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(Circulation Research. 2005;97:983.)
© 2005 American Heart Association, Inc.

Molecular Medicine

Myocardin Enhances Smad3-Mediated Transforming Growth Factor-ß₁ Signaling in a CArG Box-Independent Manner

Smad-Binding Element Is an Important cis Element for SM22 Transcription In Vivo

Ping Qiu^*, Raquel P. Ritchie^*, Zhiyao Fu, Dongsun Cao, Jerry Cumming, Joseph M. Miano, Da-Zhi Wang, Hui J. Li, Li Li

From the Department of Internal Medicine (P.Q., R.R., Z.F., J.C., L.L.) and Center for Molecular Medicine and Genetics (P.Q., R.R., L.L.), Wayne State University, Detroit, Mich; Carolina Cardiovascular Biology Center (D.C., D.-Z.W.), Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill; Center for Cardiovascular Research in the Aab Institute of Biomedical Sciences (J.M.M.), University of Rochester School of Medicine, New York; and the Molecular Cardiology Research Institute (H.J.L.), Tufts University School of Medicine, Boston, Mass.

Correspondence to Li Li, 421 E Canfield Ave, #1107, Detroit, MI 48201. E-mail lili{at}med.wayne.edu

Transforming growth factor (TGF)-ß₁ is an important cytokine involved in various diseases. However, the molecular mechanism whereby TGF-ß₁ 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-ß₁ 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-ß₁ induces Smad3 binding to the SM22 promoter in vivo. A multimerized SBE promoter responsive to TGF-ß₁ 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-ß₁ 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-ß₁ 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.

Key Words: myocardin • SM22 or transgelin • Smad-binding site (SBE) • Smad3 • transforming growth factor-ß₁ • smooth muscle transcription

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