Transforming Growth Factor-β1 (TGF-β1) Utilizes Distinct Pathways for the Transcriptional Activation of MicroRNA 143/145 in Human Coronary Artery Smooth Muscle Cells
Transforming Growth Factor-β1 (TGF-β1) Utilizes Distinct Pathways for the Transcriptional Activation of MicroRNA 143/145 in Human Coronary Artery Smooth Muscle Cells
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DOI：
10.1074/jbc.m111.258814
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发表时间：
2011-08-26
影响因子：
4.8
通讯作者：
Miano, Joseph M.
中科院分区：
文献类型：
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作者：
Long, Xiaochun;Miano, Joseph M.
microRNA 143/145（miR 143/145）仅限于成体平滑肌细胞（SMC）谱系，并部分介导几种SMC收缩基因的表达。尽管miR 143/145的功能已开始阐明，但其响应于各种信号输入的转录调控知之甚少。为了确定SMC分化的miR特征，我们筛选了人冠状动脉SMC中受TGF-β 1（一种已知的SMC分化刺激物）调节的miR。阵列分析揭示了许多TGF-β 1诱导的miR，包括miR 143/145。验证研究表明，TGF-β 1以剂量和时间依赖性方式刺激miR 143/145表达。我们使用了几种化学抑制剂，发现SB 203580，一种p38 MAPK的特异性抑制剂，显著降低TGF-β 1诱导的miR 143/145表达。siRNA研究表明，TGF-β 1对miR 143/145的作用依赖于心肌蛋白和血清反应因子转录开关以及SMAD 4。TGF-β 1刺激了580 bp的人miR 143/145增强子，诱变研究揭示了一个已知的CArG盒和一个相邻的SMAD结合元件对增强子的完全TGF-β 1依赖性激活的关键作用。染色质免疫沉淀分析记录了TGF-β 1介导的SMAD 3和SMAD 4结合在含有SMAD结合元件的增强子区域上的富集。前miR 145强烈促进SMC分化，而抗miR 145部分阻断TGF-β 1诱导的SMC分化。这些结果证明了TGF-β 1诱导miR 143/145转录的双重途径，从而揭示了TGF-β 1诱导人血管SMC分化的新机制。
MicroRNA 143/145 (miR143/145) 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, we screened human coronary artery SMCs for miRs modulated by TGF-beta 1, a known stimulus for SMC differentiation. Array analysis revealed a number of TGF-beta 1-induced miRs, including miR143/145. Validation studies showed that TGF-beta 1 stimulated miR143/145 expression in a dose-and time-dependent manner. We utilized several chemical inhibitors and found that SB203580, a specific inhibitor of p38MAPK, significantly decreased TGF-beta 1-induced miR143/145 expression. siRNA studies demonstrated that the effect of TGF-beta 1 on miR143/145 was dependent upon the myocardin and serum response factor transcriptional switch as well as SMAD4. TGF-beta 1 stimulated a 580-bp human miR143/145 enhancer, and mutagenesis studies revealed a critical role for both a known CArG box and an adjacent SMAD-binding element for full TGF-beta 1-dependent activation of the enhancer. Chromatin immunoprecipitation assays documented TGF-beta 1-mediated enrichment of SMAD3 and SMAD4 binding over the enhancer region containing the SMAD-binding element. Pre-miR145 strongly promoted SMC differentiation, whereas an anti-miR145 partially blocked TGF-beta 1-induced SMC differentiation. These results demonstrate a dual pathway for TGF-beta 1-induced transcription of miR143/145, thus revealing a novel mechanism underlying TGF-beta 1-induced human vascular SMC differentiation.