Smooth Muscle Differentiation Marker Gene Expression Is Regulated by RhoA-mediated Actin Polymerization*

Smooth muscle cell (SMC) differentiation is regulated by a complex array of local environmental cues, but the intracellular signaling pathways and the transcription mechanisms that regulate this process are largely unknown. We and others have shown that serum response factor (SRF) contributes to SMC-specific gene transcription, and because the small GTPase RhoA has been shown to regulate SRF, the goal of the present study was to test the hypothesis that RhoA signaling is a critical mechanism for regulating SMC differentiation. Coexpression of constitutively active RhoA in rat aortic SMC cultures significantly increased the activity of the SMC-specific promoters, SM22 and SM α-actin, whereas coexpression of C3 transferase abolished the activity of these promoters. Inhibition of either stress fiber formation with the Rho kinase inhibitor Y-27632 (10 μm) or actin polymerization with latrunculin B (0.5 μm) significantly decreased the activity of SM22 and SM α-actin promoters. In contrast, increasing actin polymerization with jasplakinolide (0.5 μm) increased SM22 and SM α-actin promoter activity by 22-fold and 13-fold, respectively. The above interventions had little or no effect on the transcription of an SRF-dependent c-fos promoter or on a minimal thymidine kinase promoter that is not SRF-dependent. Taken together, the results of these studies indicate that in SMC, RhoA-dependent regulation of the actin cytoskeleton selectively regulates SMC differentiation marker gene expression by modulating SRF-dependent transcription. The results also suggest that RhoA signaling may serve as a convergence point for the multiple signaling pathways that regulate SMC differentiation.

Vascular smooth muscle cell (SMC) 1 differentiation is an important process during vasculogenesis and angiogenesis, and it is recognized that alterations in SMC phenotype play a role in the progression of several prominent cardiovascular disease states including atherosclerosis, hypertension, and restenosis (1)(2)(3). It is well established that SMC do not terminally differentiate and that SMC phenotype is regulated by a complex array of local environmental cues including humoral factors, cell-cell and cell-matrix interactions, inflammatory stimuli, and mechanical stresses (reviewed in Refs. 4 and 5). However, the mechanisms by which these diverse signals regulate SMC phenotype and the transcription mechanisms that ultimately regulate SMC differentiation are largely unknown. It is clear though that the identification of the signaling and transcription pathways that control SMC-specific gene expression will be an important step toward our understanding of blood vessel development and the role played by SMC during the development of cardiovascular disease.
To date, no transcription factors have been identified that specify SMC lineage or by themselves can explain SMC-specific gene expression. However, serum response factor (SRF), a MADS box transcription factor, has been shown to contribute to the regulation of most SMC differentiation marker genes and may be a key transcriptional regulator of SMC differentiation (6 -9). Interestingly, CArG cis-elements (CC(A/T) 6 GG) that bind SRF are present in the promoters of virtually all of the SMC-specific marker genes including SM ␣-actin, SM myosin heavy chain, SM22, h-caldesmon, calponin, and telokin. The CArGs within any given SMC-specific promoter are highly conserved among species and have been shown to be required for the high level of activity that these promoters exhibit in SMC cultures (7, 8, 10 -14). Results from transgenic studies demonstrate that CArG elements within the SM22 and SM ␣-actin promoters were also required for the expression of these genes in vivo (8 -10). Indeed, we have shown that interactions among multiple CArG elements are important for the in vivo expression of SM ␣-actin and that a CArG element within the SM ␣-actin first intron functions as a SMC-specific enhancerlike element (10). Because SRF is ubiquitously expressed, the mechanisms whereby it contributes to SMC differentiation are somewhat unclear. However, evidence suggests that other factors including additional cis-and trans-regulatory elements may mediate cell-type-specific regulation by SRF (6,(15)(16)(17)(18)(19)(20)(21)(22).
The CArG motif was first identified as the core sequence of the serum response element (SRE) within early response genes such as c-fos, and much of what is known regarding the signaling pathways that regulate SRF-dependent transcription resulted from studies on the c-fos promoter (reviewed in Ref. 23). During serum-or growth factor-mediated stimulation, SRF forms an initiation complex with a protein from the ternary complex factor family whose members (Elk-1, SAP-1, and Net/ Erp) are regulated by MAP kinase phosphorylation (24 -26). The ternary complex is stabilized by protein interactions between SRF and the ternary complex factor and by interactions between the ternary complex factor and the Ets cis-element that flanks the SRE CArG (27). Recent evidence suggests that SRF-dependent transcription is also regulated by the small GTP-binding protein RhoA, which is required for serum-induced c-fos expression, and may be important for regulating CArG-dependent genes in the skeletal muscle as well (28 -31). The effects of RhoA on SRF-dependent transcription are not dependent upon the presence of an Ets domain or on the known SRF accessory proteins suggesting that RhoA activates SRFdependent transcription in a ternary complex-independent manner. Importantly, the CArG elements within the SMCspecific promoters are not flanked by a consensus Ets domain and do not appear to be regulated by an Ets-dependent mechanism.
Recently, Sotiropoulos et al. (32) have shown that in NIH 3T3 cells, the effects of RhoA on SRF-mediated transcription may be secondary to the actions of RhoA on the actin cytoskeleton. These results are of particular importance because they are the first to suggest that SRF-dependent transcription is positively regulated through increases in actin polymerization. The exact mechanisms for SRF activation remain to be identified, and the significance of these findings in regard to the expression of SRF-dependent gene targets is unclear because an artificial c-fos promoter (lacking a consensus Ets domain) was used as the primary measure of Rho-dependent SRF activation.
In the present studies, we extend the observations of Sotiropoulos et al. (32) by demonstrating in SMC that multiple SMCspecific promoters are regulated via RhoA-dependent changes in actin dynamics. In addition, we demonstrate that these RhoA-induced changes are at least partially mediated by Rho kinase. Because virtually all of the SMC-specific promoters identified to date are regulated by SRF, these results suggest that RhoA signaling regulates SMC phenotype. Moreover, because many of the environmental factors that regulate SMC phenotype have also been shown to activate RhoA, these results also suggest that RhoA may help to integrate multiple environmental signals that regulate SMC differentiation.

MATERIALS AND METHODS
Cell Culture Transient Transfections and Reporter Assays-SMC from rat thoracic aorta were isolated, cultured, and transfected as described previously (33,34). In short, cells were maintained in 10% serum and were transfected 24 h after plating at 70 -80% confluency with 2 g of reporter plasmid DNA, using the transfection reagent Superfect (Qiagen) as per protocol. The SM22 promoter (from Ϫ450 to ϩ88), the SM ␣-actin promoters (p2600Int, from Ϫ2560 to ϩ2784, and p125, from Ϫ125 to ϩ44), and the c-fos promoter (from Ϫ356 to ϩ109) that were used in this study have been described previously (8 -10, 35). Coexpression experiments were carried out by including 0.25 g of the following expression constructs: empty Prk5 vector; Prk5 containing constitutively active RhoA (L63), Cdc42 (L61), or Rac (L61); or Prk5 containing C3 transferase (expression plasmids were a generous gift from Alan Hall, University College, London, United Kingdom). Cells expressing C3 were maintained in 10% serum, whereas cells expressing the small G proteins were maintained in defined serum-free medium. After 48 h, cell lysates were prepared for the measurement of CAT activity using standard methods (34). The CAT activity of each sample was normalized to the protein concentration of each cell lysate as measured by the Bradford assay. A promoterless CAT construct was also transfected to serve as a base-line indicator of CAT activity, and the activity of each promoter construct was expressed relative to promoterless activity. Additionally, an SV40 promoter-CAT construct with enhancer (Promega) served as a positive control of transfection and CAT activity. All CAT activities represent at least three independent experiments with each construct tested in triplicate per experiment. Relative CAT activity data are expressed as the means Ϯ S.D. computed from the results obtained from each set of transfection experiments. We did not cotransfect a viral promoter/LacZ construct as a control for transfection efficiency because we have previously shown that such constructs exhibit unknown and variable squelching effects on the SM-specific promoters, presumably due to competition for common transcription factors (34). Moreover, we have found that the inclusion of such controls is unnecessary in that variations in transfection efficiency among independent experimental samples are routinely very small (Ͻ10%) (34).
Drug Treatment and F-actin Visualization-Latrunculin B and cytochalasin D were purchased from Calbiochem (San Diego, CA). Jasplakinolide was purchased from Molecular Probes (Eugene, OR). Y-27632 was a generous gift from Akiko Yoshimura (Welfide Corporation, Osaka, Japan). Before drug treatment, SMC were transfected with SMC-specific and c-fos reporter constructs as described above. Immediately after the removal of the Superfect transfection reagent, cells were treated with either latrunculin B (0.5 M), jasplakinolide (0.5 M), cytochalasin D (2.0 M), Y-27632 (10 M), or vehicle. SMC treated with latrunculin B or Y-27632 were maintained in 10% serum, and those treated with jasplakinolide or cytochalasin D were maintained in defined serum-free medium. After 12 h, cells were harvested, and reporter assays were performed as described above. Drug-induced effects on actin polymerization were measured in parallel experiments for all groups. At 0, 5, 10, and 24 h after drug treatment, cells were rinsed three times with ice-cold phosphate-buffered saline, fixed with 4% paraformaldehyde, stained with FITC-phalloidin, and visualized with a fluorescent microscope. SMC transfected with a constitutively active Myc-tagged L63 RhoA were immunostained with an anti-c-Myc antibody and counterstained with FITC-phalloidin.
Measurement of Endogenous Actin Expression-Control-or Y-27632treated SMC were maintained in minimal methionine medium containing 10% fetal calf serum for 12 h. Cells were then switched to fresh medium containing 50 Ci/ml [ 35 S]methionine and were incubated for an additional 12 h. Cells were then rinsed three times with ice-cold phosphate-buffered saline and lysed in loading buffer. Equal amounts of DNA were loaded onto tube gels, and actin isoforms were visualized by standard two-dimensional gel electrophoresis as described previously (36). Multiple labeling experiments were performed, and three separate gels were run for each treatment group per experiment. The ratio of radiolabeled SM ␣-actin to nonmuscle ␤-actin was determined by densitometry using ImageQuant software (Molecular Dynamics, Sunnydale, CA).

SMC-specific Promoter Activity Was Regulated by RhoA Signaling-Current evidence suggests that signaling through
RhoA is required for SRE-dependent c-fos expression and regulates several CArG-containing genes in the skeletal muscle (28 -31). To determine whether RhoA was important for CArGdependent SMC-specific gene regulation, we cotransfected CAT reporter constructs driven by the SM22 and SM ␣-actin promoters into rat aortic SMC along with an expression vector containing C3 transferase, which specifically ADP-ribosylates and irreversibly inhibits RhoA. The SM22 promoter (from Ϫ450 to ϩ88) and the SM ␣-actin promoter (from Ϫ2600 to ϩ2754) used in these studies were shown to direct SMC-specific regulation of these genes in transgenic models (8 -10). C3 transferase expression completely abolished the activity of both promoters, indicating that RhoA activity is required for the expression of these constructs in SMC (Fig. 1A). To test the involvement of RhoA more directly, we also cotransfected a constitutively active RhoA (L63) into serum-starved SMC. L63 RhoA trans-activated the SM22 and the full-length SM ␣-actin promoters by 10-fold and 6-fold, respectively (Fig. 1B). Constitutively active RhoA also dramatically increased the activity of a minimal SM ␣-actin promoter (Ϫ125 to ϩ44) that is regulated by two CArG elements within this short promoter region but had no effect on the same construct that contained mutations to the CArG elements (data not shown). These results indicate that the effects of RhoA on SMC-specific gene expression were mediated by a CArG/SRF-dependent mechanism. Significantly, L63 RhoA had relatively modest effects on the activity of an Ets-containing SRE-dependent c-fos promoter in SMC.
Because the Rho family members Cdc42 and Rac have also been shown to up-regulate the SRF activity of c-fos, we also tested whether the constitutively active forms of these proteins would regulate SMC differentiation marker gene expression. Although all three small GTPases slightly but significantly increased c-fos promoter activity (p Ͻ 0.01), SM ␣-actin promoter activity was not affected by constitutively active Rac and was slightly inhibited by constitutively active Cdc42 (Fig. 1C).
RhoA regulated Stress Fiber Formation in SMC-Sotiropoulos et al. (32) have suggested that RhoA activation regulates gene expression through its effects on the actin cytoskeleton. Therefore, to test the effects of RhoA activation on actin polymerization, we transfected a Myc-tagged L63 RhoA expression plasmid into SMC that were then stained for F-actin stress fibers with FITC-phalloidin. Fig. 2B shows that untransfected SMC maintained in 10% serum had fairly well developed stress fibers (small arrows). However, SMC expressing L63 RhoA had dramatically increased actin stress fiber networks (large arrows), demonstrating that RhoA activation regulates stress fiber formation in SMC.
SMC Differentiation Marker Gene Transcription Was Regulated by Actin Polymerization-Our results suggested that RhoA activation regulates both SMC-specific gene transcription and stress fiber formation in SMC. To test whether the effects of RhoA on SMC-specific promoter activity were mediated by its effects on actin dynamics, we treated SMC with latrunculin B, a toxin that disrupts actin polymerization. Latrunculin B treatment (0.5 M) of SMC in 10% serum disrupted stress fiber formation for up to 12 h (Fig. 3A). After this time, actin polymerization gradually returned, and by 24 h SMC treated with latrunculin B were virtually indistinguishable from untreated cells. In addition, latrunculin B also had little effect on total cellular protein, which indicated that this drug was not overtly cytotoxic under these conditions. Latrunculin B did not affect c-fos promoter activity but dramatically attenuated the activities of the SM22 and SM ␣-actin promoters (Fig.  3B). Importantly, latrunculin B also inhibited trans-activation of the SM ␣-actin promoter by L63 RhoA as well as L63 RhoAinduced stress fiber formation.
The results presented in Fig. 3 suggest that RhoA activation induces SMC-specific gene expression by increasing actin polymerization. Therefore, to further test whether SM ␣-actin transcription was sensitive to actin dynamics, we treated SMC with jasplakinolide, a toxin known to increase actin polymerization (37). SMC treated with 0.5 M jasplakinolide and labeled with FITC-phalloidin had very disorganized actin structures and contained large amorphous actin aggregates (data not shown). These effects are consistent with the known actions of this toxin, which severs F-actin, creating new nucleation sites that support actin polymerization (37). Jasplakinolide increased SM22 and SM ␣-actin promoter activities by 22-fold and 14-fold, respectively, but increased c-fos promoter activity by only 3-fold. Cytochalasin D had similar effects increasing the activities of the SM22 and SM ␣-actin promoters but having no effect on the c-fos promoter. The fact that cytochalasin D (which disrupts actin polymerization) increased SMC-specific promoter activity suggests that cytochalasin-induced SRF activity is not directly regulated by increased actin polymerization per se and that additional mechanisms that sense actin dynamics in SMC may be involved in regulating SMC-specific transcription. Interestingly, Sotiropoulos et al. (32) made similar observations in their studies on the c-fos SRE and suggested that SRF-dependent transcription may be negatively regulated by monomeric G-actin and that cytochalasin D may sequester monomeric G-actin in a manner that effectively reduces the free pool. To determine whether the effects of cytochalasin D were independent of those mediated by RhoA, we treated SMC transfected with L63 RhoA with cytochalasin D. The treatment of SMC with cytochalasin D prevented both the L63 RhoA-induced increase in differentiation marker gene expression (Fig. 4B) and L63 RhoA-induced stress fiber formation (data not shown).
Transcriptional Regulation by RhoA in SMC Was Partially Mediated by Rho Kinase-It is well known that RhoA-dependent activation of Rho kinase induces actin polymerization and stress fiber formation in many cell types (reviewed in Ref. 38). However, it is unclear whether the activity of Rho kinase correlates with the effects of RhoA on the transcription of c-fos (39 -42). Our results indicated that in SMC, the SMC-specific promoters were significantly more sensitive than the c-fos promoter was to activation by RhoA signaling. Therefore, to test whether SMC-specific transcription was regulated through the activation of Rho kinase, we treated SMC with the highly specific Rho kinase inhibitor Y-27632 (43). The micrograph in Fig. 5A shows that 10 M Y-27632 significantly inhibited stress fiber formation in SMC and also led to the appearance of long thin cellular extensions. Y-27632 also reduced the activity of transiently transfected SMC-specific promoters by 50 -60% (Fig. 5B), although it had little effect on the c-fos promoter or on the minimal thymidine kinase (TK) promoter that does not contain CArG elements. Fig. 5C is an autoradiograph from a representative two-dimensional gel that illustrates the effects of Y-27632 on the expression of endogenous actin isoforms. Interestingly, the results showed that all of these CArG-containing actin genes were down-regulated to some extent. However, the synthesis of the SM-specific ␣-actin isoform was inhibited to a greater extent than the ubiquitously expressed ␤-actin isoform (␣:␤ synthesis ratios were reduced by greater than 40% in Y-27632-treated SMC, p Ͻ 0.01). Taken together, these results suggest that Rho kinase plays an important role in the regulation of SMC differentiation marker gene expression and that ubiquitously expressed CArG-dependent genes, such as c-fos and nonmuscle ␤-actin, may be less sensitive to this regulatory pathway. DISCUSSION A major goal of our laboratory has been to elucidate control processes that regulate SMC differentiation by identifying the mechanisms that coordinately control the transcription of SMC differentiation marker genes. The focus of the present study was to investigate the role of RhoA signaling in the regulation of SMC-specific gene transcription. Because RhoA activates SRF, a transcription factor that has been shown to regulate virtually all of the SMC-specific marker genes identified to date (7, 8, 10 -14), we hypothesized that RhoA signaling contributes to the coordinate regulation of SMC-specific gene expression. The data presented indicate that: 1) RhoA activation is important for the expression of several SMC-specific marker genes; 2) regulation of actin polymerization by RhoA (but not by Cdc42 or Rac) may be an important determinant of SMC-specific gene transcription; 3) RhoA-dependent regulation of SMC-specific gene transcription is mediated at least in part by Rho kinase; and 4) in SMC, the CArG-dependent SMC-specific genes (i.e. SM22, SM ␣-actin) and the CArG-dependent genes (i.e. c-fos, ␤-actin) that are ubiquitously expressed are differentially regulated by RhoA.
Results presented in Fig. 1 provide strong evidence that RhoA activity is required for SMC-specific promoter activity because C3 transferase, which ADP-ribosylates and irrevers-ibly inactivates RhoA, completely inhibited the activities of the SM22 and SM ␣-actin promoters. Constitutively active RhoA up-regulated several SMC-specific CArG-dependent genes, further indicating that RhoA activation is important for this response. RhoA activation has been shown to have similar effects on the SRF-dependent activity of several CArG-dependent skeletal muscle-specific genes (30,31), suggesting that RhoA activation may be a common signaling pathway that regulates muscle-specific gene expression. In fact, RhoA also regulates SRF expression (probably through CArG elements within the SRF promoter) and may be part of an important positive feedback mechanism for maintaining high levels of SRF expression in all three muscle cell types (44,45). Although our results indicate that RhoA-dependent activation of SRF plays a major role in SMC-specific transcription, other mechanisms must be involved that distinguish SRF-dependent transcriptional regulation in SMC from SRF-dependent transcriptional regulation in the other muscle cell types. For example, it is well known that in cardiac and skeletal muscles, SRF interacts with other . After 12 h, cells were fixed in 4% paraformaldehyde and stained with FITC-phalloidin to visualize actin stress fibers. B, SMC were transfected with the indicated reporter constructs and immediately treated with Y-27632. After 24 h, CAT activity was measured and expressed relative to a promoterless CAT construct (pless) set to 1. Note that Y-27632 had little effect on the c-fos promoter or the minimal thymidine kinase promoter (TK). C, rat aortic SMC maintained in 10% serum were treated with Y-27632 for 12 h. Cells were then switched to [ 35 S]methionine containing medium and restimulated with Y-27632 for an additional 12 h. Equal amounts of control-and drug-treated SMC lysates were run on two-dimensional gels using standard protocols for visualizing actin isoforms. A representative autoradiograph is shown. cardiac and skeletal muscle-specific factors, such as myoD, GATA-4, and Nkx2.5, to regulate cell-type-specific expression (22, 46 -48). However, factors that confer SMC-specific gene regulation have not been identified in SMC. We have recently provided evidence that an SRF-associated protein that is selectively expressed in SMC may contribute to SMC-specific gene regulation by SRF (6). Several splice variants of SRF have also been described recently, but a detailed description of the expression patterns and activities of these variants has not been reported, which makes it difficult to determine whether alternative splicing of SRF contributes to cell-type-specific gene regulation (49,50).
Our results also indicate that multiple SMC-specific differentiation marker genes are regulated by RhoA-induced changes in the actin cytoskeleton. Constitutively active RhoA trans-activated the SM22 and SM ␣-actin promoters but had little effect on the CArG-containing c-fos promoter. Inhibiting actin polymerization with latrunculin B blocked trans-activation by constitutively active RhoA, which supports a model in which RhoA-mediated increases in F-actin promote SMC differentiation marker gene transcription. Our observation that jasplakinolide, an actin polymerizing agent, dramatically increased SMC-specific promoter activity is also consistent with such a model. The constitutively active forms of Cdc42 and Rac did not stimulate SMC-specific promoter activity, suggesting that actin polymerization at the cell periphery does not regulate SMC-specific transcription and suggests that separate pools of actin may differentially regulate SMC function. Our data also indicate that signaling through Rho kinase is significant, but because Y-27632 only inhibited promoter activity by 50 -60%, we cannot rule out the involvement of other Rho effectors, such as PKN, citron kinase, rhotekin, and the diaphanous family of proteins.
The precise mechanisms by which actin dynamics affect SRFdependent transcription are currently unclear. Based in part on the observed effects of cytochalasin D, Sotiropoulos (32) has suggested a model whereby G-actin inhibits SRF directly or sequesters cofactors required for SRF activation. Our data indicate that cytochalasin D-induced activation of SRF-dependent SMC-specific transcription does not require an intact actin stress fiber network. However, we did show that the expression of constitutively active RhoA in cytochalasin D-treated SMC did not result in stress fiber formation or in an increase in SM ␣-actin promoter activity above that seen with cytochalasin D alone. These data indicate that in SMC these interventions probably converge at some level and that RhoA must affect actin polymerization to increase SMC marker gene transcription. There is some evidence to suggest that nuclear actin may regulate transcription by helping to modify chromatin structure (51), and it has been shown that G-actin shuttles through the nucleus (52) and binds specifically to important nuclear proteins including the SWI⅐SNF complex (53,54). It is also important to consider that the observed effects of RhoA activation (and of the cytoskeletal inhibitors) may be partially due to secondary changes in cell morphology that occur under these conditions. In addition, the pharmacological mechanisms of the cytoskeletal inhibitors used in this study are only partially understood, and undescribed side effects of these drugs may have affected SMC differentiation marker gene expression.
The signal transduction pathways that regulate SMC differentiation are only partially understood, but results from the present studies suggest that RhoA activation may serve as a convergence point for multiple environmental factors known to regulate SMC-specific gene expression and differentiation. For example, integrin-matrix interactions, mechanical stretch, and contractile agonists, such as endothelin-1 and angiotensin II, are known to regulate SMC-specific gene expression as well as the activity of RhoA (40,(55)(56)(57). Whether these environmental factors regulate SMC differentiation through RhoA-dependent signaling mechanisms and/or changes in actin dynamics will be an important area for future studies.
RhoA activation is also required for growth factor-induced expression of c-fos, and recent evidence suggests that RhoA activation may mediate the growth response of SMC to angiotensin II, thrombin, and mechanical stretch (40,55,58). The data presented in this manuscript, however, demonstrate that c-fos expression in SMC is not dramatically upregulated by constitutively active RhoA or by drug-induced modulation of the actin cytoskeleton, indicating that the RhoA signaling pathways that regulate SMC differentiation marker gene expression diverge from those that regulate cell growth and proliferation. SMC proliferation and differentiation are not necessarily mutually exclusive (4), and it will be very important to further clarify the specific signals that differentiate these two SRF-dependent pathways. Indeed, Seasholtz et al. (58) found that constitutively active RhoA only potentiated SMC proliferation in the presence of activated Ras, and results from Alberts et al. (28) indicate that additional signaling through the SAPK/JNK pathway may be important for the Rho-mediated regulation of c-fos. Integrin signaling also requires Rho activation (59 -61), and it interesting to speculate that Rho and integrin pathways may converge (perhaps at the level of the actin cytoskeleton) to regulate SMC growth and/or differentiation.
In summary, the data presented herein indicate that RhoA signaling plays a major role in the SRF-dependent regulation of SMC differentiation marker gene expression. Although the exact mechanisms involved in controlling the transcriptional activation of SRF are not completely clear, the activation of Rho kinase and RhoA-specific changes in actin polymerization seem to be involved. Interestingly, most SMC-specific differentiation marker genes identified to date code for contractile or contractile-associated proteins (4). Therefore, RhoA signaling may also help to determine SMC phenotype (and possibly the phenotype of other muscle cell types as well) by increasing contractile protein gene expression. Moreover, when coupled with extensive evidence that RhoA increases SMC contractility (43,62), our data suggest that short term regulation of SMC contractile force may be coupled to long term regulation of SMC contractile-associated gene expression by a RhoA-dependent mechanism. Finally, given the importance of SMC growth and differentiation during blood vessel development and cardiovascular pathophysiology, further studies to identify the mechanisms that regulate RhoA-dependent changes in SMC differentiation marker gene expression may provide novel insights regarding the mechanisms that contribute to the abnormal regulation of SMC differentiation characteristic of vascular diseases such as atherosclerosis.