CRBN-E3泛素连接酶调控肾小管间质纤维化机制的实验研究

Chronic kidney disease (CKD) commonly leads to kidney fibrosis and results the end stage renal disease (ESRD), therefore, blocking or delaying the progression of kidney fibrosis is a major strategy to prevent ESRD. TGF-β1 is a major factor to promote renal fibrosis. In all tissues, the majority of intracellular proteins are degraded by the ubiquitin–proteasome system (UPS). It is reportedly that UPS can regulate the expression of a variety of growth factors inclued TGF-β1 by degrading their transcription factors or transcription repressor. During this process, E3 Ub-protein ligase is responsible for recognizing the substrate in a highly specific manner. Our preliminary results indicated that knockdown CRBN blocks the expression of TGF-β1. This response is associated with anincrease in Ski, a transcription repressor of TGF-β1. Thus, we hypothesized that inhibition of CRBN-E3 ligases blocks the degradation of Ski leading to suppression of TGF-β1. To examine our hypothesis, we plan to knockdown CRBN or to overexpress Ski in primary cultured renal tubular cells and investigate the molecular mechanisms by which they regulate TGF-β1 expression. By generating conditional CRBN knockout mouse, we will also examine whether inhibition of CRBN-E3 ligase suppresses the kidney fibrosis induce by unilateral ureteral obstruction (UUO). In addition, we plan to administrate Thalidomide, a potent CRBN inhibitor, to UUO mice and to examine whether this intervention would prevent or slow down kidney fibrosis。This proposal will identify a new pathway which regulates TGF-β1 expression during kidney fibrosis, namely, inhibition of CRBN-E3 ligase blocks the degradation of Ski in UPS, thereby represses the transcription of TGF-β1 and hence kidney fibrosis. We believe that the results of our proposal will provide new insight to the prevention and treatment of renal fibrosis.

TGF-β1过表达是导致肾小管间质纤维化及终末期肾病的主要原因，减少其过表达是延缓或阻断肾纤维化的重要手段。泛素-蛋白酶体系统(UPS)是细胞内蛋白降解的主要途径，可通过降解相应的转录因子，调节TGF-β1的表达。CRBN-E3连接酶是UPS中识别并降解转录因子的重要成员之一，有关CRBN-E3连接酶与肾小管间质纤维化的研究尚未见报道，我们前期研究表明抑制上述连接酶可减少TGF-β1的表达，而TGF-β1的转录抑制因子Ski表达增加，提示CRBN-E3连接酶可能通过降解Ski调控TGF-β1。本项目拟应用敲抑CRBN、过表达Ski技术及CRBN敲除小鼠模型，阐明抑制CRBN-E3连接酶，减少对Ski的降解，抑制TGF-β1表达，减轻肾间质纤维化的分子生物学机制。并应用沙利度胺抑制CRBN-E3连接酶，观察其对肾小管间质纤维化的影响，为老药新用及临床防治肾小管间质纤维化提供新的理论基础。

TGF-β1过表达是导致肾小管间质纤维化及终末期肾病的主要原因，减少其过表达是延缓或阻断肾纤维化的重要手段。泛素-蛋白酶体系统(UPS)是细胞内蛋白降解的主要途径，可通过降解相应的转录因子，调节TGF-β1的表达。CRBN-E3连接酶是UPS中识别并降解转录因子的重要成员之一，有关CRBN-E3连接酶与肾小管间质纤维化的研究尚未见报道，我们前期研究表明抑制上述连接酶可减少TGF-β1的表达，而TGF-β1的转录抑制因子Ski表达增加，提示CRBN-E3连接酶可能通过降解Ski调控TGF-β1。本项目应用敲抑CRBN、过表达Ski技术及CRBN敲除小鼠模型，阐明了抑制CRBN-E3连接酶，减少对Ski的降解，抑制TGF-β1表达，减轻肾间质纤维化的分子生物学机制。并应用沙利度胺抑制CRBN-E3连接酶，证明了其对肾小管间质纤维化的影响，为老药新用及临床防治肾小管间质纤维化提供了新的理论基础。