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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:1831.)
© 2005 American Heart Association, Inc.

Vascular Biology

Signal-Crosstalk Between Rho/ROCK and c-Jun NH₂-Terminal Kinase Mediates Migration of Vascular Smooth Muscle Cells Stimulated by Angiotensin II

Haruhiko Ohtsu; Mizuo Mifune; Gerald D. Frank; Shuichi Saito; Tadashi Inagami; Shokei Kim-Mitsuyama; Yoh Takuwa; Terukatsu Sasaki; Jeffrey D. Rothstein; Hiroyuki Suzuki; Hidekatsu Nakashima; Elethia A. Woolfolk; Evangeline D. Motley; Satoru Eguchi

From the Cardiovascular Research Center (H.O., M.M., H.S., H.N., S.E.), Temple University School of Medicine, Philadelphia, Penn; the Department of Biochemistry (G.D.F., S.S., T.I.), Vanderbilt University School of Medicine, Nashville, Tenn; the Department of Pharmacology and Molecular Therapeutics (S.K.-M.), Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; the Department of Physiology (Y.T.), Kanazawa University School of Medicine, Ishikawa, Japan; the Department of Biochemistry (T.S.), Sapporo Medical University, Sapporo, Japan; the Department of Neurology and Neuroscience (J.D.R.), Johns Hopkins University, Baltimore, Md; and the Department of Anatomy and Physiology (E.A.W., E.D.M.), Meharry Medical College, Nashville, Tenn.

Correspondence to Satoru Eguchi, MD, PhD, FAHA, Cardiovascular Research Center and Department of Physiology, Temple University School of Medicine, 3420 N Broad St, Philadelphia, PA 19140. E-mail seguchi{at}temple.edu

	Abstract

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Background— Rho and its effector Rho-kinase/ROCK mediate cytoskeletal reorganization as well as smooth muscle contraction. Recent studies indicate that Rho and ROCK are critically involved in vascular remodeling. Here, we tested the hypothesis that Rho/ROCK are critically involved in angiotensin II (Ang II)-induced migration of vascular smooth muscle cells (VSMCs) by mediating a specific signal cross-talk.

Methods and Results— Immunoblotting demonstrated that Ang II stimulated phosphorylation of a ROCK substrate, regulatory myosin phosphatase targeting subunit (MYPT)-1. Phosphorylation of MYPT-1 as well as migration of VSMCs induced by Ang II was inhibited by dominant-negative Rho (dnRho) or ROCK inhibitor, Y27632. Ang II–induced c-Jun NH₂-terminal kinase (JNK) activation, but extracellular signal-regulated kinase (ERK) activation was not mediated through Rho/ROCK. Thus, infection of adenovirus encoding dnJNK inhibited VSMC migration by Ang II. We have further demonstrated that the Rho/ROCK activation by Ang II requires protein kinase C- (PKC) and proline-rich tyrosine kinase 2 (PYK2) activation, but not epidermal growth factor receptor transactivation. Also, VSMCs express PDZ-Rho guanine nucleotide exchange factor (GEF) and Ang II stimulated PYK2 association with tyrosine phosphorylated PDZ-RhoGEF.

Conclusions— PKC/PYK2-dependent Rho/ROCK activation through PDZ-RhoGEF mediates Ang II–induced VSMC migration via JNK activation in VSMCs, providing a novel mechanistic role of the Rho/ROCK cascade that is involved in vascular remodeling.

By using vascular smooth muscle cells (VSMCs) in culture, we have investigated signal cross-talk in mediating VSMC migration induced by angiotensin II. We found that Rho-kinase/ROCK activated by PYK2 and PKC-delta specifically mediate angiotensin II–induced VSMC migration via JNK activation, providing a potential cascade in mediating vascular remodeling.

Key Words: angiotensin II • Rho kinase/ROCK • c-jun NH₂-terminal kinase • vascular smooth muscle cells • migration

	Introduction

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Angiotensin II (Ang II) has been implicated in various cardiovascular diseases such as hypertension, atherosclerosis, and restenosis after angioplasty.¹ Therefore, there has been considerable interest in defining the functional significance of signaling pathways of the Ang II type 1 receptor (AT₁), which is dominantly expressed in vascular smooth muscle cells (VSMCs).^1–3 Through this receptor, Ang II stimulates hypertrophy and hyperplasia of VSMCs.² The AT₁ receptor primarily couples to G_q leading to elevation of intracellular Ca²⁺ and activation of protein kinase C (PKC).² In addition, tyrosine kinase activation by Ang II is linked to downstream mitogen-activated protein kinase (MAPK) activation, thereby mediating the growth promoting response in VSMCs.^2,4,5 In this regard, several key tyrosine kinases have been identified that may contribute to the growth-promoting effects of the AT₁ receptor. These kinases include epidermal growth factor receptor (EGFR) and proline-rich tyrosine kinse 2 (PYK2).^4,5

In addition to its growth responses, VSMC migration by Ang II is strongly implicated in various cardiovascular diseases,⁶ whereas the detailed signaling mechanisms by which the AT₁ receptor mediates migration are insufficiently characterized. At least, a MAPK, ERK appears to be required for Ang II–induced migration of VSMCs.⁷ Recently, Zahn et al showed that 3 major MAPKs, ERK, p38MAPK, and c-Jun NH₂-terminal kinase (JNK), are all required for VSMC migration induced by platelet-derived growth factor (PDGF),⁸ suggesting that these MAPKs coordinately mediate VSMC migration.

A small G protein, Rho, and its effector Rho-kinase/ROCK, are involved in many aspects of cell motility, from smooth muscle contraction to cell migration.⁹ Selective ROCK inhibitors appear not only to reduce blood pressure but also prevent experimental restenosis, atherosclerosis, and vascular hypertrophy.¹⁰ In VSMCs, Rho is activated by Ang II,¹¹ and activation of Rho and ROCK is implicated in migration stimulated by a G protein–coupled receptor (GPCR) agonist, thrombin.¹² These data suggest a strong functional significance of Rho/ROCK activation in mediating a pathophysiological response of Ang II such as VSMC migration.

Based on the above information, we hypothesized that Rho and ROCK are critically involved in Ang II–induced VSMC migration through a specific signal transduction cascade. Here, we demonstrate several lines of evidence indicating that activation of Rho/ROCK through PKC and PYK2 activation is specifically required for Ang II–induced JNK activation and subsequent VSMC migration. These data will provide a novel role of the Rho/ROCK cascade that is involved in vascular remodeling associated with cardiovascular diseases.

	Materials and Methods

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The materials and methods used in this study are described in the online supplement (available at http://atvb.ahajournals.org).

	Results

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Activation of Rho/ROCK Is Required for Ang II–Stimulated Migration
ROCK on activation phosphorylates the substrate, myosin phosphatase target subunit-1 (MYPT-1), at Thr⁶⁹⁶.¹³ As shown in Figure 1A, Ang II induced a rapid and sustained phosphorylation of MYPT-1 at Thr⁶⁹⁶ in VSMCs. Pretreatment of VSMCs with a selective AT₁ receptor antogonist, RNH6270, (10 µmol/L, 30 minutes) completely inhibited the MYPT-1 phosphorylation induced by Ang II (data not shown). Also, the Ang II–induced MYPT-1 phosphorylation was completely blocked by infection of adenovirus encoding dominant negative Rho (dnRho) (Figure 1B) or treatment of a ROCK inhibitor, Y27632 (Figure 1C). These results indicate that activation of the AT₁ receptor by Ang II leads to Rho/ROCK-dependent Thr⁶⁹⁶-phosphorylation of MYPT-1 that could represent a selective marker of the Rho and ROCK activation in VSMCs.

View larger version (17K): [in this window] [in a new window]   Figure 1. MYPT-1 phosphorylation induced by Ang II represents Rho/ROCK activation in VSMCs and activation of Rho/ROCK is required for migration. A, VSMCs were stimulated with Ang II (100 nmol/L) for indicated durations. B, VSMCs infected with adenovirus encoding dnRho (100 moi) or control empty vector (100 moi) for 48 hours were stimulated with Ang II (100 nmolM) for 2 minutes. C, VSMCs pretreated with or without Y27632 (10 µmol/L) for 30 minutes were stimulated with Ang II (100 nmol/L) for 2 minutes. Immunoblotting (IB) was performed with anti-Thr696–phosphorylated MYPT-1 (MYPY-p) antibody, anti-MYPT-1 antibody, or anti-RhoA antibody as indicated. The extent of MYPT-1 phosphorylation was quantified by densitometry. Data shown are the mean±SEM from 3 independent experiments. D, VSMCs infected dnRho (100 moi) for 48 hours or pretreated with Y27632 (10 µmol/L) for 30 minutes were stimulated with Ang II (100 nmol/L) for 8 hours. Migration activities were shown as fold increase relative to basal activity. n=3 in triplicate determination, mean±SE. *P<0.05 compared to the basal control. P<0.05 compared to the stimulated control.

Because Rho/ROCK has been implicated in VSMC migration induced by thrombin,¹² we have examined whether Rho and ROCK are required for VSMC migration induced by Ang II. As shown in Figure 1D, migration of VSMCs induced by Ang II was completely blocked by dnRho or Y27632. These data suggest that activation of Rho and ROCK is required for VSMC migration induced by Ang II.

Signal Cross-Talk Between Rho/ROCK and JNK Mediates Ang II–Induced VSMC Migration
Ang II activates 3 major MAPKs in VSMCs,^3,14 and activation of ERK and JNK is indispensable for VSMC migration stimulated by Ang II.^7,15 Therefore, we have investigated the possible signaling cross-talk between Rho/ROCK activation and the MAPK family activation by Ang II. As shown in Figure 2A, Ang II–induced ERK and p38MAPK phosphorylations were not affected by dnRho. In contrast, Ang II–induced JNK phosphorylation was completely inhibited by dnRho (Figure 2B). Moreover, pretreatment of Y27632 completely blocked Ang II–induced JNK phosphorylation but not ERK phosphorylation (Figure 2C). In rat VSMCs, p54JNK phosphorylation by Ang II was dominantly observed and comigrated with the protein band recognized by anti-JNK2 antibody.¹⁴ Infection of adenovirus encoding dnJNK but not the control empty vector inhibited migration of VSMCs induced by Ang II (Figure 2D). These results suggest a unique signaling crosstalk between Rho/ROCK and JNK in mediating migration of VSMCs induced by Ang II.

View larger version (23K): [in this window] [in a new window]   Figure 2. Selective JNK activation through Rho/ROCK is required for VSMC migration induced by Ang II. A and B, VSMCs infected with adenovirus encoding dnRho (100 moi) or control empty vector (100 moi) for 48 hours were stimulated with Ang II (100 nmol/L) for 10 minutes (A) or 30 minutes (B). C, VSMCs pretreated with or without Y27632 (10 µmol/L) for 30 minutes were stimulated with Ang II (100 nmol/L) for indicated durations. Cell lysates were immunoblotted with antibodies against Thr180/Tyr182-dually phosphorylated p38MAPK (p38MAPK-p), total p38MAPK, Tyr204-phosporylated ERK1/2 (ERK-p), total ERK2, Thr183/Tyr185-dually phosphorylated JNK2 (JNK-p), total JNK2, and RhoA as indicated. The extent phosphorylation of ERK at 10 minutes, p38MAPK at 10 minutes, and JNK at 30 minutes was quantified by densitometry. Data shown are the mean±SEM from 3 independent experiments. D, VSMCs infected with adenovirus encoding dnJNK (100 moi) or control empty vector (100 moi) for 48 hours were stimulated with Ang II (100 nmol/L) for 8 hours. VSMC migration was shown as fold increase relative to basal activity. n=3 in triplicate determination, mean±SE, *P<0.05 compared to basal control. P<0.05 compared to the stimulated control.

PYK2 and PDZ-RhoGEF Exist Upstream of Rho/ROCK Activation by Ang II
Some overlapping, as well as distinct signal upstreams involving a tyrosine kinase have been proposed for activation of MAPKs by Ang II in VSMCs.^3,5 PYK2 has been shown to mediate ERK and JNK activation by Ang II.^16,17 In contrast, we have reported that EGFR transactivation specifically mediates ERK and p38 MAPK activation by Ang II in VSMCs.¹⁴ Based on this previous background, we have examined whether Rho/ROCK activation requires PYK2 or EGFR activation. As shown in Figure 3A, infection of adenovirus encoding dnPYK2 prevented MYPT-1 phosphorylation induced by Ang II, whereas the infection did not affect Ang II–induced EGFR transactivation in VSMCs. In contrast, AG1478, a selective EGFR kinase inhibitor, did not affect MYPT-1 phosphorylation but markedly blocked the EGFR phosphorylation in Ang II–stimulated VSMCs (Figure 3B). Because regulator of G protein signaling (RGS)-domain containing Rho guanine nucleotide exchange factors (RhoGEFs) consisting of p115-RhoGEF, PDZ (Post-synaptic density protein, Disc Large, and Zonula occludens)-RhoGEF and leukemia-associated RhoGEF (LARG) are implicated in GPCR-operated Rho activation,^10,18,19 we have further determined the expression and possible interaction of these RhoGEFs in VSMCs. VSMCs dominantly express PDZ-RhoGEF (Figure 3C). The differences in the molecular weight of PDZ-RhoGEF observed in VSMCs and HEK cells might be attributable to a species difference or distinct posttranslational modification of the protein. Interestingly, Ang II stimulated association between PYK2 and PDZ-RhoGEF (227±47% P<0.05 compared with basal association, mean±SD, n=4) and the PDZ-RhoGEF was tyrosine-phosphorylated on Ang II stimulation (201±24%, P<0.05 compared with basal phosphorylation, mean±SD, n=4) (Figure 3D). Thus, these data support our belief that PYK2 activation is specifically required for Rho/ROCK activation via PDZ-RhoGEF in VSMCs stimulated by Ang II.

View larger version (25K): [in this window] [in a new window]   Figure 3. Ang II activates Rho/ROCK pathway through PYK2 but not EGFR. A, VSMCs infected with adenovirus encoding dnPYK2 (100 moi) or control empty vector (100 moi) for 48 hours were stimulated with Ang II (100 nmol/L) for 2 minutes. B, VSMCs pretreated with AG1478 (500 nmol/L) or with vehicle (0.1% DMSO) for 30 minutes were stimulated with Ang II (100 nmol/L) for 2 minutes. Cells lysates were analyzed by immunoblotting with antibodies as indicated. The extent of MYPT-1 phosphorylation was quantified by densitometry. Data shown are the mean±SEM from 3 independent experiments. *P<0.05 compared to basal control. P<0.05 compared to the stimulated control. C, Equal amount of cell lysates from HEK293 and VSMCs were subjected to immunoblotting with antibodies as indicated. D, VSMCs were stimulated with Ang II (100 nmol/L) for indicated durations. Cell lysates were immunoprecipitated with anti-PYK2 antibody and immunoblotted with indicated antibodies. Upper and lower arrows denote tyrosine phosphorylated PDZ-RhoGEF and PYK2, respectively.

PKC is Required for Rho/ROCK-Dependent JNK Activation and Migration by Ang II
We have previously shown that PYK2 activation specifically requires the PKC isoform in VSMCs by using a PKC inhibitor, rottlerin, and adenovirus encoding dnPKC.²⁰ Therefore, the role of PKC in Ang II–induced Rho/ROCK pathway activation was studied by using the PKC inhibitor, rottlerin (Figure I, available online at http://atvb.ahajournals.org). Rottlerin markedly attenuated MYPT phosphorylation induced by Ang II (Figure IA). Rottlerin blocked Ang II–induced JNK activation (Figure IB), whereas this inhibitor had no effect on ERK and p38MAPK activation by Ang II (Figure IC). Rottlerin inhibited Ang II–induced VSMC migration as well (Figure 1D). These pharmacological findings were further supported by the results obtained with adenovirus encoding dnPKC (Figure II, available online at http://atvb.ahajournals.org). As shown in Figure IIA, dnPKC inhibited MYPT phosphorylation induced by Ang II. Thus, dnPKC attenuated phosphorylation of JNK (Figure IIB) whereas dnPKC had no effect on ERK or p38MAPK phosphorylation by Ang II (Figure IIC). DnPKC also inhibited VSMC migration induced by Ang II (Figure IID). The specificity of the dominant-negative intervention was tested by a JNK activator, anisomycin. Overexpression of dn mutants used in this study did not affect JNK activation by anisomycin (Figure III, available online at http://atvb.ahajournals.org). Taken together, these findings provide a previously missing signaling link by which Ang II activates Rho/ROCK and suggests how Rho/ROCK specifically upregulates VSMC migration through its selective cross-talk with JNK.

	Discussion

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The major findings presented in this study are that Rho/ROCK activation by Ang II specifically mediates JNK activation leading to migration of VSMCs, and that the upstream of Rho/ROCK activation by Ang II requires PYK2 and PKC activation. This is in line with previous findings that Ang II activates RhoA in cardiac myocytes²¹ and VSMCs.^11,22 Thus, activation of Rho/ROCK represents one of the key signal transduction pathways originating from the VSMC AT₁ receptor that mediates pathophysiological functions of Ang II such as contraction,²³ atherogenic gene expression,²⁴ cardiovascular hypertrophy,^11,25 and migration as presented here.

Limited information was available previously regarding the mechanistic insights by which Ang II activates Rho/ROCK. In general, GPCR agonists have been demonstrated to activate Rho through a Rho-specific GEF such as p115RhoGEF that interacts with G₁₂ and G₁₃.²⁶ In addition to G_q, Ang II receptors in VSMCs are able to couple G₁₂ and G₁₃.²³ However, recent accumulating evidence suggest a G_12/13-independent Rho activation as an alternative route by which some GPCRs activate Rho in non-muscle cells.^27,28 This notion is supported by the recent finding of Ca²⁺ and calmodulin-dependent Rho activation in VSMCs.²⁹ In the present study, we found a participation of PKC for Rho/ROCK activation in VSMCs. In addition, attenuation of mechanical stress-induced Rho activation was reported in VSMCs derived from PKC null mice.³⁰ Therefore, a Ca²⁺-sensitive mechanism and Ca²⁺-insensitive PKC may both participate in Rho/ROCK activation in VSMCs in addition to G_12/13.

PYK2 activation by Ang II specifically requires PKC activation in VSMCs.²⁰ Also, PYK2 has been implicated in GPCR-induced Rho activation in non muscle cells.^31,32 In line with these previous publications, our data presented here suggest that PYK2 could mediate Ang II–induced Rho/ROCK activation in VSMCs. How does PYK2 participate in Rho activation? Three RGS domain-containing RhoGEFs, p115 RhoGEF, PDZ-RhoGEF, and LARG, could mediate G_12/13- or G_q/12/13-dependent Rho activation.¹⁹ Interestingly, Rho GEF activity of LARG is enhanced via tyrosine phosphorylation by a PYK2 homologue, FAK.¹⁸ Among these RhoGEFs, we found that PDZ-RhoGEF is dominantly expressed in VSMCs and that association of PYK2 with tyrosine-phosphorylated PDZ-RhoGEF is enhanced on PYK2 activation by Ang II. Taken together, our data suggest that PYK2 activated by Ang II induces PDZ-Rho GEF tyrosine phosphorylation thereby activating the Rho/ROCK cascade in VSMCs.

The molecular mechanism underlying GPCR-induced VSMC migration through Rho/ROCK remains more obscure than what is known in non-muscle cells. JNK has recently emerged as a crucial mediator of cell migration.^33,34 Moreover, mechanical stress-induced VSMC migration was diminished in VSMCs derived from PKC null mice.³⁵ Overexpression of a PYK2 inhibitory protein blocked monocyte motility,³⁶ and macrophage from PYK2 null mice showed impaired migration.³⁷ Therefore, the present findings nicely connect these essential kinases and delineate a previously unidentified signal transduction relay of VSMC migration in which Rho/ROCK represent essential intermediates. Recently, several JNK substrates such as paxillin and c-Jun were identified that mediate cell migration in response to JNK activation.^34,38 However, we could not detect JNK-dependent paxillin phosphorylation in VSMCs by using anti-phosphoserine antibody. Alternatively, JNK may mediate Ang II–induced VSMC migration through c-Jun phosphorylation, because c-Jun as well as JNK is required for VSMC migration in response to PDGF.^39,8 Also, because the ERK cascade activation operated through EGFR transactivation is required for Ang II–induced VSMC migration,⁴⁰ it is likely that parallel ERK cascade activation together with the JNK cascade activation coordinately induce the migratory responses in VSMCs.

It has been established that the primary Rho family small G proteins involved in JNK activation are Rac and Cdc42 but not Rho.²⁶ In this regard, PAK, which can interact with the active form of Rac or Cdc42, has been implicated in Ang II–induced JNK activation.⁴¹ However, in addition to our findings presented here, Rho-dependent JNK activation has been demonstrated in certain cell types.^42,43 To exclude the possibility of nonspecific inhibition of other Rho family small G protein function by dnRho infection, we confirmed that Ang II–induced JNK activation was not affected by dnRac adenovirus (100 moi) in VSMCs (S. Eguchi, unpublished observation, 2005). Marinissen et al have recently shown that active ROCK is able to stimulate JNK and its direct upstream SEK1/MKK4 in HEK293 cells.⁴⁴ Also, activation of MEKK1 by RhoA through direct interaction was reported.⁴⁵ However, preliminarily, we could not detect this interaction in VSMCs on Ang II stimulation. Therefore, the precise mechanism of Rho/ROCK in JNK activation in VSMCs will require further investigation.

Results using the ROCK inhibitor, Y27632, should be interpreted carefully. The concentration used in this study may partially inhibit PKC and PKN activity.⁴⁶ However, all of the inhibitory actions of Y27632 presented here were mimicked by dnRho in the present study, thus suggesting the inhibitory effect of Y27632 could be explained by its action on ROCK but not other kinases. In addition, several kinases other than ROCK could phosphorylate MYPT1 at Thr696.^13,47 However, participation of these kinases on Ang II–induced MYPT phosphorylation is unlikely because they are minimally inhibited by Y27632.⁴⁷ Finally, MYPT phosphorylation and the resultant inhibition of myosin phosphatase lead to enhanced phosphorylation of its substrates, such as myosin II, adducin, and moesin. These phosphorylations are implicated not only in cell contraction but also cell motility.¹³ Although we have used the MYPT phosphorylation as a readout of ROCK activation in VSMCs, it will be interesting to further investigate the role of MYPT in regulating signaling and function of Ang II in VSMCs.

Our findings presented here are limited within multi-passaged cultured VSMCs. Future studies are necessary to confirm the presence of the cascade in an in vivo condition or diseases associated with enhanced Ang II actions. In conclusion, the signal transduction of Rho/ROCK activation and its downstream signaling that mediate VSMC migration have been demonstrated in this study. VSMC migration stimulated by Ang II is considered as a potential mechanism of atherosclerosis and restenosis after vascular injury. Therefore, further clarification of this cascade could contribute to a better understanding of the molecular mechanism of cardiovascular diseases as well as to the development of better strategies for their treatment.

	Acknowledgments

 
This work was supported in part by National Institutes of Health grants, HL076770 (S.E.), HL076575 (G.D.F.), HL058205 (T.I.), and American Heart Association Scientist Development Grant, 0130053N (S.E.). We thank Kunie Eguchi and Trinita Fitzgerald for their excellent technical assistance and members of the Temple University Cardiovascular Research Center for help and advice throughout this study.

Received March 14, 2005; accepted June 16, 2005.

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