Identification and characterization of transcription factors involved in the myofibroblast formation, and its application to novel therapeutic approach of glomerulonephritis

肌成纤维细胞被认为是理解损伤组织重建和过量基质形成的关键。我们一直在研究肌成纤维细胞在肾脏疾病中的病理生理学意义。我们报道钙调蛋白和SMαA都是进行性肾脏疾病中肌成纤维细胞的敏感和有用的分子标志物。鉴于临床观察的重要性，我们开始寻找SMαA基因调控肌成纤维细胞的分子机制，以阐明肌成纤维细胞形成的潜在机制。通过对SMαA基因的体内启动子分析，我们证实了内含子CAG基序在SMαA基因转录激活中的重要作用，无论是在平滑肌细胞还是在肾成纤维细胞中。这些结果为我们进一步研究和鉴定与该区域结合的转录因子(S)奠定了基础。我们现在正在研究转录因子…的分子特征More或与SMαA基因内含子1的Carg基序结合。小分子GTP酶Rho参与细胞与底物的黏附和细胞收缩。Rho下游效应分子如Rho相关的螺旋卷曲形成蛋白激酶(ROCK)介导的Rho的这些作用可能是肾间质纤维化进展的部分原因。在体内(单侧输尿管梗阻)和体外研究了特异性ROCK抑制剂Y-27632的抗纤维化作用。为探讨Y-27632对输尿管梗阻(UUO)大鼠肾脏的治疗作用，观察了梗阻大鼠肾脏平滑肌α肌动蛋白(SMαA)的表达及巨噬细胞的浸润和纤维化情况。Northern印迹法检测SMαA、转化生长因子β1(I)胶原蛋白、骨桥蛋白、巨噬细胞趋化因子1和细胞间黏附分子1基因的表达。为了阐明Rho-ROCK通路与肾纤维化之间的联系，我们研究了Y-27632对体外细胞增殖和细胞迁移的影响。体内分析显示，Y-27632抑制SMαA表达、巨噬细胞浸润和间质纤维化；Y-27632抑制SMαA、转化生长因子-β和α1(I)胶原基因的表达。体外分析表明，Y-27632对肾成纤维细胞的增殖无抑制作用，但对巨噬细胞的迁移有抑制作用。Rho-ROCK系统可能在组织纤维化的发展中起重要作用，Rho-ROCK信号通路可能成为预防进展性肾脏疾病间质纤维化的新的治疗靶点。较少

Myofibroblasts are recognized as the key to understand the reconstruction and excess matrix formation in injured tissue. We have been investigating the pathophysiological significance of myofibroblasts in renal diseases. We reported that both caldesmon and SMαA are sensitive and useful molecular markers for myofibroblasts in progressive renal disease. In light of the importance of the clinical observation, we started to seek the molecular mechanism of SMαA gene regulation in myofibroblasts to elucidate the underlying mechanisms of myofibroblast formation. Utilizing the in vivo promoter analysis of SMαA gene, we have demonstrated the essential role of intronic CArG motif element for the transcriptional activation of SMαA gene in both smooth muscle cells and in renal myofibroblasts. These results lead us to the further investigation and identification of the transcription factor(s) that bind to this region. We are now on the way of the molecular characterization of the transcription fact … More or that binds to the CArG motif element of the SMαA gene intron 1.The small GTPase Rho is involved in cell-to-substratum adhesion and cell contraction. These actions of Rho mediated by downstream Rho effectors such as Rho-associated coiled-coil forming protein kinase (ROCK) may be partly responsible for the progression of renal interstitial fibrosis. Anti-fibrosis effects of Y-27632, a specific ROCK inhibitor, were studied both in vivo (unilateral ureteral obstruction ; UUO) and in vitro. To investigate the therapeutic efficacy of Y-27632 in UUO kidneys, smooth muscle α actin (SMαA) expression, macrophage infiltration and fibrosis in the obstructed kidneys were studied. SMαA, transforming growth factor β(TGF-β), α1(I) collagen, osteopontin, macrophage chemoattractant peptide-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) gene expression were examined by Northern blotting. To elucidate the mechanism linking the Rho-ROCK pathway with renal fibrosis, the effects of Y-27632 on in vitro cell proliferation and cell migration were studied. In vivo analysis showed that Y-27632 suppressed SMαA expression, macrophage infiltration and interstitial fibrosis, and that Y-27632 suppressed SMαA, TGF-β and α1(I) collagen mRNA expression. In vitro analysis showed that Y-27632 did not suppress proliferation of renal fibroblasts but suppressed migration of macrophages. The Rho-ROCK system may play an important role in the development of tissue fibrosis, and the Rho-ROCK signaling pathway may be a new therapeutic target for preventing interstitial fibrosis in progressive renal disease. Less