microRNA (miR) 143/145 is restricted to adult smooth muscle cell (SMC) lineages and mediates, in part, the expression of several SMC contractile genes. Although the function of miR143/145 has begun to be elucidated, its transcriptional regulation in response to various signaling inputs is poorly understood. In an effort to define a miR signature for SMC differentiation, researchers from the University of Rochester School of Medicine and Dentistry screened human coronary artery SMC (HCASM) for miRs modulated by TGF-β1, a known stimulus for SMC differentiation. Array analysis revealed a number of TGF-β1-induced miRs, including miR143/145. Validation studies showed that TGF-β1 stimulated miR-143/145 expression in a dose- and time-dependent manner. The researchers utilized several chemical inhibitors and found that SB203580, a specific inhibitor to p38MAPK, significantly decreased TGF-β1-induced miR-143/145 expression. siRNA studies demonstrated that the effect of TGF-β1 on miR143/145 was dependent upon the myocardin (MYOCD) and serum response factor (SRF) transcriptional switch as well as SMAD4. TGF-β1 stimulated a 580 bp human miR-143/145 enhancer, and mutagenesis studies revealed a critical role for both a known CArG box and an adjacent SMAD binding element (SBE) for full TGF-β1-dependent activation of the enhancer. Chromatin immunoprecipitation assays documented TGF-β1-mediated enrichment of SMAD3 and SMAD4 binding to the enhancer region containing the SBE. pre-miR145 strongly promoted SMC differentiation while an anti-miR145 partially blocked TGF-β1-induced SMC differentiation. These results demonstrate a dual pathway for TGF-β1-induced transcription of miR143/145 thus revealing a novel mechanism underlying TGF-β1-induced human SMC differentiation.

LC SciencesTGF-β1-induced miR143/145 expression in HCASM. A, heat map illustrating relative expression of a sample of miRs in triplicate control or TGF-β1 (1 ng/ml)-treated HCASM. B, qPCR validation of indicated miRs in HCASM treated with 1 ng/ml TGF-β1 for 24 h. (C) and two SMC contractile genes (D) in HCASM following 24-h treatment with varying concentrations of TGF-β1. E, HCASM were treated with TGF-β1 (1 ng/ml) for the indicated times, and qPCR was performed to measure normalized expression of miR143/145, miR21, and miR221.

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Long X, Miano JM. (2011) TGF{beta}1 utilizes distinct pathways for the transcriptional activation of microRNA 143/145 in human coronary artery smooth muscle cells. J Biol Chem [Epub ahead of print]. [article]

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