© 1999 American Heart Association, Inc.
Original Contribution |
Rho Family Small G Proteins Play Critical Roles in Mechanical Stress–Induced Hypertrophic Responses in Cardiac Myocytes
From the Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine (R.A., I.K., T.Y., Y.Z., S.K., W.Z., T.K., Y.Y.), and the Health Service Center (T.Y.), University of Tokyo, Tokyo, Japan.
Correspondence to Issei Komuro, MD, PhD, Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail komuro-tky{at}umin.u-tokyo.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—Mechanical stress induces a variety of hypertrophic responses, such as activation of protein kinases, reprogramming of gene expression, and an increase in protein synthesis. In the present study, to elucidate how mechanical stress induces such events, we examined the role of Rho family small GTP-binding proteins (G proteins) in mechanical stress–induced cardiac hypertrophy. Treatment of neonatal rat cardiomyocytes with the C3 exoenzyme, which abrogates Rho functions, suppressed stretch-induced activation of extracellular signal–regulated protein kinases (ERKs). Overexpression of the Rho GDP dissociation inhibitor (Rho-GDI), dominant-negative mutants of RhoA (DNRhoA), or DNRac1 significantly inhibited stretch-induced activation of transfected ERK2. Overexpression of constitutively active mutants of RhoA slightly activated ERK2 in cardiac myocytes. Overexpression of C-terminal Src kinase, which inhibits functions of the Src family of tyrosine kinases, or overexpression of DNRas had no effect on stretch-induced activation of transfected ERK2. The promoter activity of skeletal
-actin and c-fos
genes was increased by stretch, and these increases were completely
inhibited by either cotransfection of Rho-GDI or pretreatment with C3
exoenzyme. Mechanical stretch increased phenylalanine incorporation
into cardiac myocytes by 
1.5-fold compared with control, and this
increase was also significantly suppressed by pretreatment with C3
exoenzyme. Overexpression of Rho-GDI or DNRhoA did not affect
angiotensin II–induced activation of ERK. ERKs were
activated by culture media conditioned by stretch of
cardiomyocytes without any treatment, but not of
cardiomyocytes with pretreatment by C3 exoenzyme. These
results suggest that the Rho family of small G proteins plays critical
roles in mechanical stress–induced hypertrophic responses.
Key Words: Rho • mechanical stress • cardiac hypertrophy
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Clinical studies have demonstrated that cardiac hypertrophy is not only an adaptational state before cardiac failure but also an independent risk factor for morbidity and mortality due to heart diseases.1 Thus, it has become even more important to understand the underlying mechanisms of the development of cardiac hypertrophy. Cardiac hypertrophy is often observed in patients who have hemodynamic overload.1 We and others have developed in vitro systems in which mechanical stress can be imposed on cultured cardiac myocytes and have demonstrated that mechanical stress induces a variety of hypertrophic responses, such as activation of protein kinases,2 3 4 5 reprogramming of gene expression,6 7 and an increase in protein synthesis2 4 in cultured cardiac myocytes. It remains unclear, however, how mechanical stress induces such intracellular events.
Integrins are a family of transmembrane receptors that bind to
extracellular matrix (ECM) proteins, such as fibronectin, collagen, and
vitronectin, and mediate a variety of cell
functions.8 9 10 Integrins are heterodimeric proteins
composed of noncovalently associated - and
ß-subunits.8 Binding to the ECM and clustering of
integrins lead to the formation of focal adhesions, by which integrins
connect the ECM to intracellular actin stress fibers.10
This "scaffolding" structure also contains a number of molecules
involved in signal transduction.11 12 In the in vitro
stretch system, we culture cardiac myocytes of neonatal rats on a
silicone membrane that has been precoated with collagen.6
Under these culture condition, cardiac myocytes attach to collagen on
the silicone membrane via integrins in adhesion plaques. Therefore, it
is conceivable that integrins first receive the stretch stimulus and
then convert it into the biochemical signals. Recent studies have
demonstrated that the cytoplasmic protein kinases, including focal
adhesion kinase (FAK), are potential mediators of integrin
signaling.13 14 FAK has been predicted to play a critical
role in initiating cytoplasmic signals from sites of focal
adhesion.13 14 We have reported that stretch
activates FAK in mesangial cells cultured on
silicone dishes.13 In cardiac myocytes, however, we could
not detect significant enhancement of FAK activity by mechanical
stretch because of its high basal activity (I.K. et al, unpublished
observations, 1998). Although it has been reported that RGD
peptide does not inhibit stretch-induced c-fos gene
expression in cardiac myocytes,15 integrins that
cannot be inhibited by RGD peptide may be involved in mechanical
stretch–induced hypertrophic responses in cardiac myocytes.
The Rho family of small GTP-binding proteins, consisting of Rho, Rac, and Cdc42 subfamilies, regulates many aspects of cytoskeletal function.16 Rho has been shown to participate in the formation of focal adhesions and actin stress fibers, as well as in mediating the redistribution of cytoskeletal components.16 17 There is also strong evidence indicating that Rho plays a pivotal role in integrin-mediated signaling events. First, activated Rho stimulates stress fiber formation,18 and plating of cells onto fibronectin-coated dishes in serum-free medium results in the rapid formation of stress fibers.19 Second, both Rho and adhesion to fibronectin activate phosphatidylinositol-4-phosphate 5-kinase.19 Third, specific inhibition of Rho by the Clostridium botulinum C3 exoenzyme blocks certain cellular responses, an event that is similar to the loss of integrin-mediated cell adhesion.19 Last, overexpression of constitutively active Rho restores the ability of suspended cells to respond to growth factors.19 Recently, integrin ligation has been demonstrated to induce activation and translocation to the nucleus of extracellular-regulated kinases (ERKs).20 In addition, Rho proteins are required for the activation of ERKs in NIH 3T3 cells plated on fibronectin.21 Although the molecular mechanism by which Rho family proteins regulate integrins still remains to be determined, all of these observations suggest that Rho is critically involved in integrin-mediated intracellular signaling.
In the present study, we examined the role of Rho family small G
proteins in the development of mechanical stress–induced hypertrophic
responses. We demonstrate that Rho family proteins play important roles
in stretch-induced activation of ERKs, expression of skeletal -actin
and c-fos genes, and an increase in protein synthesis in
cardiac myocytes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
[
-32P]ATP was purchased from Du
Pont–New England Nuclear Co. Dulbecco's modified Eagle' s
medium (DMEM), FBS, and genistein were from GIBCO BRL Co. An anti-ERK
(Y91) antibody was provided by K. Tobe (University of Tokyo
Graduate School of Medicine, Tokyo, Japan).22 C3 exoenzyme
was a kind gift from S. Narumiya (Kyoto University, Graduate
School of Medicine, Kyoto, Japan).23 An anti-hemagglutinin
(HA) polyclonal antibody was from MBL, Nagoya, Japan.
Angiotensin (Ang) II, insulin, myelin basic protein (MBP),
and other reagents were purchased from Sigma Chemical Co.
cDNA Plasmids
HA-tagged ERK2 (HA-ERK2) and the dominant-negative mutant
(Asn-17) of Ras (DNRas) were kind gifts from M. Karin
(University of California, San Diego, Calif)24 and Y.
Takai (Osaka University Faculty of Medicine, Osaka,
Japan).25 Rho-GDI and various mutants of RhoA, Rac1, and
Cdc42 were from J.S. Gutkind (National Institute of Dental Research,
Bethesda, Md),26 and wild-type C-terminal Src
kinase (CSK) and the kinase-negative mutant of CSK
(CSK-) were kindly provided by H. Sabe (Kyoto
University, Kyoto, Japan).27 The
dominant-negative mutant (Ala-17) of Raf-1 (DNRaf-1) was described
previously.28 All plasmid DNA was prepared by using QIAGEN
plasmid DNA preparation kits.
Cell Culture
Primary cultures of cardiac myocytes were prepared from
ventricles of 1-day-old Wistar rats as described
previously6 according to the method of
Simpson.29 In brief, cardiomyocytes were
plated at a field density of 105
cells/cm2 on silicone dishes with 2 mL of culture
medium (DMEM with 10% FBS).6 Twenty-four hours after
seeding, the culture medium was changed to a solution of serum-free
DMEM for 48 hours before treatment.
Transfection
Twenty-four hours after plating the cells on culture dishes, we
transfected DNA by using the calcium phosphate method as described
previously.6 For each dish, 2 µg of HA-ERK2 plasmid DNA
was transfected with 6 µg of the other relevant plasmids, such as
DNRas, DNRaf-1, CSK, or empty vector. After 15 hours of transfection,
the culture medium was removed, and the cells were washed twice with
PBS and maintained in serum-free DMEM for 48 hours before stretching or
treatment with chemical reagents. For the luciferase assay, 2 µg of
c-fos or skeletal -actin promoter luciferase plasmid was
transfected into cardiomyocytes with Rho-GDI or empty
vector by using the calcium phosphate method. After 6 or 12 hours of
transfection, the culture medium was removed, and cells were washed
twice with PBS and maintained in serum-free DMEM for 48 hours before
stretching or treatment with chemical reagents.
Kinase Assay of Endogenous ERKs
Endogenous ERK activity was measured by using MBP as
a substrate.2 22 In brief, cardiomyocytes were
lysed with buffer A (25 mmol/L Tris-HCl, pH 7.4; 25 mmol/L
NaCl; 1 mmol/L sodium orthovanadate; 10 mmol/L sodium
pyrophosphate; 10 nmol/L okadaic acid; 0.5 mmol/L EGTA; and 1
mmol/L PMSF), and the lysates were incubated with an anti-ERK
polyclonal antibody for 1 hour at 4°C. After incubation, the
immunocomplex was precipitated by using protein A–Sepharose beads,
washed, resuspended, and incubated again with 25 µg MBP at 25°C for
10 minutes in 25 µL of kinase buffer (25 mmol/L Tris-HCl, pH
7.4; 10 mmol/L MgCl2; 1 mmol/L DTT;
40 µmol/L ATP; 2 µCi [-32P]ATP;
2 µmol/L protein kinase inhibitor peptide; and 0.5
mmol/L EGTA). After incubation, the reaction was terminated by adding
Laemmli sample buffer (0.002% bromophenol blue, 0.01 mol/L sodium
phosphate buffer [pH 7.0], 10% glycerol, 0.4% SDS, and 1%
2-mercaptoethanol) to the samples, and the samples were boiled for 5
minutes. The supernatants were subjected to SDS–polyacrylamide
gel electrophoresis, and then the gel was washed with 7% acetic acid
for 30 minutes and with 3% glycerol for 30 minutes, dried, and
subjected to autoradiography.
Kinase Assay of Transfected HA-ERK2
After stimulation, cardiomyocytes into which HA-ERK2
was transfected were lysed with lysis buffer A, and the lysates were
incubated with anti-HA polyclonal antibody for 1 hour at 4°C. After
incubation, the immunocomplex was precipitated on protein A–Sepharose,
resuspended in 25 µL of the kinase buffer, and incubated with 25 µg
MBP as a substrate at 25°C for 10 minutes. After incubation, the
reaction was terminated by adding the Laemmli sample buffer to the
samples. The supernatants were subjected to SDS–polyacrylamide
gel electrophoresis, and the gel was dried and subjected to
autoradiography.
c-fos and Skeletal -Actin Promoter
Analysis (Luciferase Assay)
After transfection of 1 µg c-fos or the
skeletal -actinluciferase reporter gene, cardiac myocytes were
washed twice with PBS and maintained in serum-free medium for 2 days.
Transfected cardiomyocytes were lysed with 100 µL of the
lysis buffer included in the luciferase assay kit (Toyo Inku, Inc). An
aliquot of supernatant was added to the buffer containing luciferin
according to the luciferase assay kit. Luciferase activity was measured
by a Berthold Lumat LB9501 luminometer.
Amino Acid Uptake Into Cardiac Myocytes
After starvation for 48 hours, cardiac myocytes were stretched
by 20% for 24 hours. The relative amount of protein synthesis was
determined by assessing the incorporation of radioactivity into a
trichloroacetic acid–insoluble fraction as previously
reported.3 4
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Rho Is Important for Stretch-Induced Activation of ERKs in Cardiac Myocytes
Rho family members such as RhoA, Rac1, and Cdc42 have been reported to regulate the activity of mitogen-activated protein kinase (MAPK) subfamilies such as c-Jun NH2-terminal kinase and p38 MAPK in HeLa and COS-7 cells.26 30 It has also been reported that Rho proteins are involved in the integrin-dependent activation of ERKs21 and that Rho proteins activate ERKs through activation of p21-activated kinase.31 To elucidate the role of Rho protein in the development of mechanical stress–induced hypertrophic responses in cardiac myocytes, we first examined the effect of C3 exoenzyme on mechanical stress–induced ERK activation in cardiac myocytes. C3 exoenzyme selectively ADP-ribosylates small G proteins of the Rho subfamily at Asn-41, thereby blocking their action.32 Mechanical stress was imposed on cardiac myocytes of neonatal rats cultured on an elastic silicone dish by stretching the culture dish by 20% after cells were incubated with 10 µg/mL C3 exoenzyme for 36 hours. A phase-contrast microscopy analysis revealed that the C3 exoenzyme treatment did not change the morphology of cardiac myocytes and that C3 exoenzyme–treated as well as untreated cardiomyocytes were equally (
20%) stretched (data not shown). Mechanical stress
markedly activated (4- to 5-fold) ERKs, as described
previously.2 3 Although pretreatment with the C3 exoenzyme
had no effect on basal ERK activity, the increase in ERK activity
induced by stretch was strongly (90%) inhibited by treatment with
the C3 exoenzyme (Figure 1
).
|
We further examined the role of Rho proteins in stretch-induced ERK
activation by using different methods. We transfected HA-ERK2 into
cardiomyocytes with the Rho GDP dissociation
inhibitor (Rho-GDI), and a dominant-negative mutant of RhoA
(DNRhoA), Rac1 (DNRac1), or Cdc42 (DNCdc42) and examined the activity
of transfected ERK2 after stretching the cardiomyocytes.
Mechanical stretch increased the activity of the transfected ERK2
(Figure 2), as well as of the
endogenous ERKs (Figure 1
) in cardiac myocytes.
Stretch-induced activation of ERK2 was significantly attenuated by
overexpressing Rho-GDI, DNRhoA, or DNRac1 (Figure 2). Rho-GDI
most strongly suppressed the stretch-induced activation of ERK2, and
the activity was <40% compared with the maximal activation by stretch
(Figure 2).
|
We next examined the effects of constitutively active (CA) mutants of
the Rho family on ERK activity in cardiac myocytes. CARhoA or CARac1
only slightly (<1.5-fold) activated ERK2 in unstretched
cardiac myocytes (Figure 3). Mechanical
stretch markedly (4- to 5-fold) activated ERK2, but
overexpression of CA mutants of the Rho family did not enhance the
activity of ERK2 in stretched cardiomyocytes (Figure 3).
|
The Src Family of Tyrosine Kinases or Ras Is Not Involved in
Stretch-Induced Activation of ERKs in Cardiomyocytes
Stimulation of receptor tyrosine kinases activates the
Raf-1- MAPK/ERK kinase (MEK)-ERK cascade through Ras in many cell
types.33 Activation of the Src family of tyrosine kinases
and Ras has also been reported to be required for Ang II–induced
activation of ERKs in smooth muscle cells.34 It has
recently been reported that mechanical stress stimulates the secretion
of Ang II from cardiac myocytes and that Ang II activates Src
family tyrosine kinases and Ras in cardiac myocytes.5 35
We thus examined the role of the Src family of protein kinases and the
small G protein Ras in stretch-induced ERK activation by overexpressing
C-terminal Src kinase (CSK) and DNRas, respectively. CSK has
been reported to phosphorylate and inactivate
Src family protein kinases.27 36 We transfected
HA-ERK2 into cardiomyocytes with a kinase-negative mutant
of CSK (CSK-), wild-type CSK
(CSK+), or DNRas and stretched the
cardiomyocytes by 20% for 9 minutes. Stretch-induced ERK
activation was not attenuated by overexpression of CSK or DNRas (Figure 4A). On the other hand, activation of the
transfected ERK2 by stretch was completely suppressed by cotransfection
of DNRaf-1 (Figure 4A), indicating that stretch
activates ERKs via a pathway involving Raf-1 kinase but not Src
family protein kinases or Ras proteins in cardiac myocytes. By
contrast, the insulin-induced increase in ERK2 activity was
significantly inhibited by cotransfection of CSK+
but not by CSK- and completely suppressed by
cotransfection of DNRas and DNRaf-1 (Figure 4B).
|
Rho Is Required for Stretch-Induced Expression of
c-fos and Skeletal -Actin Genes in Cardiac
Myocytes
Expression of immediate-early response genes and fetal genes is a
genetic response of cardiac myocytes to mechanical
stress.7 37 38 We examined the role of Rho proteins in
mechanical stress–induced gene expression in cardiac myocytes. A
reporter gene containing the c-fos gene promoter was
transfected into cardiac myocytes. The luciferase activity of the
c-fos reporter gene was increased by stretch, and this
increase was completely inhibited by pretreatment with the C3 exoenzyme
(Figure 5A). When Rho-GDI was transfected
with the c-fos reporter gene into cardiac myocytes,
mechanical stress did not increase reporter gene luciferase activity
(Figure 5A). Next, we examined the role of Rho proteins in
stretch-induced fetal gene expression in cardiac myocytes. Mechanical
stress–induced activation of skeletal -actin promoter was also
markedly suppressed by pretreatment with the C3 exoenzyme or
overexpression of Rho-GDI (Figure 5B). These results suggest
that Rho proteins play a critical role in the mechanical
stress–induced reprogramming of gene expression.
|
Rho Is Required for the Stretch-Induced Increase in Phenylalanine
Incorporation Into Cardiomyocytes
To determine whether Rho proteins are involved in stretch-induced
cardiac hypertrophy, we examined relative protein synthesis
after stretch in the presence or absence of the C3 exoenzyme. As
described in a previous article,3 stretch by 20% for 24
hours increased phenylalanine incorporation into cardiac myocytes by
1.5-fold compared with controls (Figure 6). This increase was significantly
(70%) suppressed by pretreatment with 10 µg/mL of the C3
exoenzyme for 36 hours, suggesting that Rho proteins play a critical
role in the mechanical stress–induced cardiac
hypertrophy.
|
Rho Proteins Are Not Involved in Ang II–Induced ERK Activation
in Cardiomyocytes
We have reported that Ang II partly mediates the development of
mechanical stress–induced hypertrophic responses.2 39
Therefore, we examined the relationship between Ang II and Rho
proteins. We first asked whether Rho proteins are involved in Ang
II–induced ERK activation in cardiomyocytes. The
transfected ERK2 was activated by 10-6
mol/L Ang II as reported previously,40 but activation of
ERK2 was not attenuated by pretreatment with the C3 exoenzyme (Figure 7). In addition, overexpression of
Rho-GDI and DNRhoA also did not have any effect on Ang II–induced ERK
activation (Figure 7). Next, we investigated whether Rho
proteins affect Ang II–induced ERK activation during stretching. The
activation of transfected ERK2 induced by stretch was significantly
(60%) inhibited by the Ang II type 1 receptor–specific
antagonist CV-11974 (10-6 mol/L), as
reported before (Figure 8). When
cardiomyocytes were transfected by Rho-GDI and pretreated
with CV-11974, activation of transfected ERK2 by stretch was more
strongly suppressed (Figure 8). These results suggest that Rho
proteins are not involved in the process of Ang II–induced activation
of ERKs during mechanical stress–induced cardiac
hypertrophy.
|
|
P<0.01, #P<0.05 versus stretch
without any inhibition of Rho and Ang II functions.
Rho Proteins May Play an Important Role in Ang II
Secretion
It has recently been reported that Ang II is secreted from
stretched cardiomyocytes.41 We examined
whether Rho proteins are involved in Ang II secretion by using the
culture media of cardiomyocytes. The culture media of
cardiomyocytes that were not stretched or stretched by 20%
for 10 minutes were transferred to cardiomyocytes of
another culture dish, and activity of ERK was measured as previously
reported.39 The addition of media conditioned by stretch
significantly increased ERK activity (Figure 9). Pretreatment of recipient cells with
CV-11974 (10-6 mol/L) potently suppressed ERK
activation induced by addition of the conditioned media, suggesting
that Ang II in the stretch-conditioned media activated ERKs
(Figure 9). When stretch-conditioned media were prepared from
cells pretreated with the C3 exoenzyme, ERK activity was not increased
(Figure 9). These results collectively suggest that Rho proteins
may play a critical role in stretch-induced Ang II secretion in cardiac
myocytes.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mechanical stress induces reprogramming of gene expression and an increase in protein synthesis in cardiac myocytes.2 4 5 6 7 We and others have reported that mechanical stretch activates the Raf-1 kinase–MEK-ERK cascade partly through secreted Ang II and endothelin-1.3 5 40 42 Moreover, c-Jun NH2-terminal kinases, a subfamily of MAPKs, are also activated by mechanical stress and Ang II in cardiomyocytes.43 44 Although activation of the protein kinase cascade of phosphorylation has been reported to be important for the development of hypertrophic responses,3 5 42 43 it remains unknown how mechanical stress is converted into these biochemical signals. Interaction of cells with the ECM via integrins plays an important role in a wide range of cellular functions, such as cell growth, movement, and differentiation.8 After cell adhesion to ECM components, many intracellular events, including phosphorylation of FAK and FAK-associated tyrosine kinase, occur.13 14 Integrin-mediated formation of focal adhesions and phosphorylation of FAK need an intact cytoskeleton.13 14 A growing body of evidence has suggested that Rho family proteins are critically involved in regulation of the cytoskeleton, such as formation of actin microfilaments and focal adhesions.18 45 Integrins have been reported to activate ERKs when bound to the ECM,11 12 21 and Rho proteins are involved in the integrin-dependent activation of ERKs.21 Therefore, we examined whether the Rho family of small G proteins is involved in mechanical stress–induced ERK activation in cardiomyocytes plated on collagen-coated, silicone membranes. In the present study, mechanical stress–induced activation of ERKs was strongly suppressed by overexpression of Rho-GDI or pretreatment with the C3 exoenzyme in cardiac myocytes. These results suggest that Rho proteins are involved in ERK activation induced by mechanical stress. In addition, overexpression of CARhoA or CARac1 had marginal effects on ERK in unstretched cardiomyocytes, and these active mutants of the Rho family did not enhance the transfected ERK2 activity in stretched cardiomyocytes (Figure 3
). These results
suggest that Rho proteins may be required for mechanical
stretch–induced hypertrophic responses by modulating the functions of
integrins.
Hypertrophy of neonatal ventricular myocytes is
associated with a number of phenotypic changes, including reprogramming
of gene expression, changes in cell shape, and organization of
contractile proteins into sarcomeric units.37 38 46 It has
recently been reported that Rho is required for gene expression that is
induced by signals through Gq and
1-adrenergic receptors and that Rho-dependent
pathways are involved in phenylephrine (PHE)-induced atrial
natriuretic factor gene expression by interacting with Ras
cascades in cardiac myocytes.47 In addition, the
activation of ERKs through Ras is required for PHE-induced gene
expression, and Ras but not Rho plays a critical role in actin
organization in cardiac myocytes.48 49 We showed that Rho
proteins are essential for mechanical stress–induced expressions of
c-fos and skeletal -actin genes (Figure 5A and 5B). These results suggest that Rho plays a critical role in the
stretch-induced reprogramming of gene expression as well as in
PHE-induced gene expression. Although inhibition of Rho protein
functions partly inhibited stretch-induced activation of ERKs,
stretch-induced gene expression was mostly suppressed by pretreatment
with the C3 exoenzyme and overexpression of Rho-GDI. These results
suggest that the role of Rho proteins in gene expression may be
different from their role in ERK activation. Recent studies have
demonstrated that Rho proteins are involved in transcriptional
activation of the c-fos gene by regulating serum response
factor (SRF) in several cell types, including cardiac myocytes, and
activation of the SRF-linked signaling pathway is not correlated with
the activation of ERKs.50 51 Moreover, because the
CArG sequence, a regulatory sequence found in many muscle-specific
promoters including the skeletal -actin gene
promoter,52 is similar to the c-fos serum
response element, the skeletal -actin gene promoter as well as the
c-fos gene promoter may be directly regulated by Rho
proteins via CArG/serum response element sequences in cardiac
myocytes.
Recently, we have demonstrated that mechanical stress activates
ERKs partly through secreted Ang II in
cardiomyocytes.39 In the present study,
however, Rho proteins had little effect on the activation of ERK2
induced by Ang II (Figure 7), as in the case of
PHE.47 49 Stretch-induced ERK activation was more strongly
suppressed by combined treatment, ie, overexpression of Rho-GDI and
treatment with an Ang II type I receptor antagonist,
compared with single treatment (Figure 8). These results suggest
that Rho proteins are not involved in the process of Ang II–induced
ERK activation during mechanical stress–induced cardiac
hypertrophy. As shown in Figure 9, pretreatment of
cardiomyocytes with the C3 exoenzyme potently suppressed
Ang II secretion from stretched cardiomyocytes. Recent
studies have indicated that Rho is involved in the release of humoral
factors such as catecholamines and gastrin in some cell
types.53 54 Taken together, although Rho proteins do not
mediate Ang II–induced hypertrophic responses, it is conceivable that
Rho proteins regulate the secretion of Ang II in stretched
cardiomyocytes.
The small G protein Ras plays a key role in a variety of cell functions
partly through Raf-1 and ERKs.55 56 We have recently
reported that oxidative stress activates ERKs through Ras in
cardiac myocytes.57 In addition, it has been reported that
hypertrophy-promoting stimuli, such as PHE and Ang II,
activate Ras in cardiac myocytes.35 58 Ras is
usually activated by tyrosine kinases, including the Src family
of tyrosine kinases,55 59 and it has also been reported
that Ang II activates Ras through Fyn, 1 of the Src family
tyrosine kinases in cardiac myocytes.35 We therefore
examined whether Ras and Src family tyrosine kinases were required for
stretch-induced activation of ERKs in cardiomyocytes.
Although insulin-induced ERK activation was suppressed by
overexpression of DNRas and CSK (Figure 4B), DNRas or CSK had no
effect on stretch-induced activation of ERKs (Figure 4A). These
results suggest that although the small G protein Ras and Src family
tyrosine kinases may be activated by mechanical stretch, they
do not play a major role in mechanical stress–induced activation of
ERKs.
The Rho family of small G proteins plays a critical role in mechanical
stress–induced hypertrophic responses, such as activation of ERKs,
expression of specific genes, and an increase in protein synthesis.
Mechanical stretch activates ERKs, possibly through Ang
II–dependent and –independent pathways (Figure 10). Because inhibition of Rho family
functions had strong effects on stretch-evoked signal transduction, the
Rho family may be involved in both pathways. The almost-complete
inhibition of stretch-induced gene expression may indicate another role
of Rho proteins. Recent studies have demonstrated that Rho proteins
regulate c-fos gene expression through an SRF-linked
signaling pathway.50 51 Recently, several studies
have also demonstrated that Rho proteins are regulated by the integrity
of the cell wall in yeast.60 61 When cell wall integrity
is changed during growth at high temperature, the MAPK cascade is
activated through Rho1, a yeast homologue of mammalian RhoA, in
yeast cells.60 61 Therefore, the phenomenon that
mechanical stress activates the MAPK family through Rho small G
proteins may be conserved in evolution. Further investigation is
required to elucidate how Rho proteins are involved in the regulation
of stretch-induced cardiac hypertrophy.
|
|
Acknowledgments |
|---|
This work was supported by a grant-in-aid for Scientific Research, Developmental Scientific Research, and Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan; a grant from the Japan Cardiovascular Foundation and the Sankyo Life Science and Study Group of Molecular Cardiology, Japan; and a grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to I.K.). We wish to thank Dr K. Tobe for advice and F. Kataguchi, M. Takano, and C. Yamamoto for their excellent technical assistance.
|
Footnotes |
|---|
This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received March 16, 1998; accepted October 23, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP. Prognostic implications of echocardiographically determined left ventricular mass in the Framingham heart study. N Engl J Med. 1990;322:1561–1566.[Abstract]
2.
Yamazaki T, Tobe K, Hoh E, Maemura K, Kaida T, Komuro
I, Tamemoto H, Kadowaki T, Nagai R, Yazaki Y. Mechanical loading
activates mitogen-activated protein kinase and S6
peptide kinase in cultured rat cardiac myocytes. J Biol
Chem. 1993;268:12069–12076.
3. Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I, Mizuno T, Takano H, Hiroi Y, Ueki K, Tobe K, Kadowaki T, Nagai R, Yazaki Y. Mechanical stress activates protein kinase cascade of phosphorylation in neonatal rat cardiac myocytes. J Clin Invest. 1995;96:438–446.
4. Takano H, Komuro I, Zou Y, Kudoh S, Yamazaki T, Yazaki Y. Activation of p70 S6 protein kinase is necessary for angiotensin II-induced hypertrophy in neonatal rat cardiac myocytes. FEBS Lett. 1996;379:255–259.[Medline] [Order article via Infotrieve]
5. Sadoshima J, Izumo S. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J. 1993;12:1681–1692.[Medline] [Order article via Infotrieve]
6.
Komuro I, Kaida T, Shibazaki Y, Kurabayashi M, Takaku
F, Yazaki Y. Stretching cardiac myocytes stimulates proto-oncogene
expression. J Biol Chem. 1990;265:3595–3598.
7. Komuro I, Yazaki Y. Control of cardiac gene expression by mechanical stress. Annu Rev Physiol. 1993;55:55–75.[Medline] [Order article via Infotrieve]
8. Hynes RO. Integrins: versatility, modulation and signaling in cell adhesion. Cell. 1992;69:11–25.[Medline] [Order article via Infotrieve]
9.
Juliano RL, Haskill S. Signal transduction from the
extracellular matrix. J Cell Biol. 1993;120:577–585.
10. Schwartz MA, Schaller MD, Ginsberg MH. Integrins: emerging paradigms of signal transduction. Annu Rev Cell Biol. 1995;11:549–599.[Medline] [Order article via Infotrieve]
11.
Miyamoto S, Teramoto H, Coso OA, Gutkind JS, Burbelo
PD, Akiyama SK, Yamada KM. Integrin function: molecular hierarchies of
cytoskeletal and signaling molecules. J Cell Biol. 1995;131:791–805.
12.
Morino N, Mimura T, Hamasaki K, Tobe K, Ueki K, Kikuchi
K, Takehara K, Kadowaki T, Yazaki Y, Nojima Y. Matrix/integrin
interaction activates the mitogen-activated protein
kinase, p44erk-1 and p42erk-2. J Biol Chem. 1995;270:269–273.
13.
Nojima Y, Morino N, Mimura T, Hamasaki K, Furuya H,
Sakai R, Sato T, Tachibana K, Morimoto C, Yazaki Y. Integrin-mediated
cell adhesion promotes tyrosine phosphorylation of
p130Cas, a Src homology 3-containing molecule having multiple Src
homology 2-binding motifs. J Biol Chem. 1995;270:15398–15402.
14. Hildebrand JD, Taylor JM, Parsons JT. An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase. Mol Cell Biol. 1996;16:3169–3178.[Abstract]
15. Dike LE, Ingber DE. Integrin-dependent induction of early growth response genes in capillary endothelial cells. J Cell Sci. 1996;109:2855–2863.[Abstract]
16. Hall A. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Biol. 1994;10:31–54.
17. Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992;70:389–399.[Medline] [Order article via Infotrieve]
18. Nobes CD, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 1995;81:53–62.[Medline] [Order article via Infotrieve]
19. Chong LD, Traynor-Kaplan A, Bokoch GM, Schwartz MA. The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. Cell. 1994;79:507–513.[Medline] [Order article via Infotrieve]
20.
Chen Q, Kinch MS, Lin TH, Burridge K, Juliano RL.
Integrin-mediated cell adhesion activates
mitogen-activated protein kinases. J Biol Chem. 1994;269:26602–26605.
21.
Renshaw MW, Toksoz D, Schwartz MA. Involvement of the
small GTPase rho in integrin-mediated activation of
mitogen-activated protein kinase. J Biol Chem. 1996;271:21691–21694.
22.
Tobe K, Kadowaki T, Hara K, Gotoh Y, Kosako H, Matsuda
S, Tamemoto H, Ueki K, Akanuma Y, Nishida E, Yazaki Y. Sequential
activation of MAP kinase activator, MAP kinases, and S6
peptide kinase in intact rat liver following insulin injection.
J Biol Chem. 1992;267:21089–21097.
23. Morii N, Narumiya S. Preparation of native and recombinant Clostridium botulinum C3 ADP-ribosyltransferase and identification of Rho proteins by ADP-ribosylation. Methods Enzymol. 1995;256:196–206.[Medline] [Order article via Infotrieve]
24.
Minden A, Lin A, Smeal T, Derijard B, Cobb M, Davis R,
Karin M. c-Jun N-terminal phosphorylation correlates
with activation of the JNK subgroup but not the ERK subgroup of
mitogen-activated protein kinases. Mol Cell Biol. 1994;14:6683–6688.
25. Kikuchi A, Kaibuchi K, Hori Y, Nonaka H, Sakoda T, Kawamura M, Mizuno T, Takai Y. Molecular cloning of the human cDNA for a stimulatory GDP/GTP exchange protein for c-Ki-ras p21 and smg p21. Oncogene. 1992;7:289–293.[Medline] [Order article via Infotrieve]
26. Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, Miki T, Gutkind JS. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell. 1995;81:1137–1146.[Medline] [Order article via Infotrieve]
27.
Hata A, Sabe H, Kurosaki T, Takata M, Hanafusa H.
Functional analysis of Csk in signal transduction through the
B-cell antigen receptor. Mol Cell Biol. 1994;14:7306–7313.
28.
Izumi T, Tamemoto H, Nagao M, Kadowaki T, Takaku F,
Kasuga M. Insulin and platelet derived growth factor stimulate
phosphorylation of the c-raf product at serine and
threonine residues in intact cells. J Biol Chem. 1991;266:7933–7939.
29.
Simpson P. Norepinephrine-stimulated
hypertrophy of cultured rat myocardial cells is an
1-adrenergic response. J Clin Invest. 1983;72:732–738.
30. Minden A, Lin A, Claret FX, Abo A, Karin M. Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell. 1995;81:1147–1157.[Medline] [Order article via Infotrieve]
31. Frost JA, Xu S, Hutchison MR, Marcus S, Cobb MH. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members. Mol Cell Biol. 1996;16:3707–3713.[Abstract]
32. Valencia A, Chardin P, Wittinghofer A, Sander C. The ras protein family: evolutionary tree and role of conserved amino acids. Biochemistry. 1991;30:4637–4648. Review.[Medline] [Order article via Infotrieve]
33.
Satoh T, Nakafuku M, Kaziro Y. Function of Ras as a
molecular switch in signal transduction. J Biol Chem. 1992;267:24149–24152.
34.
Schieffer B, Paxton WG, Chai Q, Marrero MB, Bernstein
KE. Angiotensin II controls p21ras activity via pp60c-src.
J Biol Chem. 1996;271:10329–10333.
35. Sadoshima J, Izumo S. The heterotrimeric Gq protein-coupled angiotensin II receptor activates p21ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes. EMBO J. 1996;15:775–787.[Medline] [Order article via Infotrieve]
36.
Okada M, Nada S, Yamanashi Y, Yamamoto T, Nakagawa H.
CSK: a protein-tyrosine kinase involved in regulation of src family
kinases. J Biol Chem. 1991;266:24249–24252.
37.
Komuro I, Katoh Y, Kaida T, Shibazaki Y, Kurabayashi M,
Takaku F, Yazaki Y. Mechanical loading stimulates cell
hypertrophy and specific gene expression in cultured rat
cardiac myocytes. J Biol Chem. 1991;266:1265–1268.
38.
Karns LR, Kariya K, Simpson PC. M-CAT, CArG, and Sp1
elements are required for 1-adrenergic induction of the
skeletal -actin promoter during cardiac myocyte
hypertrophy. J Biol Chem. 1995;270:410–417.
39.
Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I,
Mizuno T, Takano H, Hiroi Y, Ueki K, Tobe K, Kadowaki T, Nagai R,
Yazaki Y. Angiotensin II partly mediates mechanical
stress-induced cardiac hypertrophy. Circ Res. 1995;77:258–265.
40.
Zou Y, Komuro I, Yamazaki T, Aikawa R, Kudoh S,
Shiojima I, Hiroi Y, Mizuno T, Yazaki Y. Protein kinase C, but not
tyrosine kinases or Ras, plays a critical role in
angiotensin II-induced activation of Raf-1 kinase and
extracellular signal-regulated protein kinases in cardiac myocytes.
J Biol Chem. 1996;271:33592–33597.
41. Sadoshima J, Xu Y, Slayter HS, Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell. 1993;75:977–984.[Medline] [Order article via Infotrieve]
42.
Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I, Hiroi
Y, Mizuno T, Maemura K, Kurihara H, Aikawa R, Takano H, Yazaki Y.
Endothelin-1 is involved in mechanical stress-induced
cardiomyocyte hypertrophy. J Biol
Chem. 1996;271:3221–3228.
43. Komuro I, Kudo S, Yamazaki T, Zou Y, Shiojima I, Yazaki Y. Mechanical stretch activates the stress-activated protein kinases in cardiac myocytes. FASEB J. 1996;10:631–636.[Abstract]
44.
Kudoh S, Komuro I, Mizuno T, Yamazaki T, Zou Y, Ichiro
S, Takekoshi N, Yazaki Y. Angiotensin II stimulates c-Jun
NH2-terminal kinase in cultured cardiac myocytes
of neonatal rats. Circ Res. 1997;80:139–146.
45.
Narumiya S. The small GTPase Rho: cellular functions
and signal transduction. J Biochem. 1996;120:215–228.
46. Chien KR, Knowlton KU, Zhu H, Chien S. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J. 1991;5:3037–3046.[Abstract]
47.
Sah VP, Hoshijima M, Chien KR, Brown JH. Rho is
required for Gq and 1-adrenergic receptor signaling in
cardiomyocytes. J Biol Chem. 1996;271:31185–31190.
48.
Thorburn J, Frost JA, Thorburn A.
Mitogen-activated protein kinases mediate changes in gene
expression, but not cytoskeletal organization associated with cardiac
muscle cell hypertrophy. J Cell Biol. 1994;126:1565–1572.
49. Thorburn J, Xu S, Thorburn A. MAP kinase- and Rho-dependent signals interact to regulate gene expression but not actin morphology in cardiac muscle cells. EMBO J. 1997;16:1888–1900.[Medline] [Order article via Infotrieve]
50. Hill CS, Wynne J, Treisman R. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell. 1995;81:1159–1170.[Medline] [Order article via Infotrieve]
51.
Ueyama T, Sakoda T, Kawashima S, Hiraoka E, Hirata K,
Akita H, Yokoyama M. Activated RhoA stimulates c-fos gene
expression in myocardial cells. Circ Res. 1997;81:672–678.
52. Treisman R. The SRE: a growth factor responsive transcriptional regulator. Semin Cancer Biol. 1990;1:47–58.[Medline] [Order article via Infotrieve]
53. Seensalu R, Avedian D, Barbuti R, Song M, Slice L, Walsh JH. Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by protein kinase C, and is enhanced by disruption of rho/cytoskeletal pathways. J Clin Invest. 1997;100:1037–1046.[Medline] [Order article via Infotrieve]
54.
Gasman S, Chasserot-Golaz S, Popoff MR, Aunis D, Bader
MF. Trimeric G proteins control exocytosis in chromaffin cells: go
regulates the peripheral actin network and
catecholamine secretion by a mechanism involving the small
GTP-binding protein Rho. J Biol Chem. 1997;272:20564–20571.
55. Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, Morgenbesser SD, DePinho RA, Panayotatos N, Cobb MH, Yancopoulos GD. ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell. 1991;65:663–675.[Medline] [Order article via Infotrieve]
56. Rozakis-Adcock M, McGlade J, Mbamalu G, Pelicci G, Daly R, Li W, Batzer A, Thomas S, Brugge J, Pelicci PG, et al. Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature. 1992;360:689–692.[Medline] [Order article via Infotrieve]
57. Aikawa R, Komuro I, Yamazaki T, Zou Y, Kudoh S, Tanaka M, Shiojima I, Hiroi Y, Yazaki Y. Oxidative stress activates extracellular signal-regulated kinases through src and ras in cultured cardiac myocytes of neonatal rats. J Clin Invest. 1997;100:1813–1821.[Medline] [Order article via Infotrieve]
58.
Thorburn A, Thorburn J, Chen S-Y, Powers S, Shubeita
HE, Feramisco JR, Chien KR. HRas-dependent pathways can
activate morphological and genetic markers of cardiac muscle
cell hypertrophy. J Biol Chem. 1993;268:2244–2249.
59.
Davis RJ. The mitogen-activated protein kinase
signal transduction pathway. J Biol Chem. 1993;268:14553–14556.
60. Nonaka H, Tanaka K, Hirano H, Fujiwara T, Kohno H, Umikawa M, Mino A, Takai Y. A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae. EMBO J. 1995;14:5931–5938.[Medline] [Order article via Infotrieve]
61.
Kamada Y, Qadota H, Python CP, Anraku Y, Ohya Y, Levin
DE. Activation of yeast protein kinase C by Rho1 GTPase. J
Biol Chem. 1996;271:9193–9196.
This article has been cited by other articles:
 |
|
 |
|
  A. Zhang, J. J. David, S. V. Subramanian, X. Liu, M. D. Fuerst, X. Zhao, C. V. Leier, C. G. Orosz, R. J. Kelm Jr., and A. R. Strauch Serum response factor neutralizes Pur{alpha}- and Pur{beta}-mediated repression of the fetal vascular smooth muscle {alpha}-actin gene in stressed adult cardiomyocytes Am J Physiol Cell Physiol, March 1, 2008; 294(3): C702 - C714. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Albinsson and P. Hellstrand Integration of signal pathways for stretch-dependent growth and differentiation in vascular smooth muscle Am J Physiol Cell Physiol, August 1, 2007; 293(2): C772 - C782. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Abe, K. Ono, T. Kawamura, H. Wada, T. Kita, A. Shimatsu, and K. Hasegawa Leptin induces elongation of cardiac myocytes and causes eccentric left ventricular dilatation with compensation Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2387 - H2396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Barrientos, D. Frank, K. Kuwahara, S. Bezprozvannaya, G. C. T. Pipes, R. Bassel-Duby, J. A. Richardson, H. A. Katus, E. N. Olson, and N. Frey Two Novel Members of the ABLIM Protein Family, ABLIM-2 and -3, Associate with STARS and Directly Bind F-actin J. Biol. Chem., March 16, 2007; 282(11): 8393 - 8403. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. S. Chaturvedi, H. M. Marsh, X. Shang, Y. Zheng, and M. D. Basson Repetitive Deformation Activates Focal Adhesion Kinase and ERK Mitogenic Signals in Human Caco-2 Intestinal Epithelial Cells through Src and Rac1 J. Biol. Chem., January 5, 2007; 282(1): 14 - 28. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Otis and N. Gallo-Payet Differential Involvement of Cytoskeleton and Rho-Guanosine 5'-Triphosphatases in Growth-Promoting Effects of Angiotensin II in Rat Adrenal Glomerulosa Cells Endocrinology, November 1, 2006; 147(11): 5460 - 5469. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Warren, A. Rutkowski, and L. Cassimeris Infection with Replication-deficient Adenovirus Induces Changes in the Dynamic Instability of Host Cell Microtubules Mol. Biol. Cell, August 1, 2006; 17(8): 3557 - 3568. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Winaver, E. Ovcharenko, I. Rubinstein, K. Gurbanov, P. Pollesello, B. Bishara, A. Hoffman, and Z. Abassi Involvement of Rho kinase pathway in the mechanism of renal vasoconstriction and cardiac hypertrophy in rats with experimental heart failure Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H2007 - H2014. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Khan, S. Varadharaj, L. P. Ganesan, J. C. Shobha, M. U. Naidu, N. L. Parinandi, S. Tridandapani, V. K. Kutala, and P. Kuppusamy C-phycocyanin protects against ischemia-reperfusion injury of heart through involvement of p38 MAPK and ERK signaling Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H2136 - H2145. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hoshijima Mechanical stress-strain sensors embedded in cardiac cytoskeleton: Z disk, titin, and associated structures Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1313 - H1325. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Brown, D. P. Del Re, and M. A. Sussman The Rac and Rho Hall of Fame: A Decade of Hypertrophic Signaling Hits Circ. Res., March 31, 2006; 98(6): 730 - 742. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Torsoni, T. M. Marin, L. A. Velloso, and K. G. Franchini RhoA/ROCK signaling is critical to FAK activation by cyclic stretch in cardiac myocytes Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1488 - H1496. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kuwahara, T. Barrientos, G. C. T. Pipes, S. Li, and E. N. Olson Muscle-Specific Signaling Mechanism That Links Actin Dynamics to Serum Response Factor Mol. Cell. Biol., April 15, 2005; 25(8): 3173 - 3181. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. U. Muller, G. Lewin, H. A. Baba, P. Boknik, L. Fabritz, U. Kirchhefer, P. Kirchhof, K. Loser, M. Matus, J. Neumann, et al. Heart-directed Expression of a Human Cardiac Isoform of cAMP-Response Element Modulator in Transgenic Mice J. Biol. Chem., February 25, 2005; 280(8): 6906 - 6914. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Yatani, K. Irie, T. Otani, M. Abdellatif, and L. Wei RhoA GTPase regulates L-type Ca2+ currents in cardiac myocytes Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H650 - H659. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Yin, N. J.D. Gower, H. A. Baylis, and K. Strange Inositol 1,4,5-Trisphosphate Signaling Regulates Rhythmic Contractile Activity of Myoepithelial Sheath Cells in Caenorhabditis elegans Mol. Biol. Cell, August 1, 2004; 15(8): 3938 - 3949. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ito, T. Adachi, D. R. Pimentel, Y. Ido, and W. S. Colucci Statins Inhibit {beta}-Adrenergic Receptor-Stimulated Apoptosis in Adult Rat Ventricular Myocytes via a Rac1-Dependent Mechanism Circulation, July 27, 2004; 110(4): 412 - 418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Hoffmann, D. Podlich, B. Hahnel, W. Kriz, and N. Gretz Angiotensin II Type 1 Receptor Overexpression in Podocytes Induces Glomerulosclerosis in Transgenic Rats J. Am. Soc. Nephrol., June 1, 2004; 15(6): 1475 - 1487. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. McClung, R. W. Thompson, L. L. Lowe, and J. A. Carson RhoA expression during recovery from skeletal muscle disuse J Appl Physiol, April 1, 2004; 96(4): 1341 - 1348. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Polte, G. S. Eichler, N. Wang, and D. E. Ingber Extracellular matrix controls myosin light chain phosphorylation and cell contractility through modulation of cell shape and cytoskeletal prestress Am J Physiol Cell Physiol, March 1, 2004; 286(3): C518 - C528. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Krepinsky, A. J. Ingram, D. Tang, D. Wu, L. Liu, and J. W. Scholey Nitric Oxide Inhibits Stretch-Induced MAPK Activation in Mesangial Cells Through RhoA Inactivation J. Am. Soc. Nephrol., November 1, 2003; 14(11): 2790 - 2800. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kawamura, S. Miyamoto, and J. H. Brown Initiation and Transduction of Stretch-induced RhoA and Rac1 Activation through Caveolae: CYTOSKELETAL REGULATION OF ERK TRANSLOCATION J. Biol. Chem., August 15, 2003; 278(33): 31111 - 31117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Pikkarainen, H. Tokola, T. Majalahti-Palviainen, R. Kerkela, N. Hautala, S. S. Bhalla, F. Charron, M. Nemer, O. Vuolteenaho, and H. Ruskoaho GATA-4 Is a Nuclear Mediator of Mechanical Stretch-activated Hypertrophic Program J. Biol. Chem., June 20, 2003; 278(26): 23807 - 23816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Torsoni, P. M. Fonseca, D. P Crosara-Alberto, and K. G. Franchini Early activation of p160ROCK by pressure overload in rat heart Am J Physiol Cell Physiol, June 1, 2003; 284(6): C1411 - C1419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Akazawa and I. Komuro Roles of Cardiac Transcription Factors in Cardiac Hypertrophy Circ. Res., May 30, 2003; 92(10): 1079 - 1088. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Putnam, J. J. Cunningham, B. B. L. Pillemer, and D. J. Mooney External mechanical strain regulates membrane targeting of Rho GTPases by controlling microtubule assembly Am J Physiol Cell Physiol, March 1, 2003; 284(3): C627 - C639. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Sussman, A. McCulloch, and T. K. Borg Dance Band on the Titanic: Biomechanical Signaling in Cardiac Hypertrophy Circ. Res., November 15, 2002; 91(10): 888 - 898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D. Oldenhof, O. P. Shynlova, M. Liu, B. L. Langille, and S. J. Lye Mitogen-activated protein kinases mediate stretch-induced c-fos mRNA expression in myometrial smooth muscle cells Am J Physiol Cell Physiol, November 1, 2002; 283(5): C1530 - C1539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Indolfi, E. Di Lorenzo, C. Perrino, A. M. Stingone, A. Curcio, D. Torella, A. Cittadini, L. Cardone, C. Coppola, L. Cavuto, et al. Hydroxymethylglutaryl Coenzyme A Reductase Inhibitor Simvastatin Prevents Cardiac Hypertrophy Induced by Pressure Overload and Inhibits p21ras Activation Circulation, October 15, 2002; 106(16): 2118 - 2124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Lavelin, N. Meiri, M. Einat, O. Genina, and M. Pines Mechanical strain regulation of the chicken glypican-4 gene expression in the avian eggshell gland Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2002; 283(4): R853 - R861. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. S. Chockalingam, R. Cholera, S. A. Oak, Y. Zheng, H. W. Jarrett, and D. B. Thomason Dystrophin-glycoprotein complex and Ras and Rho GTPase signaling are altered in muscle atrophy Am J Physiol Cell Physiol, August 1, 2002; 283(2): C500 - C511. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Arai, J. A. Spencer, and E. N. Olson STARS, a Striated Muscle Activator of Rho Signaling and Serum Response Factor-dependent Transcription J. Biol. Chem., June 28, 2002; 277(27): 24453 - 24459. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Kobayashi, S. Nakano, S.-i. Mita, T. Kobayashi, T. Honda, Y. Tsubokou, and H. Matsuoka Involvement of Rho-Kinase Pathway for Angiotensin II-Induced Plasminogen Activator Inhibitor-1 Gene Expression and Cardiovascular Remodeling in Hypertensive Rats J. Pharmacol. Exp. Ther., May 1, 2002; 301(2): 459 - 466. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sotoudeh, Y.-S. Li, N. Yajima, C.-C. Chang, T.-C. Tsou, Y. Wang, S. Usami, A. Ratcliffe, S. Chien, and J. Y.-J. Shyy Induction of apoptosis in vascular smooth muscle cells by mechanical stretch Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1709 - H1716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yanazume, K. Hasegawa, H. Wada, T. Morimoto, M. Abe, T. Kawamura, and S. Sasayama Rho/ROCK Pathway Contributes to the Activation of Extracellular Signal-regulated Kinase/GATA-4 during Myocardial Cell Hypertrophy J. Biol. Chem., March 1, 2002; 277(10): 8618 - 8625. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Laufs, H. Kilter, C. Konkol, S. Wassmann, M. Bohm, and G. Nickenig Impact of HMG CoA reductase inhibition on small GTPases in the heart Cardiovasc Res, March 1, 2002; 53(4): 911 - 920. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Souchet, E. Portales-Casamar, D. Mazurais, S. Schmidt, I. Leger, J.-L. Javre, P. Robert, I. Berrebi-Bertrand, A. Bril, B. Gout, et al. Human p63RhoGEF, a novel RhoA-specific guanine nucleotide exchange factor, is localized in cardiac sarcomere J. Cell Sci., January 2, 2002; 115(3): 629 - 640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Lavelin, N. Meiri, O. Genina, R. Alexiev, and M. Pines Na+-K+-ATPase gene expression in the avian eggshell gland: distinct regulation in different cell types Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2001; 281(4): R1169 - R1176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Hill, H. Zou, S. J. Potocnik, G. A. Meininger, and M. J. Davis Signal Transduction in Smooth Muscle: Invited Review: Arteriolar smooth muscle mechanotransduction: Ca2+ signaling pathways underlying myogenic reactivity J Appl Physiol, August 1, 2001; 91(2): 973 - 983. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Takeda, T. Ichiki, T. Tokunou, N. Iino, S. Fujii, A. Kitabatake, H. Shimokawa, and A. Takeshita Critical Role of Rho-Kinase and MEK/ERK Pathways for Angiotensin II-Induced Plasminogen Activator Inhibitor Type-1 Gene Expression Arterioscler. Thromb. Vasc. Biol., May 1, 2001; 21(5): 868 - 873. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. WEI, L. WANG, J. A. CARSON, J. E. AGAN, K. IMANAKA-YOSHIDA, and R. J. SCHWARTZ {beta}1 integrin and organized actin filaments facilitate cardiomyocyte-specific RhoA-dependent activation of the skeletal {alpha}-actin promoter FASEB J, March 1, 2001; 15(3): 785 - 796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. P. van Nieuw Amerongen and V. W.M. van Hinsbergh Cytoskeletal Effects of Rho-Like Small Guanine Nucleotide-Binding Proteins in the Vascular System Arterioscler. Thromb. Vasc. Biol., March 1, 2001; 21(3): 300 - 311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y Sawada, K Nakamura, K Doi, K Takeda, K Tobiume, M Saitoh, K Morita, I Komuro, K De Vos, M Sheetz, et al. Rap1 is involved in cell stretching modulation of p38 but not ERK or JNK MAP kinase J. Cell Sci., January 3, 2001; 114(6): 1221 - 1227. [Abstract] [PDF]
|
|
|
|
|
|
U. Laufs, M. Endres, F. Custodis, K. Gertz, G. Nickenig, J. K. Liao, and M. Bohm Suppression of Endothelial Nitric Oxide Production After Withdrawal of Statin Treatment Is Mediated by Negative Feedback Regulation of Rho GTPase Gene Transcription Circulation, December 19, 2000; 102(25): 3104 - 3110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Aikawa, M. Nawano, Y. Gu, H. Katagiri, T. Asano, W. Zhu, R. Nagai, and I. Komuro Insulin Prevents Cardiomyocytes From Oxidative Stress-Induced Apoptosis Through Activation of PI3 Kinase/Akt Circulation, December 5, 2000; 102(23): 2873 - 2879. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Vatner and D. L. Kunze Prologue: low-molecular-weight GTPases in the heart and circulation Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1733 - H1735. [Full Text] [PDF]
|
|
|
|
|
|
M. Ichida and T. Finkel Ras Regulates NFAT3 Activity in Cardiac Myocytes J. Biol. Chem., January 26, 2001; 276(5): 3524 - 3530. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R. Pimentel, J. K. Amin, L. Xiao, T. Miller, J. Viereck, J. Oliver-Krasinski, R. Baliga, J. Wang, D. A. Siwik, K. Singh, et al. Reactive Oxygen Species Mediate Amplitude-Dependent Hypertrophic and Apoptotic Responses to Mechanical Stretch in Cardiac Myocytes Circ. Res., August 31, 2001; 89(5): 453 - 460. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Asakawa, H. Takano, T. Nagai, H. Uozumi, H. Hasegawa, N. Kubota, T. Saito, Y. Masuda, T. Kadowaki, and I. Komuro Peroxisome Proliferator-Activated Receptor {gamma} Plays a Critical Role in Inhibition of Cardiac Hypertrophy In Vitro and In Vivo Circulation, March 12, 2002; 105(10): 1240 - 1246. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||