Myocardin Enhances Smad3-Mediated Transforming Growth Factor-β ₁ Signaling in a CArG Box-Independent Manner

Abstract

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

Cooperation of myocardin and smad2 in inducing differentiation of mesenchymal stem cells into smooth muscle cells

“…Thus, to further assess whether myocardin participated in Smad2/Smad3-mediated TGF-b3 signal pathways, we examined the effect of myocardin overexpression on Smad2/3-induced SM22a promoter activities in COS-7 cells and primary rat bone marrow-derived MSCs. Consistent with previously reports (13), myocardin significantly induced SM22a promoter transcription, and Smad2 and Smad3 slightly activated SM22a promoter (Fig. 3A).…”

“…SM22a-luciferase reporter plasmid was provided by Olson (The University of Texas Southwestern Medical Center, Dallas, TX). Several SM22a promoter-luciferase reporter plasmids containing mutations were constructed according to the previously reported method (13) and confirmed by sequencing. The pRK5F-Smad2 (Plasmid 12623) and pRK5F-Smad3 (Plasmid 12625) expression plasmids were purchased from (Addgene, Cambridge/MA).…”

“…Several groups have explored how Smadmediated TGF-b signals regulate SM22a promoter transcription during myofibroblast differentiation (25,26). Myocardin was reported to cooperate with Smad3 to activate SM22a promoter in 10T1/2 cells (13). Thus, to further assess whether myocardin participated in Smad2/Smad3-mediated TGF-b3 signal pathways, we examined the effect of myocardin overexpression on Smad2/3-induced SM22a promoter activities in COS-7 cells and primary rat bone marrow-derived MSCs.…”

“…These results indicate that the TGF-b plays a key role in the differentiation of mesenchymal lineage cell type into SMCs. A few of studies show that myocardin, a serum response factor (SRF) cotranscriptional factor, can enhance Smad3-mediated TGF-b1 signaling in 10T1/2 cells (13), and myocardin mRNA are increased in SPC-and TGF-b3-induced differentiation of hATSCs to SMCs (4). Together, research suggests that myocardin may be associated with the TGF-b-induced MSC differentiation.…”

TGF-β signaling in vascular biology and dysfunction

“…TGF-β is a potent stimulator of vascular SMC differentiation by activating the genetic program that includes a large set of SMC differentiation marker genes [61]. Myocardin, an important coactivator of serum response factor, was shown to potently enhance TGF-β/Smad3-mediated activation of SM22α actin transcription [62]. The zinc finger E-box binding transcription factor DeltaEF1 was also shown to have an important effector role in this respect by forming a complex with serum response factor and Smad3 [63].…”