Hills Claire E, Willars Gary B, Brunskill Nigel J
Department of Infection, Immunity and Inflammation, University of Leicester, College of Medicine Biological Sciences and Psychology, Leicester, United Kingdom.
Mol Endocrinol. 2010 Apr;24(4):822-31. doi: 10.1210/me.2009-0391. Epub 2010 Mar 2.
Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or TGF-beta1 in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor beta (RARbeta), hepatocyte growth factor (HGF), cellular retinoic acid-binding protein II (CRABPII), vimentin, E-cadherin, Snail, and beta-catenin was assessed by immunoblotting. The cellular localization of vimentin and beta-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1458 genes after C-peptide exposure for 18 h or 48 h, respectively. From these, members of the antifibrotic retinoic acid (RA)- and HGF-signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARbeta, CRABPII, and HGF. We confirmed a role for RA in reversal of TGF-beta1-induced changes associated with epithelial-mesenchymal transition, including expression changes in Snail, E-cadherin, vimetin, and redistribution of beta-catenin. Importantly, these TGF-beta1-induced changes were inhibited by C-peptide. Further, effects of TGF-beta1 on Snail and E-cadherin expression were blocked by HGF, and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA-signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy.
最近发现了C肽的新信号传导作用，有证据表明它可以改善1型糖尿病的并发症。在此，我们试图确定与糖尿病肾病病理生理学相关的、受C肽调节的新途径。我们进行了微阵列分析，以确定在人近端肾小管细胞系HK-2中受C肽和/或转化生长因子-β1（TGF-β1）调节的基因。通过免疫印迹法评估视黄酸受体β（RARβ）、肝细胞生长因子（HGF）、细胞视黄酸结合蛋白II（CRABPII）、波形蛋白、E-钙黏蛋白、Snail和β-连环蛋白的表达。通过免疫细胞化学确定波形蛋白和β-连环蛋白的细胞定位。通过相差显微镜评估细胞形态的变化。基因表达谱显示，C肽暴露18小时或48小时后，分别有953个和1458个基因差异表达。从这些基因中，选择了抗纤维化视黄酸（RA）和HGF信号通路的成员。免疫印迹表明，C肽增加了RARβ、CRABPII和HGF。我们证实RA在逆转TGF-β1诱导的与上皮-间质转化相关的变化中起作用，包括Snail、E-钙黏蛋白、波形蛋白的表达变化以及β-连环蛋白的重新分布。重要的是，这些TGF-β1诱导的变化被C肽抑制。此外，HGF阻断了TGF-β1对Snail和E-钙黏蛋白表达的影响，而C肽的抑制作用通过阻断HGF活性而消除。本研究确定了HGF作为C肽效应器的新作用，可能是通过RA信号通路，突出了C肽作为糖尿病肾病潜在治疗方法的地位。
