Simvastatin Modulates Angiotensin II Signaling Pathway by Preventing Rac1-Mediated Upregulation of p27
Lixia Zeng*,
Hanshi Xu*,
Teng-Leong Chew,
Rex Chisholm,
Mehran M. Sadeghi,
Yashpal S. Kanwar and
Farhad R. Danesh*
*Division of Nephrology/Hypertension, Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois; Division of Cardiology, Yale University School of Medicine, New Haven, Connecticut; and Department of Pathology, Northwestern University, Chicago, Illinois
Correspondence to Dr. Farhad R. Danesh, Division of Nephrology/Hypertension, Feinberg School of Medicine, Northwestern University Medical School, Morton Building 2-615, 303 East Chicago Avenue, Chicago, IL 60611. Phone: 312-503-4753; Fax: 312-503-0622; E-mail: f-rahimi{at}northwestern.edu
ABSTRACT. Recent experimental observations have suggested thatstatins may exert modulatory effects on a number of pathobiologicalprocesses beyond their cholesterol-lowering properties. Someof the pleiotropic effects of statins seem to be mediated bytheir ability to block the synthesis of isoprenoid intermediates,which serve as important lipid attachments required for theproper function and activation of the small GTP-binding proteins.The current study explored the modulatory effects of simvastatin(SMV) on the angiotensin II (Ang II)-induced Rac1-mediated,upregulation of cyclin-dependent kinase inhibitor p27. Ang II(100 nM) stimulation of rat mesangial cells induced a significantincrease in p27 protein expression. Co-treatment of cells withSMV (1 µM) inhibited Ang II–induced upregulationof p27 protein. Addition of mevalonate (200 µM) or geranylgeranylpyrophosphate (5 µM) reversed the inhibitory effect ofSMV on p27 protein expression, suggesting that the effect ofSMV is geranylgeranyl dependent. This study also provides evidencefor a sequential link between Ang II stimulation and downstreamactivation of Rac1, intracellular H2O2 production, and Akt kinaseleading to upregulation of p27 protein in mesangial cells. Itwas also shown that SMV, by inhibiting Rac1 activity, reversedAng II–induced increase in intracellular H2O2 production,Akt activation, and p27 protein expression. The data presentedin this study not only elucidate Ang II–mediated signalingcascade in mesangial cells but also demonstrate for the firsttime the modulatory effects of SMV on Ang II–induced signalingpathway at the cell cycle level.
The 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, orstatins, are potent inhibitors of cholesterol biosynthesis thatare used extensively in the treatment of patients with hypercholesterolemia(1,2). Several studies have demonstrated the beneficial effectsof statins in reducing cardiovascular-related morbidity andmortality (3,4). Recently, it was also suggested that statinsmay confer renoprotection in a variety of glomerular diseases,including diabetic nephropathy (5,6). It is usually assumedthat the beneficial effects of statins result from the competitiveinhibition of cholesterol synthesis. However, stat- ins mayalso exert additional effects on cell signaling pathways bypreventing the synthesis of various isoprenoids derived fromthe mevalonate (MEV) pathway, such as farnesyl pyrophosphate(FPP) and geranylgeranyl pyrophosphate (GGPP) (7,8). Both FPPand GGPP are important lipid attachments required for the subcellularlocalization and function of a variety of proteins, includingsmall GTPase-binding proteins (9). In support of cholesterol-independentor pleiotropic properties of statins in diabetic nephropathy,we and others recently showed that some of the beneficial effectsof statin therapy in diabetic milieu may be mediated via modulationof small GTPase proteins (10,11).
The Rho family of small GTPases are 20- to 40-kD monomeric Gproteins that can cycle between two interconvertible forms:GDP-bound (inactive) and GTP-bound (active) states (12,13).Activation of transmembrane growth factor receptors can promotethe exchange of GDP to GTP on Rho proteins, causing membranetranslocation and activation of GTP-bound Rho proteins. Recentstudies in a variety of cell types have demonstrated that theRho family of small GTPases may play a crucial role in regulatingcellular hypertrophy (12–15). For instance, it has beenshown that Rac1, a member of Rho family of small GTPases, iscritical for the signal transduction leading to cardiac myocytesand mesangial cell (MC) hypertrophy via activation of reactiveoxygen species (ROS) (14,16).
Glomerular cell hypertrophy is a characteristic lesion of earlystages of diabetic nephropathy (17–19). Intrarenal angiotensinII (Ang II), hyperglycemia, and TGF- hierarchically and/or coordinatelyhave been suggested as the mediators of cell hypertrophy indiabetic nephropathy (20–22). Ang II, an octapeptide hormone,exerts both hemodynamic (leading to increased glomerular capillarypressure) and nonhemodynamic effects (stimulation of cellularhypertrophy and extracellular matrix expansion). Mediators ofAng II–induced MC hypertrophy in diabetic milieu havenot fully been identified. However, several observations haveshown the pivotal role of p27, a member of CIP/KIP family ofcyclin-dependent kinase inhibitors (CDKI), in glucose and AngII–induced cellular hypertrophy (23–27).
In the current study, we examined the modulatory effects ofsimvastatin (SMV) on a novel signaling pathway by which 3-hydroxy-3-methylglutaryl-CoAreductase inhibitors may ameliorate the detrimental effectsof Ang II, independent of their cholesterol-lowering properties,on MC hypertrophy. To this aim, we investigated the modulatoryeffect of SMV on the Ang II–induced Rac1-regulated, NADPH-dependentoxidase signaling pathway involving Akt activation and upregulationof CDKI p27.
Reagents and antibodies
DMEM/F12, FBS, and PBS were obtained from Invitrogen (Carlsbad,CA). Angiotensin, diphenylene iodonium (DPI), MEV, GGPP, andFPP were purchased from Sigma (St. Louis, MO). Anti-Akt andanti phospho-Akt (Ser473) were commercially purchased (CellSignal Technology, Beverly, MA). Akt1 cDNA allelic pack andRac Activation Assay Kits were obtained from Upstate Biotechnology(Lake Placid, NY). Anti-p27 antibody was purchased from BD BiosciencePharmingen (San Diego, CA), and fluorescence probe 2', 7'-dichlorofluororescindiacetate (DCF-DA) was obtained from Molecular Probes (Eugene,OR). SMV and MEV were chemically activated as described previously(10).
Cell Culture and Transfection
Rat glomerular mesangial cells were grown in DMEM/F12 mediumcontaining 10% FBS, 100 U/ml penicillin, and 100 µg/mlstreptomycin in a humidified incubator at 37°C under 5%CO2. Transfections of different mutants were performed as describedpreviously (10). Briefly, in transfection studies, cells weregrown to 50% confluence and then transfected with 1 µgof fusion plasmid DNA using LipofectAMINE Reagent accordingto the manufacturer’s protocol (Invitrogen). Transfectedcells were grown in 800 µg/ml G418 (Invitrogen) untilcolonies were formed. Subsequently, the colonies were grownin growth medium containing 800 µg/ml G418 and incubatedat 37°C and 5% CO2 until the cells achieve 70% confluence.The confluent cells were trypsinized and grown in growth mediumcontaining 10% FBS and 800 µg/ml G418. Plasmids containingwild-type Rac1 (pcDNA3 Rac1) and dominant negative Rac1 construct(pcDNA3 Rac1N17) were gifts of Dr. Jacob Sznajder (NorthwesternUniversity, Chicago, IL).
Western Blotting
For each experiment, a total of 5 x 105 cells were seeded, andat subconfluence (70%), cells were made quiescent for 48 h.Cells were rinsed twice with ice-cold PBS and scraped in 500µl of ice-cold lysis buffer (20 mM Tris [pH 7.5], 150mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodiumpyrophosphate, 1 mM PMSF, 10 µg/ml leupeptin, 100 kiu/mlaprotinin, and 1 mM Na3VO4). The samples were centrifuged anddissolved in SDS sample buffer (187.5 mM Tris [pH 6.8], 6% SDS,30% glycerol, 150 mM dithiothreitol, and 0.03% bromphenol blue)and boiled for 5 min. Protein concentrations were determinedby the BCA protein assay (Pierce, Rockford, IL). Equal amountsof protein were separated on SDS–polyacrylamide gels andtransferred to nitrocellulose membranes. The membranes wereprobed with primary antibodies at 4°C overnight (at a dilutionof 1:100 for p27, 1:500 for AKT, and 1:500 for Rac1) and incubatedwith the appropriate secondary antibodies for 1 h. Immunoreactivebands were visualized by enhanced chemiluminescence reaction.Each blot is representative of at least three similar experiments.
Rac1 Activation Assay
Rac1 activity was determined by a pull-down assay accordingto the instructions by the manufacturer (Rac activity assaykit; Upstate Biotechnology). In brief, 107 cells were grownin 10-cm dishes, washed in cold PBS, and lysed in ice-cold MLBbuffer (25 mM HEPES [pH 7.5], 150 mM NaCl, 1% NP-40, 10 mM MgCl2,1.0 mM EDTA, and 2% glycerol). Samples were centrifuged andincubated for 60 min at 4°C with 10 µl of PAK-1 PBD-agaroseto precipitate GTP-bound Rac1. Precipitated complexes were washedthree times in MLB buffer and resuspended in 30 µl of2x Laemmli buffer. Total lysates and precipitates were analyzedby performing SDS-PAGE and Western blot analysis using mousemonoclonal antibody against Rac1 (at a dilution of 1:500; UpstateBiotechnology).
Detection of Intracellular H2O2
The H2O2-sensitive fluorescence probe DCF-DA was used to assessthe generation of intracellular H2O2. This compound is convertedby intracellular esterases to 2',7'-dichlorofluorescin, thenoxidized by H2O2 to the highly fluorescence 2',7'-dichlorofluorescen.Cells were grown to near confluence in coverglass chambers andthen made quiescent by serum starvation for 48 h. Cells werestimulated with Ang II (100 nM) for 1 h and incubated with 10µM DCF-DA for 30 min at 37°C. The DCF fluorescencewas visualized at an excitation wavelength of 488 nm and emissionat 520 nm using a bandpass filter on Zeiss LSM510 laser scanningconfocal microscope.
Confocal Laser Scanning Fluorescence Microscopy
MC were grown on glass coverslips. The cells were fixed with3.7% formaldehyde and permeabilized with 0.3% Triton X-100 inPBS for 10 min at room temperature. Cells were then incubatedwith anti-P27 Kip1 antibody for 2 h at room temperature. Coverslipswere washed and incubated with TRITC-conjugated secondary antibody(Zymed Laboratories, South San Francisco, CA). After DAPI staining(3 µg/ml), the coverslips were mounted on glass slideswith antifade mounting media (Molecular Probes) and examinedusing a confocal fluorescence microscope (Zeiss LSM510).
[3H]Leucine Incorporation
MC were plated in 24-well plates, serum starved for 48 h, andthen exposed to Ang II (100 nM) in the presence or absence ofSMV (1 µM) for 24 h with addition of 1 µCi of [3H]leucinefor 6 h (Amersham, Piscataway, NJ) and then processed for determinationof incorporated radioactivity after precipitation with 10% TCAas described previously (10).
Statistical Analyses
ANOVA with a Student-Newman-Keuls test was used to evaluatedifferences between two or more different experimental groups.Results are expressed as mean ± SEM. P 0.05 was consideredas statistically significant.
Effect of SMV on Ang II–Induced p27 Protein Expression
Ang II–mediated MC hypertrophy is characterized by theinduction of CDKI p27 at the cell cycle level (23–27).To define the modulatory effect of SMV on Ang II–inducedupregulation of p27 protein expression, cultured rat MC weremade quiescent by serum deprivation for 48 h and exposed toAng II (100 nM) in the presence or absence of SMV (1 µM)for 6 h. As shown in Figure 1, Ang II–treated MC exhibiteda significant increase in p27 protein expression that was reversedwith co-treatment of cells with SMV as determined by Westernblot analysis. For determining the role of various isoprenoidsderived from MEV in regulating the inhibitory effect of SMVon Ang II–induced upregulation of p27 protein, Ang II–stimulatedMC were co-treated with 1 µM SMV and MEV (200 µM)or various isoprenoids intermediaries (geranylgeranyl pyrophosphate[GGPP; 5 µM], farnesyl pyrophosphate [FFP; 5 µM],and squalene [SQ; 5 µM]). As shown in Figure 1, co-treatmentof cells with MEV reversed the inhibitory effect of SMV on p27protein expression. In addition, co-treatment of cells withGGPP but not with FPP or SQ reversed the inhibitory effect ofSMV, suggesting that the modulatory effect of SMV is geranylgeranyldependent and independent of cholesterol biosynthesis as SQ,an immediate precursor of cholesterol, failed to reverse theeffect of SMV on p27 protein expression.
Figure 1. Modulatory effect of simvastatin (SMV) on angiotensin II (Ang II)-induced p27 protein expression. (A) Mesangial cells (MC) were pretreated with SMV (1 µM) before Ang II stimulation (100 nM). SMV inhibited Ang II–induced upregulation of p27 in MC. Co-treatment of cells with mevalonate (MEV) and geranylgeranyl pyrophosphate (GGPP) reversed the effect of SMV on p27 protein expression. However, co-treatment of cells with farnesyl pyrophosphate (FPP) and squalene (SQ) failed to reverse the effect of SMV, indicating that the modulatory effect of SMV is geranylgeranyl-dependent. (B) Densitometric analysis of p27 protein levels (n = 3; *P = 0.02 versus control).
Modulatory Effect of SMV on Ang II–Induced Rac1 Activation
We recently reported that SMV modulates the activation of theRho family of small GTPases in diabetic milieu (10). To decipherwhether Rac1, a member of the Rho family of small GTPases, mediatesAng II–induced increase in p27 protein expression, ratMC were exposed to Ang II (100 nM), and the temporal profileof Ang II–induced Rac1 activation was determined. Rac1activation was measured by an affinity pull-down assay usingGST fusion protein PAK1-PBD, which recognizes only the activeform of Rac1 (GTP-Rac1). Ang II stimulation of MC increasedRac1 activity by approximately threefold in MC after 5 min (Figure 2A).For studying the effect of SMV on Ang II–inducedRac1 activity, cells stimulated with Ang II were co-treatedwith SMV (1 µM). Co-treatment of Ang II–stimulatedMC with SMV reversed Ang II–induced increase in Rac1 activity,suggesting a novel role for SMV in modulating Ang II–inducedRac1-mediated signaling pathway in MC (Figure 2B). The inhibitoryeffect of SMV on Rac1 activity was reversed when cells wereco-treated with MEV (200 µM), indicating that the effectof SMV on Ang II–induced Rac1 activity is MEV dependent.As shown in Figure 2B, total Rac1 protein levels were unchanged.
Figure 2. Effects of Ang II and SMV on Rac1 activity. (A) Temporal profile of Ang II–induced Rac1 activity; Rac-GTP was pulled down with a PAK-1 fusion protein, and samples were subjected to Western blotting and detection with anti-Rac1 antibody. Rac1 activity significantly increased after 5 min of Ang II stimulation and remained elevated for 60 min. (B) SMV (1 µM) inhibited Ang II–induced Rac1 activation, and the effect of SMV was reversed with MEV (200 µM) without significant changes in total Rac1 protein expression. (C) Densitometric analysis of GTP-Rac1 (n = 3; *P < 0.05).
For establishing a sequential link between Rac1 activity andAng II–induced upregulation of p27 protein, MC were transfectedwith dominant-negative (N17Rac) and wild-type Rac1 and stimulatedwith Ang II (100 nM). As shown in Figure 3, Ang II stimulationfailed to increase p27 protein expression in dominant negativeRac1 transfected cells, indicating a critical role for Rac1activation in Ang II–induced upregulation of p27 in MC.
Figure 3. Effect of dominant-negative Rac1 on p27 protein expression. To determine whether Rac1 regulates Ang II–induced upregulation of p27, MC were transfected with dominant-negative Rac1 (N17 Rac) and wild-type Rac1 (wt Rac). Ang II stimulation in cells transfected with dominant-negative Rac1 exhibited significantly lower protein levels of p27 as compared with wild-type Rac1. The figure is representative of three separate experiments.
The protein abundance of p27 is known to be mainly regulatedby posttranslational mechanisms (28,29). Whereas P27 needs tobe transported into the nucleus to exert its effect on the cellcycle (30), degradation of p27 by a ubiquitin-dependent pathwayrequires cytoplasmic localization of p27 (31,32). Thus, thenext question that we addressed was the role of Rac1 on theAng II–induced nuclear translocation of p27. To this end,we investigated the subcellular localization of p27 in responseto Ang II using immunofluorescence microscopy. DAPI was usedas a marker for nuclear staining. As shown in Figure 4, quiescentcells exhibited nuclear and weak cytoplasmic staining of p27.Upon stimulation with Ang II, intense nuclear staining of p27was detected. However, cells transfected with dominant negativeRac1 mainly displayed diffuse cytoplasmic staining of p27, suggestingthat Ang II–induced nuclear translocation of p27 fromthe cytoplasm is Rac1 mediated. Thus, these data confirm thatRac1 plays a major role in Ang II signaling pathway in MC.
Figure 4. Effect of Rac1 on the nuclear translocation of p27. MC were treated with Ang II (100 nM), and samples were subjected to immunofluorescence microscopy. (Left) DAPI was used as a marker for nuclear staining. (Middle) Immunofluorescent microscopy of p27 using anti-p27 antibody. (Right) Merged images. Control cells exhibited nuclear and weak cytoplasmic staining of p27. After stimulation with Ang II, p27 nuclear staining became intense. However, cells transfected with dominant-negative Rac1 (N17 Rac) showed diffuse cytoplasmic staining. Images were visualized using a Zeiss LSM510 confocal laser scanning microscope. Representative of three separate experiments.
Effect of SMV on NADPH-Mediated H2O2 Production
One of the recent discoveries on the role of ROS in cell signalingpertains to the observations that ROS can act as an integralpart of signaling pathways as they fulfill the important prerequisitesfor intracellular messengers (33–37). For determiningwhether Ang II stimulates NADPH oxidase activity and intracellularH2O2 production, MC were grown and made quiescent in culturemedium that contained 0.1% FBS. Cells were then stimulated with100 nM Ang II for 1 h. Medium was replaced with Hanks’solution containing H2O2 sensitive fluorophore DCF-DA (10 µM).Ang II stimulation caused a robust increase in DCF-DA fluorescencein MC (Figure 5, A and B). For determining whether NADPH oxidaseis the source of H2O2 production, Ang II–stimulated MCwere preincubated with DPI (10 µmol/L), a molecule thatcompetitively inhibits flavin-containing enzymes such as NADPHoxidase. Preincubation with DPI resulted in complete inhibitionof Ang II–induced increase in DCF-DA fluorescence (Figure 5C),suggesting that a flavin-containing enzyme is the sourcefor intracellular H2O2. Ang II–induced increase in H2O2was also inhibited when cells were preincubated with 10 µMlosartan, a specific AT1 receptor blocker, indicating that thisinduction was AT1 receptor dependent (Figure 5D). Similar resultswere obtained when cells were treated with 1 µM SMV (Figure 5E),suggesting that SMV inhibits Ang II–induced upregulationof NADPH oxidase activity and intracellular H2O2 production.
Figure 5. Determination of the effects of Ang II and SMV on intracellular H2O2. MC were stimulated without (A) and with (B) Ang II for 1 h, followed by incubation with 10 µM 2', 7'-dichlorofluororescin diacetate for 30 min. Cells were washed in PBS, trypsinized, and resuspended in PBS, and the intensity of fluorescence was immediately visualized at an excitation wavelength of 488 nm and emission at 520 nm using a bandpass filter on Zeiss LSM510 laser scanning confocal microscope. MC were stimulated with Ang II in the presence of DPI (C), losartan (D), and SMV (E) and in cells transfected with dominant-negative Rac1 (N17 Rac; F). n = 3, in triplicate.
Because Rac1 proteins are essential in NADPH oxidase signalingpathway, its role in activation of the NADPH complex by AngII was also investigated. For determining whether Rac1 regulatesAng II–induced upregulation of H2O2 in MC, cells weretransfected with dominant-negative and wild-type Rac1 and stimulatedwith Ang II (100 nM). As shown in Figure 5F, Ang II stimulationdid not increase H2O2 production in dominant-negative Rac1 transfectedMC as determined by DCF-DA fluorescence, indicating a sequentiallink between Rac1 activity and intracellular H2O2 production.
Effect of SMV on Ang II–Induced Akt Activity
The list of redox-sensitive targets of ROS include the mitogen-activatedprotein kinase family, stress-activated protein kinases, NF-B,caspases, and Akt (37,38). Growing evidence indicates that Akt,a serine-threonine kinase, is a potential target of Ang II–inducedhypertrophy of vascular smooth muscle cell and MC (16,37,38).For studying the effect of Ang II on Akt activation and themodulatory effects of SMV on Akt signaling pathway, serum-starvedMC were stimulated with Ang II (100 nM), and total and phosphorylatedAkt (Ser473) were detected using antibodies against total andphospho-Akt (Ser473; Cell Signal Technology). Ang II–stimulatedMC showed an approximately threefold increase in the ratio ofphospho-Akt/total Akt after 20 min (Figure 6A). As shown inFigure 6B, co-treatment of cells with SMV (1 µM) inhibitedAng II–induced upregulation of phospho-Akt. The inhibitoryeffect of SMV was reversed when cells were co-treated with MEV(200 µM).
Figure 6. Effects of Ang II and SMV on phosphorylated Akt. (A, Left) MC were stimulated with Ang II and subjected to Western blotting with anti–phospho-Akt and total Akt antibodies using the same membranes. The ratio of phosphorylated/total Akt significantly increased after 20 min of Ang II stimulation and remained elevated for 60 min. (Right) Densitometric analysis of phospho Akt/total Akt ratio (n = 3; *P < 0.05). (B, Left) SMV (1 µM) inhibited Ang II–induced Akt phosphorylation, and the effect of SMV was reversed with MEV (200 µM). (Right) Densitometric analysis of phospho Akt/total Akt ratio (n = 3; *P < 0.05).
For determining whether Rac1 regulates Ang II–inducedupregulation of phospho-Akt, MC were transfected with dominant-negativeand wild-type Rac1 and stimulated with Ang II (100 nM). As shownin Figure 7, Ang II stimulation did not increase phospho-Aktprotein expression in MC transfected with dominant-negativeRac1, indicating that Ang II–induced Akt phosphorylationis mediated by a Rac1-dependent pathway.
Figure 7. Effect of dominant-negative Rac1 on Akt phosphorylation. MC were transfected with dominant-negative (N17 Rac) and wild-type Rac1 (wt Rac). Ang II stimulation failed to increase phospho-Akt expression in dominant-negative Rac1 cells, indicating a sequential link between Rac1 and Akt activation in Ang II signaling pathway in MC. Representative of three separate experiments.
For determining whether Akt signaling pathway mediates Ang II–inducedupregulation of p27, MC were transfected with dominant-negative(K179M) and wild-type Akt1 (wtAKT1) and stimulated with AngII (100 nM). As shown in Figure 8, Ang II stimulation did notincrease p27 protein expression in MC transfected with dominant-negativeAkt1 cells, indicating that Akt activation is necessary forthe Ang II–induced upregulation of p27 in MC.
Figure 8. Effect of dominant-negative Akt1 on p27 protein expression. For determining whether Akt is involved upstream of Rac1 in Ang II–induced increase in p27 protein levels, MC were transfected with dominant-negative Akt1 (K199M) and wild-type Akt1 (wtAkt) and stimulated with Ang II (100 nM). Ang II stimulation failed to increase p27 protein expression in dominant-negative Akt1 transfected cells, suggesting that Akt activation is necessary for Ang II–induced upregulation of p27 in MC (n = 3).
To gain further insights into the role of Akt on the nucleartranslocation of p27, we performed immunofluorescent microscopyof p27. As shown in Figure 9, MC stimulated with Ang II exhibitedintense nuclear staining of p27. However, cells transfectedwith dominant-negative Akt showed diffuse cytoplasmic stainingof p27, suggesting that Akt is also necessary for the Ang II–inducednuclear translocation of p27 from the cytoplasm.
Figure 9. Effect of Akt on the nuclear translocation of p27. (Left) DAPI was used as a marker for nuclear staining. (Middle) Immunofluorescent microscopy of p27 using anti-p27 antibody. (Right) Merged images. After stimulation with Ang II, MC displayed intense p27 nuclear staining of p27. MC transfected with dominant-negative Akt1(K199M) showed diffuse cytoplasmic staining. Thus, the data indicate that Akt activation promotes nuclear translocation of p27. Images were visualized using a Zeiss LSM510 confocal laser scanning microscope (n = 3).
Effect of SMV on Ang II–Induced MC Hypertrophy
Previous studies have shown the pivotal role of Ang II in MChypertrophy as measured by [3H]leucine incorporation (23–25).The role of SMV on the Ang II–induced de novo proteinsynthesis in MC was examined using [3H]leucine incorporation.Serum-starved cultured rat MC were made quiescent by serum deprivationfor 48 h and then exposed to 100 nM Ang II for 24 h. The incorporationof [3H]leucine was compared in cells exposed to Ang II in thepresence and absence of SMV (1 µM). As shown in Figure 10,exposure of MC to Ang II increased [3H]leucine incorporationby 195 ± 3% compared with control cells (P < 0.05).Co-treatment of cells with SMV attenuated Ang II–inducedincrease in [3H]leucine uptake (128 ± 2%). Cells co-treatedwith SMV and GGPP (5 µM) exhibited increased [3H]leucineincorporation (170 ± 8%), indicating that the inhibitoryeffect of SMV on Ang II–induced [3H]leucine uptake isgeranylgeranyl dependent.
Figure 10. Effect of SMV on Ang II–induced [3H]leucine incorporation. [3H]leucine-labeled MC were exposed to Ang II (100 nM) in the presence or absence of SMV (1 µM) for 24 h. Data are expressed as percentage increase in [3H]leucine incorporation induced by Ang II over the control cells incubated without Ang II. Each bar represents the mean of four experiments performed in triplicate. *P < 0.05.
This study demonstrate that Ang II stimulation in mesangialcells via activation of Rac1GTPase protein, intracellular H2O2activation, and increased Akt activity leads to increased p27protein expression. Our data also provide evidence for the modulatoryeffect of SMV on Ang II–induced Rac1-regulated, NADPH-dependentoxidase signaling pathway involving Akt activation and CDKIp27 in mesangial cells (Figure 11).
Figure 11. Proposed Ang II signaling path in MC. SMV inhibits Ang II–induced Rac1-regulated, NADPH- and Akt kinase-dependent upregulation of p27 protein by preventing geranylation of small GTPase protein Rac1.
Previous studies from our laboratory and others have indicatedthat statins modulate DNA synthesis and cellular proliferationby preventing prenylation of small GTPase proteins such as Rasand Rho GTPases (10,39,40). The posttranslational lipid modification(isoprenylation) of small GTPase proteins is necessary for thetranslocation of Rho GTPases from the cytosol to the membrane,where activation of these proteins takes place. Statins, byinhibiting isoprenylation of small GTPase proteins, preventmembrane translocation and activity of small GTPases and thusinterfere in a number of cellular processes, such as apoptosis,differentiation, cellular proliferation, and hypertrophy (41–43).
The critical role of p27 in cellular hypertrophy induced byAng II or high glucose has been shown in a variety of cell types(23,24). For instance, Wolf et al. (23) demonstrated that theexpression of p27 was upregulated in proximal tubular cellsstimulated by Ang II and that p27 antisense oligonucleotidereversed the Ang II–mediated cellular hypertrophy. Ithas also been reported that MC isolated from p27 –/–mice did not undergo cellular hypertrophy under high glucoseconditions (25,26). More recently, the significance of p27 inthe progression of diabetic nephropathy in vivo has also beenestablished (44). However, the underlying upstream mediatorsof Ang II–induced MC hypertrophy are not fully identified.Gorin et al. (24) recently suggested the involvement of Rac1GTPase in Ang II–induced signaling pathway in MC hypertrophy.Several other studies have also indicated the involvement ofRho A and Rho-kinase in Ang II–induced hypertrophy invascular smooth muscle cells (16,45). In this study, we notonly demonstrated the role of Rac1 on Ang II–induced signalingpathway at the cell cycle level, but we also showed that SMV,by inhibiting Rac1 activity, downregulates Ang II–inducedincrease in p27 protein levels. The data presented in this studyalso suggest that the effect of SMV on p27 is cholesterol independentas co-treatment of cells with SQ, an immediate precursor ofcholesterol in the MEV pathway, did not reverse the inhibitoryeffect of SMV on p27 protein expression. However, co-treatmentof MC with GGPP reversed the inhibitory effect of SMV, indicatingthat the effect of SMV on Ang II–induced upregulationof p27 protein is geranylgeranyl dependent. Furthermore, thisstudy showed that Rac1 activation is required for Ang II–inducedupregulation of p27, as MC transfected with dominant-negativeRac1 failed to increase p27 protein expression in response toAng II.
Our data also suggest that Ang II–induced signaling pathwayin MC involves ROS and increased NADPH oxidase activity. WhenNADPH oxidase activity was inhibited by DPI, the Ang II increasein H2O2 production was eliminated. Thus, our results confirmprevious observations regarding the involvement of ROS in AngII–mediated signaling in MC (24,46,47). This study, however,provides new insights by establishing a sequential link betweenAng II–induced Rac1 activity and an increase in intracellularH2O2 production as dominant-negative Rac1 transfected MC failedto increase H2O2 production. Furthermore, the data presentedin this study indicate that SMV, by inhibiting activation ofRac1, prevents Ang II–induced intracellular H2O2 productionand NADPH-dependent oxidase activity in MC.
We also assessed the contribution of Akt activation on Ang II–inducedupregulation of p27 and the effect of SMV on Ang II–inducedAkt signaling pathway. Our data indicate that Ang II increasedphospho-Akt (active form) in a time-dependent manner. SMV inhibitedthe activation of Akt, and the inhibitory effect of SMV on Aktphosphorylation was MEV dependent. Moreover, we provided furtherevidence that Akt activation is required for Ang II increasein p27 protein expression as MC transfected with dominant-negativeAkt1 did not express upregulation of p27 protein in responseto Ang II. These data support the previous reports indicatingthat Ang II stimulation induces cellular hypertrophy by activatingAkt pathway, probably through phosphorylation of p27 at serineresidues (48–50).
The expression of p27 is transcriptionally and posttranslationallyregulated (28–32). Although transcriptional regulationof p27 has been reported by Forkhead transcription factor, thep27 expression is known to be mainly regulated by posttranslationalmechanisms (30–32). The best characterized mechanism ofp27 degradation is ubiquitin-dependent degradation (28–32).It is generally believed that degradation of p27 requires cytoplasmiclocalization of p27. To provide further evidence for the pivotalrole of Rac and Akt on the Ang II–induced upregulationof p27, we performed several immunohistochemical analyses usingconfocal laser scanning fluorescence microscopy. Together, ourdata indicate that both Rac1 and Akt are necessary for the AngII–induced nuclear translocation of p27. The data presentedin this study demonstrate that dominant-negative mutants ofRac and Akt promote degradation of p27 with the subsequent decreasein p27 protein levels.
Lipophilic statins, such as SMV, are much more widely takenup by passive diffusion into a broad range of tissues and cellsas compared with hydrophilic statins. This distinction amongstatins could influence the ability of statins to exert theirpleiotropic effects on the basis of the ability of nonhepaticcells to transport the different members of the statin familyinto the cell according to their hydrophobicity. Therefore,further studies are planned to examine the class effect of variousstatins on Ang II signaling pathway.
In conclusion, on the basis of our findings, we propose thatAng II–induced MC hypertrophy involves several mediatorsthat include small GTPase protein, Rac1, activation of ROS,phosphorylation of Akt, and increased expression of CDKI p27.This study also demonstrates for the first time the modulatoryeffect of SMV on Ang II–induced Rac1-regulated, NADPHand Akt kinase-dependent signaling pathway involved in the upregulationof CDKI p27 protein expression in MC.
Acknowledgments
This study was supported by grants from the American DiabetesAssociation (1-03-RA-15), the National Institutes of Health(DK064106-01), and a Merck Medical School Award.
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Received for publication January 2, 2004.
Accepted for publication April 6, 2004.
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Top
Abstract
Introduction
hierarchically and/or coordinately have been suggested as the mediators of cell hypertrophy in diabetic nephropathy (20–22). Ang II, an octapeptide hormone, exerts both hemodynamic (leading to increased glomerular capillary pressure) and nonhemodynamic effects (stimulation of cellular hypertrophy and extracellular matrix expansion). Mediators of Ang II–induced MC hypertrophy in diabetic milieu have not fully been identified. However, several observations have shown the pivotal role of p27, a member of CIP/KIP family of cyclin-dependent kinase inhibitors (CDKI), in glucose and Ang II–induced cellular hypertrophy (23–27). 
50% confluence and then transfected with 1 µg of fusion plasmid DNA using LipofectAMINE Reagent according to the manufacturer’s protocol (Invitrogen). Transfected cells were grown in 800 µg/ml G418 (Invitrogen) until colonies were formed. Subsequently, the colonies were grown in growth medium containing 800 µg/ml G418 and incubated at 37°C and 5% CO2 until the cells achieve 
0.05 was considered as statistically significant. 
B, caspases, and Akt (37,38). Growing evidence indicates that Akt, a serine-threonine kinase, is a potential target of Ang II–induced hypertrophy of vascular smooth muscle cell and MC (16,37,38). For studying the effect of Ang II on Akt activation and the modulatory effects of SMV on Akt signaling pathway, serum-starved MC were stimulated with Ang II (100 nM), and total and phosphorylated Akt (Ser473) were detected using antibodies against total and phospho-Akt (Ser473; Cell Signal Technology). Ang II–stimulated MC showed an approximately threefold increase in the ratio of phospho-Akt/total Akt after 20 min (Figure 6A). As shown in Figure 6B, co-treatment of cells with SMV (1 µM) inhibited Ang II–induced upregulation of phospho-Akt. The inhibitory effect of SMV was reversed when cells were co-treated with MEV (200 µM). 
