Tgf-β3Induced Bone Marrow-Drived Mesenchymal Stem Cells Differen Tiation Into Smooth Muscle Cells By Activating Myocardin

Mesenchymal stem cells (MSCs) are the most advantageous seed cells in tissue engineering, and have the potentiality of self-renewal and multi-differentiation. The research of the molecular mechanism by which bone marrow MSCs differentiation into smooth muscle cells (SMCs) will offer an experimental foundation for vascular injury clinical treatment. In this paper, we found that myocardin played an important role in the SMC differentiation of BMMSCs induced by TGF-β3, and further researched the molecular mechanism.Transforming growth factor β3(TGF-β3) is a multifunctional regulator in the proliferation and differentiation processes. After treatment with TGF-P3, the SMC-specific genes mRNA levels and protein expressions are examined by semi-quantitative RT-PCR and immunocytochemistry assay. Our results demonstrated that the mRNA levels of SMC-specific markers, such as SMMHC, SM22α, a-SMA, were significantly increased in MSCs treated with TGF-p3. These results indicated that TGF-β3may induce MSCs differentiation to SMCs.Myocardin is a SRF cofactor and play an important role in regulating SMC differentiation. We determined the effect of TGF-P3on the mRNA level of myocardin in MSCs. Myocardin was significantly increased in response to treatment with TGF-β3. We blocked the function of endogenous myocardin by transfected C-terminal truncation mutant of myocardin (myocardin△TAD) into MSCs and found that TGF-β3lose the ability to increase the expression of the SMC-specific genes. Luciferase assay determined that the SM22a-luc reporter is strongly activated by myocardin. These results indicated that myocardin was able to induce SMC differentiation of rat MSCs.To further research the molecular mechanisms of SMC differentiation of MSCs induced by TGF-β3and myocardin, we examined the mRNA levels of TGF-P3downstream signal component Smad2/3by RT-PCR. The results showed that the mRNA levels of Smad2/3were significantly increased. TGF-P3elicited rapid phosphorylation of Smad2after30minutes exposure. Luciferase assay determined that the SM22a-luc reporter is strongly activated by the synergism of myocardin and Smad2. We found that after blocking the function of endogenous myocardin, Smad2lose the ability to increase the SMC-specific genes mRNA levels. Cell cycle inhibitors, such as Rb, p16, and p21, were involved in myocardin/TGF-β3-induced BMMSCs differentiation into SMCs.