We have recently reported that angiotensin II (Ang II)-induced mitogen-activated protein kinase (MAPK) activation is mainly mediated by Ca 2؉ -dependent activation of a protein tyrosine kinase through G q -coupled Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMC). In the present study, we found Ang II rapidly induced the tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and its association with Shc and Grb2. These reactions were inhibited by the EGF receptor kinase inhibitor, AG1478. The Ang II-induced phosphorylation of the EGF receptor was mimicked by a Ca 2؉ ionophore and completely inhibited by an intracellular Ca 2؉ chelator. Thus, AG1478 abolished the MAPK activation induced by Ang II, a Ca 2؉ ionophore as well as EGF but not by a phorbol ester or platelet-derived growth factor-BB in the VSMC. Moreover, Ang II induced association of EGF receptor with catalytically active c-Src. This reaction was not affected by AG1478. These data indicate that Ang II induces Ca 2؉ -dependent transactivation of the EGF receptor which serves as a scaffold for pre-activated c-Src and for downstream adaptors, leading to MAPK activation in VSMC.

In cultured rat vascular smooth muscle cells, angiotensin II (Ang II) induced a rapid increase in mitogen-activated protein kinase (MAPK) activity through the Ang II type 1 receptor, which was insensitive to pertussis toxin but was abolished by the phospholipase C inhibitor, U73122. The Ang II-induced MAPK activation was not affected by the protein kinase C inhibitor, GF109203X, and was only partially impaired by pretreatment with a phorbol ester, whereas both treatments completely prevented MAPK activation by the phorbol ester. Intracellular Ca2+ chelation by TMB-8, but not extracellular Ca2+ chelation or inhibition of Ca2+ influx, abolished Ang II-induced MAPK activation. The calmodulin inhibitor, calmidazolium, and the tyrosine kinase inhibitor, genistein, completely blocked MAPK activation by Ang II as well as by the Ca2+ ionophore A23187. Ang II caused a rapid increase in the binding of GTP to p21(ras), and this was inhibited by genistein, TMB-8, and calmidazolium but not by pertussis toxin or GF109203X. These data suggest that Ang II-induced MAPK activation through the Ang II type 1 receptor could be mediated by p21(ras)activation through a currently unidentified tyrosine kinase that lies downstream of Gq-coupled Ca2+/calmodulin signals.

The growth-promoting effect of mechanical stress on vascular smooth muscle cells (VSMCs) has been implicated in the progress of vascular disease in hypertension. Extracellular signal-regulated kinases (ERKs) have been implicated in cellular responses, such as vascular remodeling, induced by mechanical stretch. However, it remains to be determined how mechanical stretch activates ERKs. The cytoskeleton seems the most likely candidate for force transmission into the interior of the cell. Therefore, we examined (1) whether the cytoskeleton involves mechanical stretch-induced signaling, (2) whether Rho is activated by stretch, and (3) whether Rho mediates the stretch-induced signaling in rat cultured VSMCs. Mechanical stretch activated ERKs, with a peak response observed at 20 minutes, followed by a significant increase in DNA synthesis. Treatment with the ERK kinase-1 inhibitor, PD98059, inhibited the stretch-induced increase in DNA synthesis. Cytochalasin D, which selectively disrupts the network of actin filaments, markedly inhibited stretch-induced ERK activation. In the control state, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to mechanical stretch. Botulinum C3 exoenzyme, which inactivates Rho p21 (known to participate in the reorganization of the actin cytoskeleton), attenuated stretch-induced ERK activation. Inhibition of Rho kinase (p160ROCK) also suppressed stretch-induced ERK activation dose dependently. Our results suggest that mechanotransduction in VSMCs is dependent on intact actin filaments, that Rho is activated by stretch, and that Rho/p160ROCK mediates stretch-induced ERK activation and vascular hyperplasia.

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 (AT 1 ) 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 AT 1 receptor blocker candesartan but not by the Ang II type 2 (AT 2 ) receptor antagonist PD123319. Botulinum C 3 exoenzyme, which inactivated RhoA, attenuated Ang II-induced [ 3 H]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 Ⅲ hypertrophy Ⅲ G proteins P revious 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 angiotensinconverting 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 (AT 1 ) 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 w...

Abstract-PYK2, a recently identified Ca 2ϩ -sensitive tyrosine kinase, has been implicated in extracellular signal-regulated kinase (ERK) activation via several G protein-coupled receptors. We have reported that angiotensin II (Ang II) induces Ca 2ϩ -dependent transactivation of the epidermal growth factor receptor (EGFR) which serves as a scaffold for preactivated c-Src and downstream adaptors (Shc/Grb2), leading to ERK activation in cultured rat vascular smooth muscle cells (VSMC). Herein we demonstrate the involvement of PYK2 in this cascade. Ang II rapidly induced tyrosine phosphorylation of PYK2, whose effect was completely inhibited by an AT 1 receptor antagonist and an intracellular Ca 2ϩ chelator. A Ca 2ϩ ionophore also induced PYK2 tyrosine phosphorylation to a level comparable with that by Ang II, whereas phorbol ester-induced phosphorylation was less than that by Ang II. Moreover, PYK2 formed a complex coprecipitable with catalytically active c-Src after Ang II stimulation. Although a selective EGFR kinase inhibitor completely abolished Ang II-induced recruitment of Grb2 to EGFR and markedly attenuated Ang II-induced ERK activation, it had no effect on Ang II-induced PYK2 tyrosine phosphorylation or its association with c-Src and Grb2. These data suggest that the AT 1 receptor uses Ca 2ϩ -dependent PYK2 to activate c-Src, thereby leading to EGFR transactivation, which preponderantly recruits Grb2 in rat VSMC.

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 NH 2 -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. A ngiotensin 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 (AT 1 ), 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 AT 1 receptor primarily couples to G q leading to elevation of intracellular Ca 2ϩ and activation of protein kinase C (PKC). 2 In addition, tyrosine kinase activation by Ang II is linked to downstream mitogenactivated 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 AT 1 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 AT 1 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 NH 2 -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...

In cultured vascular smooth muscle cells (VSMC), the vasculotrophic factor, angiotensin II (AngII) activates three major MAPKs via the G q -coupled AT 1 receptor. Extracellular signal-regulated kinase (ERK) activation by AngII requires Ca 2؉ -dependent "transactivation" of the EGF receptor that may involve a metalloprotease to stimulate processing of an EGF receptor ligand from its precursor. Whether EGF receptor transactivation also contributes to activation of other members of MAPKs such as p38MAPK and c-Jun N-terminal kinase (JNK) by AngII remains unclear. In the present study, we have examined the effects of a synthetic metalloprotease inhibitor BB2116, and the EGF receptor kinase inhibitor AG1478 on AngII-induced activation of MAPKs in cultured VSMC. BB2116 markedly inhibited ERK activation induced by AngII or the Ca 2؉ ionophore A23187 without affecting the activation by EGF or PDGF. BB2116 as well as HB-EGF neutralizing antibody inhibited the EGF receptor transactivation by AngII, suggesting a critical role of HB-EGF in the metalloprotease-dependent EGF receptor transactivation. In addition to the ERK activation, activation of p38MAPK and JNK by AngII was inhibited by an AT 1 receptor antagonist, RNH6270. A23187 and EGF markedly activate p38MAPK, whereas A23187 but not EGF markedly activates JNK, indicating the possible contribution of the EGF receptor transactivation to the p38MAPK activation. The findings that both BB2116 and AG1478 specifically inhibited activation of p38MAPK but not JNK by AngII support this hypothesis. From these data, we conclude that ERK and p38MAPK activation by AngII requires the metalloproteasedependent EGF receptor transactivation, whereas the JNK activation is regulated without involvement of EGF receptor transactivation.

Abstract-Thrombin has been shown to activate endothelial NO synthase (eNOS) leading to endothelium-dependent vasorelaxation. In addition to its activation by Ca 2ϩ /calmodulin, eNOS has several regulatory sites. Ser 1179 phosphorylation of eNOS by the phosphatidylinositol 3-kinase-dependent Akt stimulates its catalytic activity. In this study, we have elucidated the signaling mechanism of thrombin-induced phosphorylation of eNOS in the regulation of NO production. Immunoblot analysis showed that thrombin rapidly phosphorylates eNOS at Ser 1179 in cultured bovine aortic endothelial cells. Also, thrombin was unable to stimulate eNOS if the Ser 1179 was mutated to Ala. Akt is phosphorylated in response to thrombin at Ser 473 at a later time point than eNOS. In this regard, a phosphatidylinositol 3-kinase inhibitor, LY294002, blocked Akt phosphorylation without affecting eNOS phosphorylation and cGMP production by thrombin. The Ca 2ϩ ionophore A23187 stimulated eNOS phosphorylation, as well as cGMP production, and pretreatment with intracellular or extracellular Ca 2ϩ chelators inhibited thrombin-induced eNOS phosphorylation and cGMP production. Moreover, infection of bovine aortic endothelial cell with adenovirus encoding dominant-negative mutants of protein kinase C (PKC)␣ and PKC␦ or pretreatment of bovine aortic endothelial cells with PKC inhibitors revealed that PKC␦ is indispensable for thrombin-induced eNOS phosphorylation and activation. From these data, we concluded that thrombin induces the Ser 1179 phosphorylation-dependent eNOS activation through a Ca 2ϩ -dependent, PKC␦-sensitive, but phosphatidylinositol 3-kinase/Akt-independent pathway. (Hypertension. 2007;49:577-583.)