Myocardin enhances Smad3-mediated transforming growth factor-beta1 signaling in a CArG box-independent manner: Smad-binding element is an important cis element for SM22alpha transcription in vivo.
Transforming growth factor (TGF)-beta1 is an important cytokine involved in various diseases. However, the molecular mechanism whereby TGF-beta1 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 SM22alpha promoter as one of the TGF-beta1 response elements. Here, we show that mutation of the SBE reduces the activation potential of a SM22alpha promoter in transgenic mice during embryogenesis. Chromatin immunoprecipitation assays reveal that TGF-beta1 induces Smad3 binding to the SM22alpha promoter in vivo. A multimerized SBE promoter responsive to TGF-beta1 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-beta1 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 SM22alpha, SM myosin heavy chain, and SMalpha-actin promoters; they also activate the CArG box-mutated SM22alpha 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-beta1 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.
Qiu
Ping
P
Department of Internal Medicine, Wayne State University, Detroit, Michigan, USA.
Ritchie
Raquel P
RP
Fu
Zhiyao
Z
Cao
Dongsun
D
Cumming
Jerry
J
Miano
Joseph M
JM
Wang
Da-Zhi
DZ
Li
Hui J
HJ
Li
Li
L
D000818:Animals; D001665:Binding Sites; D005786:Gene Expression Regulation; D051379:Mice; D008810:Mice, Inbred C57BL; D008840:Microfilament Proteins / Q000235:genetics*; D009124:Muscle Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D011401:Promoter Regions, Genetic; D020218:Response Elements / Q000502:physiology*; D026362:Serum Response Factor / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D051899:Smad2 Protein / Q000502:physiology; D051900:Smad3 Protein / Q000737:chemistry / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000502:physiology*; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D000099251:Myocardin
D008840:Microfilament Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D026362:Serum Response Factor; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D000099251:Myocardin; C497783:Smad2 protein, mouse; C497791:Smad3 protein, mouse; C508961:Tgfb1 protein, mouse; C051615:transgelin