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(Hypertension. 2000;35:313.)
© 2000 American Heart Association, Inc.

Scientific Contributions

Involvement of Rho-Kinase in Angiotensin II–Induced Hypertrophy of Rat Vascular Smooth Muscle Cells

Tadashi Yamakawa; Shun-ichi Tanaka; Kotaro Numaguchi; Yuko Yamakawa; Evangeline D. Motley; Sahoko Ichihara; Tadashi Inagami

From the Department of Biochemistry (T.Y., K.N., Y.Y., S.I., T.I.), Vanderbilt University School of Medicine, Nashville, Tenn; the 3rd Department of Internal Medicine (T.Y., H.S.) and Department of Dermatology (Y.Y.), Yokohama City University School of Medicine, Yokohama, Japan; Neurobiology of Aging Laboratories (S.T.), Mt. Sinai School of Medicine, New York, NY; and the Department of Anatomy and Physiology (E.D.M.), Meharry Medical College, Nashville, Tenn.

Correspondence to Dr Tadashi Yamakawa, 3rd Department of Internal Medicine, Yokohama City University School of Medicine, Fukuura 3-9, Kanazawa-ku, Yokohama, 236-0004, Japan. E-mail Yamakat{at}med.yokohama-cu.ac.jp

	Abstract

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Abstract—Angiotensin II (Ang II) is now believed to play a critical role in the pathogenesis of hypertrophy and/or hyperplasia of vascular smooth muscle cells (VSMCs). Several G_i- and G_q-coupled receptors, including the Ang II type 1 (AT₁) receptor, activate Rho and Rho-associated kinase in Swiss 3T3 cells and cardiac myocytes. However, little is known about the role of Rho-kinase in Ang II–induced vascular hypertrophy in VSMCs. In the present study, we explored the role of Rho and Rho-kinase in Ang II–induced protein synthesis in VSMCs. In unstimulated cells, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to Ang II (100 nmol/L). This effect was completely blocked by the AT₁ receptor blocker candesartan but not by the Ang II type 2 (AT₂) receptor antagonist PD123319. Botulinum C₃ exoenzyme, which inactivated RhoA, attenuated Ang II–induced [³H]leucine incorporation. The specific Rho-kinase inhibitor, Y-27632, dose-dependently abolished Ang II–induced protein synthesis and also suppressed Ang II–induced c-fos mRNA expression. On the other hand, Y-27632 had no effect on Ang II–stimulated phosphorylation of p70 S6 kinase and extracellular signal—regulated kinase 1/2, which are reported to be involved in Ang II–induced protein synthesis, nor had it any effect on the Ang II–induced phosphorylation of PHAS-I, a heat- and acid-stable eIF-4E–binding protein. The phosphorylation of PHAS-I is regulating for translation initiation. These observations suggest that the Rho, Rho-kinase, and c-fos pathways may play a role in Ang II–induced hypertrophic changes of VSMCs through a novel pathway.

Key Words: angiotensin II • hypertrophy • G proteins

	Introduction

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Previous studies have reported that medial thickening, at least in large conduit vessels, is due in part to increased vascular smooth muscle cell (VSMC) mass, which occurs primarily by enlargement or hypertrophy of preexisting VSMCs, with little or no change in the number of VSMCs.^{1 2} There is clear evidence implicating a role for angiotensin II (Ang II) in the mediation of VSMC hypertrophy during chronic hypertension. For instance, angiotensin-converting enzyme inhibitors and Ang II receptor blockers have been shown to be highly effective in inhibiting the development of VSMC medial hypertrophy in a variety of hypertensive animal models.^{3 4} Importantly, the effects of angiotensin-converting enzyme inhibitors or Ang II antagonists do not appear to be due simply to blood pressure lowering, because other antihypertensive drugs were not as efficacious in blocking hypertrophy despite equipotent reductions in blood pressure. Consistent with in vivo studies, several laboratories have shown that Ang II stimulates increased protein synthesis and cellular hypertrophy in cultured VSMCs by stimulating Ang II type 1 (AT₁) receptors. The mechanism of this pathway is not clear and seems to be complex.^{5 6} There has been considerable interest in identifying the mechanism and cellular signaling pathways whereby Ang II stimulates hypertrophy in VSMCs.

The low-molecular-weight G protein Rho is a member of the Rho family of small GTPases that also includes Rac and Cdc42. These GTPases act as molecular switches to regulate cellular functions, the best characterized of which are changes in the actin cytoskeleton. An increasing body of evidence has revealed that some heterotrimeric G-protein–coupled receptors (GPCRs) signal through small G proteins, such as Rho. GPCR agonists, such as lysophosphatidic acid⁷ and carbachol,⁸ have been shown to increase levels of membrane-associated Rho or decrease cytosolic Rho, which is indicative of Rho activation. Very recently, it has been reported that Rho and Rho-kinase mediate thrombin-stimulated VSMC DNA synthesis and migration.⁹ The AT₁ receptor is one of the GPCRs whose activation of the AT₁ receptor is also known to activate Rho. However, the roles of downstream signaling and of Rho and Rho-kinase on Ang II–induced vascular hypertrophy remain to be determined.

To gain insight into the mechanism of vascular hypertrophy induced by Ang II, we examined the roles of Rho and Rho-kinase on Ang II–induced leucine uptake in VSMCs.

	Methods

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Materials
DMEM, FCS, penicillin, and streptomycin were obtained from Life Technologies, Inc. Polyclonal antibodies to Thr202- and Tyr204-phosphorylated extracellular signal—regulated kinase (ERK) 1/2 were purchased from New England Biolabs. Polyclonal antibodies to RhoA, a heat- and acid-stable eukaryotic initiation factor (eIF)-4E–binding protein (PHAS-I), and p70 S6 kinase were obtained from Santa Cruz Biotechnology. Botulinum C₃ exoenzyme was purchased from Calbiochem. Candesartan was a generous gift from Takeda Chemical Industries, Ltd (Osaka, Japan), and PD123319 was purchased from Research Biochemicals, Inc. Y-27632 was kindly provided from Yoshitomi Pharmaceutical Industries, Ltd (Saitama, Japan).

Cell Culture
VSMCs were prepared from the thoracic aorta of 12-week-old Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, Mass) by the explant method as previously described.¹⁰ Subcultured VSMCs from passages 3 to 15, used in the experiments, showed >99% positive immunostaining against smooth muscle -actin antibodies and were negative for mycoplasma infection. For subsequent experiments, cells at 80% confluence in culture wells were used 1 day after serum depletion.

Subcellular Fractionation
Cell-free lysates were prepared by adding 100 µL hypotonic lysis buffer (per 60-mm dish) containing 20 mmol/L Tris (pH 8.0), 3 mmol/L MgCl₂, 0.4 mmol/L AEBSF, 5 µg/mL aprotinin, 2 µg/mL trypsin inhibitor, and 20 µmol/mL leupeptin. After 3 cycles of freeze and thaw, samples were centrifuged at 100 000g at 4°C for 60 minutes. The supernatant was saved as a "soluble" fraction. Pellets were washed twice with the same lysis buffer and resuspended in 100 µL of the lysis buffer supplemented with 1% Triton X-100 and 0.1% SDS. Cell debris was separated by centrifugation at 14 000 rpm for 20 minutes at 4°C, and supernatant was saved as a "particulate" fraction. The protein content of each fraction was determined by the Lowry method.

Preparation of Cell Extracts and Western Blotting
VSMCs were stimulated with agonists for a specified duration. Then cells were lysed with ice-cold lysis buffer, pH 7.4, containing 500 mmol/L HEPES, 5 mmol/L EDTA, 50 mmol/L NaCl, 1% Triton X-100, a mixture of protease inhibitors, and 1 mmol/L sodium orthovanadate. Solubilized proteins were centrifuged at 14 000g for 30 minutes, and supernatants were stored at -80°C. Proteins (25 µg) were separated by SDS-PAGE, followed by Western blot analysis with the use of indicated antibodies and the ECL detection system (Amersham).

[³H]Leucine Incorporation
Subconfluent cells in 12-well plates were incubated for 24 hours in serum-free DMEM in the absence or presence of the stimuli. During the last 6 hours of incubation, [³H]leucine at 2 µCi per well was added. Thereafter, cells were washed twice with ice-cold PBS and incubated in 1 mL of 5% trichloroacetic acid for 30 minutes at 4°C. Cells were washed twice with 5% trichloroacetic acid and solubilized in 1 mol/L NaOH for 30 minutes at 37°C. After neutralization, solubilized proteins were counted by a scintillation counter.

Isolation of Total RNA and Northern Blot Hybridization
Northern blot analysis was performed as described previously.¹⁰ VSMCs were stimulated with agonists for the indicated duration, and total RNA was isolated by a 1-step preparation. Total RNA (20 µg) was size-separated by electrophoresis on 1% agarose/formaldehyde gels and then transferred to Hybond-N membranes (Amersham). Hybridization was carried out at 65°C overnight with the solution containing 1 mol/L NaCl, 10% dextran, 1% SDS, 0.1 mg/mL denatured salmon sperm DNA, and a -³²P–labeled denatured c-fos probe. The membranes were washed and then exposed to x-ray film.

Statistics
Data are given as mean±SE. Statistical analyses were performed by ANOVA. A post hoc test was performed by the method of Bonferroni (Glantz ¹¹ ). Significance was accepted at the P<0.05 level.

	Results

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Translocation of RhoA by Ang II Stimulation in VSMCs
Rho has been shown to partially translocate from the soluble to the particulate fraction on activation.¹² To obtain evidence that RhoA is activated by Ang II in VSMCs, the subcellular localization of RhoA before and after Ang II stimulation was examined. Although a weak signal of RhoA was observed in the particulate fraction before Ang II stimulation, the treatment caused a rapid and statistically significant increase in the RhoA content of the particulate fraction (Figure 1A). A significant decrease in the RhoA content in the soluble fraction was not detectable, most likely because of the greater abundance of RhoA in the soluble fraction. Candesartan, an AT₁ receptor–specific antagonist, abolished the translocation of RhoA (data not shown), indicating that the major part of RhoA was translocated from the soluble to the particulated fraction in response to Ang II through the AT₁ receptor.

View larger version (14K): [in this window] [in a new window]   Figure 1. Involvement of Rho and Rho-kinase on Ang II–induced leucine incorporations in VSMCs. A, Ang II–induced translocation of RhoA from soluble to particulate fractions in VSMCs. VSMCs were stimulated with Ang II for indicated periods and fractionated to soluble and particulate fractions. Equal amounts of proteins were loaded in each lane. Immunoblot analyses were performed by using a specific anti-RhoA antibody. Results shown are representative of 3 independent experiments. B and C, Effect of C3 exoenzyme or Y-27632 on Ang II–induced increase in leucine incorporation. Forty-eight hours after pretreatment of C3 exoenzyme (B) or 24 hours after pretreatment of Y-27632 (C), VSMCs were stimulated with Ang II for 24 hours. [3H]Leucine was added 6 hours before harvest, and the total radioactivity incorporated into proteins was determined by liquid scintillation counting. **P<0.05.

Effect of C₃ Exoenzyme on Ang II–Induced Vascular Hypertrophy
Exoenzyme C₃ has been a useful and well-established tool in studying the function of Rho, because it causes ADP-ribosylation at Asn41 of Rho, which results in specific inactivation of Rho.¹³ To clarify the cellular mechanism by which Ang II induces vascular hypertrophy, we investigated the possible involvement of RhoA cascades because they are known to be activated by Ang II in cardiac myocytes.¹⁴ VSMCs were treated with 4 µg/mL of C₃ exoenzyme for 48 hours. Treatment with C₃ exoenzyme significantly attenuated the leucine incorporation stimulated by Ang II (Figure 1B), suggesting that the small G protein, RhoA, is involved in the vascular hypertrophy induced by Ang II.

Involvement of Rho-Kinase on Ang II–Induced Leucine Uptake
Recently, Uehata et al¹⁵ have developed the specific inhibitor of Rho-kinase, Y-27632. To investigate the targets of Rho in protein synthesis induced by Ang II, we examined the effect of 0.5 to 10 µmol/L Y-27632 on Ang II–induced leucine uptake in VSMCs. Pretreatment of the cells with Y-27632 dose-dependently suppressed the leucine incorporation induced by Ang II (Figure 1C), although Y-27632 itself had no effect on the number of the cells (data not shown).

Effect of the Rho-Kinase Inhibitor on c-fos mRNA Expression
Rapid induction of nuclear proto-oncogenes, such as c-fos, is one of the earliest transcriptional events and has been associated with cellular proliferation, differentiation, and hypertrophy.¹⁶ To gain insight into Ang II–induced proto-oncogene c-fos mRNA expression, we extracted mRNA from VSMCs stimulated by Ang II with or without pretreatment with Y-27632. As shown in Figure 2, Ang II induced a c-fos transcription, which is not expressed in quiescent cells. Ang II–induced c-fos mRNA expression was slightly, but statistically significantly, suppressed by Y-27632.

View larger version (46K): [in this window] [in a new window]   Figure 2. Effect of Y-27632 on Ang II–induced c-fos mRNA expression. VSMCs were stimulated by Ang II (10-7 mmol/L) for 1 hour with or without pretreatment of Y-27632 at indicated concentrations. Total RNA was extracted, and Northern blotting analysis was performed with use of c-fos as a probe. The filters were stained by methylene blue to check relative loading of total RNA. Results are representative of 3 individual experiments.

Effect of Y-27632 on ERK1/2 and the p70 S6 Kinase Activation Pathway
To delineate the role of Rho-kinase on Ang II–induced leucine incorporation, we examined the effect of Y-27632 on ERK1/2 and p70 S6 kinase, which are reported to be involved in vascular hypertrophy. As previously described, Ang II–induced ERK1/2 phosphorylation peaked at 7 minutes,¹⁷ and p70 S6 kinase phosphorylation peaked at 20 minutes (data not shown). Both of them rapidly declined to the basal level. Therefore, after pretreatment with Y-27632, we determined the levels of phosphorylation of ERK1/2 and p70 S6 kinase induced by Ang II only at peak points. Y-27632 had no effect on the ERK1/2 and p70 S6 kinase phosphorylations induced by Ang II (Figure 3).

View larger version (29K): [in this window] [in a new window]   Figure 3. Effect of Y-27632 on Ang II–induced phosphorylation of ERK1/2 and p70 S6 kinase. Before stimulation, VSMCs were treated with 1 to 10 µmol/L of Y-27632 for 2 hours. Cells were stimulated with Ang II for 7 minutes, and ERK1/2 (A) and p70 S6 kinase (B) phosphorylation was analyzed by immunoblotting (IB) with indicated antibodies. Results are representative of 3 individual experiments.

Effect of Y-27632 on Ang II–Induced PHAS-I Phosphorylation
PHAS-I generally appears as 3 migrating bands when separated by SDS-PAGE and analyzed by immunoblotting. The nonphosphorylated form migrates most rapidly. Two others are designated as ß and .^{18 19} Increases in phosphorylation of the intermediate ß form to the most highly phosphorylated form slow migration of PHAS-I proportionally when separated by SDS-PAGE. In unstimulated conditions, the nonphosphorylated form of PHAS-I in VSMCs is barely detectable. Figure 4A shows a rapid increase in the phosphorylation of PHAS-I after the addition of 100 nmol/L Ang II in quiescent rat cultured VSMCs, where maximal phosphorylation occurred 10 to 20 minutes after Ang II addition and then gradually decreased to basal levels. Figure 4B shows that the phosphorylation of PHAS-I after 10 and 20 minutes of exposure to Ang II was not inhibited by pretreatment with 10 µmol/LY-27632

View larger version (37K): [in this window] [in a new window]   Figure 4. Effect of Y-27632 on Ang II–induced phosphorylation of PHAS-I. A, VSMCs were stimulated by Ang II for indicated times, and PHAS-I phosphorylation was determined. B, After treatment with Y-27632, cells were stimulated by Ang II. PHAS-I phosphorylation was determined. Results are representative of 3 individual experiments.

	Discussion

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The present study has demonstrated that Rho and Rho-kinase play roles in Ang II–induced vascular hypertrophy, a novel finding that has not been previously reported to our knowledge. Rho-kinase was not involved in the reported pathways of the Ang II–induced phosphorylation of ERK1/2, p70 S6 kinase activation, and PHAS-I, suggesting that Rho-kinase regulated the vascular hypertrophy by a pathway distinct from the mechanisms already reported.

Because Rho is one of the small GTP-binding proteins, activation of Rho could be reflected in increased binding of GTP. However, neither guanine nucleotide binding assays nor determinations of the guanine nucleotide exchange activity for Rho have been technically feasible to date because the available antibodies are not suitable for immunoprecipitating the native guanine nucleotide bound form of Rho²⁰ in the presence of Mg²⁺. Because Mg²⁺ is essential for maintaining the guanine nucleotide binding of Rho,²¹ it is difficult to immunoprecipitate Rho while preserving its guanine nucleotide binding. It was recently shown that several growth factors, including norepinephrine,²² lysophosphatidic acid, endothelin,⁷ and insulin,²³ cause translocation of a fraction of Rho from the cytoplasmic to the particulate fractions in various cell types, leading to a 1.5- to 2-fold increase in the RhoA content in the particulate fraction. Ang II was also reported to induce RhoA translocation in cardiac myocytes.¹⁴ In the present study, we found that Ang II induced RhoA translocation in VSMCs, suggesting that Ang II activates Rho A in VSMCs.

Pretreatment with exoenzyme C₃ significantly attenuated the leucine incorporation stimulated with Ang II (Figure 1B), suggesting that the small G protein, RhoA, was involved in the vascular hypertrophy induced by Ang II. Although Ang II induced the translocation of RhoA (Figure 1A), the downstream signaling pathway connecting Rho to protein synthesis remains unclear. Recently, several targets of Rho have been identified, including protein kinase N,²⁴ Rho-kinase,²⁵ and citron-kinase.²⁶ However, precise roles of these proteins are not yet known. Thus, it is interesting to determine which target proteins are related to Ang II–induced protein synthesis in VSMCs. By using Y-27632, which specifically suppresses Rho-kinase,¹⁵ we showed that Rho-kinase plays a role in Ang II–induced leucine uptake in VSMCs. Figure 1C suggested that Rho-kinase is involved in Ang II–induced vascular hypertrophy. Rho-kinase may mediate plural pathways from Rho and may function in cooperation with other Rho targets. The identification of physiological substrates for Rho-kinase is expected to shed light on the molecular mechanism of Ang II–induced vascular hypertrophy. To date, several proteins, such as the myosin-binding subunit of myosin phosphatase, myosin light chain,²⁷ and glial fibrillary acidic protein,²⁸ have been identified as physiological substrates for Rho-kinase. Further studies are necessary to identify Rho-kinase substrates that might be associated with vascular hypertrophy.

Several potential mechanisms for the regulation of Ang II–induced leucine uptake were investigated in the present study. Ang II activates multiple second-messenger systems in VSMCs,²⁹ and each signaling molecule seems to mediate distinct hypertrophic responses. ERK1/2 and p70 S6 kinase have been reported to be closely related to protein synthesis in VSMCs. However, the relation between these kinases and Rho-kinase has not been clearly identified. Recently, we reported that Rho-kinase was involved in mechanical stress–induced ERK1/2 activation in VSMCs.³⁰ Because Ang II activates the mitogen-activated protein kinase pathways in VSMCs³¹ and because ERK1/2 is regarded as a critical mediator for cell growth and vascular hypertrophy, we examined whether Rho-kinase might be involved in Ang II–induced ERK1/2 activation. Y-27632 had no effect on Ang II–induced ERK1/2 phosphorylation. In addition, Y-27632 had no effect on Ang II–induced p70 S6 kinase phosphorylation. These observations suggest that Rho and Rho-kinase regulate Ang II–induced protein synthesis mediated by a pathway different from ERK1/2 or p70 S6 kinase.

It was reported that the Ang II–induced expression of the c-fos gene was mediated through activation of ERK1/2.³² However, 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 that the activation of the SRF-linked signaling pathway is not correlated with the activation of ERK1/2.^{33 34} In practice, Ang II–induced c-fos mRNA expression in VSMCs was not suppressed completely by the treatment with the MEK-1, a direct upstream of ERK1/2, inhibitor PD98059.³⁵ Thus, it is important to define the c-fos activation mechanism by Ang II and, in particular, to determine whether RhoA and Rho-kinase play roles in this signaling pathway. In the present study, we showed that Rho-kinase is partially involved in Ang II–induced expressions of the c-fos gene, although Rho-kinase was not involved in Ang II–induced ERK1/2 phosphorylation (Figure 3A). It is possible that Rho-kinase activation by Ang II affects the protein levels of SRF or SRF binding activity to the c-fos SRE. Further studies are needed to examine the effect of Y-27632 on the levels of SRF and binding activity of SRF to the c-fos SRE in VSMCs. On the basis of the present results, we submit that the Ang II–induced expression of the c-fos gene might be partially mediated through activation of Rho, Rho-kinase, and SRF, but not ERK1/2, in VSMCs.

The initiation phase of mRNA translation is generally rate limiting for protein synthesis.^{36 37} Initiation is mediated in part by the eIF-4F complex, which is composed of 3 subunits, eIF-4, eIF-4A, and eIF-4E.³⁷ The initiation factor, eIF-4E, is the least abundant component of the eIF-4F subunits, and it is generally believed that the amount of eIF-4E is limiting for translation initiation. The availability of eIF-4E is regulated by PHAS-I, initially identified in rat adipocytes.³⁸ When phosphorylated in the appropriate sites after exposure of responsive cells to insulin, PHAS-I dissociates from eIF-4E, thus allowing eIF-4E to participate in translation initiation. Ang II induces phosphorylation of eucaryotic protein synthesis initiation factor 4E in VSMCs.³⁹ In the present study, we tried to determine whether Rho-kinase is involved in Ang II–induced PHAS-I phosphorylation. Pretreatment of VSMCs with a high concentration of Y-27632 had no effect on Ang II–induced PHAS-I phosphorylation, suggesting that the role of Rho-kinase in Ang II–induced protein synthesis might not be due to the modulation of PHAS-I phosphorylation in VSMCs.

Details of the mechanisms of Rho-kinase in Ang II–induced vascular hypertrophy remain unclear. The present study provides the first evidence that Rho and Rho-kinase may play an important role in Ang II–induced vascular hypertrophy. We need to investigate the upstream and downstream Rho and Rho-kinase pathways in VSMCs.

	Acknowledgments

 
This study was supported in part by National Institutes of Health grants HL-35323, HL-58205, and DK-20593. Dr Yamakawa is supported by a fellowship from the Uehara Memorial Foundation.

Received September 14, 1999; first decision October 29, 1999; accepted November 15, 1999.

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