Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208, USA.
J Biol Chem. 2010 Jul 23;285(30):23241-50. doi: 10.1074/jbc.M109.084673. Epub 2010 May 18.
Smooth muscle cells (SMCs) retain remarkable plasticity to undergo phenotypic modulation in which the expression of smooth muscle markers is markedly attenuated while conversely expression of extracellular matrix (ECM) is dramatically up-regulated. Myocardin is perhaps the most potent transcription factor for stimulating expression of smooth muscle-specific genes; little is known, however, about whether myocardin can orchestrate ECM expression to act in concert with smooth muscle differentiation program. In this study, we demonstrated myocardin coordinate smooth muscle differentiation by inducing transcription of microRNA-143 (miR-143), which attenuates ECM versican protein expression. Previous studies have shown that versican is a chondroitin sulfate proteoglycan of the ECM that is produced by synthetic SMCs and promotes SMC migration and proliferation. Our data demonstrated that myocardin significantly represses versican expression in multiple cell lines, and this occurs through the induction of miR-143 by myocardin. By a modified reverse transcribed PCR, we found that miR-143 specifically binds to the 3'-untranslated region of versican mRNA. Reporter assays validated that miR-143 targets versican 3'-untranslated region through an evolutionarily conserved miR-143 binding site. Furthermore, overexpression of miR-143 significantly represses versican expression, whereas conversely, depletion of endogenous miR-143 results in up-regulation of versican expression. In addition, we demonstrated that myocardin represses versican through induction of miR-143. Finally, we found that the regulation of versican by miR-143 is involved in platelet-derived growth factor BB-induced SMC migration. This study provides the first evidence that myocardin, in addition to activating smooth muscle-specific genes, regulates ECM expression through induction of microRNAs during smooth muscle differentiation.
平滑肌细胞（SMCs）具有显著的可塑性，可以发生表型调节，其中平滑肌标志物的表达明显减弱，而细胞外基质（ECM）的表达则显著上调。肌球蛋白是刺激平滑肌特异性基因表达的最有效转录因子；然而，尚不清楚肌球蛋白是否可以协调 ECM 表达，与平滑肌分化程序协同作用。在这项研究中，我们证明了肌球蛋白通过诱导 microRNA-143（miR-143）的转录来协调平滑肌分化，从而减弱 ECM versican 蛋白的表达。先前的研究表明，versican 是 ECM 中的一种软骨素硫酸盐蛋白聚糖，由合成的 SMC 产生，并促进 SMC 迁移和增殖。我们的数据表明，肌球蛋白在多种细胞系中显著抑制 versican 的表达，这是通过肌球蛋白诱导 miR-143 实现的。通过改良的反转录 PCR，我们发现 miR-143 特异性结合 versican mRNA 的 3'-非翻译区。报告基因实验验证了 miR-143 通过一个进化上保守的 miR-143 结合位点靶向 versican 3'-非翻译区。此外，miR-143 的过表达显著抑制 versican 的表达，而相反，内源性 miR-143 的耗竭导致 versican 的表达上调。此外，我们证明肌球蛋白通过诱导 miR-143 来抑制 versican。最后，我们发现 miR-143 对 versican 的调节参与了血小板衍生生长因子 BB 诱导的 SMC 迁移。这项研究首次证明，肌球蛋白除了激活平滑肌特异性基因外，还通过诱导平滑肌分化过程中的 microRNAs 来调节 ECM 表达。
