PDF(Pubmed)
Abstract：
During the progression of pleural fibrosis, pleural mesothelial cells (PMCs) undergo a phenotype switching process known as mesothelial-mesenchymal transition (MesoMT). During MesoMT, transformed PMCs become myofibroblasts that produce increased extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1) that is critical to develop fibrosis. Here, we studied the mechanism that regulates FN1 expression in myofibroblasts derived from human pleural mesothelial cells (HPMCs). We found that myocardin (Myocd), a transcriptional coactivator of serum response factor (SRF) and a master regulator of smooth muscle and cardiac muscle differentiation, strongly controls FN1 gene expression. Myocd gene silencing markedly inhibited FN1 expression. FN1 promoter analysis revealed that deletion of the Smad3-binding element diminished FN1 promoter activity, whereas deletion of the putative SRF-binding element increased FN1 promoter activity. Smad3 gene silencing decreased FN1 expression, whereas SRF gene silencing increased FN1 expression. Moreover, SRF competes with Smad3 for binding to Myocd. These results indicate that Myocd activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression in HPMCs. In HPMCs, TGF-β induced Smad3 nuclear localization, and the proximity ligation signal between Myocd and Smad3 was markedly increased after TGF-β stimulation at nucleus, suggesting that TGF-β facilitates nuclear translocation of Smad3 and interaction between Smad3 and Myocd. Moreover, Myocd and Smad3 were coimmunoprecipitated and isolated Myocd and Smad3 proteins directly bound each other. Chromatin immunoprecipitation assays revealed that Myocd interacts with the FN1 promoter at the Smad3-binding consensus sequence. The results indicate that Myocd regulates FN1 gene activation through interaction and activation of the Smad3 transcription factor.NEW & NOTEWORTHY During phenotype switching from mesothelial to mesenchymal, pleural mesothelial cells (PMCs) produce extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1), critical components in the development of fibrosis. Here, we found that myocardin, a transcriptional coactivator of serum response factor (SRF), strongly activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression. This study provides insights about the regulation of FN1 that could lead to the development of novel interventional approaches to prevent pleural fibrosis.
摘要：
在胸膜纤维化的进展过程中，胸膜间皮细胞(PMCs)经历一种称为间皮-间充质转化(MesoMT)的表型转换过程.在MesoMT期间，转化的PMC成为肌成纤维细胞，产生增加的细胞外基质（ECM）蛋白，包括胶原蛋白和纤连蛋白（FN1），这对纤维化的发展至关重要。这里,我们研究了调节源自HPMC的肌成纤维细胞中FN1表达的机制。我们发现Myocardin(Myocd),血清反应因子（SRF）的转录共激活因子和平滑肌和心肌分化的主要调节因子，强控制HPMCFN1基因表达。Myocd基因沉默显著抑制FN1表达。FN1启动子分析显示，Smad3结合元件的缺失降低了FN1启动子的活性，而推定的SRF结合元件的缺失增加了FN1启动子的活性。Smad3基因沉默降低FN1表达，而SRF基因沉默增加FN1表达。此外，SRF与Smad3竞争与Myocd的绑定。这些结果表明Myocd通过Smad3激活FN1表达,而SRF抑制HPMC中的FN1表达。在HPMC中，TGF-β诱导Smad3核定位,TGF-β刺激细胞核后，Myocd和Smad3之间的邻近连接信号显着增加，提示TGF-β促进Smad3的核易位以及Smad3与Myocd之间的相互作用。此外，将Myocd和Smad3共免疫沉淀并分离Myocd和Smad3蛋白彼此直接结合。染色质免疫沉淀分析显示Myocd在Smad3结合共有序列与FN1启动子相互作用。结果表明，Myocd通过相互作用和Smad3转录因子的激活来调节FN1基因的激活。
