Myocardin Is a Critical Serum Response Factor Cofactor in the Transcriptional Program Regulating Smooth Muscle Cell Differentiation

Abstract

The SAP family transcription factor myocardin functionally synergizes with serum response factor (SRF) and plays an important role in cardiac development. To determine the function of myocardin in the smooth muscle cell (SMC) lineage, we mapped the pattern of myocardin gene expression and examined the molecular mechanisms underlying transcriptional activity of myocardin in SMCs and embryonic stem (ES) cells. The human and murine myocardin genes were expressed in vascular and visceral SMCs at levels equivalent to or exceeding those observed in the heart. During embryonic development, the myocardin gene was expressed abundantly in a precise, developmentally regulated pattern in SMCs. Forced expression of myocardin transactivated multiple SMC-specific transcriptional regulatory elements in non-SMCs. By contrast, myocardininduced transactivation was not observed in SRF ؊/؊ ES cells but could be rescued by forced expression of SRF or the SRF DNA-binding domain. Furthermore, expression of a dominant-negative myocardin mutant protein or small-interfering-RNA-induced myocardin knockdown significantly reduced SM22␣ promoter activity in SMCs. Most importantly, forced expression of myocardin activated expression of the SM22␣, smooth muscle ␣-actin, and calponin-h1 genes in undifferentiated mouse ES cells. Taken together, these data demonstrate that myocardin plays an important role in the SRF-dependent transcriptional program that regulates SMC development and differentiation.The diverse functions mediated by smooth muscle cells (SMCs) in organ systems throughout the body are ultimately dependent upon the expression of a unique set of SMC-restricted contractile and cytoskeletal proteins that distinguish this cell lineage from cardiac and skeletal myocytes. A distinguishing feature of the SMC lineage is the capacity of SMCs to reversibly modulate their phenotype and proliferate in response to a variety of stimuli during postnatal development (for a review, see reference 35). In the vasculature, SMCs in the tunica medium of arteries and veins are cell cycle arrested and express a set of lineage-restricted genes, including those for smooth muscle (SM) myosin heavy chain, SM ␣-actin, SM22␣, and calponin, which together define the unique contractile properties of this muscle cell lineage. However, in response to arterial injury, SMCs downregulate expression of contractile genes and concomitantly upregulate a set of genes required for synthetic, migratory, and proliferative functions. This phenotypic modulation has been implicated in the pathogenesis of diseases, including atherosclerosis, restenosis following coronary angioplasty and/or stent implantation, pulmonary hypertension, and asthma (14,33,39,41).Because it is expressed exclusively and abundantly in SMCs during postnatal development (23, 42), our group and others have utilized the SMC-restricted SM22␣ promoter as a model system to elucidate the molecular mechanisms that regulate SMC differentiation and modulation of the SMC phenotype (4,19,24,28,34,42,44). The 441-bp mo...

Induction and modulation of smooth muscle differentiation in Xenopus embryonic cells

“…In Xenopus animal cap cells, myocardin initiates the transcription of several SM markers, including SM22␣, SM ␣-actin, and calponin (Du et al, 2003;Small et al, 2005). We have confirmed those findings and extended it to SM-MHC.…”

“…A growing number of proteins, including GATA, MEF2, Nkx2.5 and myocardin family members have been shown to interact with SRF and modulate the transcription of target genes in a smooth muscle context (Miano, 2003). Myocardin and the myocardin-related transcription factors (MRTFs) are of particular interest because gain-and loss-of-function experiments showed that they were essential for the development and differentiation of SMC (Du et al, 2003;Li et al, 2003Li et al, , 2005Wang and Olson, 2004;Oh et al, 2005). The basic helix-loop-helix (bHLH) HAND proteins have also been implicated in the regulation of smooth muscle gene expression in mammals (Yamagishi et al, 2000;Morikawa and Cserjesi, 2004).…”

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).…”

Kruppel-like Factors (KLFs) in muscle biology

“…Using siRNA approaches, studies from the Owens' laboratory suggest that KLF4 mediates, at least in part, the repressive effects of PDGFbb on SMC gene expression. This effect may occur as a consequence of KLF4 mediated repression of myocardin -a critical transcriptional coactivator involved in SMC differentiation [85,[87][88][89][90]. In contrast to PDGF-bb, TGFβ1 is known to induce SMC gene expression.…”