Rho a negatively regulates cytokine-mediated inducible nitric oxide synthase expression in...

Original Contribution

Rho A NEGATIVELY REGULATES CYTOKINE-MEDIATED INDUCIBLENITRIC OXIDE SYNTHASE EXPRESSION IN BRAIN-DERIVED

TRANSFORMED CELL LINES: NEGATIVE REGULATION OF IKK�

RAMANDEEP RATTAN,* SHAILENDRA GIRI,* A VTAR K. SINGH,† and INDERJIT SINGH**Department of Pediatrics, Medical University of South Carolina, Charleston, SC, USA; and†Department of Pathology and

Laboratory Medicine, Medical University of South Carolina and Ralph H. Johnson VA Center, Charleston, SC, USA

(Received 4 April 2003;Revised 2 June 2003;Accepted 1 July 2003)

Abstract—The present study describes the role of RhoA as a negative regulator of iNOS expression via the inactivationof NF-�B in transformed brain cell lines [C6 glioma, human astrocytoma (T98G, A172), neuroblastoma (NEB), andimmortal rat astrocytes]. Treatment with lovastatin resulted in the induction of LPS/IFN-�-mediated iNOS mRNA andincreased nitric oxide (NO) production. The addition of mevalonate and geranylgeranylpyrophosphate (GGPP) reversedthe lovastatin-mediated effect, whereas FPP had no effect. An inhibitor of geranylgeranyltransferase inhibitor (GGTI298) further induced the cytokine and lovastatin-mediated iNOS expression, suggesting the involvement of geranylgera-nylated proteins in the regulation of iNOS. Bacterial toxin B (inactivates RhoA, B, and C; CDC42; Rac proteins), C3ADP-ribosyltransferase (C3) toxin fromC. botulinum (inactivates RhoA, B, and C proteins), and Y-27632 (selectiveinhibitor of Rho-associated kinases) increased the LPS/IFN-�-mediated iNOS expression. Lovastatin treatment inducedNO by increasing NF-�B translocation and its association with the CREB-binding protein (CBP/p300) via thedownregulation of RhoA. Inhibition of RhoA resulted in increased activation of IKK�. Cotransfection studies withdominant-negative form of RhoA and iNOS-luciferase or NF-�B-luciferase reporter constructs further support theseobservations. Taken together, these studies show that downregulation of RhoA by lovastatin resulted in increased iNOSexpression via the activation of NF-�B-CBP/p300 pathway in transformed brain cells. © 2003 Elsevier Inc.

Keywords—Statins, RhoA, Geranylgeranylation, NF-�B, iNOS, IKK�, CBP/p300, Free radicals

INTRODUCTION

Nitric oxide (NO) is involved in many physiological andpathological functions [1] including blood vessel relax-ation, neurotransmission, and macrophage cytotoxicityduring host defense, sometimes leading to tissue injury insome inflammatory neurodegenerative and autoimmunediseases. NO is produced by NO synthase (NOS) throughthe oxidation of L-arginine to L-citrulline. Three NOSisoforms exist in mammalian cells. Type I [neuronal NOsynthase (nNOS)] and type III [endothelial NO synthase(eNOS)], first identified in neurons and endothelial cells,respectively, are constitutively expressed and are cal-cium and calmodulin dependent. Type II [inducible NOsynthase (iNOS)] is induced by proinflammatory agents.

Although NO produced by iNOS accounts for bacte-ricidal and tumoricidal properties of macrophages, it alsocontributes to the pathophysiologies of inflammatory anddemyelinating disorders (e.g., multiple sclerosis, exper-imental allergic encephalopathy, X-adrenoleukodystro-phy, and globoid cell leukodystrophy), ischemia andtraumatic injuries associated with infiltrating macro-phages, and the production of proinflammatory cytokines[2–8]. Glial cells in the central nervous system alsoproduce NO in response to the induction of iNOS bybacterial lipopolysacchride (LPS) and cytokines [inter-leukin-1� (IL-1�), tumor necrosis factor-� (TNF-�), andinterferon-� (IFN-�)]. NO derived from both astrocytesand macrophages is assumed to contribute to oligoden-drocyte degeneration in demyelinating diseases and neu-ronal death during ischemia and trauma [3,4,6,8].

The cytokines used to induce iNOS expression indifferent cell models are known to activate small Gproteins of the Ras/Rho family [9], which can be in-

Address correspondence to: Dr. Inderjit Singh, Medical Universityof South Carolina, Department of Pediatrics, 96 Jonathan Lucas Street,316 Clinical Science Building, Charleston, SC 29425, USA; Tel: (843)792-7545; Fax: (843) 792-7130; E-Mail: [email protected].

Free Radical Biology & Medicine, Vol. 35, No. 9, pp. 1037–1050, 2003Copyright © 2003 Elsevier Inc.

Printed in the USA. All rights reserved0891-5849/03/$–see front matter

doi:10.1016/S0891-5849(03)00459-3

1037

volved in activation of the mitogen-activated proteinkinase (MAPK) pathways [10]. Ras protein activatespredominantly the extracellular-regulated kinase path-way (ERK1/2-MAPK). Rho proteins, on the other hand,activate the Jun-N terminal kinase (JNK) and p38 MAPK[10]. Because proteins of the Ras/Rho family can beactivated only after prenylation, inhibition of prenyl bio-synthesis by HMG CoA reductase inhibitors (statins)prevents activation of these proteins [11,12]. The in-volvement of small G proteins in iNOS regulation iscomplex.

Previously, our laboratory reported that lovastatin in-hibited LPS-induced iNOS expression in rat primaryastrocytes, macrophages, and primary microglia, whichcould be reversed by FPP (farnesylypyrophosphate) andmevalonate [13]. This indicated the involvement of theRas family of proteins, which require farnesylation foractivation [14]. Other studies have also reported that aspecific inhibitor of geranylgeranyltransferase (GGTase)leads to the superinduction of iNOS expression, whereasa farnesyltransferase (FTase) inhibitor has been shown tocause a decrease in the iNOS in rat primary pulmonaryartery smooth muscle cells and rat hepatocytes [15].Thus, Rho proteins, which need to be geranylgerany-lated, eventually emerged as a negative regulator foriNOS expression.

In the present work, we have demonstrated that lova-statin potentiated LPS/IFN-�-mediated iNOS expressionin transformed cell lines via the inactivation of the smallG protein RhoA. Downregulation of RhoA activates theNF-�B-CBP/p300 pathway, which induces iNOS expres-sion in C6 glioma cells.

MATERIAL AND METHODS

Reagents and cell culture

Recombinant rat interferon (IFN-�) was obtainedfrom R & D Systems (Minneapolis, MN, USA). DMEM/F-12 medium, fetal bovine serum (FBS), and Hank’sbalanced salt solution were obtained from Life Technol-ogies (Grand Island, NY, USA). Human IL-1� was pur-chased from Genzyme (Boston, MA, USA). Mouse re-combinant TNF-� was obtained from BoehringerMannheim (Mannheim, Germany). Lipopolysaccharide(LPS) (Escherichia coli) was obtained from SigmaChemical Co. (St. Louis, MO, USA). Antibody againstmouse macrophage iNOS was purchased from Calbio-chem (San Diego, CA, USA). The enhanced chemilumi-nescence (ECL) detecting reagent was from AmershamPharmacia Biotech (Arlington Heights, IL, USA), andthe luciferase assay system was from Promega (Madison,WI, USA). NF-�B-luciferase and iNOS-luciferasechemiluminescence kits were purchased from Clontech

(Palo Alto, CA, USA). C6 glioma, immortalized rat as-trocytes, A172, and T98G human astrocytoma cell lineswere obtained from ATCC (Rockville, MD, USA).

Induction of NO production in C6 glial cells

C6 glial cells obtained from ATCC were maintainedand induced with different stimuli as indicated inDMEM/F-12 media.

Assay for NO synthesis

Production of NO was determined by assaying culturesupernatants for nitrite, a stable reaction product of NOand molecular oxygen. Briefly, 500 �l of culture super-natant was allowed to react with 500 �l of Griess reagent[16] and incubated at room temperature for 15 min. Theoptical density of the assay samples was measured spec-trophotometrically at 570 nm. Fresh culture mediumserved as the blank in all experiments. Nitrite concentra-tions were calculated from a standard curve derived fromthe reaction of NaNO2 (sodium nitrite) in the assay.

Immunoblot analysis for iNOS

After 24 h of incubation in the presence or absence ofdifferent stimuli, the cells were scraped off, washed withHank’s buffer, and sonicated in 50 mM Tris-HCl (pH7.4) containing protease inhibitors (1 mM PMSF, 5�g/ml aprotinin, 5 �g/ml antipain, 5 �g/ml pepstatin A,and 5 �g/ml leupeptin). Proteins were resolved by SDS-PAGE and transferred onto nitrocellulose membranes.The membranes were blocked for 1 h in 5% nonfat drymilk TTBS (20 mM Tris, 500 mM NaCl, and 0.1%Tween 20, pH 7.5) and incubated for 1.5 h in primaryantisera (antimouse iNOS, 1:2000) containing 1% nonfatdry milk. The blots were then washed four times withTTBS (5 min/wash) and incubated for 45 min at roomtemperature with HRP-conjugated antimouse secondaryantibody at a dilution of 1:7000. The blots were washedthree times in TTBS and once in 0.1 M PBS (pH 7.4) atroom temperature; iNOS protein was detected with ECL,per the manufacturer’s specifications (Amersham Phar-macia Biotech).

Subcellular distribution

To determine the subcellular distribution of RhoA, C6

cells were plated in 10 cm dishes and treated with lova-statin and LPS/IFN-� as described earlier. Cultures wereincubated for 16 h in DMEM � 10% FBS and the cellswere lysed in 0.2 ml of TCM buffer (40 mM Tris-HCl,pH 7.4, 2 mM CaCl2, 10 mM MgCl2, and 1 mM phe-nylmethylsulfonyl fluoride). Soluble and particulate frac-tions were obtained by centrifuging the cell lysate at100,000 � g for 60 min at 4°C. One quarter of eachfraction was subjected to SDS-PAGE and immunoblot

1038 R. RATTAN et al.

analysis using a monoclonal antibody against the RhoA(Upstate Biotechnology, Lake Placid, NY, USA), fol-lowed by HRP-conjugated goat antimouse IgG and ECLdetection reagent (Amersham Pharmacia Biotech).

RNA isolation and Northern blot analysis

Cells were harvested from the culture dish directly byadding Ultraspec-II RNA reagent (Biotecx Laboratories,Houston, TX, USA), and the total RNA was isolatedaccording to the manufacturer’s protocol. For Northernblot analysis, 20 �g of total RNA was electrophoresed on1.2% denaturing formaldehyde-agarose gels, electro-transferred to Hybond-Nylon Membrane (AmershamPharmacia Biotech), and hybridized at 68°C with 32P-labeled cDNA probe using Express Hyb hybridizationsolution (Clontech). The cDNA probe for iNOS wasmade by polymerase chain reaction amplification usingtwo primers (forward primer, 5�-CTC CTT CAA AGAGGC AAA AAT A-3� and reverse primer, 5�-CAC TTCCTC CAG GAT GTT GT-3�). After hybridization, thefilters were washed two or three times in solution I (2xSSC, 0.05% SDS) for 1 h at room temperature, followedby solution II (0.1x SSC, 0.1% SDS) at 50°C for 1 hmore. Membranes were then dried and exposed to radio-graphic film (Eastman Kodak, Rochester, NY, USA).The same membrane was probed for �-actin to normalizethe RNA loading.

Preparation of nuclear extracts and electrophoreticmobility shift assay (EMSA)

Nuclear extracts from stimulated or unstimulated C6

glial cells were prepared using the method of Dignamand coworkers [17], with slight modification. Cells wereharvested, washed twice with ice-cold PBS, and lysed in400 �l of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl,2 mM MgCl2, 0.5 mM dithiothreitol, 1 mM PMSF, 5�g/ml aprotinin, 5 �g/ml pepstatin A, and 5 �g/mlleupeptin) containing 0.1% Nonidet P-40 for 15 min onice, vortexed vigorously for 15 s, and centrifuged at20,000 � g for 30 s. The pelleted nuclei were resus-pended in 40 �l of buffer B (20 mM HEPES, pH 7.9,25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2mM EDTA, 0.5 dithiothreitol, 1 mM PMSF, 5 �g/mlaprotinin, 5 �g/ml pepstatin A, and 5 �g/ml leupeptin).After 30 min on ice, lysates were centrifuged at 20,000 �g for 10 min. Supernatants containing the nuclear pro-teins were diluted with 20 �l of modified buffer C (20mM HEPES, pH 7.9, 20% (v/v) glycerol, 0.05 M KCl,0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mMPMSF) and stored at �70°C until use. EMSA (electronmobility shift assay) was performed as described previ-ously [18] with the NF-�B consensus sequence end-labeled with [-32P] ATP. Nuclear extracts were normal-

ized for protein (Bio-Rad protein assay, Bio-Rad,Richmond, CA, USA) and equal amounts were loaded.DNA-protein complexes were resolved by PAGE on 5%gel in 45 mM Tris, 45 mM boric acid, 1 mM EDTA (0.5xTBE), and run at 11 V/cm. The gels were dried andautoradiographed at �70°C. For competition analysis,nuclear extracts were preincubated with water or com-petitor DNA for 5 min at room temperature and thesamples were processed as for EMSA.

Transfection studies

Plasmids were purified using the endotoxin-free plas-mid midi prep kit (Qiagen, Santa Clarita, CA, USA). Fortransient transfections, C6 glioma cells were seeded in6-well plates and grown to 60–80% confluence inDMEM-F12 � 5% FBS without antibiotics and trans-fected the next day using FuGene reagent (Promega),according to the manufacturer’s protocol, with a total of1.8–2 �g/well of plasmid DNA. For transactivation ex-periments, 1.5 �g of pNOS-2-luc/NF-�B-luc or 0.2 �gRhoA DN expression vector or insertless expressionvector (pBluescript) were used. Then, 24 h after trans-fection, cells were maintained for another 24 h in serum-free medium and then treated with cytokines and/orlovastatin. Finally, 16–20 h later, cell lysates were pre-pared using lysis buffer (Promega) to measure luciferaseactivity. Protein content of the lysates was determinedusing Bradford reagent (Bio-Rad).

Cell viability

Cytotoxicity of all inhibitors was determined by mea-suring the metabolic activity of cells with (3-[4,5-di-methylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide(MTT) assay.

IKK� assay

For IKK� assay, C6 cells were pretreated with lova-statin and/or its metabolites and then stimulated withLPS/IFN-� (15 ng/2 U/ml) for 15 min. For other exper-iments, cells were transiently transfected with Rho DN orpcDNA and then treated with lovastatin and cytokines asstated before. Cells were washed with cold PBS andlysed in lysis buffer (50 mM Tris-HCl, pH 7.4, contain-ing 50 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10%glycerol, and protease inhibitor cocktail). Approximately200 �g of cell extracts were incubated with anti-IKK�antibody (Santa Cruz Biotechnology, Santa Cruz, CA,USA) and protein A/G Sepharose and incubated for 2 hto overnight at 4°C. The immune complexes werewashed twice in lysis buffer and twice in kinase buffer(20 mM HEPES, pH 7.5, and 10 mM MgCl2) and incu-bated at 30°C in 30 �l of kinase buffer containing 20mM �-glycerophosphate, 20 mM p-nitrophenyl phos-

1039Rho A negatively regulates iNOS via NF-�B

phate, 1 mM dithiothreitol, 50 �M Na3V04, 20 �M ATP,and 5 �Ci of [�-32P] ATP. Approximately 2 �g ofGST-I�B� fusion protein (Santa Cruz Biotechnology)were used as substrate in each reaction. Reactions werestopped after 30 min by denaturation in SDS loadingbuffer. Proteins were resolved by SDS-PAGE, and sub-strate phosphorylation was visualized by autoradiogra-phy. The autoradiographs were scanned by using a Bio-Rad (model GS-670) imaging densitometer.

CBP/p300 and p65 association

To determine the association between CBP/p300 andp65, nuclear extracts prepared from the treated cells wereincubated with LPS/IFN-� and/or lovastatin for 45 min.The extracts were incubated with NF-�B-sepharosebeads (Santa Cruz Biotechnology). The bound proteinswere resolved by SDS loading buffer and run on SDS-PAGE gel. The membrane blot was probed with anti-p300 antibody. The same gel was stripped and reprobedwith anti-p65 antibody.

Statistical analysis

The data was statistically analyzed by performing theStudent Newman-Keuls test.

RESULTS

Lovastatin induced the expression of iNOS andthe production of NO in cytokine-treated rat C6

glioma cells

The inhibition of HMG-CoA reductase, a key enzymecatalyzing the rate-limiting step in the cholesterol bio-synthetic pathway, prevents isoprenoid synthesis andprenylation of small GTP-binding proteins. To under-stand the mechanism of lovastatin-induced production ofNO in LPS/IFN-�-treated C6 glial cells, we examined theeffect of lovastatin on nitrite production. To measuredose dependency, various concentrations of cytokineswere used with lovastatin, and the production of NO wasmeasured. Higher concentrations of cytokines resulted insaturation (data not shown). Since we were interested inthe potentiation caused by lovastatin, we selected thelowest concentration of cytokines, 15 ng of LPS and 2 Uof IFN-�, that gave significant induction with lovastatin(10 �M). At this concentration, lovastatin potentiatedNO production in a dose- and time-dependent manner.

As shown, lovastatin induced the production of NO(Fig. 1A) and the expression of iNOS protein in LPS/IFN-�-treated C6 glial cells in a dose- (Fig. 1B) and time-(Fig. 1C) dependent manner. The lowest dose of lova-statin to induce the production of NO and the expressionof iNOS protein was observed to be 1 �M. At 10 �M, theproduction of NO and the expression of iNOS protein

were found to be maximum (Fig. 1B). Higher concen-trations of lovastatin (20 �M) lead to cell death, whereaslovastatin concentrations less than 20 �M have no effecton cell viability as judged by the MTT assay (data notshown). To understand the mechanism of the stimulatoryeffect of lovastatin on LPS/IFN-�-mediated induction ofiNOS, we examined the effect of lovastatin on the ex-pression of iNOS mRNA. Cells were treated with cyto-kine (LPS/IFN-�) with or without lovastatin (10 �M),and RNA was isolated at different time points. As shownin Fig. 1D, the highest levels of iNOS mRNA wereobserved at 9 h of treatment with LPS/IFN-� alone,followed by a decline to undetectable levels at 24 h.However, lovastatin/LPS/IFN-�-treated cells showed asignificant increase of iNOS mRNA at 9, 12, and 24 h oftreatment (Fig. 1D). This observation shows that lova-statin not only induces the LPS/IFN-�-mediated induc-tion of iNOS mRNA but may also stabilize in situ levelsof iNOS mRNA.

Therefore, consistent with the effect of lovastatin onthe production of NO, lovastatin induced the expressionof iNOS protein and mRNA (Figs. 1B–1D) in LPS/IFN-�-treated cells. In summary, lovastatin induced the LPS/IFN-�-mediated iNOS expression in C6 glioma cells.

Lovastatin induced NO production in varioustransformed brain cell lines

Our laboratory had previously reported that lovastatininhibited NO production and iNOS gene expression inrat primary glial cells (astrocytes and microglia [13]) andthat lovastatin behaved differently in C6 glioma cells.Therefore, we hypothesized that lovastatin may be dif-ferentially regulating iNOS gene expression in primaryand transformed brain cell lines. To test this hypothesis,we used other transformed brain cell lines such as humanastrocytoma A172 and T98G, rat immortal astrocytes,and human neuroblastoma cell lines to test the effect oflovastatin. As expected, lovastatin induced NO produc-tion in all tested transformed cell lines (Fig. 2). Cyto-kines variably produced NO, and lovastatin treatmentsubstantially enhanced the cytokine-mediated productionof NO. This result indicates that lovastatin regulates NOproduction differentially in primary and transformed celllines.

The effect of mevalonate and its metabolites onlovastatin-induced iNOS

Inhibition of the cholesterol biosynthetic pathway blocksthe synthesis of mevalonate, isoprenoids, and other metab-olites; thus, it was of interest to determine which metabo-lite’s depletion causes the lovastatin-induced iNOS geneexpression. To this end, we incubated cells with LPS/IFN-�and lovastatin and supplemented cells with various choles-


terol metabolites such as cholesterol, 25-hydroxy choles-terol, lanosterol, squalene, and mevalonate, and we exam-ined the effect of each on lovastatin-induced NOproduction. Induction of iNOS by lovastatin was com-pletely reversed by L-mevalonate. To evaluate which me-valonate metabolite may be important in lovastatin-inducedNO production, we studied whether other mevalonate de-rivatives could inhibit lovastatin-induced NO production(Fig. 3). Neither cholesterol, 25-hydroxy cholesterol orlanosterol, nor their precursor squalene prevented orblocked lovastatin-induced NO production, suggesting thatthese sterols do not play a role in the induction of NO underthese experimental conditions. FPP and GGPP, also metab-olites of the cholesterol biosynthetic pathway, are requiredfor the post-translational modification of small G proteins of

the Ras/Rho family. GGPP reversed lovastatin-induced NOproduction completely, whereas FPP had no effect (Fig. 3).This indicated that geranylgeranylation but not farnesyla-tion is involved in lovastatin-induced iNOS gene expres-sion, and that geranylgeranylated protein(s) negatively reg-ulate(s) iNOS expression.

Geranylgeranylation inhibited lovastatin-inducediNOS expression

To test the hypothesis that geranylgeranylation is in-volved in lovastatin-mediated induction of iNOS, weused GGTI-286 and FTI-277, selective inhibitors ofgeranylgeranyltransferase and farnesyltransferase, re-spectively. The treatment of C6 glioma with GGTI-286(10 �M) significantly induced LPS/IFN-�-mediated NO

Fig. 1. Lovastatin potentiates cytokine-mediated iNOS expression in a dose- and time-dependent manner. (A) C6 glioma cells werepretreated for 2 h with lovastatin and then with 15 ng/2 U of LPS/IFN-�. At 24 h of incubation, NO was measured in the supernatant.*p � .05 compared with untreated; # p � .001 compared with LPS/IFN-�-treated cells. For detection of iNOS protein expression byWestern blot analysis, cell lysate was prepared (B) after 24 h or (C) at indicated time points with LPS/IFN-� and/or lovastatin (10 �Mor indicated concentrations) treatment. (D) For detection of iNOS message, RNA was isolated from C6 glioma cells at various timeperiods after treatment with LPS/IFN-� and/or lovastatin and processed for Northern analysis. The blots are representatives of threeexperiments.


production. GGTI and lovastatin, when used together,produced an additive effect in the potentiation of NOproduction as shown in Fig. 4. Treatment of C6 cells withFTI-277 induced LPS/IFN-�-mediated NO productionbut had no significant effect on lovastatin-mediated NOproduction compared to GGTI-286 (Fig. 4). Prenylationis a prerequisite for the activation of small G proteins ofthe Ras/Rho proteins. Ras proteins are predominantlyfarnesylated, whereas the Rho proteins are predomi-nantly geranylgeranylated. Thus, the data indicate thatgeranylgeranylation is involved in lovastatin-inducediNOS gene expression but not farnesylation, indicating arole for the Rho family of small G proteins.

The effect of modulators of Rho GTPases oniNOS expression

The Rho family of GTP-binding proteins such as Rho,Rac, and Cdc42 are geranylgeranylated and play an

important role in cell adhesion, actin dynamics, andregulation of gene transcription [19]. To determinewhether these proteins mediate the effects induced bylovastatin, we incubated the cells with C. difficile toxin B(toxin B), an inhibitor of the different Rho GTPases [20].Treatment of C6 glioma cells for 24 h with 2 nM of toxinB induced iNOS expression as shown in Fig. 5A. Theeffect of lovastatin on iNOS expression was further pro-nounced by the treatment with toxin B.

To further confirm and refine these findings, weused C3 exoenzyme, another Rho-modifying Clostrid-ial toxin (C3 toxin), isolated from C. botulinum. Thistoxin ADP-ribosylates Rho (A, B, and C) at asparag-ines 41, but not Rac or CDC42, leading to its inacti-vation [21]. Treatment of the cells with 1 �g/ml of C3exoenzyme resulted in an induction of LPS/IFN-� aswell as lovastatin-mediated expression of iNOS (Fig.5B). Because C3 exoenzyme is specific for the inac-tivation of Rho, we evaluated the subcellular distribu-tion of RhoA in C6 glioma cells in response to cyto-kines and lovastatin treatment. As shown in Fig. 6A, inuntreated cells a significant amount of RhoA wasobserved in the membrane fraction and treatment withcytokines did not induce it further. On the other hand,in lovastatin-treated cells, RhoA was found predomi-nantly in the cytosol in the presence or absence ofcytokines. Therefore, we hypothesized that lovastatintreatment typically depleted the pool of GGPP re-quired for prenylation of RhoA protein, leading tomarked accumulation of unmodified RhoA protein inthe cytosol with a corresponding decrease in the mem-brane-associated RhoA protein.

We examined the effect of transient expression ofdominant-negative RhoA on the production of NO andthe expression of iNOS in C6 glioma cells to furtherconfirm the induction of iNOS by RhoA. Consistent withthe induction of iNOS by lovastatin, the expression ofdominant-negative forms of RhoA markedly induced theproduction of NO (data not shown) and the expression ofiNOS protein (Fig. 6B) in LPS/IFN-�- and/or lovastatin-treated cells. Also, we cotransfected dominant-negativeRhoA cDNA with iNOS-luciferase in C6 glioma cellsand assessed transcriptional activity of iNOS-luciferase.As is evident from Fig. 6C, expression of dominant-negative RhoA cDNA induced iNOS-dependent expres-sion of luciferase in cytokine- and cytokine-plus-lova-statin-treated cells, supporting the conclusion thatdownregulation of RhoA upregulates the expression ofiNOS.

Because the serine/threonine kinases ROCK (RhoA-associated kinase) are among the identified targets ofRho, we tested the effect of a selective inhibitor of theROCK I and II, Y-27632, on iNOS expression. Cellswere incubated with Y-27632 (10 �M) for 24 h along

Fig. 2. Lovastatin potentiates cytokine-mediated NO production inother transformed cell lines. Various transformed cell lines (humanastrocytoma A172 and T98G, immortal astrocytes, and NEB) weretreated with lovastatin (10 �M) and cytokine combinations as men-tioned below. After 24 h of incubation, nitrite levels were measured insupernatants. Various combinations of cytokines treatment for differentcell lines are as follows: A172 (IFN-�, 30 ng/ml; IL-1�, 10 ng/ml;TNF-�, 10 ng/ml); T98G (IFN-�, 5 ng/ml; TNF-�1, 10 ng/ml); im-mortal astrocytes (LPS/IFN-�, 0.5 �g/20 U/ml); and NEB (IFN-�/TNF-�/IL-1�, 50 U/5 ng/5 ng/ml). ***p � .001; **p � .01 comparedwith cytokine-treated cells.


with lovastatin in the presence or absence of LPS/IFN-�.Treatment with Y-27632 significantly induced the iNOSexpression in LPS/IFN-�-treated cells, further document-ing the involvement of RhoA signaling in the regulationof iNOS expression (Fig. 6D).

Lovastatin induced the expression of iNOS through theactivation of NF-�B in C6 glioma cells

Because the activation of NF-�B is necessary for theinduction of iNOS, we examined the effect of RhoA onthe activation of NF-�B in C6 glioma cells to understandthe basis of expression of iNOS. Activation of NF-�Bwas monitored by both DNA-binding and transcriptionalactivity of NF-�B. DNA-binding activity of NF-�B wasevaluated by the formation of a distinct and specificcomplex in a gel shift DNA-binding assay. Treatment ofC6 glioma cells with lovastatin (10 �M) resulted in aninduction of LPS/IFN-�-mediated DNA-binding activityof NF-�B (Fig. 7A). The effect of lovastatin on NF-�B-dependent transcription of luciferase (3xNF-�B-LUC) inC6 glioma cells was assayed, using the reporter construct,pNF�B-luciferase. Consistent with the results of theDNA-binding activity of NF-�B, lovastatin also inducedthe NF-�B-dependent transcription of luciferase in adose-dependent manner (Fig. 7B). To further confirm theactivation of NF-�B by RhoA, we transiently cotrans-fected dominant-negative RhoA along with pNF�B-lu-ciferase reporter in C6 glioma cells and examined thetranscriptional activity of NF-�B. The data in Fig. 7Cshow that the expression of dominant-negative RhoAinduced the NF-�B-dependent expression of luciferase,indicating that the observed lovastatin-induced iNOSgene expression occurs by downregulation of RhoA and

Fig. 3. The effect of various metabolites of HMG-CoA reductase pathway on lovastatin-induced, cytokine-mediated NO production.C6 glioma cells were pretreated for 2 h with lovastatin (10 �M) and different metabolites of the HMG-C0A reductase pathway suchas cholesterol (10 mM), 25-hydroxy cholesterol (10 mM), squalene (10 mM), FPP (5 �M), GGPP (5 �m), and mevalonate (10 mM).After the addition of LPS/IFN-�, cells were incubated for 24 h and NO was estimated from the supernatants. $p � .001 compared withuntreated cells; ***p � .001 compared with LPS/IFN-�-treated cells; #p � .001 compared with LPS/IFN-�- and lovastatin-treatedcells; NS � nonsignificant.

Fig. 4. Geranylgeranyltransferase inhibitor potentiates the lovastatin-induced cytokine-mediated NO production. C6 cells were treated 30min before lovastatin treatment with geranylgeranyltransferase inhibi-tor (GGTI-298) or farnesyltransferase inhibitor (FTI). Then, 2 h afterlovastatin treatment, LPS/IFN-� was added and cells were incubatedfor 24 h. NO production was measured in the supernatant of the cells.$p � .001 compared with untreated cells; *p � .001 compared withLPS/IFN-�-treated cells; #p � .001 compared with LPS/IFN-�- andlovastatin-treated cells; NS � nonsignificant.


RhoA is negatively regulating activation of NF-�B/iNOSin C6-transformed cells.

To further investigate lovastatin-induced expressionof iNOS in C6 glioma cells being dependent on theactivation of NF-�B, we examined the effect of protea-some inhibitors on lovastatin-mediated expression ofiNOS. Proteasome inhibitors (PCI and MG132) inhibitedthe ubiquitination of I�B�, thereby inhibiting the trans-location of NF-�B into the nucleus and resulting in theinhibition of lovastatin-mediated induction of iNOS.This indicates that the activation of NF-�B is necessaryfor the expression of iNOS in lovastatin-stimulated C6

glioma cells (Fig. 7D).

Lovastatin-mediated downregulation of RhoA inducedLPS/IFN-�-mediated IKK� activity

The activation of NF-�B is associated with transmi-gration of NF-�B complex (p50:p65) into the nucleus asa result of the phosphorylation and degradation of I�B�.The upstream kinase for I�B is an IKK complex con-taining catalytic subunits (IKK� and �) and the IKK� orNEMO regulatory subunit. Its activation triggers phos-phorylation of I�B and leads to the ubiquitin-dependentdegradation of I�B. Therefore, we examined the effect oflovastatin on the activity of IKK�. As shown in Fig. 8A,LPS/IFN-� slightly stimulated the IKK� activity, whichwas further potentiated significantly by lovastatin treat-ment. Lovastatin-induced IKK� activity can be partiallyreversed by the addition of GGPP, FPP, and mevalonate.GGPP and mevalonate inhibited the basal activity ofIKK�, whereas FPP had no effect on IKK� activity. Sofar, we have observed that lovastatin inhibited the trans-location of RhoA to membrane, thereby inhibiting RhoAsignaling; as such, it was considered worthwhile to ex-amine the effect of dominant-negative RhoA on LPS/IFN-�-mediated IKK activity. To this end, the dominant-negative form of RhoA was transiently transfected andthe activity of IKK� was examined. As shown in Fig.8B, LPS/IFN-� slightly induced the IKK� activity at 15min of treatment while transient transfection of the dom-inant-negative form of RhoA significantly induced theIKK� activity. A slight increase in the IKK� activity wasalso observed when cells transfected with dominant-negative RhoA were treated with lovastatin (10 �M).These observations clearly demonstrate that lovastatininduced the iNOS gene expression and NO productionby activating IKK� activity, which is mediated by thedownregulation of RhoA.

Lovastatin induced the interaction of CBP/p300with p65

NF-�B transcriptional competence requires interac-tion with the transcription cofactor CBP/p300 [22]. Toassess the physical interactions of p65 with CBP/p300,nuclear extracts were prepared from LPS/IFN-�-treatedcells in the presence or absence of lovastatin. The nuclearextracts were incubated with NF-�B gel shift oligonu-cleotides agarose conjugate to coprecipitate NF-�B com-plex. Interaction with NF-�B complex and CBP wasanalyzed by Western blot analysis with anti-p300 anti-body and reprobing with anti-p65 antibody. As shown inFig. 9, treatment with LPS/IFN-� induced the binding ofp65 as compared to untreated cells. In LPS/IFN-�-treatednuclear extract, p65 was able to trap p300, suggesting astrong recruitment of cofactor CBP/p300 to the NF-�Btranscription factor. Upon the addition of lovastatin,LPS/IFN-�-stimulated p65/p300 interaction was ob-

Fig. 5. Involvement of Rho GTPase proteins in lovastatin-inducedcytokine-mediated NO production. C6 cells were treated for 18 h with(A) toxin B (2 nM) or (B) C3 exoenzyme (1 �g/ml). After 24 h ofincubation with lovastatin and LPS/IFN-� treatment, NO was measuredin the supernatant of the cells. *p � .001 compared with untreatedcells; #p � .01 in (A) and #p � .001 in (B) compared with LPS/IFN-�-and lovastatin-treated cells.


served to be further potentiated, which directly indicatesthat lovastatin induced iNOS gene expression via therecruitment of p300 by NF-�B (Fig. 9).

DISCUSSION

We have reported here for the first time that an inhib-itor of HMG-CoA reductase, lovastatin, increases theexpression of iNOS in transformed brain cell lines. Pre-viously, our laboratory demonstrated the anti-inflamma-tory property of lovastatin in primary rat astrocytes,microglia, and peritoneal macrophages [13]. This is aninteresting observation describing the differential regu-lation of iNOS gene in primary and transformed braincells. The conclusions of negative regulation of iNOSinduction by metabolites of the mevalonate pathway(isoprenoids) in brain-derived transformed cell lines arebased on the following observations: (i) lovastatin, aninhibitor of HMG-CoA reductase, upregulated the cyto-kine-mediated induction of iNOS in transformed braincell lines tested (C6 glial cell line, A172, T98G, immor-talized astrocytes, and NEB cell line); (ii) metabolites ofHMG CoA reductase, mevalonate, and the isoprenoidGGPP reversed the induction of iNOS by statins; (iii)

similar to lovastatin, GGTI-286, an inhibitor of gera-nylgeranyltransferases, and toxin B and C3 exoenzyme(inhibitors of Rho proteins) induced iNOS; (iv) domi-nant-negative RhoA upregulates the expression of iNOS;and (v) dominant-negative Rho activates IKK� activity,leading to the activation of NF-�B and the induction ofthe iNOS gene. These results demonstrate that gera-nylgeranylated proteins are involved in the downregula-tion of iNOS expression in transformed glial cells.

Although iNOS expression is thought to be regulatedby farnesylated proteins such as H-Ras or K-Ras [15,13],farnesylation does not appear to be important in thepresent study. Other metabolites of the cholesterol bio-synthesis pathway did not modify iNOS expression, sug-gesting that regulation of cellular cholesterol level is notinvolved in this effect. Treatment with GGPP and GGTIclearly demonstrated that geranylgeranylation is in-volved in the regulation of iNOS expression in trans-formed brain cell lines. Results obtained with RhoAdominant-negative studies establish RhoA as the gera-nylgeranylated protein negatively regulating iNOS ex-pression. The Rho family (including RhoA, B, and C;Rac-1 and -2; Cdc42; Rho G; G25K; and TC10) consistsof small G proteins that play a critical role in the regu-

Fig. 6. Involvement of RhoA GTPases in lovastatin-potentiated NO production. C6 glioma cells were incubated with LPS/IFN-� and/orlovastatin as indicated. (A) After 16 h of lovastatin incubation, cytosolic and membrane fractions were isolated and processed for theimmunodetection of RhoA protein by Western blot analysis. (B) iNOS protein expression was measured in the presence of RhoA DNin the cell lysates of cytokine- and lovastatin-treated cells. (C) C6 glioma cells were transiently cotransfected with 1.5 �g ofiNOS-luciferase and 0.2 �g of RhoA dominant-negative (DN) or pcDNA3, followed by stimulation for 20 h with the indicatedtreatment. Lysates were collected and assayed for luciferase activities. *p � .001 compared with LPS/IFN-�-treated cells; #p � .001compared with LPS/IFN-�- and lovastatin-treated cells; NS � nonsignificant compared to LPS/IFN-�-treated cells. C6 cells weretreated 30 min prior to lovastatin with the specific Rho kinase inhibitor Y27632. (D) After the addition of LPS/IFN-�, cells wereincubated for 24 h and iNOS protein expression was measured in cell lysates. Blots are representatives of three experiments.


lation of cell morphology, growth, aggregation, apopto-sis, motility, and vascular smooth muscle cell contraction[19]. Recent evidence indicates that Rho GTPase pro-teins act as components in signal-transducing kinasecascades and are able to stimulate the activity of c-JunNH2-terminal kinases/stress-activated protein kinase(JNK/SAPK) and p38 kinase [19]. Various studies haveshown that the inhibition of geranylgeranylation and RhoGTPases by statins and other inhibitors (GGTI, Toxin

Band C3 exoenzyme) results in the enhancement of NOproduction in response to inflammatory stimuli [15,23–26]. Our data are consistent with these studies. In oursystem, C. difficile toxin B, selective inhibitor of all RhoGTPases, affected iNOS expression after the activationby LPS/IFN-�. This suggests that Rho GTPases partici-pate in iNOS regulation. The further demonstration thatboth C. botulinum C3 transferase and Y-27632, the se-lective inhibitors of Rho and ROCK, respectively, in-

Fig. 7. Lovastatin induces the expression of iNOS through the activation of NF-�B in C6 cells. C6 glioma cells were treated withLPS/IFN-� and/or lovastatin as before and nuclear extracts were prepared at the stipulated time points. (A) Gel shift assay wasperformed with the labeled consensus NF-�B sequence. (B) C6 glioma cells were transiently transfected with 1.5 �g of NF-�B-luciferase reporter construct and treated with different doses of lovastatin as shown. $p � .05 compared with untreated cells; *p � .05compared with LPS/IFN-�-treated cells. Lysates were collected after 20 h and assayed for luciferase assay. Cells were alsocotransfected with 1.5 �g of NF-�B-luciferase and 0.2 �g RhoA dominant-negative or pcDNA3, followed by stimulation for 20 h withthe indicated treatments. (C) Lysates were collected and assayed for luciferase activities. $p � .05 compared with untreated; *p � .05compared with LPS/IFN-� **p � .01 compared with LPS/IFN-�; #p � .05 compared with LPS/IFN-� and lovastatin. (D) Proteasomeinhibitors were used along with LPS/IFN-�/lovastatin treatments, and iNOS protein was detected by performing Western blot analysisfrom the cell lysates after 24 h. Blots are representatives of three experiments.


duced iNOS expression stressed the role of Rho proteinin the negative regulation of iNOS expression. Consis-tent with this, the level of immunoreactive RhoA wasdecreased in membrane fractions of lovastatin-treated C6

glioma. Rho (A or C) has also been shown to control theexpression of different proteins in vessels includingiNOS, eNOS, TGF-�, Cox-2, and pre-pro-endothelin-1(ET-1) expression [23,27–30].

However, few studies have examined the mechanismbehind the negative modulation of iNOS by RhoA GT-Pases [25]. Our aim was to investigate the pathway bywhich this negative regulation occurs. For this, we in-vestigated the NF-�B-mediated pathway of NO induc-tion. Activation of nuclear factor �B (NF-�B) plays acrucial role in the expression of iNOS in various celllines [31]. NF-�B is present as a homodimer of fiveidentified members of the NF-�B/Rel family. The mostabundant dimer present in most cells is composed ofp50:p65 dimer. In unstimulated cells, NF-�B is usuallysequestered in the cytoplasm by the binding of inhibitoryproteins, I�Bs. The essential step in its activation is thephosphorylation of I�B proteins, leading to their identi-fication and degradation by the ubiquitin pathway, whichleads to translocation of the p50:p65 dimer into thenucleus, finally resulting in the activation of variousgenes including the iNOS gene.

Activation of NF-�B can be achieved by numerousphysiological and nonphysiological stimuli. At present itis unclear how these various stimuli converge to triggerNF-�B, but those that are activated by proinflammatorystimuli converge on the upstream I�B kinase or IKK.The IKK complex contains two catalytic subunits (IKK�and �) and the IKK� or NEMO regulatory subunit. Ourdata clearly demonstrate that lovastatin substantially in-creased the LPS/IFN-�-induced DNA binding as well astranscriptional activity of NF-�B in C6 glioma cells. Theinhibition of lovastatin-mediated induction of iNOS byproteasome inhibitors (PCI and MG132) also indicatedthat the activation of NF-�B is necessary for the expres-sion of iNOS in lovastatin-stimulated C6 glioma cells.Furthermore, the significant induction of iNOS andNF-�B reporter activity by RhoA indicated its involve-ment in negative regulation of iNOS/NF-�B transcrip-tional activity. To determine at which point RhoA mod-ulated NF-�B activity, IKK� activity was assessed and,as shown, RhoA dominant-negative was significantlyable to enhance the IKK� activity over lovastatin andLPS/IFN-�. This clearly demonstrated that lovastatininduced the iNOS gene expression and NO productionby activating IKK� activity, which is mediated by thedownregulation of RhoA, or RhoA negatively regulatedIKK� activity. Other studies have shown that Rho pro-teins, including RhoA, stimulate activity of NF-�B byeither an IKK-independent pathway in HeLa andNIH3T3 cells [32] or by increasing the IKK� phosphor-ylation [33].

Our observation is different from these studies, whichmight be due to a different cell type being used. This isthe first report showing that RhoA can also negativelyregulate NF-�B, i.e., RhoA inhibition leads to NF-�Bactivation via IKK� activation and iNOS induction. Thehypothetical pathway occurring in our experimental con-

Fig. 8. Lovastatin induced LPS/IFN-�-mediated IKK� activity via thedownregulation of RhoA. (A) C6 cells were pretreated with lovastatin (10�M) and/or mevalonate (10 mM), GGPP (10 �M), and FPP(10 �M), andthen stimulated with LPS/IFN-� (15 ng/2 U/ml) for 15 min. Cells werewashed and lysed in lysis buffer and incubated with anti-IKK� antibodyand protein A/G sepharose. The immune complexes were washed andincubated with kinase buffer. GST-IKB� fusion protein was used assubstrate in each reaction. Reactions were stopped after 30 min andproteins were resolved by SDS-PAGE; substrate phosphorylation wasvisualized by autoradiography. The autoradiograph was scanned by den-sitometer (Bio-Rad GS-670) and the arbitary units were plotted. (B) C6cells were transiently transfected with Rho DN and or pcDNA and thentreated with lovastatin and LPS/IFN-� as before. Cell lysates were pre-pared and subjected to the same procedure as described above. The graphsare cumulative of three experiments. $p � .05 compared with untreated;*p � .01 compared with LPS/IFN-�-treated cells; #p � .05 compared withLPS/IFN-�- and lovastatin-treated cells.


ditions is depicted in Fig. 10. However, the exact path-way and the factors participating therein remain to beelucidated, including whether RhoA is directly or indi-rectly affecting IKK�.

Lovastatin also induced LPS/IFN-�-mediated NF-�Btranslocation and its association with the CBP/p300.CBP/p300 was originally discovered based on its abilityto interact with the cAMP-responsive element-binding

protein and has been demonstrated to interact with manyproteins in a cell signal-regulated manner. It has beenshown that phosphorylation of p65 promotes the inter-action with CBP and results in the enhanced transactiva-tion potential of NF-�B [22]. It acts as a bridging factorbetween NF-�B and DNA-binding sites. CBP/p300 withintrinsic enzyme activity can acetylate histone, whichallows the unwinding or loosening of chromatin, and it

Fig. 9. Lovastatin induced the interaction of p300 with P65. C6 cells were treated with LPS/IFN-� and/or lovastatin. Nuclear extractswere prepared after 45 min and incubated with NF-�B sepharose beads (Santa Cruz Biotechnology). The bound protein was resolvedby SDS loading buffer and run on SDS-PAGE gel. The membrane blot was probed with anti-p65 antibody. The same gel was strippedand reprobed with anti-p300 antibody. The gels are representative of three experiments.

Fig. 10. Proposed lovastatin- and RhoA-regulated iNOS expression pathway in C6 glioma cells. Lovastatin inhibits the mevalonatepathway, which also results in the inhibition of GGPP biosynthesis. Lack of GGPP leads to loss of Rho GTPase at the membrane.Unavailability of functional RhoA leads to the activation of the NF-�B pathway leading to iNOS expression. Dominant-negative RhoAenhances the activity of IKK�, which results in enhanced phosphorylation of I�B leading to its degradation, leaving p65:p50 free totranslocate to the nucleus and turn on the iNOS gene.


can acetylate p65 itself. A close relationship has beendemonstrated recently between CBP/p300 and p65.Phosphorylation status of p65 determines its associationwith either CBP (for histone acetylases activity) orHDAC (histone deacetylases activity) and regulates NF-�B-dependent transcriptional activity. This raises thepossibility that lovastatin may also be directly affectingNF-�B activity along with its action via the downregu-lation of the RhoA GTPases. However, the exact cross-talk taking place between the Rho and NF-�B pathwaysstill remains to be elucidated.

Lovastatin, as indicated in the Northern blot (Fig.1D), was able to induce iNOS gene expression signifi-cantly at 9 h (and also for a longer time), suggesting thatit is able to modulate either the transcription or stabilityof the iNOS mRNA. However, lovastatin had no effecton the activity of iNOS-luciferase promoter (Fig. 6C),suggesting that it is not affecting the transcription of thegene but may be stabilizing the protein. The 3�UTRregion has been shown to play a role in the stability ofmurine and human iNOS mRNA [34,35]. It will beinteresting to clone the 3�UTR region of rat iNOSmRNA in conjunction with iNOS-luciferase reporter andsee the effect of lovastatin on its activity. Lovastatin maybe affecting some protein that is responsible for thestabilization or destabilization of the iNOS mRNA.

Overall, we hypothesize that downregulating RhoAprotein by lovastatin induces iNOS gene expression intransformed glioma cells by negatively regulating theNF-�B pathway. (Fig. 10).

Acknowledgements — We gratefully acknowledge Dr. H. Zhang forproviding the 3.2 iNOS promoter luciferase construct, Dr. A. Hall forproviding the RhoA dominant-negative construct, and Dr. G. Rawadifor providing the NF-�B-luciferase promoter construct. We thank Ms.Jennifer G. Schnellmann and Ms. Anne G. Gilg for their assistance inthe preparation of the manuscript. This work was supported by grantsNS-22576, NS-34741, NS 37766, and NS-40810.

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