RhoA Influences the Nuclear Localization of Extracellular Signal–Regulated Kinases to Modulate p21Waf/Cip1 Expression
Background— The 42/44-kD mitogen-activated protein kinases (extracellular signal–regulated kinases, ERKs) regulate smooth muscle cell (SMC) cell-cycle progression and can either promote or inhibit proliferation depending on the activation status of the small GTPase RhoA. RhoA is involved in the regulation of the actin cytoskeleton and converges on multiple signaling pathways. However, the mechanism by which RhoA modulates ERK signaling is not well defined. The purpose of this investigation was to examine whether RhoA regulates ERK downstream signaling and cellular proliferation through its effects on the cytoskeleton and the nuclear localization of ERK.
Methods and Results— Treatment of SMCs with Clostridia botulinum C3 exoenzyme, which inhibits RhoA activation, decreased SMC proliferation to 24±7% of that of controls and increased p21Waf1/Cip1 transcription and protein levels. These effects of RhoA were reversed by inhibition of ERK phosphorylation. However, inactivation of RhoA did not alter levels of ERK phosphorylation but did increase nuclear localization of phosphorylated ERK. In addition, immunostaining demonstrated that phosphorylated ERK associated with the actin cytoskeleton, which was disrupted by C3 exoenzyme. Leptomycin B, an inhibitor of Crm1 that results in ERK nuclear accumulation, similarly increased p21Waf1/Cip1.
Conclusions— RhoA inhibition increased levels of phosphorylated ERK in the cell nucleus. Inhibition of RhoA or pharmacological inhibition of nuclear export resulted in increased p21Waf1/Cip1 expression and decreased SMC proliferation, effects that were partially dependent on ERK. RhoA regulation of the actin cytoskeleton may determine ERK subcellular localization and its subsequent effects on SMC proliferation.
Received January 29, 2003; revision received April 18, 2003; accepted April 21, 2003.
Smooth muscle cell (SMC) proliferation is common to the pathophysiology of many vascular diseases. Such diseases are characterized by vascular injury that results in localized inflammation with elaboration of growth factors and cytokines. These mitogens stimulate SMC cell-cycle progression and proliferation. The ERK signaling pathway has been reported to be important in a number of physiological as well as pathophysiological functions in SMCs, including either the promotion or the inhibition of proliferation.1,2
Several signaling pathways have been identified that mediate the proliferative fate of ERK signaling. The small GTPase RhoA has been demonstrated to modify ERK signaling, cell-cycle–regulatory proteins, and cellular proliferation. Olson et al3 described a role of RhoA in determining whether ERK signaling increases expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 to result in inhibition of fibroblast proliferation. Welsh et al4 illustrated that RhoA, in part, controls the timing of cyclin A and cyclin D expression to promote proliferation. Rho has recently been recognized to be an important mediator in vascular cell biology and inhibition of the posttranslational prenylation of Rho to be the mechanism by which HMG-CoA reductase inhibitors decrease cellular proliferation and increase nitric oxide synthase.2,5,6 The interaction between RhoA and ERK signaling pathways, however, remains unclear.
Other mechanisms can influence ERK activation and cell-cycle progression. These include integrin signaling, cytoskeletal rearrangement, and cell shape.7–9 Interestingly, RhoA influences these other cellular pathways and functions. Moreover, the effects of the temporal aspects of ERK activation on cell-cycle regulation have become increasingly more evident. Harada et al10 demonstrated that sustained ERK activation regulates Egr-1 and cellular proliferation. Murphy et al11 took these observations further to illustrate that induction of immediate-early genes is one mechanism by which cells sense the duration of ERK activation in the G1 phase of the cell cycle.
These above-mentioned studies suggest that RhoA, cytoskeletal proteins, and intracellular trafficking may modulate the outcome of ERK signaling. We demonstrate that RhoA signaling converges with the ERK pathway through the actin cytoskeleton to influence the intracellular localization of ERK and that increased ERK nuclear localization increases p21Waf1/Cip1 protein levels to inhibit SMC proliferation.
Aortic SMCs were cultured from explanted thoracic aortas from Sprague-Dawley rats (Harlan, Indianapolis, IN) as previously described.12
C3 Exoenzyme Treatment
C3 exoenzyme treatment was performed using the scrape loading technique described by Malcolm et al.13 Briefly, cells were gently scraped in isotonic buffer with or without 5 μg/mL C3 exoenzyme (Biomol). Cells were then replated and allowed to recover overnight in medium containing 10% FBS. SMCs were then serum-starved for 24 hours before each assay.
RhoA GTP-Binding Assay
GTP binding was assayed by use of a modification of the method previously described by Downward et al.14 Briefly, SMCs were exposed to phosphate-free medium with 0.2 mCi [32P]orthophosphate for 8 hours and then serum-stimulated (10% FBS) for 15 minutes. Cells were washed with PBS and then lysed in 250 μL buffer A (20 mmol/L Tris with 100 μmol/L phenylmethylsulfonylfluoride, 1 μmol/L leupeptin, 1 μmol/L sodium orthovanadate, 10 mmol/L MgCl2, and 1% Triton X-100) for 15 minutes. Nuclei were pelleted by centrifuging at 10 000g for 10 minutes. Lysates were immunoprecipitated with 2 μg of mouse anti-RhoA antibody (Santa Cruz Biotechnology), and the immunoprecipitate was subjected to thin-layer chromatography. Intensity was determined by phosphor imaging.
Growth-arrested SMCs were serum-stimulated in the presence of 5 μCi/mL of [3H]thymidine (NEN) for 24 hours. For some experiments, cells were treated with leptomycin B (LMB; 2 ng/mL; gift from M. Yoshida, University of Tokyo), C3 exoenzyme, Y27632 (5 μmol/L; Biomol) or U0126 (10 μmol/L; Cell Signaling Technologies). [3H]thymidine incorporation into trichloroacetic acid–precipitated DNA was quantified by scintillation counting.
Nuclear and cytosolic fractions were collected after treatment by scraping cells in buffer A (10 mmol/L HEPES, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5% NP-40, 100 μmol/L sodium orthovanadate, 50 μmol/L dithiothreitol, and protease inhibitors). Lysates were kept at 4°C for 15 minutes and then centrifuged at 5000g for 10 minutes. The supernatant contained the cytosolic fraction. The pellet was washed with buffer A without NP-40 and then centrifuged at 5000g for 10 minutes. The supernatant was discarded, and the pellet was resuspended in a 3:1 mixture of buffer C (20 mmol/L HEPES, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 200 μmol/L EDTA, 10% glycerol, 100 μmol/L sodium orthovanadate, 50 μmol/L dithiothreitol, and protease inhibitors) to buffer D (20 mmol/L HEPES, 1.5 mmol/L MgCl2, 1.6 mol/L KCl, 200 μmol/L EDTA, 10% glycerol, 100 μmol/L sodium orthovanadate, 50 μmol/L dithiothreitol, and protease inhibitors). The suspension was incubated for 60 minutes at 4°C and then centrifuged at 15 000g for 30 minutes. The final supernatant contained the nuclear proteins. Protein concentrations were quantified by bicinchoninic acid protein assay (Pierce).
Western Blot Analysis
SMC lysates were collected as described previously.12 Lysates were subjected to SDS-PAGE on 10% and 13% gels and transferred to nitrocellulose membranes (Schleicher & Schuell). Membranes were blocked in 5% nonfat milk, then hybridized with antibodies against p42/44 MAPK (1:1000; Cell Signaling Technologies), p21, p27, and HSP 90 (Santa Cruz Biotechnology; 1:1000), followed by horseradish peroxidase–linked goat anti-rabbit or goat anti-mouse antibodies (1:10 000; Pierce). Proteins were visualized using chemiluminescence reagents according to the manufacturer’s instructions (Supersignal Substrate; Pierce).
p21Waf1/Cip1 Reporter Assay
Plasmids containing full-length (−2326) p21 promoter linked to the firefly luciferase gene were as described previously by Tombes et al15 and Chinery et al16 (gift from Paul Dent, University of North Carolina). SMCs were transfected with the plasmid construct and a construct to express β-galactosidase using Lipofectamine-2000 (Invitrogen) and then recovered overnight. Six hours after serum stimulation, SMCs were lysed with the manufacturer’s lysis buffer, and luciferase assays were performed according to the manufacturer’s instructions (Promega) with a Bertold luminometer. Results were corrected for protein concentration and transfection efficiency.
After treatment, SMCs grown on glass coverslips were washed with PBS and fixed immediately in 2% paraformaldehyde for 20 minutes. SMCs were then permeabilized with Triton X-100 (0.3%) in PBS for 20 minutes. Nonspecific binding was blocked with 5% goat serum and 1.5% BSA for 30 minutes. The cells were then incubated with primary antibody (rabbit anti-p42/44 MAPK; mouse anti-phosphorylated p42/44; Cell Signaling Technologies) followed by goat secondary antibodies conjugated to Cy3 (Molecular Probes). F-actin was stained with phalloidin-rhodamine. Hoechst dye was applied as a nuclear stain. Slides were analyzed with an Olympus Provis fluorescence light microscope or confocal microscope.
Results are expressed as mean±SEM of 3 independent experiments performed in triplicate. Differences among groups were analyzed with 1-way ANOVA with Student-Newman-Keuls post hoc test for all pairwise comparisons (SigmaStat). Statistical significance was assumed at a value of P<0.05. Western blots depicted are representative of 3 independent experiments.
RhoA Activation and SMC Proliferation
SMCs treated with C3 exoenzyme or untreated were plated and allowed to recover overnight. Treatment with C3 exoenzyme decreased serum-stimulated RhoA GTP binding to 37±14% (P<0.05) of that measured in control SMCs (Fig. 1A). In addition, the effect of RhoA inactivation on SMC proliferation was confirmed by measuring [3H]thymidine incorporation. Inhibition of ERK activation by U0126 in serum-stimulated SMCs decreased DNA synthesis to ≈60% of controls, whereas C3 exoenzyme inhibited SMC DNA synthesis to 24±7% (P<0.01) of controls (Fig. 1B). This effect of RhoA was partially reversed by U0126. Inhibition of rho kinase (a downstream effector protein of RhoA) by Y27632 (5 μmol/L) had effects similar to those of C3 exoenzyme on SMC proliferation. These data suggest that the effects of RhoA inhibition on SMC proliferation are dependent, in part, on the ERK pathway. Likewise, serum-induced proliferation is also partially dependent on ERK activation.
RhoA and Cyclin-Dependent Kinase Inhibitors
To determine whether RhoA inactivation can alter the expression of the cyclin-dependent kinase inhibitors p21Waf1/Cip1 and p27Kip1, protein levels were assayed by Western blot analysis (Fig. 2A). Consistent with previous findings, serum stimulation resulted in a minimal compensatory increase in p21Waf1/Cip1 protein. C3 exoenzyme increased p21Waf1/Cip1 protein levels compared with serum-stimulated controls, and this was partially inhibited by U0126, an inhibitor of ERK phosphorylation. Baseline p27Kip1 protein levels were high in serum-starved SMCs and decreased with serum stimulation. The serum-mediated inhibition of p27Kip1 expression was reversed with U0126, indicating that p27Kip1 expression is regulated by ERK activation. In contrast to p21Waf1/Cip1, however, RhoA inhibition with C3 exoenzyme did not change p27Kip1 expression compared with serum-stimulated controls. Y27632 had effects similar to those of C3 exoenzyme on p21Waf1/Cip1 protein (Fig. 2B). To determine whether RhoA inactivation affected p21Waf1/Cip1 expression at the transcriptional level, the influence of Rho inactivation on p21Waf1/Cip1 promoter activity was measured by luciferase-reporter assay. C3 exoenzyme increased p21Waf1/Cip1 transcription by 2.95-fold that of controls (*P<0.05; Fig. 2C). Pretreatment with U0126 reversed the effects of C3 exoenzyme, resulting in only a 1.55-fold increase over controls. These findings suggest that RhoA or rho-kinase inhibition increased p21Waf1/Cip1 expression through an ERK-dependent pathway. However, RhoA does not appear to be involved in the regulation of p27Kip1 expression.
RhoA and ERK Activation/Nuclear Localization
Because the effects of RhoA on p21Waf1/Cip1 expression and SMC proliferation are at least partially dependent on ERK, the effects of RhoA inhibition on ERK phosphorylation were assayed. Phosphorylated ERK levels in whole-cell lysates of serum-stimulated SMCs (0 to 120 minutes) were not altered by C3 exoenzyme (Fig. 3A). Given the discrepancy between the dependence on ERK activation and lack of effect on ERK phosphorylation, we investigated other possible means of RhoA convergence with ERK. After mitogenic stimuli, activated ERK translocates to the nucleus to regulate the transcription of genes encoding a variety of proteins, including cell-cycle–regulatory proteins. To determine whether RhoA regulates ERK nuclear translocation, levels of phosphorylated ERK were measured by Western blot analysis in nuclear and cytosolic fractions collected from serum-stimulated SMCs treated with C3 exoenzyme or untreated. Levels of phosphorylated ERK were increased significantly in the nuclear fractions of serum-stimulated SMCs treated with C3 exoenzyme compared with that isolated from SMCs not treated with C3 exoenzyme (Fig. 3B).
RhoA mediates the assembly of stress fibers and the actin cytoskeleton, and these structural elements may be associated with ERK nuclear translocation. Fluorescence immunocytochemistry was performed on serum-stimulated SMCs in the absence or presence of C3 exoenzyme or Y27632. Immunocytochemistry for total ERK in C3 exoenzyme–treated (Fig. 4O) and control (Fig. 4N) cells demonstrated diffuse cellular staining. However, immunostaining for phosphorylated ERK revealed colocalization with the actin stress fibers in serum-treated control cells (Fig. 4, A–D). Confocal microscopy was used to confirm the association of phosphorylated ERK and F-actin (Fig. 4M). Staining for F-actin with phalloidin-rhodamine demonstrated that C3 exoenzyme or Y27632 disrupted stress fiber formation within the cells, resulting in a change in cell morphology (Fig. 4, F and J). The organized pattern of phosphorylated ERK staining was absent in the presence of C3 exoenzyme or Y27632. Instead, there was increased staining for phosphorylated ERK within the nucleus (Fig. 4, E and I), confirming the results of Western blotting. These results indicate that activated ERK associates with the actin cytoskeleton and that disruption of the organization of the cytoskeleton by RhoA inhibition leads to accumulation of nuclear ERK.
Nuclear ERK and Cyclin-Dependent Kinase Inhibitors
The quantity of nuclear ERK or the length of time that ERK spends in the nucleus may govern whether ERK signaling is proproliferative or antiproliferative. To determine whether prolonging the presence of ERK in the nucleus modulates p21Waf1/Cip1 expression, SMCs were serum-stimulated in the presence of LMB, an inhibitor of nuclear exportin Crm1. ERK nuclear export is dependent on Crm1.17 Nuclear accumulation of phosphorylated ERK was determined by immunocytochemistry (Fig. 5A). LMB decreased [3H]thymidine uptake in SMCs to 3.1±0.7% that of serum-stimulated controls (Fig. 5B). Like C3 exoenzyme treatment, LMB increased p21Waf1/Cip1 protein expression in serum-stimulated SMCs (Fig. 5C). U0126 (10 μmol/L) partially reversed these effects. LMB had no effect on ERK phosphorylation (data not shown). Furthermore, LMB increased p21Waf1/Cip1 promoter activity by 2.7-fold compared with serum-stimulated controls (P<0.05; Fig. 5D). These data suggest that not only the level of ERK activation but also ERK nuclear localization regulates the expression of p21Waf1/Cip1 and SMC proliferation. The specificity of LMB, however, is lacking, and the profound effect on cellular proliferation most likely results from the interference of Crm1-mediated nuclear export of other proteins as well.
In the present study, we report that RhoA inactivation results in upregulation of p21Waf1/Cip1 and inhibition of proliferation in SMCs through an ERK-dependent pathway. The inactivation of RhoA did not affect levels of ERK phosphorylation/activation but instead led to an increase in localization of nuclear ERK. Thus, suggesting that RhoA activation converges with ERK signaling by modulating the cellular location of activated ERK. Pharmacological inhibition of ERK nuclear export by leptomycin B also resulted in an ERK-dependent upregulation of p21Waf1/Cip1. The duration or intensity of ERK nuclear localization may modify subsequent transcriptional regulation of cell-cycle–regulatory genes. In addition, we demonstrate that phosphorylated ERK associates with the actin cytoskeleton, organization of which is disrupted by inactivation of RhoA. Influences of RhoA on the cytoskeleton may represent how RhoA modulates ERK localization.
C3 exoenzyme does not inactivate exclusively RhoA; it also has some effects on the less well-described RhoB and RhoC.18 However, inhibition of rho kinase, which is a specific downstream effector of RhoA, achieved similar experimental results. This implicates specificity of the RhoA/rho-kinase pathway in mediating these effects.
Proliferation of SMCs usually requires growth factor stimulation, cell adhesion, and an intact cytoskeleton. Previous studies revealed conflicting information about the role of the cytoskeleton in ERK signaling. Multiple studies in fibroblasts show that integrins acting through the cytoskeleton regulate growth factor–induced activation of ERK.19–21 Others have demonstrated that ERK activation occurs independently of cell shape and spreading.7,22 Fringer et al23 reported that ERK phosphorylation was not dependent on intracellular isometric tension and actin stress fiber formation but did require cells to be adherent. Our experiments in SMCs revealed similar findings in which inhibition of RhoA or rho kinase disrupts the cytoskeleton but has no effect on serum-stimulated phosphorylation of ERK. Interestingly, we demonstrated that phosphorylated ERK colocalizes with the actin cytoskeleton. ERK has recently been shown in vitro to associate with actin and the cytoskeleton. Leinweber et al24 demonstrated that ERK 1/2 interact with actin-binding proteins via calponin homology domains. In addition, phosphorylated ERK can localize to focal adhesion complexes.25 The association of ERK with the actin cytoskeleton and subsequent intracellular trafficking of ERK may influence the ultimate transcriptional regulation mediated by these activated proteins.
In addition, the cytoskeleton and RhoA have all previously been linked to the control of cell-cycle–regulatory proteins. Welsh et al4 and Bottazzi et al26 illustrated that RhoA and cytoskeletal integrity, in part, control the timing of cyclin A and cyclin D expression. Olson et al3 demonstrated that inactivation of RhoA in stimulated fibroblasts led to ERK-induced transcriptional regulation of p21Waf1/Cip1. Huang et al7 reported that cell shape governs the proliferative signal in endothelial cells. Multiple studies on SMCs have reported that RhoA inactivation is antiproliferative and increases the expression of p21Waf1/Cip1 and/or p27Kip1.5,27 Our studies suggest that RhoA converges with the ERK pathway to influence cell-cycle regulation by modulating the actin cytoskeleton and the nuclear accumulation of ERK.
Our data demonstrate that RhoA modifies ERK signaling through the regulation of nuclear localization of activated ERK; specifically, that inactivation of RhoA increases the amount of phosphorylated ERK within the nucleus. The mechanisms involved in nuclear-cytoplasmic shuttling of ERK are complex and have only recently been partially elucidated. ERK trafficking to the nucleus requires the formation of ERK homodimers,28 and nuclear export of ERK relies on the nuclear exportin Crm1.17 The interaction between RhoA and this nuclear trafficking machinery or ERK binding/scaffolding proteins to functionally compartmentalize ERK within the cell is not known. However, RhoA and the actin cytoskeleton have been directly linked to the nuclear-cytoplasmic trafficking of ERK. Aplin et al9,29 reported that integrins and cytoskeletal integrity were necessary for the nuclear transport of ERK in fibroblasts.
Studies indicate that it may be the quantity of nuclear ERK or the length of time ERK spends in the nucleus that determines whether ERK signaling is proproliferative or antiproliferative.10,11,30,31 Dent et al32 showed that prolonged activation and increased nuclear levels of ERK inhibited DNA synthesis in hepatocytes and that this was partially dependent on p21Waf1/Cip1 expression. In our studies, pharmacological inhibition of nuclear export by leptomycin inhibited proliferation and p21Waf1/Cip1 expression in SMCs. These effects were partially attenuated by inhibition of ERK phosphorylation. Taken together with our results, these studies delineate an important role for RhoA and the cytoskeleton in mediating the intensity, localization, and timing of ERK activation and cell-cycle regulation.
In conclusion, RhoA influences the nuclear localization of ERK in SMCs. The association of ERK with actin and regulation of the cytoskeleton by RhoA appears to mediate this effect. Spatial and temporal regulation of activated ERK may lead to differential regulation of growth-regulatory proteins. Additional studies are needed to better comprehend the signaling interactions between ERK and RhoA that influence subcellular localization and ultimately proliferation.
This study was supported by grants from the National Institutes of Health (R01-HL-5785405) (Dr Tzeng), the Pacific Vascular Society (Dr Tzeng), and an Ethicon/Society of University Surgeons Resident Research Award (Dr Zuckerbraun).
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