Peroxisome Proliferator-activated Receptor Ligands Inhibit Mitogenic Induction of p21 by Modulating the Protein Kinase C Pathway in Vascular Smooth Muscle Cells*

The cyclin-dependent kinase inhibitor p21 is upregulated in response to mitogenic stimulation in various cells. PPAR ligands troglitazone (TRO, 10 M) and rosiglitazone (RSG, 10 M) attenuated the induction of p21 protein by platelet-derived growth factor (PDGF) and insulin without affecting cognate mRNA levels in rat aortic smooth muscle cells (RASMC). The protein kinase C (PKC ) inhibitor rottlerin also blocked the induction of p21 protein, whereas the conventional PKC isotype inhibitor Gö 6976 had no effect. Kinetic studies using the protein synthesis inhibitor cycloheximide showed that TRO, RSG, and rottlerin shortened the half-life of p21 protein. TRO, RSG, and rottlerin inhibited PDGF-induced expression of p21, but they did not affect insulininduced expression of p21. Both ligands inhibited PKC enzymatic activity in PDGF-stimulated RASMC but not in insulin-stimulated cells. Adenovirus-mediated overexpression of PKC rescued the down-regulation of p21 expression both by TRO and RSG in PDGF-treated RASMC. These data suggested that the PKC pathway plays a critical role in PDGF-induced expression of p21 in RASMC and may be the potential target for PPAR ligand effects. Src kinase-dependent tyrosine phosphorylation of PKC was decreased substantially by TRO and RSG. Tyrosine phosphorylation and activation of c-Src in response to PDGF were unaffected by either PPAR ligand. Protein-tyrosine-phosphatase inhibitors sodium orthovanadate and dephostatin prevented PPAR ligand effects on PKC tyrosine phosphorylation and enzymatic activity. Both inhibitors also reversed PPAR ligand effects on p21 expression in PDGFtreated RASMC. PPAR ligands enhanced proteintyrosine-phosphatase activity in RASMC, which may be the mechanism for decreased PKC tyrosine phosphorylation and activity. PPAR ligands regulate p21 at a post-translational level by blocking PKC signaling and accelerating p21 turnover. Vascular smooth muscle cells (VSMC) in the normal vessel wall proliferate at a very low frequency. Injury to the endothelium results in the release of mitogens that stimulate quiescent, G0/G1-arrested VSMC to reenter the cell cycle, replicate DNA, and divide. VSMC hyperplasia is a key event in the formation of restenotic and atherosclerotic lesions in the vasculature (1, 2). Cyclin-dependent kinases (CDKs) are serinethreonine protein kinases that regulate cell cycle progression after forming complexes with and being activated by cyclins (3). Mitogenic activation of cyclin D CDK4, cyclin D CDK6, and cyclin E CDK2 during the G1 phase results in phosphorylation of retinoblastoma gene (Rb) proteins. In its hyperphosphorylated state, Rb functions as a gatekeeper for the G1 3 S transition by binding and sequestering E2F, a transcription factor that induces the expression of a battery of genes that encode the enzymatic machinery for S phase DNA synthesis. Cyclindependent kinase inhibitors (CDKIs) can negatively regulate the mitogen-induced cascade of G1 events by inhibiting CDK activity and preventing Rb phosphorylation (3, 4). Consistent with the view that CDKIs are major negative regulators of the cell cycle, overexpression of Cip/Kip family members (p21, p27) blocks the G1 exit in VSMC and other cell types (5, 6). Data emerging from recent studies, however, suggest that CDKIs p21 and p27 can also function as positive regulators during the G1 phase as assembly factors to promote formation of cyclin CDK holoenzyme complexes (3, 7). Such a role for CDKIs may explicate apparent paradoxical observations that p21 is frequently up-regulated after mitogenic stimulation of quiescent cells (8, 9). In rat VSMC cell lines, prevention of mitogen-induced p21 expression by antisense oligodeoxynucleotides attenuated both DNA synthesis and cell proliferation (10). We recently reported that ligands for the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR ) inhibited Rb phosphorylation and G1 3 S transition in rat aortic VSMC (11). Interestingly, the antiproliferative effects of PPAR ligands were accompanied by an inhibition of the induction of p21 by plateletderived growth factor (PDGF) plus insulin. This inhibition of the mitogenic induction of p21 by PPAR ligands may be a * This work was supported in part by National Institutes of Health Grant HL58328-03 (to W. A. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Supported by a fellowship from the Mary K. Iacocca Foundation. ¶ Supported by a research fellowship from Gonda (Goldschmied) Diabetes Center, UCLA. ‡‡ To whom correspondence should be addressed: Division of Endocrinology, Diabetes, and Hypertension, UCLA School of Medicine, Warren Hall, Second Floor, Suite 24-130, 900 Veteran Ave., Box 957073, Los Angeles, CA 90095. Tel.: 310-794-7555; Fax: 310-794-7654; E-mail: [email protected]. The abbreviations used are: VSMC, vascular smooth muscle cells; CDK, cyclin-dependent kinase; CDKI, cyclin-dependent kinase inhibitor; Rb, retinoblastoma; PPAR , peroxisome proliferator-activated receptor gamma; PDGF, platelet-derived growth factor; PKC, protein kinase C; PTPase, protein-tyrosine-phosphatase; RASMC, rat aortic smooth muscle cells; FBS, fetal bovine serum; TRO, troglitazone; RSG, rosiglitazone; PP2 and PP3, protein phosphatases 2 and 3, respectively; pfu, plaque-forming units; PTEN, phosphatase and tensin homolog deleted from chromosome 10. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 50, Issue of December 14, pp. 47650–47657, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.