Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 1999;84:458-466

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aikawa, R.
Right arrow Articles by Yazaki, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aikawa, R.
Right arrow Articles by Yazaki, Y.
Related Collections
Right arrow Cell signalling/signal transduction
Right arrow Gene expression
Right arrow Heart failure - basic studies
(Circulation Research. 1999;84:458-466.)
© 1999 American Heart Association, Inc.

Original Contribution

Rho Family Small G Proteins Play Critical Roles in Mechanical Stress–Induced Hypertrophic Responses in Cardiac Myocytes

Ryuichi Aikawa, Issei Komuro, Tsutomu Yamazaki, Yunzeng Zou, Sumiyo Kudoh, Weidong Zhu, Takashi Kadowaki, Yoshio Yazaki

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
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {alpha}-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 {approx}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
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {alpha}- 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 {alpha}-actin and c-fos genes, and an increase in protein synthesis in cardiac myocytes.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
[{gamma}-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 ({alpha}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 {alpha}-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 [{gamma}-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 {alpha}-Actin Promoter Analysis (Luciferase Assay)
After transfection of 1 µg c-fos or the skeletal {alpha}-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
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 ({approx}20%) stretched (data not shown). Mechanical stress markedly activated (4- to {approx}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 ({approx}90%) inhibited by treatment with the C3 exoenzyme (Figure 1Down).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. C3 exoenzyme inhibits stretch-induced activation of ERKs in cardiomyocytes. Cultured cardiac myocytes were stretched by 20% for 9 minutes. Some cardiomyocytes were pretreated with 10 µg/mL C3 exoenzyme for 36 hours (C3+Stretch). Treatment with C3 exoenzyme had no effect on the activity of ERKs (C3). ERKs were immunoprecipitated from cell lysates by using a polyclonal antibody against ERKs ({alpha}Y91), and kinase activity was assayed by using MBP as a substrate. After electrophoresis, the gel was dried and subjected to autoradiography. A representative autoradiogram from 3 independent experiments is shown.

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 2Down), as well as of the endogenous ERKs (Figure 1Up) in cardiac myocytes. Stretch-induced activation of ERK2 was significantly attenuated by overexpressing Rho-GDI, DNRhoA, or DNRac1 (Figure 2Down). Rho-GDI most strongly suppressed the stretch-induced activation of ERK2, and the activity was <40% compared with the maximal activation by stretch (Figure 2Down).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 2. Rho family small G proteins are involved in stretch-induced activation of transfected ERK2 in cardiomyocytes. After transfection of HA-ERK2 with either Rho-GDI, DNRhoA, DNRac1, or DNCdc42, cardiac myocytes were stretched by 20% for 9 minutes. HA-ERK2 was immunoprecipitated from cell lysates by using a polyclonal antibody against HA, and kinase activity was measured by using MBP as a substrate. After electrophoresis, the gel was dried and subjected to autoradiography. The data represent the mean percentage of controls (100%) from 4 independent experiments (mean±SE). *P<0.05 versus control and #P<0.05 versus stretch without any inhibition of Rho function.

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 3Down). Mechanical stretch markedly (4- to {approx}5-fold) activated ERK2, but overexpression of CA mutants of the Rho family did not enhance the activity of ERK2 in stretched cardiomyocytes (Figure 3Down).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 3. Constitutively active (CA) mutants of Rho family proteins have marginal effects on ERK2. After transfection of HA-ERK2 with CARhoA, CARac1, or CACdc42, cardiac myocytes cultured on collagen-coated silicone dishes were washed and maintained in serum-free medium for 2 days. Some cardiac myocytes were stretched by 20% for 9 minutes. The activity of transfected ERK2 was determined by using MBP as a substrate as described in the legend to Figure 2Up. The data represent the mean percentage of controls (100%) from 3 independent experiments (mean±SE). *P<0.05 versus control.

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 4ADown). On the other hand, activation of the transfected ERK2 by stretch was completely suppressed by cotransfection of DNRaf-1 (Figure 4ADown), 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 4BDown).



View larger version (45K):
[in this window]
[in a new window]
  		Figure 4. DNRaf, but not CSK or DNRas, suppresses activation of HA-ERK2 induced by stretch. Twelve hours after transfection of HA-ERK2 with kinase domain–deleted CSK (CSK-), wild-type CSK (CSK+), DNRas, or DNRaf-1, cardiac myocytes were washed twice with PBS, maintained in serum-free medium for 2 days, and then stretched by 20% for 9 minutes (A) or incubated with 10-6 mol/L insulin for 10 minutes (B). The activity of transfected ERK2 was determined by using MBP as a substrate as described in the legend to Figure 2Up. A representative autoradiogram from 4 independent experiments is shown.

Rho Is Required for Stretch-Induced Expression of c-fos and Skeletal {alpha}-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 5ADown). When Rho-GDI was transfected with the c-fos reporter gene into cardiac myocytes, mechanical stress did not increase reporter gene luciferase activity (Figure 5ADown). Next, we examined the role of Rho proteins in stretch-induced fetal gene expression in cardiac myocytes. Mechanical stress–induced activation of skeletal {alpha}-actin promoter was also markedly suppressed by pretreatment with the C3 exoenzyme or overexpression of Rho-GDI (Figure 5BDown). These results suggest that Rho proteins play a critical role in the mechanical stress–induced reprogramming of gene expression.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 5. Rho proteins are required for stretch-induced expression of c-fos and skeletal {alpha}-actin genes. c-fos (A) or the skeletal {alpha}-actin (B) luciferase reporter gene was transfected into cultured cardiac myocytes by the calcium phosphate method. Functions of Rho proteins were inhibited by either pretreatment with 10 µg/mL C3 exoenzyme for 36 hours or overexpression of Rho-GDI. Cardiac myocytes were stretched for 48 hours and lysed with 100 µL of lysis buffer. Luciferase activity was measured with a Berthold Lumat LB9501 luminometer. The data represent the mean percentage of controls (100%) from 4 independent experiments (mean±SE). *P<0.05 versus control and #P<0.05 versus stretch without any inhibition of Rho function.

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 {approx}1.5-fold compared with controls (Figure 6Down). This increase was significantly ({approx}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.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 6. Rho is required for the stretch-induced increase in protein synthesis. After pretreatment with 10 µg/mL C3 exoenzyme for 36 hours, cardiac myocytes were stretched by 20% for 24 hours, and [3H]phenylalanine (1 µCi/mL) was added 2 hours before harvest. The total radioactivity of incorporated [3H]phenylalanine into proteins was determined by liquid scintillation counting. The data represent the mean percentage of controls (100%) from 3 independent experiments (mean±SE). *P<0.05 versus control and #P<0.05 versus stretch without any inhibition of Rho function.

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 7Down). In addition, overexpression of Rho-GDI and DNRhoA also did not have any effect on Ang II–induced ERK activation (Figure 7Down). Next, we investigated whether Rho proteins affect Ang II–induced ERK activation during stretching. The activation of transfected ERK2 induced by stretch was significantly ({approx}60%) inhibited by the Ang II type 1 receptor–specific antagonist CV-11974 (10-6 mol/L), as reported before (Figure 8Down). When cardiomyocytes were transfected by Rho-GDI and pretreated with CV-11974, activation of transfected ERK2 by stretch was more strongly suppressed (Figure 8Down). 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.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 7. Rho proteins are not involved in Ang II–induced activation of transfected ERK2 in cardiomyocytes. After transfection of HA-ERK2 with either Rho-GDI or DNRhoA or after pretreatment with 10 µg/mL C3 exoenzyme for 36 hours, cardiac myocytes were treated with 10-6 mol/L Ang II. The activity of transfected ERK2 was determined by using MBP as a substrate as described in the legend to Figure 2Up. A representative autoradiogram from 3 independent experiments is shown.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 8. Ang II and Rho proteins are involved in mechanical stretch–induced ERK activation. Cardiac myocytes were transfected with Rho-GDI and/or pretreated with 10-6 mol/L CV-11974 for 30 minutes; cells were then stretched by 20% for 9 minutes. The activity of transfected ERK2 was determined by using MBP as a substrate as described in the legend to Figure 2Up. The data represent the mean percentage of controls (100%) from 4 independent experiments (mean±SE). *P<0.05 versus control and {dagger}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 9Down). 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 9Down). When stretch-conditioned media were prepared from cells pretreated with the C3 exoenzyme, ERK activity was not increased (Figure 9Down). These results collectively suggest that Rho proteins may play a critical role in stretch-induced Ang II secretion in cardiac myocytes.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 9. Rho proteins may play a critical role in Ang II secretion from stretched cardiomyocytes. Bar graph shows the activity of ERKs after addition of stretch-conditioned media. The culture media of cells that were not stretched (Non-Stretch) or stretched by 20% for 8 minutes (Stretch) were transferred to quiescent cardiac myocytes cultured in plastic dishes. Recipient cells were pretreated with CV-11974 (10-6 mol/L) for 30 minutes and stimulated with the stretch-conditioned media (CV11974+Stretch). After cardiomyocytes were incubated with 10 µg/mL C3 exoenzyme for 36 hours, cells were stretched, and the culture media were transferred to quiescent cardiac myocytes (C3 exoenzyme||Stretch). The data represent the mean percentage of controls (100%=nonstretch-conditioned media) from 4 independent experiments (mean±SE). *P<0.05 versus control and #P<0.05 versus stretch without any inhibition of Rho and Ang II functions.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
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 3Up). 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 G{alpha}q and {alpha}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 {alpha}-actin genes (Figure 5AUp and 5BUp). 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 {alpha}-actin gene promoter,52 is similar to the c-fos serum response element, the skeletal {alpha}-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 7Up), 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 8Up). 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 9Up, 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 4BUp), DNRas or CSK had no effect on stretch-induced activation of ERKs (Figure 4AUp). 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 10Down). 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.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 10. Proposed mechanism of mechanical stress–induced hypertrophic responses in cardiac myocytes. Mechanical stress induces hypertrophic responses, including activation of ERKs, reprogramming of gene expression, and an increase in protein synthesis in cardiac myocytes. The Rho family of small G proteins may play important roles in multiple steps of the development of these hypertrophic responses.


*    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
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  29. Simpson P. Norepinephrine-stimulated hypertrophy of cultured rat myocardial cells is an {alpha}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.[Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  38. Karns LR, Kariya K, Simpson PC. M-CAT, CArG, and Sp1 elements are required for {alpha}1-adrenergic induction of the skeletal {alpha}-actin promoter during cardiac myocyte hypertrophy. J Biol Chem. 1995;270:410–417.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  45. Narumiya S. The small GTPase Rho: cellular functions and signal transduction. J Biochem. 1996;120:215–228.[Abstract/Free Full Text]
  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 G{alpha}q and {alpha}1-adrenergic receptor signaling in cardiomyocytes. J Biol Chem. 1996;271:31185–31190.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  59. Davis RJ. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993;268:14553–14556.[Free Full Text]
  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.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
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]

Home page
 Am. J. Physiol. Cell Physiol.Home page
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]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
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]

Home page
 J. Biol. Chem.Home page
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]

Home page