Objective-We have previously shown that smooth muscle cell (SMC) differentiation marker gene expression is regulated by the small GTPase, RhoA. The objective of the present study was to determine the contributions of the RhoA effectors, diaphanous 1 and 2 (mDia1 and mDia2), to this regulatory mechanism. Methods and Results-mDia1 and mDia2 are expressed highly in aortic SMCs and in a number of SMC-containing organs including bladder, lung, and esophagus. Activation of mDia1/2 signaling by RhoA strongly stimulated SMC-specific promoter activity in multiple cell-types including primary aortic SMCs, and stimulated endogenous SM ␣-actin expression in 10T1/2 cells. Expression of a dominant negative Dia1 variant that inhibits both mDia1 and mDia2 significantly decreased SMC-specific transcription in SMCs. The effects of mDia1 and mDia2 required the presence of SRF and the activity of the myocardin transcription factors and were dependent on changes in actin polymerization. Importantly, stimulation of mDia1/2 signaling synergistically enhanced the activities of the myocardin-related transcription factors, MRTF-A and MRTF-B, and this effect was attributable to increased nuclear localization of these factors. Conclusions-These

Objective-Our goal was to test whether formin homology protein 1 (FHOD1) plays a significant role in the regulation of smooth muscle cell (SMC) differentiation and, if so, whether Rho kinase (ROCK)-dependent phosphorylation in the diaphanous autoinhibitory domain is an important signaling mechanism that controls FHOD1 activity in SMC. Methods and Results-FHOD1 is highly expressed in aortic SMCs and in tissues with a significant SMC component.Exogenous expression of constitutively active FHOD1, but not wild-type, strongly activated SMC-specific gene expression in 10T1/2 cells. Treatment of SMC with the RhoA activator sphingosine-1-phosphate increased FHOD1 phosphorylation at Thr1141, and this effect was completely prevented by inhibition of ROCK with Y-27632. Phosphomimetic mutations to ROCK target residues enhanced FHOD1 activity, suggesting that phosphorylation interferes with FHOD1 autoinhibition. Importantly, knockdown of FHOD1 in SMC strongly inhibited sphingosine-1-phosphate-dependent increases in SMC differentiation marker gene expression and actin polymerization, suggesting that FHOD1 plays a major role in RhoA-dependent signaling in SMC. Key Words: biology, developmental Ⅲ molecular biology Ⅲ signal transduction Ⅲ vascular biology Ⅲ vascular muscle S mooth muscle cell (SMC) differentiation is an important component of vascular development, and defective control of this process in adult animals has been shown to contribute to a variety of cardiovascular pathologies, including atherosclerosis and restenosis (see 1 for review). It is well known that SMC differentiation marker gene expression is regulated by serum response factor binding to conserved CArG cis elements within the SMC-specific promoters. The myocardin family of serum response factor cofactors (myocardin and the myocardin-related transcription factor-[MRTF]-A/ megakaryoblastic leukemia-1 and MRTF-B/megakaryoblastic leukemia-2) are also critical and have been shown to be required for SMC differentiation marker gene expression in a variety of in vitro and in vivo models. 2-5 Thus, identifying the signaling mechanisms by which extrinsic cues regulate serum response factor/myocardin factor activity will be critical for our understanding of the control of SMC phenotype. Conclusion-OurMiralles et al were the first to demonstrate that MRTF-A activity is controlled by the small GTPase RhoA and that MRTF-A nuclear localization was enhanced by RhoAdependent actin polymerization. 6 Studies from our laboratory and others have shown that regulation of MRTF-A and MRTF-B by this mechanism plays an important role in the regulation of SMC phenotype in at least some SMC subtypes. [7][8][9] Of the RhoA effector proteins, Rho kinase (ROCK) is the most well studied and has been shown to enhance actin polymerization through LIM kinase-mediated inhibition of cofilin and to stimulate contractility by inhibiting myosin phosphatase. Although the ROCK inhibitor Y-27632 attenuates SMC-specific transcription, 10,11 it is clear that other RhoA effectors are also involved.Ac...

Objective-Our goal was to identify phosphorylation sites that regulate serum response factor (SRF) activity to gain a better understanding of the signaling mechanisms that regulate SRF's involvement in smooth muscle cell (SMC)-specific and early response gene expression. Methods and Results-By screening phosphorylation-deficient and mimetic mutations in SRF Ϫ/Ϫ embryonic stem cells, we identified T159 as a phosphorylation site that significantly inhibits SMC-specific gene expression in an embryonic stem cell model of SMC differentiation. This residue conforms to a highly conserved consensus cAMP-dependent protein kinase (PKA) site, and in vitro and in vivo labeling studies demonstrated that it was phosphorylated by PKA. Results from gel shift and chromatin immunoprecipitation assays demonstrated that T159 phosphorylation inhibited SRF binding to SMC-specific CArG elements. Interestingly, the myocardin factors could at least partially rescue the effects of the T159D mutation under some conditions, but this response was promoter specific. Finally, PKA signaling had much less of an effect on c-fos promoter activity and SRF binding to the c-fos CArG. Key Words: smooth muscle Ⅲ serum response factor Ⅲ protein kinase A S mooth muscle cell (SMC) phenotypic modulation plays an important role in the progression of several prominent cardiovascular diseases, including atherosclerosis, hypertension, and restenosis. 1 Although it is well known that this process is controlled by local environmental cues (see 2 for review), the precise signaling mechanisms that regulate this process are unclear. Extensive evidence indicates that serum response factor (SRF) is a critical transcriptional regulator of SMC differentiation maker gene expression. [3][4][5][6][7][8] However, because SRF is a ubiquitously expressed gene that also regulates early response and cardiac-and skeletal musclespecific gene expression (see 9 for review), it is clear that additional mechanisms are involved. Conclusions-OurThe identification of the myocardin family of transcription factors (myocardin and the Myocardin-Related-Transcription Factors, MRTF-A and MRTF-B) was a particularly important advance because these SRF cofactors strongly transactivate SMC-specific gene expression (see 10 for review). Importantly, knock-out studies in mice have demonstrated that myocardin and MRTF-B are required for the differentiation of specific SMC subtypes, whereas MRTF-A is required for the expression of SM ␣-actin that occurs in mammary epithelial cells during lactation. 10 Early studies also demonstrated that SRF is phosphorylated at multiple residues just N-terminal to the DNA binding domain by several kinases including casein kinase II, CaM kinase IV, and MAPKAP kinase 2. 11-17 Phosphorylation of these sites (especially Ser 103) increased SRF affinity for the c-fos CArG. However, because SRF phosphorylation had only marginal effects on SRF's ability to stimulate c-fos expression and because SRF phosphorylation was not altered by serum stimulation, 18 the physiological ...

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