Role of TGF-beta-Regulated microRNAs on SMC Differentiation

Smooth muscle cells (SMC) are important in maintaining normal structure and function of blood vessels. SMC can alter their differentiated state in response to diverse stimuli, a process known as phenotypic modulation. Such phenotypic modulation is a hallmark of atherosclerosis, restenosis and hypertension. Myocardin (MYOCD) is a potent co-activator of serum response factor (SRF) and together this transcriptional switch is sufficient for directing SMC contractile phenotype. microRNAs (miRs) have emerged as important post-transcriptional regulators of SMC gene expression. miR-143/145 has drawn considerable attention as critical regulators of the SMC differentiation phenotype. As with other SMC-specific genes, both miR-143/145 are decreased in the neointima of the vessel wall after injury. Thus, miR-143/145 is part of the molecular signature for SMC differentiation. Although the functionality of miR-143/145 has begun to be elucidated, the molecular regulation and related upstream signaling pathways directing its expression in SMC is poorly understood. TGF-beta is a fundamental stimulator for SMC differentiation. In an effort to define a miR expression profile linked to differentiated SMC, I performed a miR array using TGF-beta1 treated human coronary artery smooth muscle cells (HCASM). miR-143/145 was found to be one of the most dramatically induced miRs and preliminary data show the induction of miR-143/145 is accompanied by up-regulation of MYOCD. Additional novel miRs were also identied. This application seeks to build from these exciting data and test the hypothesis that TGF-beta1 controls a subset of miRs critical for SMC differentiation. The first aim will validate TGF-beta1-induced miR-143/145 and MYOCD expression using human and rodent SMC. The second aim will define the molecular regulation of TGF-beta1-induced miR-143/145 using inhibitors and siRNA to key signaling mediators I have already identified to be important. miR-143/145 promoter studies will also be examined. The third aim will elucidate the role of novel TGF-beta1-regulated miRs in SMC phenotype in vitro and in vivo using gain- and loss-of-function approaches. Together, these studies will advance our understanding of the novel role TGF-beta1 plays in regulating SMC phenotype though microRNAs. (AHA Program: Scientist Development Grant)