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VASCULAR BIOLOGY
Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Submitted 25 November 2005 ; accepted in final form 10 August 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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cell signaling; posttranslational modification; vascular disease
Several signaling pathways have been implicated in NO-mediated inhibition of SMC proliferation. We and others have demonstrated that NO activation of the extracellular signal-regulated kinase (ERK) pathway is necessary for carrying forth the antiproliferative effect of NO on SMC. Additionally, the phosphorylation and activation of ERK as initiated by NO result in the upregulation of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 (2, 16). Furthermore, these studies show that NO-mediated ERK activation, p21 upregulation, and SMC antiproliferation are independent of cGMP signaling (2, 16).
The ERK pathway has classically been associated with proproliferative signaling. The outcome of ERK signaling appears to be linked to the activation status of the small GTPase RhoA. Olson et al. (27) showed that inactivation of RhoA in the presence of activated ERK led to increased p21Waf1/Cip1 expression and resulted in inhibition of fibroblast proliferation. Rho has recently been recognized to be an important mediator in vascular cell biology, and inhibition of posttranslational prenylation of Rho appears to be the mechanism by which 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors decrease cellular proliferation and increase endothelial NOS (eNOS) expression (23, 25).
The influence of NO on RhoA activation has not been well studied. Several groups have demonstrated that RhoA can be serine phosphorylated by protein kinase G, which can be activated by NO through cGMP (29, 30). The phosphorylation of Rho can lead to decreased membrane association of RhoA, which in turn decreases GTP binding and, therefore, inactivates the small GTPase. Additionally, NO can directly modify proteins and subsequent cell signaling via interaction with cysteines to form nitrosothiols (12, 32, 33). For example, GTP binding of the small GTPase Ras can be increased by this process of S-nitrosation (20, 21). Reversible regulation of protein function by S-nitrosation is proposed to be a form of posttranslational modification. The purpose of the present investigations is to test the hypothesis that NO-induced inhibition of SMC proliferation is dependent on inactivation of RhoA and that NO directly decreases GTP binding of RhoA via S-nitrosation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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RhoA GTP-binding assay. SMC were plated on 10-cm plates and grown to 85% confluence. Cells were treated as described above. GTP binding was assayed using thin-layer chromatography to quantitate the amount of RhoA bound to GTP or to GDP (36). Briefly, serum-free medium was changed to phosphate-free medium, and 0.2 mCi of [32P]orthophosphate was added to the cells for 8 h. This medium was removed, and the cells were exposed to medium with or without 40 mM 1H-1,2,4-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ) for 1 h and then with or without NO donor for an additional 30 min. The cells were scraped from the plates and lysed on ice in 250 µl of buffer A [20 mM Tris with 100 µM phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO), 1 µM leupeptin (Sigma), 1 µM sodium orthovanadate (Sigma), 10 mM MgCl2, and 1% Triton X-100] for 15 min. Nuclei were pelleted by centrifugation at 10,000 g for 10 min. Lysates from the supernatant fraction were immunoprecipitated with 2 µg of mouse anti-RhoA antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and the immunoprecipitate was subjected to thin-layer chromatography. Intensity was determined by phosphor imaging. Relative percent GTP binding was calculated by measurement of intensity of (2/3 GTP)/[(2/3 GTP) + GDP].
RhoA affinity precipitation. RhoA activation was assayed by affinity precipitation of GTP-bound forms of these small GTPases. SMC lysates were collected, and affinity precipitation was performed using kits per the manufacturer’s instructions (Pierce, Rockford, IL).
N-methylanthraniloyl-GTP binding. Fluorometric GTP loading was assayed using a method described by Rojas et al. (28). Briefly, in a total volume of 200 µl/reaction, 0.4 µM N-methylanthraniloyl (MANT)-GTP (Molecular Probes), 2 µM His-tagged recombinant RhoA (Cytoskeleton, Denver, CO), 20 mM Tris (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 1 mg/ml BSA, and 10% glycerol was mixed with 250 µM L-propanamine-2,3-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) or decomposed PAPA-NONOate. The MANT-GTP nucleotide will fluoresce only when associated with a protein. Fluorescence with excitation at 360 nm and emission at 440 nm was determined over time.
GTPase activity assay.
Recombinant RhoA (200 nM; Cytoskeleton) was loaded with 50 nM GTP, i.e., 1:30 [
-32P]GTP (NEN, Boston, MA)-cold GTP, in 20 µl of 20 mM Tris (pH 7.5) and 2 mM EDTA at room temperature for 15 min. The loading reaction was terminated by addition of 3 µl of 100 mM MgCl2, and the sample was placed on ice. Three microliters of loaded RhoA were added to 26 µl of reaction buffer [20 mM Tris (pH 7.5), 1 mM GTP, and 1 mg/ml BSA] containing vehicle or 250 µM PAPA-NONOate or decomposed PAPA-NONOate at 37°C. After 15 min, 4 µl of the sample were removed and added to 2 µl of elution buffer (0.2% SDS, 5 mM EDTA, 5 mM DTT, 5 mM GTP, and 5 mM GDP) to terminate the reaction. The samples were boiled for 5 min, and eluted GTP and GDP were separated by thin-layer chromatography on polyethyleneimine-cellulose with 0.75 M KH2PO4 (pH 3.5) and subjected to autoradiography and PhosphorImager quantification (Molecular Dynamics).
Adenoviral gene transfer. Adenoviral gene transfer of AdLacZ, AdiNOS, or AdRhoA63L was carried out as described previously (16).
Cellular proliferation. Growth-arrested SMC were stimulated to proliferate with 10% fetal bovine serum in the presence of 5 µCi/ml of [3H]thymidine (NEN) for 24 h. [3H]thymidine incorporation into trichloroacetic acid-precipitated DNA was quantified by scintillation counting.
Western blot analysis. SMC lysates were collected as described previously. Protein concentration was quantified using the bicinchoninic acid assay. Whole cell samples (30 µg) were subjected to SDS-PAGE on 13% gels and transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH). Membranes were blocked in 5% nonfat milk for 2 h and hybridized with antibodies against RhoA, p21Waf1/Cip1 (1:1,000 dilution; Santa Cruz Biotechnology), or S-nitrosocysteine [1:250 dilution (Alexis, Lausen, Switzerland) or 1:500 dilution (AG Scientific, San Diego, CA)] followed by horseradish peroxidase-linked goat anti-rabbit or goat anti-mouse antibody (1:10,000 dilution; Pierce). Proteins were visualized using chemiluminescence reagents according to the manufacturer’s instructions (Supersignal Substrate, Pierce).
Immunocytochemistry. SMC were grown on sterilized glass coverslips (Fisher Scientific, Pittsburgh, PA). After treatment, cells were washed twice with ice-cold PBS and fixed immediately in 2% paraformaldehyde in PBS for 20 min at 4°C. The cells were then washed with ice-cold PBS and permeabilized with 0.3% Triton X-100 in PBS for 20 min at 4°C. The cells were rinsed with PBS and then incubated with phalloidin-rhodamine in PBS. Hoechst dye was applied for 30 s to stain nuclei, the slides were rinsed in PBS, and coverslips were applied. Slides were analyzed using an Olympus Provis fluorescence light microscope.
Measurement of nitrosation by NO release.
Initially, recombinant RhoA was incubated with 10 mM DTT for 30 min at 25°C. DTT was removed via centrifugation using a Vivaspin 500 filter (10-kDa mol wt cutoff; Cole Parmer, Vernon Hills, IL); the first spin was followed by four wash cycles with Chelex-purified PBS containing 0.1 mM desferrioxamine. DTT-free RhoA was incubated with 0.3 mM S-nitrosoglutathione (GSNO) for 30 min at 20°C, and the excess GSNO was removed via ultrafiltration using a Vivaspin 500 filter with three wash cycles with PBS-desferrioxamine. In the final reaction solution, the content of RhoA-(SNO)n was assessed with a Bio-Rad protein assay kit. The number of S-nitroso functions per mole of RhoA was determined with a Sievers NO analyzer, with helium used as a gas carrier. The reaction chamber of the analyzer contained 0.1 M phosphate buffer (pH 7.4), 0.2 mM CuCl2, and 50 mM ascorbic acid (RhoA-SNO + Cu+ 
RhoA-SH + Cu2+ + NO). The experimental peaks were calibrated with standard solutions of GSNO under these conditions. No release of NO was observed with up to 0.1 mM NaNO2.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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RhoA activation and NO-induced inhibition of SMC proliferation.
Since NO decreases GTP binding to RhoA, the effect of the activation status of RhoA on NO-induced inhibition of SMC proliferation was studied. Adenoviral gene transfer of constitutively active RhoA (RhoA63L; multiplicity of infection = 100 plaque-forming units), which lacks GTPase function, was utilized in these studies. The influence of NO on this constitutively activated RhoA was evaluated by GTP-binding assays. Gene transfer of constitutively active RhoA increased GTP binding to RhoA by 41 ± 9% compared with control cells treated with an adenoviral vector carrying 
-galactosidase cDNA (AdLacZ) under serum-free conditions (P < 0.05). Although NO treatment modestly decreased GTP binding in AdRhoA63L-treated cells, GTP binding was still significantly elevated compared with AdLacZ-treated controls (Fig. 2A). To assess the influence of NO on SMC proliferation, the NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diole (DETA-NONOate) was used because of its longer half-life. Serum stimulation of SMC significantly increased proliferation compared with serum-free controls (Fig. 2B). Gene transfer using AdLacZ did not alter proliferative rates of serum-treated cells. Gene transfer of RhoA63L, however, increased proliferation of serum-stimulated SMC by 17 ± 5% compared with AdLacZ-treated cells cultured in serum. This modest increase in proliferation by gene transfer of constitutively active RhoA may be secondary to the already relatively high levels of active RhoA under serum-stimulated conditions. Treatment of AdLacZ-infected SMC with the NO donor DETA-NONOate decreased proliferation by 89 ± 6% compared with AdLacZ controls treated with decomposed NO donor (P < 0.01; Fig. 2B). SMC treated with AdRhoA63L were significantly less influenced by DETA-NONOate, with only a 32 ± 7% decrease in proliferation compared with AdLacZ controls treated with decomposed NO donor (Fig. 2B). ODQ inhibition of guanylyl cyclase had no significant influence on these antiproliferative effects (data not shown). Cell counts by trypan blue exclusion corroborated the results of the thymidine incorporation assay, demonstrating similar trends in overall cell number. Additionally, this assay did not show a significantly increased portion of dead (trypan-positive) cells, indicating that the effects of NO are due to inhibition of proliferation, rather than cell death. Furthermore, consistent with previous findings (16), NO donor treatment did not result in increased apoptosis, as assayed by flow cytometry analysis after propidium iodide and annexin V staining (data not shown). Western blot analysis of p21Waf1/Cip1 protein levels corresponded with changes in proliferation. Constitutively active RhoA prevented NO-induced upregulation of p21 and maintained proliferative activity of the SMC (Fig. 2C). These results suggest that NO-induced inhibition of SMC proliferation is, in part, dependent on decreased GTP binding to RhoA.
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320 nm. In addition to this spectrophotometric assay, recombinant RhoA protein treated in the same fashion was subjected to SDS-PAGE using a lysis buffer containing no reducing agents. Blots were then probed with an anti-nitrosocysteine antibody as well as an anti-RhoA antibody. Only the RhoA treated with NO donor produced a signal when probed with anti-RhoA antibody (Fig. 3A). The band coincided with the band detected with the anti-RhoA antibody, indicating that the nitrosothiol group was present on the recombinant RhoA protein. Furthermore, as a control, NO-treated recombinant RhoA was also incubated with 3.7 mM HgCl2. The addition of mercury causes cleavage of the S-nitroso bond of nitrosothiols. This diminished the Western blot signal for nitrosocysteine (Fig. 3A). To determine whether RhoA could be S-nitrosated within cells, SMC were treated with 250 µM PAPA-NONOate or decomposed donor compound for 1 h. Cell lysates were harvested and subjected to immunoprecipitation using anti-RhoA antibody. Immunoprecipitates were resolved by SDS-PAGE and then probed with anti-nitrosocysteine antibody. Immunoprecipitates from SMC treated with PAPA-NONOate demonstrated a nitrosocysteine signal on Western blot analysis (Fig. 3B). Additional experiments were performed in SMC treated with AdiNOS or AdLacZ (multiplicity of infection = 10 plaque-forming units). Cell lysates were collected 24 h after the addition of tetrahydrobiopterin as a required cofactor for inducible NOS (iNOS) activity. After immunoprecipitation with anti-RhoA antibody, Western blot analysis for nitrosocysteine revealed a positive signal in RhoA precipitates from lysates treated with AdiNOS (Fig. 3C). iNOS was utilized because of its increased expression after vascular injury. The addition of the sulfhydryl-reducing agent DTT (1 mM) to these in vitro cell culture experiments diminished the anti-nitrosocysteine signal (Fig. 3, B and C). These findings demonstrate that RhoA can be S-nitrosated by exogenous NO donors as well as by increased NOS expression.
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5 mol SNO per mole of RhoA (Fig. 4A). Analysis of the amino acid sequence of RhoA reveals five cysteines that could be susceptible to S-nitrosation. To ensure that the peaks were not due to excess GSNO after ultrafiltration, we injected this solution, which demonstrated no detectable SNO (injection 1, Fig. 4B) and compared the results with results from injection of the final solution containing RhoA incubated with GSNO (injection 2, Fig. 4B). Injection of a solution of untreated RhoA did not cause release of NO to any significant extent. Furthermore, we show that the increased peak observed after incubation of RhoA with GSNO is reversible with 1 mM DTT. Taken together, these data demonstrate the RhoA could be nitrosated.
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-32P]GTP. GTP-loaded RhoA was then treated with buffer alone, PAPA-NONOate, or decomposed PAPA-NONOate for 15 min. The reaction was stopped, and GTP and GDP were separated by thin-layer chromatography. The relative production of [-32P]GDP was similar between all groups (Fig. 6B). These results indicate that although S-nitrosation of RhoA decreases GTP binding, there is little effect on the intrinsic GTPase activity of the recombinant protein.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Several studies have illustrated that RhoA is a critical signaling molecule involved in the regulation of cellular proliferation. Olson et al. (27) demonstrated that inactivation of RhoA in stimulated fibroblasts led to ERK-induced transcriptional regulation of p21Waf1/Cip1. Multiple studies on SMC have reported that RhoA inactivation is antiproliferative and increases the expression of p21Waf1/Cip1 and/or p27Kip1 (26, 31). We previously reported that RhoA converges with the ERK pathway to influence cell cycle regulation by modulating the actin cytoskeleton and the nuclear accumulation of ERK (36). ERK phosphorylation and the upregulation of p21Waf1/Cip1 are known to be central in NO-mediated SMC antiproliferation. In this study, we integrate these findings and demonstrate that RhoA is a critical signaling molecule in NO-mediated SMC antiproliferation and that NO can directly inactivate RhoA, which is necessary for NO-induced upregulation of p21.
These data suggest that the mechanism by which NO regulates RhoA is, at least in part, through nitrosation of RhoA, which decreases GTP binding and, thus, RhoA activation. RhoA has been shown to regulate NOS activity. Inactivation of RhoA leads to increased expression of NOS enzymes (10, 22, 24). Pharmacological inhibition of RhoA prenylation by 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors or geranylgeranyl transferase inhibitors, as well as inhibition of Rho kinase, can upregulate eNOS and iNOS expression. Conversely, the influence of NO on RhoA expression and activation is less well described. Sauzeau et al. (30) demonstrated that NO, acting through guanylyl cyclase and cGMP-dependent protein kinase, leads to increased RhoA transcription and protein stabilization. These same investigators illustrated that cGMP-dependent protein kinase can serine phosphorylate RhoA, resulting in membrane dissociation and inactivation (30). Several other studies have similarly shown that activation of guanylyl cyclase by NO can lead to the phosphorylation and inactivation of RhoA (3, 4, 8, 19, 29). In the present study, we show that NO inhibition of RhoA is independent of cGMP and that the cGMP analog 8-bromo-cGMP has a minimal effect.
From a biochemical standpoint, nitrosation can occur via multiple intermediates or catalytic pathways after the formation of NO. Based on the literature, we speculate on a number of possibilities for the generation of nitrosating species from our in vitro cell experiments and with NONOate donors and recombinant protein. In the presence of oxygen, NO undergoes oxidation to N2O3, which is a nitrosating species with poor substrate selectivity. In parallel, NO forms metal-nitrosyl complexes that can also act as nitrosating species (5, 17). Since glutathione is the most abundant cellular thiol (15–30 nmol/mg protein), formation of GSNO is expected to parallel the activity of NOS. In fact, several studies have confirmed the formation of GSNO in biological systems (15, 18, 34).
These data demonstrate that RhoA activation status can be altered by S-nitrosation of the protein. S-nitrosation has been proposed to function as a form of posttranslational modification much like phosphorylation or acetylation (1, 11, 32, 33). It has been reported that the related GTPase Ras can be S-nitrosated, which results in an increase in GTP binding and activation (20, 21, 35). Recently, it has also been illustrated that Ran GTPase can also be nitrosylated (9). Jaffrey et al. (14) highlighted a population of proteins that are endogenously S-nitrosated. Our data suggest that RhoA can be S-nitrosated by NO and that GTP binding is subsequently reduced. However, this modification of RhoA had no effect on its intrinsic GTPase activity. S-nitrosation was independent of cGMP. Nevertheless, the contribution of nitrosation vs. cGMP-dependent phosphorylation of RhoA requires further investigation. Furthermore, RhoA contains five putative cysteine residues that may be the target(s) of nitrosothiol formation, which needs to be elucidated, and the effect of this modification on GTP binding necessitates additional studies. Interestingly, overexpression of RhoA63L, which lacks GTPase activity, was not significantly inhibited by NO. However, our studies also show that NO did not influence the GTPase activity of RhoA. Further studies with point mutations of each of the cysteine residues will yield more insight into how NO specifically decreases GTP binding and activation of RhoA.
In conclusion, our findings indicate that inactivation of RhoA plays a role in NO-mediated SMC antiproliferation and that S-nitrosation contributes to decreased GTP binding of RhoA. Nitrosation of RhoA and other proteins likely contributes to cGMP-independent effects of NO signaling.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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