Abstract-Transforming growth factor (TGF)-␤ 1 is an important cytokine involved in various diseases. However, the molecular mechanism whereby TGF-␤ 1 signaling modulates the regulatory network for smooth muscle gene transcription remains largely unknown. To address this question, we previously identified a Smad-binding element (SBE) in the SM22␣ promoter as one of the TGF-␤ 1 response elements. Here, we show that mutation of the SBE reduces the activation potential of a SM22␣ promoter in transgenic mice during embryogenesis. Chromatin immunoprecipitation assays reveal that TGF-␤ 1 induces Smad3 binding to the SM22␣ promoter in vivo. A multimerized SBE promoter responsive to TGF-␤ 1 signaling is highly activated by Smad3 but not by the closely related Smad2. Intriguingly, myocardin (Myocd), a known CArG box-dependent serum response factor coactivator, participates in Smad3-mediated TGF-␤ 1 signaling and synergistically stimulates Smad3-induced SBE promoter activity independent of the CArG box; no such synergy is seen with Smad2. Importantly, Myocd cooperates with Smad3 to activate the wild-type SM22␣, SM myosin heavy chain, and SM␣-actin promoters; they also activate the CArG box-mutated SM22␣ promoter as well as the CArG box-independent aortic carboxypeptidase-like protein promoter. Immunopreciptiation assays reveal that Myocd and Smad3 directly interact both in vitro and in vivo. Mutagenesis studies indicate that the C-terminal transactivation domains of Myocd and Smad3 are required for their functional synergy. These results reveal a novel regulatory mechanism whereby Myocd participates in TGF-␤ 1 signal pathway through direct interaction with Smad3, which binds to the SBEs. This is the first demonstration that Myocd can act as a transcriptional coactivator of the smooth muscle regulatory network in a CArG box-independent manner. (Circ Res. 2005;97:983-991.)Key Words: myocardin Ⅲ SM22␣ or transgelin Ⅲ Smad-binding site (SBE) Ⅲ Smad3 Ⅲ transforming growth factor-␤ 1 Ⅲ smooth muscle transcription

TGF-β signaling plays critical roles in the pathogenesis of aneurysms; however, it is still unclear whether its role is protective or destructive. In this study, we investigate the role of SMAD3 in the pathogenesis of calcium chloride (CaCl2)-induced abdominal aortic aneurysms (AAA) in Smad3−/−, Smad3+/− and Smad3+/+ mice. We find that loss of SMAD3 drastically increases wall thickening of the abdominal aorta. Histological analyses show significant vessel wall remodeling with elastic fiber fragmentation. Remarkably, under polarized light, collagen fibers in the hyperplastic adventitia of Smad3−/− mice show extensive reorganization accompanied by loosely packed thin and radial collagen fibers. The expressions of matrix metalloproteinases including MMP2, MMP9, and MMP12 and infiltration of macrophage/T cells are drastically enhanced in the vascular wall of Smad3−/− mice. We also observe marked increase of NF-κB and ERK1/2 signaling as well as the expression of nuclear Smad2, Smad4 and TGF-β1 in the vessel wall of Smad3−/− mice. In addition, we find that SMAD3 expression is reduced in the dedifferentiated medial smooth muscle-like cells of human AAA patients. These findings provide direct in vivo evidence to support the essential roles of SMAD3 in protecting vessel wall integrity and suppressing inflammation in the pathogenesis of AAAs.

Vascular smooth muscle cell (VSMC) phenotypic modulation is characterized by the downregulation of SMC actin cytoskeleton proteins. Our published study shows that depletion of SM22α (aka SM22, Transgelin, an actin cytoskeleton binding protein) promotes inflammation in SMCs by activating NF-κB signal pathways both in cultured VSMCs and in response to vascular injury. The goal of this study is to investigate the underlying molecular mechanisms whereby SM22 suppresses NF-κB signaling pathways under inflammatory condition. NF-κB inducing kinase (Nik, aka MAP3K14, activated by the LTβR) is a key upstream regulator of NF-κB signal pathways. Here, we show that SM22 overexpression suppresses the expression of NIK and its downstream NF-κB canonical and noncanonical signal pathways in a VSMC line treated with a LTβR agonist. SM22 regulates NIK expression at both transcriptional and the proteasome–mediated post-translational levels in VSMCs depending on the culture condition. By qPCR, chromatin immunoprecipitation and luciferase assays, we found that Nik is a transcription target of serum response factor (SRF). Although SM22 is known to be expressed in the cytoplasm, we found that SM22 is also expressed in the nucleus where SM22 interacts with SRF to inhibit the transcription of Nik and prototypical SRF regulated genes including c-fos and Egr3. Moreover, carotid injury increases NIK expression in Sm22-/- mice, which is partially relieved by adenovirally transduced SM22. These findings reveal for the first time that SM22 is expressed in the nucleus in addition to the cytoplasm of VSMCs to regulate the transcription of Nik and its downstream proinflammatory NF-kB signal pathways as a modulator of SRF during vascular inflammation.

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The mechanism underlying transcriptional coactivation by the co-repressor CtBP is not established. We previously found that CtBP co-occupies several actively transcribed endocrine genes with the transcription factor NeuroD1 to paradoxically increase transcription by recruiting KDM1A and CoREST. While the importance of the oligomeric form of CtBP for corepression is well established, the role of oligomerization in transcriptional coactivation has received little attention. Here, we examined the importance of the oligomeric state of CtBP for coactivation of NeuroD1-dependent transcription by expressing a CtBP dimerization mutant in cells depleted of endogenous CtBP. Dimerization mutants failed to increase transcription or to associate with KDM1A and CoREST, suggesting that oligomeric, but not monomeric CtBP is required to recruit other proteins needed to activate transcription.