Original Contribution |
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 |
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-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 IIinduced 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 stressinduced hypertrophic responses.
Key Words: Rho mechanical stress cardiac hypertrophy
| Introduction |
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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
stretchinduced 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 stressinduced 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 |
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-32P]ATP was purchased from Du
PontNew 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 ASepharose 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 SDSpolyacrylamide
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 ASepharose,
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 SDSpolyacrylamide
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 acidinsoluble fraction as previously
reported.3 4
| Results |
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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 IIinduced
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 stressinduced 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
stressinduced 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
stressinduced reprogramming of gene expression.
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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 stressinduced cardiac
hypertrophy.
|
Rho Proteins Are Not Involved in Ang IIInduced ERK Activation
in Cardiomyocytes
We have reported that Ang II partly mediates the development of
mechanical stressinduced 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
IIinduced 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 IIinduced ERK
activation (Figure 7
). Next, we investigated whether Rho
proteins affect Ang IIinduced ERK activation during stretching. The
activation of transfected ERK2 induced by stretch was significantly
(
60%) inhibited by the Ang II type 1 receptorspecific
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 IIinduced activation
of ERKs during mechanical stressinduced cardiac
hypertrophy.
|
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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 |
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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 G
q 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 stressinduced 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 IIinduced
ERK activation during mechanical stressinduced 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 IIinduced 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 stressinduced activation of
ERKs.
The Rho family of small G proteins plays a critical role in mechanical
stressinduced hypertrophic responses, such as activation of ERKs,
expression of specific genes, and an increase in protein synthesis.
Mechanical stretch activates ERKs, possibly through Ang
IIdependent 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.
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| Acknowledgments |
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| Footnotes |
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Received March 16, 1998; accepted October 23, 1998.
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skeletal
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q and
1-adrenergic receptor signaling in
cardiomyocytes. J Biol Chem. 1996;271:3118531190.This article has been cited by other articles:
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