1Department of Physiology, Meharry Medical College, Nashville, Tennessee; 2Cardiovascular Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania; 3Department of Biochemistry and Molecular Pathophysiology, School of Medicine, University of Occupational and Environmental Health, Japan, Kitakyushu, Japan; and 4Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada
Submitted 10 September 2004 ; accepted in final form 13 July 2005
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ABSTRACT |
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vascular remodeling; signal transduction
ANG II produces its action by binding to high-affinity G protein-coupled receptors, of which there are two subtypes, ANG II type 1 and type 2 (AT1 and AT2) receptors (3). The AT1 receptor, the primary ANG II receptor expressed in VSMCs, not only regulates most physiological responses to ANG II but also is the principal ANG II receptor involved in pathophysiological signaling in the cardiovascular system (21, 38). The AT1 receptor mediates a number of different signaling pathways through activation of the Gq/11 family of heterotrimeric G proteins (5, 18, 21, 38, 41). Thus activation of the AT1 receptor results in PLC-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating inositol trisphosphate and diacylglycerol. Formation of inositol trisphosphate and diacylglycerol leads to mobilization of Ca2+ and activation of PKC, respectively (18, 21, 38). The AT1 receptor is also coupled to the production of reactive oxygen species (ROS) (18, 38), which likely activate both downstream tyrosine and serine/threonine kinases (5, 10, 11, 18, 21, 38, 41).
Several key serine/threonine kinases have been identified in ANG II-induced signaling pathways. These kinases include the MAPK/ERK family (5, 8, 18, 21, 38, 41), Akt/PKB (7, 35), PKC (18, 21, 38), p70S6 and p90S6 kinases (7, 18, 21, 38), and p21-activated kinase (PAK) (32, 33), all of which have been implicated in ANG II-induced cardiovascular remodeling. PAKs are a family of 60- to 65-kDa serine/threonine kinases that have been demonstrated to regulate cellular proliferation, differentiation, transformation, and survival via several downstream signaling pathways (1). They are activated by GTP-bound Rac and Cdc42, protein kinases such as phosphoinositide-dependent kinase (PDK)-1, tyrosine kinase receptors, cytokine receptors, and G protein-coupled receptors (1). Currently, very few studies have reported activation of PAK by ANG II in VSMCs (32, 33); however, a role for PAK in cell migration processes has been established through overexpression of mutant PAK constructs in both endothelial cells (22) and epithelial cancer cells (24). Thus PAK may also have a significant role in ANG II-induced cardiovascular remodeling.
The purpose of the present study was to examine the signaling events in the ANG II-induced regulation of PAK1 activation. We hypothesized that ANG II mediates PAK1 activity through a signaling pathway that involves the elevation of intracellular Ca2+ and the activation of PKC. Herein we provide evidence that the G protein-coupled AT1 receptor, Ca2+, PKC
, ROS, PYK-2, and Rac are essential upstream components of the signaling pathway utilized by ANG II to phosphorylate and activate PAK1, leading to protein synthesis in VSMCs.
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MATERIALS AND METHODS |
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Cell culture.
VSMCs were prepared from the thoracic aortas of 12-wk-old Sprague-Dawley rats (Charles River Breeding Laboratories) by the explant method and cultured in DMEM containing 10% fetal calf serum, penicillin, and streptomycin as previously described (8). Subcultured cells from passages 315 were used in the experiments; they showed >99% positive immunostaining of smooth muscle -actin antibody and were negative for mycoplasma infection. For each experiment, cells were cultured to 8090% confluence and then made quiescent by incubation in serum-free medium for 23 days. VSMCs were stimulated with ANG II or other agonists at 37°C. The animal protocol (no. M/00/296) was approved by the Vanderbilt University Institutional Animal Care and Use Committee.
Adenoviral infection.
The generation of dominant-negative (dn)PKC, dnPYK-2, dnRac, and dnPAK1 adenovirus is described in detail elsewhere (2, 20, 28, 40). VSMCs were infected with adenovirus as previously described (7). Briefly, VSMCs were infected with adenovirus at a multiplicity of infection (MOI) of 100, unless otherwise stated, by incubating cells with adenovirus-containing medium for 1 h followed by incubation for 2 more days under serum-free conditions (7).
Immunoblotting. Cell lysates were separated by SDS-PAGE, and proteins were electrophoretically transferred to a nitrocellulose membrane as previously described (9). The membranes were exposed to primary antibodies overnight at 4°C and then incubated with a peroxidase-linked secondary antibody for 1 h at room temperature. An ECL Western blotting detection kit (Amersham Pharmacia Biotech) was used to visualize immunoreactive proteins.
Protein assay. Subconfluent VSMCs on 12-well culture plates were incubated with serum-free DMEM for 1 day and were infected with adenovirus encoding empty vector or dnPAK1 at MOI of 100 for 2 days. Adenovirus-infected cells were further incubated with or without 100 nM ANG II for 3 days. After aspiration of the medium, cells were washed twice with ice-cold Hanks' balanced salt solution, and the total amount of cellular protein was measured with a bicinchoninic acid protein assay kit (Pierce) before and after ANG II stimulation.
Proliferation assay. Cell proliferation was measured with a CellTiter 96 AQueous cell proliferation assay kit (Promega), following the manufacturer's protocol. In brief, after incubation with serum-free DMEM for 3 days, quiescent VSMCs in 96-well plates were stimulated with ANG II (100 nM) for 72 h. After incubation with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-nyl)-2H-tetrazolium solution provided with the kit, viable cells were determined at 490-nm absorbance with a 96-well plate reader.
Statistical analysis. Unless otherwise stated, the data presented in this study are representative of a minimum of three independent experiments yielding similar results. The data were analyzed using one-way ANOVA with posttest to indicate statistically significant differences.
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RESULTS |
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Involvement of PKC in ANG II-induced PAK activation.
The AT1 receptor is also coupled to the activation of PKC (18). As shown in Fig. 4A, PMA, a potent PKC activator, can evoke a sustained elevation of PAK phosphorylation from 5 to 20 min. To ascertain whether PKC mediates AT1 receptor activation of PAK1 and to reveal which isoform(s) of PKC might be involved in ANG II-mediated PAK phosphorylation, we pretreated the cells with rottlerin, a selective PKC
inhibitor, and Go-6976, a selective PKC
and PKC
inhibitor (Fig. 4B). Whereas rottlerin significantly reduced levels of phosphorylated PAK1 and only partially inhibited activation of ERK, Go-6976 had little effect on phosphorylated levels of either PAK or ERK.
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Cross talk between Ca2+ and PKC mediates PAK1 activation by ANG II.
To study the possible link between Ca2+ and PKC
in mediating PAK activation by ANG II, we examined the effect of the Ca2+ ionophore on PKC
phosphorylation at Tyr311. This phosphorylation site has been implicated in PKC
activation in cells treated with H2O2 (23). In VSMCs, both ANG II and A-23187 induced phosphorylation of PKC
at Tyr311 (Fig. 5, A and B). Figure 5C demonstrates that phosphorylation of PAK1 by the Ca2+ ionophore A-23187 is inhibited by pretreatment with rottlerin, a PKC
inhibitor. However, rottlerin had no effect on A-23187-induced ERK phosphorylation. Moreover, pretreatment with TMB-8 markedly inhibited PKC
phosphorylation induced by ANG II in VSMCs (Fig. 5D). Together these data suggest that Ca2+ activates PKC
through phosphorylation of Tyr311, leading to PAK1 activation in response to ANG II.
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DISCUSSION |
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Both intracellular Ca2+ elevation and activation of PKC occur as a result of AT1 receptor activation through Gq (18, 21, 38). In our study, phosphorylation of PAK1 by ANG II was markedly reduced by both BAPTA-AM and TMB-8, which suggests that Ca2+ is required for ANG II-induced activation of PAK and that release of Ca2+ from intracellular storage sites is most critical for phosphorylation of PAK by ANG II. The difference in the degree of inhibition caused by BAPTA and TMB-8 may be due to the fact that these substances used distinct mechanisms to antagonize Ca2+. In support of our findings, Lian et al. (26) implicated a major role for the Ca2+/calmodulin complex in the activation of PAK in neutrophils. Moreover, in a separate study performed in VSMCs by Schmitz et al. (32), Ca2+ was again shown to be involved in activation of PAK1 by ANG II.
In the present study, the time course for PAK phosphorylation by A-23187 appears to have been delayed compared with ANG II, perhaps because of the distinct nature of intracellular Ca2+ elevation or because ANG II has additional signaling such as PKC activation. The PMA initial time course mimics that of ANG II but is sustained. This may reflect the stronger and more sustained nature of PKC activation by PMA than ANG II. The involvement of PKC has been shown in the activation of PAK in HepG2 cells (19). Schmitz et al. (32) also previously reported PKC-dependent activation of PAK by ANG II in VSMCs. However, the PKC isoform(s) involved in this PAK activation has not been sufficiently characterized. PKC, a member of the novel class of PKC isoforms, is abundantly expressed in VSMCs (15). We found that pretreatment with rottlerin, but not Go-6976, significantly reduced activation of PAK1 by ANG II. Furthermore, overexpression of dnPKC
also diminished the level of phosphorylated PAK1 induced by ANG II. Together, our findings indicate that the PKC
isoform is essential for PAK activation by ANG II.
Although the novel PKCs have no structural site for Ca2+-dependent activation (37), we demonstrate that increasing Ca2+ levels in VSMCs by exposing them to A-23187, as well as to ANG II stimulation, can induce phosphorylation of PKC at Tyr311, which has been shown to be an important phosphorylation and activation site for PKC
in response to other agonists such as H2O2 (23). Our data further support the notion that intracellular Ca2+ is a critical upstream signal for Tyr311 phosphorylation of PKC
and subsequent activation of PAK1 through phosphorylation of Ser199/204. Thus we provide a novel PAK activation pathway involving Ca2+-dependent activation of PKC
through Tyr311 phosphorylation by ANG II in VSMCs.
An earlier study by Schmitz et al. (32) proposed the presence of a Src kinase inhibitor (PP1)-insensitive tyrosine kinase upstream of PAK activation by ANG II in VSMCs. The activation of this kinase may cause an adaptor Nck to activate PAK (33). PYK-2 could be such a kinase because ANG II-induced PYK-2 activation lies downstream of intracellular Ca2+ elevation and PKC activation in VSMCs (6, 30). Together with our previous findings (12, 14) demonstrating the requirement of PKC and ROS for ANG II-induced PYK-2 activation in VSMCs, we have investigated the roles of PYK-2 and ROS in ANG II-induced PAK phosphorylation. We found that overexpression of dnPYK-2 or antioxidants inhibits PAK1 phosphorylation by ANG II, thus strongly indicating the involvement of PYK-2 and ROS in ANG II-induced PAK1 activation. Recently, Weber et al. (39) demonstrated that phosphorylation of PAK1 at Thr423 but not Ser199/204 by PDGF is mediated by ROS and PDK1 in cultured VSMCs. We observed that Thr423 was minimally phosphorylated in response to ANG II in VSMCs, whereas ANG II-induced Ser199/204 phosphorylation was markedly inhibited by kinase-inactive dnPAK transfection, supporting our notion that Ser199/204 phosphorylation is a useful marker with which to study the regulation of PAK activation by ANG II in VSMCs. The discrepancy between ANG II and PDGF in regard to Thr423 phosphorylation and ROS requirement for Ser199/204 phosphorylation may be due to distinct downstream PAK activation mechanisms that are utilized by the AT1 and PDGF receptors in VSMCs. Studies also have demonstrated that tyrosine phosphorylation of PYK-2 by ANG II augments its association with Src (6, 30). Rybin et al. (29) found that H2O2-stimulated phosphorylation of PKC
at Tyr311 was mediated by a Src family tyrosine kinase in cultured cardiac myocytes. Thus the role of Src and its possible involvement in PAK1 activation in response to ANG II requires further investigation.
PAK1 is a well-known effector of Rac (36), and Rac has been implicated in ANG II-induced PAK1 activation in VSMCs (33). Because Rac is associated with the activation of NADPH oxidase, which is a major source of ROS in ANG II-stimulated VSMCs (34), it would follow that Rac would also play a role in ANG II-induced activation of PAK1 via ROS production. In the present study, we verified the essential role of Rac in the ANG II-induced activation of PAK1 and PKC with dnRac. Because dnRac inhibited PAK phosphorylation by ANG II without affecting PKC
phosphorylation, we submit a model in which Rac had no connection to ROS-sensitive components of PAK1's upstreams involving PKC
and PYK-2. Our findings may be partially in line with previous findings and proposals in VSMCs (34) that there are two distinct ROS production mechanisms by ANG II in which initial ROS production via NADPH oxidase is Rac independent but is essential for activation of tyrosine kinases by ANG II. Thus we propose an upstream mechanism of PAK1 activation involving Rac by ANG II, as illustrated in Fig. 8, which may need further detailed clarification in the future.
Previous studies revealed a functional role of PAK1 in mediating PDGF-induced migration in VSMCs (39) as well as in tracheal smooth muscle cells (2). In tracheal smooth muscle cells, it was further shown that PAK stimulates migration by signaling to p38 MAPK. Herein we have shown that PAK is involved in protein synthesis induced by ANG II as well. Although we have not investigated downstream of PAK1 in mediating protein synthesis, JNK could be the candidate. This is because JNK has been demonstrated as a downstream effector of PAK in VSMCs (32).
In conclusion, we have demonstrated that the signaling molecules Ca2+, PKC, PYK-2, ROS, and Rac play critical roles in ANG II-induced activation of PAK1 and that PAK1 is involved in protein synthesis in VSMCs. These data may provide novel insight into the cellular mechanisms by which ANG II mediates vascular hypertrophy. Further elucidation of this signaling pathway will reveal new therapeutic targets that, when inhibited, attenuate actions of the AT1 receptor, thereby reducing cardiovascular remodeling associated with development and progression of cardiovascular diseases.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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