Abstract：
RhoA and its effectors, the transcriptional coactivators myocardin-related transcription factor (MRTF) and serum response factor (SRF), control epithelial phenotype and are indispensable for profibrotic epithelial reprogramming during fibrogenesis. Context-dependent control of RhoA and fibrosis-associated changes in its regulators, however, remain incompletely characterized. We previously identified the guanine nucleotide exchange factor GEF-H1 as a central mediator of RhoA activation in renal tubular cells exposed to inflammatory or fibrotic stimuli. Here we found that GEF-H1 expression and phosphorylation were strongly elevated in two animal models of fibrosis. In the Unilateral Ureteral Obstruction mouse kidney fibrosis model, GEF-H1 was upregulated predominantly in the tubular compartment. GEF-H1 was also elevated and phosphorylated in a rat pulmonary artery banding (PAB) model of right ventricular fibrosis. Prolonged stimulation of LLC-PK1 tubular cells with tumor necrosis factor (TNF)-α or transforming growth factor (TGF)-β1 increased GEF-H1 expression and activated a luciferase-coupled GEF-H1 promoter. Knockdown and overexpression studies revealed that these effects were mediated by RhoA, cytoskeleton remodeling, and MRTF, indicative of a positive feedback cycle. Indeed, silencing endogenous GEF-H1 attenuated activation of the GEF-H1 promoter. Of importance, inhibition of MRTF using CCG-1423 prevented GEF-H1 upregulation in both animal models. MRTF-dependent increase in GEF-H1 was prevented by inhibition of the transcription factor Sp1, and mutating putative Sp1 binding sites in the GEF-H1 promoter eliminated its MRTF-dependent activation. As the GEF-H1/RhoA axis is key for fibrogenesis, this novel MRTF/Sp1-dependent regulation of GEF-H1 abundance represents a potential target for reducing renal and cardiac fibrosis.NEW & NOTEWORTHY We show that expression of the RhoA regulator GEF-H1 is upregulated in tubular cells exposed to fibrogenic cytokines and in animal models of kidney and heart fibrosis. We identify a pathway wherein GEF-H1/RhoA-dependent MRTF activation through its noncanonical partner Sp1 upregulates GEF-H1. Our data reveal the existence of a positive feedback cycle that enhances Rho signaling through control of both GEF-H1 activation and expression. This feedback loop may play an important role in organ fibrosis.
摘要：
RhoA及其效应物,转录共激活因子Myocardin相关转录因子（MRTF）和血清反应因子（SRF），控制上皮表型，并且对于纤维发生过程中促纤维化上皮重编程是必不可少的。RhoA的上下文依赖性控制及其调节因子的纤维化相关变化，然而,保持不完全的特征。我们先前将鸟嘌呤核苷酸交换因子GEF-H1鉴定为暴露于炎症或纤维化刺激的肾小管细胞中RhoA激活的中心介质。在这里，我们发现GEF-H1表达和磷酸化在两种纤维化动物模型中都强烈升高。在单侧输尿管梗阻小鼠肾纤维化模型中,GEF-H1主要在管状隔室中上调。GEF-H1在右心室纤维化的大鼠肺动脉带模型中也升高和磷酸化。用肿瘤坏死因子-α或转化生长因子β1延长LLC-PK1肾小管细胞的刺激增加了GEF-H1的表达并激活了荧光素酶偶联的GEF-H1启动子。敲低和过表达研究表明，这些作用是由RhoA介导的，细胞骨架重塑与MRTF,表明存在正反馈周期。的确,沉默内源性GEF-H1减弱了GEF-H1启动子的激活。重要的是,在两种动物模型中,使用CCG-1423抑制MRTF可防止GEF-H1上调.通过抑制转录因子Sp1阻止了GEF-H1的MRTF依赖性增加，而GEF-H1启动子中推定的Sp1结合位点的突变消除了其MRTF依赖性激活。由于GEF-H1/RhoA轴是纤维发生的关键，这种新的MRTF/Sp1依赖性调节GEF-H1丰度是减少肾和心脏纤维化的潜在靶点.
