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J Biol Chem, Vol. 274, Issue 31, 21926-21931, July 30, 1999

3-Hydroxy-3-methylglutaryl-CoA Reductase Inhibitors Attenuate Vascular Smooth Muscle Proliferation by Preventing Rho GTPase-induced Down-regulation of p27^Kip1*

Ulrich Laufs^§, Diego Marra^§, Koichi Node, and James K. Liao^¶

From the Cardiovascular Division, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachustts 02115

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The mechanism by which platelet-derived growth factor (PDGF) regulates vascular smooth muscle cell (SMC) DNA synthesis is unknown, but may involve isoprenoid intermediates of the cholesterol biosynthetic pathway. Inhibition of isoprenoid synthesis with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, simvastatin (Sim, 1-10 µM), inhibited PDGF-induced SMC DNA synthesis by >95%, retinoblastoma gene product hyperphosphorylation by 90%, and cyclin-dependent kinases (cdk)-2, -4, and -6 activity by 80 ± 5, 50 ± 3, and 48 ± 3%, respectively. This correlated with a 20-fold increase in p27^Kip1 without changes in p16, p21^Waf1, or p53 levels compared with PDGF alone. Since Ras and Rho require isoprenoid modification for membrane localization and are implicated in cell cycle regulation, we investigated the effects of Sim on Ras and Rho. Up-regulation of p27^Kip1 and inhibition of Rho but not Ras membrane translocation by Sim were reversed by geranylgeranylpyrophosphate, but not farnesylpyrophosphate. Indeed, inhibition of Rho by Clostridium botulinum C3 transferase or overexpression of dominant-negative N19RhoA mutant increased p27^Kip1 and inhibited retinoblastoma hyperphosphorylation. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 decreased p27^Kip1 and increased SMC DNA synthesis. These findings indicate that the down-regulation of p27^Kip1 by Rho GTPase mediates PDGF-induced SMC DNA synthesis and suggest a novel direct effect of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors on the vascular wall.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Vascular proliferative diseases such as atherosclerosis, post-angioplasty re-stenosis, and transplant arteriosclerosis are characterized by vascular smooth muscle cell (SMC)¹ DNA synthesis (1). The entry and progression of SMC into the cell cycle is stimulated by growth factors derived from inflammatory cells, platelets, and the vascular wall (2). Although these growth factors which include basic fibroblast growth factor, platelet-derived growth factor (PDGF), transforming growth factor-1, angiotensin II, and insulin-like growth factor utilize distinct signaling pathways to promote SMC DNA synthesis, these signaling pathways, however, must converge upon common regulators of the cell cycle (3). These regulators include the cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors. Indeed, gene therapy with Cdk inhibitors or treatment with a neutralizing antibody to PDGF inhibits neointimal smooth muscle DNA synthesis after balloon angioplasty (1, 4).

The transition through the cell cycle is regulated by the expression and activity of cell cycle checkpoint proteins comprising of cyclins and Cdks (5). These in turn are regulated by the family of Cdk inhibitor proteins, such as p16, p21^Waf1 and p27^Kip1. The final common pathway leading to G₀/G₁/S transition is the hyperphosphorylation of the retinoblastoma gene product (Rb), which functions as a molecular switch dedicating the cell to DNA replication. Hyperphosphorylation of Rb results in the release of the transcription factor E2F, which induces the expression of genes required for the progression through S, G₂, and M phases (5). Despite recent advances in the understanding of cell cycle regulation in proliferative vascular diseases such as atherosclerosis and post-angioplasty restenosis, therapy is still lacking which can effectively prevent SMC DNA synthesis.

Large clinical trials have shown that inhibition of cholesterol biosynthesis by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors or statins improve clinical outcomes in patients with atherosclerosis. For example, treatment with HMG-CoA reductase inhibitors reduces post-angioplasty re-stenosis, coronary bypass occlusions (6, 7), and transplant arteriosclerosis (8). Although HMG-CoA reductase inhibitors have been shown to inhibit SMC proliferation in vitro (9), the mechanism(s) by which they inhibit cell growth is not known. The HMG-CoA reductase inhibitors not only inhibit cholesterol synthesis, but also, inhibit the synthesis of important isoprenoid intermediates such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). Both FPP and GGPP are important lipid attachments for the post-translational modification of a variety of proteins, including Ras and Rho GTP-binding proteins (10, 11). We and others (12, 13) have recently shown that some of the direct effects of HMG-CoA reductase inhibitors on the vascular wall are mediated by inhibition of isoprenoid but not cholesterol synthesis. The purpose of this study, therefore, is to determine the role and mechanism of isoprenoid intermediates in regulating cell cycle progression in human vascular SMC.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents-- All standard culture reagents were obtained from JRH Bioscience (Lenexa, KS). Mevastatin (compactin), farnesylpyrophosphate, geranylgeranylpyrophosphate, and L-mevalonate were purchased from Sigma. Low density lipoprotein, FTI-276, and GGTI-286 were obtained from Calbiochem (San Diego, CA). Simvastatin was obtained from Merck Sharp and Dohme, Inc. Mevastatin and simvastatin were chemically activated by alkaline hydrolysis prior to use as described previously (13). PDGF-BB was purchased from Genzyme (Boston, MA). [-³²P]ATP, [³H]thymidine, [³H]geranylgeranyl pyrophosphate, and [³H]farnesylpyrophosphate were supplied by NEN Life Science Products Inc. The antibody detection kit (enhanced chemiluminescence) and the nylon nucleic acid (Hybond) and protein (polyvinylidene difluoride) transfer membranes were purchased from Amersham Corp. (Arlington Heights, IL). The Clostridium botulinum C3 transferase was obtained from List Biological Laboratories, Inc. (Campbell, CA). Recombinant Escherichia coli cytotoxic necrotizing factor (CNF)-1 and RhoA mutants were kindly provided by K. Aktories (University of Freiberg, Germany) and W. Moolenaar (Netherlands Cancer Institute, Netherlands), respectively.

Cell Culture-- Human vascular SMC were isolated from outgrowths of tunica media explants derived from human aortic and saphenous vein tissues as described previously (14). Cells of two to four passages were grown in culture medium containing Dulbecco's modified Eagle's medium (Life Technologies, Inc.), 10% fetal calf serum (FCS) (Hyclone, Logan, UT), and antibiotic mixture of penicillin (100 units/ml)/streptomycin (100 µg/ml)/Fungizone (1.25 µg/ml). The cells were characterized by phase-contrast microscopy and staining for SMC-specific -actin. Confluent SMC were rendered quiescent by incubation in 0.4% FCS for 48 h before experiments. Cellular viability was determined by cell count, morphology, and trypan blue exclusion.

Protein Isoprenylation-- For measurement of isoprenoid incorporation, SMC were treated with simvastatin (10 µM) for 24 h before addition of 50 µCi/ml [³H]GGPP or [³H]FPP, respectively. After 24 h, cells were washed four times with PBS. Aliquots of the cell lysates were counted in a liquid scintillation counter (Beckmann LS1800). Cell lysates were separated on SDS-PAGE as described (13). Gels were stained with Coomassie Blue to visualize protein loading and fixed according to the manufacturer's protocol (NEN Life Science Products). After vaccuum drying, autoradiography was performed for 4 weeks at 80 °C.

Western Blotting-- Cellular lysates from membrane- and cytoplasma-enriched fractions were prepared and separated on SDS-PAGE as described (13, 15). The presence of G_i2 and Rel A (p65) were used as specific markers of membrane and cytoplasmic fractions, respectively, as described previously (15). Preliminary studies using our high/low speed ultracentrifugation through sucrose gradient showed that the high-speed membrane fractions were relatively pure (i.e. presence of G_i2 and absence of Rel A). Immunoblotting was performed using monoclonal antibodies against Rb (1:250, Pharmingen 14001A) and p21^Waf1 (F-5), p27^Kip1 (C-19), p53 (DO-1), Ras (pan-Ras), RhoA (1:250 dilution,) and to c-myc tag (9E10, 1:200 dilution) (Santa Cruz Biotechnology, Inc.). Immunodetection was accomplished using a sheep anti-mouse secondary antibody (1:4000 dilution) or donkey anti-rabbit secondary antibody (1:4000 dilution) and the enhanced chemiluminescence (ECL) kit (Amersham Corp.). Autoradiography was performed at 23 °C and the appropriate exposures were quantitated by densitometry.

[³H]Thymidine Incorporation-- SMC were grown to 80% confluence and growth-arrested by incubation in Dulbecco's modified Eagle's medium containing 0.4% fetal calf serum (FCS) for 48 h. The media was then replaced with growth medium (Dulbecco's modified Eagle's medium, 10% FCS containing 6 ng/ml PDGF), and the indicated reagents were simultaneously added. For experiments using FTI-276 and GGTI-286, these reagents were added 1 h before stimulation with PDGF. After 48 h, [³H]thymidine (10 µCi/ml) was added and the cells were incubated an additional 24 h. Cells were then washed 4 times in ice-cold PBS and lysed in glycerol-SDS lysis buffer. Incorporated radioactivity was determined using a liquid scintillation counter (Beckman LS 6000IC).

Cyclin-dependent Kinase Assay-- The cyclin-dependent kinase assay was performed as described (15). Briefly, whole cellular extracts were prepared with a buffer containing 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM -glycerophosphate, 10 mM sodium fluoride, 10 mM p-nitrophenyl phosphate, 300 µM Na₃VO₄, 1 mM benzamidine, 2 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM dithiothreitol. The Cdk-2, -4, and -6 were immunopreciptiated with 3 µg of the corresponding human Cdk-specific antibody (Santa Cruz Biotechnology) from 200 µg of total cellular lysates for 1 h at 4 °C. The Cdk-antibody complex was then precipitated with protein A-agarose and washed three times with PD buffer (40 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1% Nonidet P-40, 6 mM EDTA, 6 mM EGTA, 10 mM -glycerophosphate, 10 mM sodium fluoride, 10 mM p-nitrophenyl phosphate, 300 µM Na₃VO₄, 1 mM benzamidine, 2 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM dithiothreitol) and once with kinase buffer (without ATP).

The purified enzyme was then incubated with a full-length pRb-glutathione S-transferase fusion protein (2 µg, Santa Cruz Biotech) as a substrate in 15 µl of kinase buffer containing Hepes (20 mM, pH 7.7), MgCl₂ (10 mM), ATP (10 µM), 3 µCi of [-³²P]ATP, -glycerophosphate (10 mM), sodium fluoride (10 mM), p-nitrophenyl phosphate (10 mM), Na₃VO₄ (300 µM), benzamidine (1 mM), phenylmethylsulfonyl fluoride (2 µM), aprotinin (10 µg/ml), leupeptin (1 µg/ml), pepstatin (1 µg/ml), and dithiothreitol (1 mM). The reaction was terminated by the addition of 2 times SDS-PAGE sample buffer and boiling for 5 min. Proteins were separated on 12% SDS-PAGE and autoradiography of the dried gel was performed at 80 °C.

Overexpression of RhoA Mutants-- SMC were seeded on washed microscope cover glasses placed into 6-well dishes. Cells (60-70% confluent) were transfected with 5 µg of pcDNA3-c-myc-wtRhoA (wild type RhoA) and pcDNA3-c-myc-N19RhoA (dominant-negative RhoA mutant) using LipofectAMINE (Life Technologies, Inc., Grand Island, NY) following the manufacturer's description. Both constructs contain a N-terminal c-myc tag. Approximately 48 h after transfection, cells were fixed with 3.7% formaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS. Cells were washed and incubated overnight with mouse anti-c-myc and rabbit anti-p27^Kip1 antibody (1:50 in 1% bovine serum albumin) at 4 °C. After washing with 1% bovine serum albumin, tetramethylrhodamine B isothiocyanate-conjugated goat anti-mouse IgG (red fluorescence) and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (green fluorescence) (Jackson Labs Inc., West Grove, PA) were used as secondary antibodies (1:500 in 1% bovine serum albumin) and incubated at 4 °C for 3 h in the dark. Cells were then washed five times with PBS and mounted onto glass slides using the ProLong Antifade Kit (Molecular Probes, Eugene, OR). Immunofluorescence was visualized using an Olympus BX 60F inverted fluorescence microscope. Photographic images were taken from five random fields.

Data Analysis-- Band intensities from Western blots and kinase assays were analyzed densitometrically by the National Institutes of Health Image Program (16). All values are expressed as mean ± S.E. compared with controls and among separate experiments. Paired and unpaired Student's t tests were employed to determine the significance of changes in densitometric measurements. A significant difference was taken for p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Smooth Muscle Cell Viability-- Relatively pure (>98%) human SMC were confirmed by phase-contrast microscopy and staining for SMC-specific -actin (data not shown). There was no observable adverse effects of mevastatin (<20 µM), simvastatin (<10 µM), FPP, GGPP, C3 transferase, or CNF-1 on cellular viability. However, higher concentrations of mevastatin (>50 µM) or simvastatin (Sim, >20 µM) were avoided because of toxicity.

Protein Isoprenylation-- For measurement of isoprenoid incorporation, SMC were treated with simvastatin (10 µM) for 24 h before addition of 50 µCi/ml [³H]GGPP or 50 µCi/ml [³H]FPP. SDS-PAGE and scintillation counting of cell lysates after 24 h revealed that 18 ± 2% of [³H]GGPP and 17 ± 3% of [³H]FPP were taken up by SMC. SDS-PAGE showed multiple bands in the [³H]FPP-treated cell lysates including one at 21-23 kDa, whereas [³H]GGPP-treated cell lysates showed labeling of only one prominent band at 21-23 kDa (data not shown). These findings suggest that both GGPP and FPP are taken up by SMC under our experimental condition. Similar results were also obtained using radiolabeled geranylgeranol and farnesol.

Effects of HMG-CoA Reductase Inhibitors on SMC DNA Synthesis-- To determine the contribution of isoprenoid synthesis to PDGF-stimulated SMC cell cycle progression, cells were treated with increasing concentrations of Sim (1-10 µM) for 24 h. By [³H]thymidine incorporation, Sim inhibited SMC DNA synthesis by 94 ± 0.3% which was completely reversed by co-treatment with mevalonate (200 µM) (Fig. 1A). Mevalonate alone, however, had no effect on [³H]thymidine uptake (not shown). This decrease in [³H]thymidine incorporation by Sim correlated with a concentration-dependent decrease in Rb hyperphosphorylation by 98 ± 4% (Fig. 1B). Similar effects were oberved with another HMG-CoA reductase inhibitor, mevastatin (data not shown). Inhibition of SMC DNA synthesis was independent of extracellular cholesterol concentration since all cells were cultured in 10% fetal calf serum under cholesterol-clamped conditions. Furthermore, addition of low density lipoprotein-cholesterol (1 mg/ml) did not reverse the effects of Sim on [³H]thymidine incorporation (not shown). These findings suggest that isoprenoid synthesis is important in PDGF-induced SMC DNA synthesis.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of simvastatin (Statin, 1-10 µM) alone or in combination with L-mevalonate (Meva, 200 µM) on PDGF (6 ng/ml)-induced: A, SMC DNA synthesis and B, Rb hyperphosphorylation (Rb-P) at 24 h. Thymidine incorporation experiments were performed three times in duplicate. *, p < 0.05 as compared with PDGF alone. The immunoblots were representative of three seperate experiments.

Effects of HMG-CoA Reductase Inhibitors on Cdk Activity-- The entry into and progression of the cell cycle is regulated by holoenzyme complexes consisting of cyclins and Cdks (5). To determine whether isoprenoids can regulate Cdk activity, we measured the effect of Sim on PDGF-induced Cdk-2, Cdk-4, and Cdk-6 activity. Using glutathione S-transferase-Rb fusion protein as substrate for Cdk, we found that quiescent SMC have little or no Cdk-2, -4, and -6 activity (Fig. 2). Stimulation with PDGF (6 ng/ml, 24 h) increased Cdk-2, -4, and -6 activity. Co-treatment with Sim (5 µM) inhibited PDGF-induced Cdk-2, -4, and -6 kinase activity by 80 ± 5, 60 ± 3, and 48 ± 3%, respectively. Thus, inhibition of isoprenoid synthesis down-regulated Cdk activity with the greatest decrease observed with Cdk-2.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effects of PDGF (6 ng/ml) and simvastatin (Statin, 5 µM) on cyclin-dependent kinase (Cdk)-2, -4, and -6 activity at 24 h. Equal amounts of Cdk-2, -4, and -6 were immunoprecipitated from 200 µg of total cellular lysate for each lane. The kinase assays were performed three times with similar results.

Effects of HMG-CoA Reductase Inhibitors on Cdk Inhibitors-- The Cdk inhibitors such as p16, p21^Waf1 and p27^Kip1 bind to and inhibit the activation of Cdk·cyclin complexes. The tumor suppressor gene p53 regulates cell cycle progression, in part, by up-regulating the expression of p21^Waf1 (1). To determine whether inhibition of isoprenoid synthesis can affect the expression of Cdk inhibitors, we investigated the effects of Sim (5 µM) on p16, p21^Waf1, p27^Kip1, and p53 levels in SMC stimulated with PDGF (6 ng/ml) for 12, 24, and 36 h. Compared with PDGF alone, we find that Sim did not affect the levels of p16 and p21^Waf1 (Fig. 3). In contrast, stimulation with PDGF decreased p27^Kip1 by 80 ± 7, 95 ± 3, and 75 ± 8% after 12, 24, and 36 h, respectively. Co-treatment with Sim (5 µM) reversed the down-regulation of p27^Kip1 by PDGF resulting in 20-fold increase in p27^Kip1 after 24 h compared to that of PDGF. Although p53 levels decline in a time-dependent manner after PDGF stimulation, the levels were not different in the presence or absence of Sim. These findings indicate that inhibition of isoprenoid synthesis by Sim preferentially up-regulates p27^Kip1 levels and suggest that p27^Kip1 may be involved in the inhibition of Cdk-2 activity.

View larger version (70K): [in this window] [in a new window]   Fig. 3.   Immunoblots showing the time-dependent effects of PDGF (6 ng/ml) with and without simvastatin (Statin, 5 µM) on the levels of p53 and Cdk inhibitors p16, p21Waf1, and p27Kip1. The blots are representative of three separate experiments.

Effects of Isoprenoids on Rho and Ras Membrane Localization-- The small GTPases of the Ras and Rho family have been shown to regulate entry into the cell cycle (17, 18). Ras and Rho are post-translationally modified by isoprenylation which is necessary for their membrane localization and function (11). We have previously shown that Ras farnesylation and Rho geranylgeranylation are required for their membrane-associated GTPase activity (13). Stimulation with PDGF (6 ng/ml, 24 h) increased the level of membrane-bound RhoA by 420 ± 20% without significantly affecting the total amount of RhoA in smooth muscle cells (Fig. 4). Inhibition of isoprenoid synthesis by Sim (5 µM) completely reversed and prevented PDGF-induced RhoA membrane localization. Interestingly, in the presence of PDGF, Sim increased the total cellular expression of RhoA possibly to compensate for decreases in membrane-associated RhoA activity. Indeed, co-treatment with GGPP, but not FPP reversed the effect of Sim on RhoA membrane localization.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Immunoblots showing the effects of PDGF (6 ng/ml), simvastatin (Statin, 5 µM), GGPP (5 µM), and FPP (5 µM) on RhoA and Ras protein expression in cell membranes and total cell lysates after 24 h. Each blot is representative of three separate experiments.

Interestingly, the expression of Ras in SMC membranes was not significantly altered by treatment with PDGF or Sim after 24 h (Fig. 4). In total cell lysates, however, treatment with Sim resulted in the appearance of a higher molecular weight non-farnesylated Ras. Previous studies have demonstrated that farnesylated Ras migrates slightly faster on SDS-PAGE than unmodified Ras (19, 13). Indeed, the shifted band corresponding to non-farnesylated Ras in SMC treated with PDGF and Sim is absent in the presence of FPP, but not GGPP. These findings confirm that inhibition of geranylgeranylation by Sim specifically prevents PDGF-induced membrane localization of Rho, but not Ras.

Effects of Rho on p27^Kip1 Expression-- To determine which isoprenoid intermediate down-regulates p27^Kip1, PDGF (6 ng/ml)-induced SMC were treated with Sim (5 µM) in the presence or absence of GGPP (5 µM) and FPP (5 µM). Co-treatment with GGPP, but not FPP, completely reversed the up-regulation of p27^Kip1 by Sim (Fig. 5A). Since Rho is a major target for geranylgeranylation, we investigated whether inhibition of Rho up-regulates p27^Kip1. Treatment of SMC with C. botulinum C3 transferase (50 µg/ml, 24 h), an exoenzyme which specifically inactivates Rho by ADP-ribosylation (20), completely prevented the PDGF-induced down-regulation of p27^Kip1 (Fig. 5B).

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Immunoblots showing: A, the effects of PDGF (6 ng/ml), simvastatin (Statin, 5 µM), GGPP (5 µM), FPP (5 µM) and, B, C. botulinum C3 transferase (C3, 50 µg/ml) on Cdk inhibitor p27Kip1 after 24 h. Each blot is representative of three separate experiments.

To determine whether changes in p27^Kip1 are specific to inhibition of Rho, we transfected SMC with wild-type RhoA (wtRhoA) and a dominant-negative RhoA (N19RhoA). Both of these RhoA constructs contain a N-terminal c-myc tag. The transfection efficiency as measured by -galactosidase fluorescence and c-myc immunofluorescence is approximately 8-10%. Using dual immunofluoresecent microscopy, overexpression of wtRhoA in PDGF-induced SMC did not change p27^Kip1 expression relative to non-transfected cells (i.e. cells without c-myc immunofluorescence) (Fig. 6). In contrast, SMC overexpressing the dominant-negative N19RhoA mutant, as identified by co-staining for c-myc, showed increased p27^Kip1 expression. Taken together, these findings indicate that RhoA negatively regulates p27^Kip1 expression and suggest that Sim up-regulates p27^Kip1 by inhibiting Rho membrane localization and activity.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Dual immunofluorescence of c-myc-tagged wild-type RhoA (wtRhoA) or dominant-negative RhoA (N19RhoA) in transfected SMC stimulated with PDGF (6 ng/ml, 24 h). The red immunofluorescence for c-myc identifies only transfected cells while the green immunofluorescence for p27Kip1 identifies both transfected and non-transfected cells. The same cells are shown for the corresponding panels for c-myc and p27Kip1 immunofluorescence. This is representative of five fields chosen at random.

Effects of Rho on Smooth Muscle Cell Cycle Progression-- To determine the effects of Rho on cell cycle progression, we investigated the effects of PDGF (6 ng/ml), Sim (5 µM), GGPP (5 µM), FPP (5 µM), or C3 transferase (50 µg/ml) on Rb hyperphosphorylation in SMC after 24 h. Co-treatment with GGPP, and to a much lesser extent FPP, reversed the inhibitory effect of Sim on PDGF-induced Rb hyperphosphorylation in SMC (Fig. 7A). Direct inactivation of Rho by C3 transferase decreased Rb hyperphosphorylation by 80 ± 7%. This decrease in Rb hyperphosphorylation by C3 transferase was not reversed by GGPP or FPP (data not shown). When SMC was treated with E. coli CNF-1 (200 ng/ml) which is known to directly activate Rho by blocking Rho GTP hydrolysis (21, 22), Rb became hyperphosphorylated in the absence of fetal calf serum (Fig. 7B). Furthermore, in the presence of 10% fetal calf serum in the culture medium, CNF-1 augmented Rb hyperphosphorylation by approximately 2-fold.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   A, immunoblots showing the effects of PDGF (6 ng/ml), simvastatin (Statin, 5 µM), GGPP (5 µM), FPP (5 µM), and C3 transferase (C3, 50 µg/ml) on Rb phosphorylation and hyperphosphorylation (Rb-P) after 24 h. The blots are representative of three seperate experiments. B, immunoblots showing the effects of CNF (200 ng/ml) on Rb hyperphosphorylation in the presence and absence of 10% FCS.

Compared with treatment with PDGF (6 ng/ml), thymidine incorporation was inhibited in the presence of Sim (6 ± 0.3% incorporation, p < 0.001) (Fig. 8A). Co-treatment with GGPP (5 µM) reversed the inhibitory effect of Sim (88 ± 6% incorporation, p > 0.05 compared with PDGF), where as FPP (5 µM) only partially reversed the effects of Sim (48 ± 4% incorporation, p < 0.01 compared with PDGF). Co-treatment with low density lipoprotein-cholesterol (1 mg/ml) did not reverse the inhibitory effects of simvastatin (p > 0.05). Co-treatment with CNF-1 (200 ng/ml) increased thymidine incorporation by 42 ± 2% while co-treatment with C3 transferase (50 µg/ml) reduced thymidine incorporation by 56 ± 1% (p < 0.05 for both compared with PDGF). To determine the degree that geranylgeranylation and farnesylation contribute to PDGF-induced SMC DNA synthesis, we investigated the effects of inhibitors of geranylgeranyltransferase and farnesyltransferase on PDGF-induced thymidine incorporation. The geranylgeranyltransferase inhibitor (GGTI-286, 30 µM) decreased PDGF (6 ng/ml)-induced thymidine incorporation by 91 ± 5% (p < 0.01) while the farnesyltransferase inhibitor (FTI-276, 10 nM) decreased PDGF (6 ng/ml)-induced thymidine incorporation by 37 ± 4% (p < 0.05) (Fig. 8B).

View larger version (15K): [in this window] [in a new window]   Fig. 8.   A, effects of simvastatin (Statin, 5 µM), GGPP (5 µM), FPP (5 µM), low-density lipoprotein-cholesterol (LDL-C, 1 mg/ml), CNF-1 (200 ng/ml), and C3 transferase (C3, 50 µg/ml) on PDGF (6 ng/ml, 24 h)-induced SMC DNA synthesis. Experiments were performed three times in duplicates. *, p < 0.05 compared with PDGF. B, effects of farnesyltransferase inhibitor (FTI-276, 10 nM) or geranylgeranyltransferase inhibitor (GGTI-286, 30 µM) on PDGF (6 ng/ml, 24 h)-induced SMC DNA synthesis. Experiments were performed two times in quadruplicates. *, p < 0.05 compared with PDGF.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have shown that the isoprenoid, geranylgeranyl, and the protein that it post-translationally modifies, Rho, mediates PDGF-induced cell cycle progression by down-regulating the expression of the Cdk inhibitor p27^Kip1 and stimulating the activity of Cdk-2 and hyperphosphorylation of Rb. Treatment with the HMG-CoA reductase inhibitor, simvastatin, decreases Rho geranylgeranylation and membrane localization, inhibits Cdk activity and Rb hyperphosphorylation, and prevents SMC DNA synthesis. Indeed, Rho-induced Rb hyperphosphorylation and DNA synthesis are necessary and sufficient for serum- or PDGF-induced SMC DNA synthesis and are associated with decreases in p27^Kip1 expression. These findings indicate an important role of Rho in PDGF-stimulated mitogenesis and are consistent with studies showing that p27^Kip1 accumulation mediates cell cycle arrest (23, 24) and that the degradation of p27^Kip1 facilitates the growth of rat FRTL-5 cells (25). Our findings, however, are in contrast to previous studies which suggest that PDGF-induced SMC DNA synthesis is mediated predominantly by Ras (17).

The Rho GTPase family which includes RhoA, RhoB, Rac, Cdc42 are major substrates for post-translational modification by geranylgeranylation. Geranylgeranyl modification is important for cellular trafficking and targets these Rho GTPases to the cellular membrane (10, 26). The membrane translocation of inactive or GDP-bound Rho involves the release of its cytoplasmic inhibitor, Rho guanine nucleotide dissociation inhibitor, and activation of Rho through GDP/GTP exchange in the presence of guanine nucleotide exchange factor (27-29). Since most of the cytoplasmic Rho proteins are inactive (GDP-bound state), their GTP binding activity remains invariably low even in the presence of HMG-CoA reductase inhibitor treatment. Thus, inhibition of Rho geranylgeranylation and membrane translocation by HMG-CoA reductase inhibitors leads to a greater accumulation of inactive Rho in the cytoplasm. This is consistent with our finding that in the presence of HMG-CoA reductase inhibitors, addition of GGPP, but not FPP, restores membrane expression of Rho, decreases p27^Kip1 levels, and enhances cell cycle progression. Interestingly, in addition to increasing RhoA activity by GTP loading, we find that stimulation with PDGF increases the amount of membrane-associated RhoA. The mechanism by which PDGF increases RhoA in the membrane, however, remains to be determined.

The importance of Rho in SMC DNA synthesis was further confirmed by studies showing that inhibition of Rho by C. botulinum C3 transferase also inhibits SMC DNA synthesis. The C3 transferase ADP-ribosylates asparagine 41 of RhoA and RhoB and renders them biologically inactive in the GDP-bound state (20). Although under certain circumstances, the C3 transferase may also ADP-ribosylate Rac-1 or Cdc42 (20, 21), our results showing that the overexpression of a dominant-negative RhoA mutant, N19RhoA, also increases p27^Kip1 expression are consistent with the inhibitory effects of C3 transferase on Rho. In contrast, direct activation of Rho by E. coli CNF-1 reversed the inhibitory effects of HMG-CoA reductase inhibitors. Interestingly, CNF-1 alone was sufficient to augment and induce SMC cell cycle progression in the presence and absence of serum, respectively. These findings indicate that Rho GTPase is a predominant mediator of PDGF-induced SMC DNA synthesis.

Our results suggest that cell cycle progression is mediated by Rho-dependent down-regulation of the Cdk inhibitor p27^Kip1. In contrast, Rho has been shown to decrease p21^Waf1 but not p27^Kip1 levels in NIH 3T3 cells (30). Indeed, another HMG-CoA reductase inhibitor, lovastatin, has been shown to increase the level of p21^Waf1 in prostate cancer cells (31). Our studies with human vascular SMC, however, showed no effect of HMG-CoA reductase inhibitors on p21^Waf1 or p16 expression. Thus, cell cycle arrest in SMC induced by HMG-CoA reductase inhibitors appears to be mediated by mechanisms which are independent of p21^Waf1 and p16 levels. Another important tumor suppressor gene which is involved in cell cycle regulation is p53 (2, 32). Since up-regulation of p53 was not observed after inhibition of isoprenoid synthesis, SMC growth arrest by HMG-CoA reductase inhibitors probably also occurred by mechanisms which are independent of p53 levels. These results are consistent with the absence of changes in p21^Waf1 levels which has been shown in previous studies to be induced by p53 (32). In contrast to p16 and p21^Waf1, p27^Kip1 levels are increased by treatment with HMG-CoA reductase inhibitors and the up-regulation of p27^Kip1 correlated with the inhibition of Cdk-2 activity, and to a lesser extent, Cdk-4 and -6 activity.

Both Ras and Rho have been shown to play pivotal roles in the regulation of growth in a variety of cell types. In contrast to GGPP, addition of FPP only partially reversed the inhibitory effects of HMG-CoA reductase inhibitors on SMC DNA synthesis by 50%, suggesting that Ras farnesylation alone may not be the predominant mediator of PDGF-induced SMC DNA synthesis. This may be due to the fact that after 24 h of treatment, there was no effect of PDGF, GGPP, or FPP on the level of Ras in the cellular membrane. These findings are consistent with recent studies showing that the translocation of Rho, but not Ras, to the membrane corresponds with cell cycle progression from G₁ to S in FRTL-5 cells (33, 34) and that GGPP completely reversed the up-regulation of p27^Kip1 by HMG-CoA reductase inhibitors, whereas FPP had little or no effect on p27^Kip1. However, a recent study demonstrates that Ras and Rho GTPases both interact to regulate p21^Waf1 and cell cycle progression in NIH 3T3 cells (30). Further studies, therefore, are needed to determine the precise role of Ras in PDGF-induced SMC cell cycle progression.

Recent clinical data suggest that some of the beneficial effects of treatment with HMG-CoA reductase inhibitors may occur independent of changes in serum cholesterol levels (35). Our data indicate that there is a direct effect of these agents on the vessel wall via inhibition of Rho geranylgeranylation. We propose that Rho is a necessary and sufficient mediator of SMC cell cycle progression and that inhibition of Rho in SMC may have beneficial effects in vascular proliferative diseases such as atherosclerosis and post-angioplasty re-stenosis. The precise mechanism(s) by which Rho up-regulates p27^Kip1 expression, however, remains to be determined.

	ACKNOWLEDGEMENTS

We thank K. Aktories, W. Moolenaar, and P. Libby for kindly providing CNF-1, RhoA mutants, and human vascular smooth muscle cells, respectively. We are also grateful to Todd Bourcier for assistance in the GGPP/FPP labeling assays.

	FOOTNOTES

^* This work was supported in part the National Institutes of Health Grant HL-52233 and the Deutsche Forschungsgemeinschaft.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.

Present address: Klinik III für Innere Medizin, University of Cologne, Germany.

^§ Contributed equally to the results of this paper.

^¶ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Vascular Medicine & Atherosclerosis Unit, Brigham & Women's Hospital, 221 Longwood Ave., LMRC-322, Boston, MA 02115. Tel.: 617-732-6538; Fax: 617-264-6336; E-mail: jliao@rics.bwh.harvard.edu.

	ABBREVIATIONS

The abbreviations used are: SMC, smooth muscle cell; PDGF, platelet-derived growth factor; Rb, retinoblastoma; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; FPP, farensylpyrophosphate; GGPP, geranylgeranylpyrophosphate; CNF-1, cytotoxic necrotizing factor-1; FCS, fetal calf serum; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; Cdk, cyclin-dependent kinase.

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