Smad3-mediated myocardin silencing: A novel mechanism governing the initiation of smooth muscle differentiation

Both TGF-β and myocardin (MYOCD) are important for smooth muscle cell (SMC) differentiation, but their precise role in regulating the initiation of SMC development is less clear. In TGF-β-induced SMC differentiation of pluripotent C3H10T1/2 progenitors, we found that TGF-β did not significantly induce Myocd mRNA expression until 18 h of stimulation. On the other hand, early SMC markers such as SM α-actin, SM22α, and SM calponin were detectable beginning 2 or 4 h after TGF-β treatment. These results suggest that Myocd expression is blocked during the initiation of TGF-β-induced SMC differentiation. Consistent with its endogenous expression, Myocd promoter activity was not elevated until 18 h following TGF-β stimulation. Surprisingly, Smad signaling was inhibitory to Myocd expression because blockade of Smad signaling enhanced Myocd promoter activity. Overexpression of Smad3, but not Smad2, inhibited Myocd promoter activity. Conversely, shRNA knockdown of Smad3 allowed TGF-β to activate the Myocd promoter in the initial phase of induction. Myocd was activated by PI3 kinase signaling and its downstream target Nkx2.5. Interestingly, Smad3 did not affect PI3 kinase activity. However, Smad3 physically interacted with Nkx2.5. This interaction blocked Nkx2.5 binding to the Myocd promoter in the early stage of TGF-β induction, leading to inhibition of Myocd mRNA expression. Moreover, Smad3 inhibited Nkx2.5-activated Myocd promoter activity in a dose-dependent manner. Taken together, our results reveal a novel mechanism for Smad3-mediated inhibition of Myocd in the initiation phase of SMC differentiation.