Short-Term Pravastatin Mediates Growth Inhibition andApoptosis, Independently of Ras, via the Signaling Proteinsp27Kip1 and PI3 Kinase

ROBERT H. WEISS,*†‡ AL RAMIREZ,* and ADRIANE JOO**Division of Nephrology, Department of Internal Medicine, and†Cell and Developmental Biology GraduateGroup, University of California, Davis, California; and‡Department of Veterans’ Affairs Northern CaliforniaHealth Care System, Mather, California.

Abstract.Growth factor-stimulated DNA synthesis in a varietyof cell lines has been shown to be decreased after overnight (orlonger) treatment with the 3-hydroxy-3-methylglutaryl CoAreductase inhibitors, the statins. Although this anti-mitogeniceffect had been presumed to be the result of the impairment ofRas lipidation, a stable modification (T1/2 approximately 20 h),this study provides new data demonstrating that brief (approx-imately 1 h) pretreatment of rat vascular smooth muscle cellswith 100 mM pravastatin before platelet-derived growth fac-tor-BB (PDGF-BB) stimulation results in attenuation of DNAsynthesis through a Ras-independent mechanism. PDGF-BB-stimulated PDGF-b receptor tyrosine phosphorylation, Rasactivity, and mitogen-activated protein/extracellular signal-regulated kinase activity are unaffected by from 10 min to 1 hof pravastatin incubation, while Raf activity is markedly in-creased after 1 h of pravastatin. Phosphatidylinositol-3 kinaseactivity and phosphorylation of its downstream effector Akt are

decreased after 1 h pravastatin incubation. Rho is stabilized bypravastatin, and ADP-ribosylation of Rho by C3 exoenzymedecreases PDGF-stimulated phosphatidylinositol-3 kinase ac-tivity, mimicking the effect of pravastatin on this signalingprotein. Levels of the cyclin-dependent kinase inhibitorp27Kip1 are increased when cells were preincubated with prav-astatin for 1 h and then exposed to PDGF, and apoptosis isinduced by pravastatin incubation times as short as 1 to 4 h.Thus, short-term, high-dose pravastatin inhibits vascularsmooth muscle cell growth and induces apoptosis indepen-dently of Ras, likely by means of the drug’s effect on p27Kip1,mediated by Rho and/or phosphatidylinositol-3 kinase. Thiswork demonstrates for the first time that the statins may betherapeutically useful when applied for short periods of timesuch that potential toxicity of long-term statin use (such aschronic Ras inhibition) may be avoided, suggesting futuretherapeutic directions for statin research.

The 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductaseinhibitors, or statins, block mevalonate production, the rate-limiting step in cholesterol biosynthesis. As a salutary butunexpected consequence of the use of these medications inhyperlipidemic patients, a decrease in vasculopathy has beenobserved in renal and cardiac transplant patients. This impor-tant clinical observation has been shown to involve inhibitionof vascular smooth muscle (VSM) cell proliferation, indepen-dent of serum LDL. Such an effect, at least in part, could havebeen anticipated by the work of many investigators who foundthat inhibition of the formation of isoprenoids, precursors ofsterol synthesis, played an important role in altering the pro-cessing of signaling proteins that require lipidation, such asRas (1). However, while the statins appear to have some effecton transplant coronary vasculopathy (2), unsuccessful effortshave been made to use statins to block other processes char-

acterized by VSM proliferation, such as restenosis after coro-nary angioplasty (3). Furthermore, the chronic use of theseagents is also limited by both real (in the case of skeletalmuscle damage) and potential toxicity, when used over longperiod of time, likely due to inhibition of lipidation of keysignaling proteins in a variety of somatic cells.

The available studies on statin inhibition of mitogenesishave focused on long-term (overnight or longer) statin incuba-tion, where modification of signaling protein lipidation (par-ticularly of Ras) likely comes into play (1). Examination ofearly events that may be affected by short-term (less thanapproximately 20 h) exposure to HMG-CoA reductase inhibi-tors has not been reported. We asked whether short-term prav-astatin incubation can cause growth inhibition and, if so,whether such inhibition occurs through signaling mechanismsthat have been established for the anti-mitogenic effects oflong-term statin exposure. We now show that short-term, high-dose application of pravastatin in a tissue culture VSM cellmodel has no effect on Ras-GTP loading, but rather causesmodification of a signaling pathway that implicates the cyclin-dependent kinase (cdk) inhibitors, as well as phosphatidylino-sitol-3 kinase (PI3K) and the small GTPase Rho, as the medi-ators of the anti-mitogenic and pro-apoptotic effects of short-term pravastatin.

Received December 18, 1998. Accepted March 15, 1999.Correspondence to Dr. Robert H. Weiss, Division of Nephrology, TB 136,Department of Internal Medicine, University of California, Davis, CA 95616.Phone: 530-752-4010; Fax: 530-752-3791; E-mail: rhweiss@ucdavis.edu

1046-6673/1009-1880Journal of the American Society of NephrologyCopyright © 1999 by the American Society of Nephrology

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Materials and MethodsMaterials

Recombinant human PDGF-BB was obtained from Upstate Bio-technology, Inc. (Lake Placid, NY). Rabbit polyclonal anti-PDGF-breceptor and anti-C-Raf, mouse monoclonal anti-phosphotyrosine andanti-p27Kip1, and sheep polyclonal anti-Rho (recognizing both RhoAand RhoB) antibodies were obtained from Santa Cruz Biotechnology(Santa Cruz, CA). Rabbit polyclonal anti-phospho-Akt (Thr308) an-tibody and the mitogen-activated protein (MAP) kinase assay com-ponents were obtained from New England Biolabs (Beverly, MA).Goat anti-mouse and anti-rabbit horseradish peroxidase-linked IgGwere obtained from BioRad (Richmond, CA). Rat monoclonal anti-v-H-Ras was obtained from Calbiochem (San Diego, CA). C3 exoen-zyme was obtained from List Biological (Campbell, CA). Reagentsfor the enhanced chemiluminescence system, [3H]-thymidine,g-[32P]-ATP, and [35S]-methionine, were obtained from Amersham(Arlington Heights, IL). Pravastatin was obtained from Bristol-MyersSquibb (Princeton, NJ) and solubilized in water. The Apo-TACSreagents forin situ terminal deoxynucleotidyl transferase (TdT)-me-diated nick end labeling (TUNEL) assays were obtained from Trevi-gen (Gaithersburg, MD). All other reagents were from Sigma (St.Louis, MO).

CellsCultures of A10 aortic VSM cells were obtained from American

Type Culture Collection (Rockville, MD). The cells were maintainedas described (4) and were used between passages 15 and 25. The cellswere growth-arrested by placing them in serum-free quiescence me-dium containing Dulbecco’s modified Eagle’s medium, 20 mMHepes, pH 7.4, 5mg/ml transferrin, 0.5 mg/ml bovine serum albumin,penicillin (50 U/ml), and streptomycin (50 U/ml).

DNA SynthesisCultured VSM cells were harvested, plated onto 24-well plates, and

grown to confluence. The confluent monolayers were then placed inquiescence medium for an additional 2 d. Cells were then exposed togrowth factors as indicated, and [3H]-thymidine incorporation wasassessed as described previously (5).

Immunoprecipitations and Western BlotsCells were grown to confluence in 6-cm culture dishes and serum-

deprived. After treatment with pravastatin and PDGF-BB, the cellswere washed with phosphate-buffered saline (PBS) and lysed in lysisbuffer and the supernatant was immunoprecipitated and/or Western-blotted as described in the study by Weisset al. (4).

MAP Kinase and Raf Kinase Activity AssayCells were grown to confluence on 10-cm culture dishes and

serum-deprived. PDGF-BB and/or pravastatin were added at the in-dicated concentrations and times to the medium. The cells werewashed three times with PBS, and Raf and MAP kinase activity assayswere performed as described (4).

Ras Activity AssayCells were grown to confluence on 6-cm culture dishes, serum-

deprived, and incubated in medium containing carrier-free [32P]-orthophosphate (10 mCi/ml per dish) for 2 h and then pravastatin wasadded for the last hour. After stimulation with PDGF-BB for 1 to 5min, the cells were lysed in solubilization buffer (50 mM Tris-HCl,pH 7.5, 20 mM MgCl2, 150 mM NaCl, 0.5% [vol/vol] Nonidet P-40,

20 mg/ml aprotinin, 1 mM Na3VO4), removed, and, after pre-clearingwith charcoal, incubated with agarose-linked anti-v-H-Ras antibody.The samples were centrifuged and the pellet was washed three timesin solubilization buffer and two times in wash buffer (50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 150 mM NaCl). The samples were elutedwith elution buffer (40 mM Tris-HCl, pH 7.5, 40 mM ethylenedia-minetetra-acetic acid, 4% [wt/vol] sodium dodecyl sulfate, 1 mMGDP, 1 mM GTP), spotted on cellulose thin-layer chromatography(TLC) plates, and developed in 0.75 M KH2PO4, pH 3.4 (6). Ras-GTPand Ras-GDP were identified using nonradioactive GTP and GDPstandards run on the same plate and visualized by ultraviolet light.

PI3K Activity AssayVSM cells were grown to confluence on 6-cm culture dishes and

serum-deprived. The cells were preincubated with pravastatin at thetimes indicated before stimulation for 10 min with PDGF-BB. Thecells were washed three times in PBS, and PI3K activity was assessedas described (7).

Scrape Loading of C3 ExoenzymeVSM cells were grown to confluence in 10-cm tissue culture dishes

and washed with PBS. Scraping buffer (114 mM KCl, 15 mM NaCl,5.5 mM MgCl2, and 10 mM Tris-HCl, pH 7.4) was added in thepresence or absence of C3 exoenzyme (5mg/ml), and the cells werescraped with a rubber policeman and centrifuged at 2000 rpm for 5min at room temperature. The pellets were resuspended in completeculture media in 6-cm tissue culture dishes and incubated overnight,and PI3K activity was assessed as described above.

RhoA StabilityVSM cells were grown to confluence on 6-cm culture dishes and

serum-deprived. Then cells were incubated for 2 h with 110mCi of[35S]-methionine per ml in Dulbecco’s modified Eagle’s mediumlacking methionine that was supplemented with 2% fetal bovineserum. Pravastatin was added to cells 1 h after [35S]-methionineaddition. After an additional hour, the cells were washed twice withwarm PBS and then quiescent media with PDGF-BB (30 ng/ml) wasadded to chase the [35S]-methionine label for various times. At theindicated time intervals (after complete medium addition), the cellswere washed three times with PBS and lysed in lysis buffer (50 mMHepes, 1% Triton X-100, 10 mM sodium pyrophosphate, 100 mMNaF, 4 mM ethylenediaminetetra-acetic acid, 1mg/ml aprotinin, 2mM phenylmethylsulfonylfluoride, 2 mM Na3VO4). The lysate wasremoved with a rubber spatula and passed through a 25-gauge syringeto ensure complete lysing. The lysate was centrifuged at 12,000 rpm,and the protein concentration of the supernatant was determined byabsorbance at A595. Equal protein concentrations of lysate were in-cubated with Rho antibody overnight. The immunocomplex was in-cubated with protein A-Sepharose beads, centrifuged, and washedtwice with lysis buffer. The pellet was resuspended and electropho-resed. The gel was then fixed (glacial acetic acid:methanol:water,10:20:70, vol/vol/vol), soaked overnight (20% methanol, 3% glycer-ol), dried, and visualized with a PhosphorImager.

ApoptosisNon-serum-starved VSM cells were harvested from 12-mm glass

coverslips. After addition of 20ml (100 mM) of pravastatin at theindicated incubation times, the cells were fixed in 3.7% bufferedformaldehyde for 10 min, washed twice with PBS, dried, and storedin CytoPore (Trevigen) at 4°C overnight. The coverslips were washedtwice with water, immersed in quenching solution (45 ml of methanol,

J Am Soc Nephrol 10: 1880–1890, 1999 Short-Term Pravastatin Inhibition of Growth 1881

5 ml of 30% hydrogen peroxide) for 5 min, washed again with water,and immersed in TdT labeling buffer (Trevigen). The cells werecovered with 50ml of labeling reaction mix (B-dNTP, TdT, TdTlabeling buffer) and incubated at 37°C for 1 h in ahumidity chamber.The coverslips were immersed in 13 TdT stop buffer (Trevigen) andwashed. The cells were covered with 50ml of Detection Mix A(Trevigen) and incubated for 1 h in a humidity chamber. Afterwashing slips twice in PBST (PBS, 0.05% Tween 20) and once inwater, the samples were covered with 50ml of TACS Blue Label(Trevigen) for 4 min and washed twice in water. After staining, thecells were counterstained with Nuclear Fast Red, dehydrated in 95%ethanol, and mounted onto the glass slides. Data are expressed aspercentage of blue cells in the stained slides.

ResultsAll biologically active Ras proteins are modified by poly-

isoprenylation (8). After carboxyl methylation of its farnesy-lated cysteine, fully processed Ras is stably anchored to the cellmembrane (9) with a half-life of approximately 20 h (10).Thus, short-term (less than 20 h) incubation of cells with anHMG-CoA reductase inhibitor would not be expected to re-duce membrane-bound Ras and thus Ras catalytic activity.Surprisingly, however, we found that incubation of serum-starved VSM cells with 100mM pravastatin from 10 min to 1 hbefore PDGF-BB addition significantly inhibited PDGF-stim-ulated [3H]-thymidine incorporation (Figure 1A). As has beenshown in other systems using a variety of statins (11–13), therewas further inhibition, to 71%, when pravastatin was presentfor 24 h before PDGF stimulation (Figure 1A). The inhibitionof PDGF-BB-stimulated DNA synthesis was pravastatin dose-dependent from 1 to 100mM, when pravastatin was added 1 hbefore this growth factor (Figure 1B). To examine how muchtime was actually required for pravastatin to exert its inhibitoryeffect on growth, we removed pravastatin from the cells atvarious times after its addition (Figure 1C). Pravastatin need bepresent for only 1 h, and subsequently washed off, before theaddition of PDGF-BB to exert its inhibitory effect. The inhib-itory effect of 1 h pravastatin incubation was completely re-versed by the addition of mevalonate (1 mM), the catalyticproduct of HMG-CoA reductase on HMG-CoA, when added4 h before PDGF-BB, demonstrating that the mitogenic inhi-bition of pravastatin at times quite unlikely to be affecting Rasremain specific to the drug’s effect on HMG-CoA reductase.

We next examined elements of the PDGF mitogenic signal-ing cascade to ascertain which are being affected by short-termpravastatin administration. Early events after binding of PDGFto its receptor are set in motion by autophosphorylation of thePDGF receptor on tyrosine residues, with subsequent activa-tion of a variety of cytoplasmic signal transfer proteins (14). Torule out an effect of short-term pravastatin incubation on earlyPDGF receptor events, we examined the effect of pravastatinon receptor tyrosine phosphorylation after PDGF stimulation.Serum-starved VSM cells were preincubated with pravastatinfor 1, 3, and 24 h and then subjected to PDGF-BB stimulationfor 10 min. The cell lysates were immunoprecipitated withantibody to the PDGF-b receptor, electrophoresed, and immu-noblotted with phosphotyrosine antibody. Control cells andcells incubated with pravastatin alone, in the absence of PDGF

Figure 1. Both short- and long-term incubation of vascular smoothmuscle (VSM) cells with pravastatin decreases platelet-derivedgrowth factor-BB (PDGF-BB)-induced DNA synthesis. (A) Conflu-ent, VSM cells were serum-starved for 2 d and incubated withpravastatin (100mM) for the indicated times before addition ofPDGF-BB (30 ng/ml) for 24 h (o). Control cells (f) were incubatedwith pravastatin (where indicated) but were not stimulated withPDGF. (B) Serum-starved cells were incubated for 1 h with prava-statin at the concentrations indicated before addition of PDGF-BB (30ng/ml) for 24 h. (C) Serum-starved cells were incubated with prava-statin (100mM) for the times indicated. At the end of the indicatedtime period, the cells were washed twice with phosphate-bufferedsaline, to remove pravastatin, and PDGF-BB (30 ng/ml) was thenreadded for 24 h. PDGF-BB was added 1 h after pravastatin in allcases where pravastatin was added. In the “no wash” cells, pravastatinwas not removed after its addition. [3H]-Thymidine (1mCi/well) wasadded for the last 8 to 11 h of incubation, and TCA-precipitableradioactivity was assessed by liquid scintillation. Data are expressedas average6 SEM of three wells per data point.P , 0.05 comparedto PDGF alone. The results shown are representative of two to fourseparate experiments.

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stimulation, did not cause PDGF-b receptor tyrosine phosphor-ylation, whereas in cells stimulated with PDGF, either in thepresence or absence of 100mM pravastatin, the receptorshowed a similar degree of tyrosine phosphorylation at 1 and3 h (Figure 2). Tyrosine phosphorylation of the PDGF receptorwas reduced after 24 h of pravastatin incubation, consistentwith a previous report (15). Thus, short-term pravastatin incu-bation does not affect PDGF-b receptor tyrosine phosphoryla-tion and, by inference, receptor/ligand binding.

Upon receptor autophosphorylation, signal transfer proteinsare recruited and become activated, generally by means oftyrosine phosphorylation (14). Several of these proteins areresponsible for early activation of Ras. Ras requires farnesy-lation to remain tethered to the cell membrane, where it sub-sequently recruits Raf to the membrane for activation, leadingto subsequent activation of the mitogen-activated protein/ex-tracellular signal-regulated kinase (MAP/ERK) signaling path-way (16). Long-term exposure to the HMG-CoA reductaseinhibitors is known to inhibit lipidation of such second-mes-senger proteins as Ras, which becomes associated with GTPupon activation, but whether short-term exposure causes sim-ilar Ras changes is not known. Serum-starved VSM cells wereplaced in medium supplemented with [32P]-orthophosphate,preincubated with pravastatin for 1 h, and stimulated for 1 or 5min with PDGF-BB. After immunoprecipitation of the lysatewith monoclonal anti-v-H-Ras antibody (which recognizes thep21 translational products of the H-, K-, and N-Ras genes[(17)]), radiolabeled Ras was assessed by thin-layer chroma-tography, and spots on the chromatography plate representingGTP and GDP were identified using nonradioactive standards.PDGF-BB increased Ras-GTP association in control cells after1 min of PDGF stimulation, and there was no diminution ofRas-GTP loading after 1 h preincubation of the cells withpravastatin (Figure 3). Thus, short-term pravastatin exposurehas an anti-mitogenic effect in VSM cells without decreasingactivation of Ras.

We next asked whether another signal transfer protein that isinitiated by binding to activated receptor and is required forPDGF-induced growth is being modulated by short-term prav-astatin exposure. PI3K is activated by the stimulated PDGFreceptor and has been shown to be required for DNA synthesisand trafficking by PDGF (18–20) in several cell lines. Thereappear to be distinct binding sites on the PDGF receptor forPI3K and for upstream elements of the Ras/Raf/MAPK path-way (19–21). We thus examined the activity of PI3K as apossible mediator of growth inhibition by short-term pravasta-tin incubation. VSM cells were serum-starved for 2 d, exposedto pravastatin for 10 min to 24 h before PDGF-BB stimulationfor 10 min, immunoprecipitated with anti-phosphotyrosine an-tibody, and the resulting immunoprecipitate was assessed forPI3K activity (Figure 4A). After incubation of the cells withpravastatin for 10 min, there was a 43% decrease in PI3Kactivity, and by 1 h PI3K activity was reduced by 65% ofmaximum (Figure 4B). To confirm the PI3K assay results, wenext examined the phosphorylation of Akt, a downstreammolecule in the anti-apoptotic pathway of PI3K (22,23). Thecells were preincubated with pravastatin for 1 h and subse-quently exposed to PDGF for an additional 1 or 24 h, and thenWestern-blotted with phospho-Akt antibody. After 1 h prein-cubation with pravastatin, phospho-Akt was inhibited when thecells were exposed to PDGF for either 1 or 24 h (Figure 5),consistent with our PI3K activity assay results (Figure 4).

Figure 2.Tyrosine phosphorylation of the PDGF-b receptor is unaf-fected by short-term pravastatin incubation. VSM cells were grown toconfluence and serum-starved as in Figure 1. Cells were exposed to100 mM pravastatin (P1, P3, P24) for the times indicated (in hours)before addition of PDGF-BB (30 ng/ml) (where indicated) for 10 min.Control cells (C) did not receive pravastatin. After immunoprecipita-tion with PDGF-b receptor antibody, the lysate was electrophoresedand Western blotted with phosphotyrosine antibody. The band repre-senting the PDGF-b receptor is indicated by an arrow. The resultsshown are representative of three separate experiments.

Figure 3.PDGF-BB stimulated Ras-GTP association is not inhibitedafter 10 min and 1 h ofpravastatin incubation. VSM cells were grownto confluence and serum-starved as in Figure 1. The medium wasreplaced with phosphate-free medium for 2 h before the addition of[32P]-orthophosphate for 3 h. Pravastatin (100mM) was then addedwhere indicated for 1 h before the cells were stimulated withPDGF-BB (30 ng/ml) for 1 or 5 min before cell lysis. Control cellswere not stimulated with PDGF. The lysate was immunoprecipitatedwith Ras antibody and separated by thin-layer chromatography, andthe plate was visualized with a PhosphorImager. Mobilities of non-radioactive GTP and GDP standards were visualized by ultravioletlight. The results shown are representative of two separate experi-ments.

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Pravastatin alone increased Akt to a slight degree in both cases,probably due to autocrine secretion of growth factors for the 1or 24 h that the cells were in quiescent media supplementedwith pravastatin. Thus, PI3K inhibition may contribute to theanti-mitogenic effect of pravastatin. However, since this mol-ecule has no known farnesylation sites, we hypothesize that itis affected by the statins independently of their prenylationinhibitory property. Since PI3K is activated by proteins in theMAP/ERK cascade (e.g., MEK [(24)]), we next examined theeffect of short-term pravastatin on this signaling pathway.

To determine whether elements of the MAP/ERK kinasepathway downstream of Ras are inhibited by short-term prav-astatin incubation, we next examined MAP kinase activity afterboth short and long pravastatin incubation times. MAP kinaselies downstream of Ras and Raf in the MAP/ERK kinasepathway used for mitogenesis by receptor tyrosine kinases(25). Serum-starved VSM cells were preincubated with prav-astatin for 1 or 24 h and then exposed to PDGF-BB for 10 min.Active ERK1/2 was immunoprecipitated with phospho-spe-

Figure 4. PDGF-BB-stimulated PI3K activity is decreased by short-term incubation with pravastatin. VSM cells were grown to conflu-ence in 6-cm plates and serum-deprived as in Figure 1. Cells wereexposed to 100mM pravastatin for the times indicated (in hours)before addition of PDGF-BB (30 ng/ml) (where indicated) for 10 min.Control cells (C) did not receive pravastatin. The cell lysate wasnormalized for protein content, and equal protein quantities of eachlysate were used in each experiment. The lysate was immunoprecipi-tated with phosphotyrosine antibody. (A) Incorporation of [g32P]-ATP into phosphatidylinositol-phosphate catalyzed by the lysate wasperformed, the lipid phase reaction products were separated by thin-layer chromatography (TLC), and the TLC plate was visualized witha PhosphorImager. PIP represents the position of nonradioactive phos-phatidylinositol-3 phosphate visualized by iodine vapor. (B) Densi-tometry of the phosphatidylinositol-3 phosphate spot was determinedby a PhosphorImager and pooled from three experiments. Controlcells (s) were incubated with pravastatin (where indicated) but werenot stimulated with PDGF.o, PDGF-stimulated cells. *P , 0.05relative to PDGF alone. The results shown are representative of threeseparate experiments.

Figure 5.Akt phosphorylation is decreased by 1 h pravastatin prein-cubation. VSM cells were grown to confluence in 6-cm plates andserum-deprived as in Figure 1. Cells were exposed to 100mMpravastatin for 1 h and then PDGF-BB (PD; 30 ng/ml) was added foran additional 1 or 24 h. The cell lysate was normalized for proteincontent, and equal protein quantities of each lysate were used in eachexperiment. (A) The lysate was electrophoresed and Western blottedwith phospho-Akt antibody. (B) Densitometry of the phospho-Aktbands (representing different degrees of phosphorylation) was deter-mined with a Bioimager gel reader. In PDGF-stimulated lanes, cellsexposed to 1 h PDGF (f) and 24 h PDGF (s) are so indicated. Theresults shown are representative of two separate experiments.

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cific MAP kinase antibody and reactedin vitro with its sub-strate Elk-1 (26–28); phospho-Elk-1 was visualized by West-ern blotting (Figure 6). In agreement with our Ras data, whileMAP kinase activation was strongly induced by PDGF-BB,there was no diminution in PDGF-stimulated MAP kinaseactivity after incubation with pravastatin for 1 h.

Raf lies in the MAP/ERK kinase mitogenic signaling cas-cade for PDGF and has been shown to mediate both growthstimulation and inhibition (29), as well as apoptosis (30). Weand others have shown that increased Raf activity does notnecessarily correlate with either increased cell growth or hy-perphosphorylation on an sodium dodecyl sulfate gel (4,31),but the state of Raf activity can offer clues to its mechanism ofsignaling (vide infra). We thus examined Raf activity and gelmobility after exposure of cells to pravastatin for both shortand long periods. Serum-starved VSM cells were exposed topravastatin for 1 or 24 h and then stimulated with PDGF-BBfor 10 min. The lysate was divided in half; Raf activity, usinga cascade reaction resulting in Elk-1 phosphorylation, wasdetermined with part of the lysate, and Raf electrophoreticmobility, by Western blotting, with the other. Although the Rafhyperphosphorylation observed after PDGF stimulation wasunaffected by pravastatin incubation at all times examined(Figure 7A), Raf catalytic activity was consistently increasedby pravastatin (Figure 7B), at both short and long incubationtimes, in a manner that paralleled growth inhibition (Figure 1).Raf activity was decreased in PDGF-stimulated cells (com-pared with controls) despite increased hyperphosphorylation,consistent with other reports suggesting that the more highlymodified (slowly migrating) form of Raf is actually inactive(4,32,33): Active Raf may in this case be acting as a growthinhibitory signal (4,31), such that inactive Raf allows PDGF tobe growth stimulatory under these conditions.

We describe above our observation of increased Raf activityassociated with growth arrest, and others have shown that such

Raf-induced cell cycle arrest is mediated by such cdk inhibitorsas p21Waf1/Cip1(29,34) and p27Kip1 (31). Furthermore, since (1)we showed above that Ras inhibition does not account for theeffect of short-term pravastatin incubation on cell growth, (2)PI3K has been shown to be dependent on Rho activity (35,36),and (3) Rho is inhibitory toward cdk inhibitors (37,38), we nextdecided to examine the stability of Rho after 1 h statin expo-sure. Serum-starved VSM cells were pulsed with [35S]-methi-onine for 2 h, with pravastatin added for the last hour, andchased with quiescent media in the presence of PDGF-BB for2 to 4 h (39), and the lysate was then immunoprecipitated withRho antibody (which recognizes both RhoA and RhoB). Afterthe addition of protein A-Sepharose beads, the lysate waselectrophoresed, and the gel dried and autoradiographed. Incontrol cells, [35S]-methionine-containing Rho decreasedmarkedly by 4 h, indicating degradation of this protein as hasbeen reported for RhoB (39). However, the level of [35S]-methionine-containing Rho in the 1-h pravastatin preincubatedcells remained constant at 4 h, demonstrating that its stabilitywas increased by pravastatin (Figure 8). We suggest that theincreased stability of this molecule is due to the fact thatpravastatin allows Rho to remain bound to its inhibitor Rho-GDI (since its lipidation is inhibited), and is therefore resistantto proteolytic degradation (40).

Rho has been shown to suppress transcription of the cdkinhibitor p27Kip1 to allow Ras to increase transit through thecell cycle (38), and long-term lovastatin has been shown todecrease mesangial cell proliferation through p27Kip1 induction

Figure 6. Mitogen-activated protein (MAP) kinase activity is notreduced after 1 h of pravastatin preincubation. VSM cells were grownto confluence and serum-starved as in Figure 1. Cells were exposed to100 mM pravastatin (P1, P3, P24) for the times indicated (in hours)before addition of PDGF-BB (30 ng/ml) (where indicated) for 10 min.Control cells (C) did not receive pravastatin. Phospho-MAP wasimmunoprecipitated and reactedin vitro with Elk-1 fusion protein asdescribed in Materials and Methods; phospho-Elk-1 was identified byWestern blotting and is indicated by an arrow. The thick upper bandsrepresent IgG from the immunoprecipitation. The results shown arerepresentative of three separate experiments.

Figure 7.Raf activity is increased by pravastatin, with no change inhyperphosphorylation. VSM cells were grown to confluence in 6-cmplates and serum-deprived as in Figure 1. Cells were exposed to 100mM pravastatin for the times indicated (in hours) before addition ofPDGF-BB (30 ng/ml) (where indicated) for 10 min. Control cells (C)did not receive pravastatin. The cell lysate was normalized for proteincontent, and equal protein quantities of each lysate were used in eachexperiment. (A) The lysate was electrophoresed and Western blottedwith Raf-1 antibody. (B) The same lysate as in Panel A was immu-noprecipitated with Raf-1 antibody, and Raf catalytic activity wasdetermined as described in Materials and Methods. The results shownare representative of three separate experiments.

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(41). Other cdk inhibitors, such as p21Waf1/Cip1, as well asp27Kip1, have also been shown to mediate growth arrest insituations in which Raf is activated (29,31). We thus examinedthe effect of short-term pravastatin incubation on p27Kip1 pro-tein levels. Western blotting of cell lysate with p27Kip1 anti-body demonstrated an increase in p27Kip1 to control levelswhen cells were stimulated with PDGF 1 h and 24 h after 1 hpreincubation with pravastatin (Figure 9), consistent with thismolecule being an effector of Rho (38) and the mediator ofarrest of PDGF-induced growth observed in our cells after 1 hpravastatin exposure.

Since our initial observation was that PI3K activity wasinhibited by short-term pravastatin exposure, we next sought tocorrelate these data with the mechanism of growth inhibitionelucidated above. The C3 exoenzyme ofClostridium botuli-num has been shown to catalyze an ADP-ribosyltransferase

reaction that interferes with the biologic effects of Rho byADP-ribosylating and inactivating this small G protein (36,42).VSM cells were scrape-loaded with C3 exoenzyme as has beendescribed (a procedure that loads.80% of cells with C3[(43)]), and PI3K activity was assessed both with and withoutstimulation by PDGF-BB. Because the cells did not tolerateserum starvation after scrape loading, basal PI3K activity wasnot absent in the PDGF nonstimulated cells (since they werenot serum-starved). Although PDGF-BB increased PI3K activ-ity in the non-C3-loaded cells, those cells scrape-loaded withC3 isoenzyme demonstrated no PDGF-BB-mediated increasein PI3K activity (Figure 10), supporting our supposition thatthe decreased PI3K activity observed with short-term prava-statin incubation is in fact due to Rho inhibition.

The activated Raf (44) and PI3K/Akt (22) pathways de-scribed above have been shown to mediate apoptosis, whichmay be the mechanism of the anti-mitogenic, as well as themyopathic, effect of the statins. To examine whether short-term pravastatin is also capable of mediating this effect in ourcell line, we performedin situTUNEL assays. We consistentlyfound apoptosis with short times of pravastatin incubation,with approximately 10% apoptosis after only 4 h ofpravastatinincubation (Figure 11).

DiscussionWe report here a novel finding that PDGF-BB stimulation of

DNA synthesis is attenuated by an HMG-CoA reductase in-hibitor when VSM cells are incubated for only 1 h beforegrowth factor stimulation. We show, consistent with work fromother investigators (8,10), that this is insufficient time forabolition of Ras activation. Rather, our data indicate that thisantiproliferative effect correlates with increased Raf activityand is most likely due to increased levels of the cdk inhibitorp27Kip1 mediated by decreased Rho and/or PI3K activation

Figure 8.Stability of Rho is increased after 1 h pravastatin preincu-bation. VSM cells were grown to confluence in 6-cm plates. The cellswere pulsed with [35S]-methionine for 2 h in methionine-deficientmedium, with pravastatin added for the last hour, chased with quies-cent media in the presence of PDGF-BB (30 ng/ml) for 2 to 4 h, andthe lysate was then immunoprecipitated with Rho antibody. The cellswere harvested at the indicated times (after complete medium addi-tion), immunoprecipitated with Rho antibody, and electrophoresed,and the gel was dried and examined with a PhosphorImager. Theresults shown are representative of two separate experiments.

Figure 9.p27Kip1 protein level is increased by PDGF exposure after 1 h pravastatin preincubation. VSM cells were grown to confluence in 6-cmplates and serum-deprived as in Figure 1. Cells were exposed to 100mM pravastatin for 1 h and then PDGF-BB (PD; 30 ng/ml) was addedfor an additional 1 or 24 h. The cell lysate was normalized for protein content, and equal protein quantities of each lysate were used in eachexperiment. The lysate was electrophoresed and Western blotted with p27Kip1 antibody. The results shown are representative of two separateexperiments.

1886 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1880–1890, 1999

(Figure 12). Our finding that p27Kip1 is increased when thecells are stimulated by pravastatin is consistent with otherreports of Raf-mediated cell cycle inhibition mediated by thecdk inhibitors p27Kip1 (31,45) and p21Waf1/Cip1 (29,34). Wefound that PI3K activity is decreased after short-term prava-

statin incubation, an effect that we also show to be likelymediated by the effect of HMG-CoA reductase inhibition onRho. Since the PDGF receptor requires PI3K for its mitogeniceffect (18,19), it is unclear whether the cell cycle arrest inducedby short-term pravastatin incubation is mediated directly byp27Kip1 or through an effect on PI3K.

Our data showing that ADP-ribosylation of Rho (by C3

Figure 10.PDGF-stimulated PI3K activity is decreased by C3 exoen-zyme. VSM cells were grown to confluence in 6-cm plates andserum-deprived as in Figure 1. Non-serum-starved cells were scrape-loaded with C3 exoenzyme (where indicated) as described in Mate-rials and Methods and grown overnight. After stimulation withPDGF-BB for 10 min, cells were lysed, equal protein quantities oflysate were immunoprecipitated with anti-phosphotyrosine antibody,and PI3K activity was determined. (A) Incorporation of32P intophosphatidylinositol-phosphate, catalyzed by the lysate, was per-formed, the lipid phase reaction products were separated by TLC, andthe TLC plate was visualized with a PhosphorImager. PIP representsthe position of nonradioactive phosphatidylinositol-3 phosphate visu-alized by iodine vapor. (B) Densitometry of the phosphatidylinositol-3phosphate spot was determined by a PhosphorImager and is expressedas the fraction of control density and the range of the two experiments.o, cells stimulated with PDGF.■, control cells not stimulated. Theresults shown are representative of two separate experiments.

Figure 11.Apoptosis is induced by pravastatin at short times. Non-serum-starved VSM cells were grown on glass coverslips, incubatedwith pravastatin (100mM) for the times indicated, and subjected toinsitu terminal deoxynucleotidyl transferase-mediated nick end labelingassays. The results are mean of apoptotic cells per plate and the rangeof two separate experiments.

Figure 12.Ras-independent inhibition of growth by short-term prav-astatin is via Rho and PI3K. Short-term incubation of VSM cells withpravastatin likely inhibits growth through the scheme described in theshaded part of the figure. Short-term pravastatin increases Rho sta-bility, probably as a result of its decreased lipidation and consequentassociation with Rho-GDI. This leads to inhibition of PI3K activity,an event that is reproduced by inactivation of Rho by C3 exoenzyme.The release of tonic inhibition on p27Kip1 from Rho and/or PI3Kcauses increased p27Kip1 activity, leading to inhibition of cell cycletransit. Raf and MAP kinase were not inhibited by short-term prava-statin. PDGF-R is the PDGF receptor.

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exoenzyme) inhibits PDGF-BB stimulation of PI3K activityare consistent with work from other laboratories (35,36), butthe mechanism by which Rho influences PI3K remains obscureand was not investigated here. Because of its role as an orga-nizer of the actin cytoskeleton, it is possible that Rho may befunctioning as a membrane-directing element in the signalingcascade of PI3K, such that the non-lipidated protein mayprevent some PI3K molecules, despite functionalsrc-homol-ogy domains, to be directed to activated growth factor receptor.However, PI3K is unlikely to be the sole mediator of growthinhibition by short-term pravastatin. Based on our data show-ing that p27Kip1 protein levels, attenuated by PDGF stimula-tion, are increased to control levels after 1 h of pravastatinpreincubation, we believe that the Rho/p27Kip1 pathway mayplay a role at least equal to PI3K inhibition in the eventsleading to cell cycle arrest mediated by this drug. Our findingthat growth suppression is not complete with short-term, high-dose pravastatin is not inconsistent with our proposed mecha-nism. Many cells have evolved multiple and redundant path-ways for growth control, and we believe it is likely that someof these other cascades may come into play when both PI3Kand Rho are modulated as we have described. Of course,another possibility is that we have not completely inhibitedPI3K, or maximally stimulated p27Kip1, with the doses ofpravastatin used.

We found no effect of short-term pravastatin on bothPDGF-b receptor autophosphorylation and MAP kinase acti-vation, suggesting that the “classical” MAP/ERK pathway isnot involved in growth inhibition under these circumstances.Others have shown that tyrosine phosphorylation of the insulinreceptor and IRS-1 is inhibited by overnight lovastatin incu-bation (46). Although we did not find this to be the case for thePDGF receptor in our short-term pravastatin studies, that dataaffirm that there exist non-lipidation signaling events, such aspossibly a direct effect on PI3K, that may be affected by thestatins. Another group has shown that phosphatidylinositol-3,4,5-P3, the in vivo product of PI3K, may act to recruitSH2-containing proteins to the plasma membrane, suggesting ameans by which PI3K could be contributing to the assembly ofsignaling proteins to the membrane (47); whether this action insome way involves lipidation is not known. There are otherreports of Ras-independent mitogenic inhibition by an HMG-CoA reductase inhibitor, but this phenomenon was reported inNIH-3T3 cells transformed by oncogenes, and was examinedafter 2 to 3 d of lovastatin incubation (48). Other investigatorshave shown that newly synthesized RhoA, not Ras, is themoiety that is involved in cell cycle progression due to avariety of growth stimuli (49). These authors are in agreementwith our findings that membrane-associated Ras is not alteredby pravastatin.

Other investigators have shown that Rho processing is af-fected by the HMG-CoA reductase inhibitors, but there areconflicting data regarding the effect of pravastatin. One groupshowed that the reappearance of membrane-bound RhoA aftergrowth stimulation of FRTL-5 cells was blocked by 36 h ofpravastatin incubation (49), while another group showed thatRhoB prenylation was unaffected by 24 h incubation of rat

VSM cells with this drug (although prenylation was affected byother statins) (50). Our finding that the stability of Rho isincreased by short-term statin incubation, likely due to itscomplex with Rho-GDI and thus resistance to proteolysis (40),is consistent with a short-term pravastatin effect on this GT-Pase.

Other investigators have shown similar statin effects onp27Kip1. Pravastatin reduced p27Kip1 degradation in FRTL-5cells (45) and increased p27Kip1 protein levels in mesangialcells (41), and lovastatin increased p27Kip1 protein but notmRNA in HeLa cells (51). The latter authors show a modestincrease in p27Kip1 protein less than 2 h after release from G1,consistent with our finding of rapid induction of this proteinwith pravastatin.

Our finding of apoptosis induced by pravastatin is consistentwith other reports of statin effects in VSM cells (50,52). Thelatter authors did not observe apoptosis with pravastatin; how-ever, these investigators used only morphologic criteria andflow cytometry rather than TUNEL assays. Other investigatorshave demonstrated apoptosis induced by long-term exposure tothe farnesyltransferase inhibitors (53). In our VSM cells, webelieve that the PI3K arm of the signaling pathway affected bypravastatin may be responsible for mediating apoptosis. Ourfinding of decreased levels of Akt after both short- and long-term pravastatin incubation is consistent with other reports ofAkt mediating survival and its inhibition, apoptosis (22,23).Whether the apoptosis observed with statin incubation contrib-utes to the myopathic effect of the statins, the most commonadverse effect seen especially at high doses (54), is a distinctpossibility based on our new data and remains to be explored.

Our data thus demonstrate a novel mechanism by which thestatins are capable of inhibiting receptor tyrosine kinase growthfactor mitogenic signaling. This short-term pravastatin signal-ing pathway is probably separate and independent of long-termstatin-induced inhibition of Ras farnesylation, which has beenwell described (55,56) and is likely independent of anti-lipi-dation effects. The novelty of our results is that we find anacute inhibitory effect of pravastatin on a key, non-Ras mito-genic signaling pathway. Our finding that short-term pravasta-tin incubation at high concentrations (the concentration of 100mM used in these studies is 667 times higher than the “thera-peutic” Cmax in humans [(57)]) causes Ras-independent VSMcell growth arrest as well as apoptosis has seminal implicationsin the treatment of a number of disparate disorders. For exam-ple, pravastatin could be applied locally at high concentrationsduring angioplasty to prevent the common complication ofrestenosis without toxicity associated with long-term Ras in-hibition in normal cells. In addition, since hemodialysis accessstenosis has been shown to involve aberrant VSM cell prolif-eration, especially at the venous anastomosis, statins mayprove useful as therapy for this disorder.

Our work on the effect of short-term, high-dose statins onmitogenic signaling pathways may prove clinically useful astherapy for VSM-mediated processes such as angioplasty re-stenosis and hemodialysis vascular access failure. To confirmthe feasibility of such supra-therapeutic doses in humans, arecent Phase I clinical study showed minimal toxicity of high-

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dose pulsed lovastatin (approximately 45 times the normaltherapeutic doses [(54)]), the dose-limiting effect being myop-athy. Since we now show that the anti-mitogenic, as well aspossibly the myopathic, effect of high-dose statins are associ-ated not with Ras inhibition, but rather with cdk inhibitorinduction, PI3K inhibition, and Rho modulation, other agentsthat are more specific to these signaling pathway and that donot affect Ras could be developed using this new paradigm.

AcknowledgmentsThis work was supported by the Research Service of the U.S.

Department of Veterans Affairs, by a grant from the National KidneyFoundation, and by a gift from Dialysis Clinics, Inc. We thank AnithaToke and Tina Chang for technical assistance.

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