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J. Biol. Chem., Vol. 275, Issue 29, 22435-22441, July 21, 2000

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Peroxisome Proliferator-activated Receptor  Ligands Inhibit Retinoblastoma Phosphorylation and G₁  S Transition in Vascular Smooth Muscle Cells^*

Shu Wakino^§, Ulrich Kintscher^¶, Sarah Kim, Fen Yin, Willa A. Hsueh, and Ronald E. Law^**

From the  Division of Endocrinology, Diabetes, and Hypertension, Department of Medicine, and the  Molecular Biology Institute, UCLA, Los Angeles, California 90095 and the ^¶ Department of Medicine/Cardiology, Virchowklinikum, Humboldt University, Berlin, and German Heart Institute, Berlin D-13353, Germany

Received for publication, December 30, 1999, and in revised form, April 18, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Peroxisome proliferator-activated receptor  (PPAR) is a member of the nuclear receptor superfamily that is activated by binding certain fatty acids, eicosanoids, and insulin-sensitizing thiazolidinediones (TZD). The TZD troglitazone (TRO) inhibits vascular smooth muscle cell proliferation and migration both in vitro and in vivo. The precise mechanism of its antiproliferative activity, however, has not been elucidated. We report here that PPAR ligands inhibit rat aortic vascular smooth muscle cell proliferation by blocking the events critical for G₁  S progression. Flow cytometry demonstrated that both TRO and another TZD, rosiglitazone, prevented G₁  S progression induced by platelet-derived growth factor and insulin. Movement of cells from G₁  S was also inhibited by the non-TZD, natural PPAR ligand 15-deoxy-^12,14 prostaglandin J₂ (15d-PGJ₂), and the mitogen-activated protein kinase pathway inhibitor PD98059. Inhibition of G₁  S exit by these compounds was accompanied by a substantial blockade of retinoblastoma protein phosphorylation. TRO and rosiglitazone attenuated both the mitogen-induced degradation of p27^kip1 and the mitogenic induction of p21^cip1. 15d-PGJ₂ and PD98059 inhibited both the degradation of p27^kip1 and the induction of cyclin D1 in response to mitogens. These effects resulted in the inhibition of mitogenic stimulation of cyclin-dependent kinases activated by cyclins D1 and E. These data demonstrate that PPAR ligands are antiproliferative drugs that act by modulating cyclin-dependent kinase inhibitors; they may provide a new therapeutic approach for proliferative vascular diseases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Proliferation of vascular smooth muscle cells (VSMC)¹ plays a key role in the development of restenosis and in the progression of atherosclerosis (1, 2). Injury to the endothelium results in the migration of underlying VSMC into the intimal layer of the arterial wall, where they proliferate and synthesize extracellular matrix components. Although many growth factors induce the proliferation of VSMC (3), platelet-derived growth factor (PDGF) is an important mitogenic and chemotactic regulator of VSMC, since blocking antibodies to PDGF inhibits neointimal formation in rat models of arterial injury (4). Insulin is also a potent mitogen for VSMC, and physiologic hyperinsulinemia enhances the mitogenic effects of PDGF (5). In response to vascular injury, quiescent VSMC (G₀) must transit through the G₁ phase of the cell cycle and enter into the S phase to undergo DNA replication.

Progression through the cell cycle requires the formation and activation of cyclin and cyclin-dependent kinase (CDK) complexes (6). Progression through the G₁ phase requires cyclin D/CDK4, cyclin D/CDK6, and cyclin E/CDK2 holoenzymes. Functional cyclin A/CDK2 complexes are required for DNA synthesis (S phase), and subsequently, cyclin A/CDC2 and cyclin B/CDC2 pairs are assembled and activated during G₂ phase and mitosis (M phase), respectively. Activation of the G₁ phase cyclin·CDK complexes results in the phosphorylation of retinoblastoma gene products (Rb) (7). Rb proteins are critical negative regulators of cell cycle progression by controlling gene expression mediated by E2F transcription factors (7). E2F-regulated genes encode proteins required for S phase DNA synthesis (8). In the absence of its being phosphorylated by CDKs, Rb binds and sequesters E2F, thereby preventing transcriptional activation of target genes. CDK-phosphorylated Rb releases E2F that permits the induction of E2F-dependent genes. CDK inhibitors (CDKIs), p21^cip1 (9, 10), p27^kip1 (11-13), and p15/p16^ink4 (14), regulate this process by inhibiting cyclin/CDK activity and phosphorylation of Rb, resulting in G₁ arrest (15). Progression through the mammalian cell cycle is regulated by the balance between the levels and activities of cyclin·CDK complexes, the growth-promoting transcriptional factors they regulate, CDKIs and other growth suppressor proteins.

Thiazolidinediones (TZDs) are high-affinity ligands for peroxisome proliferator-activated receptor  (PPAR). PPAR is expressed in VSMC (16, 17), and ligands for this nuclear receptor inhibit VSMC proliferation induced by several different mitogens in vitro (18, 19) and intimal hyperplasia in vivo (18). These observations suggest that activation of PPAR interferes with the function of a fundamental component of the cell cycle machinery. The specific mechanism by which PPAR inhibits VSMC proliferation, however, remains to be determined. We have previously shown that PPAR ligands inhibit ERK MAPK-dependent mitogenic signaling pathways in VSMC at a step downstream of ERK activation (18). Induction of cyclin D and G₁  S progression of nonvascular cells has also been shown to require activation of ERK and MAPK. The purpose of this study was to examine the effect of PPAR ligands on cell cycle regulators in rat aortic smooth muscle cells and to delineate the mechanism of their antiproliferative activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Treatment with Growth Factor and Reagents-- Rat aortic smooth muscle cells (RASMC) were prepared from thoracic aorta of 2-3-month-old Harlan Sprague-Dawley rats by using the explant technique. The cells were cultured in Dulbecco's modified Eagle's medium containing 10% FBS (Irvine Scientific, Santa Ana, CA), 100 units/ml penicillin, 100 µg/ml streptomycin, and 200 mM L-glutamine. Human coronary artery smooth muscle cells (CASMC, purchased from Clonetics, San Diego, CA) were cultured in smooth muscle cell growth medium-2 containing 5% FBS, 2 ng/ml human basic fibroblast growth factor, 0.5 ng/ml human epidermal growth factor, 50 µg/ml gentamicin, 50 ng/ml amphotericin-B, and 5 µg/ml bovine insulin (all purchased from Clonetics). For all experiments, early passaged (5-8) RASMC or CASMC were grown to 60-70% confluency and made quiescent by serum starvation (0.4% FBS) for at least 24 h. Each reagent examined was added 30 min before the addition of human recombinant PDGF-BB (Sigma) and insulin (Lilly) at the final concentration of 20 ng/ml and 1 µM, respectively. For all data shown, each individual experiment was performed using an independent preparation of RASMC or CASMC. Troglitazone (TRO) was kindly provided by Parke-Davis; rosiglitazone (RSG, formerly BRL 49653) was a generous gift from Smith Kline Beecham Laboratories. 15-Deoxy-^12,14 prostaglandin J₂ (15d-PGJ₂) was obtained from Biomol (Plymouth Meeting, PA). The MEK inhibitor PD98059 was purchased from New England Biolabs (Beverly, MA).

Cell Cycle Distribution-- Flow cytometry was performed to analyze cell cycle distribution. Quiescent RASMC were pretreated for 30 min with each compound or vehicle (Me₂SO), followed by the addition of growth factors (PDGF-BB 20 ng/ml + insulin 1 µM). After 24 h cells were trypsinized, centrifuged at 1500 rpm for 3 min, washed with PBS, and then treated with 20 µg/ml RNase A (Calbiochem). DNA was stained with 100 µg/ml propidium iodide for 30 min at 4 °C and protected from light, and 1 × 10⁶ cells were then analyzed with a FACScan (Becton Dickinson). DNA histogram analysis was performed using the ModFitLT software (Becton Dickinson). Experiments were repeated at least 3 times.

Western Blots-- Cells were harvested 24 h after the addition of growth factors by scraping in cold 1× PBS. Harvested cells were lysed and sonicated in solubilization buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA, 1% Triton X-100; 2.5 mM sodium pyrophosphate; 1 mM sodium vanadate; 10 µg/ml each aprotinin and leupeptin; 2 mM phenylmethylsulfonyl fluoride). Cell lysates were cleared by centrifugation, and protein concentrations were determined by the Lowry assay (Bio-Rad). Cell lysates containing equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis. Protein was transferred electrophoretically to a nitrocellulose membrane (Hybond, Amersham Pharmacia Biotech). After blocking in 20 mM Tris-HCl, pH 7.6, containing 150 mM NaCl, 0.1% Tween 20, and 2% (w/v) non-fat dry milk, blots were incubated with specific antibodies against total Rb (14001A PharMingen), phospho-Rb Ser-807/Ser-811 (9308S, New England Biolabs), cyclin D1 (sc-481, Santa Cruz Biotechnology), cyclin E (sc-753, Santa Cruz Biotechnology), CDK2 (sc-6248, Santa Cruz Biotechnology), CDK4 (sc-749, Santa Cruz Biotechnology), CDK6 (sc-7181, Santa Cruz Biotechnology), CDKI p27^kip1 (sc-1641, Santa Cruz Biotechnology), and p21^cip1 (sc-6246, Santa Cruz Biotechnology) at a 1:200 concentration. Immunoreactive bands were visualized by incubation with peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG antibody (1:1000 dilution) (Amersham Pharmacia Biotech). The antigen-antibody complexes were detected using ECL (Amersham Pharmacia Biotech). Quantification of the Western blots was done by densitometry.

Immunocomplex Kinase Assay-- Cyclin D1·CDK complex activity and cyclin E-CDK activity were measured as described previously (20). Briefly, after appropriate treatments, cells were washed with cold PBS and solubilized on ice in lysis buffer (50 mM Tris, pH 8.0; 250 mM NaCl; 0.5% Nonidet P-40; 1 µg/ml leupeptin; and 1 mM phenylmethylsulfonyl fluoride). Insoluble materials were cleared through centrifugation at 4 °C for 10 min at 12,000 rpm. Protein concentrations were determined, and protein was suspended in 1 ml of lysis buffer and immunoprecipitated by incubating with agarose-conjugated anti-cyclin D1 (sc-450AC, Santa Cruz Biotechnology) or cyclin E rabbit IgG (sc-481AC, Santa Cruz Biotechnology) overnight. Immunoprecipitants were washed three times with kinase buffer (150 mM NaCl; 1 mM EDTA; 50 mM Tris-HCl, pH 7.5; and 0.1% Tween 20). CDK activities present in the immunoprecipitants were determined by resuspension in kinase buffer (50 mM Tris-HCl, pH 7.5; 10 mM MgCl₂; 10 mM dithiothreitol; 1 mM ATP; and 1 mM EGTA). Resuspended complexes were incubated for 15 min at 37 °C with 0.5 µg of soluble Rb (sc-4112, Santa Cruz Biotechnology) or histone-H1 (Upstate Biotechnology, Inc.) and with 3 µCi of [-³²P]ATP. Samples were analyzed by SDS-polyacrylamide gel electrophoresis, and the dried gel was exposed on film with an intensifying screen at 80 °C overnight and quantitated by densitometry.

Statistics-- Analysis of variance with paired or unpaired t tests was performed for statistical analysis, as appropriate. Values of p < 0.05 were considered to be statistically significant. Data are expressed as mean ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PPAR Ligands and PD98059 Block the Progression of VSMC into S Phase-- TZD PPAR ligands TRO and RSG, a non-TZD PPAR ligand 15d-PGJ₂, and a MEK inhibitor PD98059 all inhibited cell cycle progression, as determined by flow cytometry. Subconfluent RASMC accumulated in G₁ after serum starvation for 24 h (70.74% in G₀/G₁ phase and 16.14% in S phase; Fig. 1A). Quiescent RASMC were induced to enter S phase by stimulation with the competence factor PDGF (20 ng/ml) and a progression factor insulin (1 µM). The population of G₀/G₁ cells decreased substantially (43.91%; Fig. 1b) with a concomitant increase in RASMC in S phase (48.45%; Fig. 1b). TRO and RSG inhibited G₁  S progression as reflected by the higher percentage of G₀/G₁ cells (65.17% in Fig. 1c and 62.14% in Fig. 1d, respectively) and by the lower percentage of S phase cells (19.59% in Fig. 1c and 21.68% in Fig. 1d, respectively). Movement of cells from G₁  S was also inhibited by 15d-PGJ₂ and the MAPK pathway inhibitor PD98059, with an increase in the population of G₀/G₁ cells (66.29% in Fig. 1e and 66.34% in Fig. 1f, respectively) and with a concomitant decrease in S phase cells (19.25% in Fig. 1e and 20.66% in Fig. 1f, respectively). All PPAR ligands tested, as well as PD98059, prevented mitogen-induced G₁  S progression in a dose-dependent manner (Fig. 1B). Inhibition of ~80% was observed at 10 µM for TRO and RSG, 5 µM for 15d-PGJ₂, and 30 µM for PD98059.

View larger version (59K): [in this window] [in a new window]   Fig. 1.   PPAR ligands and PD98059 prevent mitogen-induced G1  S progression in RASMC. A, quiescent RASMC (0.4% FBS for 24 h) were stimulated by treatment with PDGF (20 ng/ml) and insulin (1 µM). Cells were preincubated with troglitazone (10 µM), rosiglitazone (10 µM), 15d-PGJ2 (5 µM), and PD98059 (30 µM) for 30 min prior to addition of mitogens. 24 h after stimulation, DNA was stained with propidium iodide (P+I), and 1 × 106 cells were analyzed by flow cytometry. A shows representative DNA histograms for quiescent RASMC (0.4% FBS for 24 h) (a), RASMC stimulated with PDGF and insulin (P + I) (b), RASMC stimulated in the presence of TRO (c), RSG (d), 15d-PGJ2 (e), and PD98059 (f), respectively. The x and y axes represent the intensity of propidium iodide fluorescence and cell number, respectively. The data are representative of three separate experiments. B, PPAR ligands and PD98059 inhibit mitogen-induced G1  S progression in a dose-dependent manner. Results are the mean of three independent experiments. Mean ± S.E. is expressed as percentage of S phase transition.

PPAR Ligands Inhibit Mitogen-induced Rb Phosphorylation-- To elucidate the mechanism by which PPAR ligands inhibit G₁  S progression, we examined their effect on Rb phosphorylation. Rb migrates in an SDS-polyacrylamide gel as multiple, closely spaced bands reflecting varying degrees of phosphorylation. After 24 h mitogenic stimulation with PDGF + insulin, a mobility shift of Rb was observed indicative of increased phosphorylation in RASMC. All PPAR ligands tested, as well as PD98059, inhibited the mobility shift (Fig. 2A).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   PPAR ligands and PD98059 inhibit phosphorylation of Rb. Quiescent RASMC (A) and human CASMC (B) were stimulated by treatment with PDGF (20 ng/ml) and insulin (1 µM). Cells were preincubated with TRO (10 µM), RSG (10 µM), 15d-PGJ2 (5 µM), PD98059 (30 µM), and Me2SO (control) for 30 min prior to addition of mitogens. A, after 24 h whole cell proteins (75 µg) were assayed by Western immunoblotting using anti-Rb antibody. B, whole cell proteins (30 µg) were assayed using anti-Rb (a) or anti-phospho-Rb Ser 807/811 (b) antibodies. Each autoradiogram is representative of three separate experiments.

In some experiments, Rb from RASMC treated with PPAR ligands appeared to migrate through gels with a slightly faster mobility than that observed for Rb in G₀/G₁-arrested cells. Enhanced mobility of Rb after PPAR activation could result from dephosphorylation of hypophosphorylated Rb present in G₀/G₁ cells. To explore this finding further, we performed similar experiments using human CASMC. An advantage of using CASMC is the availability of antibodies that recognize site-specific phosphorylations on Rb. Several of these antibodies were tried, unsuccessfully, on RASMC. In CASMC, a phospho-specific antibody was used to assess the phosphorylation status of Ser-807/Ser-811 in Rb, which mediates CDK-dependent regulation of Rb function (21, 22). PPAR ligands, as well as PD98059 at highest concentration tested, inhibited the mitogen-induced phosphorylation at Ser-807/Ser-811 (Fig. 2B, b). Importantly, even at 20 µM, TRO and RSG did not reduce Ser-807/Ser-811 phosphorylation to levels lower than that detected in G₀/G₁ cells (data not shown). No evidence of PPAR ligand-induced dephosphorylation of hypophosphorylated Rb (i.e. band migrating faster than those detected in G₀/G₁ cells) was observed when an antibody recognizing total human Rb was used to analyze CASMC (Fig. 2B, a). In combination, these experiments strongly suggest that activation of PPAR, or inhibition of ERK-MAPK activity, blocks only mitogen-induced Rb phosphorylation and has no effect on its basal phosphorylation in G₀/G₁.

Effects of PPAR Ligands on Expression of Early G₁ Cyclins and CDKs in VSMC-- To understand the mechanism by which PPAR ligands inhibit Rb phosphorylation, we examined their effect on the expression of CDKs and their cyclin partners for which Rb is a major physiological substrate. CDK2 levels were low in quiescent cells, increased after 24 h mitogenic stimulation, and did not change with any of these compounds (Fig. 3A). Quiescent RASMC expressed both CDK4 and CDK6 which did not change after either mitogenic stimulation or treatment with any of these compounds (Fig. 3A). We next examined the effect of these compounds on protein expression of G₁ phase cyclins D1 and E. Both cyclins D1 and E were expressed at low levels in quiescent RASMC and increased after 24 h stimulation with PDGF + insulin. Treatment with TRO and RSG had no effect on the induction of cyclin D1 by mitogens, whereas addition of 15d-PGJ₂ and PD98059 to mitogen-stimulated VSMC attenuated induction of cyclin D1 levels by 89 ± 3.7 and 68 ± 7.7% at the maximum concentration tested, respectively (p < 0.05 versus PDGF/insulin alone, Fig. 3, A and B). Mitogenic induction of cyclin E was not affected by any agent (Fig. 3A).

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Effects of PPAR ligands and PD98059 on expression of CDKs and G1-cyclins D1 and E. A, quiescent RASMC were stimulated by treatment with PDGF (20 ng/ml) and insulin (1 µM). Cells were preincubated with TRO (10 µM), RSG (10 µM), 15d-PGJ2 (5 µM), PD98059 (30 µM), and Me2SO (control) for 30 min prior to addition of mitogens. After 24 h whole cell proteins (30 µg) were assayed by immunoblotting using antibodies against CDK2, CDK4, CDK6, cyclin D1, and cyclin E. B, dose-dependent effect of 15d-PGJ2 and PD98059 on cyclin D1 expression. Each autoradiogram is representative of three separate experiments.

Effects of PPAR Ligands on CDKI Expression in VSMC-- The CDKI p27^kip1 inhibits the activities of cyclin E·CDK2 and cyclin D1·CDK4 complexes (11, 12). Down-regulation of p27^kip1 during G₁ in response to mitogens is important for maximal activation of G₁ cyclin/CDK holoenzymes (23). We therefore investigated the effect of PPAR ligands and PD98059 on p27^kip1 expression after mitogenic stimulation. Western analysis of quiescent RASMC revealed substantial p27^kip1 protein. Expression of p27^kip1 decreased markedly after 24 h stimulation with PDGF + insulin (PDGF + insulin alone, 32.5 ± 4.7% of quiescent cells, p < 0.01 versus quiescent cells). All PPAR ligands and PD98059 significantly attenuated mitogen-induced down-regulation of p27^kip1 (PDGF + insulin + 10 µM TRO, 63 ± 8.5% of quiescent cells; 10 µM RSG, 64 ± 8.2% of quiescent cells; 5 µM 15d-PGJ₂, 75 ± 6.0% of quiescent cells; 30 µM PD98059, 57 ± 6.1% of quiescent cells; all p < 0.01 versus PDGF + insulin alone) (Fig. 4A). All tested compounds attenuated p27^kip1 down-regulation in a dose-dependent manner (Fig. 4B). In contrast, levels of CDKIs p15^ink4b and p16^ink4a did not change after either mitogenic stimulation or treatment with any of these compounds (data not shown). Inhibition of G₁  S progression of RASMC by these agents likely results, at least in part, through their effects to block CDKI p27^kip1 degradation.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effects of PPAR ligands and PD98059 on expression of CDKIs p27kip1 and p21cip1. A, PPAR ligands and PD98059 prevent mitogen-induced down-regulation of CDKI p27kip1. Quiescent RASMC were stimulated by treatment with PDGF (20 ng/ml) and insulin (1 µM). Cells were preincubated with TRO (10 µM), RSG (10 µM), 15d-PGJ2 (5 µM), PD98059 (30 µM), and Me2SO (control) for 30 min prior to addition of mitogens. After 24 h whole cell proteins (30 µg) were assayed by immunoblotting using anti-p27kip1 antibody. B, dose-dependent effect of each compound on expression of p27kip1. C, TRO and RSG inhibit mitogenic induction of p21cip1. Quiescent RASMC were stimulated by treatment with PDGF (20 ng/ml) and insulin (1 µM). Cells were preincubated with TRO (10 µM), RSG (10 µM), 15d-PGJ2 (5 µM), PD98059 (30 µM), and Me2SO (control) for 30 min prior to addition of mitogens. After 24 h whole cell proteins (30 µg) were assayed by immunoblotting using anti-p21cip1 antibody. D, dose-dependent effect of TRO and RSG on expression of p21cip1. Each autoradiogram is representative of three separate experiments.

Previous studies have shown that mitogens can up-regulate p21^cip1 (23-26). Increased p21^cip1 levels may be important during G₁ phase because it has been recently reported that p21^cip1 is required for facilitating functional kinase complex formation of CDK4 with cyclin D1 (27). Since cyclin D1/CDK4 phosphorylates Rb in early G₁, we investigated the effect of PPAR ligands and PD98059 on p21^cip1 expression after mitogenic stimulation. Western analysis of quiescent RASMC revealed low levels of p21^cip1 protein. Expression of p21^cip1 increased markedly after 24 h stimulation with PDGF + insulin. At 10 µM, TRO and RSG inhibited mitogen-stimulated induction of p21^cip1 in RASMC by 63 ± 6.3 and 58 ± 8.4%, respectively (both p < 0.05 versus PDGF + insulin alone, Fig. 4C). This inhibition was dose-dependent for both ligands (Fig. 4D). In contrast, 15d-PGJ₂ and PD98059 had no effect (Fig. 4C).

Effects of PPAR Ligands and PD98059 on Cyclin D1-associated CDK and Cyclin E-associated CDK Activities-- To determine whether various PPAR ligands and PD98059 can regulate CDK activity, we measured the effects of these compounds on mitogen-stimulated cyclin D1-associated and cyclin E-associated CDK activities. By using glutathione S-transferase-Rb fusion protein and purified histone H1 proteins as substrates for cyclin D1-associated CDK and cyclin E-associated CDK, respectively, we found that stimulation with PDGF (20 ng/ml) + insulin (1 µM) increased activity of both CDKs (Fig. 5, A and B). All these PPAR ligands and PD98059 inhibited the induction of cyclin D1-dependent kinase activity (PDGF + insulin + 10 µM TRO, 58 ± 3.7% inhibition, p < 0.01 versus PDGF + insulin alone; 10 µM RSG, 53 ± 4.5% inhibition, p < 0.05 versus PDGF + insulin alone; 5 µM 15d-PGJ₂, 79 ± 4.6% inhibition, p < 0.01 versus PDGF + insulin alone; 30 µM PD98059, 77 ± 7.4% inhibition, p < 0.01 versus PDGF + insulin alone) (Fig. 5A). Similarly, all the compounds inhibited the induction of cyclin E-dependent kinase activity (10 µM TRO, 73 ± 7.3% inhibition; 10 µM RSG, 67 ± 7.8% inhibition; 5 µM 15d-PGJ₂, 82 ± 8.0% inhibition; 30 µM PD98059, 87 ± 9.4% inhibition; all p < 0.01 versus PDGF + insulin alone) (Fig. 5B).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   PPAR ligands and PD98059 inhibit cyclin D1- and cyclin E-dependent kinase activities. Quiescent VSMC were stimulated by treatment with PDGF (20 ng/ml) and insulin (1 µM). Cells were preincubated with TRO (10 µM), RSG (10 µM), 15d-PGJ2 (5 µM), PD98059 (30 µM), and Me2SO (control) for 30 min prior to addition of mitogens. After 24 h cyclin D1- and cyclin E-associated kinase were immunoprecipitated with anti-cyclin D1 (A) or anti-cyclin E (B) antibody from 750 (A) and 100 µg (B) of total cellular lysate. The kinases dependent on cyclin D1 (A) and anti-cyclin E (B) assays were performed as described under "Experimental Procedures" four times with similar results.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The principal finding of this study is that PPAR ligands inhibit VSMC proliferation by attenuating the activity of several key cell cycle regulators that control G₁  S progression. All PPAR ligands prevented mitogen-induced phosphorylation of Rb by inhibiting cyclin D1- and cyclin E-dependent kinase activity. Attenuation of mitogen-induced p27^kip1 degradation by PPAR ligands is likely the major mechanism ultimately resulting in the inhibition of Rb phosphorylation. Indeed, depletion of p27^kip1 by antisense oligodeoxynucleotides promotes cell growth (28), and mice with targeted disruption of the p27^kip1 gene have enhanced growth with enlargement of the pituitary, adrenals, and gonads (29, 30). A recent study demonstrated that overexpression of p27^kip1 inhibited serum-stimulated DNA synthesis in VSMC (28). Furthermore, in a porcine balloon-injury model, p27^kip1 expression was markedly reduced in the intima and media early after angioplasty, consistent with an injury-induced proliferative response (31, 32). Thus, p27^kip1 down-regulation is a necessary event for cell cycle progression. The prostanoid PPAR ligand 15d-PGJ₂ and the ERK/MAPK pathway inhibitor PD98059 exhibited a broader profile of activity toward G₁ cell cycle regulatory proteins, since they also blocked mitogenic induction of cyclin D1.

Stimulation of the Ras-ERK/MAPK pathway triggers proliferation for a variety of different cell types (33). Activation of Ras  Raf  MEK  ERK/MAPK pathway is critical for both mitogen-dependent cyclin D1 induction and p27^kip1 degradation (34, 35). In NIH-3T3 fibroblasts, constitutive expression of an activated form of MEK1, the upstream dual-specificity kinase that phosphorylates and activates ERKs, down-regulated p27^kip1 (35). In CCL39 fibroblasts and IEC-6 intestinal epithelial cells, the blockade of the MAPK cascade with the MEK inhibitor PD98059 prevented both S phase entry and p27^kip1 down-regulation (36). These observations are in concordance with our finding that blocking ERK/MAPK signaling with PD98059 attenuated mitogen-induced down-regulation of p27^kip1. Degradation of several cell cycle regulatory proteins including cyclins, p53, Rb, and p27^kip1 results from ubiquitin-mediated proteolysis in proteosomes (37-39). The mechanism by which the MAPK pathway and proteosomes interact, however, is unclear. Nevertheless, in combination, these studies support an important role for ERK/MAPKs in regulating p27^kip1 levels after mitogenic stimulation.

Similar to other cell types, the ERK/MAPK pathway is required for the growth in VSMC (40). We previously demonstrated in VSMC that the PPAR ligand TRO inhibited mitogen-induced MAPK signaling downstream of ERK phosphorylation and activation by MEK, which is associated with an inhibition of growth (18). In the present study, we find that PPAR ligands and the MAPK pathway inhibitor PD98059 inhibit mitogenic degradation of p27^kip1, thereby preventing Rb hyperphosphorylation and the exit of quiescent VSMC from G₁  S. Consistent with previous studies in nonvascular cells (34, 35), we found that blockade of MAPK signaling with PD98059 potently inhibited cyclin D1 up-regulation by mitogens. In contrast, the TZD PPAR ligands TRO and RSG had no effect on cyclin D1 expression. Thus, PPAR ligands do not globally suppress MAPK-dependent processes, but they may inhibit some transcription factors regulated by the MAPK pathway. We have reported that TRO inhibited activation of the transcription factor Elk-1, which is regulated by MAPK (18). Inhibition of MAPK-dependent Elk-1 activation was associated with an inhibition of c-Fos expression and cell proliferation (18). These data also suggest that inhibition of cyclin D1 expression by 15d-PGJ₂ may not be mediated by its activation of PPAR but rather through its binding to prostaglandin receptors.

Ras can also activate the Rho pathway, which is also important for cell proliferation. Rho signaling has been implicated in the regulation of p27^kip1 degradation (41, 42). In IIC9 hamster embryo fibroblasts, expression of dominant-negative Rho prevented PDGF-induced degradation of p27^kip1 (42). In marked contrast to results obtained in mouse fibroblasts linking ERK/MAPK activation to p27^kip1 degradation, overexpression of dominant-negative ERK in IIC9 cells had no effect on p27^kip1 levels. This study concluded that Ras activation of Rho, but not ERK, regulates p27^kip1 degradation. Rho-dependent degradation of p27^kip1 in PDGF-stimulated fibroblasts requires its phosphorylation by cyclin E-CDK2 (41). We observed that all PPAR ligands inhibited cyclin E-dependent kinase activity, which may be the mechanism by which they attenuate p27^kip1 degradation. This effect could result from an inhibition of mitogen-induced ERK signaling by PPAR as we previously identified. Alternatively, PPAR may regulate p27^kip1 turnover through a novel action to block Rho signaling. However, we found that PPAR activation did not decrease mitogen-enhanced Rho protein levels.² In addition, it is unlikely that a nuclear receptor like PPAR would affect Rho movement from the cytosol to the plasma membrane, which is important for activation of the Rho pathway (43). Additional experiments are required to establish whether the Rho pathway is targeted by PPAR.

Quiescent VSMC expressed high levels of p27^kip1, but p21^cip1 was not detectable. Mitogenic stimulation with the combination of PDGF and insulin increased expression of p21^cip1 during G₁  S transition. Up-regulation of p21^cip1 during G₁ at first glance is paradoxical, given that it and other CDKIs function to regulate negatively cyclin-CDK activity. Several recent reports, however, have revealed that CDKI modulation of the cell cycle is complex and involves both positive and negative regulation by CDKIs (23-26, 44). Threshold levels of p21^cip1 have been shown to be required for the formation of functional cyclin D1·CDK4 complexes (44). Higher concentrations of p21^cip1, however, inhibited cyclin D1-CDK4 activity consistent with its more traditionally recognized CDKI function (44). The potential for p21^cip1 to regulate CDKs positively is supported by a recent genetic study showing that primary mouse embryonic fibroblasts from p27^kip1/p21^cip1 double knockout animals failed to assemble detectable amounts of cyclin D1·CDK complexes (27). At physiological concentration (10 µM), the PPAR ligands TRO and RSG inhibited mitogen-induced p21^cip1, but 15d-PGJ₂ and the MAPK pathway inhibitor had no effect. Since we saw similar effects of TRO and RSG to inhibit p21^cip1 induction, it is possible that this also plays a role in the inhibition of G₁  S transition by PPAR. PPAR blockade of p21^cip1 induction by mitogens may result from the ability of these nuclear receptors to inhibit the function of transcription factors, such as NFB, STAT, and AP-1, via the mechanism of transrepression (45, 46).

Recently, Morrison and Farmer (47) showed that activation of ectopically expressed PPAR in 3T3-L1 fibroblasts inhibited their growth and promoted their differentiation into adipocytes, which was associated with a concomitant increase in mRNA and protein for CDKIs, p18^ink4c and p21^cip1. There was no effect of PPAR activation on p27^kip1 in these cells. Inhibition of cell cycle progression frequently is a prerequisite to terminal differentiation. Regulatory mechanisms causing cell cycle arrest during differentiation, however, appear to differ from those governing the exit of quiescent cells from G₁  S. For example, during adipocyte differentiation PPAR-mediated growth arrest did not require a functional pRB (48). In contrast, our data strongly implicate PPAR-dependent inhibition of Rb phosphorylation as the mechanism by which PPAR ligands prevent quiescent VSMC from exiting G₁. PPAR ligands also inhibit growth of tumor cells (49-51) and growth of endothelial cells to prevent angiogenesis (52). The impact of PPAR ligands on the cell cycle in these cell types remains to be determined.

Recent studies have illustrated the feasibility of targeting specific cell cycle regulators in cardiovascular cells as an alternative antiproliferative therapy (53). A wide range of anti-proliferative drugs has been tested as means to prevent restenosis and vein graft neointimal formation. One alternative to drug therapy is the use of modified viruses designed to carry a cell cycle regulatory gene directly into the arterial wall. Infection of porcine femoral or rat carotid arteries with an adenoviral vector designed to express a nonphosphorylatable, constitutively active form of Rb inhibited neointima formation in animal balloon-injury models (54). Our recent studies have shown that TRO inhibited intimal hyperplasia after balloon injury of aortae in the rat (18). The present study suggests that the prevention of the reduction of p27^kip1 levels by TRO in vivo may contribute, at least in part, to its activity to inhibit the vascular injury response. The observation that PPAR ligands inhibit important cell cycle processes activated by growth factors produced in response to vascular damage may provide a new oral therapeutic approach for proliferative vascular disease such as restenosis and atherosclerosis.

	FOOTNOTES

^* 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. The 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.

^** 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: rlaw@med1.medsch.ucla.edu.

Published, JBC Papers in Press, May 8, 2000, DOI 10.1074/jbc.M910452199

² S. Wakino, U. Kintscher, S. Kim, F. Yin, W. A. Hsueh, and R. E. Law, unpublished results.

	ABBREVIATIONS

The abbreviations used are: VSMC, vascular smooth muscle cells; PDGF, platelet-derived growth factor; CDK, cyclin-dependent kinase; CDKI, CDK inhibitors; Rb, retinoblastoma tumor suppressor protein; TZD, thiazolidinediones; PPAR, peroxisome proliferator-activated receptor ; RASMC, rat aortic smooth muscle cells; CASMC, human coronary artery smooth muscle cells; TRO, troglitazone; RSG, rosiglitazone; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; FBS, fetal bovine serum; PBS, phosphate-buffered saline; 15d-PGJ₂, 15-deoxy-^12,14 prostaglandin J₂; MEK, MAP kinase/extracellular signal-regulated kinase..

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