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 SMCspecific promoters, SM22 and SM ␣-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 M) or actin polymerization with latrunculin B (0.5 M) significantly decreased the activity of SM22 and SM ␣-actin promoters. In contrast, increasing actin polymerization with jasplakinolide (0.5 M) increased SM22 and SM ␣-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 SRFdependent. 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 (1-3). 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 ...

The aims of the present studies were to define sufficient promoter sequences required to drive endogenous expression of smooth muscle (SM) alpha-actin and to determine whether regulation of SM alpha-actin expression in vivo is dependent on CArG (CC(A/T)6GG) cis elements. Promoter deletions and site directed mutagenesis techniques were used to study gene regulation in transgenic mice as well as in smooth muscle cell (SMC) cultures. Results demonstrated that a Lac Z transgene that contained 547 bp of the 5' rat SM alpha-actin promoter was sufficient to drive embryonic expression of SM alpha-actin in the heart and in skeletal muscle but not in SMCs. Transient transfections into SMC cultures demonstrated that the conserved CArG element in the first intron had significant positive activity, and gel shift analyses demonstrated that the intronic CArG bound serum response factor. A transgene construct from -2600 through the first intron (p2600Int/Lac Z) was expressed in embryos and adults in a pattern that closely mimicked endogenous SM alpha-actin expression. Expression in adult mice was completely restricted to SMCs and was detected in esophagus, stomach, intestine, lung, and nearly all blood vessels, including coronary, mesenteric, and renal vascular beds. Mutation of CArG B completely inhibited expression in all cell types, whereas mutation of the intronic CArG selectively abolished expression in SMCs, which suggests that it may act as an SMC-specific enhancer-like element. Taken together, these results provide the first in vivo evidence for the importance of multiple CArG cis elements in the regulation of SM alpha-actin expression.

Extensive studies over the last 30 years have demonstrated that vascular smooth muscle cell (SMC) differentiation and phenotypic modulation is controlled by a dynamic array of environmental cues. The identification of the signaling mechanisms by which these environmental cues regulate SMC phenotype has been more difficult because of our incomplete knowledge of the transcription mechanisms that regulate SMC-specific gene expression. However, recent advances in this area have provided significant insight, and the goal of this review is to summarize the signaling mechanisms by which extrinsic cues control SMC differentiation.

Sphingosine 1-phosphate (S1P) is a lipid agonist that regulates smooth muscle cell (SMC) and endothelial cell functions by activating several members of the S1P subfamily of G-protein-coupled Edg receptors. We have shown previously that SMC differentiation is regulated by RhoA-dependent activation of serum response factor (SRF). Because S1P is a strong activator of RhoA, we hypothesized that S1P would stimulate SMC differentiation. Treatment of primary rat aortic SMC cells with S1P activated RhoA as measured by precipitation with a glutathione S-transferase-rhotekin fusion protein. In SMC and 10T1 ⁄2 cells, S1P treatment up-regulated the activities of several transiently transfected SMC-specific promoters, and these effects were inhibited by the Rho-kinase inhibitor, Y-27632. S1P also increased smooth muscle ␣-actin protein levels in SMC but had no effect on SRF binding to the smooth muscle ␣-actin CArG B element. Quantitative reverse transcriptase-PCR showed that S1P treatment of SMC or 10T1 ⁄2 cells did not increase the mRNA level of either of the recently identified SRF co-factors, myocardin or myocardin-related transcription factor-A (MRTF-A). MRTF-A protein was expressed highly in SMC and 10T1 ⁄2 cultures, and importantly the effects of S1P were inhibited by a dominant negative form of MRTF-A indicating that S1P may regulate the transcriptional activity of MRTF-A. Indeed, S1P treatment increased the nuclear localization of FLAG-MRTF-A, and the effect of MRTF-A overexpression on smooth muscle ␣-actin promoter activity was inhibited by dominant negative RhoA. S1P also stimulated SMC growth by activating the early growth response gene, c-fos. This effect was not attenuated by Y-27632 but could be inhibited by the MEK inhibitor, UO126. S1P enhanced SMC growth through ERK-mediated phosphorylation of the SRF cofactor, Elk-1, as measured by gel shift and Elk-1 activation assays. Taken together these results demonstrate that S1P activates multiple signaling pathways in SMC and regulates proliferation by ERK-dependent activation of Elk-1 and differentiation by RhoA-dependent activation of MRTF-A.

Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538-540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [ 3 H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals.Smooth muscle cell (SMC) proliferation and migration are essential features of vasculogenesis and blood vessel maturation and clearly play a role in the pathophysiology of several prominent cardiovascular disease states, such as atherosclerosis, restenosis following balloon angioplasty, and hypertension (15,24). These processes are regulated by a number of humoral factors, including growth factors (i.e., platelet-derived growth factor [PDGF] and fibroblast growth factor), contractile agonists (i.e., angiotensin II and thrombin), and cytokines (i.e., transforming growth factor ␤ and interleukins) (1,15,24). Extracellular matrix (ECM) interactions are also important, and it is clear that deposition of ECM components within the blood vessel wall and the specific expression of SMC integrin receptors are regulated during vascular development and under pathologic conditions (27). Thus, it is important to identify the molecular mechanisms by which growth factor-and ECMmediated signaling pathways in SMCs converge to regulate proliferation and migration of vascular SMCs.Activation of the integrin families of transmembrane cell surface receptors is mediated by contact with ECM components such as fibronectin and collagen and leads to the formation of focal adhesion structures. Focal adhesions contain not only cytoskeletal components that physically tether the integrin cytoplasmic tail to the actin cytoskeleton but also a number of signaling molecules that are activated in ...