a change of perfusion pressure in an intact blood vessel may alter the stress in the
attachment sites between native ECM, and VSMC integrins, triggering downstream
signaling events for myogenic response.
involve multiple Ca2+ influx and release pathways. Activation of integrins has been
implicated in the modulation of L-type Ca2+ channels. Early studies in rat cre-
master VSMCs showed that beads coated with fibronectin or 5 integrin antibody
enhanced, whereas v 3 ligands or 3 antibody inhibited L-type Ca2+ currents (Wu
et al., 1998). Another study showed that the activation of 4 1 integrins also trig-
gered an increase in Ca2+ influx via L-type channels (Waitkus-Edwards et al., 2002).
Leu-Asp-Val (LDV) peptide, a sequence of an alternatively spliced fibronectin vari-
ant, caused vasoconstriction and increase in [Ca2+ ]i in cremaster arterioles; and
the response could be blocked by nifedipine and anti-4 antibody. The 5 1 and
4 1 integrin induced L-type Ca2+ current potentiation is mediated through tyro-
sine phosphorylation involving Src proteins in native VSMCs (Wu et al., 2001;
Waitkus-Edwards et al., 2002). It was confirmed in a heterologous expression sys-
tem that 5 1 integrin regulates L-type Ca2+ channels (Cav 1.2) by PKA and c-Src
dependent phosphorylation of 1C C-terminal residues Ser1901 and Tyr2122 , respec-
tively (Gui et al., 2006). Furthermore, activation of 5 1 integrin using antibodies
or fibronectin also potentiated BK channel activity in rat cremaster VSMCs through
both Ca2+ and c-Src dependent mechanism (Wu et al., 2008), suggesting negative
feedback regulation of Ca2+ entry through L-type Ca2+ channel. In cerebral arteries,
TRPC6 and TRPM4 channels have been shown to be involved in myogenic response
(Welsh et al., 2002; Earley et al., 2004; Spassova et al., 2006; Reading and Brayden,
2007). But the connection between these TRP channels and integrin has not been
established.
In renal VSMCs, integrin mediated mechanotransduction triggers a global
increase in [Ca2+ ]i . Application of Arg-Gly-Asp (RGD)-containing peptide initi-
ated local increase in [Ca2+ ]i which propagated as recurrent Ca2+ waves across
renal VSMCs (Chan et al., 2001; Balasubramanian et al., 2008b). However, the
RGD-induced Ca2+ mobilization in renal VSMCs was ryanodine-sensitive, but
not inhibited by nifedipine or removal of extracellular Ca2+ . Moreover, mechani-
cal force applied by pulling freshly isolated renal VSMCs with fibronectin-coated
beads triggered ryanodine-sensitive local Ca2+ release (Ca2+ spark) which was
followed by global Ca2+ increase (Balasubramanian et al., 2007). In intact arte-
rioles, stepwise increase of pressure from 80 mm Hg to 120 mm Hg induced
an increase in the frequency of Ca2+ sparks (Yip et al., 2007; Balasubramanian
et al., 2008a). Preincubation with ryanodine or RGD-containing peptide inhibited
pressure-induced myogenic constriction (Balasubramanian et al., 2007). These find-
ings support the concept that ligation of integrin modulate myogenic constriction in
renal arterioles by activating Ca2+ release from the ryanodine-gated Ca2+ stores.
Similar to renal VSMCs, a study in VSMCs of rat pulmonary arteries showed
that the GRGDSP peptide (Gly-Arg-Gly-Asp-Ser-Pro) triggers Ca2+ release from
ryanodine-sensitive stores and lysosome-like organelles; and Ca2+ influx is not
required for the integrin mediated Ca2+ response (Umesh et al., 2006).
It is interesting that myogenic response and integrin activation trigger both global
and local Ca2+ response. Global mobilization of [Ca2+ ]i activates Ca2+ /calmodulin
dependent myosin light chain kinase to initiate actin-myosin interactions and
smooth muscle contraction; whereas local Ca2+ events may modulate ion channels
and Ca2+ effectors in local vicinity to regulate vascular reactivity. In cerebral
arterioles, increase in intraluminal pressure elicits myogenic response and activates
10 K.-P. Yip et al.
Ca2+ sparks (Nelson et al., 1995; Jaggar et al., 1998), which are local Ca2+ release
events originate from clusters of ryanodine receptors. It is well established that
Ca2+ sparks causes large increase in local [Ca2+ ] (10100 M) in subsarcolem-
mal microdomains and activate nearby Ca2+ activated K+ channels (KCa ), leading
to membrane hyperpolarization, reduction of Ca2+ influx via L-type Ca2+ channels
and vasodilatation. They operate as the negative feedback modulators of membrane
potential in cerebral arterioles. In contrast, the integrin mediated Ca2+ sparks in
renal arterioles contribute to the myogenic response because their inhibition with
ryanodine reversed the active myogenic vasoconstriction to passive vasodilatation
(Yip et al., 2007; Balasubramanian et al., 2008a). Integrin induced Ca2+ sparks in
renal arterioles may support vasoconstriction by functioning as elementary Ca2+
events underlying the global Ca2+ transients (Cheng et al., 1993). Alternatively,
Ca2+ sparks may activate Ca2+ activated Cl channels (ClCa )(Gordienko et al., 1999)
to initiate myogenic response via membrane depolarization and the subsequent Ca2+
entry through voltage gated Ca2+ channels. The latter is supported by the obser-
vations of spontaneous transient inward ClCa currents activated by Ca2+ sparks in
airway (Kotlikoff and Wang, 1998; ZhuGe et al., 1998; Bao et al., 2008), portal vein
(Mironneau et al., 1996), and corpus cavernosum smooth muscle cells (Williams
and Sims, 2007). A significant contribution of Ca2+ sparks in vasoconstriction has
also been suggested in rat pulmonary arterial smooth muscle cells (Remillard et al.,
2002; Zhang et al., 2003). Nevertheless, the roles of local Ca2+ signaling in integrin
mediated myogenic response require further investigations.
Fig. 1.1 Potential interactions among integrins, Ca2+ sparks, ion channels in myogenic response.
cADP cyclic adenosine diphosphate; ClCa calcium activated chloride channel; DEG/ENaC
degenerin/epithelial Na+ channel; ECM extracellular matrix; ERK extracellular signal-
regulated kinase; FAK focal adhesion kinase; KCa calcium activated potassium channel; KV
voltage gated potassium channel; MAPKs mitogen-activated protein kinases; NKCC sodium
potassium chloride cotransporter; NCX sodium calcium exchanger; NSCC non-selective
stretch-sensitive cation channel; RGD Arg-Gly-Asp motif; RyR ryanodine receptor; SR
sarcoplasmic reticulum; Src sarcoma family of tyrosine kinases; TRP transient receptor
potential superfamily of channels; VGCC voltage gated calcium channel
12 K.-P. Yip et al.
that ENaC subunits form the pore and interact with integrins, ECM and CSK to
form a larger mechanosensitive complex (Shakibaei and Mobasheri, 2003; Jernigan
and Drummond, 2005; OHagan et al., 2005; Drummond et al., 2008). There is
no direct evidence for these mechanosensitive complexes in VSMCs yet. However,
1 integrin subunit has been shown to co-localize with ENaC in mouse limb-bud
chondrocytes by co-immunoprecipitation and immunofluorescence (Shakibaei and
Mobasheri, 2003; Fig. 1.1).
was also inhibited with an antibody directed to 3 integrins (Bakker et al., 2004).
Since V 3 integrin is associated with myogenic response (Martinez-Lemus et al.,
2005), VSMC migration (Jones et al., 1996) and ECM remodeling (Bendeck et al.,
2000; Sajid and Stouffer, 2002), these observations are consistent with the notion
that ECM-V 3 interactions can trigger eutrophic remodeling.
In addition, the collagen receptor 1 1 integrin was upregulated in conduit
arteries of angiotensin induced hypertensive rats and mice (Brassard et al., 2006;
Louis et al., 2007). Evidence from 1 integrin knockout mice suggested that 1 1
integrin play a significant role in smooth muscle cell hypertrophy in conduit arter-
ies of hypertensive mice through the FAK and p38 MAPK signaling pathways
(Louis et al., 2007). Furthermore, the tissue-type transglutaminase, a cross-linking
enzyme that associates with 1 and 3 integrins to form co-receptors for fibronectin
(Akimov et al., 2000), also contributed significantly to enthothelin-1 induced inward
eutrophic remodeling in small resistance arteries (Bakker et al., 2005), suggesting
that associated protein partners of integrin are involved in the remodeling process.
growth factor receptor clustering and phosphorylation (Jones et al., 1997b, 1999).
The evidence that v 3 integrin blockade with selective antagonists induced apopto-
sis of pulmonary arterial smooth muscle cells and reduced medial wall thickness in
organ culture of pulmonary arteries of monocrotaline-treated rats (Merklinger et al.,
2005) supports an important role for v 3 integrin in hyperplasia and hypertrophic
remodeling.
The expression of integrins in pulmonary vasculature with respect to pul-
monary hypertension has not been systemically examined. Our recent prelim-
inary study found significant changes in the expression of multiple and
integrins in pulmonary arteries of chronic hypoxia and monocrotaline-induced
pulmonary hypertensive rats (Umesh and Sham, unpublished). These changes
in integrin expression could be related to the phenotypic changes of VSMCs
occur during vascular remodeling. It is noteworthy that mild myogenic tone
was observed in pulmonary microvessels of pulmonary hypertensive animals
(Broughton et al., 2008), but the involvement of integrins has not yet to be
determined.
Integrins are also major contributors in vascular remodeling and neointimal forma-
tion in vascular injury, atherosclerosis, and restenosis. 1 1 , 5 1 , V 3 , 7 and
3 integrin expression were increased (Gotwals et al., 1996; Stouffer et al., 1998;
Pickering et al., 2000; Kappert et al., 2001; Sajid and Stouffer, 2002; Chao et al.,
2004) and the expression of 8 and 1 integrins were reduced in vascular cells of
neointima formed after vascular injury (Gotwals et al., 1996; Zargham and Thibault,
2005). Among these integrins, V 3 integrin has attracted most attention. Increased
expression of V 3 integrin in the medial and neointimal layers have been observed
following mechanical injury in various animal models, including those in mice, rats,
rabbits, baboons, and pigs, and in human atherosclerotic graft and arteriopathic
coronary arteries (Hoshiga et al., 1995; Sajid and Stouffer, 2002; Sadeghi et al.,
2004; Zhang et al., 2005). Many potential ligands of V 3 , including osteopontin,
thrombospondin, prothrombin, fibronectin, vitronectin, MMP-2 were found at the
sites of injury with increased abundance. Studies using V 3 antagonists to inhibit
neointima formation and vascular remodeling suggested a central role of V 3 inte-
grins in neointima formation through multiple mechanisms including reduction of
VSMC proliferation and migration, MMP production, and increased smooth mus-
cle cell apoptosis (Slepian et al., 1998; van der Zee et al., 1998; Bendeck et al.,
2000; Dufourcq et al., 2002; Honda et al., 2005). Similar to V 3 antagonists, 3
integrin blockade with peptide inhibitors or anti-3 antibodies reduced neointimal
thickening, VSMC migration and proliferation, MMP activity in injured arteries
(Slepian et al., 1998; Stouffer et al., 1998; Bendeck and Nakada, 2001). The 3 inte-
grin mediated VSMC migration could be related to activation of 3 -FAK signaling
1 Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells 15
pathway by osteopontin (Yue et al., 1994; Wang et al., 1996; Han et al., 2007). In
contrast to pharmacological inhibition of 3 -integrin, deletion of 3 -integrin gene in
mice provides no protection to transluminal probe induced mechanical injury, but
inhibited VSMC accumulation in neointima after milder injury induced by carotid
ligation (Smyth et al., 2001; Choi et al., 2004). The discrepancy could be explained
by species differences in the response to vascular injury, mechanistic differences
between 3 -integrin deficiency and blockade with antagonists, and compensatory
mechanisms in 3 -integrin knockout animals.
Other integrins, such as 1 1 , 5 1 , 7 1 and 8 1 , may also contribute to neoin-
tima formation. For example, the expression of 8 1 integrin is downregulated in
the medial layer in the early stage and is upregulated in the intima later during
inward constrictive remodeling after balloon angioplasty (Zargham and Thibault,
2005; Zargham et al., 2007). It has been shown that 8 integrin is required for main-
taining the contractile, differentiated phenotype of VSMCs (Zargham and Thibault,
2006). Gene silencing of 8 integrin increased VSMC migration and expression of
smooth muscle cell de-differentiation markers; and overexpression of 8 integrin
attenuated smooth muscle cell migratory activity and restored contractile pheno-
types (Zargham et al., 2007a, b). The alteration in 8 1 integrin expression, hence,
correlated with de-differentiation of VSMCs for migration during early neoin-
tima formation, and the contractile phenotype of VSMCs during late constrictive
remodeling in restenosis.
References
Abu-Amarah I, Bidani AK, Hacioglu R, Williamson GA, Griffin KA (2005) Differential
effects of salt on renal hemodynamics and potential pressure transmission in stroke-prone
and stroke-resistant spontaneously hypertensive rats. Am J Physiol Renal Physiol 289(2):
F305F313
Akimov SS, Krylov D, Fleischman LF, Belkin AM (2000) Tissue transglutaminase is an integrin-
binding adhesion coreceptor for fibronectin. J Cell Biol 148(4):825838
Bakker EN, Buus CL, Spaan JA, Perree J, Ganga A, Rolf TM, Sorop O, Bramsen LH, Mulvany MJ,
Vanbavel E (2005) Small artery remodeling depends on tissue-type transglutaminase. Circ Res
96(1):119126
Bakker EN, Buus CL, VanBavel E, Mulvany MJ (2004) Activation of resistance arteries with
endothelin-1: from vasoconstriction to functional adaptation and remodeling. J Vasc Res
41(2):174182
Bakker EN, van der Meulen ET, van den Berg BM, Everts V, Spaan JA, VanBavel E (2002)
Inward remodeling follows chronic vasoconstriction in isolated resistance arteries. J Vasc Res
39(1):1220
Balasubramanian L, Ahmed A, Lo CM, Sham JS, Yip KP (2007) Integrin-mediated mechanotrans-
duction in renal vascular smooth muscle cells: activation of calcium sparks. Am J Physiol Regul
Integr Comp Physiol 293(4):R1586R1594
Balasubramanian L, Sham JS, Yip KP (2008a) Effects of perfusion pressure on Ca2+ sparks in rat
renal afferent arterioles. Faseb J 22:761.21
Balasubramanian L, Yip KP, Hsu TH, Lo CM (2008b) Impedance analysis of renal vascular smooth
muscle cells. Am J Physiol Cell Physiol 295(4):C954C965
Bao R, Lifshitz LM, Tuft RA, Bellve K, Fogarty KE, ZhuGe R (2008) A close association of RyRs
with highly dense clusters of Ca2+ -activated Cl channels underlies the activation of STICs by
Ca2+ sparks in mouse airway smooth muscle. J Gen Physiol 132(1):145160
Bayliss WM (1902) On the local reactions of the arterial wall to changes of internal pressure. J
Physiol 28:220231
Bendeck MP, Irvin C, Reidy M, Smith L, Mulholland D, Horton M, Giachelli CM (2000) Smooth
muscle cell matrix metalloproteinase production is stimulated via alpha(v)beta(3) integrin.
Arterioscler Thromb Vasc Biol 20(6):14671472
Bendeck MP, Nakada MT (2001) The beta3 integrin antagonist m7E3 reduces matrix metallopro-
teinase activity and smooth muscle cell migration. J Vasc Res 38(6):590599
Bendig G, Grimmler M, Huttner IG, Wessels G, Dahme T, Just S, Trano N, Katus HA, Fishman
MC, Rottbauer W (2006) Integrin-linked kinase, a novel component of the cardiac mechanical
stretch sensor, controls contractility in the zebrafish heart. Genes Dev 20(17):23612372
Bezie Y, Lamaziere JM, Laurent S, Challande P, Cunha RS, Bonnet J, Lacolley P (1998)
Fibronectin expression and aortic wall elastic modulus in spontaneously hypertensive rats.
Arterioscler Thromb Vasc Biol 18(7):10271034
Bidani AK, Schwartz MM, Lewis EJ (1987) Renal autoregulation and vulnerability to hypertensive
injury in remnant kidney. Am J Physiol 252(6 Pt 2):F1003F1010
Botney MD, Kaiser LR, Cooper JD, Mecham RP, Parghi D, Roby J, Parks WC (1992) Extracellular
matrix protein gene expression in atherosclerotic hypertensive pulmonary arteries. Am J Pathol
140(2):357364
Boudreau NJ, Jones PL (1999) Extracellular matrix and integrin signalling: the shape of things to
come. Biochem J 339 (Pt 3):481488
Brassard P, Amiri F, Thibault G, Schiffrin EL (2006) Role of angiotensin type-1 and angiotensin
type-2 receptors in the expression of vascular integrins in angiotensin II-infused rats.
Hypertension 47(1):122127
Broughton BR, Walker BR, Resta TC (2008) Chronic hypoxia induces Rho kinase-dependent
myogenic tone in small pulmonary arteries. Am J Physiol Lung Cell Mol Physiol 294(4):
L797L806
1 Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells 17
Buck CA, Edelman JM, Buck CE, Kennedy G, Baldwin HS (1996) Expression patterns of adhesion
receptors in the developing mouse lung: functional implications. Cell Adhes Commun 4(2):
6987
Chan WL, Holstein-Rathlou NH, Yip KP (2001) Integrin mobilizes intracellular Ca2+ in renal
vascular smooth muscle cells. Am J Physiol Cell Physiol 280(3):C593C603
Chao JT, Meininger GA, Patterson JL, Jones SA, Partridge CR, Neiger JD, Williams ES, Kaufman
SJ, Ramos KS, Wilson E (2004) Regulation of alpha7-integrin expression in vascular smooth
muscle by injury-induced atherosclerosis. Am J Physiol Heart Circ Physiol 287(1):H381H389
Chen HC, Appeddu PA, Isoda H, Guan JL (1996) Phosphorylation of tyrosine 397 in focal
adhesion kinase is required for binding phosphatidylinositol 3-kinase. J Biol Chem 271(42):
2632926334
Chen J, Fabry B, Schiffrin EL, Wang N (2001) Twisting integrin receptors increases endothelin-1
gene expression in endothelial cells. Am J Physiol Cell Physiol 280(6):C1475C1484
Cheng H, Lederer WJ, Cannell MB (1993) Calcium sparks: elementary events underlying
excitation-contraction coupling in heart muscle. Science 262(5134):740744
Choi ET, Khan MF, Leidenfrost JE, Collins ET, Boc KP, Villa BR, Novack DV, Parks WC,
Abendschein DR (2004) Beta3-integrin mediates smooth muscle cell accumulation in neoin-
tima after carotid ligation in mice. Circulation 109(12):15641569
Cowan KN, Heilbut A, Humpl T, Lam C, Ito S, Rabinovitch M (2000) Complete reversal of fatal
pulmonary hypertension in rats by a serine elastase inhibitor. Nat Med 6(6):698702
Dajnowiec D, Langille BL (2007) Arterial adaptations to chronic changes in haemodynamic
function: coupling vasomotor tone to structural remodelling. Clin Sci (Lond) 113(1):
1523
Dajnowiec D, Sabatini PJ, Van Rossum TC, Lam JT, Zhang M, Kapus A, Langille BL (2007)
Force-induced polarized mitosis of endothelial and smooth muscle cells in arterial remodeling.
Hypertension 50(1):255260
Damjanovich L, Albelda SM, Mette SA, Buck CA (1992) Distribution of integrin cell adhesion
receptors in normal and malignant lung tissue. Am J Respir Cell Mol Biol 6(2):197206
DAngelo G, Davis MJ, Meininger GA (1997) Calcium and mechanotransduction of the myogenic
response. Am J Physiol 273(1 Pt 2):H175H182
Davis MJ, Donovitz JA, Hood JD (1992) Stretch-activated single-channel and whole cell currents
in vascular smooth muscle cells. Am J Physiol 262(4 Pt 1):C1083C1088
Davis MJ, Hill MA (1999) Signaling mechanisms underlying the vascular myogenic response.
Physiol Rev 79(2):387423
Davis MJ, Wu X, Nurkiewicz TR, Kawasaki J, Davis GE, Hill MA, Meininger GA (2001) Integrins
and mechanotransduction of the vascular myogenic response. Am J Physiol Heart Circ Physiol
280(4):H1427H1433
Dedhar S (2000) Cell-substrate interactions and signaling through ILK. Curr Opin Cell Biol
12(2):250256
Deng JT, Van Lierop JE, Sutherland C, Walsh MP (2001) Ca2+ -independent smooth muscle
contraction. a novel function for integrin-linked kinase. J Biol Chem 276(19):1636516373
Drummond HA, Gebremedhin D, Harder DR (2004) Degenerin/epithelial Na+ channel proteins:
components of a vascular mechanosensor. Hypertension 44(5):643648
Drummond HA, Grifoni SC, Jernigan NL (2008) A new trick for an old dogma: ENaC proteins as
mechanotransducers in vascular smooth muscle. Physiology (Bethesda) 23:2331
Dufourcq P, Couffinhal T, Alzieu P, Daret D, Moreau C, Duplaa C, Bonnet J (2002) Vitronectin
is up-regulated after vascular injury and vitronectin blockade prevents neointima formation.
Cardiovasc Res 53(4):952962
Earley S, Waldron BJ, Brayden JE (2004) Critical role for transient receptor potential channel
TRPM4 in myogenic constriction of cerebral arteries. Circ Res 95(9):922929
Facemire CS, Mohler PJ, Arendshorst WJ (2004) Expression and relative abundance of short tran-
sient receptor potential channels in the rat renal microcirculation. Am J Physiol Renal Physiol
286(3):F546F551
18 K.-P. Yip et al.
Falcone JC, Granger HJ, Meininger GA (1993) Enhanced myogenic activation in skeletal muscle
arterioles from spontaneously hypertensive rats. Am J Physiol 265(6 Pt 2):H1847H1855
Falcone JC, Meininger GA (1999) Endothelin mediates a component of the enhanced myogenic
responsiveness of arterioles from hypertensive rats. Microcirculation 6(4):305313
Friedrich EB, Liu E, Sinha S, Cook S, Milstone DS, MacRae CA, Mariotti M, Kuhlencordt
PJ, Force T, Rosenzweig A, St-Arnaud R, Dedhar S, Gerszten RE (2004) Integrin-linked
kinase regulates endothelial cell survival and vascular development. Mol Cell Biol 24(18):
81348144
Glogauer M, Ferrier J, McCulloch CA (1995) Magnetic fields applied to collagen-coated fer-
ric oxide beads induce stretch-activated Ca2+ flux in fibroblasts. Am J Physiol 269(5 Pt 1):
C1093C1104
Glukhova MA, Frid MG, Koteliansky VE (1994) Phenotypic changes of human aortic
smooth muscle cells during development and in adult. J Atheroscler Thromb 1 Suppl 1:
S47S49
Goldschmidt ME, McLeod KJ, Taylor WR (2001) Integrin-mediated mechanotransduction in
vascular smooth muscle cells: frequency and force response characteristics. Circ Res 88(7):
674680
Gordienko DV, Zholos AV, Bolton TB (1999) Membrane ion channels as physiological targets for
local Ca2+ signalling. J Microsc 196(Pt 3):305316
Gotwals PJ, Chi-Rosso G, Lindner V, Yang J, Ling L, Fawell SE, Koteliansky VE (1996) The
alpha1beta1 integrin is expressed during neointima formation in rat arteries and mediates
collagen matrix reorganization. J Clin Invest 97(11):24692477
Guan Z, Pollock JS, Cook AK, Hobbs JL, Inscho EW (2009) Effect of Epithelial Sodium Channel
Blockade on the Myogenic Response of Rat Juxtamedullary Afferent Arterioles. Hypertension
54:10621069
Gui P, Wu X, Ling S, Stotz SC, Winkfein RJ, Wilson E, Davis GE, Braun AP, Zamponi GW,
Davis MJ (2006) Integrin receptor activation triggers converging regulation of Cav1.2 calcium
channels by c-Src and protein kinase A pathways. J Biol Chem 281(20):1401514025
Han M, Wen JK, Zheng B, Liu Z, Chen Y (2007) Blockade of integrin beta3-FAK signaling path-
way activated by osteopontin inhibits neointimal formation after balloon injury. Cardiovasc
Pathol 16(5):283290
Hartner A, Cordasic N, Klanke B, Muller U, Sterzel RB, Hilgers KF (2002) The alpha8 integrin
chain affords mechanical stability to the glomerular capillary tuft in hypertensive glomerular
disease. Am J Pathol 160(3):861867
Hassoun PM (2005) Deciphering the matrix in pulmonary vascular remodelling. Eur Respir J
25(5):778779
Heerkens EH, Shaw L, Ryding A, Brooker G, Mullins JJ, Austin C, Ohanian V, Heagerty AM
(2006) alphaV integrins are necessary for eutrophic inward remodeling of small arteries in
hypertension. Hypertension 47(2):281287
Honda Y, Kitano T, Fukuya F, Sato Y, Iwama S, Morie T, Notake M (2005) A Novel alphavbeta3
integrin antagonist suppresses neointima formation for more than 4 weeks after balloon injury
in rats. Arterioscler Thromb Vasc Biol 25(7):13761382
Hoshiga M, Alpers CE, Smith LL, Giachelli CM, Schwartz SM (1995) Alpha-v beta-3 integrin
expression in normal and atherosclerotic artery. Circ Res 77(6):11291135
Hu Y, Bock G, Wick G, Xu Q (1998) Activation of PDGF receptor alpha in vascular smooth muscle
cells by mechanical stress. Faseb J 12(12):11351142
Ihida-Stansbury K, McKean DM, Lane KB, Loyd JE, Wheeler LA, Morrell NW, Jones PL (2006)
Tenascin-C is induced by mutated BMP type II receptors in familial forms of pulmonary arterial
hypertension. Am J Physiol Lung Cell Mol Physiol 291(4):L694L702
Ingber DE (1990) Fibronectin controls capillary endothelial cell growth by modulating cell shape.
Proc Natl Acad Sci USA 87(9):35793583
Ingber DE (1997) Integrins, tensegrity, and mechanotransduction. Gravit Space Biol Bull 10(2):
4955
1 Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells 19
Ingber DE (2000) Opposing views on tensegrity as a structural framework for understanding cell
mechanics. J Appl Physiol 89(4):16631670
Ingber DE (2003a) Tensegrity I. Cell structure and hierarchical systems biology. J Cell Sci 116
(Pt 7):11571173
Ingber DE (2003b) Tensegrity II. How structural networks influence cellular information process-
ing networks. J Cell Sci 116(Pt 8):13971408
Inoue R, Jensen LJ, Shi J, Morita H, Nishida M, Honda A, Ito Y (2006) Transient receptor potential
channels in cardiovascular function and disease. Circ Res 99(2):119131
Intengan HD, Schiffrin EL (2000) Structure and mechanical properties of resistance arteries in
hypertension: role of adhesion molecules and extracellular matrix determinants. Hypertension
36(3):312318
Intengan HD, Thibault G, Li JS, Schiffrin EL (1999) Resistance artery mechanics, structure, and
extracellular components in spontaneously hypertensive rats : effects of angiotensin receptor
antagonism and converting enzyme inhibition. Circulation 100(22):22672275
Jaggar JH, Stevenson AS, Nelson MT (1998) Voltage dependence of Ca2+ sparks in intact cerebral
arteries. Am J Physiol 274(6 Pt 1):C1755C1761
Jernigan NL, Drummond HA (2005) Vascular ENaC proteins are required for renal myogenic
constriction. Am J Physiol Renal Physiol 289(4):F891F901
Johnson PC (1980). The Handbook of Physiology. The Cardiovascular System. Vascular Smooth
Muscle. Am. Physiol. Soc., Bethesda, MD
Jones FS, Meech R, Edelman DB, Oakey RJ, Jones PL (2001) Prx1 controls vascular smooth
muscle cell proliferation and tenascin-C expression and is upregulated with Prx2 in pulmonary
vascular disease. Circ Res 89(2):131138
Jones JI, Prevette T, Gockerman A, Clemmons DR (1996) Ligand occupancy of the alpha-V-beta3
integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth
factor. Proc Natl Acad Sci USA 93(6):24822487
Jones PL, Cowan KN, Rabinovitch M (1997a) Tenascin-C, proliferation and subendothelial
fibronectin in progressive pulmonary vascular disease. Am J Pathol 150(4):13491360
Jones PL, Crack J, Rabinovitch M (1997b) Regulation of tenascin-C, a vascular smooth muscle
cell survival factor that interacts with the alpha v beta 3 integrin to promote epidermal growth
factor receptor phosphorylation and growth. J Cell Biol 139(1):279293
Jones PL, Jones FS, Zhou B, Rabinovitch M (1999) Induction of vascular smooth muscle cell
tenascin-C gene expression by denatured type I collagen is dependent upon a beta3 integrin-
mediated mitogen-activated protein kinase pathway and a 122-base pair promoter element.
J Cell Sci 112 (Pt 4):435445
Kappert K, Blaschke F, Meehan WP, Kawano H, Grill M, Fleck E, Hsueh WA, Law RE, Graf
K (2001) Integrins alphavbeta3 and alphavbeta5 mediate VSMC migration and are elevated
during neointima formation in the rat aorta. Basic Res Cardiol 96(1):4249
Karlsen FM, Andersen CB, Leyssac PP, Holstein-Rathlou NH (1997) Dynamic autoregulation and
renal injury in Dahl rats. Hypertension 30(4):975983
Katsumi A, Orr AW, Tzima E, Schwartz MA (2004) Integrins in mechanotransduction. J Biol
Chem 279(13):1200112004
Keller M, Lidington D, Vogel L, Peter BF, Sohn HY, Pagano PJ, Pitson S, Spiegel S, Pohl U, Bolz
SS (2006) Sphingosine kinase functionally links elevated transmural pressure and increased
reactive oxygen species formation in resistance arteries. Faseb J 20(6):702704
Kirber MT, Walsh JV Jr, Singer JJ (1988) Stretch-activated ion channels in smooth mus-
cle: a mechanism for the initiation of stretch-induced contraction. Pflugers Arch 412(4):
339345
Kotlikoff MI, Wang YX (1998) Calcium release and calcium-activated chloride channels in airway
smooth muscle cells. Am J Respir Crit Care Med 158(5 Pt 3):S109S114
Lal H, Verma SK, Foster DM, Golden HB, Reneau JC, Watson LE, Singh H, Dostal DE (2009)
Integrins and proximal signaling mechanisms in cardiovascular disease. Front Biosci 14:
23072334
20 K.-P. Yip et al.
Lin MJ, Leung GP, Zhang WM, Yang XR, Yip KP, Tse CM, Sham JS (2004) Chronic hypoxia-
induced upregulation of store-operated and receptor-operated Ca2+ channels in pulmonary
arterial smooth muscle cells: a novel mechanism of hypoxic pulmonary hypertension. Circ
Res 95(5):496505
Louis H, Kakou A, Regnault V, Labat C, Bressenot A, Gao-Li J, Gardner H, Thornton SN,
Challande P, Li Z, Lacolley P (2007) Role of alpha1beta1-integrin in arterial stiffness and
angiotensin-induced arterial wall hypertrophy in mice. Am J Physiol Heart Circ Physiol
293(4):H2597H2604
MacKenna DA, Dolfi F, Vuori K, Ruoslahti E (1998) Extracellular signal-regulated kinase and
c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-
specific in rat cardiac fibroblasts. J Clin Invest 101(2):301310
Maroto R, Raso A, Wood TG, Kurosky A, Martinac B, Hamill OP (2005) TRPC1 forms the stretch-
activated cation channel in vertebrate cells. Nat Cell Biol 7(2):179185
Marsh DJ, Osborn JL, Cowley AW, Jr. (1990) 1/f fluctuations in arterial pressure and regulation of
renal blood flow in dogs. Am J Physiol 258(5 Pt 2):F1394F1400
Martinez-Lemus LA, Crow T, Davis MJ, Meininger GA (2005) alphavbeta3- and alpha5beta1-
integrin blockade inhibits myogenic constriction of skeletal muscle resistance arterioles. Am J
Physiol Heart Circ Physiol 289(1):H322H329
Martinez-Lemus LA, Hill MA, Bolz SS, Pohl U, Meininger GA (2004) Acute mechanoadap-
tation of vascular smooth muscle cells in response to continuous arteriolar vasoconstriction:
implications for functional remodeling. Faseb J 18(6):708710
Martinez-Lemus LA, Wu X, Wilson E, Hill MA, Davis GE, Davis MJ, Meininger GA (2003)
Integrins as unique receptors for vascular control. J Vasc Res 40(3):211233
Medhora MM (2000) Retinoic acid upregulates beta(1)-integrin in vascular smooth muscle cells
and alters adhesion to fibronectin. Am J Physiol Heart Circ Physiol 279(1):H382H387
Meininger GA, Davis MJ (1992) Cellular mechanisms involved in the vascular myogenic response.
Am J Physiol 263(3 Pt 2):H647H659
Merklinger SL, Jones PL, Martinez EC, Rabinovitch M (2005) Epidermal growth factor receptor
blockade mediates smooth muscle cell apoptosis and improves survival in rats with pulmonary
hypertension. Circulation 112(3):423431
Mironneau J, Arnaudeau S, Macrez-Lepretre N, Boittin FX (1996) Ca2+ sparks and Ca2+ waves
activate different Ca(2+)-dependent ion channels in single myocytes from rat portal vein. Cell
Calcium 20(2):153160
Moiseeva EP (2001) Adhesion receptors of vascular smooth muscle cells and their functions.
Cardiovasc Res 52(3):372386
Mori T, Cowley AW Jr (2004) Role of pressure in angiotensin II-induced renal injury: chronic
servo-control of renal perfusion pressure in rats. Hypertension 43(4):752759
Morita H, Honda A, Inoue R, Ito Y, Abe K, Nelson MT, Brayden JE (2007) Membrane stretch-
induced activation of a TRPM4-like nonselective cation channel in cerebral artery myocytes.
J Pharmacol Sci 103(4):417426
Muraki K, Iwata Y, Katanosaka Y, Ito T, Ohya S, Shigekawa M, Imaizumi Y (2003) TRPV2 is
a component of osmotically sensitive cation channels in murine aortic myocytes. Circ Res
93(9):829838
Muranyi A, MacDonald JA, Deng JT, Wilson DP, Haystead TA, Walsh MP, Erdodi F, Kiss E,
Wu Y, Hartshorne DJ (2002) Phosphorylation of the myosin phosphatase target subunit by
integrin-linked kinase. Biochem J 366(Pt 1):211216
Na S, Collin O, Chowdhury F, Tay B, Ouyang M, Wang Y, Wang N (2008) Rapid signal trans-
duction in living cells is a unique feature of mechanotransduction. Proc Natl Acad Sci USA
105(18):66266631
Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, Lederer WJ (1995) Relaxation
of arterial smooth muscle by calcium sparks. Science 270(5236):633637
Novotna J, Herget J (1998) Exposure to chronic hypoxia induces qualitative changes of collagen
in the walls of peripheral pulmonary arteries. Life Sci 62(1):112
1 Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells 21
Smyth SS, Reis ED, Vaananen H, Zhang W, Coller BS (2001) Variable protection of beta
3-integrindeficient mice from thrombosis initiated by different mechanisms. Blood 98(4):
10551062
Spassova MA, Hewavitharana T, Xu W, Soboloff J, Gill DL (2006) A common mechanism under-
lies stretch activation and receptor activation of TRPC6 channels. Proc Natl Acad Sci USA
103(44):1658616591
Stenmark KR, Fagan KA, Frid MG (2006) Hypoxia-induced pulmonary vascular remodeling:
cellular and molecular mechanisms. Circ Res 99(7):675691
Stenmark KR, Meyrick B, Galie N, Mooi WJ, McMurtry IF (2009) Animal models of pul-
monary arterial hypertension: the hope for etiological discovery and pharmacological cure.
Am J Physiol Lung Cell Mol Physiol 297(6):L1013L1032
Stouffer GA, Hu Z, Sajid M, Li H, Jin G, Nakada MT, Hanson SR, Runge MS (1998)
Beta3 integrins are upregulated after vascular injury and modulate thrombospondin-
and thrombin-induced proliferation of cultured smooth muscle cells. Circulation 97(9):
907915
Sun Z, Martinez-Lemus LA, Hill MA, Meininger GA (2008) Extracellular matrix-specific focal
adhesions in vascular smooth muscle produce mechanically active adhesion sites. Am J Physiol
Cell Physiol 295(1):C268C278
Umesh A, Thompson MA, Chini EN, Yip KP, Sham JS (2006) Integrin Ligands Mobilize Ca2+
from Ryanodine Receptor-gated Stores and Lysosome-related Acidic Organelles in Pulmonary
Arterial Smooth Muscle Cells. J Biol Chem 281(45):3431234323
van der Zee R, Murohara T, Passeri J, Kearney M, Cheresh DA, Isner JM (1998) Reduced intimal
thickening following alpha(v)beta3 blockade is associated with smooth muscle cell apoptosis.
Cell Adhes Commun 6(5):371379
van Dokkum RP, Sun CW, Provoost AP, Jacob HJ, Roman RJ (1999) Altered renal hemody-
namics and impaired myogenic responses in the fawn-hooded rat. Am J Physiol 276(3 Pt 2):
R855R863
Vecchione C, Carnevale D, Di Pardo A, Gentile MT, Damato A, Cocozza G, Antenucci G,
Mascio G, Bettarini U, Landolfi A, Iorio L, Maffei A, Lembo G (2009) Pressure-
Induced Vascular Oxidative Stress Is Mediated Through Activation of Integrin-Linked Kinase
1/{beta}PIX/Rac-1 Pathway. Hypertension 54(5):964965
Velling T, Nilsson S, Stefansson A, Johansson S (2004) beta1-Integrins induce phosphorylation of
Akt on serine 473 independently of focal adhesion kinase and Src family kinases. EMBO Rep
5(9):901905
Vuori K (1998) Integrin signaling: tyrosine phosphorylation events in focal adhesions. J Membr
Biol 165(3):191199
Waitkus-Edwards KR, Martinez-Lemus LA, Wu X, Trzeciakowski JP, Davis MJ, Davis GE,
Meininger GA (2002) alpha(4)beta(1) Integrin activation of L-type calcium channels in
vascular smooth muscle causes arteriole vasoconstriction. Circ Res 90(4):473480
Wang N (1998) Mechanical interactions among cytoskeletal filaments. Hypertension 32(1):
162165
Wang N, Butler JP, Ingber DE (1993) Mechanotransduction across the cell surface and through the
cytoskeleton. Science 260:11241127
Wang N, Ingber DE (1994) Control of cytoskeletal mechanics by extracellular matrix, cell shape,
and mechanical tension. Biophys J 66(6):21812189
Wang N, Ingber DE (1995) Probing transmembrane mechanical coupling and cytomechanics using
magnetic twisting cytometry. Biochem Cell Biol 73(78):327335
Wang N, Naruse K, Stamenovic D, Fredberg JJ, Mijailovich SM, Tolic-Norrelykke IM, Polte T,
Mannix R, Ingber DE (2001) Mechanical behavior in living cells consistent with the tensegrity
model. Proc Natl Acad Sci USA 98(14):77657770
Wang X, Louden C, Ohlstein EH, Stadel JM, Gu JL, Yue TL (1996) Osteopontin expression in
platelet-derived growth factor-stimulated vascular smooth muscle cells and carotid artery after
balloon angioplasty. Arterioscler Thromb Vasc Biol 16(11):13651372
1 Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells 23
Abstract Cells are capable of sensing mechanical stimuli and translating them into
biochemical signals. This ability allows cells to adapt to their physical surround-
ings by remodeling their cytoskeleton, activating various signaling pathways, and
changing their gene expression. These phenomena involve two essential processes
mechanosensing and mechanotransduction. In these processes, force or deformation
needs to be transmitted from the outside environment to the proteins and organelles
inside the cell. The actin cytoskeleton composed of actin filaments, myosin motors,
and actin crosslinking proteins plays a critical role in force propagation and in
response to deformations. Cellular adaptation to these deformations is often asso-
ciated with feedback loops, and proteins in the actin cytoskeleton accumulate and
function cooperatively in response to mechanical stimuli. Mutations in these pro-
teins cause failure in cellular mechanosensing, which eventually leads to cellular
errors associated with disease progression.
2.1 Introduction
sense the mechanical stimuli, such as force, pressure and flow speed, and trans-
mit the mechanical signals into the inside of cells. Ion channels, protein kinases,
integrins, membrane glycocalyx, G proteins, intercellular junction proteins and
other membrane-associated signal-transduction molecules are capable of sensing
mechanical stimuli as shown in Fig. 2.1 (Ingber, 2006; Vogel and Sheetz, 2006;
Wang et al., 2009). The transducers, mainly situated inside the cell membrane
or inside the cytosol, convert the mechanical signals to chemical and biological
signals.
Physiological examples of mechanosensation include hearing (Fettiplace and
Hackney, 2006), blood flow regulation in the circulatory system (Chien, 2007), and
bone remodeling (Robling et al., 2006). Auditory sensing occurs in the inner ear
in a region known as the cochlea that is covered with hair cells characterized by
stereocilia. The mechanoelectrical transduction (MET) ion channels in one stere-
ocilium are linked to a neighboring stereocilium through tip linkers. These linkages
are then coupled internally to the core actin bundles through myosin I motors. When
one stereocilium is flexed due to sound vibrations, this induces tension along the
tip link, leading to channel opening and conversion of the sound into an electrical
signal. Subsequent adaption and associated closing of the METs occurs partially
through the unbinding of myosin I from the actin bundles. In the circulatory sys-
tem, endothelial cells sense the shear and stretch forces due to blood flow and
activate a number of mechanosensors, such as membrane proteins, integrins, G
proteins and ion channels. The mechanosensing triggers a cascade of signaling
pathways and consequently modulates gene expression, resulting in cytoskeleton
remodeling and cell realignment. Bone adapts its structure to mechanical stimuli
and repairs structural damage through remodeling. At bone surfaces, osteoclasts and
osteoblasts control the bone resorption and formation, respectively. However, the
osteocytes, which are embedded deep within the bone, appear to have the primary
job of sensing where the bone needs to be remodeled and then relaying this infor-
mation to the osteoblasts and osteoclasts located on the bone surface. One appealing
model suggests that the osteocytes sense changes in fluid flow inside canaliculi and
the associated viscous drag creates tensile forces along the central actin filament
bundle in the osteocytes. The osteocytes then release second messengers such as
prostaglandins and nitric oxide, which activate the osteoblasts and osteoclasts. At
the molecular level, the bone mechanotransduction is also thought to involve ion
channels, focal adhesions, and G protein-coupled receptors, but detailed molecular
mechanisms are still unclear.
The actin cytoskeleton composed of actin filaments, actin crosslinking proteins
(ACLPs) and myosin II motors, is unambiguously involved in these mechanosens-
ing processes. To understand how mechanical stimuli are propagated through actin
filaments and how the actin cytoskeleton responds to these stimuli, it is essential
to understand the mechanical behaviors of the individual players, the reconstituted
networks in vitro and the whole network in vivo.
Fig. 2.2 Domain structure of myosin II. HMM and LMM represent heavy meromyosin and light
meromyosin, respectively
2 The Role of the Actin Cytoskeleton in Mechanosensation 29
Fig. 2.4 Schematic graph of a few actin crosslinking proteins:(a) human filamin A; (b) -
actinin; (c) cortexillin-I. In all cases, N and C represent the N-terminus and C-terminus,
respectively
and fascin, are monomeric with at least two actin-binding motifs, allowing them
to crosslink as monomers. By contrast, other ACLPs, such as filamin A, -actinin,
dynacortin, and cortexillin are dimeric, and filamin A, cortexillin, and most likely
dynacortin form parallel dimers while -actinin forms anti-parallel dimers. Actin-
binding domains are localized at the amino-terminus of filamin A and -actinin. Due
to the differences in their structures, Y-shaped filamin A dimers can only crosslink
actin filaments into nearly orthogonal networks while -actinin forms either net-
works or loose bundles. Some of the most common filamentous actin-binding
domains (ABDs) are the calponin homology (CH) domain, the Wiskott-Aldrich syn-
drome homology region 2 (WH2 ) domain, the gelsolin homology (GH) domain, and
the formin homology (FH) domain (Sjblom et al., 2008). Different ABDs generally
associate with different surfaces on the actin monomer and with different apparent
affinities.
These network structures may experience four types of deformations (Vogel,
2006; Ferrer et al., 2008): (1) deformation in actin filaments, (2) deformation of
the binding between actin filaments and myosin-II, (3) deformation of the bind-
ing between actin filaments and ACLPs and (4) intramolecular deformation of
ACLPs. In other words, intermolecular and intramolecular deformations exist at
the same time within the same network. One additional feature of how these net-
works respond to deformation is the relationship of the time-scale of the deformation
to the time-scale of the turnover/remodeling of the network. For example, pro-
filin binds monomeric actin, inducing it to exchange its nucleotide and shuttling
the actin monomer to sites where elongation has been stimulated. By contrast,
cofilin binds cooperatively to sever the actin filaments, which has a complicated
effect on the actin network. Severing provides free actin plus ends that may
grow whereas the free minus ends can disassemble. These proteins influence the
time-scales over which the actin polymers can grow and disassemble, modulat-
ing the time-scales of remodeling of the actin network. These features of network
remodeling may provide a phenomenological viscous character to the network,
allowing it to flow and remodel over the long time-scales of many biological
processes.
2 The Role of the Actin Cytoskeleton in Mechanosensation 31
D Energy
slip bond pathway catch bond pathway
bound
state
unbound unbound
state state
Force Direction
Fig. 2.5 A schematic diagram of the force dependence of protein-protein interaction strength:
(a) catch-bond model; (b) slip-bond model; (c) three typical bond behaviors; (d) two pathways
between bound state and unbound state. Red and blue lines are energy landscapes in the presence
and absence of external load, respectively
32 T. Luo and D.N. Robinson
the force direction. When the force pulls the system close to the transition state,
i.e., f x > 0, Eq. (2.1) describes the slip-bond model; however, when f x < 0,
it refers to the catch-bond model. Equation (2.1) can only be applied to a single
pathway (either catch-bond or slip-bond). However, sometimes there are multiple
pathways between the bound state and unbound state, and catch-slip and slip-catch
transitions usually occur (as shown in Fig. 2.5). In that case, the unbinding rate is a
superposition of the rates of two different pathways:
f xs f xc
koff = ks0 exp + kc0 exp , (2.2)
kB T kB T
dP (t)
= koff (f ) P (t) , (2.3)
dt
where rf is the loading rate and f (t) = rf t (Ackbarow et al., 2007). Equation (2.4)
predicts a linear relationship between the rupture force and logarithm of the constant
loading
rate. If the intermolecular interaction involves multiple bonds, the f ln
rf will show different slopes associated with different bond energies.
Intramolecular interactions include covalent bonds, hydrogen bonds, electrostatic
interactions, van der Waals interactions, and hydrophobic interactions. Mechanical
deformation can cause twisting and stretching of a single bond or the whole protein,
general conformational changes and the unfolding of domains. Since intramolecular
interactions are much more complicated than intermolecular interactions, there is no
simple mathematic model to describe how mechanical force affects the intramolec-
ular interactions even though protein unfolding can still be probed experimentally
using similar techniques. However, for a linear polyprotein, such as an actin filament
and titin, the force-extension relationship can be predicted using a worm-like-chain
(WLC) model that was originally developed to describe the mechanical behaviors of
double stranded DNA molecules (Bustamante et al., 1994; Marko and Siggia, 1995):
2 The Role of the Actin Cytoskeleton in Mechanosensation 33
kB T 1 x 1
f = 2 + , (2.5)
Lp 4 1 x Lc Lc 4
where Lp is the persistence length, Lc is the contour length, and x is the exten-
sion. Another popular model of the force-extension of proteins is the free-joint-chain
(FJC) model (Bueche, 1962):
fl kB T
x = Lc coth , (2.6)
kB T fl
where l is the Kuhn monomer size. In the WLC model, the force is written as a
function of the chain extension while in FJC model the extension is written as a func-
tion of the force. However mathematically, both models give similar force-extension
curves.
theoretical estimate implies that simply by increasing the free actin concentration,
greater forces may be generated by filament assembly. However, this process is
limited by the flexural rigidity of the filament, which sets a force limit beyond
which the filament buckles. However, actin-associated proteins may increase the
flexural rigidity, increasing the range of forces that might be generated by filament
assembly.
motor performs work as it undergoes its conformational change. For most myosin
family members (with myosin VI having some unique and exquisitely complicated
twists on this theme (Phichith et al., 2009)), the head region is highly rigid while
the lever arm is considered to be elastic. Furthermore, the ADP-release step itself
is not force-sensitive for most myosin isoforms. Rather the conformational changes
that precede the formation of the ADP-bound post-stroke configuration are force-
sensitive. Therefore, as the motor swings the elastic lever arm through its power
stroke, resistive tension can lead to deformation (strain) of the lever arm, inhibit-
ing the lever arm swing and locking the motor in the load-bearing, transition state.
Moreover, the step-size of the myosin is related to the length of its lever arm, and this
relationship has been shown to be linearly proportional to the lever arm length for
Dictyostelium myosin II. A further hypothesis is that the maximum force depends
on the length of the lever arm (Uyeda et al., 1996). Lower force is required to stall a
motor with a long lever arm whereas the same motor with a short lever arm powers
through greater loads before being stalled by load, implying that the strain on the
lever arm prevents the full conformational change needed to allow for the motor
to acquire the conformation where it can release the ADP. This load-dependency
is consistent with a catch-slip-like behavior, which can be interpreted using
Eq. (2.4).
where u (z) is the transverse deviation of the filament away from a straight con-
formation along the z-axis (MacKintosh et al., 1995). Based on the equipartition
theorem, the force-extension of a single actin filament is
Lc2 f
L = Lc , (2.9)
Lp
2 n n fb + f
2 2
n=1
where fb = 2 b Lc2 is the threshold force for the Euler buckling instability and
L is the end-to-end distance. In the low force regime, the force is approximately
38 T. Luo and D.N. Robinson
a linear function of extension, i.e. f k2 (Lc L) b TL4 (Storm et al., 2005).
2
However, the force-extension relationship diverges nonlinearly as f 1 (Lc L)
in high force regimes where L Lc . Therefore, the stress increases non-linearly
with increasing strain. That is the F-actin shows strain-stiffening at high stress or
high strain, which is an essential property of many biological materials.
Semi-flexible polymers are viscoelastic in nature, i.e., their deformation is
time-dependent (frequency-dependent) and loading-history dependent (pre-stress-
dependent). The complex modulus G () can be measured by applying an oscil-
latory shear strain sin (t) to the actin solution and by measuring the stress
sin (t + ), where is the frequency and is the phase shift in the range of 0
2 with =0 and = 2, corresponding to a Hookean solid and Newtonian fluid,
respectively. The shear and loss moduli are defined as G () = |G ()| cos ( ())
and G ()=|G ()| sin ( ()), respectively. The complex modulus of F-actin has
a very weak frequency dependency in the low frequency regime 0.0110 Hz whereas
it shows a strong frequency dependency (G 3/4 ) in the high frequency regime
(1010,000 Hz) (Gittes et al., 1997; Gisler and Weitz, 1999; Crocker et al., 2000).
The shear modulus also shows a concentration dependency in which the concentra-
tion determines the mesh size of the actin network and le is the distance between
entanglement points. The 2D density of filaments then is 2 and 0.3 acA ,
where a is the actin monomer size and cA is the actin concentration (Schmidt et al.,
1989). If the extension is assumed to be linearly proportional to le , then the shear
modulus of the actin filament network at small strains is
b
G = , (2.10)
kB T 2 le3
where and are stress and strain, respectively (MacKintosh et al., 1995).
Furthermore, the fluctuating segment of length le occupies a volume of kB TLe4 b
and the shear modulus can be written as a function of cA such that G b
2 5
b kb T / (acA )11/5 , i.e., G cA / . On the other hand, considering the excluded
11 5
law dependence breaks down when the thermal fluctuation of actin filaments is
severely depressed. For example, in a confined volume such as a spherical aque-
ous droplet, the entropic effect gives G kb Tlp/ D7/2 when lp < D, where
12
D is the diameter of the droplet (Claessens et al., 2006b). Therefore, the modu-
lus is as much a function of the size of confinement as it is a function of actin
concentration.
2 The Role of the Actin Cytoskeleton in Mechanosensation 39
x is the bundling exponent and y is the crosslinking exponent for bundled F-actin
(Shin et al., 2004). G () and G () show a weak frequency dependency in the
low frequency regime. However, both scruin and biotin-avidin crosslinked actin
networks have G () G () 3/4 in the high frequency regime (Gardel
et al., 2004b; Koenderink et al., 2006). By contrast, fascin-bundled actin shows
G () G () 0.5 (Lieleg and Bausch, 2007). Apparently, actin networks
crosslinked by scruin and biotin-avidin interactions, but not fascin, display a similar
power-law dependency of the elastic modulus on frequency at these high frequencies
as that of pure-actin gels.
40 T. Luo and D.N. Robinson
Fig. 2.7 Microstructural images of actin networks crosslinked by ACLPs seen by confocal
microscopy. The actin bundle size increases with the [scruin]/[actin] ratio R as shown in a.
Pure actin-fascin ([fascin]/[actin] = 0.05) and pure actin-filamin ([filamin]/[actin] = 0.1) are shown
in b and c, respectively. d shows the actin composite crosslinked by fascin and filamin at
[fascin]/[actin] = 0.01 and [filamin]/[actin] = 0.1. An illustration of the structural features domi-
nated by individual ACLP when its concentration is dominant is shown in e. Scale bars denote
10 m in all panels. Figure 2.7a is reproduced from Shin et al. (2004) with permission from Proc
Natl Acad Sci USA and the remaining images are reproduced from Schmoller et al. (2008) with
permission from the American Physical Society
Fig. 2.8 ACLP concentration-dependence of bundle size and mechanical properties of actin net-
works crosslinked by scruin. Panels a and b show the increasing of bundle size and mesh size as
a linear function of R ([scruin]/[actin]). The corresponding microstructures are shown in Fig. 2.7.
Elastic modulus (panel c) and critical strain (panel d) increases and decreases respectively with
increasing R. The scale of R in all panels is the same as shown in panel D. Reproduced from Shin
et al. (2004) with permission from the Proc Natl Acad Sci USA
microstructures; yet these networks display little cross-talk between the two proteins
since the corresponding mechanical properties appear to be determined by the
dominant ACLP as shown in Fig. 2.7 (Schmoller et al., 2008). By contrast, actin
networks crosslinked by -actinin and fascin displayed synergistic enhancement of
elasticity (Tseng et al., 2005). The underlying mechanism may be the complement
between bundled and orthogonal branched networks. Actin networks crosslinked
by -actinin and filamin exhibit more solid-like behaviors than those crosslinked
by the individual crosslinkers, specifically displaying increased G ( ) at low
regime with little enhancement of G () as shown in Fig. 2.9 (Esue et al., 2009).
Thus, further experiments are needed to fully elucidate the crosstalk between other
ACLPs and their synergistic effects on the mechanical properties of purified actin
networks.
Crosslinked networks can also be described as affine or nonaffine. In general,
in the high strain regime, the deformation of the whole network remains affine and
microscopically every filament has almost the same strain, whereas in the low strain
regime the deformation may be nonaffine. The transition from nonaffine to affine is
controlled by three length parameters: filament contour length Lc , distance between
crosslinkers lc (mesh size ) and material length lb that is defined as lb = b ,
where is the stretch modulus of the filaments (Wilhelm and Frey, 2003; Head et al.,
2003; Das et al., 2007; Buxton and Clarke, 2007). The transition can be measured
by = Lc / lc3 lb . Independent of the strain magnitude, nonaffine deformation
3
and affine deformation occur when < 2 and > 20, respectively, and transient
behaviors exist in the range of 2 < < 20. For a fixed contour length (actin dynam-
ics reaches steady state), elasticity is dominated by the stretch modulus under high
crosslinker density or small lc conditions (affine); the bending modulus dominates
under low crosslinker density or large lc conditions (nonaffine). The apparent strain-
stiffening during deformation is basically a nonaffine to affine transition (Onck et al.,
2005; Gardel et al., 2004b). The nonaffine-affine transition theory also successfully
Fig. 2.9 Mechanical properties of actin networks crosslinked by -actinin and fascin. The syn-
ergistic effect on the elasticity of -actinin and fascin is shown in a. The elasticity displays
[fascin]/[-actinin] dependence in b. The exponent of the elasticity as a function of frequency
is shown in c. The total concentration of ACLPs is 0.96 M in b and c and the actin concentration
is 24 M for all panels. Reproduced from Tseng et al. (2005) with permission of Elsevier
2 The Role of the Actin Cytoskeleton in Mechanosensation 43
interprets the scalings between G (or K ) and cA , and the pre-stress observed
in these experimental systems. The corresponding microstructural transition was
observed in scruin-bundled actin network by confocal microscopy (Liu et al., 2007).
Fig. 2.10 Microstructures and mechanical properties of actin meshwork with muscle myosin II
and human filamin A. Electron micrographs of pure actin, actin+myosin ([myosin]/[actin] = 0.02
and 5 mM ATP) and filamin A +myosin+actin ([filamin]/[actin] = 0.005, [myosin]/[actin] = 0.02
and 5 mM ATP) are shown in a, b and c, respectively. The corresponding fluorescent images are
found in the inserts. The illustration of an active stiffening mechanism is shown in d. Stiffening
behaviors of active networks (with myosin to actin ratio of 0.02 (blue squares), 0.005 (green
squares), and 0.001 (red squares)) and a passive network at fixed [filamin]/[actin] of 0.005 (white
triangles) are shown in e. The filamin concentration dependence of rupture force is shown in f.
Critical strain decreases as a function of the internal stress generated by myosin II. Scale bars are
10 m in all panels. Reproduced from Koenderink et al. (2009) with permission by Proc Natl Acad
Sci USA
44 T. Luo and D.N. Robinson
In the presence of ACLPs, internal tensile stresses associated with the myosin
II motor stiffen the actin network, leading to stress-stiffening such that G ()
G () 1/2 (Mizuno et al., 2007). In assembled networks, myosin II and filamin
A work together to enhance the network stiffness as shown in Fig. 2.10 (Koenderink
et al., 2009). Increasing the myosin concentration leads to higher differential mod-
ulus and increasing the filamin A concentration makes the network able to sustain
higher stresses. The critical strain displays a power-law decay of the internal stress
that is dependent on the myosin II concentration. Because ACLP binding to actin is
dynamic, myosin may break the binding between ACLPs and actin when the myosin
concentrations become high enough, resulting in the power-law decay of the critical
strain with increasing myosin concentration.
Fig. 2.11 Mechanical properties of Dictyostelium cells. a and c show the frequency dependence
of complex moduli of different cell-lines. b and d show the effective cortical tension for these cells.
wt and myoll represent wild-type and myosin II-null cells, respectively. dynhp and fimhp
refer to dynacortin-hairpin and fimbrin-hairpin, respectively. Dynacortin and fimbrin are ACLPs,
and hairpin constructs are used to silence gene expression through RNA interference. Reproduced
from Reichl et al. (2008) with permission of Elsevier
Many of the studies of living cells draw upon laser-based tracking of single or
multiple particles embedded in the living network. Because the particles may fluctu-
ate due to thermal or active forces that act on the particles, the fluctuation-dissipation
theorem (FDT) is used to extract viscoelastic parameters. However, the FDT should
only be applied to systems at equilibrium, not out of equilibrium. Therefore, by
measuring the mean square displacement (MSD) of the particles as a function of
correlation time, it was found that the FDT cannot describe the particle behaviors in
the low frequency regime (Lau et al., 2003; Bursac et al., 2005; Girard et al., 2006;
Mizuno et al., 2007; Wilhelm, 2008; Reichl et al., 2008). Furthermore, in some
contexts, the FDT is violated because the apparent diffusive behavior has a much
larger magnitude than expected, considering the viscous damping for the particle
size. This suggests that local active processes can essentially facilitate the stirring
of the cytoplasm (Brangwynne et al., 2009). It was further demonstrated that the
mechanochemistry of myosin II motors in combination with ACLPs regulates the
46 T. Luo and D.N. Robinson
MSDs in the low frequency regime (Girard et al., 2006). ACLP mutant cells also dis-
play significant effects on the complex modulus over a wide frequency range (Girard
et al., 2004). The coupled effect of myosin and ACLPs was also investigated (Girard
et al., 2006; Reichl et al., 2008). The frequency-dependent mechanical behaviors
of cells imply that microscopic processes, such as unbinding events and conforma-
tional changes of ACLPs and motors with a broad distribution of characteristic times
play important roles in regulating cell mechanics.
Based on these types of experimental observations, three major cell mechan-
ics models have been proposed: tensegrity (Ingber, 2003), soft glassy rheology
(Fabry et al., 2001; Trepat et al., 2007; Zhou et al., 2009) and the sol-gel hypothesis
(Janmey et al., 1990). In the tensegrity model, the actin network, including myosin II
and ACLPs, is considered to be primary sources of pre-stress (Wang et al., 2001),
and cell stiffness is proportional to the pre-stress that cells experience. Tensegrity
can help explain the stress-stiffening, but it does not predict the power-law behaviors
of cells. The soft glassy rheology (SGR) model considers the cells glassy mate-
rials that are microstructurally disordered and thermodynamically close to a glass
transition. The deformations in glassy materials are microscopically inelastic, local-
ized and time-dependent. The SGR model successfully accounts for the power-law
behavior in the low frequency regime but it does not capture the stress-stiffening.
The sol-gel model assumes that the cell is a gel of filamentary polymers embed-
ded within a fluid cytosol, which predicts a weaker frequency dependency in the
low frequency regime than is observed experimentally. Thus, all models capture
some aspects of cell mechanics, but a single model that accounts for all of cellular
behaviors has yet to emerge.
Dividing cells have a mechanosensory system that they use to monitor their shape
as they cleave into two daughter cells. The system could be activated in a controlled
2 The Role of the Actin Cytoskeleton in Mechanosensation 47
Fig. 2.12 Mechansensing system in Dictyostelium. a and b show the accumulation of fluores-
cently labeled cortexillin I and myosin II, respectively. Cortexillin I and myosin II are green. The
microtubules are labeled red with RFP-tubulin. The lever arm length of different myosin II mutants
is shown in c. The percentage of cells displaying mechanosensing increases with elevated pressure
as shown in d. Reproduced from Ren et al. (2009) with permission of Elsevier
fashion, using micropipette aspiration (Effler et al., 2006). Here, myosin II and the
actin crosslinker cortexillin I accumulate cooperatively in highly deformed regions
in dividing wild type cells as shown in Fig. 2.12. Both myosin-II and cortexillin-I
are necessary for this function since cell-lines devoid of either protein are unable
to respond to cellular deformations (Ren et al., 2009). Furthermore, only fully wild
type myosin-II and wild type cortexillin are able to fully restore mechanosensing
while many of the functions of each protein are expendable for cytokinesis contrac-
tility (Ren et al., 2009). Except for cortexillin-I, other ACLPs that play a major role
in cytokinesis and the microtubules may be dispensed for mechanosensing in divid-
ing Dictyostelium cells (Effler et al., 2006). In sum, these observations indicate that
the cooperative interaction between myosin II and cortexillin I provide the core of
the mechanosensor.
Myosin-II mechanochemistry appears to be the essential active component of the
mechanosensory module. Regulatory light chain phosphorylation leads to the activa-
tion of Dictyostelium myosin-II. Interestingly, this phosphorylation step is essential
for mechanosensing (Ren et al., 2009) but is not required for cytokinesis (Beach
and Egelhoff, 2009). The lack of a requirement for light chain phosphorylation in
cytokinesis is likely due to the fact that RLC phosphorylation only leads to a 3-5-
fold activation of the actin-activated ATPase activity in Dictyostelium myosin II, and
that the myosin II mechanochemistry is not rate limiting for cytokinesis over at least
a 30-fold range (Zhang and Robinson, 2005; Chen et al., 1995). By contrast, many
48 T. Luo and D.N. Robinson
thick filaments in highly deformed region by increasing the binding life time of
myosin-actin. This tight binding then promotes additional myosin accumulation
through cooperative interactions between motor domains of assembled (in BTF
form) and unassembled myosin monomers. The second mechanism is that the
binding of cortexillin and the binding of myosin to actin filaments facilitate each
other, i.e. cortexillin and myosin II may bind actin cooperatively. A third possible
mechanism is that the curvature sensitivity of phosphatidylinositol 4,5-diphosphate
(PIP2 ) lipid molecules might lead to PIP2 accumulation in the pipette, result-
ing in cortexillin accumulation since cortexillin binds PIP2 (Stock et al., 1999).
Consequently, the cortexillin accumulation increases the local stiffness of the actin
network and enhances the force propagation, which can potentially lead to myosin
II accumulation through the first mechanism. Finally, the fourth possible mech-
anism is that BTF assembly regulatory enzymes, including MHCK and myosin
heavy chain phosphatase (which is less well characterized), may be force sensitive.
The reduced phosphorylation (by inhibiting MHCK or activating the myosin heavy
chain phosphatase) of myosin heavy chains with increasing force could promote
local accumulation of the BTFs. However, force-sensitive activation or inhibition
of these enzymes may not be essential; rather the enzymes may only be required
to maintain the available free pool of myosin monomers, which is essential for
the mechanosensory response. These four mechanisms are not mutually exclu-
sive. In the next sections, we will show how force-dependent promotion of BTF
assembly and cooperativity between myosin II and cortexillin might lead to the
inter-dependent, mechanical stress-induced accumulation of these proteins.
BTFn+1 are the bipolar filaments having 6, 2n and 2(n+1) monomers, respectively.
The formation of the anti-parallel tetramer T is the nucleation step, and the subse-
quent steps are the growth phase of BTF assembly where dimers D are added to the
BTF. The reaction rates are derived from a combination of in vitro kinetic studies of
BTF assembly as well as in vivo fluorescence recovery after photobleaching exper-
iments (Mahajan and Pardee, 1996; Moores and Spudich, 1998; Reichl et al., 2008;
Ren et al., 2009). From this analysis, the most likely process that may be affected
is the ratio of k1 to k1 . Figure 2.13 shows the kinetics of assembly after this ratio
is shifted ten-fold, which could mimic inhibition of MHCK, activation of myosin
phosphatase, or a lowering of the energy barrier required for incorporating M0 into
Fig. 2.13 Accumulation of myosin II in response to force in Dictyostelium cells during anaphase:
(a) spatial distribution of myosin II intensity in pipette region normalized by the intensity at the
opposite pole of the cell; (b) averaged transient curve of myosin II accumulation where intensity
is normalized by the intensity in the cytoplasm; (c) simulation result of the kinetics of myosin II
accumulation when k1 in Table 2.1 is decreased by 10 fold to mimic the force effect; (d) the BTFs
distribution before (without force) and 60 s after 10-fold change in k1 . Reproduced from Ren et al.
(2009) with permission from Elsevier
2 The Role of the Actin Cytoskeleton in Mechanosensation 51
a pre-existing BTF. The force dependence of these steps generally can have the form
similar to Eq. (2.1).
Overall, this analysis suggests that the largest impact on the assembly mechanism
might be in the transition from assembly-incompetent to assembly-competent states.
Although force might act on MHCK or a myosin heavy chain phosphatase, this sce-
nario requires additional enzymes and steps. A very appealing mechanism is one
where motor domains found in pre-existing mini-BTFs are stabilized in the mechan-
ical transition state by force, which then leads to the local accumulation of myosin
monomers, putting them in close proximity to the BTF where they can be directly
inserted. The motors found in the HMM form of myosin II (dimeric but unassem-
bled myosin monomers; see Fig. 2.2) binds actin in a highly cooperative fashion
but only during the transition state of the actomyosin-ADP+Pi complex (Tokuraku
et al., 2009) or if the actin structure has been altered such as by being assembled
with Ca2+ cations (Ca2+ ATP-actin as opposed to Mg2+ ATP-actin found in cells)
(Orlova and Egelman, 1997). This mechanism has the appeal that no additional
enzymes are required, and that all of the necessary parts are included directly in
the myosin motor and thick filament assembly region. In this model, the MHCK
and myosin heavy chain phosphatase are still required to maintain the pool of avail-
able myosin monomers M0 and to ensure that the system relaxes back once the
mechanical signal subsides. However, more experiments are required to see if this
mechanism accounts for mechanosensitive BTF assembly.
dCM M dCC
= g (CC ) kon CM koff
M
and = h (CM ) kon
C
CC koff
C
. (2.11)
dt dt
both g and h always have non-negative values. Furthermore, because of the force-
dependence of myosin-actin M is assumed to be a function of force, i.e.
binding, koff
M (f ) = k0 exp f x
koff off kB T .
two more VBSs appear when the talin rod is stretched by relatively low (12 pN)
forces (del Rio et al., 2009). In the absence of force, these two force-sensitive VBSs
are thought to remain buried in adjacent amphipathic helices through hydrophobic
interactions.
Talin then links to vinculin, a 116 kDa actin-binding protein, which links the core
FA proteins to the actin cytoskeleton. The vinculin head domain consists of seven
-helices arranged as two four-helical bundles (eight -helices), and its tail domain
has five -helices that form an anti-parallel bundle. The strong interaction between
the head and the tail masks the binding sites for other proteins such as talin, F-actin,
-actinin and paxillin and keeps vinculin in its inactive states. Upon talin-binding
to -actinin, the tail domain of vinculin is displaced away from the head domain,
which activates vinculin (Izard et al., 2004) and enables its binding to F-actin and
other molecules.
The mechanical stretching of FAs triggers many downstream signaling pathways
by activating the SH2 domain-containing phosphatase SHP-2 and non-receptor pro-
tein tyrosine kinases, such as Src and FAK (Tamada et al., 2004; Giannone and
Sheetz, 2006). These enzymes regulate the assembly/disassembly of FAs by con-
trolling the actin stress fiber (actin bundles with myosin II thick filaments) formation
(Vicente-Manzanares et al., 2009). Activity of some of these kinases, such as FAK
kinase, requires actin and myosin II-dependent tension (Tilghman and Parsons,
2008). Furthermore, stretching of p120Cas in cells or using an in vitro reconsti-
tution system exposes more Src kinase binding sites and leads to its local activation
(Sawada et al., 2006). This stretch-induced activation can be very fast as Src at
remote sites may be activated within 0.3 s, demonstrating just how fast signals can
propagate through the elastic cytoskeleton (Na et al., 2008).
Both chemical and mechanical signals can be transmitted over long distances.
Propagation of chemical signals occurs mainly through the diffusion of molecules
in the cytosol and its speed is limited by the chemical reaction (such as phospho-
rylation) rates, unbinding/binding rates and diffusion rates. Diffusion is usually the
limiting step since the diffusion coefficient of molecules in cells is in the range of
0.01100 m2 s1 , depending on the molecular size and shape and the viscosity of
cytoplasm on the length-scale of the diffusing particle (Howard, 2001). For exam-
ple, it can take one molecule 1100 s to travel 10-m using diffusion alone. On
the other hand, mechanical signals may be transmitted through the deformation of
cytoskeleton along actin filaments, microtubules and intermediate filaments. The
speed of transmission depends on the elastic modulus of the cytoskeleton where
signals may propagate over a 10-m distance on sub-second time-scales, indicating
that proteins in cells can sense mechanical stimuli much more rapidly than chem-
ical signals (Forgacs, 1995). This propagation of mechanical deformations likely
54 T. Luo and D.N. Robinson
depends on the pre-stress as well as the elastic modulus of the cytoskeleton, and
the magnitude of deformation decays exponentially in space with a characteristic
length that is comparable to or larger than the size of the cell (Wang and Suo,
2005).
One consequence of signal propagation through the integrated elastic actin
cytoskeleton is that signals can be transmitted over long distances and broad areas
and to a range of organelles. For example, actin filaments are connected to the
nuclear envelope through a complex of SUN and nesprin proteins (Wang et al.,
2009), to the plasma membrane through proteins such as ezrin, radixin, and moesin
(ERM proteins) (Sato et al., 1992), to stretch-activated channels (SACs) by myosin
I motor proteins (Fettiplace and Hackney, 2006), and to mitochondria by mitochon-
drial ABPs (Boldogh et al., 1998). Forces transmitted by the actin cytoskeleton
to the nucleus alter gene expression, which may in turn regulate actin remodel-
ing. The SACs can also be activated or deactivated by cytoskeleton stretching,
resulting in ion flux, regulating stress fiber formation and orientation and myosin
II bipolar thick filament assembly. Mechanical stimuli propagated to membrane-
bound or associated proteins through the actin-membrane connections can lead
to changes in activity of membrane-bound signaling molecules and other ion
channels.
uncapping of the actin filament barbed ends, and can enhance the linkages between
the actin cytoskeleton and the plasma membrane by activating ERM proteins
(Nebl et al., 2000). PIP2 can also activate vinculin, promoting FA assembly
(Sechi and Wehland, 2000), and WASP, increasing actin polymerization (Pollard,
2007).
system with the cortical actin network. In Drosophila melanogaster, cappuchino (an
FH2 containing protein) and spire (a WH2 containing protein) can crosslink micro-
tubules and actin filaments (Rosales-Nieves et al., 2006). In Schizosaccharomyces
pombe, tea4p and tea1p also localize to the plus ends of microtubules, and a com-
plex of tea1p, tea4p and the formin for3p is necessary for the establishment of cell
polarity and actin nucleation at new cell ends (Martin et al., 2005). Thus, several
linkages between the actin and microtubule networks promote their integration and
may facilitate force-propagation, and therefore signal-propagation, through these
systems.
In addition to traditional micropipette aspiration (MPA), the past few decades have
witnessed the development of various new techniques using the combinations of
nanomanipulation, microfabrication, magnetic techniques, and optical techniques
(Bao and Suresh, 2003; Addae-Mensah and Wikswo, 2008). These methods include
atomic force microscopy (AFM), magnetic tweezers, optical tweezers, magnetic
twisting cytometry (MTC), particle-tracking microrheology (PTM), microfluidic
devices, stretching devices, traction force microscopy (TFM) and MEMS-based
devices. Classic MPA still offers a number of advantages in that it is relatively easy
to implement and can be readily adapted to a broad array of cell-types, particularly
those which are not highly adherent. MPA can be used to measure effective tension
and elastic and viscous properties of the cell. Perhaps more significantly, MPA is
very useful for imposing deformations to cells so that the cells response may be
monitored using an array of fluorescence methods. However, the major limitation of
MPA is that it is difficult to measure properties occurring on fast sub-second time-
scales or to measure frequency-dependent features. For these sorts of measurements,
many of the other methods, for example AFM, MTC and PTM, are more suitable
(Girard et al., 2004; Hoffman et al., 2006). PTM can be categorized into single-
bead, two-bead and multiple-bead modes (Wirtz, 2009). Depending on the driving
force, PTM has two working modes: passive and active. To measure the mechanical
properties of single molecules, AFM is commonly used for the high force ranges
(>10 pN) whereas optical tweezers and magnetic tweezers are commonly used
for relatively low forces (Neuman and Nagy, 2008; Finer et al., 1994). AFM and
optical tweezers also allow three-dimensional manipulation of molecules. AFM is
often used to study protein folding/unfolding and protein-protein interactions while
tweezers are usually used to study biological motors, including cytoskeletal motors
and DNA and RNA polymerases. Additionally, many of these methods have been
combined with fluorescence microscopy techniques, such as total internal reflection
fluorescence (TIRF) imaging and fluorescence resonance energy transfer (FRET)
(Sarkar et al., 2004; Moffitt et al., 2008; del Rio et al., 2009).
Microfluidic devices and flow chambers are used to study the cell responses to
shear flow (del lamo et al., 2008; Wang and Levchenko, 2009). Stretching devices
2 The Role of the Actin Cytoskeleton in Mechanosensation 57
are usually coupled with fluorescence imaging methods to quantify the effects of
stretch on actin cytoskeleton rearrangement, activation of kinases, gene expression
and cell differentiation (Sawada et al., 2006; Kurpinski et al., 2006). TFM was
invented to investigate the traction force that cells apply to the substrates (Pelham
and Wang, 1997). Initially, TFM methods utilized fluorescent beads embedded in
polymeric substrates so that traction forces exerted by adherent cells on the substrate
could be calculated from the displacements of the beads. Recently, micropatterned
substrates by soft photolithography have been used to control the cell adhesion areas
and cell shapes (Balaban et al., 2001; Tan et al., 2003; Thry and Bornens, 2006).
Therefore, a broad range of mechanical measurements and manipulations are now
possible across a broad array of length- and time-scales and from the molecule to
cellular levels.
Mechanical inputs must have been among the first signals that cells received and had
to respond to, and the ability of cells to sense and react to these inputs likely evolved
at a very early time point. Thus, it is not surprising that there are numerous overlaps
between the mechanotransduction and traditional chemical signal transduction
pathways. Because of the overlap between these pathways, mechanical-chemical
coupling and feedback loops are a natural consequence of this system integra-
tion. Because the actin cytoskeleton is structurally integrated with nearly every
aspect of the cell, mechanical inputs can be transmitted quickly throughout the cell.
Furthermore, individual proteins may be involved in multiple pathways and con-
tributing multiple functions. Therefore, to fully understand the roles of individual
proteins and the cooperativity among them in the actin cytoskeleton, in vitro exper-
iments involving single molecule measurements, reconstituted actin networks, and
computational simulations of protein folding/unfolding and protein-protein inter-
actions have to be combined with quantitative in vivo observations. Challenges
for understanding mechanosensation through the actin cytoskeleton include reveal-
ing how proteins function cooperatively over short nanometer-length-scales and
fast sub-second time-scales. Direct observations of force propagation in cells and
eventually between cells within tissues will be essential. Novel designs of mechan-
ical strain sensors using fluorescence readouts such as FRET pairs and inventive
imaging setups will be needed to fulfill these demands. Applying a repertoire of
these approaches to a genetically tractable organism, such as Dictyostelium cells
as they perform physiologically and medically important processes like cell divi-
sion, will continue to provide unique insights into cellular mechanosensing and
mechanotransduction.
Glossary List
Affine Describes an actin network, which is co-linear during stretching and
shearing
Non-affine Describes an actin network in which co-linearity is absent during
stretching and shearing
Stress The force applied on a unit area
Strain The ratio between the length-change associated with deformation and
the original length (no deformation)
ABD Actin-binding domain
ABP Actin-binding protein
ACLP Actin-crosslinking protein
BTF Bipolar thick filament
ELC Essential light chain
FJC Free-joint-chain
HMM Heavy meromyosin
RLC Regulatory light chain
WLC Worm-like-chain
D Bundle size
E Youngs modulus: the proportionality between stress and the resulting
strain
fb Bending modulus: the proportionality between bending momentum
and the resulting curvature
G Complex modulus
G Shear modulus the real part of G : the proportionality between shear
stress and the shear strain
G Loss modulus the imaginary part of G
K Differential shear modulus: the differential proportionality between
shear stress and the shear strain
Lc Contour length: the integrated length along the polymer chain
Lp Persistence length: the length over which correlations in the direction
of the tangent are lost
Le Distance between entanglements
Mesh size
Crosslinking density: the ratio between the cross-linker concentration
and actin concentration
References
Ackbarow T, Chen X, Keten S, Buehler MJ (2007) Hierarchies, multiple energy barriers, and
robustness govern the fracture mechanics of -helical and -sheet protein domains. Proc Natl
Acad Sci USA 104:1641016415
2 The Role of the Actin Cytoskeleton in Mechanosensation 59
Addae-Mensah KA, Wikswo JP (2008) Measurement techniques for cellular biomechanics in vitro.
Exp Biol Med 233:792809
Altmann SM, Grnberg RG, Lenne PF, Ylnne J, Raae A, Herbert K, Saraste M, Nilges M,
Hrber JK (2002) Pathways and intermediates in forced unfolding of spectrin repeats. Structure
10:10851096
Balaban NQ, Schwarz US, Riveline D, Goichberg P, Tzur G, Sabanay I, Mahalu D,
Safran S, Bershadsky A, Addadi L, Geiger B (2001) Force and focal adhesion assem-
bly: a close relationship studied using elastic micropatterned substrates. Nat Cell Biol 3:
466472
Bauelos S, Saraste, M, Djinovic-Caruga K (1998) Structural comparisons of calponin homology
domains: implications for actin binding. Structure 6:14191431
Bao G, Suresh S (2003) Cell and molecular mechanics of biological materials. Nat Mater 2:
715725
Bausch AR, Kroy K (2006) A bottom-up approach to cell mechanics. Nat Phys 2:231238
Beach JR, Egelhoff TT (2009) Myosin II recruitment during cytokinesis independent of
centralspindlin-mediated phosphorylation. J Biol Chem 284:2737727383
Bell GI (1978) Models for the specific adhesion of cells to cells. Science 200:618627
Bertz M, Wilmanns M, Rief M (2009) The titin-telethonin complex is a directed, superstable
molecular bond in the muscle Z-disk. Proc Natl Acad Sci USA 106:13307133310
Boldogh I, Vojtov N, Karmon S, Pon LA (1998) Interaction between mitochondria and the actin
cytoskeleton in budding yeast requires two integral mitochondrial outer member proteins,
Mmm1p and Mdm10p. J Cell Biol 141:13711381
Brakebusch C, Fssler R (2003) The integrin-actin connection, an eternal love affair. EMBO J
22:23242333
Brangwynne CP, Koenderink GH, MacKintosh FC, Weitz DA (2009) Intracellular transport by
active diffusion. Trends Cell Biol 19(9):423427
Brockwell DJ, Paci E, Zinober RC, Beddard GS, Olmsted PD, Smith DA, Perham RN, Radford
SE (2003) Pulling geometry defines the mechanical resistance of a -sheet protein. Nat Struct
Biol 10:731737
Buchsbaum RJ (2007) Rho activation at a glance. J Cell Sci 120:11491152
Bueche F (1962) Physical properties of polymers 37. Interscience, New York, NY
Buehler MJ, Keten S (2008) Ealsticity, strength and resilience: a comparative study on mechanical
signatures of -helix, -sheet and tropocollagen domains. Nano Research 1:6371
Bursac P, Lenormand G, Fabry B, Oliver M, Weitz DA, Viasnoff V, Butler JP, Fredberg JJ (2005)
Cytoskeletal remodelling and slow dynamics in the living. Nat Mater 4:557561
Bustamante C, Marko JF, Siggia ED, Smith S (1994) Entropic elasticity of lambda-phage DNA.
Science 265:15991600
Buxton GA, Clarke N (2007) Bending to stretching transition in disordered networks. Phys Rev
Lett 98:238103238106
Carrion-Vazquez M, Li H, Lu H, Marszalek P, Oberhauser AF, Fernandez JM (2003) The
mechanical stability of ubiquitin is linkage dependent. Nat Struct Biol 10:738743
Chaudhuri O, Parekh SH, Fletcher DA (2007) Reversible stress softening of actin network. Nature
445:295298
Chen TL, Kowalczyk PA, Ho G, Chisholm RL (1995) Targeted disruption of the Dictyostelium
myosin essential light chain gene produces cells defective in cytokinesis and morphogenesis. J
Cell Sci 108:32073218
Chen Y, Yan B, Chalovich JM, Brenner B (2001) Theoretical kinetic studies of models for bind-
ing myosin subfragment-1 to regulated actin: Hill model versus Geeves model. Biophys J 80:
23382349
Chien S (2007) Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell. Am
J Physiol Heart Circ Physiol 292:H1209H1224
Claessens MM, Bathe M, Frey E, Bausch AR (2006a) Actin-binding proteins sensitively mediate
F-actin bundle stiffness. Nat Mater 5:748753
Claessens MM, Tharmann R, Kroy K, Bausch AR (2006b) Microstructure and viscoelasticity of
confined semiflexible polymer networks. Nat Phys 2:186189
60 T. Luo and D.N. Robinson
Geiger B, Spatz JP, Bershadsky AD (2009) Environmental sensing through focal adhesions. Nat
Rev Mol Cell Biol 10:2133
Giannone G, Sheetz MP (2006) Substrate rigidity and force define form through tyrosine
phosphatase and kinase pathways. Trends Cell Biol 16:213223
Girard KD, Chaney C, Delannoy M, Kuo SC, Robinson DN (2004) Dynacortin contributes
to cortical viscoelasticity and helps define the shape changes of cytokinesis. EMBO J 23:
15361546
Girard KD, Kuo SC, Robinson DN (2006) Dictyostelium myosin II mechanochemistry pro-
motes active behavior of the cortex on long time scales. Proc Natl Acad Sci USA 103:
21032108
Gisler T, Weitz DA (1999) Scaling of the microrheology of semidilute F-actin solutions. Phys Rev
Lett 82:16061609
Gittes F, Mickey B, Nettleton J, Howard J (1993) Flexural rigidity of microtubules and actin
filaments measured from thermal fluctuations in shape. J Cell Biol 120:923934
Gittes F, Schnurr B, Olmsted PD, MacKintosh FC, Schmidt CF (1997) Microscopic viscoelastic-
ity: shear moduli of soft materials determined from the thermal fluctuations. Phys Rev Lett
79:32863289
Geeves MA, Halsall DJ (1987) Two-step ligand binding and cooperativity. A model to
describe the cooperative binding of myosin subfragment 1 to regulated actin. Biophys J 52:
215220
Guo B, Guilford WH (2006) Mechanics of actomyosin bonds in different nucleotide states are
tuned to muscle contraction. Proc Natl Acad Sci USA 103:98449849
Head DA, Levine AJ, MacKintosh FC (2003) Deformation of cross-linked semiflexible polymer
networks. Phys Rev Lett 91:108102108105
Hill TL, Eisenberg E, Greene L (1980) Theoretical model for the cooperative equilibrium binding
of myosin subfragment 1 to the actin-troponin-tropomyosin complex. Proc Natl Acad Sci USA
77:31863190
Hinner B, Tempel M, Sackmann E, Kroy K, Frey E (1998) Entanglement, elasticity, and viscous
relaxation of actin solutions. Phys Rev Lett 81:26142617
Hoffman BD, Massiera G, van Citters KM, Crocker JC (2006) The consensus mechanics of
cultured mammalian cells. Proc Natl Acad Sci USA 103:1025910264
Holmes KC, Popp D, Gebhard W, Kabsch W (1990) Atomic model of the actin filament. Nature
347:4449
Hostetter D, Rice S, Dean S, Altman D, McMahon PM, Sutton S, Tripathy A, Spudich JA (2004)
Dictyostelium myosin bipolar thick filament formation: importance of charge and specific
domains of the myosin rod. PLoS Biol 2:18801892
Howard J (2001) Mechanics of motor proteins and the cytoskeleton. Sinauer Associates,
Sunderland, MA
Humphrey D, Duggan C, Saha D, Smith D, Ks J (2002) Active fluidization of polymer networks
through molecular motors. Nature 416:413416
Huveneers S, Danen EH (2009) Adhesion signaling-crosstalk between integrins, Src and Rho. J
Cell Sci 122:10591069
Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110:673687
Ingber DE (2003) Tensegrity I: Cell structure and hierarchical systems. J Cell Sci 116:11571173
Ingber DE (2006) Cellular mechanotransduction: putting all the pieces together again. FASEB J
20:811827
Izard T, Evans G, Borgon RA, Rush CL, Bricogne G, Bois PR (2004) Vinculin activation by talin
through helical bundle conversion. Nature 427:171175
Janmey PA, Hvidt S, Lamb J, Stossel TP (1990) Resemblance of actin-binding protein/actin gels
to covalently crosslinked networks. Nature 345:8992
Katsumi A, Naoe T, Matsushita T, Kaibuchi K, Schwartz MA (2005) Integrin activation and matrix
binding mediate cellular response to mechanical stretch. J Biol Chem 280:1654616549
Kaufmann S, Ks J, Goldmann WH, Sackmann E, Isenberg G (1992) Talin anchors and nucleates
actin filaments at lipid membranes. A direct demonstration. FEBS Lett 314:203205
62 T. Luo and D.N. Robinson
Neuman KC, Nagy A (2008) Single-molecule force spectroscopy: optical tweezers, magnetic
tweezers and atomic force microscopy. Nat Methods 5:491505
Nome RA, Zhao JM, Hoff WD, Scherer NF (2007) Axis-dependent anisotropy in protein unfolding
from integrated nonequilibrium single-molecule experiments, analysis, simulation. Proc Natl
Acad Sci USA 104:2079920804
Onck PR, Koeman T, van Dillen T, van der Giessen E (2005) Alternative explanation of stiffening
in cross-linked semiflexible networks. Phys Rev Lett 95:178102178105
Orlova A, Egelman EH (1997) Cooperative rigor binding of myosin to actin is a function of F-actin
structure. J Mol Biol 265:469474
Owen C, DeRosier D (1994) A 13- map of the actin-scruin filament from the limulus acrosomal
process. J Cell Biol 123:337344
Pascual J, Pfuhl M, Walther D, Saraste M, Nilges M (1997) Solution structure of the
spectrin repeat: a left-handed antiparallel triple-helical coiled-coil. J Mol Biol 273:
740751
Pelham RJ Jr, Wang Y (1997) Cell locomotion and focal adhesions are regulated by substrate
flexibility. Proc Natl Acad Sci USA 94:1366113665
Phichith D, Travaglia M, Yang Z, Liu X, Zong AB, Safer D, Sweeney HL (2009) Cargo binding
induces dimerization of myosin VI. Proc Natl Acad Sci USA 106:1732017324
Pollard TD (2007) Regulation of actin filament assembly by Arp2/3 complex and formins. Ann
Rev Biophys Biomol Struct 36:451477
Puklin-Faucher E, Sheetz MP (2009) The mechanical integrin cycle. J Cell Sci 122:179186
Reichl EM, Ren Y, Morphew MK, Delannoy M, Effler JC, Girard KD, Divi S, Iglesias PA, Kuo SC,
Robinson DN (2008) Interactions between myosin and actin crosslinkers control cytokinesis
contractility dynamics and mechanics. Curr Biol 18:471480
Ren Y, Effler JC, Norstrom M, Luo T, Firtel RA, Iglesias PA, Rock RS, Robinson DN (2009)
Mechanosensing through cooperative interactions between myosin II and the actin crosslinker
cortexillin I. Curr Biol 19:14211428
Revenu C, Athman R, Robine S, Louvard D (2004) The co-workers of actin filaments: from cell
structure to signals. Nat Rev Mol Cell Biol 5:112
Rief M, Pascual J, Saraste M, Gaub HE (1999) Single molecule force spectroscopy of spectrin
repeats: low unfolding forces in helix bundles. J Mol Biol 286:553561
Riveline D, Zamir E, Balaban NQ, Schwarz US, Ishizaki T, Narumiya S, Kam Z, Geiger B,
Bershadsky AD (2001) Focal contacts as mechanosensors: externally applied local mechan-
ical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent
mechanism. J Cell Biol 153:11751186
Robinson DN, Spudich JA (2000) Towards a molecular understanding of cytokinesis. Trends Cell
Biol 10:228237
Robinson DN, Spudich JA (2004) Mechanics and regulation of cytokinesis. Curr Opin Cell Biol
16:182188
Robling AG, Castillo AB, Turner CH (2006) Biomechanical and molecular regulation of bone
remodeling. Annu Rev Biomed Eng 8:455498
Rohs R, Etchebest C, Lavery R (1999) Unraveling proteins: a molecular mechanics study. Biophy
J 76:27602768
Rosales-Nieves AE, Johndrow JE, Keller LC, Magie CR, Pinto-Santini DM, Parkhurst SM (2006)
Coordination of microtubule and microfilament dynamics by Drosophila Rho1, spire and
cappuccino. Nat Cell Biol 8:367376
Rosenblatt J, Cramer LP, Baum B, McGee KM (2004) Myosin II-dependent cortical movement
is required for centrosome separation and position during mitotic spindle assembly. Cell 117:
361372
Sarkar A, Robertson RB, Fernandez JM (2004) Simultaneous atomic force microscope and flu-
orescence measurements of protein unfolding using a calibrated evanescent wave. Proc Natl
Acad Sci USA 101:1288212886
64 T. Luo and D.N. Robinson
Sato N, Funayama N, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S (1992) A gene family con-
sisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane
association sites. J Cell Sci 103:131143
Sawada Y, Tamada M, Dubin-Thaler BJ, Cherniavskaya O, Sakai R, Tanaka S, Sheetz MP (2006)
Force sensing by mechanical extension of the src family kinase substrate p130Cas. Cell
127:0151026
Schmidt CF, Brmann M, Isenberg G, Sackmann E (1989) Chain dynamics, mesh size, and
diffusive transport in networks of polymerized actin: a quasielastic light scattering and
microfluorescence study. Macromolecules. 22:36383649
Schmoller KM, Lieleg O, Bausch AR (2008) Cross-linking molecules modify composite actin
networks independently. Phys Rev Lett 101:118102118105
Schwaiger I, Kardinal A, Schleicher M, Noegel AA, Rief M (2004) A mechanical unfolding
intermediate in an actin-crosslinking protein. Nat Struct Mol Biol 11:8185
Sechi AS, Wehland J (2000) The actin cytoskeleton and plasma membrane connection:
Ptdlns(4,5)P2 influences cytoskelton protein activity at the plasma membrane. J Cell Sci
113:36853695
Shin JH, Gardel ML, Mahadevan L, Matsudaira P, Weitz DA (2004) Relating microstructure to
rheology of a bundled and cross-linked f-actin network in vitro. Proc Natl Acad Sci USA
101:96369641
Siegrist SE, Doe CQ (2007) Microtubule-induced cortical cell polarity. Genes Dev 21:483496.
Sjblom B, Ylnne J, Djinovic-Carugo K (2008) Novel structural insights into F-actin-binding and
novel functions of calponin homology domains. Curr Opin Struct Biol 18:702708
Spudich JA (2001) The myosin swinging cross-bridge model. Nat Rev Mol Cell Biol 2:387292
Stock A, Steinmetz MO, Janmey PA, Aebi U, Gerisch G, Kammerer RA, Weber I, Faix J (1999)
Domain analysis of cortexillin I: actin-bundling, PIP2 -binding and the rescue of cytokinesis.
EMBO J 18:52745284
Storm C, Pastore JJ, Mackintosh FC, Lubensky TC, Janmey PA (2005) Nonlinear elasticity in
biological gels. Nature 435:191194
Stossel TP, Fenteany G, Hartwig JH (2006) Cell surface actin remodeling. J Cell Sci 119:
32613264
Tadokoro S, Shattil SJ, Eto K, Tai V, Liddington RC, de Pereda JM, Ginsberg MH, Calderwood DA
(2003) Talin binding to integrin beta tails: a final common step in integrin activation. Science
302:103106
Tamada M, Sheetz MP, Sawada Y (2004) Activation of a signaling cascade by cytoskeleton stretch.
Dev Cell 7:709718
Tan JL, Tien J, Pirone DM, Gray DS, Bhadriraju K, Chen CS (2003) Cells lying on a bed
of microneedles: an approach to isolate mechanical force. Proc Natl Acad Sci USA 100:
14841489
Tharmann R, Claessens MM, Bausch AR (2007) Viscoelasticity of isotropically cross-linked actin
networks. Phys Rev Lett 98:088103088106
Thry M, Bornens M (2006) Cell shape and cell division. Curr Biol 18:648657
Thomas W (2008) Catch bond in adhesion. Ann Rev Biomed Eng 10:3957
Tilghman RW, Parsons JT (2008) Focal adhesion kinase as a regulator of cell tension in the
progression of cancer. Semin Cancer Biol 18:4552
Tokuraku K, Kurogi R, Toya R, Uyeda TQ (2009) Novel mode of cooperative binding between
myosin and Mg2+ -actin filaments in the presence of low concentrations of ATP. J Mol Biol
386:149162
Trepat X, Deng L, An SS, Navajas D, Tschumperlin DJ, Gerthoffer WT, Butler J, Fredberg JJ
(2007) Universal physical responses to stretch in the living cells. Nature 447:592595
Tseng Y, Kole TP, Lee JS, Fedorov E, Almo SC, Schafer BW, Wirtz D (2005) How actin
crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response.
Biochem Biophys Res Comm 334:183192
2 The Role of the Actin Cytoskeleton in Mechanosensation 65
Qiang Fu, Yiping Zhang, Yajuan Xu, Yourui Li, Ling Guo, and Minfeng Shao
Abstract Mechanosensitivity is the ability of tissues and cells to detect and make
response to mechanical stimuli. Osteoblast is a kind of important mechanosen-
sitive cell in bone tissue, while cytoskeleton plays an important role in the
mechanotransduction in osteoblasts. This article reviewed the roles of cytoskeleton
on the mechanotransduction of genes in osteoblasts, summarized that cytoskele-
ton integrity is essential for the expression of bone formationrelated genes in
osteoblasts, and concluded that cytoskeleton reorganization inhibition can enhance
the mechanosensitivity of some genes in osteoblasts. Further researches on the
specific mechanisms of cytoskeleton will shed new light on the transmission mech-
anisms of mechanical stress in osteoblasts, and will lay the foundation for the
further investigations on the biomechanical behaviors of bone cells and even bone
tissues.
3.1 Introduction
Cytoskeleton is the protein fiber network system in eukaryotic cells. The general
concept of cytoskeleton includes nucleus cytoskeleton (intranuclear cytoskele-
ton, nuclear lamina, and chromosome cytoskeleton in mitotic state), cytoplasm
cytoskeleton (microfilament, microtubule, intermediate fibre, and microtrabecular
lattice), cell membrane cytoskeleton and extracellular matrix. The narrow sense
of cytoskeleton only refers to the cytoplasm cytoskeleton. Cytoplasm cytoskele-
ton, which is a fibrous structure system mainly distributed in the eukaryotic cells,
consists of three basic filaments: microtubule, microfilament and intermediate fibre.
Q. Fu (B)
Department of Prosthodontics, Guanghua School & Hospital of Stomatology, Sun Yat-Sen
University, Guangzhou 510055, Guangdong, China
e-mail: fuqiangds@yahoo.com.cn; fuqiangds@gmail.com
These three filaments are polymerized from different protein subunits (skelemin)
in different specific ways (Qian et al., 2009). The cytoskeleton we reported in this
review is mainly about the cytoplasm cytoskeleton.
The cytoskeleton plays a crucial role not only in maintaining the cellular morphol-
ogy, bearing loads and keeping the intracellular structures in order, but also in lots of
life activities, for examples: guiding the chromosome segregation during cell divi-
sion, transporting kinds of vesicles and organelles along it in a directional transport.
Recently, the increasing evidences indicate that several cellular activities, includ-
ing activation of several signal transduction pathways and transcriptional activity of
many genes, are based on the reorganization of the cytoskeleton. The cytoskeleton
has the function of transferring and dispersing stress, as well as responding to the
mechanical signals (Higuchi et al., 2009).
makes come true the transformation from mechanical signals to biological signals
and chemical signals. The transmission and transduction of mechanical signals can
be completed by the cooperation of these two ways (Charras and Horton, 2002).
The function of cytoskeleton, directly as the conductive track to transmit mechan-
ical signals into cells, can be explained by the tensegrity model. In this model, actin
microfilaments are the major part to bear the tension, meanwhile, microtubules can
be viewed as the load-bearing element for compression, and intermediate fibres,
as the potent integrated components, connect the microtubules with the inotropic
microfilaments and fix them to the membrane surface and the nucleus. If the con-
ducting system is based on the tensegrity, it must be instant for the mechanical
signals to be transmitted into cells, and faster than any signal conducting system
based on diffusion, which enhances the conducting efficiency of the mechanical
signals (Ingber, 2003). Mechanical stimulus can be transmitted to cytoskeleton
through integrin-microfilament pathway and cadherins-microtubule pathway, which
can change the distribution and polymerization of cytoskeleton, increase the perme-
ability of ion channels coupled with cytoskeleton and the activity of some surface
receptors, and alter the function of the proteins and functional enzymes in many
signal conducting systems coupled with cytoskeleton, then produces the second
messengers and transmits the signals to nucleus, and finally promotes the metabolic
activities of the proliferation and differentiation of cells and the synthesis and
secretion of proteins (Sawada and Sheetz, 2002). The extracellular signals can be
transmitted to cells through this continuum structure, vice versa. Such is the major
way for microfilaments to participate in signal transduction (Chou et al., 2001).
Bone formation-related genes, which could promote the proliferation and activa-
tion of osteoblasts, include c-fos, early growth response gene-1 (egr-1), osteopontin
(OPN), cyclooxygenase-2 (COX-2), extracellular signal-regulated kinase (ERK),
and so on. When these genes are activated, some bone formation-related proteins
are expressed, and then the proliferation and differentiation of osteoblasts and new
bone formation are promoted (Kapur et al., 2004; Lee et al., 2008).
activity of these genes. AP-1 plays a significant role both in the proliferation and
differentiation of osteoblasts and the promotion of bone fracture healing (Cowles
et al., 2000; Zayzafoon et al., 2005). The DNA sequences of the important bone
formation-related genes that are known so far, such as OPN (Nomura and Takano-
Yamamoto, 2000), COX-2 (Ogasawara et al., 2001) and so on, have AP-1 binding
sites. That means, the expression of these genes would be regulated by c-fos gene.
Studies have found that tensile stress and compressive stress could activate
c-fos gene expression of osteoblasts, but when the cytoskeleton is destructed by
Cytochalasin D, the cytoskeleton would depolymerize, which could interrupt the
c-fos gene expression of osteoblasts induced by tensile stress and compressive stress
(Nomura and Takano-Yamamoto, 2000; Ogasawara et al., 2001). Our previous work
has showed that Cytochalasin D used to destruct the cytoskeleton could also inter-
rupt the induction of c-fos gene in osteoblasts under fluid shear stress (Chen and Fu,
2008).
3.3.2 Egr-1
3.3.3 OPN
sequences, seven to ten consecutive Asp sequences, RGD sequences and tyro-
sine kinase-phosphorylation recognition sequences. The RGD (arginine-glycine-
aspartic acid) sequence, a special structure of OPN, could combine with integrin,
and then facilitate the bone cells to adhere to the extracellular mineralized matrix.
So, it mediates lots of significant physiological functions.
Many studies showed that mechanical stress could induce the expression of OPN
in osteoblasts (Toma et al., 1997). Morinobu et al. (2003) have established an osteo-
genesis model induced by stress to observe the role of OPN in bone formation
with stress. They found that the osteogenesis under stress was obvious in wild-type
mouse, and the expression of OPN protein was significant in new bone formation
area, but not in OPN knock-out mice. Fujihara et al. (2006) found that bone remod-
eling induced by mechanical stress was significantly inhibited in OPN knock-out
mice, which suggested that OPN also plays an important role in bone remodeling.
But other studies showed that OPN is also an important regulatory factor in bone
loss (disuse atrophy) (Ishijima et al., 2006). So, OPN may play important roles in
both bone formation and bone resorption.
After studying the OPN signaling pathway induced by mechanical stress, both
Toma et al. (1997) and Carvalho et al. (2002) have found that the cytoskele-
ton destruction with Cytochalasin D could interrupt the induction of OPN under
mechanical stress.
3.3.4 ERK
3.3.5 COX-2
calcified tissue in bone, accelerate fracture healing, enhance the proliferation and
differentiation of osteoblasts (Norrdin and Shih, 1988), and also mobilize the pre-
cursor cells of osteoblasts to differentiate into osteoblasts (Suponitzky and Weinreb,
1998). Cyclooxygenase includes two isoenzyme types: Conservative COX (COX-1)
and induction COX (COX-2). COX-1 is widely distributed in various tissues and is
maintained at a stable level under physiological conditions to regulate the physio-
logical function of organisms, while COX-2, as an inducible enzyme, which can be
induced by mechanical stress, is a rate-limiting enzyme for PGE2 synthesis under
mechanical stress. Studies showed that COX-2 plays a significant role in the PGE2
expression induced by fluid shear stress (Bakker et al., 2003).
Fluid shear stress could induce the expression of COX-2 and the release of PGE2
in osteoblasts, and both of them are necessary for mechanical stress to stimulate
bone formation (Bakker et al., 2001; Klein-Nulend et al., 1997; Reich et al., 1997;
Smalt et al., 1997; Chow and Chambers, 1994). The selective inhibition of COX-2
gene expression could interrupt the bone formation in vivo induced by mechanical
stress (Chow and Chambers, 1994; Forwood, 1996).
FU et al. had indicated that cytoskeleton integrity plays an important role in the
expression of cox-2 gene in osteoblasts induced by fluid shear stress (Wu and Fu,
2009).
To sum up, cytoskeleton integrity is necessary for the expressions of bone
formation-related genes (such as c-fos, egr-1, OPN, ERK, COX-2, and so on)
induced by fluid shear stress.
Mechanosensitivity refers to the ability that tissues or cells detect and make response
to mechanical stimuli.
Many studies showed that the response of bone cells to external stress could be
changed (Turner, 1999; Schriefer et al., 2005; Jaasma et al., 2007). Cytoskeleton
plays an important role in the biotransformation of mechanical signals transmitted
to bone tissues. The previous work of Fu et al. has proved that LIMK2 gene plays
a crucial role in cytoskeleton reorganization of osteoblasts induced by fluid shear
stress (Fu et al., 2008; Fig. 3.1).
Presently, Fu et al. have studied the effects of cytoskeleton reorganization inhi-
bition on c-fos and cox-2 genes in osteoblasts induced by fluid shear stress. They
found that cytoskeleton reorganization inhibition with RNAi (RNA interference)
could enhance the expression of c-fos and cox-2 genes induced by fluid shear stress
(Chen and Fu, 2008; Wu and Fu, 2009). Thus, Fu et al. concluded that on condition
of maintaining the cytoskeleton integrity, cytoskeleton reorganization inhibition can
promote the expression of c-fos and cox-2 genes in osteoblasts induced by fluid
shear stress. In other words, cytoskeleton reorganization inhibition can enhance the
mechanosensitivity of c-fos and cox-2 genes in osteoblasts.
74 Q. Fu et al.
References
Bakker AD, Klein-Nulend J, Burger EH (2003) Mechanotransduction in bone cells proceeds via
activation of COX-2, but not COX-1. Biochem Biophys Res Commun 305:677683
Bakker AD, Soejima K, Klein-Nulend J, Burger EH (2001) The production of nitric oxide and
prostaglandin E(2) by primary bone cells is shear stress dependent. J Biomech 34:671677
3 Effect of Cytoskeleton on the Mechanosensitivity of Genes in Osteoblasts 75
Carvalho RS, Bumann A, Schaffer JL, Gerstenfeld LC. (2002) Predominant integrin ligands
expressed by osteoblasts show preferential regulation in response to both cell adhesion and
mechanical perturbation. J Cell Biochem 84:497508
Charras GT, Horton MA (2002) Single cell mechanotransduction and its modulation analyzed by
atomic force microscope indentation. Biophys J 82:29702981
Chen NX, Ryder KD, Pavalko FM, Turner CH, Burr DB, Qiu J, Duncan RL (2000) Ca(2+) regu-
lates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts. Am J
Physiol Cell Physiol 278:C989C997
Chen R, Fu Q (advisor) (2008) Effects of cytoskeleton reorganization inhibition on the expression
of c-fos in osteoblasts induced by fluid shear stress. Masters Thesis, Sun Yat-Sen University,
Guangzhou
Chou YH, Helfand BT, Goldman RD (2001) New horizons in cytoskeletal dynamics: transport of
intermediate filaments along microtubule tracks. Curr Opin Cell Biol 13:106109
Chow JW, Chambers TJ (1994) Indomethacin has distinct early and late actions on bone formation
induced by mechanical stimulation. Am J Physiol 267:E287E292
Cowles EA, Brailey LL, Gronowicz GA (2000) Integrin-mediated signaling regulates AP-1
transcription factors and proliferation in osteoblasts. J Biomed Mater Res 52:725737
Forwood MR (1996) Inducible cyclo-oxygenase (COX-2) mediates the induction of bone forma-
tion by mechanical loading in vivo. J Bone Miner Res 11:16881693
Fu Q, Wu C, Shen Y, Zheng S, Chen R (2008) Effect of LIMK2 RNAi on reorganization of the
actin cytoskeleton in osteoblasts induced by fluid shear stress. J Biomech 41:32253228
Fujihara S, Yokozeki M, Oba Y, Higashibata Y, Nomura S, Moriyama K (2006) Function and
regulation of osteopontin in response to mechanical stress. J Bone Miner Res 21:956964
Guzman C, Jeney S, Kreplak L, Kasas S, Kulik AJ, Aebi U, Forro L (2006) Exploring the mechan-
ical properties of single vimentin intermediate filaments by atomic force microscopy. J Mol
Biol 360:623630
Higuchi C, Nakamura N, Yoshikawa H, Itoh K (2009) Transient dynamic actin cytoskeletal change
stimulates the osteoblastic differentiation. J Bone Miner Metab 27:158167
Ingber DE (2003) Tensegrity II. How structural networks influence cellular information processing
networks. J Cell Sci 116:13971408
Ishijima M, Ezura Y, Tsuji K, Rittling SR, Kurosawa H, Denhardt DT, Emi M, Nifuji A,
Noda M (2006) Osteopontin is associated with nuclear factor kappaB gene expression during
tail-suspension-induced bone loss. Exp Cell Res 312:30753083
Jaasma MJ, Jackson WM, Tang RY, Keaveny TM (2007) Adaptation of cellular mechanical
behavior to mechanical loading for osteoblastic cells. J Biomech 40(9):19381945
Kapur S, Baylink DJ, Lau KH (2003) Fluid flow shear stress stimulates human osteoblast pro-
liferation and differentiation through multiple interacting and competing signal transduction
pathways. Bone 32:241251
Kapur S, Chen ST, Baylink DJ, Lau KH (2004) Extracellular signal-regulated kinase-1 and -2 are
both essential for the shear stress-induced human osteoblast proliferation. Bone 35:525534
Klein-Nulend J, Burger EH, Semeins CM, Raisz LG, Pilbeam CC (1997) Pulsating fluid flow
stimulates prostaglandin release and inducible prostaglandin G/H synthase mRNA expression
in primary mouse bone cells. J Bone Miner Res 12:4551
Lammerding J, Kamm RD, Lee RT (2004). Mechanotransduction in cardiac myocytes. Ann NY
Acad Sci 1015:5370
Lau LF, Nathans D (1987) Expression of a set of growth-related immediate early genes in
BALB/c 3T3 cells: coordinate regulation with c-fos or c-myc. Proc Natl Acad Sci USA 84:
11821186
Lee DY, Yeh CR, Chang SF, Lee PL, Chien S, Cheng CK, Chiu JJ (2008) Integrin-mediated expres-
sion of bone formation-related genes in osteoblast-like cells in response to fluid shear stress:
roles of extracellular matrix, Shc, and mitogen-activated protein kinase. J Bone Miner Res
23:11401149
Li J, Chen G, Zheng L, Luo S, Zhao Z (2007) Osteoblast cytoskeletal modulation in response to
compressive stress at physiological levels. Mol Cell Biochem 304:4552
76 Q. Fu et al.
Liu J, Liu T, Zheng Y, Zhao Z, Liu Y, Cheng H, Luo S, Chen Y (2006) Early responses
of osteoblast-like cells to different mechanical signals through various signaling pathways.
Biochem Biophys Res Commun 348:11671173
McMahon AP, Champion JE, McMahon JA, Sukhatme VP (1990) Developmental expression of
the putative transcription factor Egr-1 suggests that Egr-1 and c-fos are coregulated in some
tissues. Development 108:281287
Moalli MR, Caldwell NJ, Patil PV, Goldstein SA (2000). An in vivo model for investigations of
mechanical signal transduction in trabecular bone. J Bone Miner Res 15:13461353
Morinobu M, Ishijima M, Rittling SR, Tsuji K, Yamamoto H, Nifuji A, Denhardt DT, Noda M
(2003) Osteopontin expression in osteoblasts and osteocytes during bone formation under
mechanical stress in the calvarial suture in vivo. J Bone Miner Res 18:17061715
Nomura S, Takano-Yamamoto T (2000) Molecular events caused by mechanical stress in bone.
Matrix Biol 19:9196
Norrdin RW, Shih MS (1988) Systemic effects of prostaglandin E2 on vertebral trabecular
remodeling in beagles used in a healing study. Calcif Tissue Int 42:363368
Ogasawara A, Arakawa T, Kaneda T, Takuma T, Sato T, Kaneko H, Kumegawa M, Hakeda Y
(2001) Fluid shear stress-induced cyclooxygenase-2 expression is mediated by C/EBP beta,
cAMP-response element-binding protein, and AP-1 in osteoblastic MC3T3-E1 cells. J Biol
Chem 276:70487054
Ogata T (1997) Fluid flow induces enhancement of the Egr-1 mRNA level in osteoblast-like cells:
involvement of tyrosine kinase and serum. J Cell Physiol 170:2734
Qian A, di S, Gao X, Zhang W, Tian Z, Li J, Hu L, Yang P, Yin D, Shang P (2009) cDNA
microarray reveals the alterations of cytoskeleton-related genes in osteoblast under high
magneto-gravitational environment. Acta Biochim Biophys Sin (Shanghai) 41:561577
Reich KM, McAllister TN, Gudi S, Frangos JA (1997) Activation of G proteins mediates flow-
induced prostaglandin E2 production in osteoblasts. Endocrinology 138:10141018
Sawada Y, Sheetz MP (2002) Force transduction by Triton cytoskeletons. J Cell Biol 156:609615
Schriefer JL, Warden SJ, Saxon LK, Robling AG, Turner CH (2005) Cellular accommodation and
the response of bone to mechanical loading. J Biomech 38:18381845
Smalt R, Mitchell FT, Howard RL, Chambers TJ (1997) Mechanotransduction in bone cells: induc-
tion of nitric oxide and prostaglandin synthesis by fluid shear stress, but not by mechanical
strain. Adv Exp Med Biol 433:311314
Stein GS, van Wijnen AJ, Stein JL, Lian JB, Pockwinse SH, McNeil S (1999) Implications
for interrelationships between nuclear architecture and control of gene expression under
microgravity conditions. FASEB J 13 Suppl:S157S166
Suponitzky I, Weinreb M (1998) Differential effects of systemic prostaglandin E2 on bone mass in
rat long bones and calvariae. J Endocrinol 156:5157
Thiel G, Cibelli G (2002) Regulation of life and death by the zinc finger transcription factor Egr-1.
J Cell Physiol 193:287292
Toma CD, Ashkar S, Gray ML, Schaffer JL, Gerstenfeld LC (1997) Signal transduction of
mechanical stimuli is dependent on microfilament integrity: identification of osteopontin as
a mechanically induced gene in osteoblasts. J Bone Miner Res 12:16261636
Turner CH (1999). Toward a mathematical description of bone biology: the principle of cellular
accommodation. Calcif Tissue Int 65:466471
Wadhwa S, Godwin SL, Peterson DR, Epstein MA, Raisz LG, Pilbeam CC (2002) Fluid flow
induction of cyclo-oxygenase 2 gene expression in osteoblasts is dependent on an extracellular
signal-regulated kinase signaling pathway. J Bone Miner Res 17:266274
Wu CJ, Fu Q (advisor) (2009). Effect of cytoskeleton reorganization inhibition on the mechanosen-
sitivity of COX-2 in osteoblasts induced by fluid shear stress. Masters Thesis, Sun Yat-Sen
University, Guangzhou
Zayzafoon M, Fulzele K, McDonald JM (2005) Calmodulin and calmodulin-dependent kinase
IIalpha regulate osteoblast differentiation by controlling c-fos expression. J Biol Chem
280:70497050
Chapter 4
Involvement of the Cytoskeletal Elements
in Articular Cartilage Mechanotransduction
Emma J. Blain
4.1 Introduction
4.1.1.1 Actin
Actin, a 43-kDa globular protein, is reported to be the most abundant protein in
eukaryotic cells (Disanza et al., 2005) and comprises three different monomers (,
and ). Under physiological conditions, the actin monomers (globular/G-actin)
assemble into long, highly organised filaments (filamentous/F-actin) (Fig. 4.1), and
it is the formation of these F-actin filaments which are critical to the activities of
many fundamental cellular events including cell migration (Heath and Holifield,
1991) and adhesion (Turner and Burridge, 1991), movement of organelles (Simon
and Pon, 1996), secretion (Sontag et al., 1988; Koukouritaki et al., 1996), alteration
of cell shape (Sims et al., 1992) and extracellular matrix assembly (Hayes et al.,
1999).
Actin assembly/disassembly is coupled with continuous ATP hydrolysis; a cleft
containing the nucleotide ATP (or ADP) and the cations Mg2+ and Ca2+ exists
within the G-actin moiety to meet the energy demands of actin polymerisation.
This ATP-dependent process of actin filament dis/assembly is referred to as tread-
milling, and it is the mechanism controlling cell locomotion, morphogenetic
movements and intracellular transport (Pollard and Borisy, 2003). Actin filaments
are polarised structures comprising a fast-growing (plus or barbed) end and a
slow-growing (minus or pointed) end (Ono, 2007). Under physiological conditions,
Mg2+ -ATP-bound G-actin is incorporated into growing filaments at the barbed end
(Cooper and Schafer, 2000; Ono, 2007). ATP is hydrolysed during F-actin poly-
merisation and a phosphate moiety is released. The resulting ADPactin filaments
are disassembled through the loss of ADPG-actin from the pointed end (Cooper
and Schafer, 2000; Ono, 2007). The released ADPG-actin undergoes nucleotide
exchange to produce ATPG-actin enabling continuation of F-actin polymerisation
at the barbed end.
Globular Actin
59nm
(G-actin)
Filamentous Actin
(F-actin)
(1) promoting nucleation of actin e.g. Arp2/3 complex (Goley and Welch, 2006),
(2) inducing actin filament depolymerisation e.g. the actin-depolymerising factor
family including ADF and cofilin (Ono, 2007),
(3) associating with G-actin moieties e.g. profilin and thymosin 4 (Yarmola and
Bubb, 2004),
(4) capping the F-actin filament end e.g. gelsolin (Cooper and Schafer, 2000).
The intermediate filaments (1012 nm: intermediate in size between the micro-
filaments and microtubules) are highly organised fibrous protein structures which
connect the plasma with the nuclear membrane. Although intermediate filament
function(s) has not been completely characterised to date, they, in addition to the
other cytoskeletal elements, have been implicated in intracellular mRNA transport
(Glotzer and Ephrussi, 1996; Lopez de Heredia and Jansen, 2004) and distribution of
organelles and proteins (Herrmann and Aebi, 2000). The intermediate filaments are
involved in the localisation of organelles including the Golgi apparatus, endosomes,
lysosomes and nuclei in the cell cytoplasm (Styers et al., 2005).
However, compelling evidence exists to demonstrate their involvement in signal
transduction (Traub, 1995), and in particular, they fulfil a critical role in mechan-
otransduction i.e. the propagation of a mechanical stimulus into a downstream
biochemical response(s) (Lazarides, 1980; Wang et al., 1993). The role of interme-
diate filaments in mechanotransduction has been verified in several human genetic
diseases (Omary et al., 2004) and in knockout studies (Eckes et al., 1998; Broers
et al., 2004) where cells lacking their usual complement of intermediate filaments
have been demonstrated to be mechanically fragile. These studies infer that the
intermediate filaments confer mechanical integrity to the cells and tissues.
Five classes of intermediate filaments exist including: class I cytokeratins
(epithelia), class II desmin (muscle cells), class III vimentin (mesenchymal
cells), class IV glial fibrillar acidic protein (glial cells) and neurofilaments (neu-
rons), and the class V nuclear lamins which are present in the nuclei of all
cell types. Unlike the other two major cytoskeletal elements, intermediate fila-
ment expression can be both developmentally regulated and cell-type specific i.e.
80 E.J. Blain
only vimentin class III intermediate filaments are observed in chondrocytes, and
may reflect their diversity in biological function. Intermediate filaments all con-
tain common highly-conserved structural features including a central -helical
rod domain flanked by non--helical N-(head) and C-terminal (tail) domains. The
highly-conserved central -helical rod primarily consists of seven-residue repeats
which configures a coiled-coil structure (Parry et al., 2007). Intermediate filaments
assemble through the association of two monomeric subunits aligned in parallel and
in register to form a homodimer. Two homodimers associate to form anti-parallel,
half-staggered tetramers, and eight of these tetramers associate laterally to form a
supercoiled sheet which has the characteristic appearance of a rope-like filamen-
tous structure (Fuchs and Weber, 1994; Sokolova et al., 2006) (Fig. 4.2). However,
it is the degree of heterogeneity in the non--helical head and tail domains (size
and sequence can differ) which provides the array of intermediate filament classes
indicated above.
The intracellular organisation of intermediate filament networks i.e. the dynam-
ics of assembly and disassembly is under the control of specific protein kinases
and phosphatases (Izawa and Inagaki, 2006). Phosphorylation of the intermedi-
ate filament protein occurs on specific serine and threonine residues, resulting
in filament disassembly; most of these phosphorylation sites are located in the
N-terminal non--helical head domain (Beuttenmuller et al., 1994; Izawa and
Inagaki, 2006) (Fig. 4.3). As a consequence of phosphorylation, filament disas-
sembly generates a pool of soluble subunits in preparation for the next phase of
polymerisation. Conversely, dephosphorylation of the intermediate filament insti-
gates filament assembly, and the architecture undergoes a rearrangement (Herrmann
and Aebi, 2000).
The direct involvement of intermediate filaments in signal transduction has been
demonstrated in many cell types, as many of the regulatory proteins appear to be
dependent upon their interaction with the filaments. Several studies have demon-
strated that protein kinases phosphorylate intermediate filaments at specific sites in
vitro (reviewed in (Izawa and Inagaki, 2006; Minin and Moldaver, 2008)). These
phosphorylation sites are located mainly on the N-terminal protein domains, which
are imperative for intermediate filament assembly. In vivo, vimentin is one of
the predominant phosphoproteins in the cytoplasm, and in vitro provides a good
substrate for second messenger protein kinases (Fig. 4.3). The dynamic status of
vimentin de/phosphorylation is critical in numerous signal transduction pathways
812nm
Monomer Dimer Tetramer Rope-like filament
(8 tetramers)
Fig. 4.2 Schematic representation of the assembly of the vimentin intermediate filaments
4 Involvement of the Cytoskeletal Elements 81
N- L1 L1-2 L2 -C
1A 1B 2A 2B
Fig. 4.3 Schematic representation of the phosphorylation sites in the vimentin head domain.
Serine residues are phosphorylated by PKC (protein kinase C), PKA (protein kinase A), PAK
(p21-activated kinase), Rho-kinase, CaM kinase II (Ca2+ /calmodulin-dependent protein kinase II,
cdc2 kinase, Plk1 (polo-like kinase 1) and Aurora-B (adapted from Izawa and Inagaki, 2006; Minin
and Moldaver, 2008)
including the MAP kinases ERK 1/2 (Perlson et al., 2006); Blain, unpublished
observations), various 14-3-3 signalling protein isoforms and apoptotic factors (Kim
and Coulombe, 2007).
(Valiron et al., 2001), that self-assemble to form the polymeric microtubular struc-
ture. Many functions have been attributed to the microtubules including cell motility,
mitosis, cell morphogenesis, organisation and transport of organelles. The principal
function of microtubules occurs during mitosis when they rearrange to form the
mitotic spindle to orientate the plane of cell cleavage (Mitchison et al., 1986).
The microtubules also operate as an intracellular supramolecular motor to segregate
the chromosomes to the cell poles during anaphase (Mitchison et al., 1986). The
ability of the microtubules to act as a supramolecular motor allows them to also act
as a highway for bi-directional transport: protein trafficking and secretion results
from the delineation of microtubule tracks along which the cargo are shuttled during
endo- and exocytosis (Vale, 1987; Thyberg and Moskalewski, 1999). Microtubules
organise the cell interior and are also involved in assembling intracellular organelles
e.g. endoplasmic reticulum and the Golgi apparatus (Rogalski and Singer, 1984).
Microtubules are also involved in sensing and responding to mechanical stimuli as
they are one of the principal constituents of the axonemal structures found in cilia
(Gibbons, 1981; Poole et al., 2001) and flagella (Woolley, 2000).
Tubulin assembly/disassembly is coupled with GTP hydrolysis; -tubulin binds
a GTP molecule in a non-exchangeable manner whereas GTP--tubulin binding is
exchangeable. GTP hydrolysis occurs upon the addition of a tubulin heterodimer
to the microtubule end, with the -tubulin subunit of the heterodimer exposed on
the faster growing end (plus end) and the -tubulin subunit exposed on the slow
growing end (minus end) (Valiron et al., 2001). Microtubules organise into linear
protofilaments that assemble to form 24 nm-wide cylindrical structures (Fig. 4.4).
The numbers of protofilaments which constitute a microtubule is generally consid-
ered to be 13 in vivo (Wade and Hyman, 1997), although the number may vary
depending on assembly conditions. As a result of the unique organisation of the
- and -tubulin heterodimers, the microtubule protofilament is polarised result-
ing in differing kinetic and structural properties. Microtubule polarity is critical
for the correct implementation of the microtubule motor proteins (comprehensively
reviewed in (Mallik and Gross, 2004; Wu et al., 2006)). Recognition of microtubule
Subunit
Protofilament 24nm
8nm
polarity by the motor proteins enables the specific motor employed to determine
the direction of transport along the microtubule tracks (Mallik and Gross, 2004).
Dynein shuttles cargo toward the microtubule minus-end (Hirokawa, 1998), sus-
taining microtubule-dependent trafficking of newly synthesised proteins from the
endoplasmic reticulum to the cis side of the golgi stacks, and providing vesicular
transport for endosomal clearance (Thyberg and Moskalewski, 1999). In contrast,
the motor protein kinesin moves toward the plus-end of the microtubules bringing
cargo toward the cell periphery (Hirokawa, 1998), e.g. by trafficking membrane and
secretory proteins (following post-translational modifications) from the trans side
of the golgi stacks. Kinesin is also involved in propagating the return transport of
endoplasmic reticulum components from the cis side of the golgi stacks (Thyberg
and Moskalewski, 1999).
The major load-bearing tissue of the synovial joint is articular cartilage which pro-
vides a smooth and resilient surface for joint locomotion. It is unique in being
avascular, aneural and alymphatic and contains a sparse population of cells the
chondrocyte interspersed in a dense extracellular matrix. The ability of articu-
lar cartilage to withstand highly repetitive stresses, brought about by movement,
depends on the structure and composition of this extracellular matrix.
The unique mechanical properties of articular cartilage are dependent on the organ-
isation (Fig. 4.5a) and composition of the tissue (Fig. 4.5b). Morphologically, there
are four distinct zones including the superficial, transitional, deep, and calcified zone
which merges into the subchondral bone. Articular cartilage (wet weight) comprises
approximately 6580% water, with 80% being in the superficial zone and 65% in the
deep zone; the highly hydrated nature of the tissue allows for load-dependent defor-
mation, as well as providing a medium for lubrication to maintain a low-friction
bearing surface zone (Buckwalter and Mankin, 1998). Collagens comprise 1020%
of the wet weight of articular cartilage with type II forming the principal component
(9095%); parallel arrangement of the collagen fibrils in the surface zone is respon-
sible for providing the greatest tensile and shear strength (Duance et al., 1998).
The other principal constituent of articular cartilage is proteoglycan (1020%); two
major classes of proteoglycans are found in articular cartilage: large aggregating
monomers e.g. aggrecan and the small leucine-rich proteoglycans e.g. decorin and
biglycan (Hardingham et al., 1992). Aggrecan is most abundant in the transitional
and deep zone, where due to its viscoelastic nature (hydrated gel) provides cartilage
with its function of compressibility under load. Articular cartilage also contains
a multitude of other extracellular matrix macromolecules which contribute to its
unique structural/mechanical integrity, but this lies outside the scope of this review.
84 E.J. Blain
hyaluronate
link protein
A. B.
aggrecan
decorin
SZ HA receptor COMP
glycosaminoglycan
TZ
biglycan
DZ type IX collagen
chondrocyte
SB
type II collagen
fibromodulin
core type XI collagen
Fig. 4.5 Organisation and composition of articular cartilage. (a) Haematoxylin and Eosin staining
of articular cartilage from the carpal metacarpal joint of a 7-day-old bovine calf. (b) Schematic car-
toon illustrating the interactions between the major components of articular cartilage (adapted from
Silver and Glasgold, 1995). Collectively the composition and organisation of these matrix macro-
molecules provides the tissue with its unique biomechanical properties to withstand compressive
load
loading which is primarily applied perpendicular to the surface of the articular car-
tilage (Armstrong et al., 1984). Dynamic, compressive loading of articular cartilage
induces cell deformation, changes in hydrostatic or fluid pressure and deformation
of the charged extracellular matrix, resulting in alterations in osmolality and pH
(Urban, 1994; Hall et al., 1996a), as depicted (Fig. 4.6). Chondrocytes can detect
these physico-chemical signals, and respond by altering their metabolism via
the activation of specific intracellular signalling pathways (Palmoski et al., 1980;
Palmoski and Brandt, 1984; Kiviranta et al., 1988; Sah et al., 1989).
soluble factors
territorial matrix
fluid flow
hydrostatic pressure
51 integrin
IL-4
F-actin FAK
Ca2+
iNOS
NO vimentin
ERK
tubulin
transcriptional
ECM responses
macromolecules Stretch-activated
macromolecule synthesis channels
Fig. 4.6 A schematic representation of how mechanical deformation, acting on articular cartilage,
can alter both the intra- and extracellular environment of the cells. Exposure of cells to mechan-
ical stimulation disseminates further effects across the extracellular matrix including opening of
stretch-activated channels, focal adhesion kinase signalling via the integrins, and rearrangement of
the cytoskeletal elements to induce changes in gene and protein expression (adapted from Urban,
1993)
86 E.J. Blain
The architecture of the three major cytoskeletal networks in chondrocytes have been
characterised in articular cartilage explants (Durrant et al., 1999; Langelier et al.,
2000), in primary chondrocytes cultured in agarose (Idowu et al., 2000; Knight et al.,
2001; Trickey et al., 2004; Sasazaki et al., 2008) and in high-density monolayer
cultures (Blain et al., 2006). Key morphological features are described (Table 4.1A).
F-actin is predominantly cortical in localisation and distributed at the periph-
ery of the chondrocyte (Idowu et al., 2000; Langelier et al., 2000; Knight et al.,
2001; Trickey et al., 2004; Blain et al., 2006; Sasazaki et al., 2008). In contrast,
the vimentin intermediate filaments extend throughout the cytoplasm and form a
highly organised architecture in chondrocytes, traversing from the plasma to the
nuclear membrane (Idowu et al., 2000; Langelier et al., 2000; Holloway et al.,
2004; Trickey et al., 2004; Blain et al., 2006; Sasazaki et al., 2008). Tubulin micro-
tubules are uniformly distributed throughout the chondrocyte cytoplasm and exhibit
a loose mesh-like network (Jortikka et al., 2000; Langelier et al., 2000; Trickey
et al., 2004; Blain et al., 2006). Zonal differences are also evident in the organ-
isation of the cytoskeletal networks in articular cartilage chondrocytes (Langelier
et al., 2000). A comparison of cytoskeletal element distribution through the depth of
4 Involvement of the Cytoskeletal Elements 87
Table 4.1 Organisation of the three major cytoskeletal elements: actin, vimentin and tubulin in
[A] normal and [B] osteoarthritic articular chondrocytes. All three structures are highly organised
in normal chondrocytes and their localisation in the cytoplasm reflects their biological functions
within the cell. However, in chondrocytes from osteoarthritic cartilage there is a general loss of
actin, vimentin and tubulin organisation, and a reduction in protein amounts. The loss of vimentin
organisation in osteoarthritic cartilage may be attributed to an increase in vimentin degradation
products which are derived from N-terminal domain cleavage i.e. that which is responsible for
filament assembly
Table 4.1B
Cytoskeletal
element Organisation osteoarthritic chondrocytes Reference
F-Actin less well-defined and localised diffusely in the Fioravanti et al. (2003)
cytoplasm or limited to cell periphery [Human]
Vimentin vimentin-rich multiple, elongated processes Holloway et al. (2004)
(correlating with greatest histological and
macroscopic signs of OA) [Human]
loss of expression 37.1% reduction. Capin-Gutierrez et al.
Predominantly localised to the superficial zone; (2004)
ranged from simple disorganisation to total
disruption of architecture [Rat partial
menisectomy model]
diffuse cytoplasmic distribution [Human] Lambrecht et al. (2008)
vimentin degradation products derived from Lambrecht et al. (2008)
cleavage of N-terminal domain (responsible for
filament assembly) [Human]
Tubulin often absent [Human] Fioravanti et al. (2003)
loss of expression 20.1% reduction [Rat partial Capin-Gutierrez et al.
menisectomy model] (2004)
88 E.J. Blain
Tubulin microtubules
skeletally mature bovine articular cartilage demonstrated that F-actin was uniformly
distributed; in contrast, both vimentin and tubulin were predominantly detected
in cells of the superficial zone (Langelier et al., 2000). Interestingly, there was
an increasing gradient in both vimentin and tubulin network organisation in car-
tilage situated in proximity to weight-bearing regions (Langelier et al., 2000). The
exhibition of specific structural characteristics in chondrocyte cytoskeletal network
organisation reflects their distinct functions within the cell.
4 Involvement of the Cytoskeletal Elements 89
matrix synthesis may reflect alterations to the normal functioning of the Golgi
apparatus, or its disruption of normal routes of intracellular transport.
in itself required to maintain the ability of the cartilage to withstand the forces
that are associated with tissue deformation (Urban, 1994). However, abnormal non-
physiological loads including disuse (Palmoski et al., 1980; Palmoski and Brandt,
1984) and high, repetitive loads (Vasan, 1983; Radin et al., 1984) inhibit extracel-
lular matrix synthesis, and is detrimental to the properties of the tissue in vivo.
Altering the biomechanical properties of the chondrocyte influences cell-matrix
interactions (Guilak and Mow, 2000), supporting the role of the cytoskeleton in
mechanical signal transduction. Collectively, the three major cytoskeletal networks
play a fundamental role in maintaining the phenotype of chondrocytes and act-
ing as a physical interface between the chondrocyte and the extracellular matrix
in sensing mechanical stimuli (Table 4.2).
Table 4.2 The effect of mechanical load on the expression and organisation of cytoskeletal
elements in articular cartilage chondrocytes
Cytoskeletal
element Effect of load on chondrocyte cytoskeleton Reference
F-Actin Loss of actin stress fibres with increasing hydrostatic Parkkinen et al. (1995)
pressure (1530 MPa); reversible after 2 h
Compressive strain (1015%) induced punctuate Knight et al. (2006)
reorganisation of F-actin replacing uniform
cortical distribution in unloaded cells
Actin reorganisation in response to osmotic stress Erickson et al. (2003)
loss of cortical F-actin distribution due to severing and Chao et al.
and detachment from cell periphery (2006)
Actin- Increased thymosin 4 mRNA: 0.5 MPa, 1 Hz, Blain et al. (2002) and
binding 10 min reorganisation of thymosin 4 and F-actin Blain et al.(2003)
proteins architecturesa
Increased cofilin and destrin mRNA: 15%, 1 Hz, Campbell et al. (2007)
10 min temporal disassembly of cortical F-actin
Actin-Binding Proteins
Actin remodelling in cartilage chondrocytes subjected to a mechanical stimulus has
been shown to involve the differential expression of the actin-binding proteins (Blain
et al., 2002, 2003; Knight et al., 2006; Campbell et al., 2007). We have previously
shown that thymosin 4, an actin-binding protein, is regulated by mechanical load
in articular chondrocytes (Blain et al., 2002, 2003). Thymosin 4 mRNA levels
were significantly elevated (20-fold) in articular cartilage explants subjected to a
dynamic low-physiological load (0.5 MPa, 1 Hz, 10 min); the response was tran-
sient as the thymosin 4 mRNA signal returned to basal levels after 60 min of load
(Blain et al., 2003). In unloaded explants chondrocytes, thymosin 4 protein was
cytoplasmically diffuse, but after 60 min of load, the protein was organised into
punctate loci throughout the cytoplasm; the distribution of thymosin 4 coincided
with a more abundant immunolabelling of F-actin in the loaded explant chondro-
cytes (Blain et al, unpublished data). Thymosin 4, an actin sequestering protein,
actively disassembles the actin filaments by binding in a 1:1 complex with G-
actin, thereby preventing its availability for polymerisation (Weber et al., 1992).
Other actin-disassembling proteins are also up-regulated by cyclic compression in
chondrocytes. mRNA levels of cofilin and destrin were significantly increased after
10 min of mechanical stimulation (15% strain, 1 Hz) in chondrocytes embedded in
agarose, concomitant with a temporal disassembly of cortical F-actin (Campbell
4 Involvement of the Cytoskeletal Elements 95
often absent in OA cells (Fioravanti et al., 2003). Fioravanti et al. postulated that
the loss of cytoskeletal element assembly in OA chondrocytes could compromise,
not only the metabolic activities of the cells, but also their biomechanical function
(Fioravanti et al., 2003). Disparities in cytoskeletal element expression and/or organ-
isation are not exclusive to human pathology but have also been reported in animal
models of OA. In a rat partial menisectomy model of OA, there was a reduction
in the number of OA chondrocytes immuno-positive for F-actin (4.7%) and tubulin
(20.1%) expression (Capin-Gutierrez et al., 2004).
Concomitant with this was the observed reduction in vimentin expression
(37.1%). Vimentin filaments which predominantly localised to the superficial zone,
ranged from a simple disorganisation to a total disruption of cytoplasmic archi-
tecture; this contrasted with the uniform distribution of vimentin filaments as
perinuclear bundles in normal rat chondrocytes (Capin-Gutierrez et al., 2004). The
vimentin cytoskeleton presented as a complex fibrous structure extending through-
out the cytoplasm in both normal and OA human chondrocytes (Holloway et al.,
2004). Interestingly, multiple elongated processes containing elaborate vimentin fil-
ament networks, extending up to 30 m into the surrounding matrix, were also
observed in the OA cartilage which corresponded to sites with the greatest histo-
logical and macroscopic signs of OA (Holloway et al., 2004). Thus, the aberrant
arrangement of the cytoskeletal networks in OA cells will impact on the mechano-
sensitivity of the cell, and the downstream signalling cascades activated as a
consequence.
Chondrocyte stiffness i.e. the mechanical properties of the cell, is an important indi-
cator for how a cell will respond to mechanical perturbations in the surrounding
matrix environment (refer to Section 4.1). The observed aberration in cytoskeletal
element organisation in OA chondrocytes impacts on the mechanical properties of
the cell. Significant increases in elastic and viscous properties were observed in
OA chondrocytes from end-stage pathology (Trickey et al., 2000). Interestingly,
a recent study reported that the mechanical properties of cartilage chondrocytes
changed profoundly with ageing, and not OA pathology (Steklov et al., 2009).
Viscoelastic properties increased in aged cartilage (< 55 years of age), but there were
no apparent differences between the aged and age-matched OA cartilage. Clearly,
the biomechanical properties of the cartilage are dependent on cytoskeletal element
organisation irrespective of whether age and/or pathology are/is the confounding
influence.
An alteration to the biomechanical properties of the cell i.e. as a conse-
quence of cytoskeletal element aberrations, undoubtedly affects mechanotransduc-
tion responses. Cyclical pressure (15 MPa, 0.25 Hz, 3 h) did not affect F-actin
or tubulin distribution in OA chondrocytes, although the elements were subject to
reorganisation in normal chondrocytes (Fioravanti et al., 2005). It was suggested
98 E.J. Blain
that the cytoskeletal structures, which are not assembled appropriately in OA carti-
lage (Fioravanti et al., 2003), were unable to respond to similar loading conditions,
possibly becoming desensitised. This could compromise the metabolic activities
of the chondrocytes and hence the biomechanical integrity of the cartilage lead-
ing to pathology (Fioravanti et al., 2005). However, it was demonstrated in OA
tissue that the incidence and length of cilia, detected using an anti-acetylated tubu-
lin antibody, increased at the eroding articulating surface, resulting in an overall
increased proportion of ciliated cells with OA severity (McGlashan et al., 2008).
This study (McGlashan et al., 2008) would argue against the hypothesis that the
tubulin cytoskeleton becomes desensitised to mechanical stimulation in pathological
tissue (Fioravanti et al., 2003).
The mechanisms of cartilage chondrocyte mechanotransduction have also been
demonstrated to differ between normal and OA cells. As previously discussed
(Section 4.2.1), a mechanical stimulus is perceived by the chondrocyte and 5 1
integrin-mediated signalling is activated (Salter et al., 2001). This involves the
opening of stretch-activated channels, an alteration in membrane potential and
the rapid phosphorylation of focal adhesion proteins e.g. FAK125 , paxillin and
-catenin concomitant with F-actin treadmilling and the activation of downstream
signalling cascades e.g. PKC (Lee et al., 2000b). However, although the initial
response of OA chondrocytes to mechanical stimulation involved activation of 5 1
integrin, the mechanotransduction route was independent of the F-actin cytoskele-
ton (Millward-Sadler et al., 2000), which may imply an adaptation by the cell
to circumvent an aberrant F-actin network. Compressive stimulation activated an
integrin-dependent interleukin-4 (IL-4) autocrine/paracrine loop in normal chon-
drocytes, whereas mechanotransduction was mediated via the activation of an IL-1
autocrine/paracrine loop in OA cells (Salter et al., 2002). Interestingly, IL-1 has been
shown to increase F-actin amounts in chondrocytes (Pritchard and Guilak, 2006)
suggesting that a finely balanced interplay of soluble factors e.g. cytokines may exist
to regulate cytoskeletal element dynamics and hence organisation. Recently, a whole
genome array demonstrated that IL-1 inhibited the mRNA expression of both tubu-
lin and vimentin, as well as the LIM protein FHL2 which is associated with the actin
cytoskeleton (Joos et al., 2008). Therefore in an inflamed or pathological environ-
ment i.e. in an OA synovial joint, the organisation and expression levels of these
cytoskeletal proteins may be compromised which, in turn, will adversely influence
the biomechanical properties preventing normal modes of cartilage chondrocyte
mechano-signalling.
The cytoskeleton acts as a physical interface between the chondrocyte and the extra-
cellular matrix in sensing mechanical stimuli, and the cytoskeletal elements are
themselves subject to load-induced reorganisation. Although several mechanisms
of mechanotransduction involving the three major cytoskeletal elements have been
4 Involvement of the Cytoskeletal Elements 99
References
Armstrong CG, Lai WM, Mow VC (1984) An analysis of the unconfined compression of articular
cartilage. J Biomech Eng 106:165173
Banes AJ, Tsuzaki M, Yamamoto J, Fischer T, Brigman B, Brown T, Miller L (1995)
Mechanoreception at the cellular level: the detection, interpretation, and diversity of responses
to mechanical signals. Biochem Cell Biol 73:349365
Ben-Zeev A (1991) Animal cell shape changes and gene expression. Bioessays 13:207212
Benjamin M, Archer CW, Ralphs JR (1994) Cytoskeleton of cartilage cells. Microsc Res Tech
28:372377
Benya PD, Shaffer JD (1982) Dedifferentiated chondrocytes reexpress the differentiated collagen
phenotype when cultured in agarose gels. Cell 30:215224
Beuttenmuller M, Chen M, Janetzko A, Kuhn S, Traub P (1994) Structural elements of the amino-
terminal head domain of vimentin essential for intermediate filament formation in vivo and in
vitro. Exp Cell Res 213:128142
Blain EJ, Gilbert SJ, Hayes AJ, Duance VC (2006) Disassembly of the vimentin cytoskeleton
disrupts articular cartilage chondrocyte homeostasis. Matrix Biol 25:398408
Blain EJ, Mason DJ, Duance VC (2002) The effect of thymosin beta4 on articular cartilage
chondrocyte matrix metalloproteinase expression. Biochem Soc Trans 30:879882
Blain EJ, Mason DJ, Duance VC (2003) The effect of cyclical compressive loading on gene
expression in articular cartilage. Biorheology 40:111117
Broers JL, Peeters EA, Kuijpers HJ, Endert J, Bouten CV, Oomens CW, Baaijens FP, Ramaekers
FC (2004) Decreased mechanical stiffness in LMNA/ cells is caused by defective nucleo-
cytoskeletal integrity: implications for the development of laminopathies. Hum Mol Genet
13:25672580
Brown PD, Benya PD (1988) Alterations in chondrocyte cytoskeletal architecture during pheno-
typic modulation by retinoic acid and dihydrocytochalasin B-induced reexpression. J Cell Biol
106:171179
Buckwalter JA, Mankin HJ (1998) Articular cartilage: tissue design and chondrocyte-matrix
interactions. Instr Course Lect 47:477486
Burke B, Stewart CL (2006) The laminopathies: the functional architecture of the nucleus and its
contribution to disease. Annu Rev Genomics Hum Genet 7:369405
Buschmann MD, Hunziker EB, Kim YJ, Grodzinsky AJ (1996) Altered aggrecan synthesis cor-
relates with cell and nucleus structure in statically compressed cartilage. J Cell Sci 109
(Pt 2):499508
100 E.J. Blain
Campbell JJ, Blain EJ, Chowdhury TT, Knight MM (2007) Loading alters actin dynamics and
up-regulates cofilin gene expression in chondrocytes. Biochem Biophys Res Commun 361:
329334
Capin-Gutierrez N, Talamas-Rohana P, Gonzalez-Robles A, Lavalle-Montalvo C, Kouri JB (2004)
Cytoskeleton disruption in chondrocytes from a rat osteoarthrosic (OA) -induced model: its
potential role in OA pathogenesis. Histol Histopathol 19:11251132
Chao PH, West AC, Hung CT (2006) Chondrocyte intracellular calcium, cytoskeletal organiza-
tion, and gene expression responses to dynamic osmotic loading. Am J Physiol Cell Physiol
291:C718C725
Cohen TV, Hernandez L, Stewart CL (2008) Functions of the nuclear envelope and lamina in
development and disease. Biochem Soc Trans 36:13291334
Colucci-Guyon E, Portier MM, Dunia I, Paulin D, Pournin S, Babinet C (1994) Mice
lacking vimentin develop and reproduce without an obvious phenotype. Cell 79:
679694
Cooper C, McAlindon T, Snow S, Vines K, Young P, Kirwan J, Dieppe P (1994) Mechanical
and constitutional risk factors for symptomatic knee osteoarthritis: differences between medial
tibiofemoral and patellofemoral disease. J Rheumatol 21:307313
Cooper JA, Schafer DA (2000) Control of actin assembly and disassembly at filament ends. Curr
Opin Cell Biol 12:97103
Dechat T, Pfleghaar K, Sengupta K, Shimi T, Shumaker DK, Solimando L, Goldman RD (2008)
Nuclear lamins: major factors in the structural organization and function of the nucleus and
chromatin. Genes Dev 22:832853
Disanza A, Steffen A, Hertzog M, Frittoli E, Rottner K, Scita G (2005) Actin polymerization
machinery: the finish line of signaling networks, the starting point of cellular movement. Cell
Mol Life Sci 62:955970
Duance VC, Vaughan-Thomas A, Wardale RJ, Wotton SF (1998) Collagens of articular, growth
plate and meniscal cartilages. In: Caterson B, Archer CW, Benjamin M, Ralphs JR (eds)
Biology of the synovial joint. Harwood Academic Press, London, pp 135163
Durrant LA, Archer CW, Benjamin M, Ralphs JR (1999) Organisation of the chondrocyte
cytoskeleton and its response to changing mechanical conditions in organ culture. J Anat 194
(Pt 3):343353
Eckes B, Dogic D, Colucci-Guyon E, Wang N, Maniotis A, Ingber D, Merckling A, Langa F,
Aumailley M, Delouvee A, Koteliansky V, Babinet C, Krieg T (1998) Impaired mechanical
stability, migration and contractile capacity in vimentin-deficient fibroblasts. J Cell Sci 111
(Pt 13):18971907
Eggli PS, Hunziker EB, Schenk RK (1988) Quantitation of structural features characterizing
weight- and less-weight-bearing regions in articular cartilage: a stereological analysis of medial
femoral condyles in young adult rabbits. Anat Rec 222:217227
Erickson GR, Northrup DL, Guilak F (2003) Hypo-osmotic stress induces calcium-dependent actin
reorganization in articular chondrocytes. Osteoarthritis Cartilage 11:187197
Fioravanti A, Benetti D, Coppola G, Collodel G (2005) Effect of continuous high hydrostatic pres-
sure on the morphology and cytoskeleton of normal and osteoarthritic human chondrocytes
cultivated in alginate gels. Clin Exp Rheumatol 23:847853
Fioravanti A, Nerucci F, Annefeld M, Collodel G, Marcolongo R (2003) Morphological and
cytoskeletal aspects of cultivated normal and osteoarthritic human articular chondrocytes after
cyclical pressure: a pilot study. Clin Exp Rheumatol 21:739746
Fuchs E, Weber K (1994) Intermediate filaments: structure, dynamics, function, and disease. Annu
Rev Biochem 63:345382
Gibbons IR (1981) Cilia and flagella of eukaryotes. J Cell Biol 91:107s124s
Glotzer JB, Ephrussi A (1996) mRNA localization and the cytoskeleton. Semin Cell Dev Biol
7:357365
Goldring MB (2000) Osteoarthritis and cartilage: the role of cytokines. Curr Rheumatol Rep
2:459465
4 Involvement of the Cytoskeletal Elements 101
Goley ED, Welch MD (2006) The ARP2/3 complex: an actin nucleator comes of age. Nat Rev Mol
Cell Biol 7:713726
Grodzinsky AJ, Levenston ME, Jin M, Frank EH (2000) Cartilage tissue remodeling in response
to mechanical forces. Annu Rev Biomed Eng 2:691713
Guilak F (1995) Compression-induced changes in the shape and volume of the chondrocyte
nucleus. J Biomech 28:15291541
Guilak F, Mow VC (2000) The mechanical environment of the chondrocyte: a biphasic finite
element model of cell-matrix interactions in articular cartilage. J Biomech 33:16631673
Guilak F, Ratcliffe A, Lane N, Rosenwasser MP, Mow VC (1994) Mechanical and biochemi-
cal changes in the superficial zone of articular cartilage in canine experimental osteoarthritis.
J Orthop Res 12:474484
Guilak F, Ratcliffe A, Mow VC (1995) Chondrocyte deformation and local tissue strain in articular
cartilage: a confocal microscopy study. J Orthop Res 13:410421
Hall AC (1999) Differential effects of hydrostatic pressure on cation transport pathways of isolated
articular chondrocytes. J Cell Physiol 178:197204
Hall AC, Horwitz ER, Wilkins RJ (1996a) The cellular physiology of articular cartilage. Exp
Physiol 81:535545
Hall AC, Starks I, Shoults CL, Rashidbigi S (1996b) Pathways for K+ transport across the
bovine articular chondrocyte membrane and their sensitivity to cell volume. Am J Physiol 270:
C1300C1310
Hardingham TE, Fosang AJ, Dudhiah J (1992) Aggrecan, the chondroitin sulphate/keratan sulphate
proteoglycan from cartilage. In: Kuettner KE, Schleyerback R, Peyron JG, Hascall VC (eds)
Articular cartilage and osteoarthritis. Raven Press, New York, NY
Haudenschild DR, DLima DD, Lotz MK (2008a) Dynamic compression of chondrocytes induces
a Rho kinase-dependent reorganization of the actin cytoskeleton. Biorheology 45:219228
Haudenschild DR, Nguyen B, Chen J, DLima DD, Lotz MK (2008b) Rho kinase-dependent
CCL20 induced by dynamic compression of human chondrocytes. Arthritis Rheum 58:
27352742
Hayes AJ, Benjamin M, Ralphs JR (1999) Role of actin stress fibres in the development of
the intervertebral disc: cytoskeletal control of extracellular matrix assembly. Dev Dyn 215:
179189
Heath JP, Holifield BF (1991) Cell locomotion: new research tests old ideas on membrane and
cytoskeletal flow. Cell Motil Cytoskeleton 18:245257
Henrion D, Terzi F, Matrougui K, Duriez M, Boulanger CM, Colucci-Guyon E, Babinet C, Briand
P, Friedlander G, Poitevin P, Levy BI (1997) Impaired flow-induced dilation in mesenteric
resistance arteries from mice lacking vimentin. J Clin Invest 100:29092914
Henson FM, Vincent TA (2008) Alterations in the vimentin cytoskeleton in response to single
impact load in an in vitro model of cartilage damage in the rat. BMC Musculoskelet Disord
9:94
Herrmann H, Aebi U (2000) Intermediate filaments and their associates: multi-talented structural
elements specifying cytoarchitecture and cytodynamics. Curr Opin Cell Biol 12:7990
Hirokawa N (1998) Kinesin and dynein superfamily proteins and the mechanism of organelle
transport. Science 279:519526
Holloway I, Kayser M, Lee DA, Bader DL, Bentley G, Knight MM (2004) Increased presence of
cells with multiple elongated processes in osteoarthritic femoral head cartilage. Osteoarthritis
Cartilage 12:1724
Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:1125
Idowu BD, Knight MM, Bader DL, Lee DA (2000) Confocal analysis of cytoskeletal organisation
within isolated chondrocyte sub-populations cultured in agarose. Histochem J 32:165174
Ingber DE, Dike L, Hansen L, Karp S, Liley H, Maniotis A, McNamee H, Mooney D, Plopper G,
Sims J, et al. (1994) Cellular tensegrity: exploring how mechanical changes in the cytoskele-
ton regulate cell growth, migration, and tissue pattern during morphogenesis. Int Rev Cytol
150:173224
102 E.J. Blain
Izawa I, Inagaki M (2006) Regulatory mechanisms and functions of intermediate filaments: a study
using site- and phosphorylation state-specific antibodies. Cancer Sci 97:167174
Janmey PA (1998) The cytoskeleton and cell signaling: component localization and mechanical
coupling. Physiol Rev 78:763781
Jansen HW, Bornstein P (1974) Effects of antimicrotubular agents on glycosaminoglycan synthesis
and secretion by embryonic chick cartilage and chondrocytes. Biochim Biophys Acta 362:
150159
Jeffrey JE, Thomson LA, Aspden RM (1997) Matrix loss and synthesis following a single impact
load on articular cartilage in vitro. Biochim Biophys Acta 1334:223232
Joos H, Albrecht W, Laufer S, Reichel H, Brenner RE (2008) IL-1beta regulates FHL2 and other
cytoskeleton-related genes in human chondrocytes. Mol Med 14:150159
Jortikka MO, Parkkinen JJ, Inkinen RI, Karner J, Jarvelainen HT, Nelimarkka LO, Tammi MI,
Lammi MJ (2000) The role of microtubules in the regulation of proteoglycan synthesis in
chondrocytes under hydrostatic pressure. Arch Biochem Biophys 374:172180
Karjalainen HM, Sironen RK, Elo MA, Kaarniranta K, Takigawa M, Helminen HJ, Lammi MJ
(2003) Gene expression profiles in chondrosarcoma cells subjected to cyclic stretching and
hydrostatic pressure. A cDNA array study. Biorheology 40:93100
Kim S, Coulombe PA (2007) Intermediate filament scaffolds fulfill mechanical, organizational,
and signaling functions in the cytoplasm. Genes Dev 21:15811597
Kiviranta I, Tammi M, Jurvelin J, Saamanen AM, Helminen HJ (1988) Moderate running exercise
augments glycosaminoglycans and thickness of articular cartilage in the knee joint of young
beagle dogs. J Orthop Res 6:188195
Knight MM, Ghori SA, Lee DA, Bader DL (1998) Measurement of the deformation of isolated
chondrocytes in agarose subjected to cyclic compression. Med Eng Phys 20:684688
Knight MM, Idowu BD, Lee DA, Bader DL (2001) Temporal changes in cytoskeletal organisation
within isolated chondrocytes quantified using a novel image analysis technique. Med Biol Eng
Comput 39:397404
Knight MM, McGlashan SR, Garcia M, Jensen CG, Poole CA (2009) Articular chondrocytes
express connexin 43 hemichannels and P2 receptors a putative mechanoreceptor complex
involving the primary cilium? J Anat 214:275283
Knight MM, Toyoda T, Lee DA, Bader DL (2006) Mechanical compression and hydrostatic pres-
sure induce reversible changes in actin cytoskeletal organisation in chondrocytes in agarose.
J Biomech 39:15471551
Koukouritaki SB, Theodoropoulos PA, Margioris AN, Gravanis A, Stournaras C (1996)
Dexamethasone alters rapidly actin polymerization dynamics in human endometrial cells:
evidence for nongenomic actions involving cAMP turnover. J Cell Biochem 62:251261
Kouri JB, Arguello C, Luna J, Mena R (1998) Use of microscopical techniques in the study
of human chondrocytes from osteoarthritic cartilage: an overview. Microsc Res Tech 40:
2236
Lahiji K, Polotsky A, Hungerford DS, Frondoza CG (2004) Cyclic strain stimulates proliferative
capacity, alpha2 and alpha5 integrin, gene marker expression by human articular chondrocytes
propagated on flexible silicone membranes. In Vitro Cell Dev Biol Anim 40:138142
Lambrecht S, Verbruggen G, Verdonk PC, Elewaut D, Deforce D (2008) Differential proteome
analysis of normal and osteoarthritic chondrocytes reveals distortion of vimentin network in
osteoarthritis. Osteoarthritis Cartilage 16:163173
Langelier E, Suetterlin R, Hoemann CD, Aebi U, Buschmann MD (2000) The chondrocyte
cytoskeleton in mature articular cartilage: structure and distribution of actin, tubulin, and
vimentin filaments. J Histochem Cytochem 48:13071320
Lazarides E (1980) Intermediate filaments as mechanical integrators of cellular space. Nature
283:249256
Lee DA, Knight MM, Bolton JF, Idowu BD, Kayser MV, Bader DL (2000a) Chondrocyte defor-
mation within compressed agarose constructs at the cellular and sub-cellular levels. J Biomech
33:8195
4 Involvement of the Cytoskeletal Elements 103
Lee HS, Millward-Sadler SJ, Wright MO, Nuki G, Al-Jamal R, Salter DM (2002) Activation of
Integrin-RACK1/PKCalpha signalling in human articular chondrocyte mechanotransduction.
Osteoarthritis Cartilage 10:890897
Lee HS, Millward-Sadler SJ, Wright MO, Nuki G, Salter DM (2000b) Integrin and mechanosen-
sitive ion channel-dependent tyrosine phosphorylation of focal adhesion proteins and beta-
catenin in human articular chondrocytes after mechanical stimulation. J Bone Miner Res
15:15011509
Lohmander S, Madsen K, Hinek A (1979) Secretion of proteoglycans by chondrocytes. Influence
of colchicine, cytochalasin B, and beta-D-xyloside. Arch Biochem Biophys 192:148157
Lohmander S, Moskalewski S, Madsen K, Thyberg J, Friberg U (1976) Influence of colchicine
on the synthesis and secretion of proteoglycans and collagen by fetal guinea pig chondrocytes.
Exp Cell Res 99:333345
Lopez de Heredia M, Jansen RP (2004) mRNA localization and the cytoskeleton. Curr Opin Cell
Biol 16:8085
Loty S, Forest N, Boulekbache H, Sautier JM (1995) Cytochalasin D induces changes in cell shape
and promotes in vitro chondrogenesis: a morphological study. Biol Cell 83:149161
Madsen K, Moskalewski S, Thyberg J, Friberg U (1979) Comparison of the in vitro effects of
colchicine and its derivative colchiceine on chondrocyte morphology and function. Experientia
35:15721573
Mallik R, Gross SP (2004) Molecular motors: strategies to get along. Curr Biol 14:R971982
Maniotis AJ, Chen CS, Ingber DE (1997) Demonstration of mechanical connections between inte-
grins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc Natl Acad
Sci USA 94:849854
McGlashan SR, Cluett EC, Jensen CG, Poole CA (2008) Primary cilia in osteoarthritic chondro-
cytes: from chondrons to clusters. Dev Dyn 237:20132020
McGlashan SR, Jensen CG, Poole CA (2006) Localization of extracellular matrix receptors on the
chondrocyte primary cilium. J Histochem Cytochem 54:10051014
McGlashan SR, Knight MM, Chowdhury TT, Joshi P, Jensen CG, Kennedy S, Poole CA (2010)
Mechanical loading modulates chondrocyte primary cilia incidence and length. Cell Biol Int
34(5):441446
Millward-Sadler SJ, Wright MO, Lee H, Caldwell H, Nuki G, Salter DM (2000) Altered
electrophysiological responses to mechanical stimulation and abnormal signalling through
alpha5beta1 integrin in chondrocytes from osteoarthritic cartilage. Osteoarthritis Cartilage
8:272278
Minin AA, Moldaver MV (2008) Intermediate vimentin filaments and their role in intracellular
organelle distribution. Biochemistry (Mosc) 73:14531466
Mitchison T, Evans L, Schulze E, Kirschner M (1986) Sites of microtubule assembly and
disassembly in the mitotic spindle. Cell 45:515527
Moskalewski S, Thyberg J, Lohmander S, Friberg U (1975) Influence of colchicine and vinblastine
on the golgi complex and matrix deposition in chondrocyte aggregates. An ultrastructural study.
Exp Cell Res 95:440454
Muir H (1995) The chondrocyte, architect of cartilage. Biomechanics, structure, function and
molecular biology of cartilage matrix macromolecules. Bioessays 17:10391048
Newman P, Watt FM (1988) Influence of cytochalasin D-induced changes in cell shape on
proteoglycan synthesis by cultured articular chondrocytes. Exp Cell Res 178:199210
Nofal GA, Knudson CB (2002) Latrunculin and cytochalasin decrease chondrocyte matrix
retention. J Histochem Cytochem 50:13131324
Ofek G, Wiltz DC, Athanasiou KA (2009) Contribution of the cytoskeleton to the compressive
properties and recovery behavior of single cells. Biophys J 97:18731882
Omary MB, Coulombe PA, McLean WH (2004) Intermediate filament proteins and their associated
diseases. N Engl J Med 351:20872100
Ono S (2007) Mechanism of depolymerization and severing of actin filaments and its significance
in cytoskeletal dynamics. Int Rev Cytol 258:182
104 E.J. Blain
Palmoski MJ, Brandt KD (1984) Effects of static and cyclic compressive loading on articular
cartilage plugs in vitro. Arthritis Rheum 27:675681
Palmoski MJ, Colyer RA, Brandt KD (1980) Joint motion in the absence of normal loading does
not maintain normal articular cartilage. Arthritis Rheum 23:325334
Parkkinen JJ, Lammi MJ, Inkinen R, Jortikka M, Tammi M, Virtanen I, Helminen HJ (1995)
Influence of short-term hydrostatic pressure on organization of stress fibers in cultured
chondrocytes. J Orthop Res 13:495502
Parry DA, Strelkov SV, Burkhard P, Aebi U, Herrmann H (2007) Towards a molecular description
of intermediate filament structure and assembly. Exp Cell Res 313:22042216
Perlson E, Michaelevski I, Kowalsman N, Ben-Yaakov K, Shaked M, Seger R, Eisenstein M,
Fainzilber M (2006) Vimentin binding to phosphorylated Erk sterically hinders enzymatic
dephosphorylation of the kinase. J Mol Biol 364:938944
Pollard TD, Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin
filaments. Cell 112:453465
Poole CA, Jensen CG, Snyder JA, Gray CG, Hermanutz VL, Wheatley DN (1997) Confocal anal-
ysis of primary cilia structure and colocalization with the Golgi apparatus in chondrocytes and
aortic smooth muscle cells. Cell Biol Int 21:483494
Poole CA, Zhang ZJ, Ross JM (2001) The differential distribution of acetylated and detyrosi-
nated alpha-tubulin in the microtubular cytoskeleton and primary cilia of hyaline cartilage
chondrocytes. J Anat 199:393405
Pritchard S, Guilak F (2006) Effects of interleukin-1 on calcium signaling and the increase of
filamentous actin in isolated and in situ articular chondrocytes. Arthritis Rheum 54:21642174
Pullikuth AK, Catling AD (2007) Scaffold mediated regulation of MAPK signaling and cytoskele-
tal dynamics: a perspective. Cell Signal 19:16211632
Radin EL, Martin RB, Burr DB, Caterson B, Boyd RD, Goodwin C (1984) Effects of mechanical
loading on the tissues of the rabbit knee. J Orthop Res 2:221234
Reginato AM, Olsen BR (2002) The role of structural genes in the pathogenesis of osteoarthritic
disorders. Arthritis Res 4:337345
Rober RA, Weber K, Osborn M (1989) Differential timing of nuclear lamin A/C expression
in the various organs of the mouse embryo and the young animal: a developmental study.
Development 105:365378
Roberts SR, Knight MM, Lee DA, Bader DL (2001) Mechanical compression influences intracellu-
lar Ca2+ signaling in chondrocytes seeded in agarose constructs. J Appl Physiol 90:13851391
Rogalski AA, Singer SJ (1984) Associations of elements of the Golgi apparatus with microtubules.
J Cell Biol 99:10921100
Sah RL, Kim YJ, Doong JY, Grodzinsky AJ, Plaas AH, Sandy JD (1989) Biosynthetic response of
cartilage explants to dynamic compression. J Orthop Res 7:619636
Salter DM, Millward-Sadler SJ, Nuki G, Wright MO (2001) Integrin-interleukin-4 mechanotrans-
duction pathways in human chondrocytes. Clin Orthop Relat Res:S49S60
Salter DM, Millward-Sadler SJ, Nuki G, Wright MO (2002) Differential responses of chon-
drocytes from normal and osteoarthritic human articular cartilage to mechanical stimulation.
Biorheology 39:97108
Sasazaki Y, Seedhom BB, Shore R (2008) Morphology of the bovine chondrocyte and of its
cytoskeleton in isolation and in situ: are chondrocytes ubiquitously paired through the entire
layer of articular cartilage? Rheumatology (Oxford) 47:16411646
Schiffers PM, Henrion D, Boulanger CM, Colucci-Guyon E, Langa-Vuves F, van Essen H, Fazzi
GE, Levy BI, De Mey JG (2000) Altered flow-induced arterial remodeling in vimentin-deficient
mice. Arterioscler Thromb Vasc Biol 20:611616
Seghatoleslami MR, Roman-Blas JA, Rainville AM, Modaressi R, Danielson KG, Tuan RS (2003)
Progression of chondrogenesis in C3H10T1/2 cells is associated with prolonged and tight
regulation of ERK1/2. J Cell Biochem 88:11291144
Silver FH, Glasgold AI (1995) Cartilage wound healing. An overview. Otolaryngol Clin North Am
28:847864
4 Involvement of the Cytoskeletal Elements 105
Wu X, Xiang X, Hammer JA, 3rd (2006) Motor proteins at the microtubule plus-end. Trends Cell
Biol 16:135143
Yarmola EG, Bubb MR (2004) Effects of profilin and thymosin beta4 on the critical concentra-
tion of actin demonstrated in vitro and in cell extracts with a novel direct assay. J Biol Chem
279:3351933527
Part II
Molecular Mechanisms
of Mechanotransduction
and Ion Channels Modulation
Chapter 5
The Role of Nitric Oxide in the Regulation
of Mechanically Gated Channels in the Heart
A. Kamkin (B)
Department of Fundamental and Applied Physiology, Russian State Medical University,
Ostrivitjanova 1, Moscow 117997, Russia
e-mail: Kamkin.A@g23.relcom.ru
5.1 Introduction
Since the first report on NO effects on myocardial contractility in 1991 (Smith et al.,
1991) a lot of data, often contradicting, have been accumulated on NO effects on
heart functions. In general it can be said that the final effect of this regulator depends
112 V. Kazanski et al.
on the following: (1) the cellular source of NO, (2) amount of NO release, (3) pre-
vailing redox balance and antioxidant status, (4) stimuli such as coronary flow rate
and heart rate, (5) the target tissue, (6) interaction with neurohumoral and other
stimuli, (7) activity level of immune system or disease, and (8) activation of intracel-
lular cGMP-dependent and independent subcellular cascades (Shah and MacCarthy,
2000).
NO takes part in regulating cardiac functions already at the embryogenesis stage.
NO affects the growth and development of cardiac cells in fetal and postnatal lives
(Strijdom et al., 2009). Moreover, several authors have considered NO as the key
signaling molecule in early embryonic development of the heart, at least in rodents
(Malan et al., 2004).
NO is cyclically secreted in the working heart with fast growing concentration
during early diastolic filling, which can be connected to the autoregulation during
the cardiac cycle (Pinsky et al. 1997). Endogenous NO causes different effects in
the heart, one of which is early relaxation of the left ventricle, that was shown on
isolated heart and isolated cardiomyocytes (Shah et al., 1994; Casadei and Sears,
2003). It was discovered that specific inhibition of NOS1 or destruction of NOS1
genes results in increased left ventricular contractility in vitro and in vivo (Ashley
et al., 2002; Casadei and Sears, 2003; Dawson et al., 2005). Clinical studies have
also shown that under NO there were changes in the onset time of left ventricle
relaxation, that manifests itself in an earlier onset of relaxation and decreased peak
and end-diastolic pressure of the left ventricle (Paulus et al., 1994). Stimulation of
NOS3 activity in healthy patients is also associated with changes in left ventricular
end-diastolic pressurevolume relationship. This suggests a sharp decrease of left
ventricular stiffness (Paulus et al., 1994).
NO also has a pronounced effect on myocardial contractility and heart rate,
and these NO effects have biphasic and concentration-dependent character. Low
concentrations of NO-donors (0.110 M) increase myocardial contractility and
heart rate, while high concentrations (over 100 M) produce negative inotropic and
chronotropic effects (Koja at al., 1996, 1997; Vila-Petroff et al., 1999; Brady et al.,
1993; Shah and MacCarthy, 2000; Casadei and Sears, 2003). It should be noted that
the positive inotropic effect is close to maximum, within the range of NO concentra-
tions corresponding to those in the normal heart with intact endothelium (e.g., 13
M, according to Pinsky et al., 1997) and therefore reflects the physiological role
of NO in healthy hearts (Casadei and Sears, 2003). Supporting this hypothesis is the
fact that coronary infusion of certain concentrations of NO-synthase inhibitors into
isolated rat hearts and healthy persons causes a small but reliable decrease of left
ventricle contractility (Kojda et al., 1999; Cotton et al., 2001).
NO plays an important role in regulating myocardium reaction to mechanical
stimuli, in particular to stretch. Myocardium response to stretch includes signif-
icant and fast increases of contractile force (Frank-Starling response), followed
by a slower and less pronounced increase of contractility (Anrep effect). NO can
be involved in the realization of both effects. Endogenous NO strengthens the
Frank-Starling response probably by increasing diastolic distensibility (Shah and
MacCarthy, 2000; Casadei and Sears, 2003; Zhang et al., 2009). It was shown that
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 113
death depends on its concentration inside the cell and interaction with other bio-
logical molecules, such as oxygen and superoxide (Tsang et al., 2004; Calabrese
et al., 2009). In low concentrations NO produces cardioprotective, anti-apoptotic,
anti-hypertrophic effects protecting heart cells from damage caused by pathological
influences, like, for example, ischemia. In high concentrations NO is harmful when
it is presented in excessive amounts: pro-apoptotic, pro-necrotic effect on cardiomy-
ocytes (Casadei and Sears, 2003; Strijdom et al., 2009; Calabrese et al., 2009).
NO is involved in the regulation of cardiac activity in pathophysiological con-
ditions. Thus, expression and activity of NO-synthases increases in rat and mouse
myocardium under experimental infarction, as well as in humans with heart dis-
eases (Wildhirt et al., 1995; Casadei and Sears, 2003). NOS1 and NOS3 produced
NO influences contractility, energy balance and gene expression in myocardium
(e.g., with cardiomyopathies, infarctions), that can be an adaptive effect protecting
the injured heart from harmful influences of excessive catecholamine stimulation,
as well as from free radicals (Michel and Smith, 1993; Paulus, 2001). NOS2 par-
ticipates in developing immune-mediated heart diseases, e.g., infarction. NOS2
produced NO, on the contrary, causes apoptosis and is involved in inflammatory
processes under functional pathology of heart (Michel and Smith, 1993; Wildhirt
et al., 1995).
Fig. 5.1 Voltage dependence of membrane currents in a ventricular myocyte from young guinea-
pig in control and under cell stretch. a I-V curves recorded with repolarizing ramp commands
before (IC ) and during 10-m stretch (IS ). The main changes in the I-V curve during cell stretch
include reduction of V0 (intersection of the curve with zero-current-axis), induction of a negative
current at EK (equilibrium potential of potassium). The arrows show the direction of the I-V curve
shift. Compared to the control, 10-m stretch significantly reduces V0 , activates Ins and deactivates
IK1 . b Stretch-induced difference current (ISC : the difference of IS minus IC ). Modified from
Kazanski et al., 2010a with permission
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 117
rectifying K+ -current IK1 . At potentials positive to 40 mV, curve fit required the
superimposition of outwardly rectifying current components.
As we have shown, the 10 m stretch (1 min) shifted the I-V curve down-
wardly (Fig. 5.1a: IS ), reduced the hump of IK1 and depolarized V0 to 42 mV
(415 mV, n = 11). At the potassium equilibrium potential the potassium current
IK1 is zero. Therefore, the stretch-induced negative current I(EK ) should be inter-
preted as stretch-induced non-selective cation current Ins (Dyachenko et al., 2008),
otherwise called current through stretch activated channels ISAC (Hu and Sachs,
1997; Kamkin et al., 2000), or current through mechanically gated channels IMGC .
(Zhang Y and Hamill OP, 2000; Hamill and Martinac, 2001; White, 2006; Kamkin
and Kiseleva, 2008). I(EK ), the current at EK = 89 mV (61 mV log[5.4/155] =
89 mV), yielded a first estimate of the stretch-activated current Ins . In absence of
mechanical deformation, Ins (89 mV) was in the order of 0,03 nA (Dyachenko
et al., 2009a).
The stretch-induced difference current Isc (Fig. 5.1b) reversed polarity at
15 mV (11 2 mV, n = 11). Fit of the net and difference current according
to Ins = Gns (V Ens ) and Ins = Gns (V Ens ) with a voltage-independent Gns
yielded a straight line. At potentials negative to 20 mV mechanosensitivity is com-
posed of both Gns and GK1 . Gns has been reported to be voltage-independent
and to operate with a reversal potential Ens =10 mV (measurements with blocked
K+ -currents (Kamkin et al., 2000, 2003). The deviation of the difference current
from the straight line was attributed to the stretch-deactivation of GK1 because it
was abolished by substituting extracellular K+ by Cs+ ions (Kamkin et al., 2000,
2003). At potentials positive to 20 mV, mechanosensitive currents are carried by
K+ ions through both (Dyachenko et al., 2009a) TRPC6 outwardly rectifying chan-
nels (Hofmann et al., 1999; Spassova et al., 2006; Onohara et al., 2006) and TREK
(1 and 2) K2P 2.1 and K2P 10.1 leak channels (Honor et al., 2006; Li et al.,
2006; Patel and Honor, 2005) which are mechanosensitive.
Dyachenko et al. (2009a) proposed, that together with stretch-activated Gns
(Kamkin et al., 2000, 2003), stretch-induced deactivation of Kir2.3 and activation
of TRPC6 can destabilize the diastolic membrane, eventually leading to pacemaker-
like depolarizations and extra systoles (Kamkin et al., 2000, 2003; Lozinsky and
Kamkin, 2010; Zeng et al., 2000). After block of TRPC6 channels, stretch-induced
depolarizations remained small suggesting that stretch-activation of TRPC6 (and
not deactivation of Kir2.3 channels) is the key event Dyachenko et al. (2009a).
Thus, due to mechanosensitive currents carried by K+ ions, the difference cur-
rent Isc can only conditionally be called the MG-curent. Due to the simultaneous
activation of Gns , deactivation of Kir2.3, activation of TRPC6 and mechanosen-
sitivity of TREK Dyachenko et al. (2008) have introduced the term stretch
modulation of ion currents (SMIC: ISM ). But later these authors used the term
MG-current (Dyachenko et al., 2009b). Not to complicate the terminology here
and further on, in Figures and legends, we stick to the standard definition of
a difference current as Isc , and in presenting the data we use the term MG-
current, as MGC make the main contribution to control I-V curve modulation
under cell stretch (Dyachenko et al., 2009a). In our experiments the changes in
118 V. Kazanski et al.
MG-current increased with the amplitude of stretch and were reversible, i.e. they
have disappeared upon relaxation from stretch. These data coincide with the data
obtained under ramp-like repolarizations of ventricular myocyte Dyachenko et al.
(2008).
Since during cellular stretch (for example 10 m) both Ins (Ins(Vo) = 0.50 nA:
Fig. 5.1) and mechanosensitive currents carried by K+ ions contribute to the stretch-
induced difference current Isc , we calculated the value of stretch-induced currents
at the level of the holding potential, referring to it as IMGC(45 mV) (IMGC(45 mV) =
40 nA: meanS.D. IMGC(45 mV) = 0.410.02. Fig. 5.1). It seemed to represent
both MG-current and MG-like current.
First of all, we studied the reaction of the cell to PTIO. After whole-cell access of
the patch pipette, a glass stylus was attached to the membrane but no stretch of the
cell was conducted. After registering the control I-V curve we started perfusing the
cell with PTIO solution (500 mol/L). Such concentration of PTIO was used, for
example, for studying the regulation of cardiac calcium current by NO (Gallo et al.,
2001). Figure 5.3 shows the control I-V curves (IC ) as compared to I-V curves under
PTIO (IPTIO ) without stretching the ventricular myocyte from guinea-pig after 1 min
(Fig. 5.3a) and 3 min (Fig. 5.3b) of perfusion, and besides that the cells reaction
to washing out PTIO (W IPTIO ) for the period of 5 min (Fig. 5.3c). As the crossing
point of the I-V curve characterizes the diastolic membrane potential, it is obvious
that under the PTIO effect V0 increased by approximately 10 mV from 84 mV
(845 mV; n = 10) to 94 mV (942 mV; n = 10). PTIO shifted the I-V
curve downwardly, reduced the hump of outward current and shifted the hump from
Fig. 5.3 Voltage dependence of membrane currents in ventricular myocytes from guinea-pig under
the perfusion of unstretched cell by PTIO solution. a I-V curve measured in control (IC ) and after
1 min PTIO perfusion (IPTIO ). b After 3 min PTIO perfusion (IPTIO ) as compared to the control
(IC ). c After 5 min of PTIO wash out (W IPTIO ) as compared to control (IC ). Note: In panels (a)
and (v) the arrows show the direction of the I-V curve shift under PTIO perfusion of the cell after
1 and 3 min after the beginning of the perfusion accordingly. Modified from Kazanski et al., 2010b
with permission
120 V. Kazanski et al.
Fig. 5.4 Voltage dependence of membrane currents in ventricular myocyte from young guinea-
pig under PTIO perfusion on the background of cell stretch. a I-V curve measured before stretch
(IC ) and during 10 m stretch (IS ). The arrows show the direction of the I-V curve shift. b , c
I-V curve modifications under PTIO perfusion with continued stretch for 2 and 8 min accordingly
(IS,PTIO ). The arrows show the direction of I-V curve shift at the given cell stretch under PTIO
perfusion. For better perception the panel D combines the control I-V curve (IC ) and the I-V curve
under 8 m cell stretch after 8 min of PTIO perfusion (IS,PTIO ). It is obvious that during the PTIO
perfusion the activity of MGCs is inhibited. Modified from Kazanski et al., 2010b with permission
and the authors data
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 121
young guinea-pig in control, under cell stretch and under further PTIO perfusion.
The voltage dependency of IC and its modulation by stretch IS is shown in Fig. 5.4a.
Before stretch (IC ), the I-V curve was N-shaped and crossed the voltage axis at
85 mV (86 5 mV, n = 10). The 10-m stretch shifted the I-V curve down-
wardly (Fig. 5.4a: IS ), reduced the hump of inwardly rectifying K+ -current IK1 and
depolarized V0 to 42 mV (39 4 mV, n = 10). Close to 10 mV, the I-V
curves recorded before and during stretch crossed each other, and at positive poten-
tials IS was increased. The stretch-induced difference current (ISC ) the difference
of IS minus IC (Fig. 5.5a corresponding to Fig. 5.4a) reversed polarity at 15 mV
(123 mV, n=10) and equaled 0.41 nA (IMGC(45 mV) = 0.40 0.02 nA, n =
10). At potentials negative to 20 mV mechanosensitivity is composed of both Gns
and GK1 . The deviation of the difference current from the straight line was attributed
to the stretch-deactivation of GK1 . Thus, stretch increases the MG-currents and
depolarizes the cell (Fig. 5.4a). Figure 5.4b shows the change of IS on the back-
ground of 10 m cell stretch 2 min after beginning the PTIO perfusion (IS,PTIO ), and
Fig. 5.4c demonstrates IS,PTIO under the same stretch but after 8 min of PTIO perfu-
sion. Disregarding the continuous cell stretch PTIO perfusion returns the I-V curve
to the original state, at which the curve got back to N-shaped and crossed the volt-
age axis at 84 mV (864 mV, n=10). The stretch-induced difference current
current during stretch minus current during stretch with PTIO ISS,PTIO (Fig. 5.5b
corresponding to Fig. 5.4c) reversed polarity at 10 mV and showed deviation from
the straight line and equals (+)0.33 nA IMGC(45 mV) = (+)0.35 0.03 nA, Erev =
Fig. 5.5 Stretch-induced difference currents from ventricular myocyte of young guinea-pig. a
Different current activated by 10 m of stretch (the difference of IS minus IC ), corresponding to
Fig. 5.4a (reversal potential Erev = 15 mV, IMGC(45 mV) = 0.41 nA. b Different current
under PTIO perfusion on the background of myocyte stretch: the difference of the current during
stretch (IS ) by 10 m minus same stretch with PTIO (ISS,PTIO ). Panel (b) corresponds to Fig. 5.4c
(reversal potential Erev = 5 mV, IMGC(45 mV) = (+)0.33 nA). Note: two curves for stretch of
10 m (a) and stretch of 10 m plus PTIO (b) show a nearly linear voltage dependence. Modified
from Kazanski et al., 2010b with permission
122 V. Kazanski et al.
5.3.2 NO Donors
5.3.2.1 NO-Donor SNAP
The NO-donor SNAP or S-Nitroso-N-acetylpenicillamine was used for testing the
involvement of NO in regulating MG-current activities. First of all, we demonstrate
the reaction of an undeformed cell to SNAP. After registering the control I-V curve
we started perfusing the cell with SNAP (100 mol/L). Figure 5.7a demonstrates
the control I-V curve (IC ) registered in mouse ventricular myocyte and the I-V curve
received 1 min after the SNAP perfusion started (ISNAP ) without any cell stretch.
Figure 5.7b shows, as compared to the control curve, the I-V curve received 2 min
after SNAP perfusion started (ISNAP ). Before perfusion of SNAP (IC ), the I-V curve
was N-shaped and crossed the voltage axis (zero current potential V0 ) at 74 mV.
It is obvious that SNAP perfusion shifts V0 from 74 mV (85 4 mV, n = 8)
1 Here we must make the following note. The differential current that occurs under cell stretch as
a result of I increase (e.g., the difference of IS minus IC ) or the differential current occurring as
compared to the control under the drugs effect (the difference of IDrug minus IC ) was marked with
a minus sign. To make it easier for the reader and to avoid confusion with the differential current
under cell relaxation after stretch or the differential current resulting from I return registered in a
stretched cell to the control under the drugs effect, the latter we mark conventionally with a plus
sign in brackets (+).
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 123
Fig. 5.6 Voltage dependence of MG currents in ventricular myocyte from mouse under PTIO
perfusion of the cell and its wash out. a I-V curve measured before stretch (IC ) and during 10 m
stretch (IS ). b-, c- modified I-V curve under PTIO perfusion with continuous stretch for 2 and 3 min
accordingly (IS,PTIO ). In both panels IS characterizes MG-currents during 10 m stretch. d 5 min
of PTIO wash out under stretch (W IS,PTIO ). Note that the I-V curve W IS,PTIO did not change as
compared to I-V curve IS,PTIO in panel C. The authors data
Fig. 5.7 Voltage dependence of membrane currents in ventricular cardiomyocytes from mouse
under SNAP perfusion of unstretched cells. a I-V curve measured in control (IC ) and after 1 min
SNAP perfusion (ISNAP ). b After 2 min SNAP perfusion (ISNAP ) as compared to the control
(IC ). c Difference current activated by SNAP ISNAP-C (the difference of ISNAP minus IC ), cor-
responding to Panel A 1 min after the beginning of perfusion (reversal potential Erev = 2 mV,
IMGC(45 mV) = 0.13 nA). d ISNAP-C , corresponding to Panel B 2 min after the beginning of
perfusion (reversal potential Erev = 2 mV, IMGC(45 mV) = 0.38 nA). Note 1: In panels (a) and
(b) the arrows show the direction of the I-V curve shift under SNAP perfusion of the cell. Note 2:
After 2 min the different MG-like current (Panel d) corresponds to 10 m cell stretch. Modified
from Kazanski et al., 2010a with permission and the authors data
After 2 min of SNAP perfusion the wash out for 2 min results in an I-V curve
shift towards the control, i.e. the unstretched cell shows removal of SNAP effect
during registration time (Fig. 5.8a). After 5 min of wash out the control I-V curve
and the I-V curve received after SNAP wash out coincide completely, and the latter
crosses the voltage axis at 72 mV (82 5 mV, n = 8) (Fig. 5.8b). The value
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 125
Fig. 5.8 Voltage dependence of membrane currents in ventricular cardiomyocytes from mouse
after SNAP wash out. a After 2 min of SNAP wash out (W ISNAP ) as compared to the control (IC )
and to the perfusion of SNAP during 2 min (ISNAP ). b After 5 min SNAP wash out (W ISNAP ) as
compared to the control (IC ). The authors data
Fig. 5.9 Voltage dependence of membrane currents in ventricular myocyte from mouse under
SNAP perfusion on the background of the cell stretch. a Current-voltage relation (I-V curve)
measured before stretch (IC ) and during 10 m stretch (IS ). The arrows show the direction of
the I-V curve shift at the given cell stretch. b modified I-V curves on the background of
SNAP perfusion at continuous stretch during 2 min (IS,SNAP )and 3 min accordingly (IS,SNAP ).
The arrows show the direction of the I-V curve shift at the given cell stretch under SNAP
perfusion. It is obvious that during SNAP perfusion the activity of MGCs is inhibited. c
Difference current activated by 10 m of stretch (ISC ), corresponding to panel A (reversal
potential Erev = 10 mV, IMGC(45 mV) = 0.46 nA). d Difference current (IS-S,SNAP ): the
difference of the IS minus same stretch with SNAP IS,SNAP , corresponding to Fig. 9B (reversal
potential Erev = 3 mV, IMGC(53 mV) = (+)0.22 nA). Modified from Kazanski et al., 2010a with
permission and the authors data
(ISS,SNAP ) with SNAP 3 min after perfusion of SNAP is plotted in the I-V relations
in Fig. 5.9d and equals (+)0.53 nA (IMGC(45 mV) = (+)0.48 0.05 nA, n = 8 vs
control, P < 0.001; Erev = 3 1 mV). Thus, SNAP perfusion under 10 m cell
stretch resulted in inhibited MG-currents, probably due to the blocking of MGCs.
The I-V curve received in these conditions practically coincided after 2 min with
the I-V curve registered in the control. But the following SNAP perfusion already
at 3 min shifts the membrane potential value to 78 mV, which happens, in our
opinion, as a result of inhibiting MGCs that have background activity. Similar data
were obtained on ventricular cardiomyocytes from guinea-pig and rat.
Fig. 5.10 Voltage dependence of membrane currents in ventricular cardiomyocytes from rat under
DEA-NO perfusion of unstretched cell. a I-V curve measured in control (IC ) and after 2 min
DEA-NO perfusion (IDEA-NO ). b after 1 and 2 min of DEA-NO wash out (W IDEA-NO ) as com-
pared to IDEA-NO . Note: In panels (a) and (b) the arrows show the direction of the I-V curve
shift under DEA-NO perfusion and under cell wash out. c Difference current activated by
DEA-NO (IDEA-NOC : the difference of IDEA-NO minus IC ), corresponding to Panel a 2 min after
the beginning of perfusion (reversal potential Erev = 5 mV, IMGC(45 mV) = 0.46 nA). At
2 min IDEA-NOC corresponds to 10 m cell stretch. d IDEO-NOC activated by DEA-NO without
stretch, 1 min after the beginning of wash out (reversal potential Erev = 4 mV, IMGC(45 mV) =
(+)0.22 nA. Modified from Kazanski et al., 2010a with permission and the authors data
Fig. 5.11 Voltage dependence of membrane currents in ventricular myocyte from mouse under
DEA-NO perfusion of the cell on the background of its stretch. a I-V curve measured before
stretch (IC ) and during 10 m stretch (IS ). The arrows show the direction of the I-V curve shift
at the given cell stretch. b modified I-V curves on the background of DEA-NO perfusion under
continuous stretch during 3 min accordingly (IS,DEA-NO ). The arrows show the direction toward
which the I-V curve shifts in response to DEA-NO application to prestretched cell. It is obvi-
ous that in the process of DEA-NO perfusion the activity of MGCs is inhibited. c Difference
current (ISC ) activated by 10 m of stretch corresponding to Fig. 5.11a (reversal potential
Erev = 20 mV, IMGC(45 mV) = 0.31 nA). d Difference current (ISS,DEA-NO ): the differ-
ence of IS minus same stretch with DEA-NO IS,DEA-NO , corresponding to Fig. 5.11b (reversal
potential Erev = 20 mV, IMGC(45 mV) = (+)0.34 nA). Modified from Kazanski et al., 2010a
with permission and the authors data
Despite the continuing cell stretch, the DEA-NO perfusion returns the I-V curve to
the state close to the original, at which the curve turns again N-shaped and crosses
the voltage axis at 75 mV (873 mV, n = 9). The difference of IS minus during
stretch with DEA-NO (IS,DEA-NO ) 3 min after perfusion is plotted in the I-V relations
in Fig. 5.11d and equals (+)0.34 nA (IMGC(45 mV) = (+)0.40 0.03 nA, n = 9 vs.
130 V. Kazanski et al.
Fig. 5.12 Voltage dependence of stretch-induced membrane currents, K+ currents not suppressed.
Ventricular myocyte from guinea pig. Current-voltage relation (I-V curve) measured before stretch
(IC ) and during 10 m stretch (IS ) after preincubation of cells in L-NAME solution (20 mol/L,
2 h). Please, note that the cell preincubation in L-NAME solution changes the shape of the con-
trol I-V curve. See Fig. 5.3 compared to Fig. 5.4. Modified from Kazanski et al., 2010b with
permission
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 131
The data presented in Part 4 about the involvement of NO into regulating the activity
of MG-currents come down to several basic issues. In ventricular myocytes from
guinea pig, mouse and rat in all experimental conditions the NO scavenger PTIO
produces complete inhibition of stretch-activated MG currents and deactivation of
K1-currents. The extent of MG current block depends on incubation duration. The
wash-out of PTIO does not reverse the blockade of MG current. Wash-out stops the
development of the blockade. We believe that application of PTIO scavengers NO,
thus blocking MG currents. However during the beginning of application of PTIO,
its removal prevents the development of the MG current blockade. PTIO blockade
of MG current cannot be reversed by its washout.
The NO-donors SNAP and DEO-NO in undeformed cell result in activation of
MG-like currents. Wash out removes these currents. On the contrary, SNAP and
DEO-NO inhibit MG currents in a previously stretched cell. This is an interest-
ing phenomenon that correlates with described bimodal effects of NO. It is known
that the effect of NO is bimodal, with a positive inotropic effect at low amounts
of NO exposure but a negative one at higher amounts (Massion et al., 2003).
Admittedly, defining what low or high amounts really mean is difficult, both in
terms of actual quantity of bioactive NO delivered (e.g., with different exogenous
NO donors) as well as the correspondence with amounts endogenously produced
in vivo. Similarly, Kojda et al. (1996) observed that low concentrations of the NO
donors SNAP and DEA/NO caused a moderate positively inotropic effect in adult
rat ventricular myocytes. Higher concentrations of either SNAP or DEA/NO sup-
pressed myocyte contractile function. Mohan et al. (1996) have reported a similar
biphasic inotropic response to NO donors in isolated feline papillary muscle strips
that appeared to be dependent on a GMP-mediated signaling pathway. Since the
direction of NO donors mediated effects can be dose dependent we suggest that
NO donors open MGCs in ventricular cardiomyocytes, which underlie MG-like
currents. We also register MG currents when stretching the cell, but with that we
increase the concentration of endogeneous NO. We speculate that in both first and
second cases we have that low concentration of exogenous or endogenous NO, that
activates MGCs. In case the cell, when stretched, shows higher endogenous NO
concentration, then an additional increase of exogenous concentration using NO
donors results in increased summary concentration, which probably is the reason for
inhibiting MGCs. As we have no data on MGCs ion channel structure in ventricular
myocytes, our speculation is based on analogues with voltage gated L-type calcium
channels which also show dose dependent activatory and inhibitory effects of NO
donors on ICaL . Thus, e.g., Mery et al. (1993) originally reported that SIN-1 (NO
donor 3-morpholino-sydnonimine), the active metabolite of molsidomine, has a
biphasic effect on ICaL in enzymatically dissociated frog ventricular myocytes. The
data of these authors suggest that the activatory and inhibitory effects of NO donors
on ICaL result from an inhibition of the cGMP-inhibited cAMP-phosphodiesterase
and an activation of the cGMP-stimulated cAMP-phosphodiesterase, respectively,
both linked to the activation of guanylyl cyclase, possibly a membrane form of the
enzyme.
The use of NOS inhibitors L-NAME and LNMMA results in MG currents that
do not appear as a response to cell stretch. In NOS1/ the reaction to stretch is
present, whereas in NOS3/ there is no reaction to stretch. This allows to consider
NOS3 as the dominant source of NO involved in MGCs and also define that NOS3
is activated by stretch.
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 133
5.4 Cell Signaling of Nitric Oxide in the Heart and Possible Role
in Regulation of MG-Currents
NO has a wide range of physiological effects, but the main intracellular target for
NO is considered soluble guanylyl cyclase that causes an increase of the concen-
tration of cGMP (Fig. 5.14), which can modulate the activity of cGMP-dependent
cyclic nucleotide phosphodiesterases and, respectively cAMP level, as well as
cGMP-dependent protein kinase G, that causes phosphorylation of a number of
proteins (Moncada et al., 1991; Murad, 1998; Lane and Gross, 1999; Shah and
MacCarthy, 2000; Bryan, 2009). The cGMP-mediated NO effect also includes influ-
ence on such effectors as cyclooxygenases, phosphatases, phospholipase S (Murad,
1998; Lane and Gross, 1999; Bryan, 2009).
The effect of the cGMP-dependent pathway on the function of the myocardium
consists of modulating Sa2+ influx from the sarcolemma, lowering myofilaments
sensitivity to Sa2+ , changing the functional activity of sarcoplasmic reticulum,
changing the action potential, changing cell volume, and decreasing oxygen con-
sumption (Ji et al., 1999; Shah and MacCarthy, 2000). cGMP-dependent NO effects
in the heart are: (1) early onset of relaxation mediated by troponin I phosphory-
lation by protein kinase G, resulting in lower myofilament sensitivity to Sa2+ ; (2)
positive inotropic effect of NO at low concentrations by affecting cGMP-inhibited
cyclic nucleotide phosphodiesterase resulting in increased levels of intracellular
cAMP, but cGMP participation can not be excluded, (3) negative inotropic effect
of higher NO doses, realized probably by activating protein kinase G, (4) mod-
ulation of -adrenergic and cholinergic responses, and (5) protective effect on
cardiomyocites under myocardial hypoxia at the expense cGMP-mediated opening
Fig. 5.14 NO-cGMP-mediated signal transduction in myocardium (Reproduced from Shah and
MacCarthy, 2000 with permission from Elsevier.)
134 V. Kazanski et al.
of KATP -channels (Ji et al., 1999; Casadei and Sears, 2003; Shah and MacCarthy,
2000).
The cGMP-independent mechanisms for realizing NO effects include its reac-
tions with amino, thiol (SH), diazo and tyrosyl groups in proteins, as well as haem-,
iron- or sulphur centers of proteins (Mateo and De Artiano, 2000; Landar and
Darley-Usmar, 2003; Hughes, 2008; Bryan et al., 2009). The most important fact is
the direct interaction of NO with chemically active thiol protein groups causing their
posttranslational modifications, which results in significant functional changes.
S-nitrosylation of reactive thiol groups can affect the activity of ion channels,
transporters and Sa2+ -binding proteins participating in the regulation of Sa2+ -
cycling in myocytes. Possible targets are ryanodine receptor Ca2+ release channels
(RYR) of sarcoplasmic reticulum, SERCA 2a, phospholambam, Sa2+ -ATP-ase of
the sarcolemma (Shah and MacCarthy, 2000; Casadei and Sears, 2003; Lim et al.,
2008; Petroff et al., 2001; Jaffrey et al., 2001). A NO mediated increase of Sa2+
concentration in cardial myocytes is the main mechanism of NO modulation of
myocardial excitationcontraction coupling and realization of cardiac cell response
to mechanical impacts (Shah and MacCarthy, 2000; Seddon et al., 2007; Lim et al.,
2008; Fig. 5.15).
One more important mechanism for realizing NO effects is its interaction with
active peroxide radicals. NO, especially under pathophysiological conditions, can
interact with high reactivity superoxide, which in norm is tied by superoxide
dismutase. As a result a high-activity compound is formed, such as peroxynitrite
Fig. 5.15 Regulation of cardiomyocyte functions by nitric oxide in normal heart (Reproduced
from Seddon et al., 2007 with permission from Elsevier)
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 135
(ONOO ) that can modulate ion channel functions, oxidizing, like NO itself, the
regulatory thiol groups of channel proteins (Mateo and De Artian, 2000; Landar
and Darley-Usmar, 2003; Hughes, 2008). Also shown was the effect of peroxynitrite
on the physiological activity of a number of enzymes, such as phospholipases of dif-
ferent types (Yuen et al., 2000; Guidarelli et al., 2000). Protonation of peroxynitrite,
especially under low rN, observed in heart pathologies, results in formation of per-
oxynitrous acid. As a result, highly reactive hydroxyl-like species are formed that
can cause toxic effects related to nitration and oxidation of not only functional but
also structural proteins (reviewed in Shah and MacCarthy, 2000).
References
Ashley EA, Sears CE, Bryant SM, Watkins HC, Casadei B (2002) Cardiac nitric oxide synthase
1 regulates basal and beta-adrenergic contractility in murine ventricular myocytes. Circulation
105(25):30113016
136 V. Kazanski et al.
Balligand JL, Kobzik L, Han X, Kaye DM, Belhassen L, OHara DS, Kelly RA, Smith TW,
Michel T (1995) Nitric oxide-dependent parasympathetic signaling is due to activation of
constitutive endothelial (type III) nitric oxide synthase in cardiac myocytes. J Biol Chem
270(24):1458214586
Brady AJ, Warren JB, Poole-Wilson PA, Williams TJ, Harding SE (1993) Nitric oxide attenuates
cardiac myocyte contraction. Am J Physiol 265(1 Pt):H176H182
Bruckdorfer R (2005) The basics about nitric oxide. Mol Aspects Med 26(12):331
Bryan NS, Bian K, Murad F (2009) Discovery of the nitric oxide signaling pathway and targets for
drug development. Front Biosci 14:118
Butler AR, Flitney FW, Williams DL (1995) NO, nitrosonium ions, nitroxide ions, nitrosothiols
and iron-nitrosyls in biology: a chemists perspective. Trends Pharmacol Sci 16(1):1822
Calabrese V, Cornelius C, Rizzarelli E, Owen JB, Dinkova-Kostova AT, Butterfield DA (2009)
Nitric oxide in cell survival: a janus molecule. Antioxid Redox Signal 11(11):27172739
Casadei B, Sears CE (2003) Nitric-oxide-mediated regulation of cardiac contractility and stretch
responses. Prog Biophys Mol Biol 82(13):6780
Cotton JM, Kearney MT, MacCarthy PA, Grocott-Mason RM, McClean DR, Heymes C,
Richardson PJ, Shah AM (2001) Effects of nitric oxide synthase inhibition on Basal function
and the force-frequency relationship in the normal and failing human heart in vivo. Circulation
104(19):23182323
Damy T, Ratajczak P, Robidel E, Bendall JK, Oliviro P, Boczkowski J, Ebrahimian T, Marotte
F, Samuel JL, Heymes C 2003 Up-regulation of cardiac nitric oxide synthase 1-derived nitric
oxide after myocardial infarction in senescent rats. FASEB J 17(13):19341936
Dawson D, Lygate CA, Zhang MH, Hulbert K, Neubauer S, Casadei B (2005) nNOS gene dele-
tion exacerbates pathological left ventricular remodeling and functional deterioration after
myocardial infarction. Circulation 112(24):37293737
Dyachenko V, Christ A, Gubanov R, Isenberg G (2008) Bending of z-lines by mechanical stimuli:
an input signal for integrin dependent modulation of ion channels? Prog Biophys Mol Biol
97(23):196216
Dyachenko V, Husse B, Rueckschloss U, Isenberg G (2009a) Mechanical deformation of ventric-
ular myocytes modulates both TRPC6 and Kir2.3 channels. Cell Calcium 45(1):3854
Dyachenko V, Rueckschloss U, Isenberg G (2009b) Modulation of cardiac mechanosensitive ion
channels involves superoxide, nitric oxide and peroxynitrite. Cell Calcium 45(1):5564
Feron O, Belhassen L, Kobzik L, Smith TW, Kelly RA, Michel T (1996) Endothelial nitric oxide
synthase targeting to caveolae. Specific interactions with caveolin isoforms in cardiac myocytes
and endothelial cells. J Biol Chem 271(37):2281022814
Gallo MP, Malan D, Bedendi I, Biasin C, Alloatti G, Levi RC (2001) Regulation of cardiac calcium
current by NO and cGMP-modulating agents. Pflugers Arch 2001 Feb;441(5):621628
Gillis KD (2000) Techniques for membrane capacitance measurements. In: Sakmann BNE (ed.)
Single-channel recording. London: Plenum, pp 155198
Gmez R, Caballero R, Barana A, Amors I, Calvo E, Lpez JA, Klein H, Vaquero M, Osuna L,
Atienza F, Almendral J, Pinto A, Tamargo J, Delpn E. (2009) Nitric oxide increases cardiac
IK1 by nitrosylation of cysteine 76 of Kir2.1 channels. Circ Res. 105(4):383392
Guidarelli A, Cantoni O (2002) Pivotal role of superoxides generated in the mitochondrial res-
piratory chain in peroxynitrite-dependent activation of phospholipase A2. Biochem J 366
(Pt 1):307314
Hamill OP, Martinac B (2001) Molecular basis of mechanotransduction in living cells. Physiol
Revs 81:685740
Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T, Schultz G (1999) Direct
activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397:259263
Honor E, Patel AJ, Chemin J, Suchyna T, Sachs F (2006) Desensitization of mechano-gated K2P
channels. Proc Natl Acad Sci USA 103(18):68596864
Hu H, Sachs F (1997) Stretch-activated ion channels in the heart. J Mol Cell Cardiol 29:15111523
Hughes MN (2008) Chemistry of nitric oxide and related species. Methods Enzymol 436:319
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 137
Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G (1987) Endothelium-derived relaxing
factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci USA.
84(24):92659269
Isenberg G, Klockner U (1982) Calcium tolerant ventricular myocytes prepared by pre-incubation
in a Kb medium. Pflugers Archiv Europ J Physiol 395: 618
Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, Snyder SH (2001) Protein S-nitrosylation:
a physiological signal for neuronal nitric oxide Nat Cell Biol 3(2):193197
Ji GJ, Fleischmann BK, Bloch W, Feelisch M, Andressen C, Addicks K, Hescheler J (1999)
Regulation of the L-type Ca2+ channel during cardiomyogenesis: switch from NO to adenylyl
cyclase-mediated inhibition. FASEB J 13(2):313324
Kamkin A, Kiseleva I (2008) Mechanically gated channels and mechanosensitive channels. In:
Kamkin A, Kiseleva I (eds) Mechanosensitivity in Cells and Tissues 1. Mechanosensitive Ion
Channels. Springer, pp xiiixviii.
Kamkin A, Kiseleva I, Isenberg G (2000) Stretch-activated currents in ventricular myocytes:
amplitude and arrhythmogenic effects increase with hypertrophy. Cardiovasc Res 48:
409420
Kamkin A, Kiseleva I, Isenberg G (2003) Ion selectivity of stretch-activated cation currents in
mouse ventricular myocytes. Pflugers Arch Europ J Physiol 446(2):220231
Kazanski VE, Kamkin A, Makarenko EYu, Lysenko NN, Sutiagin PV, Tian B, Kiseleva I (2010a)
The role of the Nitric Oxide in regulation of mechanically gated channels activity in cardiomy-
ocytes: Investigation by means of the application of NO-donors. Bulletin of Experimental
Biology and Medicine 7:49. English, Russian. (See PubMed for details of English version
pages).
Kazanski VE, Kamkin A, Makarenko EYu, Lysenko NN, Sutiagin PV, Kiseleva I (2010b) The role
of the Nitric Oxide in regulation of mechanically gated channels activity in cardiomyocytes:
Investigation of NO-synthatases contribution. Bulletin of Experimental Biology and Medicine
8:228232. English, Russian. (See PubMed for details of English version pages).
Kelly RA, Balligand JL, Smith TW (1996) Nitric oxide and cardiac function. Circ Res 79(3):
363380
Kojda G, Kottenberg K (1999) Regulation of basal myocardial function by NO. Cardiovasc Res
41(3):514523
Kojda G, Kottenberg K, Nix P, Schluter KD, Piper HM, Noack E (1996) Low increase in
cGMP induced by organic nitrates and nitrovasodilators improves contractile response of rat
ventricular myocytes. Circ Res 78:91101
Kojda G, Kottenberg K, Nix P, Schlter KD, Piper HM, Noack E (1996) Low increase in
cGMP induced by organic nitrates and nitrovasodilators improves contractile response of rat
ventricular myocytes. Circ Res 78(1):91101
Kojda G, Kottenberg K, Noack E (1997) Inhibition of nitric oxide synthase and soluble guany-
late cyclase induces cardiodepressive effects in normal rat hearts. Eur J Pharmacol 334(23):
181190
Korth HG, Sustmann R, Thater C, Butler AR, Ingold KU (1994) On the mechanism of the nitric
oxide synthase-catalyzed conversion of N omega-hydroxyl-L-arginine to citrulline and nitric
oxide. J Biol Chem 269(27):1777617779
Kuebler W.M., Uhlig U., Goldmann T., et al. (2003) Stretch activates nitric oxide production in
pulmonary vascular endothelial cells in situ. Am J Respir Crit Care Med 168:13911398
Landar A, Darley-Usmar VM (2003) Nitric oxide and cell signaling: modulation of redox tone and
protein modification. Amino Acids 25(34):313321
Lane P, Gross SS (1999) Cell signaling by nitric oxide. Semin Nephrol 19(3):215229
Li XT, Dyachenko V, Zuzarte M, Putzke C, Preisig-Mller R, Isenberg G, Daut J (2006) The
stretch-activated potassium channel TREK-1 in rat cardiac ventricular muscle. Cardiovasc Res
69(1):8697
Liaudet L, Soriano FG, Szab C (2000) Biology of nitric oxide signaling. Crit Care Med
28(4):N37N52
138 V. Kazanski et al.
Lim G, Venetucci L, Eisner DA, Casadei B (2008) Does nitric oxide modulate cardiac ryanodine
receptor function? Implications for excitation-contraction coupling. Cardiovasc Res 77(2):
256264
Lozinsky I, Kamkin A (2010)Mechanosensitive alterations of action potentials and membrane cur-
rents in healthy and diseased cardiomyocytes: Cardiac tissue and isolated cell. In: Kamkin A,
Kiseleva I (eds.) Mechanosensitivity in Cells and Tissues 3. Mechanosensitivity of the Heart.
Springer, pp 185238
Malan D, Ji GJ, Schmidt A, Addicks K, Hescheler J, Levi RC, Bloch W, Fleischmann BK
(2004) Nitric oxide, a key signaling molecule in the murine early embryonic heart. FASEB
J 18(10):11081110
Marletta MA (1994) Nitric oxide synthase: aspects concerning structure and catalysis. Cell
78(6):927930
Massion PB, Balligand JL (2003) Modulation of cardiac contraction, relaxation and rate by the
endothelial nitric oxide synthase (eNOS): lessons from genetically modified mice. J Physiol
546(Pt 1):6375
Massion PB, Feron O, Dessy C, Balligand J-L (2003) Nitric oxide and cardiac function. ten years
after, and continuing. Circ Res 93:388398
Mateo AO, De Artiano AAM (2000) Nitric oxide reactivity and mechanisms involved in its
biological effects. Pharmacol Res 42(5):421427
Mayer B, Hemmens B (1997) Biosynthesis and action of nitric oxide in mammalian cells. Trends
Biochem Sci 22(12):477481
Mery P-F, Pavoine C, Belhassen L, Pecker F, Fischmeister R (1993) Nitric oxide regulates car-
diac Ca2+ current: involvement of cGMP-inhibited and cGMP-stimulated phosphodiesterases
through guanylyl cyclase activation. J Biol Chem 268:2628626295
Michel T, Smith TW (1993) Nitric oxide synthases and cardiovascular signaling. Am J Cardiol
72(8):33C-38C
Mohan P, Brutsaert DL, Paulus WJ, Sys SU (1996) Myocardial contractile response to nitric oxide
and cGMP. Circulation 93:12231229
Moncada S, Higgs A (1993) The L-arginine-nitric oxide pathway. N Engl J Med 329(27):
20022012
Moncada S, Palmer RM, Higgs EA (1991) Nitric oxide: physiology, pathophysiology, and
pharmacology. Pharmacol Rev 43(2):109142
Murad F (1998) Nitric oxide signaling: would you believe that a simple free radical could be a
second messenger, autacoid, paracrine substance, neurotransmitter, and hormone? Recent Prog
Horm Res 53:4360
Nathan C, Xie Q II (1994) Nitric oxide synthases: roles, tolls, and controls. Cell 78:915918
Onohara N, Nishida M, Inoue R, Kobayashi H, Sumimoto H, Sato Y, Mori Y, Nagao T, Kurose H
(2006) TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy. EMBO
J 25(22):53055316
Papapetropoulos A, Rudic RD, Sessa WC (1999) Molecular control of nitric oxide synthases in the
cardiovascular system. Cardiovasc Res 43(3):509520
Patel AJ, Honor E (2005) Potassium-selective cardiac mechanosensitive ion channels. In: Kohl P,
Sachs F, Franz MR (eds.) Cardiac mechano-electrical feedback and arrhythmias. From Pipette
to Patient.. Elsevier Sounders, Philadelphia, PA, pp 1120
Paulus WJ (2001) The role of nitric oxide in the failing heart. Heart Fail Rev 6(2):105118
Paulus WJ, Vantrimpont PJ, Shah AM (1994) Acute effects of nitric oxide on left ventricular relax-
ation and diastolic distensibility in humans. Assessment by bicoronary sodium nitroprusside
infusion. Circulation 89(5):20702078
Paulus WJ, Vantrimpont PJ, Shah AM (1995) Paracrine coronary endothelial control of left
ventricular function in humans. Circulation 92(8):21192126
Petroff MG, Kim SH, Pepe S, Dessy C, Marbn E, Balligand JL, Sollott SJ (2001) Endogenous
nitric oxide mechanisms mediate the stretch dependence of Ca2+ release in cardiomyocytes.
Nat Cell Biol 3(10):867873
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 139
Zhang YH, Dingle L, Hall R, Casadei B (2009) The role of nitric oxide and reactive oxygen
species in the positive inotropic response to mechanical stretch in the mammalian myocardium.
Biochim Biophys Acta. 2009 Jul;1787(7):811817
Ziolo MT, Kohr MJ, Wang HJ (2008) Nitric oxide signaling and the regulation of myocardial
function Mol Cell Cardiol 45(5):625632
Chapter 6
Role of Signaling Pathways in the Myocardial
Response to Biomechanical Stress and in
Mechanotransduction in the Heart
6.1 Introduction
Biomechanical signaling involves complex interactions between intracellular and
extracellular components. While whole organisms respond to external stimuli,
organs, tissues, and cells respond to mechanical signals from the extracellular space.
Common cellular responses to such signals include alterations in morphology,
intracellular structure, and extracellular structure. This extracellular transmission
of mechanical force followed by the subsequent intracellular conversion into bio-
chemical signals is termed mechanotransduction (Fig. 6.1). This process describes
the dynamic process of a mutually dependent relationship between cells and their
surrounding extracellular matrix (ECM). Responding to mechanical stress allows
cells to acclimate and adapt to changing environments. However, the benefits from
this intricate process are reversed if the carefully balanced equilibrium is tilted,
often leading to abnormal signaling and pathophysiological remodelling of cells
and tissues.
By detecting and responding to hemodynamic changes in the environment via
cell-ECM interactions, the heart develops compensatory responses utilizing intracel-
lular signaling cascades to maintain adequate function (Frey et al., 2004; Heineke
and Molkentin, 2006; Ruwhof and van der Laarse, 2000). Even at rest, the heart
is constantly exposed to much biomechanical and biochemical signals, causing it
to continuously adapt various aspects of its morphology such as the infrastructure
of its cytoskeleton as well as the composition of cell-ECM adhesion complexes.
This chapter will discuss several sub-cellular processes and signaling pathways
involved in mechanotransduction and the interactions between cardiomyocytes and
fibroblasts with the ECM.
Fig. 6.1 A conceptual framework of the key players involved in the detection of and the translation
of mechanical signals in the heart
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 143
Fig. 6.2 A schematic of integrin based complexes and their associated signaling pathways.
Integrin complexes linked to the Z-disc are called costameres. The ones directly linked to actin
are called focal adhesions
with cytoskeletal actin filaments (Ervasti, 2003; Laser et al., 2000; Samarel, 2005;
Sharp et al., 1997). To some degree, both types of integrin-linked complexes share
overlapping signaling cascades with maintaining some key differences in signaling.
Integrins moderate cellular behaviour partially through physically bridging the
ECM to intracellular components. Nevertheless, the formation of mutli-protein com-
plexes at their intracellular domains is necessary for mechanotransduction (Berrier
and Yamada, 2007; Hynes, 2002). Because integrins do not exhibit inherent enzy-
matic activity, they must recruit signaling proteins to their cytoplasmic tails to
convert mechanical stimuli into biochemical signals (Berrier and Yamada, 2007).
In general, the proteins recruited to integrin-linked complexes are categorized into
3 major groups (Berrier and Yamada, 2007). The first group is integrin regulating
proteins such as talin, which bind to integrins and regulate their signaling and activ-
ity (Anastasi et al., 2009; Calderwood, 2004; Chen et al., 1995; Ulmer et al., 2003).
The second group is adaptor proteins, which act as scaffolds and link integrins to
structural and enzymatic proteins (Berrier and Yamada, 2007). An example is pax-
illin, a -integrin binding protein that recruits enzymes to integrin-linked complexes
(Schlaepfer et al., 1999; Tachibana et al., 1995). Another example is vinculin, which
in response to mechanical stimulation relocates to costameres and connect integrin-
linked complexes to cytoskeletal actin (Sharp et al., 1997; Wood et al., 1994). The
third group is enzymatic proteins such as focal adhesion kinase (FAK), integrin-
linked kinase (ILK) and phosphoinositide-3-kinase (PI3K) and which can initiate
a broad spectrum of chemical signaling cascades (Berrier and Yamada, 2007; Ross
et al., 1998).
of VEGF suggesting that VEGF increases ERK phosphorylation via paracrine sig-
naling (Seko et al., 1999). The FAK mediated release of VEGF also up-regulates
the gap junction protein connexin-43 in neighbouring cells via paracrine signal-
ing (Li et al., 1997; Yamada et al., 2005). Increased expression of connexin-43
improves overall electrical signaling in the heart (Yamada et al., 2005). FAK-Src can
also up-regulate the expression of cell-cell adhesion proteins, such as N-cadherin,
desmoplakin, and plakoglobin (Yamada et al., 2005). Because stretch induced local-
ization of FAK to integrin-linked complexes increases with the magnitude and the
duration of stretch (Katz et al., 2002; Simpson et al., 1999), up-regulation of the
aforementioned cell-cell adhesion proteins in cardiomyocytes is likely proportional
to increases in work load. Cardiomyocyte adhesion to fibronectin stimulates a higher
level of N-cadherin and connexin-43 expression than adhesion to collagen suggest-
ing that matrix composition may alter focal adhesion binding and signaling (Shanker
et al., 2005). Together, these demonstrate that mechanical stimulation of FAK is
involved in the maintenance of both electrical and mechanical connectivity in the
heart.
Contrary to mechanical stimulation, chemical stimulation in cardiomyocytes
recruits FAKs to focal adhesions instead (Fig. 6.2) (Torsoni et al., 2003). For
instance, application of Ang II stimulates FAK mediated hypertrophy (Salazar and
Rozengurt, 2001). In concordance, application of AT1 receptor antagonists impairs
Ang II stimulation of FAK signaling without inhibiting cyclic stretch stimulation
of FAK signaling (Torsoni et al., 2003). This bimodal activation of FAK is one
of many examples that demonstrate the specificity of mechanotransduction and its
associated signaling cascades. While costameric and focal adhesion FAKs are, on
the most part, distinct in their respective signaling cascades, it should be noted that
there are circumstances of overlap. For instance, studies have shown that FAK acti-
vates phosphoinositide-3-kinase (PI3K) and its subsequent effectors by binding to
PI3Ks p85 adaptor subunit regardless of FAKs location (Chen and Guan, 1994;
Thamilselvan et al., 2007). Similarly, when activated, both costameric and focal
adhesion FAKs can bind to the mitogen activated protein kinases, ERK, JNK, and
p38, via Grb2 and Sos (Schlaepfer et al., 1999; Torsoni et al., 2003).
Focal-adhesion kinase importance to cardiac development is illustrated by
the heart-specific FAK-deficient embryonic mice (Table 6.1). These mutant mice
develop right ventricular eccentric hypertrophy due to ineffective FAK mediated
stimulation of physiological hypertrophic programs, ultimately resulting in decom-
pensation and maladaptive right ventricular remodelling (Peng et al., 2008). On the
other hand, after birth, these mice gradually develop left ventricular eccentric hyper-
trophy overtime due to age (Peng et al., 2008). Similar pathologies are observed
acutely when these mice are treated with Ang II or pressure overload (Peng et al.,
2008). This shift from right to left ventricular eccentric hypertrophy is likely due to
differences in ventricular dependence and workload between embryonic and postna-
tal mice (Peng et al., 2008). This model implies that FAK signaling is necessary for
myocardial tolerance of mechanical stress, possibly through eliciting hypertrophy
via the regulation of various transcription factors such as NF-B (Crosara-Alberto
et al., 2009; Gupta et al., 2002).
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 147
Table 6.1 Mutations in various genes involved in mechanotransduction and their effects on
signalling
Tu et al., 2001). The mutually exclusive binding of -parvin and -parvin respec-
tively increases and reduces ILKs prohypertrophic activity (Hannigan et al., 2005).
Interestingly, formation of either -parvin-ILK or -parvin-ILK complexes have
been suggested to increase cell-ECM adhesion complexes (Hannigan et al., 2005;
Mongroo et al., 2004; Tu et al., 2001; Yamaji et al., 2001) demonstrating ILKs
importance as a scaffold in mediating the formation of integrin-linked complexes.
This also puts forth the idea that the enzymatic and scaffolding functions of ILK
may be independent of each other (Hannigan et al., 2005).
Structural analysis of ILK reveals a highly conserved Pleckstrin homology
domain, which activates ILK by binding to phosphoinositide-3,4,5-phosphate (PIP3 )
(Delcommenne et al., 1998), the primary product of PI3K activity (Vanhaesebroeck
et al., 1997) positing that activation of PI3K can subsequently activate ILK. In
addition, inactivation of PTEN, which is a lipid phosphatase that dephosphorylates
PIP3 into PIP2 (Oudit and Penninger, 2009; Oudit et al., 2004; Sun et al., 1999a),
results in constitutive activation of ILK (Edwards et al., 2005). Previous studies
have also shown that both enhancing PI3K activity and impairing PTEN activity
can enhance intracellular PIP3 levels, leading to cardiac hypertrophy (Crackower
et al., 2002; Shioi et al., 2000; Sun et al., 1999a). Together, these suggest that
PI3K activity can directly modulate ILK activity. Finally, ILK also directly phos-
phorylates AKT and GSK-3 (Troussard et al., 1999), two well studied downstream
effectors of FAK/PI3K signaling, supporting the notion that ILK may contribute to
the myocardial compensatory response to mechanical stress.
Since PI3K is involved in mediating acute responses to both chemical stimuli
via RTK and GPCR receptors (Oudit and Penninger, 2009; Oudit et al., 2004) and
mechanical stimuli via FAK (Chen and Guan, 1994), it is possible that ILK may also
be activated by chemical agonist stimulation. For instance, the application of Ang
II induces concentric hypertrophy in wildtype mice (Sadoshima and Izumo, 1993;
Sadoshima et al., 1993), an effect that is mitigated in ILK kinase dead mutants (Lu
et al., 2006). This partial attenuation of hypertrophy in the mutant strain suggests
that ILK amplifies, but is not necessary for, PI3K signaling. On the other hand,
one study suggested that ILK activation may be entirely PI3K dependent, as phar-
macological inhibitors of PI3K fully inhibited mechanically induced ILK activity
(Khwaja et al., 1997). However, an in vivo study stated that during maladaptive
cardiac hypertrophy, increased ILK association with Rac1 was observed without
phosphorylation of the PI3K signaling cascade (Lu et al., 2006). The same study
also demonstrated that ILK kinase dead mutants are unable to develop ventricular
hypertrophy in response to mechanical stress (Lu et al., 2006). Similarly, another
study postulated that ILKs can directly respond to RTK stimulation irrespective of
PI3K, further implicating that ILK and PI3K pathways may be independent (Knoll
et al., 2007). Though the results from these studies appear to conflict, their discrep-
ancies could be attributed to differences in the hearts condition; while ILK activity
may be PI3K independent during pathological states, ILK is likely PI3K dependent
during physiological conditions.
As one might expect, ILK is necessary for survival as mice with cardiomyocyte-
specific ablation of ILK develop heart failure and sudden death within 6 weeks
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 149
of age and develop spontaneous dilated cardiomyopathy (DCM) without the need
for a secondary stressor such as pressure overload or Ang II (Table 6.1) (White
et al., 2006). Similarly, a single mutation in the human ILK gene (A262V) has
been correlated with contractile dysfunction, ventricular dysmorphism, and DCM
(Knoll et al., 2007). As mice with cardiomyocyte specific ablation of PINCH1 do
not deviate from the wildtype phenotype (Liang et al., 2005), it is likely that ILK
has other physiological targets of interaction in cardiomyocytes allowing it to sus-
tain its signaling even in the absence of PINCH1. Interestingly, mice lacking ILK
have impaired FAK signaling indicating that ILK either contributes to FAK activa-
tion or mediates a part of FAKs downstream signaling (White et al., 2006). One
possible mechanism is that FAK indirectly stimulates ILK activity via PI3K/PIP3 ,
which is not impossible due to their close spatial proximity at integrin-linked com-
plexes (Chen and Guan, 1994; Thamilselvan et al., 2007). This is further supported
by FAK and ILKs overlapping downstream signaling (Antos et al., 2002; Bueno
and Molkentin, 2002; Delcommenne et al., 1998; Shioi et al., 2002; Troussard et al.,
1999).
that a weakened energy supply may be responsible for its intolerance to stress.
However, since the overall cellular AMP to ATP ratios in the mdx cardiomyocytes
are not different from wildtypes (Khairallah et al., 2007), it is possible that mdx
cardiomyocytes either utilize a less efficient pattern of ATP allocation to sustain
normal contractile function or require more ATP to sustain physiological conditions
resulting in energy deficiency.
It is interesting to note that mdx hearts have decreased activation of AKT in vivo
(Khairallah et al., 2007) indicating that DGCs, like integrins, may associate with the
PI3K signaling cascade. Consistent with the mdx phenotype, AKT has been shown
to be necessary for physiological growth and cellular survival in the heart (DeBosch
et al., 2006). Moreover, since DGCs are located closely to integrin-linked complexes
within the cell (Rybakova et al., 2000), they could very likely interact with ILKs or
FAKs, posing a potential cause for the decreased activation of AKT observed in mdx
mice.
6.3.1 PI3K
Fig. 6.3 The PI3K signaling cascade. PI3K signals through phosphatidylinositol-3,4,5-
triphosphate (PIP3 )/Akt pathway and stimulates growth, proliferation, and survival. RTK = rece-
ptor tyrosine kinase, GPCR = G-protein coupled receptor
mode of maladaptive ECM remodelling and a likely cause of heart failure in pres-
sure overloaded PI3K-KO hearts. Despite their redundant kinase activity, PI3K
isoforms demonstrate unique effects on cardiac hypertrophy. For instance, while
PI3K activity was shown to mitigate maladaptive hypertrophy (McMullen et al.,
2007), PI3K activity may exacerbate it. Studies of PI3K-KO and PI3K-DN
cardiomyocytes demonstrated that PI3K and PI3K respectively activate and
inhibit ERK (McMullen et al., 2007; Patrucco et al., 2004), whose activation has
been associated with pathological hypertrophy (Ainscough et al., 2009; Lorenz
et al., 2009).
6.3.2 PTEN
Fig. 6.4 A schematic of adherens junctions (a) and gelsolin activity (b). The intracellular domain
of the transmembrane protein N-cadherin is anchored to actin filaments by - and -catenin while
gelsolin cleaves actins and prevents repolymerization by capping the actin filament
154 D. Guo et al.
via intercalated discs, it seems unlikely that cardiomyocytes do not experience lat-
eral strain from the surrounding ECM or from neighbouring cells, or exert lateral
force during systole (Schoenberg, 1980). In fact, a study elegantly demonstrated that
roughly 80% of N-cadherin junctions between adjacent cardiomyocytes are located
in the peripheral (lateral) membrane (Pedrotty et al., 2008). Furthermore, these lat-
eral junctions allow cardiomyocytes to adhere to other cell types as intercalated
discs are only formed between cardiomyocytes.
Aside from propagating mechanical force, adherens junctions have also been
postulated to interact with PI3K to mediate AKT signaling (Tran et al., 2002).
Increased and decreased adherens junctions were shown to respectively improve
and weaken basal AKT phosphorylation (Tran et al., 2002). However, it is possible
that this loss of AKT stimulation could be indirectly due to weakened mechanosen-
sitivity as there is still little evidence of direct interactions between cadherins and
the PI3K pathway.
While the conversion of mechanical signals into chemical signals occurs in the
intracellular compartment, the spreading of this chemical signal from cell to cell
occurs through autocrine and paracrine signaling; hence, these processes play key
roles in stretch induced cardiac hypertrophy (Cingolani et al., 2001; van Wamel
et al., 2001). For instance, Ang II, ET-1, and TGF- have all been implicated in
mediating cardiac hypertrophy in hemodynamic overload models (Arai et al., 1995;
Ishiye et al., 1995; Ito et al., 1994; Ruzicka et al., 1995; Takahashi et al., 1994). In
response pressure overload, the heart increases expression of both endogenous ET-1
and ET-1 receptors appropriating ventricular hypertrophy (Arai et al., 1995), which
can be attenuated by applying ET-1 receptor blockers (Ito et al., 1994). Similarly, in
response to volume overload, the heart develops ventricular hypertrophy as a result
of increased Ang II levels (Ruzicka et al., 1995), but can be reversed via pharma-
cological inhibition of Ang II receptors (Ishiye et al., 1995). TGF- is primarily
expressed by non-cardiomyocytes during basal condition, but in response to pres-
sure overload, cardiomyocytes surprisingly become the primarily secretor of TGF-
(Takahashi et al., 1994). TGF- paracrine signaling also stimulates excess colla-
gen deposition by fibroblasts leading to fibrosis and stiffness of the myocardium
(Eghbali et al., 1991).
Cardiac hypertrophy following mechanically induced paracrine signaling has
also been demonstrated in vitro. Cultured cardiomyocytes exposed to stretch release
ET-1 and TGF-, which in turn causes an increased expression of the hypertrophy
marker ANP (van Wamel et al., 2001). Whether cardiomyocytes express ET-1 in
the absence of stretch appears to be under debate (Suzuki et al., 1993; van Wamel
et al., 2001), but in either case, the level of ET-1 excretion in the absence of stretch
is likely insufficient for stimulating the expression of hypertrophic markers (van
Wamel et al., 2001). Similarly, studies investigating whether Ang II is expressed
by acutely stretched cardiomyocytes have yielded mixed results (Sadoshima et al.,
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 155
1993; van Wamel et al., 2001; Yamazaki et al., 1999). Nevertheless, multiple studies
have confirmed that endogenous secretion of Ang II mediates, but is not neces-
sary for inducing, hypertrophy in stretched cardiomyocytes (Sadoshima and Izumo,
1993; Sadoshima et al., 1993; van Wamel et al., 2001; Yamazaki et al., 1999).
Furthermore, it should be noted that Ang II mediated hypertrophy occurs partially
via increased production and excretion of ET-1 (Cingolani et al., 2001).
The release of Ang II by stretched cardiac fibroblasts can also induce hypertrophy
in unstretched cardiomyocytes (van Wamel et al., 2001, 2000). Similarly, stretched
cardiac fibroblasts also produce TGF-, altering gene expression in neighbouring
cardiomyocytes (van Wamel et al., 2001). Recently, a co-culture study demonstrated
that paracrine signaling from cardiac fibroblasts can induce cardiomyocyte prolif-
eration (Ieda et al., 2009). Together, these in vitro and in vivo studies describe the
prominent potential of intercellular dependence and regulation between cardiomy-
ocytes and cardiac fibroblasts in mechanosensitivity. An array of other factors, such
as ANP, is also excreted to mediate cellular changes in response to mechanical stress
(described in Sections 6.3.1 and 6.3.2) (Ruwhof et al., 2000; van Wamel et al., 2001,
2000). Hence, given the current knowledge of autocrine and paracrine effectors in
mechanically stimulated pathways in the heart, it is clear that diffusible chemical
factors are crucial in mediating mechanotransduction.
The loss of cell-ECM linkage proteins in the heart almost always leads to cardiac
dysfunction due to the inability to tolerate stressful conditions. Similar results are
observed from the loss of signaling proteins such as FAK and ILK. As previously
discussed, the inability to detect and to respond to mechanical stress can incapacitate
the hearts ability to initiate proper compensatory mechanisms ultimately leading
to heart failure. Increased mechanical work load and hemodynamic stress is often
closely correlated with concomitant intracellular and extracellular remodelling. In
the heart, like in many other organs and tissues, extracellular remodelling is primar-
ily regulated by MMPs (Kassiri and Khokha, 2005; Spinale, 2007). MMPs are a
family of potent proteolytic enzymes that digest, among others, collagen, laminin
and fibronectin and are inhibited by interactions with tissue inhibitors of metallo-
proteinases (TIMPs) (Kassiri and Khokha, 2005). Indeed, loss of TIMP3 leads to
an exacerbation of pressure-overload induced adverse myocardial remodelling and
heart failure (Kassiri et al., 2005).
Several mechanically induced signaling pathways, specifically PI3K pathways,
stimulate MMP expression and activation (Grote et al., 2003; Hess et al., 2003;
Ispanovic and Haas, 2006; Zahradka et al., 2004). For instance, stretch induces
NAD(P)H mediated formation of reactive oxygen species (ROS), increasing expres-
sion and activation of pro-MMP2 and the release of active MMP2 (Grote et al.,
156 D. Guo et al.
6.5.2.1 Actin
Intracellular remodelling of the actin cytoskeleton plays an important role in
mechanotransduction because many cell-cell and cell-ECM adhesion complexes
rely on coupling to cytoskeletal actin filaments to gain anchorage and to main-
tain location. Cytoskeletal actin regulation is mediated via a combination of
polymerization, depolymerisation, and modifications of actin crosslinking proteins
(Pollard, 2007; Vicente-Manzanares et al., 2005), maintaining actin structures in
a dynamic equilibrium. Cytoskeletal actin can also relay extracellular force to the
nucleus, altering its morphology (Maniotis et al., 1997). This immediate transmis-
sion of mechanical stimulus to the nucleus has been demonstrated to be capable
of directly inflicting changes in gene expression (Guilak et al., 2000), though this
hypothesis still remains to be elucidated. More details of the mechanical properties
of ECM-nuclear connectivity are described elsewhere (Dahl et al., 2008).
Disruption of actin polymerization prevents cell adhesion (Sanger and Holtzer,
1972), whereas cellular adhesion to matrix stimulates actin reorganization (DeMali
et al., 2003); albeit, the resulting actin infrastructure depends on the cells external
environment (DeMali et al., 2003; Hannigan et al., 2005). For instance, fibrob-
lasts in 2D cultures spread out and cover a large surface area while fibroblasts in
3D cultures have enhanced migration and adopt a star like shape (DeMali et al.,
2003) suggesting that environmental differences detected via cell-ECM adhesion
help direct migration and modulate morphology. Interestingly, the formation of focal
adhesions and the association of FAK at focal adhesions are much more prominent
in 3D cultures than 2D cultures (Berrier and Yamada, 2007; DeMali et al., 2003)
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 157
6.5.2.2 Zyxin
Zyxins are subcellular proteins found at the cellular substratum attached to cell
adhesion complexes, such as focal adhesions, as well as to cytoskeletal actin
(Beckerle, 1998; Bershadsky et al., 2003; Hoffman et al., 2003). Zyxins molec-
ular structure reveals its role as both a scaffold for focal adhesion complexes as
well as a docking site for the ENA/VASP family of proteins (Hoffman et al., 2003;
Krause et al., 2003), which are abundantly found in intercalated discs of cardiomy-
ocytes (Brancaccio et al., 2006). Under resting conditions, zyxins are found bound to
vinculin and -actinin at focal adhesions close to Z-discs (Crawford and Beckerle,
1991; Yoshigi et al., 2005). Interestingly, unlike other integrin complex proteins
such as FAK, ILK, vinculin, or paxillin, (Li et al., 1999; Sawada and Sheetz, 2002;
Sharp et al., 1997; Torsoni et al., 2003), zyxins move away from integrin-linked
complexes toward actin filaments in response to stretch, causing realignment of
actin and thickening of actin filaments according to the direction of stretch (Yoshigi
et al., 2005). Interestingly, zyxin can localize between the nucleus and the cytoplas-
mic membrane and can thus potentially mediate gene regulation (Nix and Beckerle,
1997; Nix et al., 2001). For instance, exposure of cardiomyocytes to atrial natriuretic
peptide accumulates zyxin at the nucleus inducing anti-apoptotic effects (Kato et al.,
2005).
Zyxin is not necessary for survival as zyxin knockouts are both viable and fertile
(Hoffman et al., 2003), yet the lack of zyxin compromises the tolerance to mechani-
cal stimuli (Yoshigi et al., 2005). When stretched, zyxin mutant cells have impaired
thickening, but not reorganization of actin filaments, which can be rescued via the
addition of exogenous zyxin (Yoshigi et al., 2005). Reorganization of actin filaments
was likely not impaired due to compensation from other actin remodelling proteins
such as gelsolin.
6.5.2.3 Gelsolin
Gelsolin is a calcium dependent monomeric protein that regulates actin reorganiza-
tion by severing and capping actin filaments (Fig. 6.4b) (Liepina et al., 2003; Sun
et al., 1999b; Yu et al., 1992). Increases in intracellular calcium concentrations stim-
ulates gelsolin to sever actin, following by its binding to the truncated actin filament
acting as a cap to prevent subsequent regeneration and repolymerization (Sun et al.,
1999b). The removal of the gelsolin cap requires the binding of PIP2 (Liepina et al.,
2003; Yu et al., 1992). Cell adhesion causes gelsolin to co-localize with members
158 D. Guo et al.
of the integrin complex such as FAK, PI3K, c-Src, paxillin, vinculin, and talin sug-
gesting that mechanical stimulation recruits gelsolin to integrin-linked complexes
(Chellaiah et al., 2001).
Gelsolin is up-regulated in DCM, ventricular hypertrophy and heart failure (Yang
et al., 2000). Moreover, gelsolin null mice have better contractile function post
infarction compared to wildtypes (Li et al., 2009) suggesting that gelsolin activity
may be necessary in mediating ventricular damage and various forms of cardiomy-
opathy (Nishio and Matsumori, 2009). However, gelsolin activity has been argued
to be beneficial to the survival of cardiac cells as it can stimulate anti-apoptotic sig-
naling (Azuma et al., 2000; Koya et al., 2000). Considering gelsolins potency in
cardiac cells as well as other cell types and the central role of actin in both cell-
cell and cell-ECM adhesion complexes, Gelsolins role in cardiac remodelling and
mechanotransduction poses an important target for future investigation.
References
Ainscough JF, Drinkhill MJ, Sedo A, Turner NA, Brooke DA, Balmforth AJ, Ball SG (2009)
Angiotensin II type-1 receptor activation in the adult heart causes blood pressure-independent
hypertrophy and cardiac dysfunction. Cardiovasc Res 81:592600
Anastasi G, Cutroneo G, Gaeta R, Di Mauro D, Arco A, Consolo A, Santoro G, Trimarchi
F, Favaloro A (2009) Dystrophin-glycoprotein complex and vinculin-talin-integrin system in
human adult cardiac muscle. Int J Mol Med 23:149159
Antos CL, McKinsey TA, Frey N, Kutschke W, McAnally J, Shelton JM, Richardson JA, Hill JA,
Olson EN (2002) Activated glycogen synthase-3 beta suppresses cardiac hypertrophy in vivo.
Proc Natl Acad Sci USA 99:907912
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 159
Arai M, Yoguchi A, Iso T, Takahashi T, Imai S, Murata K, Suzuki T (1995) Endothelin-1 and
its binding sites are upregulated in pressure overload cardiac hypertrophy. Am J Physiol
268:H2084H2091
Azuma T, Koths K, Flanagan L, Kwiatkowski D (2000) Gelsolin in complex with phosphatidyli-
nositol 4,5-bisphosphate inhibits caspase-3 and -9 to retard apoptotic progression. J Biol Chem
275:37613766
Beckerle MC (1998) Spatial control of actin filament assembly: lessons from Listeria. Cell 95:
741748
Berni R, Savi M, Bocchi L, Delucchi F, Musso E, Chaponnier C, Gabbiani G, Clement S, Stilli D
(2009) Modulation of actin isoform expression before the transition from experimental com-
pensated pressure-overload cardiac hypertrophy to decompensation. Am J Physiol Heart Circ
Physiol 296:H1625H1632
Berrier AL, Yamada KM (2007) Cell-matrix adhesion. J Cell Physiol 213:565573
Bershadsky AD, Balaban NQ, Geiger B (2003) Adhesion-dependent cell mechanosensitivity. Annu
Rev Cell Dev Biol 19:677695
Bock-Marquette I, Saxena A, White MD, Dimaio JM, Srivastava D (2004) Thymosin beta4 acti-
vates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair.
Nature 432:466472
Bowman JC, Steinberg SF, Jiang T, Geenen DL, Fishman GI, Buttrick PM (1997) Expression
of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in
neonates. J Clin Invest 100:21892195
Brancaccio M, Hirsch E, Notte A, Selvetella G, Lembo G, Tarone G (2006) Integrin signalling: the
tug-of-war in heart hypertrophy. Cardiovasc Res 70:422433
Bueno OF, Molkentin JD (2002) Involvement of extracellular signal-regulated kinases 1/2 in
cardiac hypertrophy and cell death. Circ Res 91:776781
Calderwood DA (2004) Integrin activation. J Cell Sci 117:657666
Calderwood DA, Shattil SJ, Ginsberg MH (2000) Integrins and actin filaments: reciprocal
regulation of cell adhesion and signaling. J Biol Chem 275:2260722610
Carver W, Price RL, Raso DS, Terracio L, Borg TK (1994) Distribution of beta-1 integrin in the
developing rat heart. J Histochem Cytochem 42:167175
Chellaiah MA, Biswas RS, Yuen D, Alvarez UM, Hruska KA (2001) Phosphatidylinositol 3,4,5-
trisphosphate directs association of Src homology 2-containing signaling proteins with gelsolin.
J Biol Chem 276:4743447444
Chen H, Huang XN, Yan W, Chen K, Guo L, Tummalapali L, Dedhar S, St-Arnaud R, Wu
C, Sepulveda JL (2005) Role of the integrin-linked kinase/PINCH1/alpha-parvin complex in
cardiac myocyte hypertrophy. Lab Invest 85:13421356
Chen HC, Appeddu PA, Parsons JT, Hildebrand JD, Schaller MD, Guan JL (1995) Interaction of
focal adhesion kinase with cytoskeletal protein talin. J Biol Chem 270:1699516999
Chen HC, Guan JL (1994) Association of focal adhesion kinase with its potential substrate
phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 91:1014810152
Cingolani HE, Perez NG, Camilion de Hurtado MC (2001) An autocrine/paracrine mechanism
triggered by myocardial stretch induces changes in contractility. News Physiol Sci 16:8891
Covington MD, Burghardt RC, Parrish AR (2006) Ischemia-induced cleavage of cadherins in NRK
cells requires MT1-MMP (MMP-14). Am J Physiol Renal Physiol 290:F43F51
Crackower MA, Oudit GY, Kozieradzki I, Sarao R, Sun H, Sasaki T, Hirsch E, Suzuki A, Shioi
T, Irie-Sasaki J, et al (2002) Regulation of myocardial contractility and cell size by distinct
PI3K-PTEN signaling pathways. Cell 110:737749
Crawford AW, Beckerle MC (1991) Purification and characterization of zyxin, an 82,000-dalton
component of adherens junctions. J Biol Chem 266:58475853
Crosara-Alberto DP, Inoue RY, Costa CR (2009) FAK signalling mediates NF-kappaB activation
by mechanical stress in cardiac myocytes. Clin Chim Acta 403:8186
Dahl KN, Ribeiro AJ, Lammerding J (2008) Nuclear shape, mechanics, and mechanotransduction.
Circ Res 102:13071318
160 D. Guo et al.
Danialou G, Comtois AS, Dudley R, Karpati G, Vincent G, Des Rosiers C, Petrof BJ (2001)
Dystrophin-deficient cardiomyocytes are abnormally vulnerable to mechanical stress-induced
contractile failure and injury. FASEB J 15:16551657
DeBosch B, Treskov I, Lupu TS, Weinheimer C, Kovacs A, Courtois M, Muslin AJ (2006) Akt1
is required for physiological cardiac growth. Circulation 113:20972104
Delcommenne M, Tan C, Gray V, Rue L, Woodgett J, Dedhar S (1998) Phosphoinositide-3-OH
kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the
integrin-linked kinase. Proc Natl Acad Sci USA 95:1121111216
DeMali KA, Wennerberg K, Burridge K (2003) Integrin signaling to the actin cytoskeleton. Curr
Opin Cell Biol 15:572582
Domingos PP, Fonseca PM, Nadruz W Jr, Franchini KG (2002) Load-induced focal adhesion
kinase activation in the myocardium: role of stretch and contractile activity. Am J Physiol Heart
Circ Physiol 282:H556H564
Dwivedi A, Slater SC, George SJ (2009) MMP-9 and -12 cause N-cadherin shedding and thereby
beta-catenin signalling and vascular smooth muscle cell proliferation. Cardiovasc Res 81:
178186
Edwards LA, Thiessen B, Dragowska WH, Daynard T, Bally MB, Dedhar S (2005) Inhibition of
ILK in PTEN-mutant human glioblastomas inhibits PKB/Akt activation, induces apoptosis, and
delays tumor growth. Oncogene 24:35963605
Eghbali M, Tomek R, Sukhatme VP, Woods C, Bhambi B (1991) Differential effects of transform-
ing growth factor-beta 1 and phorbol myristate acetate on cardiac fibroblasts. Regulation of
fibrillar collagen mRNAs and expression of early transcription factors. Circ Res 69:483490
Ervasti JM (2003) Costameres: the Achilles heel of Herculean muscle. J Biol Chem 278:
1359113594
Ervasti JM (2007) Dystrophin, its interactions with other proteins, and implications for muscular
dystrophy. Biochim Biophys Acta 1772:108117
Frey N, Katus HA, Olson EN, Hill JA (2004) Hypertrophy of the heart: a new therapeutic target?
Circulation 109:15801589
Grote K, Flach I, Luchtefeld M, Akin E, Holland SM, Drexler H, Schieffer B (2003) Mechanical
stretch enhances mRNA expression and proenzyme release of matrix metalloproteinase-2
(MMP-2) via NAD(P)H oxidase-derived reactive oxygen species. Circ Res 92:e80e86
Gu J, Tamura M, Pankov R, Danen EH, Takino T, Matsumoto K, Yamada KM (1999) Shc and
FAK differentially regulate cell motility and directionality modulated by PTEN. J Cell Biol
146:389403
Gu J, Tamura M, Yamada KM (1998) Tumor suppressor PTEN inhibits integrin- and growth
factor-mediated mitogen-activated protein (MAP) kinase signaling pathways. J Cell Biol 143:
13751383
Guilak F, Tedrow JR, Burgkart R (2000) Viscoelastic properties of the cell nucleus. Biochem
Biophys Res Commun 269:781786
Gupta S, Purcell NH, Lin A, Sen S (2002) Activation of nuclear factor-kappaB is necessary for
myotrophin-induced cardiac hypertrophy. J Cell Biol 159:10191028
Hanft LM, Rybakova IN, Patel JR, Rafael-Fortney JA, Ervasti JM (2006) Cytoplasmic gamma-
actin contributes to a compensatory remodeling response in dystrophin-deficient muscle. Proc
Natl Acad Sci USA 103:53855390
Hannigan G, Troussard AA, Dedhar S (2005) Integrin-linked kinase: a cancer therapeutic target
unique among its ILK. Nat Rev Cancer 5:5163
Hannigan GE, Coles JG, Dedhar S (2007) Integrin-linked kinase at the heart of cardiac contractility,
repair, and disease. Circ Res 100:14081414
Heineke J, Molkentin JD (2006) Regulation of cardiac hypertrophy by intracellular signalling
pathways. Nat Rev Mol Cell Biol 7:589600
Hess AR, Seftor EA, Seftor RE, Hendrix MJ (2003) Phosphoinositide 3-kinase regulates mem-
brane Type 1-matrix metalloproteinase (MMP) and MMP-2 activity during melanoma cell
vasculogenic mimicry. Cancer Res 63:47574762
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 161
Hoffman LM, Nix DA, Benson B, Boot-Hanford R, Gustafsson E, Jamora C, Menzies AS, Goh
KL, Jensen CC, Gertler FB, et al (2003) Targeted disruption of the murine zyxin gene. Mol
Cell Biol 23:7079
Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:1125
Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110:673687
Ieda M, Tsuchihashi T, Ivey KN, Ross RS, Hong TT, Shaw RM, Srivastava D (2009) Cardiac
fibroblasts regulate myocardial proliferation through beta1 integrin signaling. Dev Cell 16:
233244
Ishiye M, Umemura K, Uematsu T, Nakashima M (1995) Angiotensin AT1 receptor-mediated
attenuation of cardiac hypertrophy due to volume overload: involvement of endothelin. Eur J
Pharmacol 280:1117
Ispanovic E, Haas TL (2006) JNK and PI3K differentially regulate MMP-2 and MT1-MMP mRNA
and protein in response to actin cytoskeleton reorganization in endothelial cells. Am J Physiol
Cell Physiol 291:C579C588
Ito H, Hiroe M, Hirata Y, Fujisaki H, Adachi S, Akimoto H, Ohta Y, Marumo F (1994) Endothelin
ETA receptor antagonist blocks cardiac hypertrophy provoked by hemodynamic overload.
Circulation 89:21982203
Ito K, Okamoto I, Araki N, Kawano Y, Nakao M, Fujiyama S, Tomita K, Mimori T, Saya H
(1999) Calcium influx triggers the sequential proteolysis of extracellular and cytoplasmic
domains of E-cadherin, leading to loss of beta-catenin from cell-cell contacts. Oncogene 18:
70807090
Kassiri Z, Khokha R (2005) Myocardial extra-cellular matrix and its regulation by metallopro-
teinases and their inhibitors. Thromb Haemost 93:212219
Kassiri Z, Oudit GY, Sanchez O, Dawood F, Mohammed FF, Nuttall RK, Edwards DR, Liu PP,
Backx PH, Khokha R (2005) Combination of tumor necrosis factor-alpha ablation and matrix
metalloproteinase inhibition prevents heart failure after pressure overload in tissue inhibitor of
metalloproteinase-3 knock-out mice. Circ Res 97:380390
Kato T, Muraski J, Chen Y, Tsujita Y, Wall J, Glembotski CC, Schaefer E, Beckerle M, Sussman
MA (2005) Atrial natriuretic peptide promotes cardiomyocyte survival by cGMP-dependent
nuclear accumulation of zyxin and Akt. J Clin Invest 115:27162730
Katz BZ, Miyamoto S, Teramoto H, Zohar M, Krylov D, Vinson C, Gutkind JS, Yamada KM
(2002) Direct transmembrane clustering and cytoplasmic dimerization of focal adhesion kinase
initiates its tyrosine phosphorylation. Biochim Biophys Acta 1592:141152
Khairallah M, Khairallah R, Young ME, Dyck JR, Petrof BJ, Des Rosiers C (2007) Metabolic and
signaling alterations in dystrophin-deficient hearts precede overt cardiomyopathy. J Mol Cell
Cardiol 43:119129
Khwaja A, Rodriguez-Viciana P, Wennstrom S, Warne PH, Downward J (1997) Matrix adhesion
and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt
cellular survival pathway. EMBO J 16:27832793
Knoll R, Postel R, Wang J, Kratzner R, Hennecke G, Vacaru AM, Vakeel P, Schubert C, Murthy
K, Rana BK, et al (2007) Laminin-alpha4 and integrin-linked kinase mutations cause human
cardiomyopathy via simultaneous defects in cardiomyocytes and endothelial cells. Circulation
116:515525
Koya RC, Fujita H, Shimizu S, Ohtsu M, Takimoto M, Tsujimoto Y, Kuzumaki N (2000) Gelsolin
inhibits apoptosis by blocking mitochondrial membrane potential loss and cytochrome c
release. J Biol Chem 275:1534315349
Krause M, Dent EW, Bear JE, Loureiro JJ, Gertler FB (2003) Ena/VASP proteins: regulators of the
actin cytoskeleton and cell migration. Annu Rev Cell Dev Biol 19:541564
Kumar A, Chaudhry I, Reid MB, Boriek AM (2002) Distinct signaling pathways are activated in
response to mechanical stress applied axially and transversely to skeletal muscle fibers. J Biol
Chem 277:4649346503
Kumar A, Khandelwal N, Malya R, Reid MB, Boriek AM (2004) Loss of dystrophin causes
aberrant mechanotransduction in skeletal muscle fibers. FASEB J 18:102113
162 D. Guo et al.
Nishio R, Matsumori A (2009) Gelsolin and cardiac myocyte apoptosis: a new target in the
treatment of postinfarction remodeling. Circ Res 104:829831
Nix DA, Beckerle MC (1997) Nuclear-cytoplasmic shuttling of the focal contact protein, zyxin: a
potential mechanism for communication between sites of cell adhesion and the nucleus. J Cell
Biol 138:11391147
Nix DA, Fradelizi J, Bockholt S, Menichi B, Louvard D, Friederich E, Beckerle MC (2001)
Targeting of zyxin to sites of actin membrane interaction and to the nucleus. J Biol Chem
276:3475934767
Oudit GY, Kassiri Z, Zhou J, Liu QC, Liu PP, Backx PH, Dawood F, Crackower MA, Scholey JW,
Penninger JM (2008) Loss of PTEN attenuates the development of pathological hypertrophy
and heart failure in response to biomechanical stress. Cardiovasc Res 78:505514
Oudit GY, Penninger JM (2009) Cardiac Regulation by Phosphoinositide 3-kinases and PTEN.
Cardiovasc Res 82(2):250260
Oudit GY, Sun H, Kerfant BG, Crackower MA, Penninger JM, Backx PH (2004) The role of
phosphoinositide-3 kinase and PTEN in cardiovascular physiology and disease. J Mol Cell
Cardiol 37:449471
Patrucco E, Notte A, Barberis L, Selvetella G, Maffei A, Brancaccio M, Marengo S, Russo G,
Azzolino O, Rybalkin SD, et al (2004) PI3Kgamma modulates the cardiac response to chronic
pressure overload by distinct kinase-dependent and -independent effects. Cell 118:375387
Pedrotty DM, Klinger RY, Badie N, Hinds S, Kardashian A, Bursac N (2008) Structural cou-
pling of cardiomyocytes and noncardiomyocytes: quantitative comparisons using a novel
micropatterned cell pair assay. Am J Physiol Heart Circ Physiol 295:H390H400
Peng X, Wu X, Druso JE, Wei H, Park AY, Kraus MS, Alcaraz A, Chen J, Chien S, Cerione
RA, et al (2008) Cardiac developmental defects and eccentric right ventricular hypertrophy in
cardiomyocyte focal adhesion kinase (FAK) conditional knockout mice. Proc Natl Acad Sci
USA 105:66386643
Pollard TD (2007) Regulation of actin filament assembly by Arp2/3 complex and formins. Annu
Rev Biophys Biomol Struct 36:451477
Qian Y, Zhong X, Flynn DC, Zheng JZ, Qiao M, Wu C, Dedhar S, Shi X, Jiang BH (2005) ILK
mediates actin filament rearrangements and cell migration and invasion through PI3K/Akt/Rac1
signaling. Oncogene 24:31543165
Quinlan JG, Hahn HS, Wong BL, Lorenz JN, Wenisch AS, Levin LS (2004) Evolution of the
mdx mouse cardiomyopathy: physiological and morphological findings. Neuromuscul Disord
14:491496
Quinlivan RM, Lewis P, Marsden P, Dundas R, Robb SA, Baker E, Maisey M (1996) Cardiac
function, metabolism and perfusion in Duchenne and Becker muscular dystrophy. Neuromuscul
Disord 6:237246
Rigor DL, Bodyak N, Bae S, Choi JH, Zhang L, Ter-Ovanesyan D, He Z, McMullen JR, Shioi T,
Izumo S, et al (2009) Phosphoinositide 3-kinase Akt signaling pathway interacts with protein
kinase Cbeta2 in the regulation of physiologic developmental hypertrophy and heart function.
Am J Physiol Heart Circ Physiol 296:H566H572
Ross RS, Pham C, Shai SY, Goldhaber JI, Fenczik C, Glembotski CC, Ginsberg MH, Loftus JC
(1998) Beta1 integrins participate in the hypertrophic response of rat ventricular myocytes. Circ
Res 82:11601172
Ruwhof C, van der Laarse A (2000) Mechanical stress-induced cardiac hypertrophy: mechanisms
and signal transduction pathways. Cardiovasc Res 47:2337
Ruwhof C, van Wamel AE, Egas JM, van der Laarse A (2000) Cyclic stretch induces the release of
growth promoting factors from cultured neonatal cardiomyocytes and cardiac fibroblasts. Mol
Cell Biochem 208:8998
Ruzicka M, Skarda V, Leenen FH (1995) Effects of ACE inhibitors on circulating versus car-
diac angiotensin II in volume overload-induced cardiac hypertrophy in rats. Circulation 92:
35683573
Rybakova IN, Patel JR, Ervasti JM (2000) The dystrophin complex forms a mechanically strong
link between the sarcolemma and costameric actin. J Cell Biol 150:12091214
164 D. Guo et al.
Wood CK, Turner CE, Jackson P, Critchley DR (1994) Characterisation of the paxillin-binding
site and the C-terminal focal adhesion targeting sequence in vinculin. J Cell Sci 107 (Pt 2):
709717
Yamada K, Green KG, Samarel AM, Saffitz JE (2005) Distinct pathways regulate expression of
cardiac electrical and mechanical junction proteins in response to stretch. Circ Res 97:346353
Yamaji S, Suzuki A, Sugiyama Y, Koide Y, Yoshida M, Kanamori H, Mohri H, Ohno S, Ishigatsubo
Y (2001) A novel integrin-linked kinase-binding protein, affixin, is involved in the early stage
of cell-substrate interaction. J Cell Biol 153:12511264
Yamazaki T, Komuro I, Shiojima I, Yazaki Y (1999) The molecular mechanism of cardiac
hypertrophy and failure. Ann NY Acad Sci 874:3848
Yang J, Moravec CS, Sussman MA, DiPaola NR, Fu D, Hawthorn L, Mitchell CA, Young JB,
Francis GS, McCarthy PM, et al (2000) Decreased SLIM1 expression and increased gel-
solin expression in failing human hearts measured by high-density oligonucleotide arrays.
Circulation 102:30463052
Yart A, Roche S, Wetzker R, Laffargue M, Tonks N, Mayeux P, Chap H, Raynal P (2002) A func-
tion for phosphoinositide 3-kinase beta lipid products in coupling beta gamma to Ras activation
in response to lysophosphatidic acid. J Biol Chem 277:2116721178
Yoshigi M, Hoffman LM, Jensen CC, Yost HJ, Beckerle MC (2005) Mechanical force mobilizes
zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement. J Cell
Biol 171:209215
Yu FX, Sun HQ, Janmey PA, Yin HL (1992) Identification of a polyphosphoinositide-binding
sequence in an actin monomer-binding domain of gelsolin. J Biol Chem 267:1461614621
Zahradka P, Harding G, Litchie B, Thomas S, Werner JP, Wilson DP, Yurkova N (2004) Activation
of MMP-2 in response to vascular injury is mediated by phosphatidylinositol 3-kinase-
dependent expression of MT1-MMP. Am J Physiol Heart Circ Physiol 287:H2861H2870
Zamir E, Geiger B (2001) Molecular complexity and dynamics of cell-matrix adhesions. J Cell Sci
114:35833590
Zelarayan LC, Noack C, Sekkali B, Kmecova J, Gehrke C, Renger A, Zafiriou MP, van der Nagel
R, Dietz R, de Windt LJ, et al (2008) Beta-Catenin downregulation attenuates ischemic cardiac
remodeling through enhanced resident precursor cell differentiation. Proc Natl Acad Sci USA
105:1976219767
Zuppinger C, Eppenberger-Eberhardt M, Eppenberger HM (2000) N-Cadherin: structure, function
and importance in the formation of new intercalated disc-like cell contacts in cardiomyocytes.
Heart Fail Rev 5:251257
Chapter 7
Atomistic Molecular Simulation of Gating
Modifier Venom Peptides Two Binding
Modes and Effects of Lipid Structure
Kazuhisa Nishizawa
K. Nishizawa (B)
Department of Laboratory Medicine, Teikyo University School of Medical Technology, Kaga,
Itabashi, Tokyo 173-8605, Japan
e-mail: kazunet@med.teikyo-u.ac.jp
7.1 Introduction
Since the identification and characterization of the tarantula toxin GsMTx4 by Sachs
and coworkers (Chen et al., 1996; Suchyna et al., 2000), this cationic hydropho-
bic polypeptide has been utilized as a specific blocker of stretch-activated cation
channels (SACs). As such, GsMTx4 has proven to be a valuable tool in studies
analyzing the involvement of SACs in various phenomena. The seminal papers by
Chen et al. (1996) and Suchyna et al. (2000) have reported a role for SACs in hypo-
tonic cell swelling-induced Ca2+ increase with the use of the venom from which
GsMTx4 was later isolated. More recently, several additional targets of GsMTx4,
including TRPC6 (Spassova et al., 2006) and MscK and MscS of E. coli have
been reported (Hurst et al., 2009). The number of studies showing pharmacolog-
ical potentials of GsMTx4 is also increasing (for review, Bowman et al., 2007). In
addition to such medical interests, GsMTx4 has drawn much interest from biophysi-
cists after the elucidation of a wide variety of target channels of different phyla of
organisms. GsMTx4 inhibits cationic SACs in a variety of vertebrate cell types such
as chick heart, rat astrocytes and skeletal muscle and human smooth muscle. How
does it modify the gating of such different channels with different kinetic proper-
ties and presumably different structures? It is likely that, after GsMTx4 adsorbs to
lipid bilayer membranes, it modifies their mechanical properties, thereby modifying
the energy from bilayer tension required for gating SACs. It is also likely that the
effects of GsMTx4 on membrane proteins are dependent on its partition into the
interface between channels and lipid molecules. However, structural and dynamical
features enabling these effects on various channels and motor molecules are not well
understood.
This review primarily focuses on recent studies using MD simulation analyses
and experimental results of GsMTx4 and related gating modifier peptides includ-
ing HaTx, a gating modifier of Kv channels. Based on these findings, we propose a
model of membrane behaviors of these toxins and the mechanisms by which such
toxins affect the gating of channels. A limited amount of information on the poten-
tial medical applications of GsMTx4 can be covered here, thus interested readers are
referred to more comprehensive reviews on GsMTx4 and ICK peptides. Bowman
et al. (2007) reviewed topics of history, properties, mechanisms and pharmacology
of GsMTx4. HaTx and other gating modifier toxins are detailed in Swartz (2007).
Recent reviews on ICK peptide gating modifiers include Sollod et al. (2005) and
Escoubas and Rash (2004). Computational methods for ion channels, including
homology modeling and MD simulations, have been reviewed by Tai et al. (2008).
Computer modeling and simulation studies of bacterial mechanosensitive channels
have been reviewed by Corry and Martinac (2008a, b). Recent progress on MD
simulations of membrane proteins is covered by Lindahl and Sansom (2008).
GsMTx4 is a 34 amino acid residue peptide isolated from the venom of a spider,
the Grammostola spatulata tarantula. The inhibitory activity of the crude venom on
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 169
SACs was originally discovered in a study to evaluate the importance of L-type Ca2+
channels and SACs in the hypotonic cell swelling-induced Ca2+ increase (HICI)
in GH3 cells (Chen et al., 1996). In their study, the finding that the extracellular
Ca2+ is the source of the Ca2+ influx of HICI led the authors to determine the route
for the Ca2+ influx; both L-type Ca2+ channels and SACs were likely involved in
the HICI. Importantly, the venom was found to block the HICI without blocking
L-type Ca2+ channels, suggesting the sensory role for SACs in the events of HICI.
Sachs and coworkers continued their study and isolated the molecule responsible
for the inhibition and named it GsMTx4 (Grammostola mechanotoxin number 4)
(Suchyna et al., 2000). They further showed that GsMTx4 inhibits SACs in astro-
cytes and heart cells (Suchyna et al., 2000). The group also determined the structure
of GsMTx4 in solution using NMR (Oswald et al., 2002).
Important insights into the mechanism for gating modification by GsMTx4
were provided by an experiment using the enantiomer. GsMTx4 and its enan-
tiomer, enGsMTx4, exhibit similar efficacy in inhibiting stretch-activated channels
of rat astrocyte, suggesting that membrane distortion, rather than specific molecular
recognition, plays an important role (Suchyna et al., 2004). Despite that GsMTx4 is
unlikely related to gramicidin A, GsMTx4 causes a 1025 fold increase in the chan-
nel appearance rate of gramicidin A, leading the authors to conclude that GsMTx4
exerts its effect on the gramicidin A channel-formers by altering the lipid packing at
the bilayer/solution/channel interface. Clearly, the action of GsMTx4 is not depen-
dent on the traditional lock-and-key model of ligand protein interactions postulated
for a number of inhibitors of enzymes (Suchyna et al., 2004). This finding also drew
the interest of researchers with respect to how GsMTx4 can modify the mechanical
properties of bilayer membranes.
While the potential pharmacological value of GsMTx4 cannot be fully covered
here, notable findings include GsMTx4-dependent inhibition of atrial fibrillation
(Bode et al., 2001) and Yeung et al. (2005) have shown that GsMTx4 inhibits
Ca2+ elevation in muscle fibers from the mdx mouse, a mouse model of Duchenne
Muscular Dystrophy. This suggests that GsMTx4 may be effective in protecting
dystrophic muscle cells in which SACs activation, leading to Ca2+ influx, is easily
triggered due to muscle cells defective in the dystroglycan complex (Yeung et al.,
2005).
As reviewed in the next section, GsMTx4 is a member of a peptide family named
ICK (inhibitory cysteine knot) peptides (Narasimhan et al., 1994). Different ICK
peptides modify the gating property of a variety of channels (Swartz, 2007). Among
ICK peptides, GsMTx4 is unique in its high specificity to SACs. Recently, several
genes belonging to the TRP (transient response potential) channel family have been
cloned and a consensus is emerging that TRP channels form an important group of
SACs (Yin and Kuebler, 2009). A recent example of the specificity of GsMTx4 for
SACs was also obtained from the TRC family channels. GsMTx4 inhibits TRPC1
(Maroto et al., 2005, Bowman et al., 2007), although the presence of endogenous
TRPC1-like SACs confounded the effect of GsMTX4 on TRPC1 (Bowman et al.,
2007). On the other hand, inhibitory effects of GsMTx4 on TRPC6 channels were
reported in a more reliable system (Spassova et al., 2006). In their study, HEK293
cells were transfected with the TRPC6 cDNA under the control of an inducible
170 K. Nishizawa
SGTx1, another member of the ICK peptide family (see below) acts only with
anionic lipids. However, this study used POPC (palmitooleoyl-phosphatidylcholine)
and POPC-POPG (palmitooleoyl-phosphatidylglycerol) membranes that did not
contain DPPC (dipalmitoyl-phosphatidylcholine), sphingomyelin or other lipid
mixtures that have potential to create domains rich in saturated lipid tails. As consid-
ered below, our MD simulation study showed that the use of DPPC has a dramatic
increase of the binding energy ( G = 100 kJ/mol) compared with POPC,
indicating the necessity to take the lipid tail structure into account.
Table 7.1 Target and time for onset of ICK peptide toxins
Note that this list is not exhaustive, but focuses on the toxins/targets for which the time course of effect can be inferred from the
literature, at least approximately. For quick reference, readers are referred to the SwissProt file (http://au.expasy.org/sprot/) for each
Atomistic Molecular Simulation of Gating Modifier Venom Peptides
toxin using the accession number shown in the Fig. 7.1 legend
a Targetchannel or current shown along with the expression system used for the experiment. Artificial bilayer system. b Time
constant, , based on exponential curve fitting is shown along with the toxin concentration in the experiment. Some data are rough
estimations from the reference figure. c The time at which the steady state was reached
173
174 K. Nishizawa
Fig. 7.1 (ac) Structure of GsMTx4 and HaTx. The bottom and side view of GsMTx4 (a) and
HaTx1 (b) and the initial orientation used for simulations (c). The hydrophobic patch is oriented
around the center of the normal view and at the bottom of the side view. (ac) are similar to
Figure 1 of Nishizawa and Nishizawa (2007), in which the following coloring scheme is used;
Hydrophobic residues (Ala, Cys, Ile, Leu, Met, Phe, Pro, Trp, Tyr and Val) are green; basic (Arg
and Lys) are blue and acidic (Asp and Glu) residues are red. (d) Amino acid sequence alignment
of ICK peptides. SwissProt accession numbers are as follows: GsMTx4 (Grammostola mechan-
otoxin number 4, Q7YT39), GxTx-1E (Guangxitoxin-1E, P84835), -Aga4A (P30288), CcoTx2
(P84508), HaTx1 (P56852), SGTx1 (P56855), VSTX1 (voltage sensor toxin 1, P60980), ProTx2
(Beta-theraphotoxin-Tp2a, P83476), HmTx1 (heteroscordatoxins, P60992), ScTx1 (stromatoxin
1, P60991), HpTx3 (heteropodatoxin3, P58427), PaTx1 (phrixotoxin1, P61230), -GrTx-SIA
(Omega-grammotoxin-SIA, P60590)
(1) Slow action. They have small apparent association and dissociation rate con-
stants, 103 104 times slower than the pore blocking toxins (Swartz and
MacKinnon, 1995; Ruta et al., 2003). In the case of HaTx, despite of its
high affinity Kd = 42 nM, action is on the order of minutes. The action
tends to become slow especially when the concentration is low (Table 7.1).
One example is the slow equilibration rate of VSTx1 in inhibition of KvAP
(60 min for 20 nM VSTx1) reconstituted in a planer membrane (POPE
(palmitooleoyl-phosphatidylethanolamine): POPG=3:1) (Lee and MacKinnon,
2004). Intriguingly, in a recent experiment with the Xenopus oocyte expression
system, after the removal of HaTx from the aqueous solution, the Kv2.1 chan-
nel activity recovered very slowly, requiring 1,000 s (Milescu et al., 2007),
suggesting the presence of an energy barrier between the aqueous phase and the
action site.
(2) Small binding site and high affinity. It is puzzling why the apparent bind-
ing affinity of HaTx (and grammotoxin and VSTx1) to the voltage sensor is
so high, considering the binding site on the channel consists of only a few
amino acids on S3b and S4. However, from the VSTx1 study by MacKinnon
and coworkers, a consensus is emerging that, not only the toxin partition into
the lipid membrane enriches the toxin in the membrane, the tertiary com-
plex toxin/channel/lipid is created and this strengthens the binding of toxin to
channel proteins (Schmidt and MacKinnon, 2008).
(3) Not activation, but inhibition. Unlike -scorpion toxin that stabilizes activated
states of Nav channels (Cestle et al., 1998), HaTx and VSTx1 (and most other
ICK peptide toxins) inhibit channel activation. It is unknown how they do so.
(4) Effects of lipid mechanical states. Lipid mechanical state or structure influ-
ences the inhibition efficacy of these peptides. A recent study by Schmidt and
MacKinnon (2008) employing a Kv1.2 channel with a voltage sensor paddle
from Kv2.1 showed that VsTx1 activity depends on the mechanical state of
the membrane. While VSTx1 has no effect on channel analyzed in whole cell
recording of oocytes, VSTx1 causes a slowing of voltage activation similar to
the effect observed on the channel embedded in planar bilayers. This suggests
that voltage sensor toxins exert their effect by perturbing the interaction between
the channel and the membrane lipid. The importance of lipid head groups is also
shown by Milescu et al. (2007) in the finding that sphingomyelinase D, which
removes choline from sphingomyelin, dramatically reduces the inhibitory effect
of HaTx1.
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 177
With respect to (1) The membrane diffusion coefficient for a molecule like ICK
peptides is probably 108 cm2 s1 (Lee and Mackinnon, 2004). The authors also
speculate that the collision frequency between VSTX1 and the KvAP channel is
likely to be orders of magnitude faster than the observed kinetics of inhibition in
their experimental system. Also with respect to (1), note that the time for action
depends on the concentration of toxin; the lower concentration leads to slower action
(e.g., HaTx, AgaIVA and SGTx in Table 7.1). Although it is speculative, one
possibility is that the toxin concentration influences the location of toxin, which in
turn influences the rate of its access to the site of action. From (5) (concentration-
dependent effects of GsMTx4) and the consideration given below, it is possible that
HaTx at high concentration may be located at a distinctly different position from
the position preferred at low concentration. On the other hand, even with the same
concentration, the time for action differs significantly among the systems examined.
For example, while GsMTx4 at 5 M inhibits SACs on outside-out patch of astro-
cyte within 0.6 s, the effect of the same concentration of GsMTx4 on the OHC
motor takes 17.6 s and the Kd is also high. It is currently difficult to address this
issue because both lipid composition and peptide-protein interaction are influence
the dynamics.
With respect to (5), a recent study by Martinac and colleague (Hurst et al., 2009)
using E. coli spheroplast is of particular interest. They measured two channel activi-
ties MscS (small conductance) and MscK (small conductance, K+ dependent), both
activities being mediated by mechanosensitive channels but with distinct gating
characteristics where the membrane tension required for activation is different. For
both MscS and MscK, when GsMTx4 is applied to the outer leaflet of the bilayer
at concentrations of 24 M, channel activity is inhibited, while at higher con-
centrations of 1220 M, channel activity is potentiated. Moreover they reported
that the initial inhibiting effect of 24 M GsMTx4 becomes less pronounced with
increasing time of exposure. They repeated the pressure application three times,
each lasting 30 min including lag-times of 20 min. Their free-energy parameter
analyses showed that the initially inhibiting 4 M GsMTx4 had only a minimal
degree of effect during the third pressure cycle (Hurst et al., 2009). As the authors
discuss, these puzzling findings can be accounted for by considering the two binding
modes, which we proposed based on our MD simulation analyses (see below).
leaflet of the membrane (Nishizawa and Nishizawa, 2006). This result prompted us
to apply the free energy analysis to HaTx and GsMTx4 in lipid membranes. In this
and the next sections, technical issues for such free energy analyses are considered.
However, unlike the mean force on a water molecule in a membrane (Marrink and
Berendsen, 1994), the mean force acting on a peptide in a membrane depends on the
strong electrostatic interactions between the peptide and the lipid head groups and
on the energy associated with lipid packing. Therefore, how to avoid the membrane
deformation represents a major problem, as discussed in the next section. Another
weakness of this method is that the requirement of a long equilibration time for each
position necessitates the coarse discretization, which may lead to inaccuracy in the
free energy analyses.
In addition, the difference in the density of lipid molecules between the two
leaflets may cause inaccurate measurement of the mean force. In our first paper
in ICK peptides (Nishizawa and Nishizawa, 2006), a set of DPPC bilayers, namely
58/64, (number of DPPC of the upper/lower leaflets) was used. We also tested 61/64
but this alteration did not have a significant effect on the result. Although we have
not tested this extensively, embedding the GsMTx4 in one of the leaflets a 64/64
DPPC membrane may lead to too high a density of lipid on the side containing the
GsMTx4, which may in turn artificially promote the deep positioning of the toxin.
To minimize such a density effect, we also tried a 6 nm 12 nm wide membrane
and embedded two GsMTx4 molecules on the two sides of the membrane to approx-
imate the C2 symmetry. In this way the lipid density of the two leaflets become the
same. This consideration also has a bearing on the issue of the local lateral pres-
sure of the membrane and the effect of peptide toxins on the local pressure profile.
Lindahl and Edholm (2000) developed an important method to calculate the pres-
sure profile of membranes, which shows the lateral pressure at different depths of
the lipid membrane. Gullingsrud and Schulten (2004) applied this method to sev-
eral membranes with and without the applied tension. In the future, the effect of
GsMTx4 on the pressure profile may well be investigated. In such studies, the num-
ber of lipid molecules in two leaflets may have to be carefully varied. The flip-flop
of lipid molecules (i.e., the translocation of lipid molecules from one leaflet to the
other leaflet of the bilayer) is a slow process (minutes) even with the help of flli-
pase (Zachowski and Devaux, 1990) and therefore, the uneven distribution of the
lateral pressure among the two leaflets may be of relevance also in the real experi-
mental system. After the binding of GsMTx4 to the membrane, the lateral pressure
profile of the membrane may slowly change during the experiment as such flip-flop
events occur.
Fig. 7.2 (continued) Our measurement showed that, with the presence of GsMTx4 in the shallow
mode, the membrane thinning (from l1 (1.4 nm) to l2 (1.2 nm)) requires 15 kJ/mol of energy for
DPPC and 14 kJ/mol for POPC (Supplementary File 6 of Nishizawa and Nishizawa, 2007). This
value is much smaller than the energy difference between the shallow and deep binding modes
(Fig. 7.2). (c) The mean force measurement. The z-position of the center of mass (COM) of the
sidechain atoms of K8 and K28 was restrained and the mean force experienced by the COM was
measured. (d) Deep mode binding. (e) Free energy profile of GsMTx4 and HaTx in a DPPC mem-
brane. For GsMTx4, K8/K28 COM was used as the reaction coordinate (Nishizawa and Nishizawa,
2006). For HaTx, R24/K26 COM was used as the reaction coordinate. (f) Free energy profile same
as (e) but with the POPC membrane (Nishizawa and Nishizawa, 2007). Cartoons below (e) and (f)
represent our hypothesis on the factors influencing the stability of the deep mode relative to the
shallow mode and our hypothesis on the transition from the shallow to the deep mode. Movements
of GsMTx4 toward saturated acyl chain-rich domains and/or movements of such lipids toward
GsMTx4 may promote the transition toward the deep binding mode. The transition may also be
facilitated by binding of GsMTx4 to the membrane at a high density. (g) Two mode-based model
for the GsMTx4 biphasic effect on E.coli MscK and MscS (Hurst et al., 2009). Note that in this
illustration the Msc channel is represented by a simple tetramer composed of four transmembrane
cylinders. (h) A deep mode-based model for the inhibition of the voltage sensor movement by
HaTx
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 181
Fig. 7.2 Free energy analyses and two-binding-mode model of GsMTx4 effects. (ad) Steps
assumed for energy analyses of GsMTx4 binding and penetration into the membrane. A simple
illustration of the GsMTx4 structure is used; the shaded area represents the charge band, whereas
the Lys and Arg residues are shown by long sidechains (zigzag lines). (a) GsMTx4 binds to the
membrane from bulk water, forming the shallow mode. l1 is the distance from the bilayer center
based on the average positions of phosphorus of DPPC for representative simulations. In our study,
l1 was 1.4 nm. (b) A mildly deformed membrane was subjected to the membrane fixation, which
was useful for the mean force measurement (see text). To make the interpretation easy, a mem-
brane with a mild degree of thinning (l2 = 1.2 nm) was used. Of note, the approximate free energy
associated with the membrane thinning can also be measured by the mean force measurement.
182 K. Nishizawa
POPC membrane favored the two modes equally (Fig. 7.2f). (3) It is possible that,
after several minutes, the toxin moves into the domains rich in the saturated acyl
chains and/or the toxin attract such lipids in its vicinity (Fig. 7.2e). In this way,
deep mode binding may acquire stability. (4) Then, the toxin may interact with the
binding site on the channel, if such a site is accessible (Fig. 7.2 g, h). In the case
of HaTx, primarily the effect of (3) drives the toxin downward and this force may
inhibit the S4 upward movement (Fig. 7.2 h).
The concentration-dependent (biphasic) effect of GsMTx4 can be explained by
our two mode-based model (Hurst et al., 2009). As we hypothesize in Fig. 7.2 g,
GsMTx4 in the shallow mode may inhibit the opening of SACs. In the deep
mode, further thinning of the membrane induced by the GsMTx4 molecules may
facilitate opening the SACs. As our free energy analyses have shown, an energy
barrier between the two binding modes explains the slow equilibration and recovery
(after removal) for GsMTx4, HaTx and VSTx1 (Nishizawa and Nishizawa, 2007).
Moreover, given the recent finding showing the low position of S4 in the resting
state (Brjesson and Elinder, 2008) and the finding that VSTx1 interact at S4 of the
KvAP, the deep binding mode appears to be more suitable to stabilize the resting
state than the shallow mode. Finally, extensive chimera analyses showed that, for
the Kv2.1 channel containing KvAP S4, in the presence of VSTx1, strong depo-
larization (100 mV) allows channel opening to some extent and this opening is
accompanied by the dissociation of VSTx1 from the S4 of the chimera channel
(Alabi et al., 2007). This finding is also consistent with the view that the deep
mode is the action mode of VSTx1; considering that the partial channel opening
is accompanied by upward movement of S4, it is difficult to explain, based only on
the shallow mode, why VSTx1 exhibits the higher affinity in the resting (down) state
than in the activated state. Shiau et al. (2002) carried out modeling by the docking
method and suggested a binding model for HaTx1 and the Kv2.1 channel, although
their analyses were based on a lipid-free system (for review, Huang et al., 2007).
Intriguingly, our preliminary analyses show that the model by Shiau et al. (2002) is
more consistent with the deep binding mode (our manuscript in preparation). Further
simulation analyses are underway. In general, for ICK peptides other than GsMTx4,
more experimental and modeling studies are necessary before a model based on the
two modes of interaction can be proposed.
(2008), and those on MscS include Anishkin et al. (2008a) and Anishkin et al.
(2008b).
Recently, Yefimov et al. (2008) used the CG model of protein and lipid (DOPE).
In the study, starting from the closed crystal structure, considerable opening of the
channel was observed close to the rupture tension of the bilayer. This demonstrates
the potential usefulness of the CG approach, but it should be noted that the CG rep-
resentation introduces artificial smoothness into the channel gating. In fact, in the
atomistic simulations by Jeon and Voth (2008), the membrane tension and/or the
structural asymmetry (i.e., due to the difference in the number of lipid molecules
between two leaflets) of the membrane was rather ineffective in inducing MscL
structural changes within 20 ns. Recent determination of the structure of a truncated
MscL from Staphylococcus aureus is illuminating because it likely corresponds
to the pre-expanded state based on the tilted transmembrane helices and overall
flattening of the channel (Liu et al., 2009). This flattening is accompanied by an
increase in the cross sectional area of the MscL channel. Flattening accompany-
ing channel opening has also been pointed out in several simulation studies (e.g.,
Anishkin et al. 2008a, b). These studies should deepen our understanding of the gat-
ing mechanism by membrane stress, and also provide opportunities for simulation
studies of the tertiary complex ICK peptide/channel/lipid membrane.
In future studies, lipid composition should also be varied, since different compo-
sitions will likely alter the relative stability of the two binding modes. It is possible
that the presence of ICK peptides influences the formation of microdomains con-
taining acyl chains with a distinct degree of saturation. Besides acyl chains, lipid
headgroup structure/composition may also be influenced by ICK peptides. Such
considerations are interesting because the phospho-headgroups of membrane lipids,
together with certain acidic channel residues, are believed to provide the necessary
counter-charges for the positively charged residues of S4 during individual steps of
the voltage-sensor movement (Xu et al., 2008; Ramu et al., 2006). Therefore, some
ICK peptides may have direct effects on lipid headgroups, which then modify the
gating property of channels.
Besides such potential effects of ICK peptides on channel-lipid interaction, the
effect of the peptides on the lateral pressure profile of the membrane could exerts
indirect effects on the gating of mechanosensitive channels. The binding of GsMTx4
to the membrane may change the lateral pressure profile. For the pure bilayers of
POPE or POPC, the layer of the lipid headgroups has a tension trough that has a
weak lateral pressure (Lindahl and Edholm, 2000; Gullingsrud and Schulten, 2004).
The presence of GsMTx4 in the shallow mode may nullify the tension trough. The
GsMTx4 transition to the deep mode may further alter the pressure profile, thereby
changing the free energy required to open the gate. Clearly, many more analyses are
necessary to discuss this issue quantitatively.
To improve future simulation studies of ICK peptides, the importance of compar-
ing simulations starting from various positions/orientations is again emphasized. In
particular, when studying the deep binding mode, it is important to take into account
that various combinations of two or three charged residues may interact with the
inner leaflet of the membrane to form the deep mode. Therefore, reorientation may
be necessary until the peptide finds the orientation/position with the lowest free
186 K. Nishizawa
energy. Although, for both experiments and simulations, the reorientation may be
a time-consuming step because interaction between the charged residues and lipid
headgroup has to be remodeled.
Notwithstanding such difficulties, homology modeling and MD simulations pro-
vide useful measures to study many themes of mechanosensitive channels. In many
channel studies, it is difficult to say that MD simulations can stand alone as a
method, yet they further the understanding of how ion channels work in atom-
istic details. At the same time, ICK peptide analyses suggest that understanding
the mechanics of mechanosensitive channels at the atomic level will help us design
molecules that can modify gating dynamics in a channel-specific manner. On the
other hand, it can be anticipated that many future MD simulation studies suffer
from discrepancies between the simulation results and experimental findings. As is
the case with large membrane proteins in general, careful consideration is necessary
when applying computational methods to ion channels; for example, how close is
the simulation system to reality, or how robust is the result? As with the experi-
mental approaches, deliberate designing of a series of simulations should always be
helpful.
References
Alabi AA, Bahamonde MI, Jung HJ, Kim JI, Swartz KJ (2007) Portability of paddle motif function
and pharmacology in voltage sensors. Nature 450:370375
Anishkin A, Akitake B, Sukharev S (2008a) Characterization of the resting MscS: modeling and
analysis of the closed bacterial mechanosensitive channel of small conductance. Biophys J
94:12521266
Anishkin A, Kamaraju K, Sukharev S (2008b) Mechanosensitive channel MscS in the open
state: modeling of the transition, explicit simulations, and experimental measurements of
conductance. J Gen Physiol 132:6783
Bass RB, Strop P, Barclay M, Rees DC (2002) Crystal structure of Escherichia coli MscS, a
voltage-modulated and mechanosensitive channel. Science 298:15821587
Bemporad D, Sands ZA, Wee CL, Grottesi A, Sansom MS (2006) Vstx1, a modifier of Kv channel
gating, localizes to the interfacial region of lipid bilayers. Biochemistry 45:1184411855
Bode F, Sachs F, Franz MR (2001) Tarantula peptide inhibits atrial fibrillation. Nature 409:3536
Bond PJ, Sansom MS (2007) Bilayer deformation by the Kv channel voltage sensor domain
revealed by self-assembly simulations. Proc Natl Acad Sci USA 104:26312636
Brjesson SI, Elinder F (2008) Structure, function, and modification of the voltage sensor in
voltage-gated ion channels. Cell Biochem Biophys 52: 149174
Bosmans F, Rash L, Zhu S, Diochot S, Lazdunski M, Escoubas P, Tytgat J (2006) Four novel taran-
tula toxins as selective modulators of voltage-gated sodium channel subtypes. Mol Pharmacol
69:419429
Bowman CL, Gottlieb PA, Suchyna TM, Murphy YK, Sachs F (2007) Mechanosensitive ion chan-
nels and the peptide inhibitor GsMTx-4: history, properties, mechanisms and pharmacology.
Toxicon 49:249270
Catterall WA, Cestle S, Yarov-Yarovoy V, Yu FH, Konoki K, Scheuer T (2007) Voltage-gated ion
channels and gating modifier toxins. Toxicon 49:124141
Cestle S, Qu Y, Rogers JC, Rochat H, Scheuer T, Catterall WA (1998) Voltage sensor-trapping:
enhanced activation of sodium channels by beta-scorpion toxin bound to the S3S4 loop in
domain II. Neuron 21:919931
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 187
Lee CW, Kim S, Roh SH, Endoh H, Kodera Y, Maeda T, Kohno T, Wang JM, Swartz KJ, Kim
JI (2004) Solution structure and functional characterization of SGTx1, a modifier of Kv2.1
channel gating. Biochemistry 43:890897
Lee HC, Wang JM, Swartz KJ (2003) Interaction between extracellular Hanatoxin and the resting
conformation of the voltage-sensor paddle in Kv channels. Neuron 40:527536
Lee SY, MacKinnon R (2004) A membrane-access mechanism of ion channel inhibition by voltage
sensor toxins from spider venom. Nature 430:232235
Lindahl E, Edholm O (2000) Spatial and energetic-entropic decomposition of surface tension in
lipid bilayers from molecular dynamics simulations. J. Chem. Phys. 113: 38823893
Lindahl E, Hess B, van der Spoel D (2001) GROMACS 3.0: a package for molecular simulation
and trajectory analysis. J Mol Mod 7: 306317
Lindahl, E, Sansom MS (2008) Membrane proteins: molecular dynamics simulations. Curr Opin
Struct Biol 18:425431
Li-Smerin Y, Swartz KJ (1998) Gating modifier toxins reveal a conserved structural motif in
voltage-gated Ca2+ and K+ channels. Proc Natl Acad Sci USA 95:85858589
Li-Smerin Y, Swartz KJ (2000) Localization and molecular determinants of the Hanatoxin
receptors on the voltage-sensing domains of a K(+) channel. J Gen Physiol. 115:
673684
Liu Z, Gandhi CS, Rees DC (2009) Structure of a tetrameric MscL in an expanded intermediate
state. Nature 461:120124
MacKinnon R, Miller C (1989) Mutant potassium channels with altered binding of charybdotoxin,
a pore-blocking peptide inhibitor. Science 245:13821385
Maroto R, Raso A, Wood TG, Kurosky A, Martinac B, Hamill OP. (2005) TRPC1 forms the stretch-
activated cation channel in vertebrate cells. Nat Cell Biol. 7:179185
Marrink S-J, Berendsen HJC (1994) Simulation of water transport through a lipid membrane- J
Phys Chem 98: 41554168
Marrink SJ, de Vries AH, Mark AE (2004) Coarse Grained Model for Semiquantitative Lipid
Simulations. J Phys Chem B 108: 750760
Marvin L, De E, Cosette P, Gagnon J, Molle G, Lange C (1999) Isolation, amino acid sequence
and functional assays of SGTx1. The first toxin purified from the venom of the spider scodra
griseipes. Eur J Biochem 265:572579
Mtyus E, Kandt C, Tieleman DP (2007) Computer simulation of antimicrobial peptides. Curr
Med Chem 14: 27892798
Middleton RE, Warren VA, Kraus RL, Hwang JC, Liu CJ, Dai G, Brochu RM, Kohler MG, Gao
YD, Garsky VM, Bogusky MJ, Mehl JT, Cohen CJ, Smith MM. (2002) Two tarantula peptides
inhibit activation of multiple sodium channels. Biochemistry 41:1473414747
Milescu M, Vobecky J, Roh SH, Kim SH, Jung HJ, Kim JI, Swartz KJ (2007) Tarantula toxins
interact with voltage sensors within lipid membranes. J Gen Physiol 130:497511
Mintz IM, Venema VJ, Swiderek KM, Lee TD, Bean BP, Adams ME (1992) P-type calcium
channels blocked by the spider toxin omega-Aga-IVA. Nature 355:827829
Narasimhan L, Singh J, Humblet C, Guruprasad K, Blundell T (1994) Snail and spider toxins share
a similar tertiary structure and cystine motif. Nat Struct Biol 1:850852
Nishizawa M, Nishizawa K (2006) Interaction between K+ channel gate modifier hanatoxin
and lipid bilayer membranes analyzed by molecular dynamics simulation. Eur Biophys J 35:
373381
Nishizawa M, Nishizawa K (2007) Molecular dynamics simulations of a stretch-activated channel
inhibitor GsMTx4 with lipid membranes: two binding modes and effects of lipid structure.
Biophys J 92: 42334243
Oswald RE, Suchyna TM, McFeeters R, Gottlieb P, Sachs F (2002) Solution structure of peptide
toxins that block mechanosensitive ion channels. J Biol Chem 277:3444334450
Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kale
L, Schulten K (2005a) Scalable molecular dynamics with NAMD. J Comput Chem 26:
17811802
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 189
Phillips LR, Milescu M, Li-Smerin Y, Mindell JA, Kim JI, Swartz KJ (2005b) Voltage-sensor
activation with a tarantula toxin as cargo. Nature 436:857860
Posokhov YO, Gottlieb PA, Morales MJ, Sachs F, Ladokhin AS (2007) Is lipid bilayer binding a
common property of inhibitor cysteine knot ion-channel blockers? Biophys J 93:L20L22
Ramu Y, Xu Y, Lu Z (2006) Enzymatic activation of voltage-gated potassium channels. Nature
442:696699
Roux, B. (1995) The calculation of the potential of mean force using computer simulations. Comp
Phys Commun 91:275282
Ruta V, Jiang Y, Lee A, Chen J, MacKinnon R. (2003) Functional analysis of an archaebacterial
voltage-dependent K+ channel. Nature 422:180185
Sanguinetti MC, Johnson JH, Hammerland LG, Kelbaugh PR, Volkmann RA, Saccomano NA,
Mueller AL (1997) Heteropodatoxins: peptides isolated from spider venom that block Kv4.2
potassium channels. Mol Pharmacol 51:491498
Schmidt D, MacKinnon R (2008) Voltage-dependent K+ channel gating and voltage sensor toxin
sensitivity depend on the mechanical state of the lipid membrane. Proc Natl Acad Sci USA
105:1927619281
Shiau YS, Lin TB, Liou HH, Huang PT, Lou KL, Shiau YY (2002) Molecular simulation
reveals structural determinants of the hanatoxin binding in Kv2.1 channels. J Mol Model 8:
253257
Sollod BL, Wilson D, Zhaxybayeva O, Gogarten JP, Drinkwater R, King GF (2005) Were arachnids
the first to use combinatorial peptide libraries? Peptides 26:131139
Spassova MA, Hewavitharana T, Xu W, Soboloff J, Gill DL, (2006) A common mechanism under-
lies stretch activation and receptor activation of TRPC6 channels. Proc Natl Acad Sci USA
103:1658616591
Suchyna TM, Johnson JH, Hamer K, Leykam JF, Gage DA, Clemo HF, Baumgarten CM, Sachs F
(2000) Identification of a peptide toxin from Grammostola spatulata spider venom that blocks
cation-selective stretch-activated channels. J Gen Physiol 115:583598
Suchyna TM, Tape SE, Koeppe RE 2nd, Andersen OS, Sachs F, Gottlieb PA (2004) Bilayer-
dependent inhibition of mechanosensitive channels by neuroactive peptide enantiomers. Nature
430:235240
Swartz KJ (2007) Tarantula toxins interacting with voltage sensors in potassium channels. Toxicon
49:213230
Swartz KJ (2008) Sensing voltage across lipid membranes. Nature 456:891897
Swartz KJ, MacKinnon R (1995) An inhibitor of the Kv2.1 potassium channel isolated from the
venom of a Chilean tarantula. Neuron 15:941949
Swartz KJ, MacKinnon R (1997) Hanatoxin modifies the gating of a voltage-dependent K+ channel
through multiple binding sites. Neuron 18:665673
Tai K, Fowler P, Mokrab Y, Stansfeld P, Sansom MS (2008) Molecular modeling and simu-
lation studies of ion channel structures, dynamics and mechanisms. Methods Cell Biol 90:
233265
Takahashi H, Kim JI, Min HJ, Sato K, Swartz KJ, Shimada I (2000) Solution structure of hana-
toxin1, a gating modifier of voltage-dependent K+ channels: common surface features of gating
modifier toxins. J Mol Biol 297:771780
Wang JM, Roh SH, Kim S, Lee CW, Kim JI, Swartz KJ (2004) Molecular surface of tarantula
toxins interacting with voltage sensors in Kv channels. J Gen Physiol 123: 455467
Wee CL, Gavaghan D, Sansom MS (2008) Lipid bilayer deformation and the free energy of
interaction of a Kv channel gating-modifier toxin. Biophys J 95:38163826
Winterfield JR, Swartz KJ (2000) A hot spot for the interaction of gating modifier toxins with
voltage-dependent ion channels. J Gen Physiol 116: 637644
Xu Y, Ramu Y, Lu Z (2008) Removal of phospho-head groups of membrane lipids immobilizes
voltage sensors of K+ channels. Nature 451:826829
Yefimov S, van der Giessen E, Onck PR, Marrink SJ (2008) Mechanosensitive membrane channels
in action. Biophys J 94:29943002
190 K. Nishizawa
Yeung EW, Whitehead NP, Suchyna TM, Gottlieb PA, Sachs F, Allen DG (2005) Effects of
stretch-activated channel blockers on [Ca2+ ]i and muscle damage in the mdx mouse. J Physiol
562:367380
Yin J, Kuebler WM (2009) Mechanotransduction by TRP Channels: General Concepts and Specific
Role in the Vasculature. Cell Biochem Biophys. [Epub ahead of print] doi: 10.1007/s12013-
009-9067-2
Zachowski A, Devaux PF (1990) Transmembrane movements of lipids. Experientia 46:644656
Zheng J, Shen W, He DZ, Long KB, Madison LD, Dallos P (2000) Prestin is the motor protein of
cochlear outer hair cells. Nature 405:149155
Part III
Mechanosensitivity
and Mechanotransduction
in Vascular Cells
Chapter 8
Cellular and Molecular Effects of Mechanical
Stretch on Vascular Cells
Kou-Gi Shyu
8.1 Introduction
In addition to interacting with blood constituents and circulating cells, the vascular
endothelium is exposed to a distinct mechanical environment consisting of haemo-
dynamic shear stress and mechanical stretch from blood pressure. Disturbance
of normal haemodynamic load can contribute to cardiovascular diseases includ-
ing hypertension, intimal hyperplasia, vascular restenosis, and atherosclerosis
(Cummins et al., 2007). Laminar shear stress, the frictional force created by the
flowing blood, exerts a variety of cellular and molecular effects on endothelial
structure and functions. The molecular mechanism of shear stress on ECs has
been extensively reviewed (Cummins et al., 2007; Kakisis et al., 2004; Lehoux,
2006; Lehoux et al., 2006; Li et al., 2005) In addition to anti-apoptotic and anti-
atherosclerotic effects of laminar shear stress on ECs, laminar shear stress has a
profound impact on endothelial metabolism and can alter gene expression lead-
ing to changes in the endothelial phenotype and vessel wall homeostasis. The
genes regulated by shear stress can modulate several endothelial functions including
vessel diameter, cell proliferation, migration and angiogenesis, cell-cell commu-
nication, coagulation and fibrinolysis, anti-inflammation, and immune modulation
(Cummins et al., 2007; Kakisis et al., 2004; Lehoux, 2006; Li et al., 2005). In this
book chapter, additional novel findings about the impact of shear stress on ECs are
discussed.
The vasculoprotective effects of shear stress on ECs have been documented more
than one decade ago (Traub and Berk, 1998). Novel findings extending this aspect
have been reported recently. Bone morphogenetic protein (BMP) is a TGF- family
member cytokine that exerts proinflammatory effect on the endothelium and plays
a role in atherogenesis. BMP is upregulated at athero-prone regions in blood ves-
sels and may contribute to vascular calcification and development of atherosclerotic
plaques (Csiszar et al., 2006). Csiszar et al. have reported that laminar shear stress
activates cAMP and cAMP-dependent protein kinase and downregulates the BMP-4
expression in coronary artery ECs (Csiszar et al., 2007). This finding supports the
196 K.-G. Shyu
Fig. 8.1 Schematic summary of vasculoprotective effects of laminar shear stress on endothelial
cells. A diagram summarizing the laminar shear stress-induced mechanosensing and intracellu-
lar signaling that lead to the modulation of gene expression and cellular function, resulting in
vasculoprotection, as discussed in the text
vascular ECs adhesion, migration and survival, and angiogenesis. The shear-induced
cAMP-dependent protein kinase A-dependent 4 integrin phosphorylation may be
an important regulatory step in endothelial function during vascular development
and remodeling (Goldfinger et al., 2008). The reorientation of ECs after cyclic strain
has been related to activation of transient receptor potential vanilloid 4 (TRPV4)
ion channels (Thodeti et al., 2009). TRPV4 is one of the stretch-activated channels.
Cyclic stretch of capillary ECs activates mechanosensitive TRPV4 ion channels that
stimulate PI3K-dependent activation and binding of additional 1 integrin receptors,
which promotes cytoskeletal remodeling and cell reorientation. Simmers et al. have
also demonstrated that shear stress-induced directed migratory polarity is modulated
by exogenous growth factors and dependent on Par6 activity, a major downstream
effector of Cdc-42-induced polarity, and shear stress direction (Simmers et al.,
2007). Shear stress regulates ECs bulk migratory characteristics as well as mor-
phology. Lee et al. reported adaptation of ECs shape in arteries under axial stretch
using an organ culture model. They demonstrated that ECs were initially elon-
gated by the axial stretch but eventually adapted to the axial stretch, regaining their
normal shape (Lee et al., 2008). The capillary ECs reorientation after mechani-
cal stress is important for angiogenesis. These data indicate that mechanical stress
regulates ECs bulk migratory characteristics as well as morphology. These mechan-
ical sensitive ion channels may represent new targets for therapeutic intervention in
angiogenesis-dependent disease.
STAT3 binding to the eNOS promoter. Laminar shear stress up-regulates antioxidant
genes and activates the transcriptional factor, NF-E2-related factor-2, which is a
major transcriptional factor for EC redox homeostasis (Dai et al., 2007). Laminar
shear stress alters the functions of NF-B by inhibiting its capacity to induce pro-
inflammatory molecules such as VCAM-1, e-selectin, interleukin-8, and ICAM-1
and simultaneously enhancing the induction of NF-B-dependent cytoprotective
transcripts such as manganese superoxide dismutase, Bcl-2 and A1 (Patridge et al.,
2007). High glucose and arachidonic acid synergistically decrease cell viability
and increase glutathione oxidation and lipid peroxidation in human umbilical ECs,
while laminar shear stress attenuates the oxidative stress induced by high glucose
and arachidonic acid (Mun et al., 2008). The antioxidant effect of laminar shear
stress could be attributed to increased biosynthesis of tetrahydrobiopterin and glu-
tathione. These findings strongly support the crucial role of laminar shear stress as
anti-inflammatory and anti-oxidative force. The mechanism and signal pathways for
anti-inflammatory and anti-oxidative effects of laminar shear stress on endothelial
cells are summarized in Fig. 8.2.
High wall shear stress haemodynamics mediate adaptive outward remodeling and
cerebral aneurysm development. High wall shear stress stimulates ECs proliferation
and suppresses apoptosis (Metaxa et al., 2008). The stimulation of EC proliferation
by high wall shear stress is dependent on NO signaling pathways. The mechanism
by which ECs sense high wall shear stress is through stretch-activated calcium chan-
nels on the luminal surface of the endothelium (Brakemeier et al., 2002). High wall
shear stress by stimulation of ECs proliferation plays an important role in maintain-
ing continuity of the endothelium in regions where the vessel wall is subjected to
high levels of haemodynamic stress.
Fig. 8.2 Schematic summary of anti-inflammatory and anti-oxidative effects of laminar shear
stress on endothelial cells. A diagram summarizing the laminar shear stress-induced mechanosen-
sing and intracellular signaling that lead to the modulation of gene expression and cellular function,
resulting in anti-inflammatory and anti-oxidative stress, as discussed in the text
200 K.-G. Shyu
Fig. 8.4 Schematic summary of atherogenic effects of low shear stress on endothelial cells. A
diagram summarizing the low shear stress-induced mechanosensing and intracellular signaling
that lead to the modulation of gene expression and cellular function, resulting in atherogenesis, as
discussed in the text
Endothelial progenitor cells (EPCs), mobilized from bone marrow, contribute post-
natal neovascularization. EPCs are exposed to shear stress generated by flowing
blood and tissue fluid flow during the process of EPC incorporation into tissues and
neovascularization (Obi et al., 2009). EPCs appear to be responsible to shear stress
and the vasculogenic activities of EPCs may be modulated by shear stress. The effect
of shear stress on progenitor cell fate has been reviewed by Stolberg and McCloskey
(Stolberg and McCloskey, 2009). Shear stress can accelerate the proliferation, dif-
ferentiation, and capillary-like tube formation of EPCs (Yamamoto et al., 2003).
The endothelial differentiation of EPCs by shear stress has been demonstrated by
activation of Akt (Ye et al., 2008) and by increasing ephrinB2 expression and Sp1
activation (Obi et al., 2009). Shear stress increases the gene expression of arterial
endothelial markers in EPCs, such as ephrin B2, Notch1/3, Hey1/2 and ALK1 but
decreases the gene expression of venous endothelial markers such as EphB4 and
NRP2 (Obi et al., 2009). Shear stress within physiological range can enhance tis-
sue type plasminogen activator and prostaglandin I2 secretion (Yang et al., 2007)
and decrease plasminogen activator inhibitor-1 (Yang et al., 2006) in human EPCs,
which improves the antithrombogenic potential of human EPCs. Shear stress has
also been demonstrated to increase Cu/Zn superoxide dismutase activity and NO
production, as well as superoxide dismutase and eNOS mRNA expression (Tao
8 Cellular and Molecular Effects of Mechanical Stretch on Vascular Cells 203
et al., 2007). Shear stress also induces endothelial differentiation in a mouse mes-
enchymal progenitor cell line (Wang et al., 2005). Wang et al. reported that TGF-1
was down-regulated in mesenchymal progenitor cell line by shear stress (Wang
et al., 2008). The negative regulation of the TGF-1 system may be involved in
shear-induced endothelial cell differentiation in the mouse mesenchymal progenitor
cell line.
Arterial SMCs are aligned primarily in the circumferential direction in the media
of artery. The mechanical stretch from pulsatile blood flow is one of the key fac-
tors in regulating vascular remodeling (Taber, 1998). Mechanical environment in
vivo modulates the distinct patterns of VSMC orientation in the arterial wall. The
predominant mechanical force influencing VSMCs structural organization and sig-
naling is cyclic stretch (Halka et al., 2008). There are at least three elements
included in the cyclic stretch: magnitude, frequency, and duration. Cultured VSMCs
in vitro can be induced to reorient to a uniform alignment almost perpendicular
to stretch vector alignment by mechanical stretch (Lehoux et al., 2000; Standly
et al., 2002). The response of cell reorientation depends on the stretching mag-
nitude and frequency (Dartsch et al., 1986; Liu et al., 2008; Wang et al., 1995).
The signaling pathways involved in the stretch-induced VSMCs alignment include
p38 mitogen-activated protein (MAP) kinase (Liu et al., 2008), nitric oxide and
reactive oxygen species (Chen et al., 2003; Standly et al., 2002). The mechanosen-
sor and outside-in signal of integrin-1 is also involved in the stretch-induced
VSMCs alignment (Chen et al., 2003). An intact cyctoskeleton is important for
the stretch-induced VSMCs alignment. Destroying the actin filament system by
cytochalasin D inhibits the effect of stretch-induced alignment (Liu et al., 2008).
H1-calponin, a family of actin-associated protein, is a specifically differentiated
204 K.-G. Shyu
marker in SMCs. Cyclic strain can upregulate the expression of Rac and down-
regulate its negative regulator Rho-GD dissociation inhibitor alpha in a nonlinear
frequency-dependent pattern, then cause the activation of p38 pathway followed
by increasing expression of h1-calponon, which marked VSMC differentiation (Qu
et al., 2008). Rho-GD dissociation inhibitor alpha, a member of Rho-GD disso-
ciation inhibitor, can negatively regulate the activities of small G proteins of the
Rho family by shutting off their GDP/GTP cycling and cytosol/membrane translo-
cation. Not only VSMCs alignment is affected by stretch frequency, but also the
phenotype of VSMCs. Cyclic strain increases smooth muscle and decreases non-
muscle myosin expression in VSMCs (Reusch et al., 1996). Mechanical stretch
increases both smooth muscle -actin protein expression and promoter activity
(Tock et al., 2003). The induction of smooth muscle -actin is mediated by acti-
vation of JNK and p38 MAP kinase pathways. Mechanical stretch could promote
a frequency-dependent redifferentiation of synthetic VSMCs in vitro, mediated at
least in part by the activation of p38 MAP kinase (Qu et al., 2007). Mechanical
stretch modulates cell shape, cytoplasmic organization and intracellular processes
leading to migration, proliferation, or contraction. Rho and intact actin filaments
play an important role in mechanical stretch-induced extracellular signal-regulated
kinase (ERK) activation and cell growth (Numaguchi et al., 1999). RhoA signal-
ing plays a major role in the serum responsive factor (SRF)-dependent regulation
of SMC differentiation marker gene expression (Mack et al., 2001). The primary
genes encoding SMCs contractile proteins are regulated by the stretch-induced
RhoA pathway and associated transcription factors, most importantly the SRF
(Hellstrand and Albinsson, 2005). In vitro, RhoA enhances actin polymerization and
stimulates the SRF homodimer binding to their CArG boxes (Mack et al., 2001).
SRF binds to the serum response element region containing the 10-bp CArG-box
sequence facilitating activation of this motif alone or as a macromolecule bound to
myocardin, its specific co-activator (Halka et al., 2008). Myocardin increases the
promoter activity of the CArG-dependent VSMCs contractile markers. Stretch of
the vascular wall can stimulate increased actin polymerization, activating synthesis
of smooth muscle-specific proteins via Rho-associated kinase and cofilin down-
stream of Rho (Albinsson et al., 2004). Rho/Rho kinase, p44/p42 MAP kinase and
phosphatidylinositol-3 (PI-3) kinase pathways are all involved in the stretch-induced
human saphenous vein SMC proliferation and inhibition of either of them prevents
stretch-induced SMCs proliferation (Kozai et al., 2005). The effect of mechani-
cal stretch on SMCs phenotype has been reviewed by Halka et al. (Halka et al.,
2008).
(Liu, 1999). In a vein graft model of SMC study, vortex blood flow induces VSMCs
migration and neointimal hyperplasia in control vein graft, whereas reduction of
vortex blood flow in the vein graft strongly suppresses the migration and hyperpla-
sia (Goldman et al., 2007). In this model, VSMCs migration is regulated through the
mediation of ERK1/2 and myosin light chain kinase. In vitro, mechanical stretch of
arterial VSMCs translocates PKC from membrane to cytoskeleton and increases
the migration of VSMCs (Li et al., 2003). In our laboratory, we could also demon-
strate that mechanical stretch increased migration of VSMCs (Shyu et al., 2005).
The increased migration of VSMCs by mechanical stretch involves p38 MAP kinase
and transforming growth factor-1 (TGF-1) (Li et al., 2003). Inhibition of p38
MAP kianse and TGF-1 activity decreased the migration activity. Qi et al. used
proteomic analysis to demonstrate that low shear stress-induced VSMC migra-
tion and apoptosis are mediated by a down-regulation of Rho-GDP dissociation
inhibitor alpha (Qi et al., 2008). The effect of Rho-GDP dissociation inhibitor
alpha on VSMCs migration is dependent on the PI3K/Akt signal transduction
pathway.
The effect of mechanical stretch on survival and apoptosis of VSMCs has been
extensively reviewed by Kakisis et al. (2004) and Haga et al. (2007). In this chapter, I
would like to extend these studies by data published recently. In a mouse SMCs cul-
tured model, Cheng et al. have reported that mechanical stretch prevents apoptosis
of VSMCs in response to oxidized low-density lipoprotein (Cheng et al., 2007). The
mechanism of increased survival of VSMCs induced by mechanical stretch includes
V3 integrin expression, stabilization of PINCH-1, a survival protein that is linked
with integrin and the cytoskeleton (Xu et al., 2005), and remodeling of cytoskeleton.
Small interfering RNA (siRNA) against integrin 3 as well as VSMC isolated from
integrin 3 knockout mice abolishes the antiapoptotic effect of mechanical stretch.
Down-regulation of PINCH-1 by siRNA enhanced the ability of oxidized low den-
sity lioporotein to cause apoptosis of VSMCs, while up-regulation of integrin 3
stabilizes PINCH-1 and protects VSMCs from apoptosis. Disruption of cytoskeleton
also abolishes the antiapoptotic effect of stretch. In cultured rabbit VSMCs, mechan-
ical stretch also stimulates SMCs growth and hypertrophy (Richard et al., 2007).
The increased SMCs survival by mechanical stretch is induced by nuclear protein
import and nuclear pore protein expression that is mediated via MAP kinase. In
cultured bovine pulmonary artery VSMCs, mechanical stretch stimulates prolifera-
tion of VSMCs and RhoA is essential for stretch-induced VSMCs proliferation (Liu
et al., 2007). Blocking Rho completely inhibits the proliferation of VSMCs induced
by stretch. In cultured vein VSMC, mechanical stretch stimulates proliferation of
venous SMCs (Cheng and Du, 2007). The proliferation of venous SMCs induced
by mechanical stretch is mediated by activation of insulin-like growth factor-1
(IGF-1) and IGF-1 receptor. When IGF-1 receptor is knocked out, the mechanical
206 K.-G. Shyu
Fig. 8.6 Schematic summary of various effects of mechanical stretch on VSMCs. A diagram
summarizing mechanical stretch-induced mechanosensing and intracellular signaling that lead to
the modulation of gene expression and cellular function, resulting in migration, differentiation,
proliferation, and survival of VSMCs, as discussed in the text
under mechanical stretch needs further investigation. The mechanism and signal
pathways for effects of mechanical stretch on migration, proliferation, and survival
in VSMCs are summarized in Fig. 8.6.
208 K.-G. Shyu
up-regulation of endothelial HSPG using in vitro and ex vivo studies. Net arterial
remodeling to haemodynamic forces is controlled by a dynamic interplay between
growth stimulatory signals from VSMCs and growth inhibitory signals from ECs.
Fig. 8.7 Schematic summary of different effects of mechanical stretch on VSMCs. A diagram
summarizing mechanical stretch-induced mechanosensing and intracellular signaling that lead
to the modulation of gene expression and cellular function, resulting in vascular remodeling,
autocrine, and paracrine effects in VSMCs, as discussed in the text
210 K.-G. Shyu
under mechanical stretch (Cheng et al., 2008). The intracellular signaling path-
ways of autocrine and paracrine effect of TGF- under mechanical stress involve
ROS and NFB. The intracellular signaling pathways of stretch-induced GADD153
expression involve JNK and AP-1 pathway. AngII and TGF-1 play autocrine and
paracrine action on discoidin domain receptor 2 (DDR2) expressions in VSMC
under mechanical stretch (Shyu et al., 2005). The stretch-induced DDR2 is mediated
by p38 MAP kinase and Myc-Max pathway. DDR2 can regulate cell proliferation
and extracellular matrix remodeling mediated by MMP activities. The intracellu-
lar signaling pathways of stretch-induced DDR2 involve p38 MAP kinase and Myc
pathway. The mechanism and signaling pathways for effects of mechanical stretch
on vascular remodeling, autocrine and paracrine effects in VSMCs are summarized
in Fig. 8.7.
In an ECs-SMCs co-culture system, SMCs secrete interleukin-1 and
interleukin-6 after application of shear stress resulting in inhibition of E-
selectin expression (Chiu et al., 2007). In this model, SMC induces endothelial
E-selectin expression, while shear stress inhibits the SMC-induced E-selectin
expression via the inhibition in SMCs activation of interlukin-1 receptor associ-
ated kinase/glycoprotein-130, JNK/p38 MAP kinase, and NF-K (Haga et al., 2007;
Lehoux, 2006).
References
Albinsson S, Hellstrand P (2007) Integration of signal pathways for stretch-dependent growth and
differentiation in vascular smooth muscle cell. Am J Physiol Cell Physiol 293:C772C782
Albinsson S, Nordstrom I, Hellstrand P (2004) Stretch of the vascular wall induces smooth muscle
differentiation by promoting actin polymerization. J Biol Chem 279:3484934855
Andersson M, Karisson L, Svensson PA, Ulfhammer E, Ekman M, Jerns M, Carlsson LM, Jern
S (2005) Differential global gene expression response patterns of human endothelium exposed
to shear stress and intraluminal pressure. J Vasc Res 42:441452
Baker AB, Jonas M, Nugent MA, Iozzo RV, Edelman ER (2008) Endothelial cells provide feed-
back control for vascular remodeling through a echanosensitive autocrine TGF-beta signaling
pathway. Circ Res 103:289297
Brakemeier S, Eichler I, Hopp H, Kohler R, Hoyer J (2002) Up-regulation of endothelial stretch-
activated cation channels by fluid shear stress. Cardiovasc Res 53:209218
Brown TD (2000) Techniques for mechanical stimulation of cells in vitro: a review. J Biomech
33:314
Campos LCG, Miyakawa AA, Barauna VG, Cardoso L, Borin TF, Dallan LA, Krieger JE (2009)
Induction of CRP3/MLP expression during vein arterialization is dependent on stretch rather
than shear stress. Cardiovasc Res 83:140147
Chang H, Shyu KG, Wang BW, Kuan P (2003) Regulation of hypoxia-inducible factor-1 by
cyclical mechanical stretch in rat vascular smooth muscle cells. Clin Sci 105:447456
Chen Q, Li W, Quan Z, Sumpio BE (2003) Modulation of vascular smooth muscle cell alignment
by cyclic strain is dependent on reactive oxygen species and p38 mitogen-activated protein
kinase. J Vasc Surg 37:660668
Chen Z, Peng IC, Sun W, Su MI, Hsu PH, Fu Y, Zhu Y, DeFea K, Pan S, Tsai MD, Shyy YJ (2009)
AMP-activated protein kinase functionally phosphorylates endothelial nitric oxide synthase
Ser633. Circ Res 104:496505
Cheng J, Du J (2007) Mechanical stretch stimulates proliferation of venous smooth muscle cells
through activation of the insulin-like growth factor-1 receptor. Arterioscler Thromb Vasc Res
27:17441751
Cheng J, Zhang J, Merched A, Zhang L, Zhang P, Truong L, Boriek AM, Du J (2007) Mechanical
stretch inhibits oxidized low density lipoprotein-induced apoptosis in vascular smooth mus-
cle cells by upregulating integrin V3 and stabilization of PINCH-1. J Biol Chem 282:
3426834275
Cheng M, Wu J, Li Y, Nie Y, Chen H (2008) Activation of MAPK participates in low shear stress-
induced IL-8 gene expression in endothelial cells. Clin Biomech 23:S96S103
Cheng WP, Hung HF, Wang BW, Shyu KG (2008) The molecular regulation of GADD153 in
apoptosis of cultured vascular smooth muscle cells by cyclic mechanical stretch. Cardiovasc
Res 77:551559
Chien S (2007) Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell. Am
J Physiol Heart Circ Physiol 292:H1209H1224
Chien S. Li S, Shiu YT, Li YS (2005) Molecular basis of mechanical modulation of endothelial
cell migration. Front Biosci 10:9852000
Chiu JJ, Chen LJ, Lee CI, Lee PL, Lee DY, Tsai MC, Lin CW, Usami S, Chien S (2007)
Mechanisms of induction of endothelial cell E-selectin expression by smooth muscle cell and
its inhibition by shear stress. Blood 110:519528
Csiszar A, Ahmad M, Smith KE, Labinskyy N, Gao Q, Kaley G, Edwards JG, Wolin MS, Ungvari
G (2006) Bone morphogenetic protein-2 induces proinflammatory endothelial phenotype. Am
J Pathol 168:629638
Csiszar A, Labinskyy N, Smith KE, Rivera A, Bakker EN, Jo H, Gardner J, Orosz Z, Ungvari
Z (2007) Down-regulation of bone morphogenetic protein4 expression in coronary arterial
endothelial cells: role of shear stress and the cAMP/protein kinase A pathway. Arterioscler
Thromb Vasc Biol 27:776782
212 K.-G. Shyu
Cummins PM, Sweeney NO, Killeen MT, Birney YA, Redmond EM, Cahill PA (2007) Cyclic
strain-mediated matrix metalloproteinase regulation within the vascular endothelium: a force
to be reckoned with. Am J Physiol Heart Circ Physiol 292:H28H42
Dai G, Vaughn S, Zhang Y, Wang ET, Garcia-Cardena G, Gimbrone MA (2007) Biomechanical
forces in atherosclerosis-resistant vascular regions regulate endothelial redox balance via
phosphoinositiol 3-kinase/Akt-dependnet activation of Nrf2. Circ Res 101:723733
Dartsch P, Hammerle CH, Betz E (1986) Orientation of cultured arterial smooth muscle cells
growing on cyclically stretched substrate. Acta Anat 125:15431552
Dekker RJ, van Thienen JV, Rohlena J, de Jager SC, Elderkamp YW, Seppen J, de Vries CJ,
Biessen EA, van Berkel TJ, Pannekoek H, Horrevoets AJ (2005) Endothelial KLF2 links local
arterial stress levels to the expression of vascular tone-regulating genes. Am J Pathol 167:
609618
Di Francesco L, Totani L, Dovizio M, Piccoli A, Di Francesco A, Salvatore T, Pandolfi A,
Evangelista V, Dercho RA, Seta F, Patrignani P (2009) Induction of prostacyclin by steady
laminar shear stress suppresses tumor necrosis factor- biosynthesis via heme oxygenase-1 in
human endothelial cells. Circ Res 104:506513
Fitzgerald TN, Shepherd BR, Asada H, Teso D, Muto A, Fancher T, Pimiento J, Maloney SP,
Dardik A (2008) Laminar shear stress stimulates vascular smooth muscle cell apoptosis via the
Akt pathway. J Cell Physiol 216:389395
Goldfinger LE, Tzima E, Stockton R, Kiosses WB, Kinbara K, Tkachenko E, Gutierrez E,
Groisman A, Nguyen P, Chien S, Ginsberg MH (2008) Localized alpha4 integrin phospho-
rylation directs shear stress-induced endothelial cell alignment. Circ Res 103:177185
Goldman J, Zhong L, Liu SQ (2007) Negative regulation of vascular smooth muscle cell migration
by blood shear stress. Am J Physiol Heart Circ Physiol 292:H928H938
Gunningham KS, Gotlieb A (2005) The role of shear stress in the pathogenesis of atherosclerosis.
Lab Invest 85:923
Haga JH, Li YJ, Chien S (2007) Molecular basis of the effects of mechanical stretch on vascular
smooth muscle cells. J Biomech 40:947960
Haga JH, Kaunas R, Radeff-Huang J, Weems JM, Estrada KD, Chien S, Brown JH, Seasholtz
TM (2008) Pulsatile equibiaxial stretch inhibits thrombin-induced RhoA and NF-B activation.
Biochem Biophys Res Commun 372:216220
Hahn C, Orr W, Sanders JM, Jhaveri KA, Schwartz MA (2009) The subendothelial extracellular
matrix modulates JNK activation by flow. Circ Res 104:9951003
Halka AT, Turner NJ, Carter A, Ghosh J, Murphy MO, Kirton JP, Kielty CM, Walker MG (2008)
The effects of stretch on vascular smooth muscle cells phenotype in vitro. Cardiovasc Pathol
17:98102
Harrison DG, Widder J, Grumbach I, Chen W, Webber M, Searles C (2006) Endothelial
mechanotransduction, nitric oxide and vascular inflammation. J Int Med 259:351363
Healey JS, Baranchuk A, Crystal K, Morillo CA, Garfinkle M, Yusuf S, Connolly SJ (2005)
Prevention of atrial fibrillation with angiotensin-converting enzyme inhibitors and angiotensin
receptor blockers: a meta-analysis. J Am Coll Cardiol 45:18321839
Hellstrand P, Albinsson S (2005) Stretch-dependent growth and differentiation in vascular smooth
muscle: role of the actin cytoskeleton. Can J Physiol Pharmacol 83:869875
Iizuka K, Machida T, Kawaguchi H, Hirafuji M (2008) Pulsatile mechanical pressure promotes
angiotensin-converting enzyme expression in aortic smooth muscle cells. Cardiovasc Drugs
Ther 22:383390
Kinderlerer AR, Ali F, Johns M, Lidington EA, Leung V, Boyle JJ, Hamdulay SS, Evans PC,
Haskard DO, Mason JC (2008) KLF-dependent, shear stress-induced expression of CD59:
a novel cytoprotective mechanism against complement-mediated injury in the vasculature. J
Biol Chem 283:1463614644
Julien MA, Haller CA, Wang P, Wen J, Chaikof EL (2007a) Mechanical strain induces a per-
sistent upregulation of syndecan-1 expression in smooth muscle cells. J Cell Physiol 211:
167173
8 Cellular and Molecular Effects of Mechanical Stretch on Vascular Cells 213
Julien MA, Wang P, Haller CA, Wen J, Chaikof EL (2007b) Mechanical strain regulates syndecan-4
expression and shedding in smooth muscle cells through differential activation of MAP kinase
signaling pathways. Am J Physiol Cell Physiol 292:C517C525
Kakisis JD, Liapis CD, Sumpio BE (2004) Effects of cyclic strain on vascular cells. Endothelium
11:1728
Kouri FM, Eickelberg O (2006) Transforming growth factor-, a novel mediator of strain-induced
vascular remodeling. Circ Res 99:348350
Korff T, Aufgebauer K, Hecker M (2007) Cyclical stretch controls the expression of CD40
in endothelial cells by changing their transforming growth factor-1 response. Circulation
116:22882297
Kozai T, Eto M, Yang Z, Shimokawa H, Luscher TF (2005) Statins prevent pulsatile stretch-
induced proliferation of human saphenous vein smooth muscle cells via inhibition of
Rho/Rho-kinase pathway. Cardiovasc Res 68:475482
Lee YU, Drury-Stewart D, Vito RP, Han HC (2008) Morphologic adaptation of arterial endothelial
cells to longitudinal stretch in organ culture. J Biomech 41:32743277
Lehoux S (2006) Redox signaling in vascular responses to shear and stretch. Cardiovasc Res
71:269279
Lehoux S, Castier Y, Tedgui A (2006) Molecular mechanism of the vascular response to
haemodynamic forces. J Int Med 259:381392
Lehoux S, Esposito B, Merval R, Loufrani L, Tedgui A (2000) Pulsatile stretch-induced extracel-
lular signal-regulated kinase 1/2 activation in organ culture of rabbit aorta involves reactive
oxygen species. Arterioscler Thromb Vasc Biol 20:23662372
Lemarie CA, Tharaux P, Esposito B, Tedgui A, Lehoux S (2006) Transforming growth factor-
mediates nuclear factor B activation in strained arteries. Circ Res 99:434441
Li C, Wernig F, Leitges M, Hu Y, Xu Q (2003) Mechanical stress-activated PKC regulates smooth
muscle cell migration. FASEB J 17:21062108
Li C, Xu Q (2007) Mechanical stress-initiated signal transduction in vascular smooth muscle cells
in vitro and in vivo. Cell Signal 19:881891
Li L, Takake RJ, Natarajan K, Taba Y, Garin G, Tai C, Leung E, Surapisitchat J, Yoshizumi
M, Yan C, Abe J, Berk BC (2008) Fluid shear stress inhibits TNF-mediated JNK
activation via MRK5-BMK1 in endothelial cells. Biochem Biophys Res Commun 370:
159163
Li YSJ, Haga JH, Chien S (2005) Molecular basis of the effects of shear stress on vascular
endothelial cells. J Biomech 38:19491971
Lin Z, Kumar A, SenBanerjee S, Staniszewski K, Parmar K, Vaughan DE, Gimbrone MA Jr,
Balasubramanian V, Garca-Cardea G, Jain MK (2005) Kruppel-like factor 2 (KLF2) regulates
endothelial thrombotic function. Circ Res 96:e48e57
Liu B, Qu MJ, Qin KR, Li H, Li H, Li ZK, Shen BR, Jiang ZL (2008) Role of cyclic strain
frequency in regulating the alignment of vascular smooth muscle cells in vitro. Biophys J
94:14971507
Liu SQ (1999) Focal expression of angiotensin II type 1 receptor and smooth muscle cell prolif-
eration in the neointima of experimental vein grafts: relation to eddy blood flow. Arterioscler
Thromb Vasc Biol 19:26302639
Liu WF, Nelson CM, Tan JL, Chen CS (2007) Cadherins, RhoA, and Rac1 are differentially
required for stretch-mediated proliferation in endothelial versus smooth muscle cells. Circ Res
101:e44e52
Mack CP, Somlyo AV, Hautmann M, Somlyo AP, Owens GK (2001) Smooth muscle differentiation
marker gene expression is regulated by RhoA-mediated actin polymerization. J Biol Chem
276:341347
McCue S, Dajnowiec D, Xu F, Zhang M, Jackson MR, Langille BL (2006) Shear stress regulates
forward and reverse planar cell polarity of vascular endothelium in vivo and in vitro. Circ Res
98:939946
214 K.-G. Shyu
Metaxa E, Meng H, Kaluvala SR, Szymanski MP, Paluch RA, Kolega J (2008) Nitric oxide-
dependent stimulation of endothelial cell proliferation by sustained high flow. Am J Physiol
Heart Circ Physiol 295:H736H742
Mowbray AL, Kang DH, Rhee SG, Kang SW, Jo H (2008) Laminar shear stress up-regulates
peroxiredoxins (PRX) in endothelial cells: PRX1 as a mechanosensitive antioxidant. Biol Chem
283:16221627
Mun GI, An SM, Park H, Jo H, Boo YC (2008) Laminar shear stress inhibits lipid oxidation
induced by high glucose plus arachidonic acid in endothelial cells. Am J Physiol Heart Circ
Physiol 295:H1966H1973
Nikolovski J, Kim B, Mooney DJ (2003) Cyclic strain inhibits switching of smooth muscle cells
to an osteoblast-like phenotype. FASEB J 17:455457
Nishimura K, Li W, Hoshino Y, Kadohama T, Asada H, Ohgi S, Sumpio BE (2006) Role of AKT
in cyclic strain-induced endothelial cell proliferation and survival. Am J Physiol. Cell Physiol
290:C812C821
Ntambi JM, Miyazaki M (2004) Regulation of stearoyl-CoA desaturases and role in metabolism.
Prog Lipid Res 43:91104
Numaguchi K, Eguchi S, Yamakawa T, Motley ED, Inagami T (1999) Mechanotransduction of
rat aortic vascular smooth muscle cells require RhoA and intact actin filaments. Circ Res 85:
511
Obi S, Yamamoto K, Shimizu N, Kumagaya S, Masumura T, Sokabe T, Asahara T, Ando J (2009)
Fluid shear stress induces arterial differentiation of endothelial progenitor cells. J Appl Physiol
106:203211
OrrAW, Hahn C, Blackman BR, Schwartz MA (2008) p21-activated kinase signaling regulates
oxidant-dependent NF-B activation by flow. Circ Res 103:671679
Orr AW, Helmke BP. (2006) Mechanisms of mechanotransduction. Dev Cell 10:1120
Oyadomari S, Mori M (2004) Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell
Death Differ 11:381389
Patridge J, Carlsen H, Enesa K, Chaudhury H, Zakkar M, Luong L, Kinderlerer A, Johns M,
Blomhoff R, Mason JC, Haskard DO, Evans PC (2007) Laminar shear stress acts as a switch
to regulate divergent functions of NF-B in endothelial cells. FASEB J 21:35533561
Pyle AL, Atkinson JB, Pozzi A, Reese J, Eckes B, Davidson JM, Crimmins DL, Young PP
(2008) Regulation of the atheroma-enriched protein, SPRR3, in vascular smooth muscle cells
through cyclic strain is dependent on integrin 11/collagen interaction. Am J Pathol 173:
15771588
Qi YX, Qu MJ, Long DK, Liu B, Yao QP, Chien S, Jiang ZL (2008) Rho-GDP dissociation
inhibitor alpha down-regulated by low shear stress promotes vascular smooth muscle cell
migration and apoptosis: a proteomic analysis. Cardiovasc Res 80:114122
Qin X, Tian J, Zhang P, Fan Y, Chen L, Guan Y, Fu Y, Zhu Y, Chien S, Wang N (2007) Laminar
shear stress up-regulates the expression of stesroyl-CoA desaturase-1 in vascular endothelial
cells. Cardiovasc Res 74:506514
Qu MJ, Liu B, Wang HQ, Yan ZQ, Shen BR, Jiang ZL (2007) Frequency-dependent phenotype
modulation of vascular smooth muscle cells under cyclic mechanical strain. J Vasc Res 44:
345353
Qu MJ, Liu B, Qi YX, Jiang ZL (2008) Role of Rac and Rho-GDI alpha in the frequency-dependent
expression of h1-calponin in vascular smooth muscle cells under cyclic mechanical strain. Ann
Biomed Engin 36:14811488
Reinhart-King C, Fujiwara K, Berk BC (2008) Physiologic stress-mediated signaling in the
endothelium. Method Enzymol 443:2544
Reusch P, Wagdy H, Reusch R, Wilson E, Ives HE (1996) Mechanical strain increases smooth
muscle and decreases nonmuscle myosin expression in rat vascular smooth muscle cells. Circ
Res 79:10461053
Richard MN, Deniset JF, Kneesh AL, Blackwood D, Pierce GN (2007) Mechanical stretching
stimulates smooth muscle cell growth, nuclear protein import, and nuclear pore expression
through mitogen-activated protein kinase activation. J Biol Chem 282:2308123088
8 Cellular and Molecular Effects of Mechanical Stretch on Vascular Cells 215
Zhang Y, Lee TS, Kolb EM, Sun K, Lu X, Sladek FM, Kassab GS, Garland T Jr, Shyy JY (2006)
AMP-activated protein kinase is involved in endothelial NO synthase activation in response to
shear stress. Arterioscler Thromb Vasc Biol 26:12811287
Zhu M, Fu Y, Hou Y, Wang N, Guan Y, Tang C, Shyy JY, Zhu Y (2008) Laminar shear stress
regulates liver X receptor in vascular endothelial cells. Arterioscler Thromb Vasc Biol 28:
527533
Chapter 9
Role of Proteoglycans in Vascular
Mechanotransduction
Aaron B. Baker
9.1 Introduction
in tissue metabolism the system must adapt to seek a new level of homeostasis. For
example, hypertension is the most common clinical finding in the United States and
is associated with increased risk of stroke, atherosclerosis and myocardial infarction
(Whisnant, 1996; Rosendorff, 2007). While the fundamental mechanisms causing
essential hypertension remain elusive, the consequences to the vascular system are
profound and include arterial stiffening and vascular smooth muscle cell hypertro-
phy (van den Akker et al., 2009). Fundamental to the processes vascular adaptation
are the mechanosensing mechanisms of the vascular cells themselves. Numerous
in-vitro studies have been done examining vascular cell mechanotransduction in
response to alterations in shear stress or mechanical stretch (Davies, 2009). Vascular
cells respond to mechanical forces and are able to translate them into biochem-
ical events including gene expression and protein secretion. Various mechanisms
have been proposed to explain vascular mechanotransduction at the cellular and
molecular levels. Vascular cells have a variety of cell surface receptors including the
integrins that allow the artery detect and respond to mechanical forces (Hahn and
Schwartz, 2009). In addition, studies have implicated signaling mechanisms such as
receptor tyrosine kinases, mechanosensitive ion channels, and G proteins in cellu-
lar mechanotransduction (Li et al., 2005; Orr et al., 2006). Also implicated are the
cytoskeleton and other structural components that can transmit and modulate cellu-
lar tension through integrins/focal adhesion complexes that link to the extracellular
environment (Li et al., 2005; Orr et al., 2006).
This chapter focuses on the role of proteoglycans in vascular mechanotransduc-
tion. Proteoglycans are complex biomolecules that consist of a core protein that
is post-translationally modified to have one or more glycosaminoglycan chains.
Glycosaminoglycans are linear polymers of repeating disaccharides that is mod-
ified by multiple enzymes to have a complex sulfation and acetylation pattern
(Sasisekharan and Venkataraman, 2000). The addition of the glycosaminoglycan
chains occurs in the transgolgi and proceeds in a processive manner to create a het-
erogeneous structure and with intricate microdomains. These complex molecules
are known to play an important role in controlling vascular homeostasis and have
an emerging role in mechanobiology. We first describe the various types of pro-
teoglycans in the artery and then discuss how these molecules are involved in the
sensing and adaptation of the vascular system to mechanical stimuli. Finally, future
directions in examining the role of proteoglycans in vascular mechanobiology are
discussed.
9.2.1 Glycosaminoglycans
The most common types of glycosaminoglycans in the artery include heparan
sulfate, chondroitin sulfate and hyaluronic acid (Fig. 9.1). Of these four, only
hyaluronic acid is synthesized without being attached to a protein core. The other
9 Role of Proteoglycans in Vascular Mechanotransduction 221
Fig. 9.1 The chemical structure of glycosaminoglycans found in the vascular system. Shown in
include one potential structure for: (a) heparan sulfate, (b) chondroitin sulfate and (c) hyaluronan
9.2.2.1 Perlecan
In the vascular system heparan sulfate is found on several core proteins including
perlecan, glypicans, and syndecans. Perlecan is a large heparan sulfate proteogly-
can found in the basement membrane (Fig. 9.2a). In humans it is the product of the
HSPG2 gene with a molecular weight of 470 kDa and approximately 800 kDa after
222 A.B. Baker
Fig. 9.2 The predominant proteoglycans of the vascular system: (a) perlecan, (b) glypican-1 (c)
the syndecans (d) versican
rotary shadowing electron microscopy (Noonan et al., 1991). Perlecan has been
shown to have a role in angiogenesis, atherosclerosis and vascular injury (Nugent
et al., 2000; Iozzo and San Antonio, 2001). Heparin and heparan sulfate have also
been shown to inhibit neointimal proliferation in animal models of vascular injury
and disease (Clowes and Karnovsky, 1978; Guyton et al., 1980; Hoover et al., 1980;
Lindner et al., 1992; Edelman et al., 1993; Volker et al., 1995). This inhibition is
dependant on heparan sulfate proteoglycans but also requires a protein component
(Ettenson et al., 2000).
9.2.2.2 Glypicans
The glypicans are a family of cell surface heparan sulfate proteoglycans having a
globular structure with membrane association due to a glycosylphosphatidylinositol
(GPI) anchor (Fig. 9.2b) (Fransson, 2003). Predominantly heparan sulfate groups
are attached to serine residues in consensus sequences located between the central
domain and the C-terminal GPI-anchor. The GPI anchor can be cleaved phospholi-
pase C or D and leads to shedding of glypican from the cell surface. The GPI anchor
also localizes the protein to cholesterol and sphingolipid-rich lipid rafts within the
cell membrane (Fransson, 2003).
9.2.2.3 Syndecans
The syndecans are a family of transmembrane heparan sulfate proteoglycans found
on the cell surface and shed in a soluble form (Bernfield et al., 1999). Each synde-
can consists of an extracellular domain that contains glycosaminoglycan attachment
sites, a single pass transmembrane domain, and a short cytoplasmic domain with
multiple phosphorylation sites (Fig. 9.2c). The heparan sulfate and chondroitin sul-
fate glycosaminoglycan chains allow syndecans to interact with a large number of
ligands including FGF-2, VEGF, PDGF and TGF- (Tkachenko et al., 2005). The
interaction of syndecans with FGF-2 is probably the most characterized of these
interactions. Syndecans and the attached heparan sulfate proteoglycan are essen-
tial for effective binding and signaling of the FGF receptor (Nugent and Iozzo,
2000). On the cell surface syndecans stabilize the FGF-2/FGFR complex and are
essential for downstream signaling (Nugent and Iozzo, 2000). When shed from the
surface, syndecan-1 can inhibit FGF-2 induced cell proliferation (Mali et al., 1993).
However, physiologic degradation of syndecan by heparanase may lead to heparan
sulfate fragments that enhance FGF-2 signaling (Kato et al., 1998). Syndecan-4 has
also been shown to interact with FGF-2 and promote FGF-2 signaling (Volk et al.,
1999). Recent work has also found that syndecans can act independently of the FGF
receptor to act as a transmembrane receptor of FGF (Chua et al., 2004).
The transmembrane domain and two regions of the short cytoplasmic domain
of the syndecans are highly conserved. Conserved region 1 and 2 (C1 and C2)
are separated by a variable domain (V) that is specific to each syndecan type
(Fig. 9.2c). The C1 region has been found to bind src (Kinnunen et al., 1998),
ezrin (Granes et al., 2000), and tubulin (Brockstedt et al., 2002). The C2 region
224 A.B. Baker
contains a binding site for PDZ-domain proteins linking the syndecans to CASK,
syntenin and other molecules (Grootjans et al., 1997; Cohen et al., 1998; Hsueh
et al., 1998; Ethell et al., 2000; Gao et al., 2000). This region has been shown to
control trafficking of syndecan-1 in epithelial cells (Maday et al., 2008). The vari-
able region has differential functions in each syndecan type. The variable region
of syndecan-4, in particular, is involved in focal adhesion formation, protein kinase
C activation and binding of -actinin (Couchman, 2003). The cytoplasmic variable
region of syndecan-1 is known to affect cellular adhesion, migration and spreading
(Chakravarti et al., 2005).
Transforming growth factor- (TGF-) has been shown to interact with the hep-
aran sulfate chains on syndecans. Syndecan-2, in particular, has been shown to
interact with TGF- via a protein-protein interaction (Chen et al., 2004). The exact
nature and role of this interaction is complex and still remains to be elucidated. The
presence of syndecan-2 may serve to compete with betaglycan (TGF receptor type
III) for the binding of synectin. Synectin stabilizes betaglycan on the cell surface
and, consequently, syndecan-2 may serve to reduce signaling in the TGF pathway
(Chen et al., 2004; Tkachenko et al., 2005).
The syndecans have an intricate role in orchestrating development and are known
to be involved in cell-cell and cell-matrix adhesion. Syndecan-1 has been shown
to be important for cell adherence to type-I collagen (Sanderson et al., 1989). In
addition, syndecan-1 stabilizes the interactions of vitronectin with v3 integrin
(Beauvais et al., 2004). During migration syndecan-1, syndecan-4 and calveolin are
directed to the region of cell contraction (Baciu and Goetinck, 1995; Borset et al.,
2000; Beardsley et al., 2005). Syndecan-4 has also been shown to be an essen-
tial component for the activation of focal adhesion kinase and is known to bind
fibronectin with its heparan sulfate chains (Mukai et al., 2002; Wilcox-Adelman
et al., 2002; Hsia et al., 2003). In vascular smooth muscle cells exposed to shear
stress syndecan-4 has been shown to dissociate from focal adhesions (Li and
Chaikof, 2002).
Several studies have revealed differential regulation of syndecans by various
growth factors and cytokines. Fibroblast growth factor-2 (FGF-2) has been shown
to increase syndecan-4 expression in vascular smooth muscle cells (Cizmeci-Smith
et al., 1997). Arterial injury and myocardial infarction have also been shown to
increase syndecan-4 expression (Geary et al., 1995; Li et al., 1997; Li and Chaikof,
2002). Stimulation with tumor necrosis factor- (TNF-) increases syndecan-2
expression and decreases syndecan-1 in endothelial cells (Halden et al., 2004).
Transforming growth factor 2 increases syndecan-4 and decreases syndecan-1 in
epithelial cells (Dobra et al., 2003).
9.2.3.1 Versican
Versican is the major extracellular chondroitin sulfate bearing proteoglycan in arter-
ies (Wight and Merrilees, 2004). This molecule has multiple sites for chondroitin
9 Role of Proteoglycans in Vascular Mechanotransduction 225
sulfate attachment and reaches a molecular weight exceeding 1000 kDa (Fig. 9.2d).
Versican has been linked to controlling the viscoelastic behavior in many tissues
(Ludwig, 2007). It also has a pro-migratory affect in vascular smooth muscle cell-
sand various tumor cell types. Versican has multiple molecular interactions with
other extracellular matrix components including hyaluronan, CD44, tenascin R and
the fibulins (Wu et al., 2005).
9.2.3.2 Biglycan
Biglycan is the predominant chondroitin sulfate-bearing proteoglycan found on the
endothelial cell surface. It has been found to be associated with lipid deposition and
atherosclerosis. Biglycan binds to TGF-, collagen I/IV, fibronectin and other extra-
cellular matrix molecules. It is thought to have pro-inflammatory activity and has
a role in activating TNF- and MIP-2 in macrophages. Biglycan can be positively
regulated by TGF- in response to abnormal mechanical stresses. In addition, nitric
oxide (NO) can down regulate biglycan and can thereby link bilgycan expression to
shear stress-induced NO levels (OBrien et al., 1998; Williams, 2001).
9.2.3.3 Decorin
Decorin is another arterial proteoglycan that is similar to biglycan in that it is a mem-
ber of the small leucine-rich proteoglycan (SLRP) gene family. Decorin, howver,
has different biological activity in comparison to biglycan. It can bind to IGF-R and
IGF-I and regulate this pathway in endothelial cells. In addition, decorin induces
expression of p21WAF1, a cyclin-dependent kinase inhibitor, that can arrest the cell
cycle (Williams, 2001).
9.2.4 Hyaluronan
(von Gunten and Bochner, 2008). While sialic acid is a term that encompasses either
the N- or O-substituted derivatives of neuraminic acid, N-acetylneuraminic acid is
the predominant form found in mammalian cells. Sialic acid modification is found
in gangliosides and glycoproteins located at the cell membrane. Gangliosides are
glyocolipids anchored in the cell membrane that have been found to mediate cell-
cell recognition, inflammation and growth factor signaling. Sialic acid groups serve
as ligands for sialic acid-binding, immunoglobulin-like lectins (Siglecs). These
lectins bind to specific forms of sialic acids mediating a number of immunological
processes (von Gunten and Bochner, 2008).
Fig. 9.3 Arterial distribution of proteoglycan in the artery. Boxes show magnified regions of the
artery including the composition for the basement membrane underlying the endothelial layer and
the glycocalyx that extends into the lumen of the artery
9 Role of Proteoglycans in Vascular Mechanotransduction 227
Damiano, 2008; Chappell et al., 2009). The glycocalyx interacts directly with blood
flow and is thus a prime candidate for the mechanotransduction of shear forces in
the endothelial cells. In-vivo, this relatively thick structure is sufficient to reduce the
fluid shear forces to nearly zero at the endothelial surface (Weinbaum et al., 2003).
neuraminidase digestion did not attenuate the shear stress dependent release of PGI2
(Hecker et al., 1993).
In a parallel study, Pohl et al. examined diameter changes in response to flow
and pressure changes in the mesenteric arteries of the rabbit (Pohl et al., 1991).
They found that in-situ perfused mesenteric arteries had a three stage response to
changes in perfusion flow and pressure. The first phase was a passive distention
due to increased transmural pressure. The second phase is the myogenic response
to the arterial stretch (Johnson, 1986). The myogenic response is the propensity of
blood vessels to respond to transmural pressure elevation with constriction and to
pressure reduction with dilation. This behavior is inherent to smooth muscle and
is most pronounced in arterioles but can be demonstrated occasionally in arteries,
veins, and lymphatics (Johnson, 1986). The late phase of the response to changes
in perfusion is a flow-dependent dilation that overcomes the myogenic constriction
leading to increased vessel diameter. Neuraminidase digestion completely inhibited
the late phase of flow-induced vasodilation and enhanced the myogenic constriction
of the artery (Pohl et al., 1991).
More recently, Florian et al. studied the production of NO in cultured endothe-
lial cells under shear stress (Florian et al., 2003). The cells were treated with an
enzyme to digest heparan sulfate groups (heparinase) and then exposed to various
shear stress regimes. This study demonstrated that heparan sulfate digestion leads
to about a three-fold decrease in the NO production after exposure to steady shear
stress as well as a four-fold decrease in the NO production following treatment with
oscillatory shear stress. Similar studies found that pre-digestion with heparinase
did not inhibit bradykinin-induced increases in NO production. Additional stud-
ies on endothelial cells in culture from this group also demonstrated that digestion
with neuraminidase and hyaluronidase both attenuated shear induced NO release but
digestion with chondroitinase did not. In addition, shear induced increases in PGI2
were not affected by removal of heparan sulfate, sialic acid, chondroitin sulfate or
hyaluronan (Pahakis et al., 2007).
In a recent study, VanTeefelen et al. hypothesized that the binding of heparin to
the glycocalyx would alter the mechanotransduction of shear stress in the endothe-
lium (Van Teeffelen et al., 2007). They found that treatment with clinical levels of
heparin led to a reduction in the duration of vasodilation in response to occlusion-
induced reactive hyperemia in mice. This again adds to the evidence that heparan
sulfate is involved in NO-mediated vasodilation in response to changes in fluid flow.
While there are multiple studies supporting a role for glycocalyx proteins in the
arterial mechanotransduction of shear forces, the mechanisms remain to be uncov-
ered. The majority of studies have used digestive enzymes whose activity is not
necessarily restricted to the luminal surface or to endothelial cells versus vascular
smooth muscle cells. Thus, it remains unclear if the model of fluid shear acting
on the glycocalyx is correct in the case of heparan sulfate proteoglycans or if these
effects are due to alterations in heparan sulfate mediated extracellular matrix interac-
tion. Another notable complexity is that heparan sulfate can mediate the vasodilatory
effects of released factors such as FGF and can consequently have indirect effects
on these processes (Cuevas et al., 1991). One recent study examined the role of
9 Role of Proteoglycans in Vascular Mechanotransduction 229
the glycocalyx and the reactive oxygen species in the shear-induced NO release
(Kumagai et al., 2009). In this work, it was found that the superoxide dismutase
mimic superoxide scavenger (Tempol) restored the vasodilatory response and NO
increase in heparinase or neuraminidase digested arteries. However, Tempol did not
inhibit the vasodilation/NO response in hyaluronidase digested arteries. Endothelial
superoxide dismutase (SOD) catalyses the dismutation of superoxide into oxygen
and hydrogen peroxide and thus has anti-oxidative properties. Heparan sulfate binds
to endothelial SOD and can localize in the extracellular matrix. It has an important
role in maintaining endothelial NO as it diffuses to the vascular smooth muscle
cells in the arterial wall. Thus, the SOD binding properties of heparan sulfate may
represent a potential mechanism of involvement of heparan sulfate in flow-induced
vasodilatory responses.
Weinberg and colleagues have proposed a general model of fluid glycocalyx interac-
tion and force transmission (Weinbaum et al., 2003). Their model assumed that the
glycocalyx has a quasiperiodic structure based on experimental observations (Squire
et al., 2001). These studies revealed the ultrastructural details of the glycocalyx as
a network with a characteristic spacing of 20 nm with periodic centers spaced at
1012 nm. In their model the glycocalyx elements are linked to the cortical layer of
actin that links the dense peripheral actin band. Their model predicts that the gly-
cocalyx is fairly rigid and reduced the fluid shear stress at the surface of the cell to
almost zero. As fluid flows over the surface of the glycocalyx the resultant torque
is applied through first ~25 nm of the glycocalyx. This torque is transmitted to the
cortical actin and applied to the dense peripheral actin band causing disruption of
this actin network. Their theory was consistent with the observations of fluid stress
causing disruption of this actin network (Weinbaum et al., 2003).
mitogenesis (Clowes and Karnowsky, 1977; Ettenson et al., 2000; Nugent et al.,
2000). Heparan sulfate-induced regulation is growth state dependent, with sub-
confluent cultures of endothelial cells stimulating vascular smooth muscle cell
growth and postconfluent cultures inhibiting vascular smooth cell growth (Ettenson
et al., 2000). Consistent with these findings, both, perlecan and endothelial-derived
heparan sulfate proteoglycans have been shown to be essential in inhibiting the
neointimal response to vascular injury (Nugent et al., 2000). A recent study from our
group aimed to examine how mechanical forces alter the dynamic interplay of reg-
ulatory signals between endothelial and vascular smooth muscle cells (Baker et al.,
2008). In this work, we applied mechanical stretch to cultured endothlelial cells
and examined their production of growth-inhibitor heparan sulfate proteoglycans.
Mechanical stretch stimulated increase endothelial inhibition of vascular smooth
cell proliferation. Heparan sulfate proteoglycans and the core protein perlecan were
both in creased by exposure to mechanical stretch. Knock down studies demon-
strate that perlecan was required to increase vascular smooth muscle cell inhibition.
Experiments using small molecule inhibitors and neutralizing antibodies demon-
strated that a complex autocrine signaling cascade controlled the mechanical-load
induced production of the growth inhibitory heparan sulfate proteoglycan perlecan
(see Fig. 9.4). This pathway involved autocrine TGF-, ERK and p38 MAPK signal-
ing to induce perlecan expression in endothelial cells and lead to increased vascular
smooth muscle growth inhibition. Using ex-vivo arteries exposed to high pressure
under physiological flow conditions, these pathways were found to regulate the
production of heparan sulfate proteoglycans in intact arterial segments.
Fig. 9.4 Mechanical feedback control mechanisms of arterial structure in response to mechanical
stretch. The molecular signaling pathway shown refers to the pathways involved in controlling the
mechanical load-induced endothelial production of growth inhibitory heparan sulfate proteogly-
cans. These factors are increased with mechanical load and partially inhibit the pro-growth signals
in vascular smooth muscle cells
232 A.B. Baker
The finding that endothelial cells produce more inhibitory factors to vascular
smooth muscle cells with mechanical load is in contrast with the behavior of vas-
cular smooth muscle cells under similar mechanical strain conditions. Mechanical
stretch induces vascular proliferation (Hishikawa et al., 1997; Morita et al., 2004),
release of FGF (Cheng et al., 1997) production of PDGF (Ma et al., 1999), and pro-
duction of cell surface associated heparan sulfate proteoglycans (Li and Chaikof,
2002) (Fig. 9.4). Thus, under mechanical stimulation the endothelium provides
negative feedback control inhibiting the progrowth signals produced by vascular
smooth muscle cells under mechanical strain. Consequently, in the absence of an
intact endothelium due to disease of injury mechanical stretch would induce vascu-
lar smooth muscle cells to produce growth stimulatory factors that are unchecked by
load-enhanced endothelial cell paracrine growth inhibition. With endothelial repair,
the inhibitory responsiveness to mechanical strain is restored as well as additional
means of responding to hemodynamic forces can be applied including regulation of
vascular tone and inhibition of vascular smooth muscle cell proliferation.
References
Ainslie KM, Garanich JS, Dull RO, Tarbell JM (2005) Vascular smooth muscle cell glycocalyx
influences shear stress-mediated contractile response. J Appl Physiol 98:242249
Baciu PC, Goetinck PF (1995) Protein kinase C regulates the recruitment of syndecan-4 into focal
contacts. Mol Biol Cell 6:15031513
Baker AB, Ettenson DS, Jonas M, Nugent MA, Iozzo RV, Edelman ER (2008) Endothelial Cells
Provide Feedback Control for Vascular Remodeling Through a Mechanosensitive Autocrine
TGF-{beta} Signaling Pathway. Circ Res 103(3):289297
Beardsley A, Fang K, Mertz H, Castranova V, Friend S, Liu J (2005) Loss of caveolin-1 polarity
impedes endothelial cell polarization and directional movement. J Biol Chem 280:35413547
Beauvais DM, Burbach BJ, Rapraeger AC (2004) The syndecan-1 ectodomain regulates alphav-
beta3 integrin activity in human mammary carcinoma cells. J Cell Biol 167:171181
Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J et al. (1999) Functions of
cell surface heparan sulfate proteoglycans. Annu Rev Biochem 68:729777
9 Role of Proteoglycans in Vascular Mechanotransduction 233
Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO, Tarbell JM (2003) Heparan sulfate proteoglycan
is a mechanosensor on endothelial cells. Circ Res 93:e136142
Fransson LA (2003) Glypicans. Int J Biochem Cell Biol 35:125129
Fraser JR, Laurent TC, Laurent UB (1997) Hyaluronan: its nature, distribution, functions and
turnover. J Intern Med 242:2733
Gao Y, Li M, Chen W, Simons M (2000) Synectin, syndecan-4 cytoplasmic domain binding PDZ
protein, inhibits cell migration. J Cell Physiol 184:373379
Geary RL, Koyama N, Wang TW, Vergel S, Clowes AW (1995) Failure of heparin to inhibit
intimal hyperplasia in injured baboon arteries. The role of heparin-sensitive and -insensitive
pathways in the stimulation of smooth muscle cell migration and proliferation. Circulation 91:
29722981
Granes F, Urena JM, Rocamora N, Vilaro S (2000) Ezrin links syndecan-2 to the cytoskeleton. J
Cell Sci 113 (Pt 7):12671276
Grootjans JJ, Zimmermann P, Reekmans G, Smets A, Degeest G, Durr J et al. (1997) Syntenin,
a PDZ protein that binds syndecan cytoplasmic domains. Proc Natl Acad Sci U S A 94:
1368313688
Guyton JR, Rosenberg RD, Clowes AW, Karnovsky MJ (1980) Inhibition of rat arterial smooth
muscle cell proliferation by heparin. In vivo studies with anticoagulant and nonanticoagulant
heparin. Circ Res 46:625634
Hahn C, Schwartz MA (2009) Mechanotransduction in vascular physiology and atherogenesis. Nat
Rev Mol Cell Biol 10:5362
Halden Y, Rek A, Atzenhofer W, Szilak L, Wabnig A, Kungl AJ (2004) Interleukin-8 binds to
syndecan-2 on human endothelial cells. Biochem J 377:533538
Hecker M, Mulsch A, Bassenge E, Busse R (1993) Vasoconstriction and increased flow: two
principal mechanisms of shear stress-dependent endothelial autacoid release. Am J Physiol
265:H828833
Hishikawa K, Oemar BS, Yang Z, Luscher TF (1997) Pulsatile stretch stimulates superoxide pro-
duction and activates nuclear factor-kappa B in human coronary smooth muscle. Circ Res
81:797803
Hoover RL, Rosenberg R, Haering W, Karnovsky MJ (1980) Inhibition of rat arterial smooth
muscle cell proliferation by heparin. II. In vitro studies. Circ Res 47:578583
Hsia DA, Mitra SK, Hauck CR, Streblow DN, Nelson JA, Ilic D et al. (2003) Differential regulation
of cell motility and invasion by FAK. J Cell Biol 160:753767
Hsueh YP, Yang FC, Kharazia V, Naisbitt S, Cohen AR, Weinberg RJ et al. (1998) Direct inter-
action of CASK/LIN-2 and syndecan heparan sulfate proteoglycan and their overlapping
distribution in neuronal synapses. J Cell Biol 142:139151
Humphrey JD (2008) Vascular adaptation and mechanical homeostasis at tissue, cellular, and sub-
cellular levels. Cell Biochem Biophys 50:5378
Iozzo RV (2005) Basement membrane proteoglycans: from cellar to ceiling. Nat Rev Mol Cell Biol
6:646656
Iozzo RV, San Antonio JD (2001) Heparan sulfate proteoglycans: heavy hitters in the angiogenesis
arena. J Clin Invest 108:349355
Iozzo RV, Zoeller JJ, Nystrom A (2009) Basement membrane proteoglycans: modulators Par
Excellence of cancer growth and angiogenesis. Mol Cells 27:503513
Johnson PC (1986) Autoregulation of blood flow. Circ Res 59:483495
Kamiya A, Bukhari R, Togawa T (1984) Adaptive regulation of wall shear stress optimizing
vascular tree function. Bull Math Biol 46:127137
Kato M, Wang H, Kainulainen V, Fitzgerald ML, Ledbetter S, Ornitz DM et al. (1998)
Physiological degradation converts the soluble syndecan-1 ectodomain from an inhibitor to
a potent activator of FGF-2. Nat Med 4:691697
Kinnunen T, Kaksonen M, Saarinen J, Kalkkinen N, Peng HB, Rauvala H (1998) Cortactin-Src
kinase signaling pathway is involved in N-syndecan-dependent neurite outgrowth. J Biol Chem
273:1070210708
9 Role of Proteoglycans in Vascular Mechanotransduction 235
Kuchan MJ, Frangos JA (1994) Role of calcium and calmodulin in flow-induced nitric oxide
production in endothelial cells. Am J Physiol 266:C628636
Kuchan MJ, Jo H, Frangos JA (1994) Role of G proteins in shear stress-mediated nitric oxide
production by endothelial cells. Am J Physiol 267:C753758
Kumagai R, Lu X, Kassab GS (2009) Role of glycocalyx in flow-induced production of nitric oxide
and reactive oxygen species. Free Radic Biol Med 47:600607
Li J, Brown LF, Laham RJ, Volk R, Simons M (1997) Macrophage-dependent regulation of
syndecan gene expression. Circ Res 81:785796
Li L, Chaikof EL (2002) Mechanical stress regulates syndecan-4 expression and redistribution in
vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 22:6168
Li YS, Haga JH, Chien S (2005) Molecular basis of the effects of shear stress on vascular
endothelial cells. J Biomech 38:19491971
Lindner V, Olson NE, Clowes AW, Reidy MA (1992) Inhibition of smooth muscle cell proliferation
in injured rat arteries. Interaction of heparin with basic fibroblast growth factor. J Clin Invest
90:20442049
Ludwig MS (2007) Proteoglycans and pathophysiology. J Appl Physiol 103:735736
Ma YH, Ling S, Ives HE (1999) Mechanical strain increases PDGF-B and PDGF beta recep-
tor expression in vascular smooth muscle cells. Biochem Biophys Res Commun 265:
606610
Maday S, Anderson E, Chang HC, Shorter J, Satoh A, Sfakianos J et al. (2008) A PDZ-binding
motif controls basolateral targeting of syndecan-1 along the biosynthetic pathway in polarized
epithelial cells. Traffic 9:19151924
Mali M, Elenius K, Miettinen HM, Jalkanen M (1993) Inhibition of basic fibroblast growth factor-
induced growth promotion by overexpression of syndecan-1. J Biol Chem 268:2421524222
Moon JJ, Matsumoto M, Patel S, Lee L, Guan JL, Li S (2005) Role of cell surface heparan sul-
fate proteoglycans in endothelial cell migration and mechanotransduction. J Cell Physiol 203:
166176
Morgan MR, Humphries MJ, Bass MD (2007) Synergistic control of cell adhesion by integrins and
syndecans. Nat Rev Mol Cell Biol 8:957969
Morita N, Iizuka K, Murakami T, Kawaguchi H (2004) N-terminal kinase, and c-Src are activated
in human aortic smooth muscle cells by pressure stress. Mol Cell Biochem 262:7178
Mukai M, Togawa A, Imamura F, Iwasaki T, Ayaki M, Mammoto T et al. (2002) Sustained
tyrosine-phosphorylation of FAK through Rho-dependent adhesion to fibronectin is essential
for cancer cell migration. Anticancer Res 22:31753184
Murray CD (1926) The physiological principle of minimum work. I. The vascular system and the
cost of blood volume. Proc Natl Acad Sci U S A 12:207214
Noonan DM, Fulle A, Valente P, Cai S, Horigan E, Sasaki M et al. (1991) The complete sequence
of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity
with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule.
J Biol Chem 266:2293922947
Nugent MA, Iozzo RV (2000) Fibroblast growth factor-2. Int J Biochem Cell Biol 32:115120
Nugent MA, Nugent HM, Iozzo RV, Sanchack K, Edelman ER (2000) Perlecan is required
to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated
inhibition of intimal hyperplasia. Proc Natl Acad Sci U S A 97:67226727
OBrien KD, Olin KL, Alpers CE, Chiu W, Ferguson M, Hudkins K et al. (1998) Comparison
of apolipoprotein and proteoglycan deposits in human coronary atherosclerotic plaques:
colocalization of biglycan with apolipoproteins. Circulation 98:519527
Orr AW, Helmke BP, Blackman BR, Schwartz MA (2006) Mechanisms of mechanotransduction.
Dev Cell 10:1120
Pahakis MY, Kosky JR, Dull RO, Tarbell JM (2007) The role of endothelial glycocalyx components
in mechanotransduction of fluid shear stress. Biochem Biophys Res Commun 355:228233
Pohl U, Herlan K, Huang A, Bassenge E (1991) EDRF-mediated shear-induced dilation opposes
myogenic vasoconstriction in small rabbit arteries. Am J Physiol 261:H20162023
236 A.B. Baker
Potter DR, Damiano ER (2008) The hydrodynamically relevant endothelial cell glycocalyx
observed in vivo is absent in vitro. Circ Res 102:770776
Pure E, Assoian RK (2009) Rheostatic signaling by CD44 and hyaluronan. Cell Signal 21:651655
Rosenberg RD, Shworak NW, Liu J, Schwartz JJ, Zhang L (1997a) Heparan sulfate proteoglycans
of the cardiovascular system. Specific structures emerge but how is synthesis regulated? J Clin
Invest 100:S6775
Rosenberg RD, Shworak NW, Liu J, Schwartz JJ, Zhang L (1997b) Heparan sulfate proteoglycans
of the cardiovascular system. Specific structures emerge but how is synthesis regulated? J Clin
Invest 99:20622070
Rosendorff C (2007) Hypertension and coronary artery disease: a summary of the American Heart
Association scientific statement. J Clin Hypertens (Greenwich) 9:790795
Sanderson RD, Lalor P, Bernfield M (1989) B lymphocytes express and lose syndecan at specific
stages of differentiation. Cell Regul 1:2735
Sasisekharan R, Venkataraman G (2000) Heparin and heparan sulfate: biosynthesis, structure and
function. Curr Opin Chem Biol 4:626631
Smiesko V, Kozik J, Dolezel S (1985) Role of endothelium in the control of arterial diameter by
blood flow. Blood Vessels 22:247251
Squire JM, Chew M, Nneji G, Neal C, Barry J, Michel C (2001) Quasi-periodic substructure in
the microvessel endothelial glycocalyx: a possible explanation for molecular filtering? J Struct
Biol 136:239255
Tkachenko E, Rhodes JM, Simons M (2005) Syndecans: new kids on the signaling block. Circ Res
96:488500
van den Akker J, Schoorl MJ, Bakker EN, Vanbavel E (2009) Small Artery Remodeling: Current
Concepts and Questions. J Vasc Res 47:183202
Van Teeffelen JW, Brands J, Stroes ES, Vink H (2007) Endothelial glycocalyx: sweet shield of
blood vessels. Trends Cardiovasc Med 17:101105
Volk R, Schwartz JJ, Li J, Rosenberg RD, Simons M (1999) The role of syndecan cytoplas-
mic domain in basic fibroblast growth factor-dependent signal transduction. J Biol Chem
274:2441724424
Volker W, Bohm A, Schmidt A, Svahn CM, Gellerbring AK, Mattsson C et al. (1995) Inhibition
of smooth muscle cell proliferation and neointimal growth by low-anticoagulant heparin.
Arzneimittelforschung 45:546550
von Gunten S, Bochner BS (2008) Basic and clinical immunology of Siglecs. Ann N Y Acad Sci
1143:6182
Weinbaum S, Tarbell JM, Damiano ER (2007) The structure and function of the endothelial
glycocalyx layer. Annu Rev Biomed Eng 9:121167
Weinbaum S, Zhang X, Han Y, Vink H, Cowin SC (2003) Mechanotransduction and flow across
the endothelial glycocalyx. Proc Natl Acad Sci U S A 100:79887995
Whisnant JP (1996) Effectiveness versus efficacy of treatment of hypertension for stroke preven-
tion. Neurology 46:301307
Wight TN, Merrilees MJ (2004) Proteoglycans in atherosclerosis and restenosis: key roles for
versican. Circ Res 94:11581167
Wilcox-Adelman SA, Denhez F, Goetinck PF (2002) Syndecan-4 modulates focal adhesion kinase
phosphorylation. J Biol Chem 277:3297032977
Williams KJ (2001) Arterial wall chondroitin sulfate proteoglycans: diverse molecules with distinct
roles in lipoprotein retention and atherogenesis. Curr Opin Lipidol 12:477487
Wu YJ, La Pierre DP, Wu J, Yee AJ, Yang BB (2005) The interaction of versican with its binding
partners. Cell Res 15:483494
Yao Y, Rabodzey A, Dewey CF, Jr. (2007) Glycocalyx modulates the motility and prolifera-
tive response of vascular endothelium to fluid shear stress. Am J Physiol Heart Circ Physiol
293:H10231030
Part IV
Mechanotransduction in the Lung
Chapter 10
Control of TRPV4 and Its Effect on the Lung
(1998a) reported a 3.7-fold increase in the filtration coefficient (Kf ), a sensitive mea-
sure of lung endothelial permeability, in isolated perfused rat lungs ventilated with
30 cmH2 O peak inflation pressure (PIP). This mechanical stretch-induced injury
was completely prevented after treatment with gadolinium, a blocker of stretch
activated cation channels. Subsequent studies confirmed that the increases in Kf
and lung edema induced by mechanical ventilation were prevented by gadolinium
(Parker and Yoshikawa, 2002). Although these studies implicated a mechanically
gated Ca2+ permeable channel, the molecular nature of this stretch activated channel
was unknown at the time.
Previous studies had also implicated several signaling pathways now acknowl-
edged as important for regulation of TRPV4 channel gating. Townsley et al. (1990)
observed a synergistic impact of arachidonic acid and high vascular pressure on
increases in Kf in isolated, perfused dog lungs. In both intact mice and isolated
mouse lungs, the permeability lesion and edema resulting from high PIP ventila-
tion were attenuated after blockade of cytosolic phospholipase A2 (Miyahara et al.,
2008; Yoshikawa et al., 2005). Alvarez et al. (2004) reported an increase in Kf in
isolated rat lungs of approximately 2.5-fold after infusion of 5,6- and 14,15-EET,
whereas 8,9- and 11,12-EET had no significant impact. The permeability responses
to 5,6- and 14,15-EET were attenuated by low extracellular Ca2+ but not by phos-
pholipase A2 or P450 epoxygenase inhibitors. Figure 10.1 summarizes the impact of
extracellular Ca2+ on Kf measurements in isolated rat lungs treated with the TRPV4
Fig. 10.1 Filtration coefficients (Kf ) in isolated rat lungs showing the effect of treatment with 4-
phorbol-12,13-didecanoate (4PDD), epoxyeicosatrienoic acids (EETs), and thapsigargin (TG).
During baseline (BL) Kf and the initial Kf measurement after addition of channel agonists, a low
Ca2+ perfusate was used. Subsequently, Ca2+ addback in the same lungs brought extracellular Ca2+
to a physiologic concentration (Alvarez et al., 2006)
242 J.C. Parker and M.I. Townsley
The TRPV4 channel has now been identified as a stretch-activated cation channel
responsible for lung injury produced by both high vascular and airway pressures.
Hamanaka et al. (2007) observed that high airway pressure increased Kf in isolated
lungs of TRPV4+/+ mice (solid bars) but not in lungs of TRPV4-/- mice (shaded
bars). Heat (40 C) amplified the mechanical injury only in TRPV4+/+ mice. The
endothelial Ca2+ transients observed in TRPV4+/+ lungs during distention by high
airway pressure were absent in TRPV4/ mice and in TRPV4+/+ mice treated with
ruthenium red, a TRPV channel antagonist. Lung injury was also blocked by inhi-
bition of anandamide hydrolysis or cytochrome P450 epoxygenases. Figure 10.2
indicates the effects of high PIP ventilation, increased temperature, and inhibition
of these pathways on Kf in isolated mouse lungs from both phenotypes.
Jian et al. (2008) also measured a 4.5-fold increase in Kf in isolated lungs
from TRPV4+/+ mice after venous pressure elevations of 30 cmH2 O, which was
attenuated by approximately 80% in lungs from TRPV4/ mice and ruthe-
nium red-treated lungs from TRPV4+/+ mice. Significant attenuation of Kf was
also achieved by phospholipase A2 and cytochrome P450 epoxygenase inhibitors.
Endothelial Ca2+ transients and alveolar edema volumes were attenuated after pres-
sure stress in TRPV4/ lungs and by inhibition of Ca2+ entry, TRPV channels,
and P450 epoxygenases. Figure 10.3 shows the increase in intracellular Ca2+ after
an increase in pulmonary venous pressure in isolated mouse lungs from wild type
mice untreated, treated with ruthenium red or treated with the P450 epoxygenase
inhibitor (PPOH), and lungs from TRPV4 knockout mice either untreated or treated
with gadolinium.
10 Control of TRPV4 and Its Effect on the Lung 243
In situ fluorescence studies of lung endothelium by Yin et al. (2008) also indi-
cated an increase in intracellular Ca2+ after a venous pressure increase of only 15
cmH2 O. Calcium entry was enhanced by blockade of soluble guanylate cyclase
(sGC) or nitric oxide synthase (NOS), but decreased by the nitric oxide generator,
S-nitrosoglutathione (GSNO), a sGC activator drug, or the cGMP analog, 8Br-
cGMP. Kf measurements in the isolated rat lungs mirrored the changes in endothelial
Ca2+ . Using a nitric oxide (NO) sensitive dye, endothelial production of NO also
244 J.C. Parker and M.I. Townsley
increased as venous pressure was increased, but was attenuated by the sGC activator
or cGMP analogue. The vascular pressure-induced increase in Kf was attenuated by
gadolinium or the NO donor GSNO, but was further increased by blockade of nitric
oxide synthase (NOS). The increase in endothelial Ca2+ elicited by 4PDD was
also blocked by both ruthenium red and the cGMP analogue. Sildenafil, an inhibitor
of PDE5, also attenuated the pressure-induced increases in lung Kf , protein leak
and edema formation. These data indicate a TRPV4-mediated Ca2+ increase in lung
endothelium after venous pressure elevation which increased vascular permeabil-
ity, but which simultaneously activates a negative feedback control of the Ca2+ and
permeability increases mediated by cGMP.
Activation of Src family kinases or myosin light chain kinase (MLCK) during
mechanical distention of the lung appear to mediate the increased vascular per-
meability. Yin et al. (2008) found that inhibition of myosin light chain kinase
(MLCK) protected against the permeability response to increased vascular pressure
in isolated rat lung. Inhibition of MLCK or tyrosine kinases also attenuated air-
way pressure induced injury, whereas inhibition of tyrosine phosphatase augmented
injury (Miyahara et al., 2007; Parker et al., 1998b). The mechanism by which
MLCK amplifies TRPV4-mediated Ca2+ entry and lung injury is unknown, but may
relate to the dependence of TRPV4 channel activity on actin cytoskeleton attach-
ments and tension within the cytoskeleton (Becker et al., 2009; Ramadass et al.,
2007). Alternatively, direct phosphorylation of TRPV4 may play a role. For exam-
ple, the Src family kinases have been shown to amplify TRPV4 activity through
phosphorylation of tyrosine 253 (Xu et al., 2003). Involvement of other kinases
such as Akt/PKB may play a protective role. In isolated mouse lung, inhibition of
Akt amplified the high airway pressure vascular permeability lesion (Miyahara et al.,
2007), suggesting a protective pathway that likely acts through NO feedback.
10 Control of TRPV4 and Its Effect on the Lung 245
Fig. 10.5 Photomicrographs of toluidine blue-stained sections of rat lung showing preferential
distribution of lung edema in perivascular cuffs induced by thapsigargin (a) and in alveoli after
activation of TRPV4 with 14,15-EET (b). Pa, pulmonary artery; AF, alveolar fluid
alveolar flooding and further noted that TRPV4-mediated lung injury was associ-
ated with little perivascular cuff formation. In contrast, thapsigargin- treated lungs
had cuff formation without alveolar flooding. Although the lungs in both groups
gained approximately the same amount of edema, the thapsigargin-treated lungs
had a threefold greater decrease in dynamic lung compliance compared to 4PDD-
treated lungs. Perivascular cuff formation presumably facilitated a collapse of the
small airways to reduce compliance. Figure 10.5 contrasts the cuff formation in
isolated rat lungs treated with thapsigargin (A) with the alveolar flooding result-
ing from TRPV4 activation via 14,15-EET (B), even when the increase in Kf was
similar in these lungs. In addition to specificity for segmental vascular permeabil-
ity, the TRPV4 channel also exhibits functional specificity within the alveolar septal
endothelium. Wu et al. (2009) observed that TRPV4 activation increased endothelial
cytosolic Ca2+ and Kf in isolated mouse lungs, without causing P-selectin expres-
sion. The same increase in intracellular Ca2+ elicited by a high potassium perfusate
and activation of T-type Ca2+ channels resulted in P-selectin expression without any
increase in permeability.
rat and human lung, 14,15- and 11,12-EET proved to be the predominant products
(Zeldin et al., 1996), though all were present in some amount. Thus, availability
of EETs during mechanotransduction in lung cells will depend upon the epoxyge-
nase expressed in the lung cell exposed to mechanical stress, as well as expression
of the key enzyme responsible for EET degradation soluble epoxide hydrolase
(Spector, 2009). Interestingly, epoxygenase expression is itself up-regulated by
mechanical stress (Fisslthaler et al., 2001). While in vitro studies have identified
a number of regulatory domains in TRPV4, the molecular mechanisms underly-
ing mechanotransduction-initiated and EET-dependent activation of TRPV4 have
not been resolved. The TRPV4 channel consists of 871 amino acids with 6 trans-
membrane spanning segments and cytoplasmic C and N termini. The pore region
lies between transmembrane segments 5 and 6, as shown in Fig. 10.6. EETs could
potentially impact TRPV4 gating via a number of mechanisms.
thus by direct binding to the channel. However, based on evidence derived from
studies of EET-dependent regulation of other ion channels, the mechanism of action
could be more complex. In coronary artery smooth muscle cells, EETs activate the
large conductance Ca2+ -activated potassium channel (BKCa) in inside-out patches
via a mechanism which requires GTP and Gs (Li and Campbell, 1997). Further,
EETs appear to elicit responses via binding to G protein-coupled receptors (Wong
et al., 1997, 2000; Yang et al., 2008), suggesting the possibility for outside-in sig-
naling. EETs activate Gs , resulting in synthesis of cAMP and activation of PKA
(Yang et al., 2008; Carroll et al., 2006; Node et al., 2001; Spector and Norris, 2007).
While PKA-mediated phosphorylation has been implicated in TRPV4 activation
with hypotonicity (see below), a role for G protein-coupled receptors, Gs activa-
tion or cAMP synthesis has not been evaluated in lungs response to mechanical
stress.
10.2.4 Phosphorylation
Domain mapping of the TRPV4 sequence has highlighted a number of poten-
tial phosphorylation sites (Nilius et al., 2004; Everaerts et al., 2010; Fan et al.,
2009) for PKC, PKA and Src family tyrosine kinases. Kuebler and colleagues have
recently reported that NO- and cGMP-dependent negative feedback regulation of
TRPV4 gating in lung endothelium exposed to hydrostatic stress (Yin et al., 2008).
However, this feedback control may not be direct, as there does not appear to be a
PKG phosphorylation site in TRPV4. Most relevant to mechanical stress-induced
EET-dependent regulation of TRPV4 are PKA and the Src kinases. EET-induced
activation of Gs leads to recruitment of cAMP-PKA signaling (Yang et al., 2008;
Carroll et al., 2006; Node et al., 2001; Spector and Norris, 2007; Imig et al., 2008).
While EETs are known to activate a number of tyrosine kinase cascades via Src
kinases, MAPK or PI3 kinase (Spector and Norris, 2007), a role for tyrosine phos-
phorylation of TRPV4 in response to mechanical stress remains controversial. Xu
et al. surveyed a number of Src kinase family members, investigating a role for tyro-
sine kinase-mediated phosphorylation of TRPV4 in the response to hypotonicity
(Xu et al., 2003). Using a mutagenesis approach, these investigators mapped the Src
kinase-mediated phosphorylation to Y253, within the first ankyrin repeat domain.
While others found no role for tyrosine phosphorylation in hypotonicity-induced
TRPV4 activation (Vriens et al., 2004), a recent study utilizing a mass spectromet-
ric analysis has implicated Y110 in regulation of TRPV4 function (Wegierski et al.,
2009). While translation of these observations to the in vivo setting is limited, the
available evidence does support a role for tyrosine phosphorylation in the response
to mechanical stress. Parker and colleagues have observed tyrosine phosphoryla-
tion in lung following high pressure mechanical ventilation and have implicated
TRPV4, PI3K, Src kinases and Akt in the resultant ventilator-induced lung injury
(Parker et al., 1998b; Hamanaka et al., 2007; Miyahara et al., 2007). Similarly,
Src tyrosine kinases and TRPV4 have been implicated in mechanical hyperalgesia
(Alessandri-Haber et al., 2008).
250 J.C. Parker and M.I. Townsley
References
Alessandri-Haber N, Dina OA, Joseph EK, Reichling DB, Levine JD (2008) Interaction of transient
receptor potential vanilloid 4, integrin, and Src tyrosine kinase in mechanical hyperalgesia.
J Neurosci 28:10461057
Alvarez DF, Gjerde EA, Townsley MI (2004) Role of EETs in regulation of endothelial permeabil-
ity in rat lung. Am J Physiol Lung Cell Mol Physiol 286:L445L451
Alvarez DF, King JA, Weber D, Addison E, Liedtke W, Townsley MI (2006) Transient receptor
potential vanilloid 4-mediated disruption of the alveolar septal barrier: a novel mechanism of
acute lung injury. Circ Res 99:988995
Arniges M, Fernandez-Fernandez JM, Albrecht N, Schaefer M, Valverde MA (2006) Human
TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and
trafficking. J Biol Chem 281:15801586
Becker D, Muller M, Leuner K, Jendrach M (2008) The C-terminal domain of TRPV4 is essential
for plasma membrane localization. Mol Mem Biol 25:139151
Becker D, Bereiter-Hahn J, Jendrach M (2009) Functional interaction of the cation channel tran-
sient receptor potential vanilloid 4 (TRPV4) and actin in volume regulation. Eur J Cell Biol
88:141152
Carroll MA, Doumad AB, Li J, Cheng MK, Falck JR, McGiff JC (2006) Adenosine2A recep-
tor vasodilation of rat preglomerular microvessels is mediated by EETs that activate the
cAMP/PKA pathway. Am J Physiol Renal Physiol 291:F155F161
Cayouette S and Boulay G. (2007) Intracellular trafficking of TRP channels. Cell Calcium 42:
225232
Cheng W, Yang F, Takanishi CL, Zheng J (2007) Thermosensitive TRPV channel subunits
coassemble into heteromeric channels with intermediate conductance and gating properties.
J Gen Physiol 129:191207
Chetham PM, Babal P, Bridges JP, Moore TM, Stevens T (1999) Segmental regulation of
pulmonary vascular permeability by store operated Ca2+ entry. Am J Physiol 276:L41L50
Cioffi DL, Lowe K, Alvarez DF, Barry C, Stevens T (2009) TRPing on the lung endothelium.
Calcium channels that regulate barrier function. Antioxid Redox Signal. 11:765776
Cohen DM (2005) TRPV4 and the mammalian kidney. Pflugers Arch 451:168175
Cuajungco MP, Grimm C, Oshima K, D hoedt D, Nilius B, Mensenkamp AR, Bindels RJ, Plomann
M, Heller S (2006) PACSINs bind to the TRPV4 cation channel. PACSIN 3 modulates the
subcellular localization of TRPV4. J Biol Chem 281:1875318762
Dhoedt D, Owsianik G, Prenen J, Cuajungco MP, Grimm C, Heller S, Voets T, Nilius B (2008)
Stimulus-specific modulation of the cation channel TRPV4 by PACSIN 3. J Biol Chem
283:62726280
252 J.C. Parker and M.I. Townsley
Everaerts W, Nilius B, Owsianik G (2010) The vallinoid transient receptor potential channel Trpv4:
from structure to disease. Prog Biophys Mol Biol. 103:217
Fan HC, Zhang X, McNaughton PA (2009) Activation of the TRPV4 ion channel is enhanced by
phosphorylation. J Biol Chem 284:2788427891
Fernandes J, Lorenzo IM, Andrade YN, Garcia-Elias A, Serra SA, Fernandez-Fernandez JM,
Valverde MA (2008) IP3 sensitizes TRPV4 channel to the mechano- and osmotransducing
messenger 5 -6 -epoxyeicosatrienoic acid. J Gen Physiol 131:i2
Fisslthaler B, Popp R, Michaelis UR, Kiss L, Fleming I, Busse R (2001) Cyclic stretch enhances
the expression and activity of coronary endothelium-derived hyperpolarizing factor synthase.
Hypertension 38:14271432
Garcia-Elias A, Lorenzo IM, Vicente R, Valverde MA (2008) IP3 receptor binds to and sen-
sitizes TRPV4 channel to osmotic stimuli via a calmodulin-binding site. J Biol Chem 283:
3128431288
Hamanaka K, Jian M-Y, Weber DS, Alvarez DF, Townsley MI, Al Mehdi AB, King JA, Liedtke W,
Parker JC (2007) TRPV4 initiates the acute calcium-dependent permeability increase during
ventilator-induced lung injury in isolated mouse lungs. Am J Physiol Lung Cell Mol Physiol
293:L923L932
Hartmannsgruber V, Heyken WT, Kacik M, Kaistha A, Grgic I, Harteneck C, Liedtke W, Hoyer J,
Kohler R (2007) Arterial response to shear stress critically depends on endothelial TRPV4
expression. PLoS ONE 2:e827
Hellwig N, Albrecht N, Harteneck C, Schultz G, Schaefer M (2005) Homo- and heteromeric
assembly of TRPV channel subunits. J Cell Sci 118:917928
Imig JD, Dimitropoulou C, Reddy DS, White RE, Falck JR (2008) Afferent arteriolar dilation
to 11,12-EET analogs involves PP2A activity and Ca2+ -activated K+ Channels. Microcirc 15:
137150
Ito S, Suki B, Kume H, Numaguchi Y, Ishii M, Iwaki M, Kondo M, Naruse K, Hasegawa Y, Sokabe
M (2010) Actin cytoskeleton regulates stretch-activated Ca2+ influx in human pulmonary
microvascular endothelial cells. Am J Respir Cell Mol Biol 43:2634
Jian MY, King JA, Al Mehdi AB, Liedtke W, Townsley MI (2008) High vascular pressure-induced
lung injury requires P450 epoxygenase-dependent activation of TRPV4. Am J Respir Cell Mol
Biol 38:386392
Kttgen M, Buchholz B, Garcia-Gonzalez MA, Kotsis F, Fu X, Doerken M, Boehlke C, Steffl D,
Tauber R, Wegierski T, Nitschke R, Suzuki M, Kramer-Zucker A, Germino GG, Watnick T,
Prenen J, Nilius B, Kuehn EW, Walz G (2008) TRPP2 and TRPV4 form a polymodal sensory
channel complex. J Cell Biol 182:437447
Kuebler WM, Uhlig U, Goldmann T, Schael G, Kerem A, Exner K, Martin C, Vollmer E, Uhlig S
(2003) Stretch activates nitric oxide production in pulmonary vascular endothelial cells in situ.
Am J Resp Crit Care Med 168:13911398
Kuebler WM, Ying X, Bhattacharya J (2002) Pressure-induced endothelial Ca2+ oscillations in
lung capillaries. Am J Physiol Lung Cell Mol Physiol 282:L917L923
Kuebler WM, Ying X, Singh B, Issekutz AC, Bhattacharya J (1999) Pressure is proinflammatory
in lung venular capillaries. J Clin Invest 104:495502
Li PL and Campbell WB (1997) Epoxyeicosatrienoic acids activate K+ channels in coronary
smooth muscle through a guanine nucleotide binding protein. Circ Res 80:877884
Liedtke W (2006) TRPV channels function in osmo- and mechanotransduction. In: Liedtke W,
Heller S (eds) TRP ion channel function in sensory transduction and cellular signaling cascades.
CRC Press-Taylor & Francis, Boca Raton, FL, pp 303318
Loot AE, Popp R, Fisslthaler B, Vriens J, Nilius B, Fleming I (2008) Role of cytochrome P450-
dependent transient receptor potential V4 activation in flow-induced vasodilatation. Cardiovasc
Res 80:445452
Lowe K, Alvarez D, King J, Stevens T (2007) Phenotypic heterogeneity in lung capillary and
extra-alveolar endothelial cells. Increased extra-alveolar endothelial permeability is sufficient
to decrease compliance. J Surg Res 143:7077
10 Control of TRPV4 and Its Effect on the Lung 253
11.1 Introduction
Like in many other solid organs, physical forces play an important role in the lung
physiology, and are involved in the regulation of fetal lung development (Liu and
Post, 2000), airway branching and alveolarization, and the regulation of pulmonary
physiology (Liu et al., 1999). The lung is unique from other organs in that it is
M. Liu (B)
Faculty of Medicine, University of Toronto, 101 College Street Toronto Medical Discovery Tower,
Room 2-814, Toronto, ON, M5G 1L7 Canada
e-mail: mingyao.liu@utoronto.ca
constantly expanding and contracting to facilitate the flow of air into and out of
a complex network of airways. The physical force associated with normal breath-
ing does not lead to aberrant side effects. Abnormal physical forces however, are
known to contribute to the pathogenesis of lung diseases, such as asthma, pulmonary
hypertension, chronic obstructive pulmonary disease, etc. Mechanical ventilation as
a therapeutic modality can be used to provide life support during surgical operation,
or to patients with respiratory distress. This externally applied physical force which
ventilators use to force air into the lungs can lead to tissue damage and is associated
with several side effects known as ventilator-induced lung injury (VILI) (Girard and
Bernard, 2007). Before clinicians can address VILI, we need to recognize and under-
stand more about the mechanisms and pathways of mechanotrasduction initiated
both by normal breathing and by mechanical ventilation.
Acute lung injury (ALI), and its most severe form, acute respiratory distress syn-
drome (ARDS), is defined by The American-European Consensus Conference on
ARDS as severe gas exchange dysfunction and radiographic abnormalities after
a predisposing injury in the absence of heart failure (Bernard et al., 1994). The
age-adjusted incidence of ALI is around 86.2 per 100,000 person-years. There are
approximately 190,600 patients diagnosed with ALI each year in the United States
which is associated with 74,500 deaths and 3.6 million hospital days, represent-
ing a significant cost to the healthcare system (Rubenfeld et al., 2005). It has been
known for some time that physical force associated with high tidal volume ven-
tilation contributes to the progression of ALI/ARDS (Liu, 2007). Since the first
recommendations from the ARDS network were published in 2000 (ARDSNetwork,
2000), the importance of controlled mechanical ventilation conditions has been real-
ized. Physicians are required to perform a balancing act between using mechanical
ventilation to support the lives of critically ill patients and limiting the injurious side
effects that this treatment includes.
The acute inflammatory response associated with ARDS is thought to be one of
the major mechanisms associated with lung injury (Suter, 2006). Injurious mechan-
ical ventilation leading to VILI is a contributor to this inflammatory reaction. There
has been growing interest in the role that mechanotransduction plays in VILI in an
effort to better understand how physical forces are transmitted into biochemical sig-
nals in the lung and how these signals can be controlled to reduce or eliminate the
deleterious effects of mechanical ventilation while keeping the benefit (Liu, 2007;
Oeckler and Hubmayr, 2007).
There are a variety of mechanisms that cells can use to detect mechanical force
including stretch mediated ion channels (Hamill and Martinac, 2001), extracellular
matrix (ECM)-cytoskeleton perturbations (Ingber, 1991), and disruptions in cell-
cell adhesions (Ko et al., 2001). This chapter will focus on the role that protein-
protein interactions play in mechanosensation and mechanotransduction. There is
evidence to show that multiple pathways can be activated by mechanical ventilation
and researchers have begun looking at blocking the signals in the lung initiated
by the mechanical force associated with injurious mechanical ventilation. We will
discuss the possible therapeutic application of blocking Src protein tyrosine kinase
(PTK) pathway to reduce ALI/ARDS associated with VILI.
11 The Role of Protein-protein Interactions in Mechanotransduction 257
expansion of the lung there is shear stress between neighboring regions of different
compliance which is not seen in normal lungs (Mead et al., 1970).
mechanical ventilation are sensed by cells in the lung and converted to signalling
event is the focus of this chapter. Several basic mechanisms for mechanosensa-
tion and mechanotransduction have been discussed in this book. We will focus on
one of the novel mechanisms, protein-protein interaction in mechanosensation and
mechanotransduction.
more evident and the linker/organizer model for adaptor proteins appear to be the
exception rather than the rule (Burns K, 1998). Adaptor proteins themselves can be
dynamically regulated, adding an extra layer of complexity to the signaling cascade
(Pawson, 2007). It has been demonstrated that the function of adaptor proteins can
even be regulated by intracellular signals such as DNA damage (Stucki et al., 2005).
The association of signaling proteins with adaptors can act cooperatively to enhance
signaling or even antagonistically to gate a signal cascade (Hoshi et al., 2005).
The complexity of signal adaptor proteins doesnt end there. A single adaptor
protein can be associated with different proteins depending on the signaling pathway
functioning at any given time. In this way they serve to link different signaling pro-
teins to different downstream effectors depending on the cell type or even subcellu-
lar compartment being investigated. A classic example of this is the intracellular pro-
tein trafficking of signaling proteins. This important cellular function is dependent
on the diversity and specificity of adaptors involved in endocytosis and sorting in
the endosomes (Traub, 2003). The various capabilities of adaptors demonstrate that
the interaction subunits of these proteins are capable of much more than the simple
beads on a string model with which they were originally labeled (Pawson, 2007).
Knowing that protein-protein interaction can mediate and coordinate signal trans-
duction pathways in the cell, we would like to know whether protein-protein
interaction can be the first step to activate signal transduction, especially to con-
vert physical forces to activation of protein tyrosine kinase for signal transduction.
Several mechanosensory complexes have been defined and demonstrated that adap-
tor proteins can act as the actual mechanosensor or play an integral role in linking
the mechanosensor to the downstream signaling pathways.
Previously it was thought that the force sensing function of cells occurs at the
cytoplasm membrane via mechanisms such as calcium movement and ion chan-
nel gating (Hamill and Martinac, 2001), activation of cell surface receptors by their
ligands (Correa-Meyer et al., 2002; Tschumperlin et al., 2004), ECM-integrin inter-
actions on basolateral side of the cells by mechanical stretch (Chen et al., 1999;
Ingber, 1991), or shear stress on adhesion molecules on the apical membrane of the
cells (Tzima et al., 2005). Through the aforementioned studies it was unclear as to
whether cells can sense mechanical force without intact cellular membranes.
It is difficult to separate the role of ion channel and membrane protein function
from that of the cytoskeleton cells use to bear much of the tension and physical
force. One interesting way to solve this problem is to remove the non-cytoskeletal
components of the cell through Triton X-100 treatment of cells cultured on a col-
lagen coated matrix, leaving behind the Triton insoluble cytoskeleton. This process
removes transmembrane ion channels as well as minimizes the influence of ion flux
on mechanotransduction. In this manner, any measurable changes in protein status
(i.e. protein phosphorylation) resulting from mechanical stretch can only result from
interactions between proteins associated with the cytoskeleton. Sawada and Sheetz
11 The Role of Protein-protein Interactions in Mechanotransduction 261
used this method to remove the cellular membrane and soluble proteins from the
cytoplasm, and stretched the remaining cytoskeleton attached to the extracellular
matrix. Detergent soluble proteins were then replaced for a short period of time,
during which some were found to undergo tyrosine phosphorylation (Sawada and
Sheetz, 2002). This interesting study suggests that in the absence of membrane
related proteins and local changes in ion concentration mechanical force still can
be sensed by cells.
It has been reported that small GTPases can be involved in the mechanical
force induced signal cascade (Sawada et al., 2001). Biaxial stretch was found to
inhibit Ras activity, decrease lamellipodia formation, and activate Rap1. C3G (Rap1
guanine nucleotide exchange factor) is known to contain multiple SH3 domain
binding motifs that are able to associate with the Crk family of adaptor proteins
(Knudsen et al., 1994; Tanaka et al., 1994). It is believed that the association of
the C3G-Crk complex with other proteins containing a phosphotyrosine residue
allows for the release of cis-acting negative regulatory domain of C3G, facilitat-
ing its activation (Ichiba et al., 1999). Interestingly, Crk, C3G, and Rap1 have
all been shown to be associated with Src PTK in the regulation of cell adhesion
(Li et al., 2002), prompting researchers to suspect that this pathway may be
associated with mechanotransduction.
Using the model of cytoskeletal stretch, Tamada et al. showed that the Crk-C3G-
Rap1 signaling pathway was activated. In response to stretch, Crk was recruited to
ECM-cell contacts where it is believed to sense force applied to the cytoskeleton
(via ECM stretch) and facilitates the activation of this signaling pathway. In addi-
tion, they showed that Src PTK was associated with the cytoskeleton after stretch,
confirming the hypothesis that mechanical force can recruit Src for local activation
by protein-protein interactions (Tamada et al., 2004).
These studies showed that protein-protein interactions are able to facilitate
mechanotransduction in the absence of transmembrane ion channels or cytoplasmic
ion flux; however the question of what is the actual mechanosensor? remained
unanswered. It has been reported that p130Cas, when tyrosine phosphorylated on
its multiple internal YXXP repeats, can bind to the N-terminal SH2 domain of
Crk. Interestingly, under normal conditions, the YXXP repeats of p130Cas are
hidden through its folded tertiary structure and can be exposed only under cer-
tain conditions. p130Cas contains an N-terminal SH3 domain that can bind FAK
as well as a C-terminal binding site for Src PTK. These two binding domains
help to link p130Cas to focal adhesions where it is believed to play a role in
mechanotransduction. When cells are mechanically stretched, p130Cas underwent
extensive tyrosine phosphorylation at its multiple internal YXXP repeats (Sawada
et al., 2006). Phosphorylation of p130Cas allows for Crk binding, C3G activation,
and increased GTP-loading of Rap1. It was also shown that this effect was not the
result of an increase of Src PTK activity, implying the possibility that under mechan-
ical stretch, p130Cas is unfolded exposing internal tyrosine phosphorylation targets
for Src that are not apparent under non-stretch conditions (Sawada et al., 2006).
In this way, p130Cas can be considered a dynamic mechanosensor that can detect
a cell stretch force, and facilitate the beginning of a signal cascade through the
aforementioned protein-protein interactions (Pawson, 2007) (Fig. 11.1).
262 M. Rubacha and M. Liu
Fig. 11.2 Proteins can be considered as molecules built with functional modules. (a). The multiple
modular domain structure of Grb2 is typical for that of an adaptor protein. (b). Molecular struc-
ture of c-Src protein tyrosine kinase. (c). Molecular structure of actin filament associated protein
(AFAP)
264 M. Rubacha and M. Liu
Fig. 11.3 Mechanical stretch-induced Src activation via competitive binding with AFAP. In the
inactive state, phosphorylation at Y527 in the C-terminus of c-Src binds to its own SH2 domain and
the linker region between the SH2 and kinase domains binds to its own SH3 region. This intra-
protein folding locks c-Src in a closed confirmation (left). When cells are stretched, deformation
of F-actin stress fiber presents AFAP to c-Src. The SH3 and SH2 domain binding motifs of AFAP
bind c-Src with high affinity, disrupting the intra-protein inhibitory interactions of c-Src (middle).
While stabilized in the open confirmation by interactions with AFAP, c-Src can be phosphorylated
at the Y416 residue, locking it in the open and active confirmation (right). Reproduced from J Biol
Chem 2004;279:5479354801. Permission granted from the Journal of Biological Chemistry
AFAP was first discovered through research directed at discovering the mech-
anism by which constitutively activated v-Src could induce morphological trans-
formations in cells. AFAP was found to form a stable complex with v-Src through
its SH2 or SH3 domain binding motifs. The observation that AFAP was normally
associated with stress fibers and with rosette-like structures in v-Src transformed
chicken embryonic fibroblast cells indicated that AFAP may be involved in Src
activity-dependent alteration of actin filaments (Baisden et al., 2001a; Kanner et al.,
1991).
The avian form of AFAP was first cloned by Flynn et al. who reported the
sequence (Flynn et al., 1993). The mammalian form was first cloned in rats (Lodyga
et al., 2002) and later in humans (Han et al., 2004). The mammalian sequences
have a >80% homology with the avian form (Han et al., 2004; Lodyga et al., 2002).
Importantly, the putative Src binding and activation motifs and actin binding domain
described above are well conserved from avian to mammalian forms (Han et al.,
2004). It has been shown that AFAP can interact with c-Src through both its SH2
and SH3 domain binding motifs. Mutation of five putative tyrosine phosphorylation
sites or a single amino acid mutation from proline to alanine in the SH3 domain bid-
ing motif significantly reduced the ability of AFAP to bind to c-Src and to lead to its
activation. Han et al. further over-expressed these mutant forms of AFAP as dom-
inant negative inhibitors and demonstrated these mutants blocked stretch-induced
c-Src activation in multiple cell types (Han et al., 2004). Based on these results, Han
et al. have proposed that deformation of the cytoskeleton increases the competitive
binding between AFAP and c-Src SH2 and SH3 domains. The interaction between
AFAP and c-Src leads to a conformational change in Src, facilitating its activation.
Activation of Src allows for the initiation of a downstream signaling cascade (Han
et al., 2005) (Fig. 11.3).
ultimately leading to edema formation and the interruption of gas exchange across
the air-blood barrier. It has been shown that after only 20 min of injurious venti-
lation, vascular permeability is significantly increased in isolated, perfused mouse
lungs, much sooner than expected if this was the result of cytokine influence alone.
These results implied a rapid mechanism for mechanotransduction leading to lung
injury independent of cytokine response (Miyahara et al., 2007).
The force associated with mechanical ventilation is not limited to the airways of
the lung. This force is also transmitted to the vasculature, where a different type of
mechanical force is experienced. During normal breathing the airway and alveolar
pressure remains within an accepted range, however during mechanical ventilation,
the airway and alveolar pressure increase during inspiration to facilitate air flow
(von Bethmann et al., 1998). As the airway and alveolar pressure increases from
mechanical ventilation, the lung vasculature is stretched causing vessel narrowing
and an increase in pulmonary circulation pressure. This increase in pressure may
increase shear stress, a force parallel to the plane of reference, which is known
inducer of mechanotransduction in endothelial cells (Uhlig, 2002).
Observations from studies directed at determining the role of -catenin in the
development of increases in vascular permeability have implicated Src PTKs as
upstream modifiers of this process. Normally found at adherens junctions, -catenin
can be tyrosine phosphorylated by Src, a process leading to the re-distribution of
-catenin to the cytoplasm where it is degraded, resulting in an increase in vascular
permeability, possibly thorough the destabilization of the adherens junction (Mehta
and Malik, 2006).
The PI3K-Akt signaling pathway has also been shown to act on -catenin.
Activation of this signaling pathway can lead to the negative regulation of GSK3,
which when active, can phosphorylate -catenin on serine residues, tagging it for
degradation. PI3K signaling, however, can also activate Src. Depending on the
strength of these signaling pathways, PI3K signaling could be both a positive and
negative regulator of vascular permeability (Miyahara et al., 2007). PI3K is a diverse
kinase that is activated in response to many different stimuli (Uhlig and Uhlig,
2004). Mechanical ventilation of isolated, perfused mouse lungs leads to the activa-
tion of PI3K. Inhibition of PI3K during mechanical ventilation leads to a decrease
in vascular permeability and a similar effect is seen when Src is inhibited directly.
Interestingly, inhibition of Akt, a downstream kinase of PI3K, lead to the augmenta-
tion of VILI induced vascular permeability. Further study revealed that mechanical
ventilation induced activation of PI3K signaling may lead to a two pronged sig-
naling cascade. One prong activates Src leading to the removal of -catenin from
adherens junctions and an increase in vascular permeability. The other prong acti-
vates the Akt-GSK3 signaling pathway leading to the preservation of -catenin at
the adherens junction and the maintenance of endothelial barrier function (Miyahara
et al., 2007). It appears that during high pressure or injurious mechanical ventilation,
activation of Src is stronger than Akt signaling leading to VILI.
A recent paper by Tzima et al. provides a very comprehensive overview of
endothelial mechanotransduction (Tzima et al., 2005). They focused on investigat-
ing how integrins became activated in response to shear stress. Through a series of
11 The Role of Protein-protein Interactions in Mechanotransduction 267
inhibitor experiments, they were able to show that the Src-PI3K-integrin pathway
was responsible for the intracellular changes associated with shear force. It is known
that shear stress induces a conformational change in integrins and cell alignment
with the direction of flow, as well as the activation of NF-B (Tzima et al., 2002;
Tzima et al., 2001). The addition of a PI3K inhibitor (LY294002 or wortmannin) is
able to block integrin activation. PI3K itself has been found to become rapidly acti-
vated in response to mechanical force (as measured through phosphorylation of the
p85 regulatory subunit of PI3K, the increase of PtdIns(3,4,5)P3 and kinase activity
(Tzima et al., 2005). Src is also known to become active within seconds of the onset
of shear stress (Okuda et al., 1999; Pampori et al., 1999), even faster than PI3K. Src
inhibition with PP2 or SU6656 prevented p85 phosphorylation as well as integrin
activation (Tzima et al., 2005).
To determine what exactly is the mechanosensor in shear stress-induced
mechanotransduction, endothelial cells lacking VE-cadherin or PECAM-1
(VE-cadherin/ and PECAM-1/ respectively) were tested for their ability to
respond to shear stress. Both types of cells were unable to undergo integrin activa-
tion in response to shear stress. When VE-cadherin and PECAM-1 were ectopically
reexpressed in these cells, normal functionality was restored. Further studies demon-
strated that PECAM-1 is upstream of VE-cadherin and is required for Src activation,
and VE-cadherin acts as an adaptor protein to link Src to PI3K allowing for the Src
mediated activation of the PI3K signal cascade (Tzima et al., 2005).
2007; Sawada et al., 2006). As mentioned above, Src is believed to be directly acti-
vated by AFAP in response to mechanical stretch (Han et al., 2004) and is thought
to phosphorylate p130Cas on its YXXP repeats when it is unfolded in response
to mechanical force (Pawson, 2007; Sawada et al., 2006), it is also one of the
first signaling proteins activated in shear stress induced mechanostransduction in
endothelial cells (Tzima et al., 2005). Therefore, Src PTKs are an attractive target
for the treatment of VILI.
The role of protein-protein interactions in mechanotransduction is likely not lim-
ited to adaptor proteins. Interactions between proteins and other molecules, such as
membrane lipids, calcium and other ions, RNA and DNA molecules may also be
affected by physical forces. At every level of the signal cascade, there is a complex
and largely unknown mechanism for the interaction of signaling molecules. These
interactions may occur locally in a particular sub-cellular compartment to deter-
mine a specific cellular function; they may also be affected further by other factors
cells are exposed, such as hyperoxia, hypoxia, inflammatory mediators and environ-
mental conditions, etc. To further explore the role of protein-protein interaction and
interaction between proteins and other molecules will improve our understanding of
mechanosensitivity and mechanotransduction under physiological and pathophys-
iological conditions and reveal new targets for clinical therapies, such as VILI in
ALI/ARDS.
References
ARDSNetwork (2000) Ventilation with lower tidal volumes as compared with traditional tidal
volumes for acute lung injury and the acute respiratory distress syndrome. The acute respiratory
distress syndrome network. N Engl J Med 342:13011308
Bachofen H, Gerber U, Schurch S (2002) Effects of fixatives on function of pulmonary surfactant.
J Appl Physiol 93:911916
Baisden JM, Gatesman AS, Cherezova L, Jiang BH, Flynn DC (2001a) The intrinsic ability of
AFAP-110 to alter actin filament integrity is linked with its ability to also activate cellular
tyrosine kinases. Oncogene 20:66076616
Baisden JM, Qian Y, Zot HM, Flynn DC (2001b) The actin filament-associated protein AFAP-
110 is an adaptor protein that modulates changes in actin filament integrity. Oncogene 20:
64356447
Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, Hudson L et al. (1994) The American-
European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and
clinical trial coordination. Am J Respir Crit Care Med 149:818824
Burns K MF, Esslinger C, Pahl H, Schneider P, Bodmer JL, Di Marco F, French L, Tschopp J.
(1998) MyD88, an adapter protein involved in interleukin-1 signaling. J Biol Chem 15:
1220312209
Chen KD, Li YS, Kim M, Li S, Yuan S, Chien S et al. (1999) Mechanotransduction in response
to shear stress. Roles of receptor tyrosine kinases, integrins, and Shc. J Biol Chem 274:
1839318400
Correa-Meyer E, Pesce L, Guerrero C, Sznajder JI (2002) Cyclic stretch activates ERK1/2 via
G proteins and EGFR in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 282:
L883891
Desai LP, Chapman KE, Waters CM (2008) Mechanical stretch decreases migration of alveolar
epithelial cells through mechanisms involving Rac1 and Tiam1. Am J Physiol Lung Cell Mol
Physiol 295:L958965
Desai LP, White SR, Waters CM (2009) Mechanical stretch decreases FAK phosphorylation and
reduces cell migration through loss of JIP3-induced JNK phosphorylation in airway epithelial
cells. Am J Physiol Lung Cell Mol Physiol 297:L520529
Feller SM (2001) Crk family adaptors-signalling complex formation and biological roles.
Oncogene 20:63486371
270 M. Rubacha and M. Liu
Fischer S, Simon AR, Welte T, Hoeper MM, Meyer A, Tessmann R et al. (2006) Bridge to lung
transplantation with the novel pumpless interventional lung assist device NovaLung. J Thorac
Cardiovasc Surg 131:719723
Flynn DC (2001) Adaptor proteins. Oncogene 20:62706272
Flynn DC, Leu TH, Reynolds AB, Parsons JT (1993) Identification and sequence analysis of
cDNAs encoding a 110-kilodalton actin filament-associated pp60src substrate. Mol Cell Biol
13:78927900
Franchini KG, Torsoni AS, Soares PH, Saad MJ (2000) Early activation of the multicomponent
signaling complex associated with focal adhesion kinase induced by pressure overload in the
rat heart. Circ Res 87:558565
Girard TD, Bernard GR (2007) Mechanical ventilation in ARDS: a state-of-the-art review. Chest
131:921929
Hamill OP, Martinac B (2001) Molecular basis of mechanotransduction in living cells. Physiol Rev
81:685740
Han B, Bai XH, Lodyga M, Xu J, Yang BB, Keshavjee S et al. (2004) Conversion of mechanical
force into biochemical signaling. J Biol Chem 279:5479354801
Han B, Lodyga M, Liu M (2005) Ventilator-induced lung injury: role of protein-protein interaction
in mechanosensation. Proc Am Thorac Soc 2:181187
Hoshi N, Langeberg LK, Scott JD (2005) Distinct enzyme combinations in AKAP signalling
complexes permit functional diversity. Nat Cell Biol 7:10661073
Ichiba T, Hashimoto Y, Nakaya M, Kuraishi Y, Tanaka S, Kurata T et al. (1999) Activation of C3G
guanine nucleotide exchange factor for Rap1 by phosphorylation of tyrosine 504. J Biol Chem
274:1437614381
Ingber D (1991) Integrins as mechanochemical transducers. Curr Opin Cell Biol 3:841848
Kanner SB, Reynolds AB, Wang HC, Vines RR, Parsons JT (1991) The SH2 and SH3 domains of
pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110. EMBO
J 10:16891698
Khadaroo RG, He R, Parodo J, Powers KA, Marshall JC, Kapus A et al. (2004) The role of the Src
family of tyrosine kinases after oxidant-induced lung injury in vivo. Surgery 136:483488
Knudsen BS, Feller SM, Hanafusa H (1994) Four proline-rich sequences of the guanine-nucleotide
exchange factor C3G bind with unique specificity to the first Src homology 3 domain of Crk.
J Biol Chem 269:3278132787
Ko KS, Arora PD, McCulloch CA (2001) Cadherins mediate intercellular mechanical signal-
ing in fibroblasts by activation of stretch-sensitive calcium-permeable channels. J Biol Chem
276:3596735977
Li L, Okura M, Imamoto A (2002) Focal adhesions require catalytic activity of Src family kinases
to mediate integrin-matrix adhesion. Mol Cell Biol 22:12031217
Liu M (2007) Ventilator-induced lung injury and mechanotransduction: why should we care? Crit
Care 11:168
Liu M, Post M (2000) Invited review: mechanochemical signal transduction in the fetal lung. J Appl
Physiol 89:20782084
Liu M, Qin Y, Liu J, Tanswell AK, Post M (1996) Mechanical strain induces pp60src activation
and translocation to cytoskeleton in fetal rat lung cells. J Biol Chem 271:70667071
Liu M, Skinner SJ, Xu J, Han RN, Tanswell AK, Post M (1992) Stimulation of fetal rat lung cell
proliferation in vitro by mechanical stretch. Am J Physiol 263:L376383
Liu M, Tanswell AK, Post M (1999) Mechanical force-induced signal transduction in lung cells.
Am J Physiol 277:L667683
Liu M, Xu J, Liu J, Kraw ME, Tanswell AK, Post M (1995a) Mechanical strain-enhanced fetal lung
cell proliferation is mediated by phospholipase C and D and protein kinase C. Am J Physiol
268:L729738
Liu M, Xu J, Souza P, Tanswell B, Tanswell AK, Post M (1995b) The effect of mechanical strain
on fetal rat lung cell proliferation: comparison of two- and three-dimensional culture systems.
In Vitro Cell Dev Biol Anim 31:858866
11 The Role of Protein-protein Interactions in Mechanotransduction 271
Xu W, Doshi A, Lei M, Eck MJ, Harrison SC (1999) Crystal structures of c-Src reveal features of
its autoinhibitory mechanism. Mol Cell 3:629638
Yamamoto H, Teramoto H, Uetani K, Igawa K, Shimizu E (2002) Cyclic stretch upregu-
lates interleukin-8 and transforming growth factor-beta1 production through a protein kinase
C-dependent pathway in alveolar epithelial cells. Respirology 7:103109
Yu H, Braun P, Yildirim MA, Lemmens I, Venkatesan K, Sahalie J et al. (2008) High-quality binary
protein interaction map of the yeast interactome network. Science 322:104110
Part V
Mechanosensing
and Mechanotransduction in Bone
and Joint Tissues
Chapter 12
Cellular Mechanisms of Mechanotransduction
in Bone
Abstract Bone is a dynamic tissue that adjusts its structure over time to adapt to
changes in mechanical load. This adaptive ability is critical to skeletal development
and maintenance of optimal skeletal health throughout life. Imbalances in the abil-
ity of bone to keep pace with demands placed on it by mechanical loading results
in bone that is fragile and susceptible to fracture. The cells within bone, i.e. osteo-
cytes and osteoblasts, are responsible for detecting and responding to mechanical
loading. Understanding how bone cells sense and respond to mechanical signals
(the process known as mechanotransduction) has been the focus of considerable
research. Despite excellent progress over the past two decades, the precise mech-
anisms by which bone cells perceive and mediate proper responses to mechanical
loading are not fully understood. When bone is loaded, movement of interstitial
fluid is generated within the small spaces that surround bone cells. This movement
generates fluid shear stress (FSS) that stimulates bone cells, resulting in enhanced
anabolic activity. Most research on cellular mechanisms of mechanotransduction is
now focused on understanding the mechanisms through which bone cells sense FSS
and generate a proper biochemical response. Mechanisms of cellular mechanotrans-
duction likely involve the adhesive junctions, integrins, mechanically-sensitive ion
channels, purinergic receptors, gap junctions and primary cilia. The integrin fam-
ily of cell adhesion molecules may, as their name implies, integrate mechanical
signals across the cell surface. It is also likely that ion channels, particularly cal-
cium changes, play a critical role in mediating mechanical responses to FSS. This
review will focus on recent progress in understanding how bone cells detect and
respond to mechanical stimuli using novel multi-protein signaling complexes called
mechanosomes.
12.1 Introduction
Cellular mechanotransduction is the process by which cells sense and respond to
physical cues from their surrounding environment. This mechanical sensitivity is
evident from the simplest isolated cells on two-dimensional substrates to cells inte-
grated within the most complex tissues. Understanding the biological processes that
mediate cellular mechanotransduction has been a growing area of interest during
the past 20 years and is particularly important in understanding how bone responds
to mechanical load (Allori et al., 2008; Chen et al., 2010; Huang et al., 2004;
Papachristou et al., 2009; Papachroni et al., 2009; Robling and Turner, 2009a).
Bone is an extremely dynamic tissue that normally undergoes constant turnover,
a process that defines bone remodeling (Turner and Pavalko, 1998). The mechan-
ical responsiveness of the cells that reside in bone, including osteocytes, the most
abundant cells, and osteoblasts, the cells that secrete new osteoid, has been a pri-
mary focus in efforts to understand how bone turnover is regulated in response to
the mechanical demands placed on the skeleton (Bonewald, 2006; Bonewald and
Johnson, 2008; Turner et al., 1994, 2009). This article will discuss recent progress in
the identification and characterization of molecules at the bone cell membrane that
are responsible for detecting mechanical signals. We then discuss progress in iden-
tifying intracellular signaling pathways that are responsible for propagating those
signals inside the cell and regulate bone cell function.
Focal adhesions are sites of attachment between the cell and the extracellular
matrix and have been proposed as cell mechanosensors. Focal adhesions appear
to be integral for the detection and propagation of mechanical signals across the
plasma membrane and into the cytoplasm and nucleus. Direct and indirect connec-
tions between the extracellular matrix (ECM), integrin cell adhesion molecules, and
focal adhesion-associated signal transduction molecules play an important role in
mechanotransduction in many types of cells, including bone cells. Each of these
three key elements (matrix, integrins and intracellular signaling molecules) con-
verges at these sites of structural attachment between cells and the ECM. Although
there is strong evidence that focal adhesions are involved in mechanical signaling,
the molecular mechanisms through which mechanical signaling via focal adhesions
occurs is not completely understood. Focal adhesions are characterized by the pres-
ence of integrin cell adhesion molecules that link the ECM outside the cell to the
cytoskeleton and signaling molecules inside the cell (Burridge and Chrzanowska-
Wodnicka, 1996). Focal adhesions serve as important sites of structural attachment
between cells and the ECM (Burridge and Chrzanowska-Wodnicka, 1996), how-
ever, focal adhesions also function as important organizing centers for signal
12 Cellular Mechanisms of Mechanotransduction in Bone 279
(Pavalko et al., 1998). With longer periods of shear, osteoblasts increase formation
of fibrillar adhesions which contain 5/1 integrins localized more toward the inte-
rior of the cell (Ponik and Pavalko, 2004). Consistent with results in osteoblasts,
studies using endothelial cells demonstrate increased mRNA and protein levels of
5 and 1 integrins in response to FSS (Urbich et al., 2000). Thus, considerable
evidence supports the hypothesis that focal adhesions function as mechanosensors
that are critical in regulating the response of mechanically sensitive cells to FSS
(reviewed in, (Geiger and Bershadsky, 2002)).
When bone cells are subjected to mechanical stimulation, either by fluid shear stress
or by strain, one of the earliest responses that can be detected experimentally is a
rapid increase in intracellular Ca2+ that occurs within 30 s of exposure to shear stress
or strain (Hung et al., 1995; Jones et al., 1991). This mechanically-induced cytoplas-
mic Ca2+ spike involves both entries of extracellular Ca2+ via surface membrane ion
channels and intracellular Ca2+ stores (Hung et al., 1996). This rapid Ca2+ response
to mechanical loading has led investigators to focus on the role of ion channels in
mechanotransduction. Duncan and colleagues found that intracellular Ca2+ release
from internal stores affects gene expression through activation of phospholipase C
(PLC) and the resulting production of IP3 (Chen et al., 2003). These studies sup-
port a role for ion channel-mediated mechanical signaling in bone and highlight the
important question of how fluid shear stress results in such a rapid Ca2+ spike and
activation of PLC. Specifically, this work suggests that fluid shear-induced changes
in gene expression in osteoblasts are dependent on Ca2+ release from intracellu-
lar stores that is mediated by IP3 . In addition, numerous studies suggest that Ca2+
entry through ion channels plays an important role in mechanically-induced Ca2+
increases. A number of different ion channels have been described in osteoblasts
(for review, see (Duncan et al., 1998)). Two of these channels have been directly
linked to the mechanical response of bone cells. One is the L-type voltage-sensitive
Ca2+ channel (L-VSCC) (Li et al., 2002a) and the other is the mechanosensitive,
cation-selective channel (MSCC) (Ryder and Duncan, 2001; Zhang et al., 2006).
Experimental evidence supporting an important role for MSCCs during mechan-
otransduction includes the demonstration that inhibition of MSCCs blocks FSS-
induced release of prostaglandins in osteocytes (Ajubi et al., 1999), of TGF-1 in
osteoblastic cells (Sakai et al., 1998) and of nitric oxide (NO) in organ cultures
(Rawlinson et al., 1996). Inhibition of the L-VSCC, which has been proposed to
control growth and development of bone (Duriez et al., 1993) and affects prolifer-
ation of osteoblasts (Loza et al., 1994), significantly reduces mechanically-induced
bone formation in rodents (Li et al., 2002a). The increase in mechanically-induced
intracellular Ca2+ mediated by MSCCs and L-VSCCs is thought to promote activa-
tion of several important signaling pathways in bone cells including ERK, JNK and
p38 MAP kinases (Liu et al., 2008). In general, MAP kinases, which are activated
by phosphorylation of tyrosine and threonine residues, play a ubiquitous role in
12 Cellular Mechanisms of Mechanotransduction in Bone 281
Many cells express a single cilium that extends from the cell surface and is gen-
erally referred to as the primary cilium. Unlike other cilia, the primary cilium is
not motile but has been proposed to play a unique function as a mechanosensor
and/or chemosensor. (Wheatley et al., 1996). Primary cilia have been suggested
to be involved in several signaling pathways that are important in development
(Christensen et al., 2007, 2008) and are implicated in a number of human diseases,
such as polycystic kidney disease (Tobin and Beales, 2007; Yoder, 2007). Over the
past several years, primary cilia have gained significant attention for their potential
282 S.R.L. Yong and F.M. Pavalko
role in development and maintenance of the skeleton (Koyama et al., 2007; Malone
et al., 2007a; McGlashan et al., 2007; Whitfield, 2008; Xiao et al., 2006).
Primary cilia were shown to function as sensors of fluid flow in kidney epithelial
cells (Liu et al., 2005). Movement of fluid has been suggested to physically bend the
primary cilium leading directly to an increase in the level of intracellular calcium
(Schwartz et al., 1997) but primary cilia also appear to function independently of
calcium in bone (Malone et al., 2007b). Primary cilia have also been visualized in
bone cells, including osteocytes and osteoblasts (Matthews and Martin, 1971; Xiao
et al., 2006). Future studies using mice in which the function of primary cilia are
targeted specifically in bone cells are likely to provide more definitive evidence on
the role of primary cilia on regulation of mechanically-induced bone formation in
vivo.
Because integrins do not possess intrinsic kinase activity, signals from various
sources including mechanical stimulation must be transduced indirectly into the
cytoplasm, and eventually the nucleus. Focal adhesion kinase (FAK) is one of many
proteins recruited as part of a multiprotein complex in focal adhesions and functions
as a key intermediate in various integrin-dependent signal transduction pathways
(reviewed in, (Abbi and Guan, 2002; Hauck et al., 2002; Parsons et al., 2000)).
FAK has been shown to play a role in mediating the downstream activation of
mitogen activated protein kinase (MAPK) in response to FSS in endothelial cells
(Li et al., 1997; Takahashi et al., 1997) and our group has evidence of FAK activation
in response to FSS in osteoblasts.
Upon activation, FAK is autophosphorylated at tyrosine 397 (Tyr397). Activated
FAK can associate with various other signaling molecules including the tyrosine
kinase c-Src. As a result of this association between FAK and c-Src, c-Src becomes
capable of phosphorylating two additional focal adhesion proteins, paxillin and
p130cas . The association of phosphorylated FAK, paxillin, and p130cas serves as a
scaffold for recruitment of adaptors and signaling intermediates including the adap-
tor protein Crk and the guanine nucleotide exchange for Rap1, C3G, which can lead
to the activation of EFK (reviewed recently in, (Panetti, 2002; Turner, 2000). Thus,
FAK plays a key role in signaling through focal adhesions.
The dominant negative FAK mutant, FAK-Related Non-Kinase, or FRNK, con-
tains only the C-terminal domain of FAK and lacks kinase activity (Cooley et al.,
2000). FRNK can localize to focal adhesions, inhibit autophosphorylation of and
displace endogenous FAK from focal adhesions, thereby negatively regulating the
function of endogenous FAK (Lin et al., 1997). There is significant experimen-
tal evidence that FAK mediates integrin-dependent fluid shear induced signals via
focal adhesions from several laboratories including our own. First, in addition
to activation of FAK by FSS (Li et al., 1997), other focal adhesion components
12 Cellular Mechanisms of Mechanotransduction in Bone 283
including c-Src and p130cas which are substrates of FAK, are also rapidly acti-
vated by FSS in endothelial cells (Berk et al., 1995; Chen et al., 1999; Okuda
et al., 1999) . Second, expression of dominant negative FAK and c-Src mutants in
endothelial cells inhibits shear induced activation of ERK (Li et al., 1997). Third, a
role for FAK has been implicated in the shear induced mechanotaxis of endothe-
lial cells based on studies using GFP-tagged FAK which revealed that FAK is
recruited to newly formed focal adhesions at the leading edge of cells migrating
in the direction of fluid flow (Li et al., 2002b). p130cas is rapidly phosphorylated
in response to FSS. Determining whether shear induced p130 cas phosphoryla-
tion is dependent on the activity of FAK remains to be determined. FAK plays an
essential function in vivo as evidenced by the fact the FAK / mouse is embryonic
lethal (Ilic et al., 1995). Studies using fibroblasts isolated from FAK / embryos
support a role for FAK in several cellular processes related to cell adhesion, per-
haps via FAK-mediated inhibition of Rho GTPase activity (Chen et al., 2002; Ilic
et al., 2003; Sieg et al., 2000; Sokabe et al., 1997). Alteration of cell motility in
FAK / fibroblasts (Sieg et al., 1999), another focal adhesion regulated process,
further suggests a critical role for FAK in signaling pathways mediated through
focal adhesions. This hypothesis is supported by a study showing that mechanical
stimulation of trabecular bone tissue activates FAK in vivo (Moalli et al., 2001).
Recently the role of FAK in bone differentiation and bone regeneration was
examined using a conditional fak/ mouse. It was determined that FAK is not
required for osteoblast differentiation in vivo, but FAK is needed for appropriate
bone regeneration in adult mice (Kim et al., 2007). Although these data implicate a
role for FAK in bone biology, there is very little known regarding the role of FAK
during mechanotransduction. We have investigated the role of FAK as an important
mechanosensory component for FSS-induced signaling in osteoblasts (Young et al.,
2009). We examined both early and late FSS-induced signaling pathways as mea-
sured by activation of extracellular signal-related kinases (ERK) phosphorylation,
COX-2 up-regulation, PGE2 release, c-fos up-regulation, and osteopontin (OPN)
upregulation. Furthermore, we used osteoblasts from multiple sources and three
different methods to disrupt FAK activity to test our hypothesis. Using siRNA tech-
nology to target FAK expression, we found that reduced FAK expression resulted in
a decrease in both the early and late FSS-induced signaling pathways in osteoblasts.
In addition, we used dominant negative FRNK, which is endogenously expressed
in osteoblasts, to inhibit FAK activity. Over expression of FRNK disrupted FAK
activation, which also resulted in a reduced response to FSS in osteoblasts. FAK/
osteoblasts were also examined and they exhibited a diminished response to FSS.
Importantly, re-expression of FAK in the FAK/ osteoblasts rescued FSS-induced
mechanotransduction as measured by COX-2 up-regulation and OPN expression.
Nuclear factor-kappa B (NF-B) activity also plays a key role in post-natal bone
remodeling by inhibiting bone formation in mice (Chang et al., 2009), and NF-B
nuclear translocation is activated by FSS in osteoblasts (Chen et al., 2003). However
it has also been reported that oscillatory fluid shear stress inhibited TNF- induced
NF-B activation in osteoblast-like cells (Kurokouchi et al., 2001). Our laboratory
has also evaluated the role of FAK in regulation of fluid shear-induced activation
284 S.R.L. Yong and F.M. Pavalko
12.3.2 Wnt/-Catenin/Sclerostin
Fluid shear stress and parathyroid hormone (PTH) both promote anabolic bone
formation. Interestingly, both fluid shear and PTH also promote translocation of
-catenin from the cytoplasm to the nucleus, a critical aspect of mechanosome activ-
ity (Kulkarni et al., 2005; Norvell et al., 2004; Robinson et al., 2006; Santos et al.,
2010; Tobimatsu et al., 2006). Because -catenin plays a role in some load-induced
changes in osteoblast gene expression (Case et al., 2008) and is sensitive to mechani-
cal load and PTH (Wang et al., 2008), it may play a key role in mechanically-induced
bone formation, reviewed in (Williams and Insogna, 2009). A breakthrough in
understanding of the role of -catenin signaling in bone occurred when it was real-
ized that the low-density lipoprotein receptor-related protein 5 (LRP5) played a key
role in regulating the skeleton (Gong et al., 2001; Little et al., 2002). Patients with
loss of function mutations in LRP5 have severely reduced bone mass (Gong et al.,
2001). Turner and colleagues investigated the role of LRP5 on bone strength using
mice engineered with a loss-of-function mutation in Lrp5 (Sawakami et al., 2006).
They found that that Lrp5 is critical for mechanotransduction in osteoblasts. These
and other studies, reviewed recently in (Robling and Turner, 2009b), strongly sug-
gest that LRP5 plays a key role in transduction of mechanical signals into a proper
skeletal response.
Lrp5 appears to play a critical role in the Wnt/-catenin signaling pathway.
Secreted Wnt proteins bind a complex of proteins at the cell surface including Lrp5
and an associated transmembrane co-receptor called Frizzled. Activation of this
complex at the cell surface leads to the translocation of -catenin to the nucleus,
which complexes with members of the TCF/LEF transcription factor family to
regulate gene transcription. Several cytoplasmic proteins control the accumulation
of -catenin in the cytoplasm including glycogen synthase 3 (GSK3), casein
kinase 1 (Ck1), the protein disheveled (Dsh), Axin, and adenomatous polyposis coli
gene product (Apc). When Wnt signaling activity is low, a complex of proteins
including axin, Ck1 and Gsk3i interact with -catenin resulting in the phospho-
rylation and ubiquitination of -catenin, which targets the -catenin protein for
proteosomal degradation. Increased Wnt signaling via Lrp5 disrupts this complex
causing stabilization and accumulation of -catenin in the cytoplasm and allows
12 Cellular Mechanisms of Mechanotransduction in Bone 285
for its accumulation in the nucleus. The observation that mechanical loading pro-
motes -catenin nuclear translocation and activity in bone cells strongly supports
a link between loading and Wnt/-catenin signaling in bone (Norvell et al., 2004;
Robinson et al., 2006).
Although there is considerable evidence to suggest mechanical loading of bone
involves Wnt signaling and regulation of -catenin activity via Lrp5, less clear is
precisely how mechanical stimulation alters this pathway. Most thought in this area
considers whether a decrease in antagonistic signals (Wnt inhibitors) or an increase
in agonist activity (Wnt secretion or stimulation of Wnt signaling) is involved. A key
molecule receiving a great deal of attention is the protein product of the SOST gene,
sclerostin, reviewed recently in detail (Robling and Turner, 2009b). Sclerostin has
been identified as a modulator of Lrp5 activity in response to mechanical loading
in bone. Sclerostin is expressed exclusively in osteocytes, the most abundant cells
in bone and the cells most uniquely positioned to detect and respond to changes
in mechanical loading in bone. This protein is a potent inhibitor of bone forma-
tion and several mutations in the SOST gene have been associated with high bone
mass (Balemans et al., 2001, 2002). Sclerostin binds Lrp5 and mechanical load-
ing of bone results in a dramatic decrease in sclerostin protein levels and in SOST
transcripts (Robling et al., 2008). Significantly, regions of the bone that showed the
most dramatic decreases in sclerostin were those that experienced the highest levels
of strain and exhibited the most new bone formation in response to loading. Thus,
the level of expressed sclerostin by osteocytes appears to tightly control the bal-
ance between mechanical load and bone formation, potentially through inhibition
of Wnt/Lrp5/-catenin signaling.
Gap junctions, formed by the association of connexin molecules within cell mem-
branes, also clearly play a role in the propagation of mechanical signals between
bone cells in response to loading (Bonewald, 2006; Donahue, 2000; Jiang et al.,
2007). The role of gap junctions in oscillatory fluid flow-induced changes in cal-
cium and prostaglandin production was demonstrated in osteoblast-like MC3T3-E1
cells with normal or defective gap junctional communication (Saunders et al., 2001).
Connexin 43 (Cx43) plays a particularly important role in bone and expression of a
dominant-negative Cx43 disrupts gap junction communication in and demonstrated
that prostaglandin production in bone cells required functional gap junctions, while
shear-induced cytosolic calcium spikes did not. A recent study investigated whether
inhibition of gap junction communication between bone cells affected the response
to fluid flow (Jekir and Donahue, 2009). These studies showed that in the pres-
ence of the gap junction inhibitor 18 beta-glycyrrhetinic acid (BGA), MC3T3-E1
cells exposed to oscillatory fluid flow failed to upregulate expression of osteopontin
mRNA 24 h after flow, in contrast to flowed cells that did not receive the inhibitor.
Surprisingly, however, osteopontin protein secretion was still increased 48 h after
flow despite the inhibition of gap junctions. Gap junction hemichannels which are
286 S.R.L. Yong and F.M. Pavalko
formed by connexins on a cell that are not in contact with connexins on an adjacent
cell also mediate the response of bone cells to loading (Cherian et al., 2005; Siller-
Jackson et al., 2008). For example, fluid flow induces translocation of Cx43 to the
membrane surface where unapposed hemichannels are formed. These hemichan-
nels function to promote mechanically-induced prostaglandin secretion (Cherian
et al., 2005). Release of prostaglandins by fluid shear was inhibited by an antibody
that specifically blocks Cx43-hemichannels, but not gap junctions or other channels
(Burra and Jiang, 2009).
12.3.4 NFAT
Recent studies suggest a novel mechanism through which mechanical signals may
be propagated in bone. Nuclear factor of activated T cells (NFAT), a transcription
factor well known in inflammatory signaling pathways, was shown to be activated in
response to mechanical stimulation and mediated cyclooxygenase-2 (Cox-2) expres-
sion (Celil Aydemir et al., 2010; Riddle et al., 2007). Application of fluid shear
stress or tensile strain resulted in translocation of NFAT from the cytoplasm to
the nucleus in bone cells. This pathway could be blocked by a peptide inhibitor of
NFAT signaling. It has been suggested that ATP is required for fluid flow-induced
increases in intracellular calcium concentration, activation of calcineurin, and the
nuclear translocation of NFAT (Riddle et al., 2007). Together, these findings sup-
port a novel role for NFAT in mechanotransduction in bone and are consistent with
the tenants of the mechanosome hypothesis which suggests that mechanical load-
ing propagates signals from the cell membrane, through the cytoplasm, and into the
nucleus to regulate transcription of mechanically-sensitive genes.
MC3T3-E1 cells, Rangaswami et al. (2009) have reported that cyclic GMP-
dependent (cGMP) protein kinase (PKG II) was activated in response to fluid shear
stress via a NO-dependent mechanism. PKG II was shown to play a role in fluid
shear stress induced expression of several genes important in skeletal regulation
including c-fos, fra-1, fra-2, and fosB. When cells were treated with pharmacologi-
cal inhibitors of the NO/cGMP/PKG pathway, shear-induced expression of each of
these genes was blocked. Furthermore, shear-induced MAP kinase activation was
shown to be dependent on this NO/cGMP/PKG pathway. Interestingly, the effects
of fluid shear stress on gene expression and MAP activity could be mimicked by
treating cells with a membrane-permeable cGMP analog.
12.3.6 Nmp4/CIZ
transcription factors that associate with adhesion complex proteins (p130cas and
-catenin, respectively) and appear to translocate between adhesion sites at the
membrane and the nucleus. In a recent study we examined the relationship between
Nmp4/CIZ and -catenin signaling in response to mechanical stimulation. We
found that translocation of -catenin to the nucleus in osteoblasts that is normally
induced by oscillatory fluid shear stress (OFSS) is attenuated in the presence of
Nmp4/CIZ. Furthermore, we found that other aspects of OFSS-induced mechan-
otransduction that are associated with the -catenin pathway, including ERK, Akt
and GSK3 activity, as well as expression of the -catenin-responsive protein cyclin
D1 (Tetsu and McCormick, 1999) are enhanced in cells lacking Nmp4/CIZ. Finally,
we found that in the absence of Nmp4/CIZ, OFSS-induced reorganization of the
actin cytoskeleton and formation of focal adhesions, as has been described previ-
ously (Ponik and Pavalko, 2004) is qualitatively enhanced, further suggesting that
Nmp4/CIZ may reduce the sensitivity of bone cells to mechanical stimuli. Together,
these results support the concept that Nmp4 normally plays an inhibitory role in
bone cell mechanotransduction.
It is unclear how Nmp4/CIZ inhibits nuclear translocation of -catenin. One
potential convergence point between the p130cas /Nmp4/CIZ and Lef1/-catenin
pathways may be the SMAD proteins (Childress et al., 2010). R-SMAD activity
is upregulated by both PTH and load, and these proteins are important components
of the anabolic pathways in bone (Li, 2008; Ogita et al., 2008; Sowa et al., 2003;
Tobimatsu et al., 2006). The R-SMADs physically interact with the Lef1/-catenin
proteins and together synergistically enhance osteoblast gene expression (Guo et al.,
2008; Labbe et al., 2000; Sato et al., 2009). Nmp4/CIZ attenuates R-SMAD activ-
ity (Shen et al., 2002) but it is not yet clear whether this occurs in the cytoplasm,
the nucleus or both. Additionally, p130cas , a major binding partner of Nmp4/CIZ,
suppresses R-SMAD activation (Kim et al., 2008). Elucidating the components and
interactions comprising this putative mechanosome network is necessary for under-
standing the cellular and molecular basis of the skeletons response to PTH and
loading.
Mechanical loading clearly plays a key role in the regulation of the mammalian
skeleton. This review has discussed several of the key mechanisms used by bone
cells to convert mechanical signals into altered biochemical responses. Recent
research has resulted in significant progress toward the identification of the key
molecular components used by cells to detect mechanical stimuli and to propagate
those signals through the cytoplasm and into the nucleus. Future work will undoubt-
edly focus on gaining a better understanding of how these individual molecular
pathways are integrated into functional tissue responses to mechanical loading.
Acknowledgements This work was supported by NIH AR052682 and AR056188
12 Cellular Mechanisms of Mechanotransduction in Bone 289
References
Abbi S, Guan JL (2002) Focal adhesion kinase: protein interactions and cellular functions. Histol
Histopathol 17(4):11631171
Ajubi NE, Klein-Nulend J, Alblas MJ, Burger EH, Nijweide PJ (1999) Signal transduction path-
ways involved in fluid flow-induced PGE2 production by cultured osteocytes. Am J Physiol
276(1 Pt 1):E171178
Alahari SK, Reddig PJ, Juliano RL (2002) Biological aspects of signal transduction by cell
adhesion receptors. Int Rev Cytol 220:145184
Allori AC, Sailon AM, Pan JH, Warren SM (2008) Biological basis of bone formation, remodeling,
and repair-part III: biomechanical forces. Tissue Eng Part B Rev 14(3):285293
Alvarez M, Long H, Onyia J, Hock J, Xu W, Bidwell J (1997) Rat osteoblast and osteosarcoma
nuclear matrix proteins bind with sequence specificity to the rat type I collagen promoter.
Endocrinology 138(1):482489
Alvarez M, Thunyakitpisal P, Morrison P, Onyia J, Hock J, Bidwell JP (1998) PTH-responsive
osteoblast nuclear matrix architectural transcription factor binds to the rat type I collagen
promoter. J Cell Biochem 69(3):336352
Balemans W, Ebeling M, Patel N, Van Hul E, Olson P, Dioszegi M, Lacza C, Wuyts W, Van
Den Ende J, Willems P, Paes-Alves AF, Hill S, Bueno M, Ramos FJ, Tacconi P, Dikkers FG,
Stratakis C, Lindpaintner K, Vickery B, Foernzler D, Van Hul W (2001) Increased bone density
in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum Mol Genet
10(5):537543
Balemans W, Patel N, Ebeling M, Van Hul E, Wuyts W, Lacza C, Dioszegi M, Dikkers FG,
Hildering P, Willems PJ, Verheij JB, Lindpaintner K, Vickery B, Foernzler D, Van Hul W
(2002) Identification of a 52 kb deletion downstream of the SOST gene in patients with van
Buchem disease. J Med Genet 39(2):9197
Berk BC, Corson MA, Peterson TE, Tseng H (1995) Protein kinases as mediators of fluid shear
stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent
and calcium-independent events activated by flow. J Biomech 28(12):14391450
Bidwell JP, Alvarez M, Feister H, Onyia J, Hock J (1998) Nuclear matrix proteins and osteoblast
gene expression. J Bone Miner Res 13(2):155167
Bonewald LF (2006) Mechanosensation and Transduction in Osteocytes. Bonekey Osteovision
3(10):715
Bonewald LF, Johnson ML (2008) Osteocytes, mechanosensing and Wnt signaling. Bone
42(4):606615
Burra S, Jiang JX (2009) Connexin 43 hemichannel opening associated with Prostaglandin E(2)
release is adaptively regulated by mechanical stimulation. Commun Integr Biol 2(3):239240
Burridge K, Chrzanowska-Wodnicka M (1996) Focal adhesions, contractility, and signaling. Annu
Rev Cell Dev Biol 12:463518
Carvalho RS, Kostenuik PJ, Salih E, Bumann A, Gerstenfeld LC (2003) Selective adhesion of
osteoblastic cells to different integrin ligands induces osteopontin gene expression. Matrix Biol
22(3):241249
Case N, Ma M, Sen B, Xie Z, Gross TS, Rubin J (2008) Beta-catenin levels influence rapid
mechanical responses in osteoblasts. J Biol Chem 283(43):2919629205
Cattaruzza M, Lattrich C, Hecker M (2004) Focal adhesion protein zyxin is a mechanosen-
sitive modulator of gene expression in vascular smooth muscle cells. Hypertension 43(4):
726730
Celil Aydemir AB, Minematsu H, Gardner TR, Kim KO, Ahn JM, Lee FY (2010) Nuclear factor
of activated T cells mediates fluid shear stress- and tensile strain-induced Cox2 in human and
murine bone cells. Bone 46(1):167175
Chang J, Wang Z, Tang E, Fan Z, McCauley L, Franceschi R, Guan K, Krebsbach PH, Wang CY
(2009) Inhibition of osteoblastic bone formation by nuclear factor-kappaB. Nat Med 15(6):
682689
290 S.R.L. Yong and F.M. Pavalko
Guo W, Flanagan J, Jasuja R, Kirkland J, Jiang L, Bhasin S (2008) The effects of myostatin on
adipogenic differentiation of human bone marrow-derived mesenchymal stem cells are medi-
ated through cross-communication between Smad3 and Wnt/beta-catenin signaling pathways.
J Biol Chem 283(14):91369145
Hauck CR, Hsia DA, Schlaepfer DD (2002) The focal adhesion kinase--a regulator of cell
migration and invasion. IUBMB Life 53(2):115119
Hino K, Nakamoto T, Nifuji A, Morinobu M, Yamamoto H, Ezura Y, Noda M (2007) Deficiency
of CIZ, a nucleocytoplasmic shuttling protein, prevents unloading-induced bone loss through
the enhancement of osteoblastic bone formation in vivo. Bone 40(4):852860
Huang H, Kamm RD, Lee RT (2004) Cell mechanics and mechanotransduction: pathways, probes,
and physiology. Am J Physiol Cell Physiol 287(1):C111
Hung CT, Allen FD, Pollack SR, Brighton CT (1996) Intracellular Ca2+ stores and extracellu-
lar Ca2+ are required in the real-time Ca2+ response of bone cells experiencing fluid flow.
J Biomech 29(11):14111417
Hung CT, Pollack SR, Reilly TM, Brighton CT (1995) Real-time calcium response of cultured
bone cells to fluid flow. Clin Orthop Relat Res(313):256269
Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S, Fujimoto J, Okada M,
Yamamoto T (1995) Reduced cell motility and enhanced focal adhesion contact formation in
cells from FAK-deficient mice. Nature 377(6549):539544
Ilic D, Kovacic B, McDonagh S, Jin F, Baumbusch C, Gardner DG, Damsky CH (2003)
Focal adhesion kinase is required for blood vessel morphogenesis. Circ Res 92(3):
300307
Jalali S, del Pozo MA, Chen K, Miao H, Li Y, Schwartz MA, Shyy JY, Chien S (2001) Integrin-
mediated mechanotransduction requires its dynamic interaction with specific extracellular
matrix (ECM) ligands. Proc Natl Acad Sci U S A 98(3):10421046
Jekir MG, Donahue HJ (2009) Gap junctions and osteoblast-like cell gene expression in response
to fluid flow. J Biomech Eng 131(1):011005
Jessop HL, Rawlinson SC, Pitsillides AA, Lanyon LE (2002) Mechanical strain and fluid move-
ment both activate extracellular regulated kinase (ERK) in osteoblast-like cells but via different
signaling pathways. Bone 31(1):186194
Jiang JX, Siller-Jackson AJ, Burra S (2007) Roles of gap junctions and hemichannels in bone cell
functions and in signal transmission of mechanical stress. Front Biosci 12:14501462
Johnson DL, McAllister TN, Frangos JA (1996) Fluid flow stimulates rapid and continuous release
of nitric oxide in osteoblasts. Am J Physiol 271(1 Pt 1):E205208
Jones DB, Nolte H, Scholubbers JG, Turner E, Veltel D (1991) Biochemical signal transduction of
mechanical strain in osteoblast-like cells. Biomaterials 12(2):101110
Ke HZ, Qi H, Weidema AF, Zhang Q, Panupinthu N, Crawford DT, Grasser WA, Paralkar VM,
Li M, Audoly LP, Gabel CA, Jee WS, Dixon SJ, Sims SM, Thompson DD (2003) Deletion of
the P2X7 nucleotide receptor reveals its regulatory roles in bone formation and resorption. Mol
Endocrinol 17(7):13561367
Kim JB, Leucht P, Luppen CA, Park YJ, Beggs HE, Damsky CH, Helms JA (2007) Reconciling
the roles of FAK in osteoblast differentiation, osteoclast remodeling, and bone regeneration.
Bone 41(1):3951
Kim W, Seok Kang Y, Soo Kim J, Shin NY, Hanks SK, Song WK (2008) The integrin-coupled
signaling adaptor p130Cas suppresses Smad3 function in transforming growth factor-beta
signaling. Mol Biol Cell 19(5):21352146
Koyama E, Young B, Nagayama M, Shibukawa Y, Enomoto-Iwamoto M, Iwamoto M, Maeda Y,
Lanske B, Song B, Serra R, Pacifici M (2007) Conditional Kif3a ablation causes abnormal
hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage
formation during mouse skeletogenesis. Development 134(11):21592169
Kulkarni NH, Halladay DL, Miles RR, Gilbert LM, Frolik CA, Galvin RJ, Martin TJ, Gillespie MT,
Onyia JE (2005) Effects of parathyroid hormone on Wnt signaling pathway in bone. J Cell
Biochem 95(6):11781190
292 S.R.L. Yong and F.M. Pavalko
Kurokouchi K, Jacobs CR, Donahue HJ (2001) Oscillating fluid flow inhibits TNF-alpha -induced
NF-kappa B activation via an Ikappa B kinase pathway in osteoblast-like UMR106 cells. J Biol
Chem 276(16):1349913504
Labbe E, Letamendia A, Attisano L (2000) Association of Smads with lymphoid enhancer binding
factor 1/T cell-specific factor mediates cooperative signaling by the transforming growth factor-
beta and wnt pathways. Proc Natl Acad Sci U S A 97(15):83588363
Labrador V, Chen KD, Li YS, Muller S, Stoltz JF, Chien S (2003) Interactions of mechanotrans-
duction pathways. Biorheology 40(13):4752
Li B (2008) Bone morphogenetic protein-Smad pathway as drug targets for osteoporosis and cancer
therapy. Endocr Metab Immune Disord Drug Targets 8(3):208219
Li J, Duncan RL, Burr DB, Turner CH (2002a) L-type calcium channels mediate mechanically
induced bone formation in vivo. J Bone Miner Res 17(10):17951800
Li J, Liu D, Ke HZ, Duncan RL, Turner CH (2005) The P2X7 nucleotide receptor mediates skeletal
mechanotransduction. J Biol Chem 280(52):4295242959
Li J, Meyer R, Duncan RL, Turner CH (2009) P2X7 nucleotide receptor plays an important role in
callus remodeling during fracture repair. Calcif Tissue Int 84(5):405412
Li S, Butler P, Wang Y, Hu Y, Han DC, Usami S, Guan JL, Chien S (2002b) The role of the
dynamics of focal adhesion kinase in the mechanotaxis of endothelial cells. Proc Natl Acad Sci
U S A 99(6):35463551
Li S, Kim M, Hu YL, Jalali S, Schlaepfer DD, Hunter T, Chien S, Shyy JY (1997) Fluid shear
stress activation of focal adhesion kinase. Linking to mitogen-activated protein kinases. J Biol
Chem 272(48):3045530462
Lin TH, Aplin AE, Shen Y, Chen Q, Schaller M, Romer L, Aukhil I, Juliano RL (1997) Integrin-
mediated activation of MAP kinase is independent of FAK: evidence for dual integrin signaling
pathways in fibroblasts. J Cell Biol 136(6):13851395
Little RD, Carulli JP, Del Mastro RG, Dupuis J, Osborne M, Folz C, Manning SP, Swain PM,
Zhao SC, Eustace B, Lappe MM, Spitzer L, Zweier S, Braunschweiger K, Benchekroun Y,
Hu X, Adair R, Chee L, FitzGerald MG, Tulig C, Caruso A, Tzellas N, Bawa A, Franklin B,
McGuire S, Nogues X, Gong G, Allen KM, Anisowicz A, Morales AJ, Lomedico PT, Recker
SM, Van Eerdewegh P, Recker RR, Johnson ML (2002) A mutation in the LDL receptor-
related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am J Hum
Genet 70(1):1119
Liu D, Genetos DC, Shao Y, Geist DJ, Li J, Ke HZ, Turner CH, Duncan RL (2008) Activation of
extracellular-signal regulated kinase (ERK1/2) by fluid shear is Ca(2+)- and ATP-dependent in
MC3T3-E1 osteoblasts. Bone 42(4):644652
Liu W, Murcia NS, Duan Y, Weinbaum S, Yoder BK, Schwiebert E, Satlin LM (2005)
Mechanoregulation of intracellular Ca2+ concentration is attenuated in collecting duct of
monocilium-impaired orpk mice. Am J Physiol Renal Physiol 289(5):F978988
Liu Y, Chen BP, Lu M, Zhu Y, Stemerman MB, Chien S, Shyy JY (2002) Shear stress activation
of SREBP1 in endothelial cells is mediated by integrins. Arterioscler Thromb Vasc Biol 22(1):
7681
Lobo M, Zachary I (2000) Nuclear localization and apoptotic regulation of an amino-terminal
domain focal adhesion kinase fragment in endothelial cells. Biochem Biophys Res Commun
276(3):10681074
Loza J, Stephan E, Dolce C, Dziak R, Simasko S (1994) Calcium currents in osteoblastic cells:
dependence upon cellular growth stage. Calcif Tissue Int 55(2):128133
Malone AM, Anderson CT, Stearns T, Jacobs CR (2007a) Primary cilia in bone. J Musculoskelet
Neuronal Interact 7(4):301
Malone AM, Anderson CT, Tummala P, Kwon RY, Johnston TR, Stearns T, Jacobs CR (2007b)
Primary cilia mediate mechanosensing in bone cells by a calcium-independent mechanism.
Proc Natl Acad Sci U S A 104(33):1332513330
Matsuda N, Morita N, Matsuda K, Watanabe M (1998) Proliferation and differentiation of
human osteoblastic cells associated with differential activation of MAP kinases in response to
12 Cellular Mechanisms of Mechanotransduction in Bone 293
epidermal growth factor, hypoxia, and mechanical stress in vitro. Biochem Biophys Res
Commun 249(2):350354
Matthews JL, Martin JH (1971) Intracellular transport of calcium and its relationship to homeosta-
sis and mineralization. An electron microscope study. Am J Med 50(5):589597
McAllister TN, Frangos JA (1999) Steady and transient fluid shear stress stimulate NO release in
osteoblasts through distinct biochemical pathways. J Bone Miner Res 14(6):930936
McGarry JG, Maguire P, Campbell VA, OConnell BC, Prendergast PJ, Jarvis SP (2008)
Stimulation of nitric oxide mechanotransduction in single osteoblasts using atomic force
microscopy. J Orthop Res 26(4):513521
McGlashan SR, Haycraft CJ, Jensen CG, Yoder BK, Poole CA (2007) Articular cartilage and
growth plate defects are associated with chondrocyte cytoskeletal abnormalities in Tg737orpk
mice lacking the primary cilia protein polaris. Matrix Biol 26(4):234246
Moalli MR, Wang S, Caldwell NJ, Patil PV, Maynard CR (2001) Mechanical stimulation induces
pp125(FAK) and pp60(src) activity in an in vivo model of trabecular bone formation. J Appl
Physiol 91(2):912918
Morinobu M, Nakamoto T, Hino K, Tsuji K, Shen ZJ, Nakashima K, Nifuji A, Yamamoto H,
Hirai H, Noda M (2005) The nucleocytoplasmic shuttling protein CIZ reduces adult bone mass
by inhibiting bone morphogenetic protein-induced bone formation. J Exp Med 201(6):961970
Nakamoto T, Yamagata T, Sakai R, Ogawa S, Honda H, Ueno H, Hirano N, Yazaki Y, Hirai H
(2000) CIZ, a zinc finger protein that interacts with p130(cas) and activates the expression of
matrix metalloproteinases. Mol Cell Biol 20(5):16491658
Nix DA, Fradelizi J, Bockholt S, Menichi B, Louvard D, Friederich E, Beckerle MC (2001)
Targeting of zyxin to sites of actin membrane interaction and to the nucleus. J Biol Chem
276(37):3475934767
North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82(4):10131067
Norvell SM, Alvarez M, Bidwell JP, Pavalko FM (2004) Fluid shear stress induces beta-catenin
signaling in osteoblasts. Calcif Tissue Int 75(5):396404
Ogawa M, Hiraoka Y, Aiso S (2003) Nuclear translocation of Xenopus laevis paxillin. Biochem
Biophys Res Commun 304(4):676683
Ogita M, Rached MT, Dworakowski E, Bilezikian JP, Kousteni S (2008) Differentiation and
proliferation of periosteal osteoblast progenitors are differentially regulated by estrogens and
intermittent parathyroid hormone administration. Endocrinology 149(11):57135723
Okuda M, Takahashi M, Suero J, Murry CE, Traub O, Kawakatsu H, Berk BC (1999) Shear stress
stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation.
J Biol Chem 274(38):2680326809
Panetti TS (2002) Tyrosine phosphorylation of paxillin, FAK, and p130CAS: effects on cell
spreading and migration. Front Biosci 7:d143150
Papachristou DJ, Papachroni KK, Basdra EK, Papavassiliou AG (2009) Signaling networks and
transcription factors regulating mechanotransduction in bone. Bioessays 31(7):794804
Papachroni KK, Karatzas DN, Papavassiliou KA, Basdra EK, Papavassiliou AG (2009)
Mechanotransduction in osteoblast regulation and bone disease. Trends Mol Med 15(5):
208216
Parsons JT, Martin KH, Slack JK, Taylor JM, Weed SA (2000) Focal adhesion kinase: a regulator
of focal adhesion dynamics and cell movement. Oncogene 19(49):56065613
Pavalko FM, Chen NX, Turner CH, Burr DB, Atkinson S, Hsieh YF, Qiu J, Duncan RL (1998)
Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-
integrin interactions. Am J Physiol 275(6 Pt 1):C15911601
Pavalko FM, Norvell SM, Burr DB, Turner CH, Duncan RL, Bidwell JP (2003) A Model
for mechanotransduction in bone cells: The load-bearing mechanosomes. J Cell Biochem
88(1):104112
Pitsillides AA, Rawlinson SC, Suswillo RF, Bourrin S, Zaman G, Lanyon LE (1995) Mechanical
strain-induced NO production by bone cells: a possible role in adaptive bone (re)modeling?
FASEB J 9(15):16141622
294 S.R.L. Yong and F.M. Pavalko
Ponik SM, Pavalko FM (2004) Formation of focal adhesions on fibronectin promotes fluid shear
stress induction of COX-2 and PGE2 release in MC3T3-E1 osteoblasts. J Appl Physiol
97(1):135142
Rangaswami H, Marathe N, Zhuang S, Chen Y, Yeh JC, Frangos JA, Boss GR, Pilz RB (2009) Type
II cGMP-dependent protein kinase mediates osteoblast mechanotransduction. J Biol Chem
284(22):1479614808
Rawlinson SC, Pitsillides AA, Lanyon LE (1996) Involvement of different ion chan-
nels in osteoblasts and osteocytes early responses to mechanical strain. Bone 19(6):
609614
Riddle RC, Taylor AF, Rogers JR, Donahue HJ (2007) ATP release mediates fluid flow-induced
proliferation of human bone marrow stromal cells. J Bone Miner Res 22(4):589600
Robinson JA, Chatterjee-Kishore M, Yaworsky PJ, Cullen DM, Zhao W, Li C, Kharode Y, Sauter
L, Babij P, Brown EL, Hill AA, Akhter MP, Johnson ML, Recker RR, Komm BS, Bex FJ (2006)
Wnt/beta-catenin signaling is a normal physiological response to mechanical loading in bone.
J Biol Chem 281(42):3172031728
Robling AG, Childress P, Yu J, Cotte J, Heller A, Philip BK, Bidwell JP (2009) Nmp4/CIZ
suppresses parathyroid hormone-induced increases in trabecular bone. J Cell Physiol 219(3):
734743
Robling AG, Niziolek PJ, Baldridge LA, Condon KW, Allen MR, Alam I, Mantila SM, Gluhak-
Heinrich J, Bellido TM, Harris SE, Turner CH (2008) Mechanical stimulation of bone in vivo
reduces osteocyte expression of Sost/sclerostin. J Biol Chem 283(9):58665875
Robling AG, Turner CH (2009a) Mechanical Signaling for Bone Modeling and Remodeling. Crit
Rev Eukaryot Gene Expr 19(4):319338
Robling AG, Turner CH (2009b) Mechanical signaling for bone modeling and remodeling. Crit
Rev Eukaryot Gene Expr 19(4):319338
Ryder KD, Duncan RL (2001) Parathyroid hormone enhances fluid shear-induced [Ca2+]i sig-
naling in osteoblastic cells through activation of mechanosensitive and voltage-sensitive Ca2+
channels. J Bone Miner Res 16(2):240248
Sakai K, Mohtai M, Iwamoto Y (1998) Fluid Shear Stress Increases Transforming Growth Factor
Beta 1 Expression in Human Osteoblast-like Cells: Modulation by Cation Channel Blockades.
Calcif Tissue Int 63(6):515520
Santos A, Bakker AD, Zandieh-Doulabi B, de Blieck-Hogervorst JM, Klein-Nulend J (2010)
Early activation of the beta-catenin pathway in osteocytes is mediated by nitric oxide, phos-
phatidyl inositol-3 kinase/akt, and focal adhesion kinase. Biochem Biophys Res Commun
391(1):364369
Sato MM, Nakashima A, Nashimoto M, Yawaka Y, Tamura M (2009) Bone morphogenetic
protein-2 enhances Wnt/beta-catenin signaling-induced osteoprotegerin expression. Genes
Cells 14(2):141153
Saunders MM, You J, Trosko JE, Yamasaki H, Li Z, Donahue HJ, Jacobs CR (2001) Gap
junctions and fluid flow response in MC3T3-E1 cells. Am J Physiol Cell Physiol 281(6):
C19171925
Sawada Y, Tamada M, Dubin-Thaler BJ, Cherniavskaya O, Sakai R, Tanaka S, Sheetz MP (2006)
Force sensing by mechanical extension of the Src family kinase substrate p130Cas. Cell
127(5):10151026
Sawakami K, Robling AG, Ai M, Pitner ND, Liu D, Warden SJ, Li J, Maye P, Rowe DW, Duncan
RL, Warman ML, Turner CH (2006) The Wnt co-receptor LRP5 is essential for skeletal
mechanotransduction but not for the anabolic bone response to parathyroid hormone treatment.
J Biol Chem 281(33):2369823711
Schwartz EA, Leonard ML, Bizios R, Bowser SS (1997) Analysis and modeling of the primary
cilium bending response to fluid shear. Am J Physiol 272(1 Pt 2):F132138
Shen ZJ, Nakamoto T, Tsuji K, Nifuji A, Miyazono K, Komori T, Hirai H, Noda M (2002) Negative
regulation of bone morphogenetic protein/Smad signaling by Cas-interacting zinc finger protein
in osteoblasts. J Biol Chem 277(33):2984029846
12 Cellular Mechanisms of Mechanotransduction in Bone 295
Sieg DJ, Hauck CR, Ilic D, Klingbeil CK, Schaefer E, Damsky CH, Schlaepfer DD (2000) FAK
integrates growth-factor and integrin signals to promote cell migration. Nat Cell Biol 2(5):
249256
Sieg DJ, Hauck CR, Schlaepfer DD (1999) Required role of focal adhesion kinase (FAK) for
integrin-stimulated cell migration. J Cell Sci 112 ( Pt 16):26772691
Siller-Jackson AJ, Burra S, Gu S, Xia X, Bonewald LF, Sprague E, Jiang JX (2008) Adaptation
of connexin 43-hemichannel prostaglandin release to mechanical loading. J Biol Chem
283(39):2637426382
Sokabe M, Naruse K, Sai S, Yamada T, Kawakami K, Inoue M, Murase K, Miyazu M (1997)
Mechanotransduction and intracellular signaling mechanisms of stretch- induced remodeling
in endothelial cells. Heart Vessels Suppl 12:191193
Sowa H, Kaji H, Canaff L, Hendy GN, Tsukamoto T, Yamaguchi T, Miyazono K, Sugimoto T,
Chihara K (2003) Inactivation of menin, the product of the multiple endocrine neoplasia type
1 gene, inhibits the commitment of multipotential mesenchymal stem cells into the osteoblast
lineage. J Biol Chem 278(23):2105821069
Stewart A, Ham C, Zachary I (2002) The focal adhesion kinase amino-terminal domain localises
to nuclei and intercellular junctions in HEK 293 and MDCK cells independently of tyrosine
397 and the carboxy-terminal domain. Biochem Biophys Res Commun 299(1):6273
Takahashi M, Ishida T, Traub O, Corson MA, Berk BC (1997) Mechanotransduction in endothelial
cells: temporal signaling events in response to shear stress. J Vasc Res 34(3):212219
Tetsu O, McCormick F (1999) Beta-catenin regulates expression of cyclin D1 in colon carcinoma
cells. Nature 398(6726):422426
Thunyakitpisal P, Alvarez M, Tokunaga K, Onyia JE, Hock J, Ohashi N, Feister H, Rhodes SJ,
Bidwell JP (2001) Cloning and functional analysis of a family of nuclear matrix transcription
factors (NP/NMP4) that regulate type I collagen expression in osteoblasts. J Bone Miner Res
16(1):1023
Tobimatsu T, Kaji H, Sowa H, Naito J, Canaff L, Hendy GN, Sugimoto T, Chihara K (2006)
Parathyroid hormone increases beta-catenin levels through Smad3 in mouse osteoblastic cells.
Endocrinology 147(5):25832590
Tobin JL, Beales PL (2007) Bardet-Biedl syndrome: beyond the cilium. Pediatr Nephrol
22(7):926936
Turner CE (2000) Paxillin interactions. J Cell Sci 113 Pt 23:41394140
Turner CH, Forwood MR, Otter MW (1994) Mechanotransduction in bone: do bone cells act as
sensors of fluid flow? FASEB J 8(11):875878
Turner CH, Pavalko FM (1998) Mechanotransduction and functional response of the skeleton to
physical stress: the mechanisms and mechanics of bone adaptation. J Orthop Sci 3(6):346355
Turner CH, Warden SJ, Bellido T, Plotkin LI, Kumar N, Jasiuk I, Danzig J, Robling AG (2009)
Mechanobiology of the skeleton. Sci Signal 2(68):pt3
Tzima E, del Pozo MA, Shattil SJ, Chien S, Schwartz MA (2001) Activation of integrins in
endothelial cells by fluid shear stress mediates Rho-dependent cytoskeletal alignment. EMBO
J 20(17):46394647
Urbich C, Dernbach E, Reissner A, Vasa M, Zeiher AM, Dimmeler S (2002) Shear stress-induced
endothelial cell migration involves integrin signaling via the fibronectin receptor subunits
alpha(5) and beta(1). Arterioscler Thromb Vasc Biol 22(1):6975
Urbich C, Walter DH, Zeiher AM, Dimmeler S (2000) Laminar shear stress upregulates
integrin expression: role in endothelial cell adhesion and apoptosis. Circ Res 87(8):
683689
Wang H, Li M, Lin PH, Yao Q, Chen C (2008) Fluid shear stress regulates the expression of TGF-
beta1 and its signaling molecules in mouse embryo mesenchymal progenitor cells. J Surg Res
150(2):266270
Watanuki M, Sakai A, Sakata T, Tsurukami H, Miwa M, Uchida Y, Watanabe K, Ikeda K,
Nakamura T (2002) Role of inducible nitric oxide synthase in skeletal adaptation to acute
increases in mechanical loading. J Bone Miner Res 17(6):10151025
296 S.R.L. Yong and F.M. Pavalko
Wheatley DN, Wang AM, Strugnell GE (1996) Expression of primary cilia in mammalian cells.
Cell Biol Int 20(1):7381
Whitfield JF (2008) The solitary (primary) cilium--a mechanosensory toggle switch in bone and
cartilage cells. Cell Signal 20(6):10191024
Williams BO, Insogna KL (2009) Where Wnts went: the exploding field of Lrp5 and Lrp6 signaling
in bone. J Bone Miner Res 24(2):171178
Xiao Z, Zhang S, Mahlios J, Zhou G, Magenheimer BS, Guo D, Dallas SL, Maser R,
Calvet JP, Bonewald L, Quarles LD (2006) Cilia-like structures and polycystin-1 in
osteoblasts/osteocytes and associated abnormalities in skeletogenesis and Runx2 expression.
J Biol Chem 281(41):3088430895
Yi XP, Wang X, Gerdes AM, Li F (2003) Subcellular redistribution of focal adhesion kinase and
its related nonkinase in hypertrophic myocardium. Hypertension 41(6):13171323
Yoder BK (2007) Role of primary cilia in the pathogenesis of polycystic kidney disease. J Am Soc
Nephrol 18(5):13811388
You J, Jacobs CR, Steinberg TH, Donahue HJ (2002) P2Y purinoceptors are responsible for oscil-
latory fluid flow-induced intracellular calcium mobilization in osteoblastic cells. J Biol Chem
277(50):4872448729
Young SR, Gerard-ORiley R, Kim JB, Pavalko FM (2009) Focal adhesion kinase is important
for fluid shear stress-induced mechanotransduction in osteoblasts. J Bone Miner Res 24(3):
411424
Young SRL, Gerard-ORiley R, Harrington M, Pavalko FM (2010) Activation of NF-kB by Fluid
Shear Stress, but not TNF-a, requires Focal Adhesion Kinase in Osteoblasts. Bone 47:7482
Zaman G, Pitsillides AA, Rawlinson SC, Suswillo RF, Mosley JR, Cheng MZ, Platts LA,
Hukkanen M, Polak JM, Lanyon LE (1999) Mechanical strain stimulates nitric oxide produc-
tion by rapid activation of endothelial nitric oxide synthase in osteocytes. J Bone Miner Res
14(7):11231131
Zhang J, Ryder KD, Bethel JA, Ramirez R, Duncan RL (2006) PTH-induced actin depolymer-
ization increases mechanosensitive channel activity to enhance mechanically stimulated Ca2+
signaling in osteoblasts. J Bone Miner Res 21(11):17291737
Chapter 13
The Mechanosensitivity of Cells in Joint Tissues:
Role in the Pathogenesis of Joint Diseases
Abstract Joint tissues, including cartilage, bone, meniscus, tendon, ligament and
synovial membrane, are exposed to high mechanical stimulation. The response of
these tissues to mechanical strains is a key factor of the OA onset and progression.
Any little failure in this mechanical function may lead to cell phenotype alteration
and tissue damages. While mechanical responses of cartilage are well known, very
few data are available on the effects of mechanical strains on other articular tis-
sues. Mechanical loading plays a dual role in the homeostasis of joint tissues: when
applied at moderate and physiological range, mechanical stimuli are beneficial to
the good health of joint tissues whereas excessive loading initiate abnormal tis-
sue remodeling and structural changes. Appropriate mechanical load could play an
important role in the homeostasis of joint tissues. This raises the question of the role
played by exercises in the prevention and treatment of OA. This paper is a narrative
review based on selected recent literature in this field.
13.1 Introduction
Osteoarthritis (OA) is the most common form of arthritic disease, and it is a major
cause of disability and impaired quality of life in the elderly. A hallmark of the
disease is the progressive degeneration of articular cartilage and subsequent joint
space narrowing. However, OA is a global disease affecting not only the cartilage,
but also synovial membrane, subchondral bone, tendons, ligaments and menisci. OA
is characterized by a progressive loss a cartilage, meniscus tears and clefts, synovial
Y. Henrotin (B)
Bone and Cartilage Research Unit, University of Lige, Institute of Pathology Level 5, CHU Sart-
Tilman, 4000 Lige, Belgium
e-mail: yhenrotin@ulg.ac.be
and tumor necrosis factor (TNF) alpha in cartilage is observed after joint impact
of canine patellae (Pickvance et al., 1993). In vitro studies show that exposure of
cartilage explants and chondrocytes to mechanical strain of high amplitude leads to
the synthesis of inflammatory mediators. Intermittent compression (0.10.5 MPa,
0.5 Hz) and shear stress (1.6 MPa) stimulates nitric oxide (NO), prostaglandin (PG)
E2 and IL-6 productions through the activation of respectively inducible NO syn-
thase (iNOS), cyclooxygenase (COX)-2 and IL-6 gene expression (Mohtai et al.,
1996; Das et al., 1997; Fermor et al., 2002).
In contrast, biochemical signals could have anti-inflammatory effects. Cyclic ten-
sile stress (CTS) counteracts the IL-1 dependent chondrocyte catabolic response.
CTS (10% of deformation, 1 Hz) reduce the IL-1beta dependent synthesis of IL-6
and PGE2 (Mathy-Hartert M, 2008) in chondrocytes. The IL-1beta dependent stimu-
lation of NO/iNOS, PGE2 /COX-2 protein and gene expression is decreased by CTS
application (48% of deformation, 0.05 Hz) (Gassner et al., 1999; Agarwal et al.,
2004). The inhibition of Agg synthesis due to IL-1 is also reversed. These effects are
mediated by Nuclear Factor (NF)-B: its translocation to the nucleus is prevented by
CTS. When magnitude of the strain is increased to 15% of deformation, these anti-
inflammatory effects are nullified (Agarwal et al., 2004). IL-1 dependent mRNA
expression of MMP-3, 7, 8, 9, 13, 16, 17 and 19 of chondrocytes are also decreased
by CTS. Expression of MMP-2, 11, 14 and Tissue Inhibitor of Metalloproteinases
(TIMP) -1, 2 and 3 are not affected (Deschner et al., 2006).
Similar results were obtained in chondrocyte/agarose construct submitted to
dynamic compression (15%, 1 Hz). Load inhibited IL-1 stimulated NO/iNOS and
PGE2 /COX-2 synthesis and gene expression (Chowdhury et al., 2003; Chowdhury
et al., 2006) by a mechanism implicating Mitogen Activated Protein Kinase
(MAPK) and NF-B pathway.
The mechanisms by which mechanical stimuli alter the cellular metabolic functions
are known as mechanotransduction. When applied to cartilage, load causes a lot
of complex physiological modifications such as deformation of cells and matrix,
gradient in hydrostatic pressure, alteration in ionic, osmotic and pH composition,
intra-tissue fluid flow and streaming potentials. Biomechanics forces can be trans-
mitted into the cell by a variety of structures: deformation of the cell, strain on
the cytoskeleton, deformation of the nucleus and direct signaling pathways via
mechanoreceptors of the extracellular matrix.
Members of the integrin family play an important role in chondrocyte mechan-
otransduction as ECM-receptors (Loeser, 2002). The cytoplasmic domain of inte-
grin interacts with cytoskeletal proteins and mediates change in cell shape enabling
various cellular responses such as proliferation, ECM matrix synthesis and degra-
dation. In response to cyclic tension (10% of deformation, 1 Hz), chondrocytes
in a 3D fibrin construct undergo modification in their cytoskeletal organization:
loaded cells exhibit projection containing F-actin, vimentin and vinculin filaments
13 The Mechanosensitivity of Cells in Joint Tissues 301
The subchondral bone is a global term which includes the subchondral bone plate
(cortical bone) and the underlying trabecular bone and bone marrow space. The
subchondral bone plate consists of cortical bone, which is relatively nonporous and
poorly vascularised. It is separated from the overlying articular cartilage by a zone
of calcified cartilage. The so-called tide-mark, which can be distinguished based
on its metachromatic staining pattern, provides a line of demarcation between the
hyaline cartilage and the calcified cartilage (Goldring, 2009). In OA, there is an
increase of the osteochondral bone plate thickness, a process likely affecting the
302 C. Sanchez et al.
Fig. 13.2 Osteoarthritic osteochondral junction (a) Subchondral bone sclerosis, Safranin-O/Light
green staining, 20x (b): Tidemark duplication, Hematoxylin/Eosin, 20x
(Sanchez et al., 2008). Due to it being a potent stimulator of bone matrix for-
mation by osteoblasts, the local accumulation of free IGF-1 is presented as a key
feature of subchondral bone plate sclerosis in OA. Additionally, OA osteoblasts are
resistant to parathyroid hormone (PTH) stimulation, a finding that contributes to
explain the abnormal bone remodeling in OA. OA osteoblasts produce an abnormal
homotrimeric type I collagen with a low affinity for calcium, which is responsible
for the low mineralization of the collagen matrix of OA subchondral bone. These
findings suggest that abnormal osteoblasts play a critical role in subchondral bone
sclerosis. Conversely, IL-6, PGE2 and Receptor Activator for Nuclear Factor B
Ligand (RANKL) may also be responsible for the increased number of active osteo-
clasts in OA subchondral bone and bone resorption observed in the early phase of
experimental OA (Liu et al., 2006b).
The bone tissue remodeling is the result of coordinated and balanced activities
of osteoblasts and osteoclasts. Osteoblast function is intimately linked to osteoclast
activity via the production of cytokines, growth factors and prostaglandins (PGs)
by osteoblasts. The production of some of these factors is controlled by mechani-
cal stimuli. Recently, a numbers of in vitro models attempted to screen genes and
signalling pathways involved in this mechanism, mainly by stretching osteoblasts
or by submitting them to a fluid shear stress. Osteoblasts possess mechanosen-
sors which activate intracellular signals including ion channels, integrins, calveolar
membrane structure and cytoskeleton. Nevertheless, response to physical signals
may be quite different according to the type of mechanical stress applied. Fluid
shear stress applied on osteoblasts in monolayer have been shown to elicit multiple
intracellular signalling pathways involving intracellular calcium rise, ERK1/2 acti-
vation of c-Fos and nuclear NF-B translocation (Chen et al., 2003; Inoue et al.,
2004). Downstream of such signalling events, various gene expression are induced,
including type I collagen (COL1), osteopontin (OPN), insulin-like growth factor-I
(IGF-1) and COX-2 (Chen et al., 2003).
304 C. Sanchez et al.
Dynamic cyclic tensile stresses are also potent activator of the signalling cascade
formed by ERK/c-fos/NF-B (Liu et al., 2006a). Stretching increases the production
of vascular endothelial growth factor (VEGF), TGF-beta1 (Singh et al., 2007), alka-
line phosphatase activity (AP), osteocalcin (OC), osteoprotegerin (OPG), MMP-1
and -3 (Kanno et al., 2007), COX-1 and -2, prostaglandin (PG)D2 synthase, per-
oxisome proliferator-activated receptor (PPAR) gamma-1 (Siddhivarn et al., 2006),
but decreases the release of the soluble receptor activator of nuclear factor ligand
(sRANKL) by osteoblasts (Tang et al., 2006). In contrast, no significant effect has
been reported on MMP-2, tissue inhibitor of metalloproteinases (TIMP)-1 and -2,
and PPARgamma-2 synthesis (Siddhivarn et al., 2006; Sasaki et al., 2007).
One major barrier to understanding bone physiology at a cellular level is the
lack of models for studying cells in their native environment. Usually, compres-
sion is generated by a bending system (Liu et al., 2006a) or a glass cylinder and
is applied on osteoblasts cultured in monolayer on flat surfaces (Mitsui et al.,
2006). We have developed an original model of 3D-osteoblast culture, allow-
ing the study of compression on osteoblasts embedded in their own produced
extracellular matrix (Sanchez et al., 2009). In this model, cell/matrix interactions
are conserved and fluid flow through a three dimensional extracellular matrix is
allowed.
In our study, loading was applied at large amplitude (11.67 MPa) and at a fre-
quency of 1 Hz. These loading conditions are included in the physiological range
of amplitude and frequency of mechanical strains applied on bone during locomo-
tion. In our experimental conditions, we have observed a strong release of PGE2
in the culture medium of loaded 3D-osteoblasts. This result confirms previous
studies demonstrating that fluid flow, compression and stretching stimulate PGE2
production by osteoblasts (Bakker et al., 2006; Grimston et al., 2006). Further,
we demonstrated that PGE2 release probably results from an imbalance between
PGE2 synthesis and degradation. Indeed, in our experimental conditions, compres-
sion increased COX-2 expression but decreased 15-PGDH expression. 15-PGDH
is a cytosolic enzyme which catalyzes the first step in the catabolic pathway of
prostaglandins, and believed to be the key enzyme responsible for the biological
inactivation of this biologically potent eicosanoid (Cho et al., 2006). Another impor-
tant finding was that compression had no significant effects on COX-1 and mPGES
gene expression. This contrast with previously reported data showing that compres-
sion of cartilage explants increased mPGES1 expression by chondrocytes (Gosset
et al., 2006).
PGE2 is also a mediator involved in IL-6-induced osteoclast formation and bone
resorption (Liu et al., 2006b). Recently, we have shown for the first time that IL-6
is a highly mechanosensitive gene. IL-6 expression was early increased (1 h) and
IL-6 protein secretion was highly stimulated (up to 32-fold) by 4 h 1 Hz 1.67 MPa
compression. The role of IL-6 on bone physiology is complex. IL-6 clearly stimu-
lates osteoclast activation and bone resorption in vivo and in vitro (Palmqvist et al.,
2002; De Benedetti et al., 2006). Data on the in vivo and in vitro effects of IL-6 on
osteoblasts are still conflicting, and several models have shown contradictory results
(Franchimont et al., 2005). In a recent model of transgenic mice overexpressing
13 The Mechanosensitivity of Cells in Joint Tissues 305
Fig. 13.3 Hypothetic schema of effect of compression on bone remodelling. Osteocytes and
osteoblasts respond to mechanical stimuli and produce NO, PGE2 and IL-6. These mediators
modify the OPG/RANKL balance in favour to RANKL and to osteoclasts activation and matrix
degradation. In parallel, osteoblasts produce also MMPs which degrade the bone matrix, allow-
ing osteoclasts attachment and OPN, OC, TGFs release. VEGF is also produced by osteoblasts,
promoting the arrival of new blood vessel and new osteoclasts precursors. IL-6: Interleukin-6,
MMP: matrix metalloproteinase, NO: nitric oxide, PGE2 : prostaglandin E2, OPG: osteoprote-
gerin, RANKL: receptor activator of NF-B ligand, VEGF: vascular endothelial growth factor;
OPN: osteopontin; OC osteoocalcin; TGF- transforming growth factors
the two tissues (Fig. 13.3); (2) blood vessels have been observed in the calcified
cartilage; (3) hepatocyte growth factor, which is secreted by osteoblasts (not chon-
drocytes), are detected in the deep layers of OA cartilage; (4) co-culture models
have shown that conditioned media from primary osteoblasts of OA patients sig-
nificantly increases GAG release from normal cartilage explants whereas culture
medium conditioned by normal osteoblasts had no effects (Westacott et al., 1997),
and that osteoblasts from sclerotic (SC) zones of subchondral OA, but not non-
sclerotic (NSC) osteoblasts, downregulated AGG synthesis and upregulated MMPs
expression by chondrocytes (Sanchez et al., 2005). These effects were driven by
IL-6, a cytokine which is overexpressed by sclerotic osteoblasts.
Other joint tissues, including meniscus, tendon, ligament and synovial membrane,
are exposed to mechanical stimuli. However, the influence of mechanical stimuli on
these tissues remains unexplored.
Two different aspects of biomechanical involvement of tendons and ligaments
have to be considered in articular pathologies. On the one hand the importance of
13 The Mechanosensitivity of Cells in Joint Tissues 307
mechanical stimuli on the integrity of tendons and ligaments, and the other side
the importance of these tissues in maintaining the stability of the joint, and there-
fore the optimal distribution of mechanical stimuli on cartilage. Tendons connect
muscle to bone and are designed to transmit forces and withstand tension during
muscle contraction. They contain few cells, mostly represented by tenoblasts along
with endothelial cells and some chondrocytes-like cells. Tendons are dense connec-
tive tissues composed of a composite of collagens (mainly type I), proteoglycans
and other noncollagenous proteins (Cartilage Oligomeric Matrix Protein, decorin
and fibromodulin) (Kannus, 2000). Age-related tendon failure is due to cumulative
damage resulting from a combination of diminished matrix repair and fragmenta-
tion of extracellular matrix proteins induced by prolonged cyclical tensile strength
(Dudhia et al., 2007). Physiological cyclic tensile (5% strain at 1 Hz during 24 h)
induces the release of MMPs and degraded COMP by tendon cells or explants.
These changes are more pronounced in old than in young tendon (Dudhia et al.,
2007). Low dynamic tensile stimulation of tendons cells up-regulates MMP-13
expression (Lavagnino andArnoczky, 2005) whereas static loading down-regulates
the expression of this protease. (Arnoczky et al., 2008). These findings suggest that
tendon requires a tensile strain threshold to prevent the tendon extracellular matrix
resorption and its weakening (Kjaer et al., 2006).
Ligaments are bands of dense connective tissue which courses from bone to bone,
across joint. They play a crucial role in joint stability. The most studied is the ante-
rior cruciate ligament (ACL) which crosses joint cavity from the femur to the tibia.
It is composed of three zones: the proximal part, which is less solid, is highly cel-
lular, rich in round and ovoid cells, containing some fusiform fibroblasts, collagen
type II and glycoproteins such as fibronectin and laminin; the middle part, contain-
ing fusiform and spindle-shape fibroblasts, is a high density of collagen fibers, a
special zone of cartilage and fibrocartilage, and elastic, and oxytalan fibers; and the
distal part, which is the most solid, is rich in chondroblasts and ovoid fibroblasts,
and with a low density of collagen bundles (Duthon et al., 2006). Mechanical stim-
uli are important for ligament homeostasis. They have stress-oriented structures of
collagen bundles. Uni-axial cyclic stretch increases the gene expressions of type I
and III collagens by mediating the autocrine secretion of TGF-1 (Kim et al., 2002),
and by modulating the integrin V3-dependant focal adhesion in human ACL cells
(Tetsunaga et al., 2009).
The menisci are wedge-shaped semi-lunar fibrocartilaginous structures that
correct the incongruence of the femoral and tibial articular surfaces (Hellio Le
Graverand et al., 2001). Menisci provide important biomechanical functions to the
knee joint such as load bearing, load distribution, shock absorption and joint sta-
bility (Fithian et al., 1990). Meniscal tissue is composed mainly of type I collagen
and contain considerably less proteoglycan (<1%) than hyaline cartilage. ACL sec-
tion causes mechanical damage in meniscus. At 3 and 8 weeks following the ACL
section, histological examination demonstrated extensive extracellular matrix dete-
rioration. Altered cell distribution, areas depleted of cells, and areas of cell clusters
were found within the medial but not in the lateral meniscus (Hellio Le Graverand
et al., 2001). Type I and III collagen immunostaining was increased in both lateral
308 C. Sanchez et al.
and medial menisci. In contrast, type II collagen staining was overtly increased only
in the medial meniscus (Hellio Le Graverand et al., 2001).
In OA, synovial membrane is inflamed secondary to the release of degradation
products and inflammatory mediators from cartilage. Synovial inflammation plays
a key role in the clinical and structural changes occurring in cartilage degradation
through the release by synovial cells (macrophages, synoviocyte, lymphocyte T).
Mechanical tensile stress down-regulates the expression and the release of MMP-1
and -13, but doesnt change MMP-2 production by synovial fibroblasts (Wang et al.,
2009). In contrast, MMP-3 is up-regulated by cyclic tensile load (Raif el, 2008).
Mechanical loading plays a dual role in the homeostasis of joint tissues: when
applied at moderate and physiological range, mechanical stimuli are beneficial to
the good health of joint tissues whereas excessive loading initiate abnormal tis-
sue remodeling and structural changes. Appropriate mechanical load could play an
important role in the homeostasis of joint tissues. This raises the question of the
role played by exercises in the prevention and treatment of OA. Recent recommen-
dations, clearly indicate that patients with hip and knee OA should be encouraged
to undertake, regular aerobic, muscle strengthening and range of motion exercises
(Zhang et al., 2008). Future clinical trials are required to demonstrate the chon-
droprotective effects of regular exercises on cartilage metabolism to sustain these
expert consensus-based guidelines.
Acknowledgements Christelle Sanchez is a post-doctoral researcher of the National Fund for
Scientific Research (FNRS, Belgium)
References
Agarwal S, Deschner J, Long P, Verma A, Hofman C, Evans CH, Piesco N (2004) Role of NF-
kappaB transcription factors in antiinflammatory and proinflammatory actions of mechanical
signals. Arthritis Rheum 50:35413548
Arnoczky SP, Lavagnino M, Egerbacher M, Caballero O, Gardner K, Shender MA (2008) Loss
of homeostatic strain alters mechanostat set point of tendon cells in vitro. Clin Orthop Relat
Res 466:15831591
Bakker AD, Klein-Nulend J, Tanck E, Heyligers IC, Albers GH, Lips P, Burger EH (2006)
Different responsiveness to mechanical stress of bone cells from osteoporotic versus
osteoarthritic donors. Osteoporos Int 17:827833
Blain EJ, Gilbert SJ, Wardale RJ, Capper SJ, Mason DJ, Duance VC (2001) Up-regulation of
matrix metalloproteinase expression and activation following cyclical compressive loading of
articular cartilage in vitro. Arch Biochem Biophys 396:4955
Buschmann MD, Gluzband YA, Grodzinsky AJ, Hunziker EB (1995) Mechanical compres-
sion modulates matrix biosynthesis in chondrocyte/agarose culture. J Cell Sci 108(Pt 4):
14971508
Chai DH, Arner EC, Griggs DW, Grodzinsky AJ (2010) Alphav and beta1 integrins reg-
ulate dynamic compression-induced proteoglycan synthesis in 3D gel culture by distinct
complementary pathways. Osteoarthr Cartil 18:249256
13 The Mechanosensitivity of Cells in Joint Tissues 309
Chen CT, Burton-Wurster N, Borden C, Hueffer K, Bloom SE, Lust G (2001) Chondrocyte necrosis
and apoptosis in impact damaged articular cartilage. J Orth Res 19:703711
Chen NX, Geist DJ, Genetos DC, Pavalko FM, Duncan RL (2003) Fluid shear-induced NFkappaB
translocation in osteoblasts is mediated by intracellular calcium release. Bone 33:399410
Cho H, Huang L, Hamza A, Gao D, Zhan CG, Tai HH (2006) Role of glutamine 148 of human
15-hydroxyprostaglandin dehydrogenase in catalytic oxidation of prostaglandin E2. Bioorg
Med Chem 14:64866491
Chowdhury TT, Bader DL, Lee DA (2003) Dynamic compression counteracts IL-1 beta-induced
release of nitric oxide and PGE2 by superficial zone chondrocytes cultured in agarose
constructs. Osteoarthr Cartil 11:688696
Chowdhury TT, Bader DL, Lee DA (2006) Dynamic compression counteracts IL-1beta induced
iNOS and COX-2 activity by human chondrocytes cultured in agarose constructs. Biorheology
43:413429
DLima DD, Hashimoto S, Chen PC, Colwell CW, Jr., Lotz MK (2001) Human chondrocyte
apoptosis in response to mechanical injury. Osteoarthr Cartil 9:712719
Das P, Schurman DJ, Smith RL (1997) Nitric oxide and G proteins mediate the response of bovine
articular chondrocytes to fluid-induced shear. J Orth Res 15:8793
De Benedetti F, Rucci N, Del Fattore A, Peruzzi B, Paro R, Longo M, Vivarelli M, Muratori F,
Berni S, Ballanti P, Ferrari S, Teti A (2006) Impaired skeletal development in interleukin-
6-transgenic mice: a model for the impact of chronic inflammation on the growing skeletal
system. Arthritis Rheum 54:35513563
Deschner J, Rath-Deschner B, Agarwal S (2006) Regulation of matrix metalloproteinase expres-
sion by dynamic tensile strain in rat fibrochondrocytes. Osteoarthr Cartil 14:264272
Dudhia J, Scott CM, Draper ER, Heinegard D, Pitsillides AA, Smith RK (2007) Aging enhances
a mechanically-induced reduction in tendon strength by an active process involving matrix
metalloproteinase activity. Aging Cell 6:547556
Duthon VB, Barea C, Abrassart S, Fasel JH, Fritschy D, Menetrey J (2006) Anatomy of the anterior
cruciate ligament. Knee Surg Sports Traumatol Arthrosc 14:204213
Fanning PJ, Emkey G, Smith RJ, Grodzinsky AJ, Szasz N, Trippel SB (2003) Mechanical
regulation of mitogen-activated protein kinase signaling in articular cartilage. J Biol Chem
278:5094050948
Fermor B, Weinberg JB, Pisetsky DS, Misukonis MA, Fink C, Guilak F (2002) Induction of
cyclooxygenase-2 by mechanical stress through a nitric oxide-regulated pathway. Osteoarthr
Cartil 10:792798
Fithian DC, Kelly MA, Mow VC (1990) Material properties and structure-function relationships
in the menisci. Clin Orthop Relat Res: 1931
Fitzgerald JB, Jin M, Chai DH, Siparsky P, Fanning P, Grodzinsky AJ (2008) Shear-
and compression-induced chondrocyte transcription requires MAPK activation in cartilage
explants. J Biol Chem 283:67356743
Fitzgerald JB, Jin M, Dean D, Wood DJ, Zheng MH, Grodzinsky AJ (2004) Mechanical com-
pression of cartilage explants induces multiple time-dependent gene expression patterns and
involves intracellular calcium and cyclic AMP. J Biol Chem 279:1950219511
Fitzgerald JB, Jin M, Grodzinsky AJ (2006) Shear and compression differentially regulate clus-
ters of functionally related temporal transcription patterns in cartilage tissue. J Biol Chem
281:2409524103
Franchimont N, Wertz S, Malaise M (2005) Interleukin-6: An osteotropic factor influencing bone
formation? Bone 37:601606
Gassner R, Buckley MJ, Georgescu H, Studer R, Stefanovich-Racic M, Piesco NP, Evans CH,
Agarwal S (1999) Cyclic tensile stress exerts antiinflammatory actions on chondrocytes by
inhibiting inducible nitric oxide synthase. J Immunol 163:21872192
Goldring SR (2009) Role of bone in osteoarthritis pathogenesis. Med Clin North Am 93:2535, xv
Gosset M, Berenbaum F, Levy A, Pigenet A, Thirion S, Saffar JL, Jacques C (2006) Prostaglandin
E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene.
Arthritis Res Ther 8:R135
310 C. Sanchez et al.
Grimston SK, Screen J, Haskell JH, Chung DJ, Brodt MD, Silva MJ, Civitelli R (2006) Role of
connexin43 in osteoblast response to physical load. Ann N Y Acad Sci 1068:214224
Hall AC, Urban JP, Gehl KA (1991) The effects of hydrostatic pressure on matrix synthesis in
articular cartilage. J Orth Res 9:110
Hashimoto S, Nishiyama T, Hayashi S, Fujishiro T, Takebe K, Kanzaki N, Kuroda R, Kurosaka
M (2009) Role of p53 in human chondrocyte apoptosis in response to shear strain. Arthritis
Rheum 60:23402349
Helfrich MH, Nesbitt SA, Lakkakorpi PT, Barnes MJ, Bodary SC, Shankar G, Mason WT,
Mendrick DL, Vaananen HK, Horton MA (1996) Beta 1 integrins and osteoclast function:
involvement in collagen recognition and bone resorption. Bone 19:317328
Hellio Le Graverand MP, Vignon E, Otterness IG, Hart DA (2001) Early changes in lapine menisci
during osteoarthritis development: Part I: cellular and matrix alterations. Osteoarthr Cartil
9:5664
Holledge MM, Millward-Sadler SJ, Nuki G, Salter DM (2008) Mechanical regulation of proteo-
glycan synthesis in normal and osteoarthritic human articular chondrocytes roles for alpha5
and alphaVbeta5 integrins. Biorheology 45:275288
Hung CT, Henshaw DR, Wang CC, Mauck RL, Raia F, Palmer G, Chao PH, Mow VC,
Ratcliffe A, Valhmu WB (2000) Mitogen-activated protein kinase signaling in bovine artic-
ular chondrocytes in response to fluid flow does not require calcium mobilization. J Biomech
33:7380
Hunter CJ, Imler SM, Malaviya P, Nerem RM, Levenston ME (2002) Mechanical compression
alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen
I gels. Biomaterials 23:12491259
Huser CA, Peacock M, Davies ME (2006) Inhibition of caspase-9 reduces chondrocyte apoptosis
and proteoglycan loss following mechanical trauma. Osteoarthr Cartil 14:10021010
Inoue D, Kido S, Matsumoto T (2004) Transcriptional induction of FosB/DeltaFosB gene by
mechanical stress in osteoblasts. J Biol Chem 279:4979549803
Islam N, Haqqi TM, Jepsen KJ, Kraay M, Welter JF, Goldberg VM, Malemud CJ (2002)
Hydrostatic pressure induces apoptosis in human chondrocytes from osteoarthritic cartilage
through up-regulation of tumor necrosis factor-alpha, inducible nitric oxide synthase, p53,
c-myc, and bax-alpha, and suppression of bcl-2. J. Cell Biochem 87:266278
Jortikka MO, Parkkinen JJ, Inkinen RI, Karner J, Jarvelainen HT, Nelimarkka LO, Tammi MI,
Lammi MJ (2000) The role of microtubules in the regulation of proteoglycan synthesis in
chondrocytes under hydrostatic pressure. Arch Biochem Biophys 374:172180
Kanno T, Takahashi T, Tsujisawa T, Ariyoshi W, Nishihara T (2007) Mechanical stress-mediated
Runx2 activation is dependent on Ras/ERK1/2 MAPK signaling in osteoblasts. J Cell Biochem
101:12661277
Kannus P (2000) Structure of the tendon connective tissue. Scand J Med Sci Sports 10:312320
Kim SG, Akaike T, Sasagaw T, Atomi Y, Kurosawa H (2002) Gene expression of type I and type
III collagen by mechanical stretch in anterior cruciate ligament cells. Cell Struct Funct 27:
139144
Kisiday JD, Lee JH, Siparsky PN, Frisbie DD, Flannery CR, Sandy JD, Grodzinsky AJ (2009)
Catabolic responses of chondrocyte-seeded peptide hydrogel to dynamic compression. Ann
Biomed Eng 37:13681375
Kjaer M, Magnusson P, Krogsgaard M, Boysen Moller J, Olesen J, Heinemeier K, Hansen M,
Haraldsson B, Koskinen S, Esmarck B, Langberg H (2006) Extracellular matrix adaptation of
tendon and skeletal muscle to exercise. J Anat 208:445450
Lavagnino M, Arnoczky SP (2005) In vitro alterations in cytoskeletal tensional homeostasis control
gene expression in tendon cells. J Orthop Res 23:12111218
Lee CR, Grodzinsky AJ, Spector M (2003a) Biosynthetic response of passaged chondrocytes
in a type II collagen scaffold to mechanical compression. J Biomed Mater Res A 64:
560569
Lee DA, Bader DL (1997) Compressive strains at physiological frequencies influence the
metabolism of chondrocytes seeded in agarose. J Orth Res 15:181188
13 The Mechanosensitivity of Cells in Joint Tissues 311
Lee HS, Millward-Sadler SJ, Wright MO, Nuki G, Al-Jamal R, Salter DM (2002) Activation of
Integrin-RACK1/PKCalpha signalling in human articular chondrocyte mechanotransduction.
Osteoarthr Cartil 10:890897
Lee HS, Millward-Sadler SJ, Wright MO, Nuki G, Salter DM (2000) Integrin and mechanosen-
sitive ion channel-dependent tyrosine phosphorylation of focal adhesion proteins and beta-
catenin in human articular chondrocytes after mechanical stimulation. J Bone Miner Res
15:15011509
Lee JH, Fitzgerald JB, DiMicco MA, Cheng DM, Flannery CR, Sandy JD, Plaas AH, Grodzinsky
AJ (2009) Co-culture of mechanically injured cartilage with joint capsule tissue alters chon-
drocyte expression patterns and increases ADAMTS5 production. Arch Biochem Biophys
489:118126
Lee JH, Fitzgerald JB, Dimicco MA, Grodzinsky AJ (2005) Mechanical injury of cartilage explants
causes specific time-dependent changes in chondrocyte gene expression. Arthritis Rheum
52:23862395
Lee MS, Trindade MC, Ikenoue T, Goodman SB, Schurman DJ, Smith RL (2003b) Regulation of
nitric oxide and bcl-2 expression by shear stress in human osteoarthritic chondrocytes in vitro.
J Cell Biochem 90:8086
Li KW, Wang AS, Sah RL (2003) Microenvironment regulation of extracellular signal-regulated
kinase activity in chondrocytes: effects of culture configuration, interleukin-1, and compressive
stress. Arthritis Rheum 48:689699
Lin PM, Chen CT, Torzilli PA (2004) Increased stromelysin-1 (MMP-3), proteoglycan degradation
(3B3- and 7D4) and collagen damage in cyclically load-injured articular cartilage. Osteoarthr
Cartil 12:485496
Liu J, Liu T, Zheng Y, Zhao Z, Liu Y, Cheng H, Luo S, Chen Y (2006a) Early responses
of osteoblast-like cells to different mechanical signals through various signaling pathways.
Biochem Biophys Res Commun 348:11671173
Liu XH, Kirschenbaum A, Yao S, Levine AC (2005) Cross-talk between the interleukin-
6 and prostaglandin E(2) signaling systems results in enhancement of osteoclastogenesis
through effects on the osteoprotegerin/receptor activator of nuclear factor-{kappa}B (RANK)
ligand/RANK system. Endocrinology 146:19911998
Liu XH, Kirschenbaum A, Yao S, Levine AC (2006b) Interactive effect of interleukin-6 and
prostaglandin E2 on osteoclastogenesis via the OPG/RANKL/RANK system. Ann N Y Acad
Sci 1068:225233
Loening AM, James IE, Levenston ME, Badger AM, Frank EH, Kurz B, Nuttall ME, Hung HH,
Blake SM, Grodzinsky AJ, Lark MW (2000) Injurious mechanical compression of bovine
articular cartilage induces chondrocyte apoptosis. Arch Biochem Biophys 381:205212
Loeser RF (2002) Integrins and cell signaling in chondrocytes. Biorheology 39:119124
Loeser RF, Carlson CS, McGee MP (1995) Expression of beta 1 integrins by cultured articular
chondrocytes and in osteoarthritic cartilage. Exp Cell Res 217:248257
Lucchinetti E, Bhargava MM, Torzilli PA (2004) The effect of mechanical load on integrin subunits
alpha5 and beta1 in chondrocytes from mature and immature cartilage explants. Cell Tissue Res
315:385391
Lukoschek M, Boyd RD, Schaffler MB, Burr DB, Radin EL (1986) Comparison of joint degen-
eration models. Surgical instability and repetitive impulsive loading. Acta Orthop Scand
57:349353
Mathy-Hartert M BS, Sanchez C, Lambert C, Henrotin Y (2008) Lapplication de forces cycliques
dtirement diminue la production de mdiateurs de linflammation par les chondrocytes
arthrosiques. Rev Rhum 75:9991000
Mitsui N, Suzuki N, Maeno M, Yanagisawa M, Koyama Y, Otsuka K, Shimizu N (2006)
Optimal compressive force induces bone formation via increasing bone morphogenetic proteins
production and decreasing their antagonists production by Saos-2 cells. Life Sci 78:26972706
Mohtai M, Gupta MK, Donlon B, Ellison B, Cooke J, Gibbons G, Schurman DJ, Smith RL (1996)
Expression of interleukin-6 in osteoarthritic chondrocytes and effects of fluid-induced shear on
this expression in normal human chondrocytes in vitro. J Orth Res 14:6773
312 C. Sanchez et al.
Palmoski MJ, Brandt KD (1984) Effects of static and cyclic compressive loading on articular
cartilage plugs in vitro. Arthritis Rheum 27:675681
Palmqvist P, Persson E, Conaway HH, Lerner UH (2002) IL-6, leukemia inhibitory factor, and
oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF-
kappa B ligand, osteoprotegerin, and receptor activator of NF-kappa B in mouse calvariae. J
Immunol 169:33533362
Pickvance EA, Oegema TR, Jr., Thompson RC, Jr. (1993) Immunolocalization of selected
cytokines and proteases in canine articular cartilage after transarticular loading. J Orth Res
11:313323
Raif el M (2008) Effect of cyclic tensile load on the regulation of the expression of matrix met-
alloproteases (MMPs -1, -3) and structural components in synovial cells. J Cell Mol Med
12:24392448
Sah RL, Kim YJ, Doong JY, Grodzinsky AJ, Plaas AH, Sandy JD (1989) Biosynthetic response of
cartilage explants to dynamic compression. J Orth Res 7:619636
Sanchez C, Deberg MA, Bellahcene A, Castronovo V, Msika P, Delcour JP, Crielaard JM , Henrotin
YE (2008) Phenotypic characterization of osteoblasts from the sclerotic zones of osteoarthritic
subchondral bone. Arthritis Rheum 58:442455
Sanchez C, Deberg MA, Piccardi N, Msika P, Reginster JY, Henrotin YE (2005) Osteoblasts
from the sclerotic subchondral bone downregulate aggrecan but upregulate metalloproteinases
expression by chondrocytes. This effect is mimicked by interleukin-6, -1beta and oncostatin M
pre-treated non-sclerotic osteoblasts. Osteoarthr Cartil 13:979987
Sanchez C, Gabay O, Salvat C, Henrotin YE, Berenbaum F (2009) Mechanical loading highly
increases IL-6 production and decreases OPG expression by osteoblasts. Osteoarthr Cartil
17:473481
Sasaki K, Takagi M, Konttinen YT, Sasaki A, Tamaki Y, Ogino T, Santavirta S, Salo J (2007)
Upregulation of matrix metalloproteinase (MMP)-1 and its activator MMP-3 of human
osteoblast by uniaxial cyclic stimulation. J Biomed Mater Res B Appl Biomater 80:491498
Shimazaki A, Wright MO, Elliot K, Salter DM, Millward-Sadler SJ (2006) Calcium/calmodulin-
dependent protein kinase II in human articular chondrocytes. Biorheology 43:223233
Siddhivarn C, Banes A, Champagne C, Riche EL, Weerapradist W, Offenbacher S (2006)
Prostaglandin D2 pathway and peroxisome proliferator-activated receptor gamma-1 expression
are induced by mechanical loading in an osteoblastic cell line. J Periodontal Res 41:92100
Singh SP, Chang EI, Gossain AK, Mehara BJ, Galiano RD, Jensen J, Longaker MT, Gurtner GC,
Saadeh PB (2007) Cyclic mechanical strain increases production of regulators of bone healing
in cultured murine osteoblasts. J Am Coll Surg 204:426434
Smith RL, Carter DR, Schurman DJ (2004) Pressure and shear differentially alter human articular
chondrocyte metabolism: a review. Clin Orthop Relat Res:S8995
Tang L, Lin Z, Li YM (2006) Effects of different magnitudes of mechanical strain on Osteoblasts
in vitro. Biochem Biophys Res Commun 344:122128
Tetsunaga T, Furumatsu T, Abe N, Nishida K, Naruse K, Ozaki T (2009) Mechanical stretch stim-
ulates integrin alphaVbeta3-mediated collagen expression in human anterior cruciate ligament
cells. J Biomech 42:20972103
Valhmu WB, Raia FJ (2002) myo-Inositol 1,4,5-trisphosphate and Ca(2+)/calmodulin-dependent
factors mediate transduction of compression-induced signals in bovine articular chondrocytes.
Biochem J 361:689696
Vanderploeg EJ, Imler SM, Brodkin KR, Garcia AJ, Levenston ME (2004) Oscillatory tension
differentially modulates matrix metabolism and cytoskeletal organization in chondrocytes and
fibrochondrocytes. J Biomech 37:19411952
Wang P, Yang L, You X, Singh GK, Zhang L, Yan Y, Sung KL (2009) Mechanical stretch regulates
the expression of matrix metalloproteinase in rheumatoid arthritis fibroblast-like synoviocytes.
Connect Tissue Res 50:98109
Westacott CI, Webb GR, Warnock MG, Sims JV, Elson CJ (1997) Alteration of cartilage
metabolism by cells from osteoarthritic bone. Arthritis Rheum 40:12821291
13 The Mechanosensitivity of Cells in Joint Tissues 313
Wheeler CA, Jafarzadeh SR, Rocke DM, Grodzinsky AJ (2009) IGF-1 does not moderate the time-
dependent transcriptional patterns of key homeostatic genes induced by sustained compression
of bovine cartilage. Osteoarthr Cartil 17:944952
Wong M, Wuethrich P, Buschmann MD, Eggli P, Hunziker E (1997) Chondrocyte biosynthesis
correlates with local tissue strain in statically compressed adult articular cartilage. J Orth Res
15:189196
Wright MO, Nishida K, Bavington C, Godolphin JL, Dunne E, Walmsley S, Jobanputra P, Nuki G,
Salter DM (1997) Hyperpolarisation of cultured human chondrocytes following cyclical
pressure-induced strain: evidence of a role for alpha 5 beta 1 integrin as a chondrocyte
mechanoreceptor. J Orth Res 15:742747
Zhang W, Moskowitz RW, Nuki G, Abramson S, Altman RD, Arden N, Bierma-Zeinstra S,
Brandt KD, Croft P, Doherty M, Dougados M, Hochberg M, Hunter DJ, Kwoh K, Lohmander
LS, Tugwell P (2008) OARSI recommendations for the management of hip and knee
osteoarthritis, Part II: OARSI evidence-based, expert consensus guidelines. Osteoarthr Cartil
16:137162
Part VI
Mechanotransduction of Sensor System
Chapter 14
Primary Cilia are Mechanosensory Organelles
in Vestibular Tissues
Abstract Primary cilia have been observed for over a century, but their sensory
roles have only been revealed within the past decade. In this chapter, we will
describe cilia as newly recognized mechanosensory organelles. Cilia can sense bod-
ily fluid movement in all vestibular organs. These include nodal flow in Hensens
node, urine in the renal nephron, bile in the hepatic biliary system, digestive fluid
in the pancreatic duct, dentin in dental pulp, lacunocanalicular fluid in bone and
cartilage, and blood in vasculature. To exert their sensory functions, cilia require
both structural and functional proteins. Cells without ciliary function or structure
are unable to sense fluid-shear stress, but their sensitivity toward other mechanical
or pharmacological stimuli remains intact. The functional machineries found in the
cilia include mechanosensory polycystin-1, mechano-calcium channel polycystin-2,
and other interacting proteins. The roles of cilia as fluid sensors in Hensens node
as well as in the kidney, liver, pancreas, bone, and cardiovascular system will be
discussed.
14.1 Introduction
Biologists have long been studying the complexity of cellular functions of a sin-
gle cell, which signifies a fundamental unit of life. Cilia are among the organelles
possessed by cells that have been discussed extensively. Structurally, cilia can be
classified into two types, based on their microtubule arrangements of either 9+0
or 9+2 (Fig. 14.1). However, based on their motility, cilia can be functionally
Fig. 14.1 A brief introduction of a cilium. (a) A cilium is an organelle projected at the apical
membrane of many cell types. It is projected from the basal body. (b) A cilium is considered a cel-
lular organelle, which is composed of a membrane domain, soluble compartment, axoneme, and
basal body. The membrane domain contains various sensory proteins. The soluble compartment
has been shown to contain many regulatory proteins that are involved in signal transduction sys-
tems. The axoneme is surrounded by the nine pairs of microtubules that are thought to control the
length of a cilium. The basal body is the anchoring point for axonemal microtubules. (c) Based
on the central pair of the microtubules in the axoneme, a cilium can be categorized into 9+2
and 9+0 structures. It was once thought that a cilium with 9+0 axoneme was always immotile.
Classification of cilia becomes more complex, particularly since the presence of 9+4 cilia has
also been reported (Feistel and Blum, 2006)
classified as motile or non-motile organelles. Like any organelles in the cells, cilia
have many important and specialized cellular functions (Table 14.1). Classification
of cilia can thus provide a broad spectrum of understanding about their cellular
functions.
Primary cilia are usually classified as 9+0 non-motile organelles (Table 14.1).
Although they were first described as early as 1898 (Wheatley, 2005), primary cilia
were thought to be vestigial organelles. It was assumed that cilia were remnants
from flagella of a single-cell ancestral organism and no longer had any particular
function. A primary cilium is a microtubule-based, antenna-like structure and is
found in a single copy on the apical surface of fully differentiated mammalian cells.
The diameter of a cilium is approximately 0.25 m, and its length can vary from
Table 14.1 Ciliary classification, function and disease relevance in mammals
14
9+0 Motile Nodal flow generation Situs inversus; Situs ambiguus; Nonaka et al. (1998, 2002), and Essner et al.
Situs isomerism (2002)
Non-motile Nodal flow sensor Situs inversus; Situs ambiguus; McGrath et al. (2003) and Karcher et al. (2005)
Situs isomerism
Mechanosensor Polycystic kidney, liver, pancreas Nauli et al. (2003), Cano et al. (2006), and
Masyuk et al. (2006)
Shear-stress sensor Hypertension; Arthrosclerosis Nauli et al. (2008), Van der Heiden et al. (2008),
and AbouAlaiwi et al. (2009b)
Osmo sensor Respiratory diseases; Infertility Andrade et al. (2005) and Teilmann et al. (2005)
Gravitational sensor Osteoporosis; Chondroporosis Malone et al. (2007), McGlashan et al. (2007)
and Moorman and Shorr (2008)
Smell sensor Anosmia; Hyposmia Kulaga et al. (2004), Layman et al. (2009)
Light sensor Retinitis pigmentosa, Blindness Nishimura et al. (2004a), Moore et al. (2006),
Ghosh et al. (2010)
Chemo sensor Nephrocystin; Diabetes; Obesity Hearn et al. (2005), Winkelbauer et al. (2005),
and Davenport et al. (2007)
Developmental Abnormal development; Cancer (Christensen et al. (2008), Han et al. (2009), and
regulator Wong et al. (2009)
9+2 Motile Chemo sensor Chronic obstructive pulmonary Shah et al. (2009)
disease
Fluid clearance device Chronic obstructive pulmonary Salathe (2007), Zariwala et al. (2007), and Mall
Primary Cilia are Mechanosensory Organelles in Vestibular Tissues
disease (2008)
Oocyte transport Infertility Eddy and Pauerstein (1980) and Lyons et al.
(2006)
Sperm motility Infertility Brunner et al. (2008), Lee et al. (2008), and Imai
et al. (2009)
Fluid transport Hydrocephalus; Cell migration Ibanez-Tallon et al. (2004), Sawamoto et al.
(2006), and Wodarczyk et al. (2009)
Non-motile Sound wave sensor Hearing loss Littlewood Evans and Muller (2000), Grillet
et al., (2009a, b)
319
320 S.M. Nauli et al.
associated with cell division. In the fruit fly, interestingly, centriole formation does
not seem to be a prerequisite of cell divisions, although centrioles are still essential
for the formation of cilia (Basto et al., 2006).
It is estimated that a cilium has over a thousand proteins (Dutcher, 1995; Pazour
et al., 2005; Gherman et al., 2006), making this organelle very complex to study. To
date, a handful of proteins have been characterized and localized to different ciliary
domains (Table 14.2). The centrosomal proteins tend to be more convoluted because
of the recruitments of other proteins into centrosomes during cell division. As such,
knowledge about centrosomal proteins is expected to expand in the near future as
their relationships to ciliary localization and/or function are ascertained.
The assembly of primary cilia was first discovered through the unicellular green
alga Chlamydomonas. Cilia are assembled from proteins that are synthesized in the
cell body and transported to the ciliary tips by a complex process involving intraflag-
ellar transport (Rosenbaum and Witman, 2002; Pan and Snell, 2007). This process
involves bidirectional movements of nonmembrane-bound macro-molecular pro-
tein complexes or multi-meric protein particles along the axoneme (Scholey, 2003;
Scholey and Anderson, 2006). Thus, a successful cilia assembly depends on three
key components: the intraflagellar transport particles, the anterograde motors and
the retrograde motors. The anterograde proteins, mediated by complex B raft com-
ponents, carry axonemal components from the cell body to the growing tip of
cilia. Retrograde proteins, mediated by complex A components, carry the axone-
mal machineries back from the tip of the cilium to the cell body. Whereas kinesin-II
is the anterograde motor, dynein-2 is the retrograde motor. Both of these anterograde
and retrograde proteins are powered by microtubule motors.
Almost all human cell types have either a bundle of motile cilia or a solitary
immotile cilium. In general, only circulating cells or cells under suspension do not
develop cilia. Such cells include circulating red and white blood cells. As will be
described in the next section, mechano-fluid sensing is the most understood role
among the major functions of primary cilia. As such, it is not surprising that pri-
mary cilia as mechano-fluid sensors can be found in all organs that support and
sense fluid perfusion in the human body. By definition, the body tissues that have
small canal or cavity spaces (vestibules) to support perfusion of body fluid are called
vestibular organs.
The mechano-fluid sensing role of primary cilia has been demonstrated in many
vestibular organs. Such organs depend on mechanosensing cilia to sense and trans-
mit extracellular signals to intracellular biochemical reactions. Cilia can thus sense a
variety of fluid movements in the body, including blood in vasculature (AbouAlaiwi
et al., 2009a), interstitial fluid in the bone matrix (Turner et al., 1994; Whitfield,
2008), urine in kidney nephrons (Nauli and Zhou, 2004; Kolb and Nauli, 2008), bile
in the hepatic biliary system (Masyuk et al., 2008, 2009), digestive fluid in the pan-
creatic duct (Aughsteen, 2001; Kaestner, 2006), cerebral spinal fluid in the neuronal
322
Table 14.2 Structural and functional ciliary proteins based on their ciliary domains
EB1 Pedersen et al. (2003) and Schroder et al. (2007) 14-3-3 Fan et al. (2004a)
Gli Haycraft et al. (2005) and Liem et al. (2009) Adenylyl cyclase Menco (2005) and Bishop et al. (2007)
KIF7 Endoh-Yamagami et al. (2009) and Liem et al. Arl13b Cantagrel et al. (2008) and Hori et al. (2008)
(2009)
Smo Corbit et al. (2005), Haycraft et al. (2005) and Arl2l1 Sun et al. (2004)
Liem et al. (2009)
Sufu Jia et al. (2009) ATP synthase Hu and Barr (2005)
-arrestin-2 Menco (2005)
IFT20 Follit et al. (2006) and Jonassen et al. (2008) Phosphodiesterase Menco (2005)
IFT46 Gouttenoire et al. (2007) PKC Etienne-Manneville and Hall (2003) and Fan
et al. (2004a)
IFT52 Tsujikawa and Malicki (2004) and Liu et al. (2005a) pVHL Okuda et al. (1999), Lolkema et al. (2007), and
Thoma et al. (2007)
IFT57/curly Krock and Perkins (2008) and Lunt et al. (2009) STAT6 Low et al. (2006)
IFT57/hippi Tsujikawa and Malicki (2004) and Houde et al. Tubby Mukhopadhyay et al. (2005) and Mak et al.
(2006) (2006)
IFT80 Beales et al. (2007) TULP2 Stolc et al. (2005)
IFT81 Sun et al. (2004) and Lucker et al. (2005)
IFT88 Murcia et al. (2000), Pazour et al. (2000), Haycraft Ciliary membrane
et al. (2001), Qin et al. (2001), and Yoder et al.
(2002b)
Kif17 Jenkins et al. (2006) EGFR Ma et al. (2005)
Kif3A/B Marszalek et al. (1999), Takeda et al. (1999), Fibrocystin Ward et al. (2003), Wang et al. (2007)
Marszalek et al. (2000), and Lin et al. (2003)
MDHC7 Neesen et al. (2001) and Vernon et al. (2005) Mchr1 Berbari et al. (2008)
PACRG Lorenzetti et al. (2004) and Dawe et al. (2005) PDGFR Schneider et al. (2005)
PF13 Omran et al. (2008) Polycystin-1 Barr and Sternberg (1999) and Yoder et al.
Primary Cilia are Mechanosensory Organelles in Vestibular Tissues
(2002a)
PF16 Sapiro et al. (2002) and Zhang et al. (2005) Polycystin-2 Barr and Sternberg (1999), Pazour et al. (2002),
and Yoder et al. (2002a)
PF2 Rupp and Porter (2003) Somatostatin-3 receptor Schulz et al. (2000)
PF20 Zhang et al. (2004) Serotonin-6 receptor Brailov et al. (2000)
Tektin Tanaka et al. (2004) Tie-1,Tie-2 receptors Teilmann and Christensen (2005)
TRPN1 Kim et al. (2003) and Shin et al. (2005)
TRPV4 Qin et al. (2005) and Teilmann et al. (2005)
323
Table 14.2 (continued)
tube (Lang et al., 2006; Wodarczyk et al., 2009), fluid pressure in the inner ears
(Kernan, 2007; Petit and Richardson, 2009), and so on. The inability to sense fluid-
shear in these vestibular organs may thus contribute to multiple organ pathogenesis,
from hypertension to hydrocephalus and from deafness to cystic organ formation.
Fig. 14.2 Cilia are mechanosensory organelles. Cilia are sensory organelles that sense fluid-shear
stress on the apical membrane of the cells. Fluid flow that produces enough drag-force on the cells
will bend sensory cilia. These biomechanics play very crucial roles in vestibular organs to support
bodily fluid perfusion. Figure is reproduced from AbouAlaiwi, et al. with permission (AbouAlaiwi
et al., 2009a)
326 S.M. Nauli et al.
The polycystins are probably the most studied mechanosensory proteins in cilia.
The mechanosensory functions of polycystins have been independently described
in the mouse and human kidney epithelia (Chauvet et al., 2004; Nauli et al., 2006;
Xu et al., 2007, 2009), vascular endothelia (Nauli et al., 2008; AbouAlaiwi et al.,
2009b), osteochondrocytes (Xiao et al., 2006; Hou et al., 2009), cholangiocytes
(Masyuk et al., 2006) and developing nodes (McGrath et al., 2003). Polycystin-1
is an 11-transmembrane protein with an extracellular domain that is long, flexi-
ble, elastic, and of remarkable mechanical strength (Forman et al., 2005; Qian et al.,
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 327
Fig. 14.3 Complex of mechanosensory proteins in the cilia. Polycystin-1 and polycystin-2 interact
with each other at the COOH termini, forming a polycystin complex. Kif, as an adaptor protein,
bridges the interaction of between fibrocystin and polycystin complex. Figure is reproduced from
Kolb, et al. with permission (Kolb and Nauli, 2008)
328 S.M. Nauli et al.
14.3.2 Kidney
fibrocystin protein was repressed by 90%, the primarly cilia failed to develop (Mai
et al., 2005).
Polycystin-1, polycystin-2, and fibrocystin are localized to the primary cilia
in renal epithelial cells (Fig. 14. 3). This complex is involved in ciliary medi-
ated response to flow by initiating an increase of intracellular calcium. Interaction
of fibrocystin with calcium modulating cyclophilin ligand has also been shown,
suggesting an additional calcium protein might modulate intracellular calcium in
response to fluid-shear stress (Nagano et al., 2005). It is therefore not surprising that
mutation in genes responsible for polycystin-1, polycystin-2 and fibrocystin would
result in polycystic kidney disease. This further suggests that dysfunction of renal
cilia in response to urine flow would result in polycystic kidney disease.
The involvement of primary cilia in polycystic kidney disease has further pro-
vided that dysfunction in cilia would result in the abnormal planar cell polarity
(Fischer et al., 2006; Patel et al., 2008; Saburi et al., 2008). Within the kidney
anatomy, planar cell polarity is defined as an organized arrangement of cells in a
plane of tissue perpendicular to the apical-basal axis as a direction for the orienta-
tion of cell division. Using different mouse models of cystic kidney disease, defects
in any ciliary protein lead to abnormal orientation during cell division. It is there-
fore thought that inactivation of ciliary protein would result in abnormal planar cell
polarity, which in turn triggers tubular diameter in the kidney (Fig. 14.4). The net
result is cystic formation in the kidney.
14.3.3 Liver
The liver is an organ that secretes an aqueous solution called bile. Bile that is ini-
tially secreted by hepatocytes is called primary bile. Primary bile is further modified
within the interlobular bile ducts through secretory and absorptive processes. This
modified primary bile is called ductal bile. The ductal bile travels along the inter-
lobular bile ducts, which are composed of cuboidal and mucus-secreting epithelia
known as cholangiocytes.
Hepatocytes do not have primary cilia, whereas the primary cilia in cholangio-
cytes are extended from the apical plasma membrane into the lumens of interlobular
bile ducts (Huang et al., 2006). The primary cilia in the bile duct sense bile flow,
bile composition and bile formation. The primary cilia are also involved in regu-
lating bile duct formation and the size of the lumen. Similar to primary cilia in the
renal tubules (Brown and Murcia, 2003), the length of cholangiocyte cilia are het-
erogeneous (Huang et al., 2006). Cilia are longer in larger lumens and shorter in
lumens with smaller diameters.
To function as sensory organelles, cholangiocyte cilia express polycystin-1,
polycystin-2, and fibrocystin, among other sensory proteins (Masyuk et al., 2003,
2006). The activation of primary cilia by luminal fluid flow in microperfused intra-
hepatic bile ducts depends on the polycystin-1 and polycystin-2 mechanosensory
complex (Masyuk et al., 2006). Abnormalities in fibrocystin cause structurally
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 331
Fig. 14.4 Abnormal cilia function, planar cell polarity and cystic kidney disease. The illustra-
tion depicts mechanosensory function of renal tubular epithelial cilium. (a) Each cilium plays an
important role in transmitting extracellular information, such as urine flow, into renal epithelial
cells. This message may provide critical signals to the cell regarding the direction of cell division
along the tubule. (b) Insults, such as genetic disorder or random mutation, will result in abnormal
ciliary function for sensing fluid movement. (c) The functional abnormality in ciliary sensing may
result in loss of planar cell polarity. (d) Direction of cell division becomes randomized, result-
ing in increasing tubular diameter rather than tubular elongation. (e) Budding of a cyst from the
renal tubule and abnormal localizations of epidermal growth factor receptor (EGFR) and Na+ /K+
ATPase pump are typical characteristics of polycystic kidneys. (f) The cyst is eventually enlarged
and isolated. Multiple cysts from the neighboring nephrons are illustrated on the bottom left corner.
Figure is reproduced from Kolb et al. with permission (Kolb and Nauli, 2008)
shorter cilia in the bile ducts (Masyuk et al., 2003, 2004; Woollard et al., 2007).
The primary cilia are shown to be shorter, dysmorphic, and malformed, with
bulbous extensions of the ciliary tip. Mutations in cilia-associated genes in cholan-
giocytes have been referred to as cholangiociliopathies (Masyuk et al., 2008).
Cholangiociliopathies are characterized by cystic and fibrotic liver phenotypes.
332 S.M. Nauli et al.
14.3.4 Pancreas
The pancreas is a gland that produces hormones and digestive enzymes. This pan-
creatic fluid is transported to the gut via a system of ducts that is composed of
pancreatic epithelial cells. Each pancreatic epithelial cell has one primary cilium.
The primary cilia in the pancreatic ductal cells function to sense luminal flow to
maintain appropriate luminal dimensions (Cano et al., 2004).
To function as mechanosensory organelles, the pancreatic primary cilia have been
shown to express mechanosensory polycystin-2 channel (Cano et al., 2004). When
polycystin-2 is mislocalized or abnormally expressed, pancreatic cysts are formed.
Mutations in either functional or structural ciliary genes would block the relay of
the mechanosensory signals from extracellular to intracellular compartments (Cano
et al., 2004, 2006). This, in turn, results in uninhibited cell proliferation and pro-
gressive duct dilation. Primary cilia function and structure are thus required for
maturation and maintenance of proper tissue organization in the pancreas (Cano
et al., 2004, 2006). In addition, inability to regulate flow within the pancreatic duct
may lead to ductal dilation and acinar cell death (Scoggins et al., 2000).
Defects due to a reduction in cilia, aberrant ciliary architecture, and abnormal
ciliary function would provide gross pancreatic phenotypes, which include massive
acinar cell loss due to acinar-to-ductal metaplasia, fibrosis, lipomatosis, formation
of abnormal tubular structures, and appearance of endocrine cells in ducts (Cano
et al., 2004, 2006). All these phenotypes are similar to those found in patients with
chronic pancreatitis or pancreatic cystic fibrosis. Moreover, pancreatic primary cilia
may also be involved in pancreatic cancer and pancreatic intraepithelial neoplasia
lesions in human pancreatic ductal adenocarcinoma. Unlike cells in normal ducts,
intraepithelial neoplasia or ductal carcinoma cells do not have cilia (Seeley et al.,
2009).
14.3.5 Bone
Technically, bone has a very stiff structure. The structure, however, is compressible
due to its fluid-filled calcareous sponge. Osteocytes are the most abundant cell type
found in a compact bone. Cartilage, on the other hand, is a more flexible connective
tissue that joins bones together. Cartilage is primarily composed of chondrocytes
that produce a large amount of extracellular matrix.
Both osteocytes and chondrocytes possess primary cilia (Xiao et al., 2006;
Malone et al., 2007). The sensory cilia deflect during fluid flow and are required
for osteogenic and bone resorption in response to dynamic fluid flow. Although
osteocytes are more sensitive to fluid flow than osteoblasts (Klein-Nulend et al.,
1995), both osteocytes and chondrocytes respond to the strains on bones and joints
through primary cilia. Primary cilia in bone and cartilage are responsive to the
compression and cyclical pulses of lacunocanalicular fluid. Fluid flow through the
osteocyte canalicular network provides the physical stimulus for mechanosensa-
tion in bone (Klein-Nulend et al., 1995). Upon activation, primary cilia induce
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 333
intracellular calcium surges, which activate genes that maintain and strengthen bone
and cartilage.
To function as mechanosensory organelles, primary cilia in bone and cartilage
also enclose the mechanosensory polycystin-1 and polycystin-2 complex (Xiao
et al., 2006). Abnormality in these polycystin proteins results in abnormal bone and
cartilage formation (Boulter et al., 2001; Lu et al., 2001; Xiao et al., 2006, 2008;
Kolpakova-Hart et al., 2008; Hou et al., 2009). Mice with such mutations in ciliary
proteins display a decrease in gene products that maintain and strengthen bone and
cartilage. Thus, abnormalities in mechanosensory primary cilia in bone and cartilage
would result in osteoporosis or other bone related disorders (Malone et al., 2007).
The sensory primary cilia are also present in odontoblasts (Carmona, 1990;
Magloire et al., 2004; Maurin et al., 2009). Odontoblasts are cells that are located on
the outer surface of dental pulp. Activation of primary cilia in odontoblasts would
induce secretion of dentin required to form a calcified tissue under the tooth enamel.
Primary cilia are aligned parallel to the dentin walls, with the top part oriented
toward the pulp core. The cilia are positioned in very close proximity to the nerve
fibers. Thus, the role of cilia in sensing the microenvironment in odontoblasts is
thought to be crucial both for dentin formation and tooth pain transmission.
To function as mechanosensory organelles, primary cilia of odontoblasts express
mechanosensory polycystin-1 and polycystin-2 complex, including other ciliary
proteins such as OFD1 (Carmona, 1990; Maurin et al., 2009). Defects in molars
from Ofd1 knockout mice show the dysfunction of odontoblasts in the formation of
dentin. Thus, proper ciliary function is also a prerequisite during tooth development
(Ohazama et al., 2009).
14.3.6 Cardiovascular
Fig. 14.5 Mechanosensory cilia in blood vessel. Electron micrograph confirms the presence of
cilia in the lumen of embryonic aorta in vivo. Cilia as sensory organelles are projected from the
apical sides of cells into the lumen of blood vessels to sense fluid (blood) flow. Arrows indicate
the presence of cilia. Figure is reproduced from AbouAlaiwi et al. with permission (AbouAlaiwi
et al., 2009a)
Fig. 14.6 Mechanosensory cilia and nitric oxide production. The presence of cilia in vasculature
plays an important role in the biochemical production of a potent vasodilator, nitric oxide (NO).
The figure depicts production of NO in an artery. Increases in blood pressure, which are translated
to higher vascular shear stress, will be sensed by mechanosensory cilia. Bending or activation of
the cilia involves mechanosensory polycystin-1 and polycystin-2 complex and a cascade of bio-
chemical synthesis of NO. The cascade will further involve extracellular calcium influx (Ca2+ ),
followed by activation of various calcium-dependent proteins, including calmodulin (CaM) and
protein kinase C (PKC). Together with PKB, CaM and PKC are important downstream molecu-
lar components to activate endothelial nitric oxide synthase (eNOS). Figure is reproduced from
AbouAlaiwi, et al. with permission (AbouAlaiwi et al., 2009a)
diseases are still to be identified. There is no doubt that the physiological roles of
primary cilia will continue to be debated in years to come.
Acknowledgement Due to restricted space, we apologize to those whose work is not described
in this review. Authors are grateful for stimulating discussion about primary cilia given by research
assistants, graduates, undergraduates and pharmacy students in our laboratory. Authors thank
Drs. Robert Kolb, Stefan Somlo, Bradley Yoder, and Jing Zhou for valuable insights and use of
their laboratory reagents. Authors also thank Charisse Montgomery for her editorial review of the
manuscript. Work from our laboratory that is cited in this review has been supported by grants from
the NIH (DK080640), AHA (0630257 N), and University of Toledo research programs, including
deArce Memorial Endowment Fund. A. M. Nauli is supported by AHA (0825195F).
References
AbouAlaiwi WA, Lo ST, Nauli SM (2009a) Primary cilia: highly sophisticated biological sensors.
Sensors 9:70037020
AbouAlaiwi WA, Takahashi M, Mell BR, Jones TJ, Ratnam S, Kolb RJ, Nauli SM (2009b) Ciliary
polycystin-2 is a mechanosensitive calcium channel involved in nitric oxide signaling cascades.
Circ Res 104:860869
Afzelius BA (1976) A human syndrome caused by immotile cilia. Science 193:317319
Alenghat FJ, Nauli SM, Kolb R, Zhou J, Ingber DE (2004) Global cytoskeletal control of
mechanotransduction in kidney epithelial cells. Exp Cell Res 301:2330
Andrade YN, Fernandes J, Vazquez E, Fernandez-Fernandez JM, Arniges M, Sanchez TM,
Villalon M, Valverde MA (2005) TRPV4 channel is involved in the coupling of fluid viscosity
changes to epithelial ciliary activity. J Cell Biol 168:869874
Ansley SJ, Badano JL, Blacque OE, Hill J, Hoskins BE, Leitch CC, Kim JC, Ross AJ, Eichers
ER, Teslovich TM, Mah AK, Johnsen RC, Cavender JC, Lewis RA, Leroux MR, Beales
PL, Katsanis N (2003) Basal body dysfunction is a likely cause of pleiotropic Bardet-Biedl
syndrome. Nature 425:628633
Archer J, Solomon F (1994) Deconstructing the microtubule-organizing center. Cell 76:589591
Askham JM, Vaughan KT, Goodson HV, Morrison EE (2002) Evidence that an interaction between
EB1 and p150(Glued) is required for the formation and maintenance of a radial microtubule
array anchored at the centrosome. Mol Biol Cell 13:36273645
Aughsteen AA (2001) The ultrastructure of primary cilia in the endocrine and excretory duct cells
of the pancreas of mice and rats. Eur J Morphol 39:277283
Avidor-Reiss T, Maer AM, Koundakjian E, Polyanovsky A, Keil T, Subramaniam S, Zuker CS
(2004) Decoding cilia function: defining specialized genes required for compartmentalized cilia
biogenesis. Cell 117:527539
Bahe S, Stierhof YD, Wilkinson CJ, Leiss F, Nigg EA (2005) Rootletin forms centriole-associated
filaments and functions in centrosome cohesion. J Cell Biol 171:2733
Baker JD, Adhikarakunnathu S, Kernan MJ (2004) Mechanosensory-defective, male-sterile
unc mutants identify a novel basal body protein required for ciliogenesis in Drosophila.
Development 131:34113422
Barr MM, Sternberg PW (1999) A polycystic kidney-disease gene homologue required for male
mating behaviour in C. elegans. Nature 401:386389
Bartoloni L, Blouin JL, Pan Y, Gehrig C, Maiti AK, Scamuffa N, Rossier C, Jorissen M, Armengot
M, Meeks M, Mitchison HM, Chung EM, Delozier-Blanchet CD, Craigen WJ, Antonarakis SE
(2002) Mutations in the DNAH11 (axonemal heavy chain dynein type 11) gene cause one form
of situs inversus totalis and most likely primary ciliary dyskinesia. Proc Natl Acad Sci U S A
99:1028210286
Basto R, Lau J, Vinogradova T, Gardiol A, Woods CG, Khodjakov A, Raff JW (2006) Flies without
centrioles. Cell 125:13751386
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 337
Beales PL, Bland E, Tobin JL, Bacchelli C, Tuysuz B, Hill J, Rix S, Pearson CG, Kai M, Hartley
J, Johnson C, Irving M, Elcioglu N, Winey M, Tada M, Scambler PJ (2007) IFT80, which
encodes a conserved intraflagellar transport protein, is mutated in Jeune asphyxiating thoracic
dystrophy. Nat Genet 39:727729
Benzing T, Walz G (2006) Cilium-generated signaling: a cellular GPS? Curr Opin Nephrol
Hypertens 15:245249
Berbari NF, Lewis JS, Bishop GA, Askwith CC, Mykytyn K (2008) Bardet-Biedl syndrome pro-
teins are required for the localization of G protein-coupled receptors to primary cilia. Proc Natl
Acad Sci U S A 105:42424246
Bergmann C, Fliegauf M, Bruchle NO, Frank V, Olbrich H, Kirschner J, Schermer B, Schmedding
I, Kispert A, Kranzlin B, Nurnberg G, Becker C, Grimm T, Girschick G, Lynch SA, Kelehan
P, Senderek J, Neuhaus TJ, Stallmach T, Zentgraf H, Nurnberg P, Gretz N, Lo C, Lienkamp
S, Schafer T, Walz G, Benzing T, Zerres K, Omran H (2008) Loss of nephrocystin-3 function
can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-
pancreatic dysplasia. Am J Hum Genet 82:959970
Bishop GA, Berbari NF, Lewis J, Mykytyn K (2007) Type III adenylyl cyclase localizes to primary
cilia throughout the adult mouse brain. J Comp Neurol 505:562571
Blacque OE, Reardon MJ, Li C, McCarthy J, Mahjoub MR, Ansley SJ, Badano JL, Mah AK,
Beales PL, Davidson WS, Johnsen RC, Audeh M, Plasterk RH, Baillie DL, Katsanis N,
Quarmby LM, Wicks SR, Leroux MR (2004) Loss of C. elegans BBS-7 and BBS-8 pro-
tein function results in cilia defects and compromised intraflagellar transport. Genes Dev
18:16301642
Boulter C, Mulroy S, Webb S, Fleming S, Brindle K, Sandford R (2001) Cardiovascular, skeletal,
and renal defects in mice with a targeted disruption of the Pkd1 gene. Proc Natl Acad Sci USA
98:1217412179
Brailov I, Bancila M, Brisorgueil MJ, Miquel MC, Hamon M, Verge D (2000) Localization of
5-HT(6) receptors at the plasma membrane of neuronal cilia in the rat brain. Brain Res 872:
271275
Brown NE, Murcia NS (2003) Delayed cystogenesis and increased ciliogenesis associated with the
re-expression of polaris in Tg737 mutant mice. Kidney Int 63:12201229
Brunner S, Colman D, Travis AJ, Luhmann UF, Shi W, Feil S, Imsand C, Nelson J, Grimm C,
Rulicke T, Fundele R, Neidhardt J, Berger W (2008) Overexpression of RPGR leads to male
infertility in mice due to defects in flagellar assembly. Biol Reprod 79:608617
Cano DA, Murcia NS, Pazour GJ, Hebrok M (2004) Orpk mouse model of polycystic kidney
disease reveals essential role of primary cilia in pancreatic tissue organization. Development
131:34573467
Cano DA, Sekine S, Hebrok M (2006) Primary cilia deletion in pancreatic epithelial cells results
in cyst formation and pancreatitis. Gastroenterology 131:18561869
Cantagrel V, Silhavy JL, Bielas SL, Swistun D, Marsh SE, Bertrand JY, Audollent S, Attie-Bitach
T, Holden KR, Dobyns WB, Traver D, Al-Gazali L, Ali BR, Lindner TH, Caspary T, Otto
EA, Hildebrandt F, Glass IA, Logan CV, Johnson CA, Bennett C, Brancati F, Valente EM,
Woods CG, Gleeson JG (2008) Mutations in the cilia gene ARL13B lead to the classical form
of Joubert syndrome. Am J Hum Genet 83:170179
Carmona J (1990) [Application of electro-erosion in 2-stage denture construction]. Rev Fr Prothes
Dent:3346
Chauvet V, Tian X, Husson H, Grimm DH, Wang T, Hiesberger T, Igarashi P, Bennett AM,
Ibraghimov-Beskrovnaya O, Somlo S, Caplan MJ (2004) Mechanical stimuli induce cleavage
and nuclear translocation of the polycystin-1 C terminus. J Clin Invest 114:14331443
Christensen ST, Pedersen SF, Satir P, Veland IR, Schneider L (2008) The primary cilium coor-
dinates signaling pathways in cell cycle control and migration during development and tissue
repair. Curr Top Dev Biol 85:261301
Corbit KC, Aanstad P, Singla V, Norman AR, Stainier DY, Reiter JF (2005) Vertebrate Smoothened
functions at the primary cilium. Nature 437:10181021
338 S.M. Nauli et al.
Dave D, Wloga D, Sharma N, Gaertig J (2009) DYF-1 Is required for assembly of the axoneme in
Tetrahymena thermophila. Eukaryot Cell 8:13971406
Davenport JR, Watts AJ, Roper VC, Croyle MJ, van Groen T, Wyss JM, Nagy TR, Kesterson
RA, Yoder BK (2007) Disruption of intraflagellar transport in adult mice leads to obesity and
slow-onset cystic kidney disease. Curr Biol 17:15861594
Davis RE, Swiderski RE, Rahmouni K, Nishimura DY, Mullins RF, Agassandian K, Philp AR,
Searby CC, Andrews MP, Thompson S, Berry CJ, Thedens DR, Yang B, Weiss RM, Cassell
MD, Stone EM, Sheffield VC (2007) A knockin mouse model of the Bardet-Biedl syndrome 1
M390R mutation has cilia defects, ventriculomegaly, retinopathy, and obesity. Proc Natl Acad
Sci U S A 104:1942219427
Davy BE, Robinson ML (2003) Congenital hydrocephalus in hy3 mice is caused by a frameshift
mutation in Hydin, a large novel gene. Hum Mol Genet 12:11631170
Dawe HR, Farr H, Portman N, Shaw MK, Gull K (2005) The Parkin co-regulated gene prod-
uct, PACRG, is an evolutionarily conserved axonemal protein that functions in outer-doublet
microtubule morphogenesis. J Cell Sci 118:54215430
Dawe HR, Smith UM, Cullinane AR, Gerrelli D, Cox P, Badano JL, Blair-Reid S, Sriram N,
Katsanis N, Attie-Bitach T, Afford SC, Copp AJ, Kelly DA, Gull K, Johnson CA (2007) The
Meckel-Gruber Syndrome proteins MKS1 and meckelin interact and are required for primary
cilium formation. Hum Mol Genet 16:173186
De Robertis E (1956) Morphogenesis of the retinal rods; an electron microscope study. J Biophys
Biochem Cytol 2:209218
Donkor FF, Monnich M, Czirr E, Hollemann T, Hoyer-Fender S (2004) Outer dense fibre protein
2 (ODF2) is a self-interacting centrosomal protein with affinity for microtubules. J Cell Sci
117:46434651
Doughty MJ (1998) Changes in cell surface primary cilia and microvilli concurrent with measure-
ments of fluid flow across the rabbit corneal endothelium ex vivo. Tissue Cell 30:634643
Doxsey S, McCollum D, Theurkauf W (2005) Centrosomes in cellular regulation. Annu Rev Cell
Dev Biol 21:411434
Dutcher SK (1995) Flagellar assembly in two hundred and fifty easy-to-follow steps. Trends Genet
11:398404
Eddy CA, Pauerstein CJ (1980) Anatomy and physiology of the fallopian tube. Clin Obstet
Gynecol 23:11771193
Eley L, Gabrielides C, Adams M, Johnson CA, Hildebrandt F, Sayer JA (2008) Jouberin localizes
to collecting ducts and interacts with nephrocystin-1. Kidney Int 74:11391149
Endoh-Yamagami S, Evangelista M, Wilson D, Wen X, Theunissen JW, Phamluong K, Davis M,
Scales SJ, Solloway MJ, de Sauvage FJ, Peterson AS (2009) The mammalian Cos2 homolog
Kif7 plays an essential role in modulating Hh signal transduction during development. Curr
Biol 19:13201326
Essner JJ, Vogan KJ, Wagner MK, Tabin CJ, Yost HJ, Brueckner M (2002) Conserved function for
embryonic nodal cilia. Nature 418:3738
Etienne-Manneville S, Hall A (2003) Cdc42 regulates GSK-3beta and adenomatous polyposis coli
to control cell polarity. Nature 421:753756
Fan S, Fogg V, Wang Q, Chen XW, Liu CJ, Margolis B (2007) A novel Crumbs3 isoform regulates
cell division and ciliogenesis via importin beta interactions. J Cell Biol 178:387398
Fan S, Hurd TW, Liu CJ, Straight SW, Weimbs T, Hurd EA, Domino SE, Margolis B (2004a)
Polarity proteins control ciliogenesis via kinesin motor interactions. Curr Biol 14:14511461
Fan Y, Esmail MA, Ansley SJ, Blacque OE, Boroevich K, Ross AJ, Moore SJ, Badano JL,
May-Simera H, Compton DS, Green JS, Lewis RA, van Haelst MM, Parfrey PS, Baillie DL,
Beales PL, Katsanis N, Davidson WS, Leroux MR (2004b) Mutations in a member of the
Ras superfamily of small GTP-binding proteins causes Bardet-Biedl syndrome. Nat Genet
36:989993
Fawcett DW, Porter KR (1954) A study of the fine structure of ciliated epithelia. J Morphol 94:
221281
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 339
Feistel K, Blum M (2006) Three types of cilia including a novel 9+4 axoneme on the notochordal
plate of the rabbit embryo. Dev Dyn 235:33483358
Ferrante MI, Zullo A, Barra A, Bimonte S, Messaddeq N, Studer M, Dolle P, Franco B (2006)
Oral-facial-digital type I protein is required for primary cilia formation and left-right axis
specification. Nat Genet 38:112117
Fischer E, Legue E, Doyen A, Nato F, Nicolas JF, Torres V, Yaniv M, Pontoglio M (2006) Defective
planar cell polarity in polycystic kidney disease. Nat Genet 38:2123
Follit JA, Tuft RA, Fogarty KE, Pazour GJ (2006) The intraflagellar transport protein IFT20
is associated with the Golgi complex and is required for cilia assembly. Mol Biol Cell 17:
37813792
Forman JR, Qamar S, Paci E, Sandford RN, Clarke J (2005) The remarkable mechanical strength
of polycystin-1 supports a direct role in mechanotransduction. J Mol Biol 349:861871
Gaertig J, Wloga D (2008) Ciliary tubulin and its post-translational modifications. Curr Top Dev
Biol 85:83113
Gallagher BC (1980) Primary cilia of the corneal endothelium. Am J Anat 159:475484
Gerdes JM, Liu Y, Zaghloul NA, Leitch CC, Lawson SS, Kato M, Beachy PA, Beales PL,
DeMartino GN, Fisher S, Badano JL, Katsanis N (2007) Disruption of the basal body
compromises proteasomal function and perturbs intracellular Wnt response. Nat Genet 39:
13501360
Gherman A, Davis EE, Katsanis N (2006) The ciliary proteome database: an integrated community
resource for the genetic and functional dissection of cilia. Nat Genet 38:961962
Ghosh AK, Murga-Zamalloa CA, Chan L, Hitchcock PF, Swaroop A, Khanna H (2010) Human
retinopathy-associated ciliary protein Retinitis Pigmentosa GTPase Regulator (RPGR) regu-
lates cilia-dependent vertebrate development. Hum Mol Genet 19:9098
Gorden NT, Arts HH, Parisi MA, Coene KL, Letteboer SJ, van Beersum SE, Mans DA, Hikida
A, Eckert M, Knutzen D, Alswaid AF, Ozyurek H, Dibooglu S, Otto EA, Liu Y, Davis EE,
Hutter CM, Bammler TK, Farin FM, Dorschner M, Topcu M, Zackai EH, Rosenthal P, Owens
KN, Katsanis N, Vincent JB, Hildebrandt F, Rubel EW, Raible DW, Knoers NV, Chance PF,
Roepman R, Moens CB, Glass IA, Doherty D (2008) CC2D2A is mutated in Joubert syndrome
and interacts with the ciliopathy-associated basal body protein CEP290. Am J Hum Genet
83:559571
Gorivodsky M, Mukhopadhyay M, Wilsch-Braeuninger M, Phillips M, Teufel A, Kim C, Malik N,
Huttner W, Westphal H (2009) Intraflagellar transport protein 172 is essential for primary cilia
formation and plays a vital role in patterning the mammalian brain. Dev Biol 325:2432
Gouttenoire J, Valcourt U, Bougault C, Aubert-Foucher E, Arnaud E, Giraud L, Mallein-Gerin
F (2007) Knockdown of the intraflagellar transport protein IFT46 stimulates selective gene
expression in mouse chondrocytes and affects early development in zebrafish. J Biol Chem
282:3096030973
Grallert A, Hagan IM (2002) Schizosaccharomyces pombe NIMA-related kinase, Fin1, regulates
spindle formation and an affinity of Polo for the SPB. EMBO J 21:30963107
Graser S, Stierhof YD, Lavoie SB, Gassner OS, Lamla S, Le Clech M, Nigg EA (2007) Cep164,
a novel centriole appendage protein required for primary cilium formation. J Cell Biol 179:
321330
Grillet N, Kazmierczak P, Xiong W, Schwander M, Reynolds A, Sakaguchi H, Tokita J, Kachar B,
Muller U (2009a) The mechanotransduction machinery of hair cells. Sci Signal 2:pt5
Grillet N, Schwander M, Hildebrand MS, Sczaniecka A, Kolatkar A, Velasco J, Webster JA,
Kahrizi K, Najmabadi H, Kimberling WJ, Stephan D, Bahlo M, Wiltshire T, Tarantino LM,
Kuhn P, Smith RJ, Muller U (2009b) Mutations in LOXHD1, an evolutionarily conserved stere-
ociliary protein, disrupt hair cell function in mice and cause progressive hearing loss in humans.
Am J Hum Genet 85:328337
Han YG, Kim HJ, Dlugosz AA, Ellison DW, Gilbertson RJ, Alvarez-Buylla A (2009)
Dual and opposing roles of primary cilia in medulloblastoma development. Nat Med 15:
10621065
340 S.M. Nauli et al.
Haycraft CJ, Banizs B, Aydin-Son Y, Zhang Q, Michaud EJ, Yoder BK (2005) Gli2 and Gli3 local-
ize to cilia and require the intraflagellar transport protein polaris for processing and function.
PLoS Genet 1:e53
Haycraft CJ, Swoboda P, Taulman PD, Thomas JH, Yoder BK (2001) The C. elegans homolog of
the murine cystic kidney disease gene Tg737 functions in a ciliogenic pathway and is disrupted
in osm-5 mutant worms. Development 128:14931505
Hearn T, Spalluto C, Phillips VJ, Renforth GL, Copin N, Hanley NA, Wilson DI (2005) Subcellular
localization of ALMS1 supports involvement of centrosome and basal body dysfunction in the
pathogenesis of obesity, insulin resistance, and type 2 diabetes. Diabetes 54:15811587
Hierck BP, Van der Heiden K, Alkemade FE, Van de Pas S, Van Thienen JV, Groenendijk BC,
Bax WH, Van der Laarse A, Deruiter MC, Horrevoets AJ, Poelmann RE (2008) Primary cilia
sensitize endothelial cells for fluid shear stress. Dev Dyn 237:725735
Hiesberger T, Gourley E, Erickson A, Koulen P, Ward CJ, Masyuk TV, Larusso NF, Harris PC,
Igarashi P (2006) Proteolytic cleavage and nuclear translocation of fibrocystin is regulated by
intracellular Ca2+ and activation of protein kinase C. J Biol Chem 281:3435734364
Hogan MC, Manganelli L, Woollard JR, Masyuk AI, Masyuk TV, Tammachote R, Huang BQ,
Leontovich AA, Beito TG, Madden BJ, Charlesworth MC, Torres VE, LaRusso NF, Harris PC,
Ward CJ (2009) Characterization of PKD protein-positive exosome-like vesicles. J Am Soc
Nephrol 20:278288
Hori Y, Kobayashi T, Kikko Y, Kontani K, Katada T (2008) Domain architecture of the atypi-
cal Arf-family GTPase Arl13b involved in cilia formation. Biochem Biophys Res Commun
373:119124
Hou B, Kolpakova-Hart E, Fukai N, Wu K, Olsen BR (2009) The polycystic kidney disease 1
(Pkd1) gene is required for the responses of osteochondroprogenitor cells to midpalatal suture
expansion in mice. Bone 44:11211133
Hou X, Mrug M, Yoder BK, Lefkowitz EJ, Kremmidiotis G, DEustachio P, Beier DR, Guay-
Woodford LM (2002) Cystin, a novel cilia-associated protein, is disrupted in the cpk mouse
model of polycystic kidney disease. J Clin Invest 109:533540
Houde C, Dickinson RJ, Houtzager VM, Cullum R, Montpetit R, Metzler M, Simpson EM, Roy
S, Hayden MR, Hoodless PA, Nicholson DW (2006) Hippi is essential for node cilia assembly
and Sonic hedgehog signaling. Dev Biol 300:523533
Hovater MB, Olteanu D, Hanson EL, Cheng NL, Siroky B, Fintha A, Komlosi P, Liu W, Satlin
LM, Bell PD, Yoder BK, Schwiebert EM (2008) Loss of apical monocilia on collecting duct
principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and
flow-induced calcium signals. Purinergic Signal 4:155170
Hu J, Barr MM (2005) ATP-2 interacts with the PLAT domain of LOV-1 and is involved in
Caenorhabditis elegans polycystin signaling. Mol Biol Cell 16:458469
Huang BQ, Masyuk TV, Muff MA, Tietz PS, Masyuk AI, Larusso NF (2006) Isolation and
characterization of cholangiocyte primary cilia. Am J Physiol Gastrointest Liver Physiol
291:G500509
Huangfu D, Liu A, Rakeman AS, Murcia NS, Niswander L, Anderson KV (2003) Hedgehog
signalling in the mouse requires intraflagellar transport proteins. Nature 426:8387
Ibanez-Tallon I, Pagenstecher A, Fliegauf M, Olbrich H, Kispert A, Ketelsen UP, North A, Heintz
N, Omran H (2004) Dysfunction of axonemal dynein heavy chain Mdnah5 inhibits ependy-
mal flow and reveals a novel mechanism for hydrocephalus formation. Hum Mol Genet 13:
21332141
Imai H, Hakkaku N, Iwamoto R, Suzuki J, Suzuki T, Tajima Y, Konishi K, Minami S, Ichinose
S, Ishiizaka K, Shioda S, Arata S, Nishimura M, Naito S, Nakagawa Y (2009) Depletion of
selenoprotein GPx4 in spermatocytes causes male infertility in mice. J Biol Chem 284:32522
32532
Iomini C, Tejada K, Mo W, Vaananen H, Piperno G (2004) Primary cilia of human endothelial
cells disassemble under laminar shear stress. J Cell Biol 164:811817
Ishikawa H, Kubo A, Tsukita S, Tsukita S (2005) Odf2-deficient mother centrioles lack dis-
tal/subdistal appendages and the ability to generate primary cilia. Nat Cell Biol 7:517524
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 341
Jenkins PM, Hurd TW, Zhang L, McEwen DP, Brown RL, Margolis B, Verhey KJ, Martens JR
(2006) Ciliary targeting of olfactory CNG channels requires the CNGB1b subunit and the
kinesin-2 motor protein, KIF17. Curr Biol 16:12111216
Jia J, Kolterud A, Zeng H, Hoover A, Teglund S, Toftgard R, Liu A (2009) Suppressor of Fused
inhibits mammalian Hedgehog signaling in the absence of cilia. Dev Biol 330:452460
Jonassen JA, San Agustin J, Follit JA, Pazour GJ (2008) Deletion of IFT20 in the mouse kidney
causes misorientation of the mitotic spindle and cystic kidney disease. J Cell Biol 183:377384
Jurczyk A, Gromley A, Redick S, San Agustin J, Witman G, Pazour GJ, Peters DJ, Doxsey S
(2004) Pericentrin forms a complex with intraflagellar transport proteins and polycystin-2 and
is required for primary cilia assembly. J Cell Biol 166:637643
Kaestner KH (2006) Of cilia and cysts: modeling pancreatic polycystic disease. Gastroenterology
130:926928
Kaimori JY, Nagasawa Y, Menezes LF, Garcia-Gonzalez MA, Deng J, Imai E, Onuchic LF,
Guay-Woodford LM, Germino GG (2007) Polyductin undergoes notch-like processing and
regulated release from primary cilia. Hum Mol Genet 16:942956
Karcher C, Fischer A, Schweickert A, Bitzer E, Horie S, Witzgall R, Blum M (2005) Lack of
a laterality phenotype in Pkd1 knock-out embryos correlates with absence of polycystin-1 in
nodal cilia. Differentiation 73:425432
Kenney J, Karsenti E, Gowen B, Fuller SD (1997) Three-dimensional reconstruction of the mam-
malian centriole from cryoelectron micrographs: the use of common lines for orientation and
alignment. J Struct Biol 120:320328
Kernan MJ (2007) Mechanotransduction and auditory transduction in Drosophila. Pflugers Arch
454:703720
Kim J, Chung YD, Park DY, Choi S, Shin DW, Soh H, Lee HW, Son W, Yim J, Park CS, Kernan
MJ, Kim C (2003) A TRPV family ion channel required for hearing in Drosophila. Nature
424:8184
Kim J, Krishnaswami SR, Gleeson JG (2008) CEP290 interacts with the centriolar satellite com-
ponent PCM-1 and is required for Rab8 localization to the primary cilium. Hum Mol Genet
17:37963805
Kim JC, Badano JL, Sibold S, Esmail MA, Hill J, Hoskins BE, Leitch CC, Venner K, Ansley SJ,
Ross AJ, Leroux MR, Katsanis N, Beales PL (2004) The Bardet-Biedl protein BBS4 targets
cargo to the pericentriolar region and is required for microtubule anchoring and cell cycle
progression. Nat Genet 36:462470
Kim JC, Ou YY, Badano JL, Esmail MA, Leitch CC, Fiedrich E, Beales PL, Archibald JM,
Katsanis N, Rattner JB, Leroux MR (2005) MKKS/BBS6, a divergent chaperonin-like pro-
tein linked to the obesity disorder Bardet-Biedl syndrome, is a novel centrosomal component
required for cytokinesis. J Cell Sci 118:10071020
Kim S, Lee K, Rhee K (2007) NEK7 is a centrosomal kinase critical for microtubule nucleation.
Biochem Biophys Res Commun 360:5662
Kishimoto N, Cao Y, Park A, Sun Z (2008) Cystic kidney gene seahorse regulates cilia-mediated
processes and Wnt pathways. Dev Cell 14:954961
Klein-Nulend J, van der Plas A, Semeins CM, Ajubi NE, Frangos JA, Nijweide PJ, Burger EH
(1995) Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 9:441445
Kolb RJ, Nauli SM (2008) Ciliary dysfunction in polycystic kidney disease: an emerging model
with polarizing potential. Front Biosci 13:44514466
Kolpakova-Hart E, McBratney-Owen B, Hou B, Fukai N, Nicolae C, Zhou J, Olsen BR (2008)
Growth of cranial synchondroses and sutures requires polycystin-1. Dev Biol 321:407419
Kottgen M, Buchholz B, Garcia-Gonzalez MA, Kotsis F, Fu X, Doerken M, Boehlke C, Steffl D,
Tauber R, Wegierski T, Nitschke R, Suzuki M, Kramer-Zucker A, Germino GG, Watnick T,
Prenen J, Nilius B, Kuehn EW, Walz G (2008) TRPP2 and TRPV4 form a polymodal sensory
channel complex. J Cell Biol 182:437447
Krock BL, Perkins BD (2008) The intraflagellar transport protein IFT57 is required for cilia main-
tenance and regulates IFT-particle-kinesin-II dissociation in vertebrate photoreceptors. J Cell
Sci 121:19071915
342 S.M. Nauli et al.
Kulaga HM, Leitch CC, Eichers ER, Badano JL, Lesemann A, Hoskins BE, Lupski JR, Beales PL,
Reed RR, Katsanis N (2004) Loss of BBS proteins causes anosmia in humans and defects in
olfactory cilia structure and function in the mouse. Nat Genet 36:994998
Kyttala M, Tallila J, Salonen R, Kopra O, Kohlschmidt N, Paavola-Sakki P, Peltonen L, Kestila M
(2006) MKS1, encoding a component of the flagellar apparatus basal body proteome, is mutated
in Meckel syndrome. Nat Genet 38:155157
Lang B, Song B, Davidson W, MacKenzie A, Smith N, McCaig CD, Harmar AJ, Shen S
(2006) Expression of the human PAC1 receptor leads to dose-dependent hydrocephalus-related
abnormalities in mice. J Clin Invest 116:19241934
Layman WS, McEwen DP, Beyer LA, Lalani SR, Fernbach SD, Oh E, Swaroop A, Hegg CC,
Raphael Y, Martens JR, Martin DM (2009) Defects in neural stem cell proliferation and olfac-
tion in Chd7 deficient mice indicate a mechanism for hyposmia in human CHARGE syndrome.
Hum Mol Genet 18:19091923
Lee L, Campagna DR, Pinkus JL, Mulhern H, Wyatt TA, Sisson JH, Pavlik JA, Pinkus GS, Fleming
MD (2008) Primary ciliary dyskinesia in mice lacking the novel ciliary protein Pcdp1. Mol Cell
Biol 28:949957
Li G, Vega R, Nelms K, Gekakis N, Goodnow C, McNamara P, Wu H, Hong NA, Glynne R (2007)
A role for Alstrom syndrome protein, alms1, in kidney ciliogenesis and cellular quiescence.
PLoS Genet 3:e8
Li JB, Gerdes JM, Haycraft CJ, Fan Y, Teslovich TM, May-Simera H, Li H, Blacque OE, Li
L, Leitch CC, Lewis RA, Green JS, Parfrey PS, Leroux MR, Davidson WS, Beales PL,
Guay-Woodford LM, Yoder BK, Stormo GD, Katsanis N, Dutcher SK (2004) Comparative
genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease
gene. Cell 117:541552
Liem KF, Jr., He M, Ocbina PJ, Anderson KV (2009) Mouse Kif7/Costal2 is a cilia-associated
protein that regulates Sonic hedgehog signaling. Proc Natl Acad Sci U S A 106:1337713382
Lin F, Hiesberger T, Cordes K, Sinclair AM, Goldstein LS, Somlo S, Igarashi P (2003) Kidney-
specific inactivation of the KIF3A subunit of kinesin-II inhibits renal ciliogenesis and produces
polycystic kidney disease. Proc Natl Acad Sci U S A 100:52865291
Littlewood Evans A, Muller U (2000) Stereocilia defects in the sensory hair cells of the inner ear
in mice deficient in integrin alpha8beta1. Nat Genet 24:424428
Liu A, Wang B, Niswander LA (2005a) Mouse intraflagellar transport proteins regulate both the
activator and repressor functions of Gli transcription factors. Development 132:31033111
Liu W, Murcia NS, Duan Y, Weinbaum S, Yoder BK, Schwiebert E, Satlin LM (2005b)
Mechanoregulation of intracellular Ca2+ concentration is attenuated in collecting duct of
monocilium-impaired orpk mice. Am J Physiol Renal Physiol 289:F978988
Lolkema MP, Mans DA, Snijckers CM, van Noort M, van Beest M, Voest EE, Giles RH (2007) The
von Hippel-Lindau tumour suppressor interacts with microtubules through kinesin-2. FEBS
Lett 581:45714576
Lorenzetti D, Bishop CE, Justice MJ (2004) Deletion of the Parkin coregulated gene causes male
sterility in the quaking(viable) mouse mutant. Proc Natl Acad Sci U S A 101:84028407
Low SH, Vasanth S, Larson CH, Mukherjee S, Sharma N, Kinter MT, Kane ME, Obara T,
Weimbs T (2006) Polycystin-1, STAT6, and P100 function in a pathway that transduces ciliary
mechanosensation and is activated in polycystic kidney disease. Dev Cell 10:5769
Lu CJ, Du H, Wu J, Jansen DA, Jordan KL, Xu N, Sieck GC, Qian Q (2008) Non-random distri-
bution and sensory functions of primary cilia in vascular smooth muscle cells. Kidney Blood
Press Res 31:171184
Lu W, Shen X, Pavlova A, Lakkis M, Ward CJ, Pritchard L, Harris PC, Genest DR, Perez-Atayde
AR, Zhou J (2001) Comparison of Pkd1-targeted mutants reveals that loss of polycystin-1
causes cystogenesis and bone defects. Hum Mol Genet 10:23852396
Lucker BF, Behal RH, Qin H, Siron LC, Taggart WD, Rosenbaum JL, Cole DG (2005)
Characterization of the intraflagellar transport complex B core: direct interaction of the IFT81
and IFT74/72 subunits. J Biol Chem 280:2768827696
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 343
Lungarella G, de Santi MM, Tosi P (1984) Ultrastructural study of the ciliated cells from renal
tubular epithelium in acute progressive glomerulonephritis. Ultrastruct Pathol 6:17
Lunt SC, Haynes T, Perkins BD (2009) Zebrafish ift57, ift88, and ift172 intraflagellar transport
mutants disrupt cilia but do not affect hedgehog signaling. Dev Dyn 238:17441759
Lyons RA, Saridogan E, Djahanbakhch O (2006) The reproductive significance of human Fallopian
tube cilia. Hum Reprod Update 12:363372
Ma R, Li WP, Rundle D, Kong J, Akbarali HI, Tsiokas L (2005) PKD2 functions as an epidermal
growth factor-activated plasma membrane channel. Mol Cell Biol 25:82858298
Magloire H, Couble ML, Romeas A, Bleicher F (2004) Odontoblast primary cilia: facts and
hypotheses. Cell Biol Int 28:9399
Mahjoub MR, Qasim Rasi M, Quarmby LM (2004) A NIMA-related kinase, Fa2p, localizes to
a novel site in the proximal cilia of Chlamydomonas and mouse kidney cells. Mol Biol Cell
15:51725186
Mahjoub MR, Trapp ML, Quarmby LM (2005) NIMA-related kinases defective in murine models
of polycystic kidney diseases localize to primary cilia and centrosomes. J Am Soc Nephrol
16:34853489
Mai W, Chen D, Ding T, Kim I, Park S, Cho SY, Chu JS, Liang D, Wang N, Wu D, Li S, Zhao
P, Zent R, Wu G (2005) Inhibition of Pkhd1 impairs tubulomorphogenesis of cultured IMCD
cells. Mol Biol Cell 16:43984409
Mak HY, Nelson LS, Basson M, Johnson CD, Ruvkun G (2006) Polygenic control of
Caenorhabditis elegans fat storage. Nat Genet 38:363368
Mall MA (2008) Role of cilia, mucus, and airway surface liquid in mucociliary dysfunction:
lessons from mouse models. J Aerosol Med Pulm Drug Deliv 21:1324
Malone AM, Anderson CT, Tummala P, Kwon RY, Johnston TR, Stearns T, Jacobs CR (2007)
Primary cilia mediate mechanosensing in bone cells by a calcium-independent mechanism.
Proc Natl Acad Sci U S A 104:1332513330
Marshall WF (2009) Centriole evolution. Curr Opin Cell Biol 21:1419
Marshall WF, Nonaka S (2006) Cilia: tuning in to the cells antenna. Curr Biol 16:R604614
Marszalek JR, Liu X, Roberts EA, Chui D, Marth JD, Williams DS, Goldstein LS (2000)
Genetic evidence for selective transport of opsin and arrestin by kinesin-II in mammalian
photoreceptors. Cell 102:175187
Marszalek JR, Ruiz-Lozano P, Roberts E, Chien KR, Goldstein LS (1999) Situs inversus and
embryonic ciliary morphogenesis defects in mouse mutants lacking the KIF3A subunit of
kinesin-II. Proc Natl Acad Sci USA 96:50435048
Masyuk AI, Masyuk TV, LaRusso NF (2008) Cholangiocyte primary cilia in liver health and
disease. Dev Dyn 237:20072012
Masyuk AI, Masyuk TV, Splinter PL, Huang BQ, Stroope AJ, LaRusso NF (2006) Cholangiocyte
cilia detect changes in luminal fluid flow and transmit them into intracellular Ca2+ and cAMP
signaling. Gastroenterology 131:911920
Masyuk T, Masyuk A, LaRusso N (2009) Cholangiociliopathies: genetics, molecular mechanisms
and potential therapies. Curr Opin Gastroenterol 25:265271
Masyuk TV, Huang BQ, Masyuk AI, Ritman EL, Torres VE, Wang X, Harris PC, Larusso
NF (2004) Biliary dysgenesis in the PCK rat, an orthologous model of autosomal recessive
polycystic kidney disease. Am J Pathol 165:17191730
Masyuk TV, Huang BQ, Ward CJ, Masyuk AI, Yuan D, Splinter PL, Punyashthiti R, Ritman EL,
Torres VE, Harris PC, LaRusso NF (2003) Defects in cholangiocyte fibrocystin expression and
ciliary structure in the PCK rat. Gastroenterology 125:13031310
Maurin JC, Couble ML, Staquet MJ, Carrouel F, About I, Avila J, Magloire H, Bleicher F (2009)
Microtubule-associated protein 1b, a neuronal marker involved in odontoblast differentiation.
J Endod 35:992996
May SR, Ashique AM, Karlen M, Wang B, Shen Y, Zarbalis K, Reiter J, Ericson J, Peterson AS
(2005) Loss of the retrograde motor for IFT disrupts localization of Smo to cilia and prevents
the expression of both activator and repressor functions of Gli. Dev Biol 287:378389
344 S.M. Nauli et al.
McGlashan SR, Haycraft CJ, Jensen CG, Yoder BK, Poole CA (2007) Articular cartilage and
growth plate defects are associated with chondrocyte cytoskeletal abnormalities in Tg737orpk
mice lacking the primary cilia protein polaris. Matrix Biol 26:234246
McGrath J, Somlo S, Makova S, Tian X, Brueckner M (2003) Two populations of node monocilia
initiate left-right asymmetry in the mouse. Cell 114:6173
Menco BP (2005) The fine-structural distribution of G-protein receptor kinase 3, beta-arrestin-2,
Ca2+ /calmodulin-dependent protein kinase II and phosphodiesterase PDE1C2, and a Cl(-)-
cotransporter in rodent olfactory epithelia. J Neurocytol 34:1136
Mikule K, Delaval B, Kaldis P, Jurcyzk A, Hergert P, Doxsey S (2007) Loss of centrosome integrity
induces p38-p53-p21-dependent G1-S arrest. Nat Cell Biol 9:160170
Mollet G, Silbermann F, Delous M, Salomon R, Antignac C, Saunier S (2005) Characterization
of the nephrocystin/nephrocystin-4 complex and subcellular localization of nephrocystin-4 to
primary cilia and centrosomes. Hum Mol Genet 14:645656
Moore A, Escudier E, Roger G, Tamalet A, Pelosse B, Marlin S, Clement A, Geremek M,
Delaisi B, Bridoux AM, Coste A, Witt M, Duriez B, Amselem S (2006) RPGR is mutated in
patients with a complex X linked phenotype combining primary ciliary dyskinesia and retinitis
pigmentosa. J Med Genet 43:326333
Moorman SJ, Shorr AZ (2008) The primary cilium as a gravitational force transducer and a
regulator of transcriptional noise. Dev Dyn 237:19551959
Morgan GW, Denny PW, Vaughan S, Goulding D, Jeffries TR, Smith DF, Gull K, Field MC
(2005) An evolutionarily conserved coiled-coil protein implicated in polycystic kidney dis-
ease is involved in basal body duplication and flagellar biogenesis in Trypanosoma brucei. Mol
Cell Biol 25:37743783
Mukhopadhyay A, Deplancke B, Walhout AJ, Tissenbaum HA (2005) C. elegans tubby regulates
life span and fat storage by two independent mechanisms. Cell Metab 2:3542
Murayama T, Toh Y, Ohshima Y, Koga M (2005) The dyf-3 gene encodes a novel protein required
for sensory cilium formation in Caenorhabditis elegans. J Mol Biol 346:677687
Murcia NS, Richards WG, Yoder BK, Mucenski ML, Dunlap JR, Woychik RP (2000) The Oak
Ridge Polycystic Kidney (orpk) disease gene is required for left-right axis determination.
Development 127:23472355
Nachury MV, Loktev AV, Zhang Q, Westlake CJ, Peranen J, Merdes A, Slusarski DC, Scheller RH,
Bazan JF, Sheffield VC, Jackson PK (2007) A core complex of BBS proteins cooperates with
the GTPase Rab8 to promote ciliary membrane biogenesis. Cell 129:12011213
Nagano J, Kitamura K, Hujer KM, Ward CJ, Bram RJ, Hopfer U, Tomita K, Huang C, Miller RT
(2005) Fibrocystin interacts with CAML, a protein involved in Ca2+ signaling. Biochem
Biophys Res Commun 338:880889
Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, Li X, Elia AE, Lu W, Brown EM, Quinn
SJ, Ingber DE, Zhou J (2003) Polycystins 1 and 2 mediate mechanosensation in the primary
cilium of kidney cells. Nat Genet 33:129137
Nauli SM, Kawanabe Y, Kaminski JJ, Pearce WJ, Ingber DE, Zhou J (2008) Endothelial cilia
are fluid shear sensors that regulate calcium signaling and nitric oxide production through
polycystin-1. Circulation 117:11611171
Nauli SM, Rossetti S, Kolb RJ, Alenghat FJ, Consugar MB, Harris PC, Ingber DE, Loghman-
Adham M, Zhou J (2006) Loss of polycystin-1 in human cyst-lining epithelia leads to ciliary
dysfunction. J Am Soc Nephrol 17:10151025
Nauli SM, Zhou J (2004) Polycystins and mechanosensation in renal and nodal cilia. Bioessays
26:844856
Neesen J, Kirschner R, Ochs M, Schmiedl A, Habermann B, Mueller C, Holstein AF, Nuesslein
T, Adham I, Engel W (2001) Disruption of an inner arm dynein heavy chain gene results in
asthenozoospermia and reduced ciliary beat frequency. Hum Mol Genet 10:11171128
Nishimura DY, Fath M, Mullins RF, Searby C, Andrews M, Davis R, Andorf JL, Mykytyn K,
Swiderski RE, Yang B, Carmi R, Stone EM, Sheffield VC (2004a) Bbs2-null mice have neu-
rosensory deficits, a defect in social dominance, and retinopathy associated with mislocalization
of rhodopsin. Proc Natl Acad Sci U S A 101:1658816593
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 345
Pathak N, Obara T, Mangos S, Liu Y, Drummond IA (2007) The zebrafish fleer gene encodes an
essential regulator of cilia tubulin polyglutamylation. Mol Biol Cell 18:43534364
Pazour GJ, Agrin N, Leszyk J, Witman GB (2005) Proteomic analysis of a eukaryotic cilium.
J Cell Biol 170:103113
Pazour GJ, Dickert BL, Vucica Y, Seeley ES, Rosenbaum JL, Witman GB, Cole DG (2000)
Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are
required for assembly of cilia and flagella. J Cell Biol 151:709718
Pazour GJ, San Agustin JT, Follit JA, Rosenbaum JL, Witman GB (2002) Polycystin-2 localizes to
kidney cilia and the ciliary level is elevated in orpk mice with polycystic kidney disease. Curr
Biol 12:R378380
Pedersen LB, Geimer S, Sloboda RD, Rosenbaum JL (2003) The Microtubule plus end-tracking
protein EB1 is localized to the flagellar tip and basal bodies in Chlamydomonas reinhardtii.
Curr Biol 13:19691974
Pedersen LB, Miller MS, Geimer S, Leitch JM, Rosenbaum JL, Cole DG (2005) Chlamydomonas
IFT172 is encoded by FLA11, interacts with CrEB1, and regulates IFT at the flagellar tip. Curr
Biol 15:262266
Pennarun G, Escudier E, Chapelin C, Bridoux AM, Cacheux V, Roger G, Clement A, Goossens
M, Amselem S, Duriez B (1999) Loss-of-function mutations in a human gene related to
Chlamydomonas reinhardtii dynein IC78 result in primary ciliary dyskinesia. Am J Hum Genet
65:15081519
Pennekamp P, Karcher C, Fischer A, Schweickert A, Skryabin B, Horst J, Blum M, Dworniczak B
(2002) The ion channel polycystin-2 is required for left-right axis determination in mice. Curr
Biol 12:938943
Petit C, Richardson GP (2009) Linking genes underlying deafness to hair-bundle development and
function. Nat Neurosci 12:703710
Piehl M, Tulu US, Wadsworth P, Cassimeris L (2004) Centrosome maturation: measurement of
microtubule nucleation throughout the cell cycle by using GFP-tagged EB1. Proc Natl Acad
Sci U S A 101:15841588
Poole CA, Flint MH, Beaumont BW (1985) Analysis of the morphology and function of primary
cilia in connective tissues: a cellular cybernetic probe? Cell Motil 5:175193
Poole CA, Jensen CG, Snyder JA, Gray CG, Hermanutz VL, Wheatley DN (1997) Confocal anal-
ysis of primary cilia structure and colocalization with the Golgi apparatus in chondrocytes and
aortic smooth muscle cells. Cell Biol Int 21:483494
Praetorius HA, Spring KR (2001) Bending the MDCK cell primary cilium increases intracellular
calcium. J Membr Biol 184:7179
Praetorius HA, Spring KR (2003) Removal of the MDCK cell primary cilium abolishes flow
sensing. J Membr Biol 191:6976
Qian F, Wei W, Germino G, Oberhauser A (2005) The nanomechanics of polycystin-1 extracellular
region. J Biol Chem 280:4072340730
Qin H, Burnette DT, Bae YK, Forscher P, Barr MM, Rosenbaum JL (2005) Intraflagellar transport
is required for the vectorial movement of TRPV channels in the ciliary membrane. Curr Biol
15:16951699
Qin H, Rosenbaum JL, Barr MM (2001) An autosomal recessive polycystic kidney disease gene
homolog is involved in intraflagellar transport in C. elegans ciliated sensory neurons. Curr Biol
11:457461
Rana AA, Barbera JP, Rodriguez TA, Lynch D, Hirst E, Smith JC, Beddington RS (2004) Targeted
deletion of the novel cytoplasmic dynein mD2LIC disrupts the embryonic organiser, formation
of the body axes and specification of ventral cell fates. Development 131:49995007
Redeker V, Levilliers N, Vinolo E, Rossier J, Jaillard D, Burnette D, Gaertig J, Bre MH (2005)
Mutations of tubulin glycylation sites reveal cross-talk between the C termini of alpha- and
beta-tubulin and affect the ciliary matrix in Tetrahymena. J Biol Chem 280:596606
Rieder CL, Faruki S, Khodjakov A (2001) The centrosome in vertebrates: more than a microtubule-
organizing center. Trends Cell Biol 11:413419
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 347
Rohatgi R, Battini L, Kim P, Israeli S, Wilson PD, Gusella GL, Satlin LM (2008)
Mechanoregulation of intracellular Ca2+ in human autosomal recessive polycystic kidney
disease cyst-lining renal epithelial cells. Am J Physio Renal Physiol 294:F890899
Romio L, Fry AM, Winyard PJ, Malcolm S, Woolf AS, Feather SA (2004) OFD1 is a cen-
trosomal/basal body protein expressed during mesenchymal-epithelial transition in human
nephrogenesis. J Am Soc Nephrol 15:25562568
Rose MD, Biggins S, Satterwhite LL (1993) Unravelling the tangled web at the microtubule-
organizing center. Curr Opin Cell Biol 5:105115
Rosenbaum JL, Witman GB (2002) Intraflagellar transport. Nat Rev Mol Cell Biol 3:813825
Roth KE, Rieder CL, Bowser SS (1988) Flexible-substratum technique for viewing cells from the
side: some in vivo properties of primary (9+0) cilia in cultured kidney epithelia. J Cell Sci
89 (Pt 4):457466
Rupp G, Porter ME (2003) A subunit of the dynein regulatory complex in Chlamydomonas is a
homologue of a growth arrest-specific gene product. J Cell Biol 162:4757
Saburi S, Hester I, Fischer E, Pontoglio M, Eremina V, Gessler M, Quaggin SE, Harrison R,
Mount R, McNeill H (2008) Loss of Fat4 disrupts PCP signaling and oriented cell division
and leads to cystic kidney disease. Nat Genet 40:10101015
Salathe M (2007) Regulation of mammalian ciliary beating. Annu Rev Physiol 69:401422
Sapiro R, Kostetskii I, Olds-Clarke P, Gerton GL, Radice GL, Strauss IJ (2002) Male infertility,
impaired sperm motility, and hydrocephalus in mice deficient in sperm-associated antigen 6.
Mol Cell Biol 22:62986305
Satir P, Christensen ST (2007) Overview of structure and function of mammalian cilia. Annu Rev
Physiol 69:377400
Sawamoto K, Wichterle H, Gonzalez-Perez O, Cholfin JA, Yamada M, Spassky N, Murcia NS,
Garcia-Verdugo JM, Marin O, Rubenstein JL, Tessier-Lavigne M, Okano H, Alvarez-Buylla
A (2006) New neurons follow the flow of cerebrospinal fluid in the adult brain. Science 311:
629632
Sayer JA, Otto EA, OToole JF, Nurnberg G, Kennedy MA, Becker C, Hennies HC, Helou J,
Attanasio M, Fausett BV, Utsch B, Khanna H, Liu Y, Drummond I, Kawakami I, Kusakabe
T, Tsuda M, Ma L, Lee H, Larson RG, Allen SJ, Wilkinson CJ, Nigg EA, Shou C, Lillo C,
Williams DS, Hoppe B, Kemper MJ, Neuhaus T, Parisi MA, Glass IA, Petry M, Kispert A, Gloy
J, Ganner A, Walz G, Zhu X, Goldman D, Nurnberg P, Swaroop A, Leroux MR, Hildebrandt
F (2006) The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates
transcription factor ATF4. Nat Genet 38:674681
Schneider L, Clement CA, Teilmann SC, Pazour GJ, Hoffmann EK, Satir P, Christensen ST (2005)
PDGFRalphaalpha signaling is regulated through the primary cilium in fibroblasts. Curr Biol
15:18611866
Scholey JM (2003) Intraflagellar transport. Annu Rev Cell Dev Biol 19:423443
Scholey JM, Anderson KV (2006) Intraflagellar transport and cilium-based signaling. Cell
125:439442
Schroder JM, Schneider L, Christensen ST, Pedersen LB (2007) EB1 is required for primary cilia
assembly in fibroblasts. Curr Biol 17:11341139
Schulz S, Handel M, Schreff M, Schmidt H, Hollt V (2000) Localization of five somatostatin
receptors in the rat central nervous system using subtype-specific antibodies. J Physiol Paris
94:259264
Schwartz EA, Leonard ML, Bizios R, Bowser SS (1997) Analysis and modeling of the primary
cilium bending response to fluid shear. Am J Physiol 272:F132138
Scoggins CR, Meszoely IM, Wada M, Means AL, Yang L, Leach SD (2000) p53-dependent acinar
cell apoptosis triggers epithelial proliferation in duct-ligated murine pancreas. Am J Physiol
Gastrointest Liver Physiol 279:G827836
Seeley ES, Carriere C, Goetze T, Longnecker DS, Korc M (2009) Pancreatic cancer and precur-
sor pancreatic intraepithelial neoplasia lesions are devoid of primary cilia. Cancer Res 69:
422430
348 S.M. Nauli et al.
Tsujikawa M, Malicki J (2004) Intraflagellar transport genes are essential for differentiation and
survival of vertebrate sensory neurons. Neuron 42:703716
Turner CH, Forwood MR, Otter MW (1994) Mechanotransduction in bone: do bone cells act as
sensors of fluid flow? FASEB J 8:875878
Van der Heiden K, Groenendijk BC, Hierck BP, Hogers B, Koerten HK, Mommaas AM,
Gittenberger-de Groot AC, Poelmann RE (2006) Monocilia on chicken embryonic endo-
cardium in low shear stress areas. Dev Dyn 235:1928
Van der Heiden K, Hierck BP, Krams R, de Crom R, Cheng C, Baiker M, Pourquie MJ,
Alkemade FE, DeRuiter MC, Gittenberger-de Groot AC, Poelmann RE (2008) Endothelial
primary cilia in areas of disturbed flow are at the base of atherosclerosis. Atherosclerosis 196:
542550
Vernon GG, Neesen J, Woolley DM (2005) Further studies on knockout mice lacking a functional
dynein heavy chain (MDHC7). 1. Evidence for a structural deficit in the axoneme. Cell Motil
Cytoskeleton 61:6573
Vieira OV, Gaus K, Verkade P, Fullekrug J, Vaz WL, Simons K (2006) FAPP2, cilium formation,
and compartmentalization of the apical membrane in polarized Madin-Darby canine kidney
(MDCK) cells. Proc Natl Acad Sci U S A 103:1855618561
Wang S, Zhang J, Nauli SM, Li X, Starremans PG, Luo Y, Roberts KA, Zhou J (2007)
Fibrocystin/polyductin, found in the same protein complex with polycystin-2, regulates calcium
responses in kidney epithelia. Mol Cell Biol 27:32413252
Ward CJ, Yuan D, Masyuk TV, Wang X, Punyashthiti R, Whelan S, Bacallao R, Torra R,
LaRusso NF, Torres VE, Harris PC (2003) Cellular and subcellular localization of the ARPKD
protein; fibrocystin is expressed on primary cilia. Hum Mol Genet 12:27032710
Weatherbee SD, Niswander LA, Anderson KV (2009) A mouse model for Meckel Syndrome
reveals Mks1 is required for ciliogenesis and Hedgehog signaling. Hum Mol Genet
Wheatley DN (1995) Primary cilia in normal and pathological tissues. Pathobiology 63:222238
Wheatley DN (2005) Landmarks in the first hundred years of primary (9+0) cilium research. Cell
Biol Int 29:333339
White MC, Quarmby LM (2008) The NIMA-family kinase, Nek1 affects the stability of centro-
somes and ciliogenesis. BMC Cell Biol 9:29
Whitfield JF (2008) The solitary (primary) cilium--a mechanosensory toggle switch in bone and
cartilage cells. Cell Signal 20:10191024
Winkelbauer ME, Schafer JC, Haycraft CJ, Swoboda P, Yoder BK (2005) The C. elegans
homologs of nephrocystin-1 and nephrocystin-4 are cilia transition zone proteins involved in
chemosensory perception. J Cell Sci 118:55755587
Wodarczyk C, Rowe I, Chiaravalli M, Pema M, Qian F, Boletta A (2009) A novel mouse model
reveals that polycystin-1 deficiency in ependyma and choroid plexus results in dysfunctional
cilia and hydrocephalus. PLoS One 4:e7137
Wong SY, Seol AD, So PL, Ermilov AN, Bichakjian CK, Epstein EH, Jr., Dlugosz AA,
Reiter JF (2009) Primary cilia can both mediate and suppress Hedgehog pathway-dependent
tumorigenesis. Nat Med 15:10551061
Woollard JR, Punyashtiti R, Richardson S, Masyuk TV, Whelan S, Huang BQ, Lager DJ, van-
Deursen J, Torres VE, Gattone VH, LaRusso NF, Harris PC, Ward CJ (2007) A mouse model of
autosomal recessive polycystic kidney disease with biliary duct and proximal tubule dilatation.
Kidney Int 72:328336
Wu J, Du H, Wang X, Mei C, Sieck GC, Qian Q (2009) Characterization of primary cilia in human
airway smooth muscle cells. Chest 136:561570
Wu Y, Dai XQ, Li Q, Chen CX, Mai W, Hussain Z, Long W, Montalbetti N, Li G, Glynne R,
Wang S, Cantiello HF, Wu G, Chen XZ (2006) Kinesin-2 mediates physical and functional
interactions between polycystin-2 and fibrocystin. Hum Mol Genet 15:32803292
Xiao Z, Zhang S, Magenheimer BS, Luo J, Quarles LD (2008) Polycystin-1 regulates skeletogen-
esis through stimulation of the osteoblast-specific transcription factor RUNX2-II. J Biol Chem
283:1262412634
350 S.M. Nauli et al.
Xiao Z, Zhang S, Mahlios J, Zhou G, Magenheimer BS, Guo D, Dallas SL, Maser R, Calvet JP,
Bonewald L, Quarles LD (2006) Cilia-like structures and polycystin-1 in osteoblasts/osteocytes
and associated abnormalities in skeletogenesis and Runx2 expression. J Biol Chem 281:
3088430895
Xu C, Rossetti S, Jiang L, Harris PC, Brown-Glaberman U, Wandinger-Ness A, Bacallao R, Alper
SL (2007) Human ADPKD primary cyst epithelial cells with a novel, single codon deletion in
the PKD1 gene exhibit defective ciliary polycystin localization and loss of flow-induced Ca2+
signaling. Am J Physiol Renal Physiol 292:F930945
Xu C, Shmukler BE, Nishimura K, Kaczmarek E, Rossetti S, Harris PC, Wandinger-Ness A,
Bacallao RL, Alper SL (2009) Attenuated, flow-induced ATP release contributes to absence
of flow-sensitive, purinergic Cai2+ signaling in human ADPKD cyst epithelial cells. Am J
Physiol Renal Physiol 296:F14641476
Yamamoto M, Kataoka K (1986) Electron microscopic observation of the primary cilium in the
pancreatic islets. Arch Histol Jpn 49:449457
Yang J, Gao J, Adamian M, Wen XH, Pawlyk B, Zhang L, Sanderson MJ, Zuo J, Makino CL,
Li T (2005) The ciliary rootlet maintains long-term stability of sensory cilia. Mol Cell Biol
25:41294137
Yang J, Liu X, Yue G, Adamian M, Bulgakov O, Li T (2002) Rootletin, a novel coiled-coil protein,
is a structural component of the ciliary rootlet. J Cell Biol 159:431440
Yen HJ, Tayeh MK, Mullins RF, Stone EM, Sheffield VC, Slusarski DC (2006) Bardet-Biedl syn-
drome genes are important in retrograde intracellular trafficking and Kupffers vesicle cilia
function. Hum Mol Genet 15:667677
Yissachar N, Salem H, Tennenbaum T, Motro B (2006) Nek7 kinase is enriched at the centrosome,
and is required for proper spindle assembly and mitotic progression. FEBS Lett 580:64896495
Yoder BK, Hou X, Guay-Woodford LM (2002a) The polycystic kidney disease proteins,
polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia. J Am Soc Nephrol
13:25082516
Yoder BK, Tousson A, Millican L, Wu JH, Bugg CE, Jr., Schafer JA, Balkovetz DF (2002b) Polaris,
a protein disrupted in orpk mutant mice, is required for assembly of renal cilium. Am J Physiol
Renal Physiol 282:F541552
Zariwala MA, Knowles MR, Omran H (2007) Genetic defects in ciliary structure and function.
Annu Rev Physiol 69:423450
Zhang YJ, ONeal WK, Randell SH, Blackburn K, Moyer MB, Boucher RC, Ostrowski LE
(2002) Identification of dynein heavy chain 7 as an inner arm component of human cilia
that is synthesized but not assembled in a case of primary ciliary dyskinesia. J Biol Chem
277:1790617915
Zhang Z, Jones BH, Tang W, Moss SB, Wei Z, Ho C, Pollack M, Horowitz E, Bennett J, Baker ME,
Strauss JF, 3rd (2005) Dissecting the axoneme interactome: the mammalian orthologue of
Chlamydomonas PF6 interacts with sperm-associated antigen 6, the mammalian orthologue
of Chlamydomonas PF16. Mol Cell Proteomics 4:914923
Zhang Z, Kostetskii I, Moss SB, Jones BH, Ho C, Wang H, Kishida T, Gerton GL, Radice GL,
Strauss JF, 3rd (2004) Haploinsufficiency for the murine orthologue of Chlamydomonas PF20
disrupts spermatogenesis. Proc Natl Acad Sci U S A 101:1294612951
Part VII
Mechanosensitivity
and Mechanotransduction
in Blood Cells
Chapter 15
Mechanosensitive K+ Channels in Mouse
B Lymphocytes: PLC-Mediated Release
of TREK-2 from Inhibition by PIP2
Abstract Blood cells can encounter significant mechanical stimuli in variable flow.
In mouse B lymphocytes and their cell line WEHI-231, we found large-conductance
background K+ channels (LKbg ) that show significant mechanosensitivity. The
biophysical characteristics of LKbg were similar to those of TREK-2, a member
of the mechanosensitive two-pore domain K+ channels, and the genetic knock-
down of TREK-2 largely suppressed the LKbg activity in B lymphocytes. However,
there have been disputes regarding the sensitivities of LKbg and TREK-2 to phos-
phatidylinositol 4,5-bisphosphate (PIP2 ) and their mechanism of stretch-dependent
activation. Our recent results suggest a mechano-biochemical signalling mechanism
for the stretch-dependent activation of TREK-2; the mechanosensitive-activation of
LKbg is mediated by phospholipase C (PLC)-dependent degradation of PIP2 , which
relieves TREKs and LKbg from the intrinsic inhibition by PIP2 . Here we review the
characteristics of LKbg and permanently overexpressed TREKs and compare them
with previous reports about TREKs.
15.1 Introduction
Immune cells are electrically non-excitable and their ion channels are not as diverse
as those in neuronal and muscular cells (Panyi et al., 2004). However, the ion
channels in immunocytes have recently drawn attention because they are potential
Fig. 15.1 Inhibition of LKbg activity by cytoplasmic application of MgATP. (a). A representa-
tive case of cell-attached (c-a) and inside-out (i-o) patch clamp recording with KCl (140 mM)
pipette solution in B lymphocytes freshly isolated from mouse spleen. A huge increase of large-
conductance channel activity was observed after making membrane excision (i-o) into MgATP-free
KCl solution (60 mV holding). (b). A representative case of i-o patch clamp in WEHI-231 cells.
The recordings were obtained under the i-o patch clamp conditions at 60 mV with symmetrical
KCl (145 mM) solutions as shown above. The application of MgATP (2 mM) to the cytoplasmic
side of the membrane greatly reduced the activity of LKbg
solution. Furthermore, the LKbg activity is very low in cell-attached (c-a) record-
ings, while the relative activity of LKbg is enormously high in the absence of
cytoplasmic ATP (Nam et al., 2004). In contrast, the spontaneously activated LKbg
is effectively inhibited by MgATP applied to the cytoplasmic side (Fig. 15.1b).
Because the activity of LKbg is very low in the resting B cells, it was initially
questioned to classify them as background K+ channels. Albeit this confusion,
here we regard LKbg as a member of background-type K+ channels due to the
voltage-independence and Ca2+ -insensitivity (Nam et al., 2004).
In the i-o recording with MgATP or in the c-a conditions, LKbg is activated by
membrane-stretch induced by applying negative pressure through the patch pipette
(Fig. 15.2). The activation of LKbg in c-a condition is also induced by osmotic
swelling (Nam et al., 2007). The stretch effects on LKbg are basically reversible
upon relieving the mechanical stress. However, when the application of stretch is
prolonged beyond a certain duration (>35 min) in i-o patch recording, then the
activation of LKbg is irreversible, suggesting that a putative depletion of regulatory
molecules might occur (Nam et al., 2004) (see also Fig. 15.3c). It has also been
15 Mechanosensitive K+ Channels in Mouse B Lymphocytes 357
Fig. 15.2 Effects of membrane stretch on LKbg channel activity in c-a and i-o patches. (a). In
the c-a patches, the initially low open probability (Po ) of LKbg channels was markedly increased
by applying 43 mmHg of negative pressure. (b). With 1 mM MgATP on the cytoplasmic side,
a serial negative pressure was applied as indicated above each trace. The membrane stretch by
the negative pipette pressure reversibly increased the open probability (Po ) of LKbg channels in a
pressure-dependent manner at 60 mV
noted that the MKbg in B lymphocytes are not affected by membrane stretch (Nam
et al., 2004).
Apart from the membrane stretch, arachidonic acid (AA) and intracellular acid-
ification activate LKbg (Zheng et al. 2008, 2009). The mechanosensitivity and
chemosensitivity indicate that LKbg might be encoded by TREK-2 (KCNK10),
a member of the tandem two-pore domain K+ channel gene (KCNK) family.
Among the TREK subfamily of KCNK, TREK-2 shows a large but variable unitary
conductance that ranges up to 290 pS (Lotshaw 2007). Despite their common prop-
erties, we were initially reluctant to pinpoint TREK-2 as the molecular identity of
LKbg because of the differential responses to phosphatidylinositol 4,5-bisphosphate
(PIP2 ) between LKbg and cloned TREK-2 (see below).
358 S.J. Kim and J.H. Nam
Fig. 15.3 Mechanism of the inhibitory effects of MgATP and the effects of PLC activation on
LKbg . (a). Direct application of PIP2 (5 M) to the cytoplasmic side of membrane (bath solu-
tion) suppressed the LKbg activity. These results suggest that the cytoplasmic ATP-dependent
inhibition of LKbg is tightly related to PIP2 in the membrane. (b). For the direct stimulation of
membrane-bound PLC, m-3M3FBS (a chemical activator of PLC) was applied to i-o patches after
confirming the inhibition of LKbg channels by MgATP. The application of m-3M3FBS (50 M)
slowly activated LKbg channels in the presence of MgATP (1 mM)
As mentioned above, the i-o patch recording of LKbg shows remarkable spontaneous
activation that is readily inhibited by the application MgATP to the cytoplasmic
side (IC50 =0.18 mM). The inhibitory effect of ATP is phosphorylation-dependent
because non-hydrolysable adenosine 5-(,-imino)-triphosphate (AMP-PNP) has
no effect. However, the inhibition by ATP is not prevented by protein kinase
inhibitors, but is effectively blocked by PI-kinase inhibitor, wortmannin (Nam et al.,
2004). Such results strongly suggest the inhibitory regulation of LKbg by PIP2
locally produced from the membrane phospholipids and associated enzymes, a
membrane-delimited regulatory mechanism.
PIP2 is well recognized as a key physiological regulator of various types of ion
channels/transporters (Hilgemann et al., 2001; Horowitz et al., 2005; Suh and Hille,
2005). While most PIP2 -sensitive ion channels are positively regulated by the nega-
tively charged phosphoinositol head, LKbg are inhibited by PIP2 (Fig. 15.3a) (Nam
et al., 2007, 2004). Inhibition by intrinsic PIP2 could explain the low activity of
LKbg in intact B cells and the spontaneous activation after the membrane exci-
sion (i.e., inside-out patch formation). In physiological conditions, cytoplasmic ATP
and PI-kinases seem to ensure the repletion of PIP2 . To explain the membrane-
delimited reversible inhibition by ATP in i-o patches, it is strongly suggested that
the PI kinases are tightly associated with plasma membrane and LKbg . Moreover,
15 Mechanosensitive K+ Channels in Mouse B Lymphocytes 359
Fig. 15.4 Schematic model of the membrane-delimited regulation by PIP2 . Intrinsically generated
PIP2 by PI- and PIP-kinase inhibit LKbg . The mechanical stretch activates membrane-bound PLC
(PLC2) by signalling pathways not yet elucidated. The decrease of PIP2 levels by PLC relieves
the nearby LKbg from their tonic inhibition
the spontaneous activation of LKbg suggests that a lipid phosphatase and/or phos-
pholipase (PLC) that hydrolyzes PIP2 are also tightly associated in the same patch
membrane. Regarding the PLC-dependent regulation, it is also notable that a chem-
ical PLC activator, m-3M3FBS (Fig. 15.3b), and B cell receptor (BCR) ligation
increase the LKbg activity (Nam et al., 2004). Based these data, we suggest a model
for the membrane-delimited regulation of LKbg by PIP2 as shown in Fig. 15.4.
Above we mentioned that the TREK subfamily of KCNK, TREK-2, is the most
likely candidate for the LKbg gene. However, in comparison with the inhibition of
LKbg by PIP2 or by MgATP, TREK-1 was activated by PIP2 in earlier studies of
cloned TREK channels (Chemin et al., 2005). Interestingly, however, a more recent
study by the same group indicated dual effects of PIP2 on TREK-1 depending on the
basal activity of the channel (Chemin et al., 2007), which is partly consistent with
our results. Although TREK-1 is highly homologous with TREK-2, the c-terminal
of TREK-2 is longer than TREK-1, which might have caused the different responses
to PIP2 . To clarify this controversial issue, we compared the properties of rat TREK-
1 and -2 channels permanently expressed in HEK-293 cells with the properties of
LKbg in WEHI-231 cells (Zheng et al., 2009). According to this study, the spon-
taneous activation of both TREK-1 and TREK-2 in ATP-free conditions and their
inhibition by MgATP or by PIP2 were the same as those of LKbg . Furthermore,
TREK-2-specific si-RNA transfection decreased the LKbg current in B cells. In
conclusion, TREK-2 encodes LKbg that are normally suppressed by intrinsic PIP2
formed by PI-kinase and MgATP in intact cells (Zheng et al., 2009).
The question then is why there have been such differences between the studies
of TREKs in terms of their PIP2 -sensitivity. Also, no previous study has reported
the reversible inhibitory effects of ATP on TREK-2 shown in our i-o patch clamp
experiments. Although the question remains unsolved, the inconsistent results seem
to be due to differences in the expression systems for TREKs: our previous studies
were performed in mouse B lymphocytes and HEK-293 cells permanently express-
ing TREKs, whereas other studies were mostly performed in COS-7 cell lines or
Xenopus oocytes for transient overexpression of TREK-1. We recently found that
transiently overexpressed TREK-2 in HEK-293 cells showed irregular responses to
360 S.J. Kim and J.H. Nam
MgATP in the i-o patch recordings (data not shown). We hypothesize that the regu-
lation by PIP2 /PI-kinase/lipid-phosphatase is less effective in transiently transfected
TREKs, while the intrinsically expressed TREKs such as LKbg in B lymphocytes
are more tightly associated with the PIP2 -mediated regulatory complex.
Considering that the plasmalemmal concentration of PIP2 is relatively low
(Hilgemann et al., 2001), the extent of co-localization between PIP2 and TREKs
might vary according to the mode of expression (transient vs. permanent) or cell
types. A locally concentrated, inhomogeneous distribution of PIP2 and the regula-
tion of ion channels by the localized PIP2 could be suggested from the effects of
methyl--cyclodextrin (MCD). MCD is known to scavenge cholesterols, thereby
disrupting cholesterol-rich microdomains (e.g., lipid rafts) of plasma membranes.
The concentration of PIP2 in the lipid rafts is thought to be higher than that in other
regions, and interestingly, the application of MCD facilitates the recovery of LKbg
activity from the inhibition by PIP2 (Fig. 15.5).
Fig. 15.5 Irreversible inhibitory effects by the application of PIP2 (1 M), and the effects of
MCD on recovery from PIP2 -induced inhibition of LKbg channels. (a), The LKbg channel activity
was not recovered by washout of PIP2 up to 10 min by a sustained application of PIP2 (1 M). (b),
The application of methyl--cyclodextrin (MCD, 2 mM), a cholesterol scavenger that disrupts
lipid rafts (Kilsdonk et al., 1995), significantly facilitated the recovery of LKbg channels from the
inhibition of PIP2
15 Mechanosensitive K+ Channels in Mouse B Lymphocytes 361
application (Chemin et al., 2005). After the first report about the positive effects of
PIP2 on TREK-1, Chemin et al. also demonstrated dual effects of PIP2 on TREK-1
(Chemin et al., 2007). According to the more recent paper, exogenous PIP2 appli-
cation activated TREK-1 when the basal channel activity was low and, vice versa,
PIP2 application inhibited TREK-1 when the control activity was high.
Interestingly, in our own experiments, the concentration-dependent dual effects
of PIP2 were also demonstrated after poly-L treatment (Fig. 15.6). After confirming
the spontaneous activation of TREKs under i-o conditions, we found that poly-L
(30 g/ml) treatment almost completely inhibited TREKs. The inhibitory effect of
poly-L was inconsistent with our initial hypothesis that TREKs are simply inhibited
by PIP2 . These contradictory responses led us to modify our model to include the
dual effects of PIP2 : the activities of TREKs are positively and negatively regulated
by relatively low and high levels of PIP2 , respectively.
In our experience, however, only an excessive experimental condition such as
poly-L treatment seemed to decrease PIP2 below the level that is minimally required
Fig. 15.6 Dual effects of PIP2 on TREKs and LKbg in the presence of poly-L-lysine (poly-L).
AC, i-o recordings of TREK-1, -2 (overexpressed in HEK-293) and LKbg (WEHI-231) with
symmetrical KCl solution at 60 mV holding voltage. The application of poly-L (30 g/ml) to
the intracellular side (bath solution) completely inhibited the channel activities, and the inhibition
was not reversed by washout of poly-L. After confirming steady-state inhibition, 8 M and 20 M
PIP2 were applied sequentially. Note that 20 M PIP2 initially activated and then inhibited the
channels
362 S.J. Kim and J.H. Nam
for the activity of TREKs. After spontaneous activation of LKbg /TREK-2, only
inhibitory effects on LKbg /TREK-2 are observed in response to ATP and PIP2 . The
removal of ATP and the spontaneous degradation process actually lower the mem-
brane PIP2 level. But the level might still be greater than the putative minimum level
required to activate TREKs. Under such conditions, poly-L might further scavenge
the residual PIP2 that was required for the basal activity of TREKs. To test this
dual-mode hypothesis, we applied different concentrations of PIP2 after confirm-
ing the inhibition of TREKs by poly-L. At relatively low concentration (8 M),
TREK-2 are increased, while at a higher concentration (20 M), the channels are
transiently activated then go into an inhibited state (Fig. 15.6). The dual effects
of PIP2 on TREK-2 are basically consistent with the previous results in TREK-
1-overexpressing COS-7 cells (Chemin et al., 2007). From the responses of various
deletion mutants of TREK-1 to PIP2 and poly-L, Chemin et al. (2007) suggested that
the different c-terminal domains are responsible for the positive and negative effects
of PIP2 . Because the c-terminal domain proximal to the transmembrane domain
is highly conserved between TREK-1 and TREK-2 (Bang et al., 2000), the dual
modes of PIP2 action on TREK-2 might also due to the differential interaction with
the anionic phospholipids.
As a whole, it is evident that TREKs are modulated by PIP2 in a dual manner
at least in vitro. However, the LKbg /TREK-2 channels in B lymphocytes are under
tonic inhibition by physiological levels of intrinsic PIP2 . Then, what is the relation-
ship between the mechanosensitivity of LKbg /TREK-2 and the tonic inhibition by
PIP2 ?
Fig. 15.7 Inhibition of the mechanosensitivity of TREK-2 by PIP2 . (a). In cell-attached (c-a)
mode with KCl pipette solution, the application of negative pressure (27 mmHg) through the
patch pipette activated TREK-2 in a reversible manner. (b). The spontaneously activated TREK-
2 current by membrane excision was completely inhibited by 8 M PIP2 . Under this condition,
membrane stretch (27 mmHg) did not recover the channel activity
activated LKbg , and (4) the tyrosine phosphorylation of PLC and PIP2 hydrolysis
was confirmed in WEHI-231 cells under osmotic stress (Nam et al., 2007). More
recently, we confirmed that the stretch-dependent activation of cloned TREK-1 and
TREK-2 was also prevented by PLC inhibitor, U73122, but not by its negative ana-
logue, U73343 (Fig. 15.8). As a whole, we suggest that the stretch-sensitivity of
TREKs might actually be a release from the inhibition by PIP2 through the activa-
tion of PLC and PIP2 hydrolysis, i.e., a mechno-biochemical signalling hypothesis
(Fig. 15.4). The PIP2 hydrolysis by membrane stretch could also be suggested from
irreversible activation (open-locked state) of LKbg after sustained application of
negative pressure (Fig. 15.3c, see also Nam et al., 2007). Under the open-locked
state, LKbg are not affected by ATP alone but can be inhibited only when PI4P is
applied together, indicating that the substrate phosphoinositides for PIP2 generation
are actually depleted by sustained membrane stretch (Nam et al., 2007).
Although the evidence supports the mechano-biochemical coupling hypothesis,
it must also be noted that the stretch effects on TREKs showed different pharma-
cological sensitivity depending on the level of negative pressure. The activation
by relatively weak stretch (e.g. 27 mmHg via patch pipette) was prevented by
PLC inhibitor, U73122. In the presence of U73122, a higher negative pressure
(43 mmHg) could activate TREKs (Fig. 15.8). The PLC-independent activation of
TREKs by the stronger membrane stretch might indicate a more direct link between
the membrane stretch and channel gating, as has been suggested by Chemin et al.
for TREK-1 (Chemin et al., 2005). As a whole, the above concentration-dependent
effects of PIP2 might be associated with dual modes of mechanosensitivity where
both mechanisms are associated with PIP2 , i.e., mechano-biochemical signalling
(PLC-mediated) and direct physical signalling mediated by putative electrostatic
interaction between the c-terminus and the residual PIP2 . Actually, the mechanosen-
sitivity of TREK-1 is inhibited both by scavenging PIP2 with poly-L treatment
(Chemin et al., 2005) and by exogenous application of PIP2 (Chemin et al., 2007;
Nam et al., 2007). In these respects, PIP2 is a double-edged sword in terms of
mediating the mechanosensative regulation of TREKs.
364 S.J. Kim and J.H. Nam
Fig. 15.8 Effects of PLC inhibitor on the mechanosensitive activation of TREK-1 and -2. After
confirming the basal activities of TREK-1 and -2 in c-a configuration, relatively mild (27 mmHg)
or strong (43 mmHg) suction was applied as indicated above each trace. (a): Both TREK-1 and -2
were slowly activated by 27 mmHg of suction (upper panels), and these responses were signif-
icantly suppressed by pretreatment with U73122 (2 M, lower panels). (b): Summaries of the
changes in channel activity (NPo ) by relatively low membrane stretch (27 mmHg) and the effects
of pretreatment with U73122 (2 M) or with U73343 (10 M). In each patch, NPo during the
initial 15 s and the later 15 s of membrane stretch were normalized to the pre-stretch activity.
The averaged values are shown in the bar graphs (n=6). (c): Representative traces of TREK-1
and TREK-2 responses to the higher level of membrane stretch (43 mmHg). Pretreatment with
U73122 did not block the activation of TREKs by the stronger negative pressure. Representative
cases from five similar experiments are shown. (d): Summaries of the NPo changes by relatively
high membrane stretch (43 mmHg) and the effects of pretreatment with U73122 (2 M, n = 5).
This figure was adopted and modified from the figure 5 in Zheng et al. (2009) with permission
from the American Physical Society
15 Mechanosensitive K+ Channels in Mouse B Lymphocytes 365
References
Achard JM, Bubien JK, Benos DJ, Warnock DG (1996) Stretch modulates amiloride sensitivity and
cation selectivity of sodium channels in human B lymphocytes. Am J Physiol 270:C224234
Bang H, Kim Y, Kim D (2000) TREK-2, a new member of the mechanosensitive tandem-pore K+
channel family. J Biol Chem 275:1741217419
Beeton C, Chandy KG (2005) Potassium channels, memory T cells, and multiple sclerosis.
Neuroscientist 11:550562
Bortner CD, Cidlowski JA (2007) Cell shrinkage and monovalent cation fluxes: role in apoptosis.
Arch Biochem Biophys 462:176188
Cahalan MD, Wulff H, Chandy KG (2001) Molecular properties and physiological roles of ion
channels in the immune system. J Clin Immunol 21:235252
Cahalan MD, Zhang SL, Yeromin AV, Ohlsen K, Roos J, Stauderman KA (2007) Molecular basis
of the CRAC channel. Cell Calcium 42:133144
Chandy KG, Wulff H, Beeton C, Pennington M, Gutman GA, Cahalan MD (2004) K+ channels as
targets for specific immunomodulation. Trends Pharmacol Sci 25:280289
Chemin J, Patel AJ, Duprat F, Lauritzen I, Lazdunski M, Honore E (2005) A phospholipid sensor
controls mechanogating of the K+ channel TREK-1. EMBO J 24:4453
Chemin J, Patel AJ, Duprat F, Sachs F, Lazdunski M, Honore E (2007) Up- and down-regulation of
the mechano-gated K2P channel TREK-1 by PIP2 and other membrane phospholipids. Pflugers
Arch 455:97103
Fahrner M, Muik M, Derler I, Schindl R, Fritsch R, Frischauf I, Romanin C (2009) Mechanistic
view on domains mediating STIM1-Orai coupling. Immunol Rev 231:99112
Feske S, Gwack Y, Prakriya M, Srikanth S, Puppel SH, Tanasa B, Hogan PG, Lewis RS, Daly M,
Rao A (2006) A mutation in Orai1 causes immune deficiency by abrogating CRAC channel
function. Nature 441:179185
Hilgemann DW, Feng S, Nasuhoglu C (2001) The complex and intriguing lives of PIP2 with ion
channels and transporters. Sci STKE 2001:re19
Horowitz LF, Hirdes W, Suh BC, Hilgemann DW, Mackie K, Hille B (2005) Phospholipase C in
living cells: activation, inhibition, Ca2+ requirement, and regulation of M current. J Gen Physiol
126:243262
Inoue R, Jensen LJ, Jian Z, Shi J, Hai L, Lurie AI, Henriksen FH, Salomonsson M, Morita
H, Kawarabayashi Y, Mori M, Mori Y, Ito Y (2009) Synergistic activation of vascular
TRPC6 channel by receptor and mechanical stimulation via phospholipase C/diacylglycerol
and phospholipase A2/omega-hydroxylase/20-HETE pathways. Circ Res 104:13991409
King LB, Freedman BD (2009) B-lymphocyte calcium influx. Immunol Rev 231:265277
Lewis RS, Ross PE, Cahalan MD (1993) Chloride channels activated by osmotic stress in T
lymphocytes. J Gen Physiol 101:801826
15 Mechanosensitive K+ Channels in Mouse B Lymphocytes 367
Liu QH, Liu X, Wen Z, Hondowicz B, King L, Monroe J, Freedman BD (2005) Distinct cal-
cium channels regulate responses of primary B lymphocytes to B cell receptor engagement and
mechanical stimuli. J Immunol 174:6879
Lotshaw DP (2007) Biophysical, pharmacological, and functional characteristics of cloned and
native mammalian two-pore domain K+ channels. Cell Biochem Biophys 47:209256
Ma Z, Finkel TH (2010) T cell receptor triggering by force. Trends Immunol 31:16
Madsen LS, Christophersen P, Olesen SP (2005) Blockade of Ca2+ -activated K+ channels in T
cells: an option for the treatment of multiple sclerosis? Eur J Immunol 35:10231026
Mederos y Schnitzler M, Storch U, Meibers S, Nurwakagari P, Breit A, Essin K, Gollasch
M, Gudermann T (2008) Gq-coupled receptors as mechanosensors mediating myogenic
vasoconstriction. EMBO J 27:30923103
Miah SM, Sada K, Tuazon PT, Ling J, Maeno K, Kyo S, Qu X, Tohyama Y, Traugh JA, Yamamura
H (2004) Activation of Syk protein tyrosine kinase in response to osmotic stress requires
interaction with p21-activated protein kinase Pak2/gamma-PAK. Mol Cell Biol 24:7183
Moore AL, Roe MW, Melnick RF, Lidofsky SD (2002) Calcium mobilization evoked by hepato-
cellular swelling is linked to activation of phospholipase C. J Biol Chem 277:3403034035
Nam JH, Lee HS, Nguyen YH, Kang TM, Lee SW, Kim HY, Kim SJ, Earm YE (2007)
Mechanosensitive activation of K+ channel via phospholipase C-induced depletion of phos-
phatidylinositol 4,5-bisphosphate in B lymphocytes. J Physiol 582:977990
Nam JH, Woo JE, Uhm DY, Kim SJ (2004) Membrane-delimited regulation of novel background
K+ channels by MgATP in murine immature B cells. J Biol Chem 279:2064320654
Onfelt B, Nedvetzki S, Yanagi K, Davis DM (2004) Cutting edge: Membrane nanotubes connect
immune cells. J Immunol 173:15111513
Onfelt B, Purbhoo MA, Nedvetzki S, Sowinski S, Davis DM (2005) Long-distance calls between
cells connected by tunneling nanotubules. Sci STKE 2005:pe55
Panyi G, Possani LD, Rodriguez de la Vega RC, Gaspar R, Varga Z (2006) K+ channel blockers:
novel tools to inhibit T cell activation leading to specific immunosuppression. Curr Pharm Des
12:21992220
Panyi G, Varga Z, Gaspar R (2004) Ion channels and lymphocyte activation. Immunol Lett 92:
5566
Picard C, McCarl CA, Papolos A, Khalil S, Luthy K, Hivroz C, LeDeist F, Rieux-Laucat F,
Rechavi G, Rao A, Fischer A, Feske S (2009) STIM1 mutation associated with a syndrome
of immunodeficiency and autoimmunity. N Engl J Med 360:19711980
Roos J, DiGregorio PJ, Yeromin AV, Ohlsen K, Lioudyno M, Zhang S, Safrina O, Kozak JA,
Wagner SL, Cahalan MD, Velicelebi G, Stauderman KA (2005) STIM1, an essential and
conserved component of store-operated Ca2+ channel function. J Cell Biol 169:435445
Roselli F, Livrea P, Jirillo E (2006) Voltage-gated sodium channel blockers as immunomodulators.
Recent Pat CNS Drug Discov 1:8391
Ruwhof C, van Wamel JT, Noordzij LA, Aydin S, Harper JC, van der Laarse A (2001) Mechanical
stress stimulates phospholipase C activity and intracellular calcium ion levels in neonatal rat
cardiomyocytes. Cell Calcium 29:7383
Sharif-Naeini R, Dedman A, Folgering JH, Duprat F, Patel A, Nilius B, Honore E (2008) TRP chan-
nels and mechanosensory transduction: insights into the arterial myogenic response. Pflugers
Arch 456:529540
Suh BC, Hille B (2005) Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr
Opin Neurobiol 15:370378
Vig M, DeHaven WI, Bird GS, Billingsley JM, Wang H, Rao PE, Hutchings AB, Jouvin MH,
Putney JW, Kinet JP (2008) Defective mast cell effector functions in mice lacking the CRACM1
pore subunit of store-operated calcium release-activated calcium channels. Nat Immunol 9:
8996
Vig M, Kinet JP (2009) Calcium signaling in immune cells. Nat Immunol 10:2127
Wulff H, Knaus HG, Pennington M, Chandy KG (2004) K+ channel expression during B
cell differentiation: implications for immunomodulation and autoimmunity. J Immunol 173:
776786
368 S.J. Kim and J.H. Nam
Wulff H, Pennington M (2007) Targeting effector memory T-cells with Kv1.3 blockers. Curr Opin
Drug Discov Devel 10:438445
Zheng H, Ko JH, Nam JH, Earm YE, Kim SJ (2006) Differential functional expression of
clotrimazole-sensitive Ca2+ activated K+ current in Bal-17 and WEHI-231 murine B lympho-
cytes Korean J Physiol Pharmacol 10:1924
Zheng H, Nam JH, Nguen YH, Kang TM, Kim TJ, Earm YE, Kim SJ (2008) Arachidonic acid-
induced activation of large-conductance potassium channels and membrane hyperpolarization
in mouse B cells. Pflugers Arch 456:867881
Zheng H, Nam JH, Pang B, Shin DH, Kim JS, Chun YS, Park JW, Bang H, Kim WK, Earm YE,
Kim SJ (2009) Identification of the large-conductance background K+ channel in mouse B cells
as TREK-2. Am J Physiol Cell Physiol 297:C188197
Zhu P, Liu X, Labelle EF, Freedman BD (2005) Mechanisms of hypotonicity-induced calcium
signaling and integrin activation by arachidonic acid-derived inflammatory mediators in B cells.
J Immunol 175:49814989
Index
A D
Actin-crosslinking proteins, 27, 30, 4652, 156 Dystrophin, 35, 143, 147, 149150
Actin cytoskeleton, 2558, 89, 9294, 98, 152,
156, 225, 244, 264, 288, 301 E
Actin filament associated protein (AFAP), ECM, see Extracellular matrix (ECM)
262265 Endothelial cells, 7, 26, 194203, 224232,
Actin microfilaments, 27, 6970, 7778, 86, 240, 242, 245, 248, 265268, 279280,
8890, 9296 282283, 307, 333334
Acute lung injury, 240, 256, 267 Epoxyeicosatrienoic acids, 240241
Acute respiratory distress syndrome (ARDS), Extracellular matrix (ECM), 48, 1013,
256257, 267268 15, 52, 6768, 7778, 83, 85, 9193,
AFAP, see Actin filament associated protein 98, 135, 141149, 151152, 154156,
(AFAP) 158, 194, 210, 222, 225, 228229, 256,
ARDS, see Acute respiratory distress 260261, 278279, 298302, 304305,
syndrome (ARDS) 307, 332
Articular cartilage, 7799, 297298, 301, 305
F
B FAK, see Focal adhesion kinase (FAK)
Background K+ channel, 355356 Fluid flow, 27, 85, 94, 202, 228230,
B lymphocyte, 353366 282283, 285286, 300, 304, 325,
Bone, 2627, 7074, 8384, 195, 202, 329330, 332
277288, 297308, 321, 332333 Fluid shear stress, 69, 71, 73, 230, 279281,
283284, 288, 303, 325, 328330,
C 333334
Cadherin, 146147, 152154, 156, 267 Focal adhesion, 67, 11, 27, 46, 5253, 55,
Calcium (Ca), 4, 8, 11, 111, 116, 119, 85, 94, 98, 144147, 156157, 201, 220,
157, 240, 243, 250, 260, 262, 268, 224, 229, 232, 258, 261262, 278280,
281282, 285286, 301, 303, 328330, 282284, 287288, 301302, 307
333335 Focal adhesion kinase (FAK), 6, 11, 52, 55,
Calcium-activated channels, 11 85, 145147, 201, 224, 229, 258, 279,
Calcium spark, 911 282284, 301302
Cardiac myocyte, 210
Cartilage, 7799, 297302, 304308, 332333 G
Chondrocyte, 12, 80, 8399, 298302, 304, GsMTx-4 MscS, 168, 170, 177, 180, 184185
306307, 326, 328, 332
Cytoskeleton, 315, 2558, 6774, 7799, H
135, 142, 152, 156, 194, 205, 208209, HaTx, 168, 171172, 174178, 180, 182184
220, 225, 244, 256, 260261, 263265, Heart, 6, 109135, 141158, 168169, 173,
278, 288, 300301, 303, 326 193, 256257