Myocardin Is a Key Regulator of CArG-Dependent Transcription of Multiple Smooth Muscle Marker Genes

Abstract

Abstract-The interactions between serum response factor (SRF) and CArG elements are critical for smooth muscle cell (SMC) marker gene transcription. However, the mechanisms whereby SRF, which is expressed ubiquitously, contributes to SMC-specific transcription are unknown. Myocardin was recently cloned as a coactivator of SRF in the heart, but its role in regulating CArG-dependent expression of SMC differentiation marker genes has not been clearly elucidated. In this study, we examined the expression and the function of myocardin in SMCs. In adult mice, myocardin mRNA was expressed in multiple smooth muscle (SM) tissues including the aorta, bladder, stomach, intestine, and colon, as well as the heart. Myocardin was also expressed in cultured rat aortic SMCs and A404 SMC precursor cells. Of particular interest, expression of myocardin was induced during differentiation of A404 cells, although it was not expressed in parental P19 cells from which A404 cells were derived. Cotransfection studies in SMCs revealed that myocardin induced the activity of multiple SMC marker gene promoters including SM ␣-actin, SM-myosin heavy chain, and SM22␣ by 9-to 60-fold in a CArG-dependent manner, whereas myocardin short interfering RNA markedly decreased activity of these promoters. Moreover, adenovirus-mediated overexpression of a dominant-negative form of myocardin significantly suppressed expression of endogenous SMC marker genes, whereas adenovirus-mediated overexpression of wild-type myocardin increased expression. Taken together, results provide compelling evidence that myocardin plays a key role as a transcriptional coactivator of SMC marker genes through CArG-dependent mechanisms. Key Words: smooth muscle cells Ⅲ transcriptional coactivator Ⅲ serum response factor Ⅲ CArG element P henotypic modulation of vascular smooth muscle cells (SMCs) is a key factor in the development of atherosclerosis and restenosis after balloon angioplasty. 1 Dedifferentiated SMCs in the intima show a more proliferative and synthetic phenotype than differentiated medial SMCs, which are highly specialized for contraction. 2 To fully understand the control of SMC differentiation, it is important to elucidate the molecular mechanisms that control the transcription of genes encoding proteins necessary for the differentiated function of SMCs.Smooth muscle (SM) ␣-actin, SM-myosin heavy chain (MHC), and SM22␣ are useful markers for studying the control of SMC differentiation. 2 Their expression levels are high in differentiated medial SMCs, whereas they are low in dedifferentiated intimal SMCs. 2 These SMC-selective genes have a conserved DNA recognition element known as a CArG element, which has the general sequence motif, CC(A/ T-rich) 6 GG. 3 The CArG element was first identified as the core sequence of the serum response element (SRE) within the early-response gene, c-fos. 3 Whereas there is only one SRE/CArG element in the c-fos gene, each of the SMC marker genes contains at least two CArG elements located in the 5Ј promoter region and the fir...

Forced Expression of Myocardin Is Not Sufficient for Induction of Smooth Muscle Differentiation in Multipotential Embryonic Cells

“…10 In the present study, we extended these results by real-time RT-PCR. A404 cells were treated with 1 mol/L RA from day 0 to day 3 and with 0.5 g/mL puromycin from day 4 to day 6 to induce differentiation into SMCs as described previously.…”

“…22 Rat aortic SMCs, 10T1/2 cells, and mouse ES cells were cultured as previously described. 10,11 Mouse ES SRFϪ/Ϫ cells were kindly provided by Dr. Alfred Nordheim (Universität Tübingen, Germany). 23 Approximately 24 hours before adenoviral infection, cells were seeded at 10 4 cells/cm 2 .…”

“…8 Short interfering RNA-induced suppression of myocardin in cultured SMCs was associated with marked decreases in transcription of SM ␣-actin, SM-MHC, and SM22␣. 10 Knockout of myocardin in mice resulted in embryonic lethality and was associated with failed SMC investment and differentiation, 13 although the failure to form SM tissues may have been secondary to defects in formation of the extra-embyonic circulation. In addition, we recently provided evidence suggesting that myocardin served as a point of convergence in mediating effects of environmental cues on expression of SMC marker genes in that angiotensin II-induced expression of SM ␣-actin was associated with increased expression of myocardin, and short interfering RNAinduced suppression of myocardin attenuated angiotensin IIinduced transcription of this gene.…”

Transcriptional profiling of the megabladder mouse: A unique model of bladder dysmorphogenesis

“…Prior studies indicate that smooth muscle development is controlled by a complex series of genetic and epigenetic events centered on the dynamic interactions between SRF and myocardin (Chen et al, 2002;Du et al, 2003;Kumar and Owens, 2003;Miano, 2003;Wang et al, 2003;Yoshida et al, 2003;McDonald et al, 2006;Pipes et al, 2006). The most striking feature of our expression profiling analysis is the observed down-regulation of SRF, myocardin, and 22 potential downstream target genes.…”

“…Genetic studies indicate the homeobox genes play an important role in determining the global patterning of the urogenital system (Satokata et al, 1995;Sciavolino et al, 1997;Warot et al, 1997;Goodman and Scambler, 2001;de Santa and Roberts, 2002;Troy et al, 2003), while key signaling molecules like sonic hedgehog and wnts have been implicated in determining the regional patterning within specific urogenital organs (Perriton et al, 2002;Freestone et al, 2003;Mericskay et al, 2004). The differentiation program responsible for the development of smooth muscle involves the complex interaction of epigenetic and genetic events including chromatin remodeling and acetylation, the SRF-myocar-din pathway, basic helix-loop-helix factors, AP-1 complex genes, and the ternary complex factors of the ETS domain family (Manabe and Owens, 2001;Chen et al, 2002;Du et al, 2003;Kumar and Owens, 2003;Miano, 2003;Wang et al, 2003Wang et al, , 2004Yoshida et al, 2003;Buchwalter et al, 2004;Spin et al, 2004;McDonald et al, 2006;Pipes et al, 2006). In addition, a variety of growth factors and hormones have been implicated in modulating the cell-to-cell interactions important for normal bladder morphogenesis (Cunha et al, 1980;Cerro et al, 1993;Finch et al, 1995;Krongrad et al, 1995;Mizuno et al, 1996;Haughney et al, 1998;Warner et al, 1999;Ince et al, 2002;Miyazaki et al, 2003;Villalpando and LopezOlmos, 2003).…”