转化生长因子β1通过Smad3调控补体应答基因32转录的分子机制  

The molecular mechanism of TGF-β1 regulating trascription of response gene to complement 32 through Smad3 pathways

作　　者：沈云琳[1] 钮小玲[1] 刘华杰[1] 孙蕾[1] 匡新宇[1] 黄文彦[1] 

机构地区：[1]上海交通大学附属儿童医院肾脏风湿科,上海200062

出　　处：《肾脏病与透析肾移植杂志》2015年第5期447-452,共6页Chinese Journal of Nephrology,Dialysis & Transplantation

基　　金：国家自然科学基金资助项目(81370813);上海市科委(114119a1700);上海市卫生局优秀学科带头人项目(XBR2011010)

摘　　要：目的:前期工作发现补体应答基因32(RGC-32)是转化生长因子β(TGF-β)诱导的肾小管上皮细胞-间充质转化(EMT)过程中Smad信号下游的关键调控分子。本研究拟进一步明确TGF-β诱导肾小管EMT过程中RGC-32的转录调控机制。方法:体外培养NRK-52E细胞,利用荧光素酶报告基因实验检测RGC-32启动子活性以确定Smad结合元件(SBE)是否为TGF-β诱导的RGC-32转录调控位点;通过凝胶迁移率实验(EMSA)明确与之结合的转录调控分子。结果:(1)转染野生型SBE片段的NRK-52E细胞荧光素酶活性明显增高,而转染突变的SBE片段的细胞无此表现,表明SBE是调控TGF-β诱导的RGC-32转录启动的关键位点。(2)合成32P标记的包含SBE的寡核苷酸探针行EMSA可见TGF-β诱导的核蛋白与探针复合物生成,且加入包含SBE序列的竞争片段可以抑制此核蛋白-探针复合物的形成,表明有蛋白与SBE结合并调控RGC-32的转录。(3)当加入不同的Smad抗体行超迁移实验发现Smad2和Smad3抗体可以和复合物相互作用,表明Smad2和Smad3可能是TGF-β诱导的转录复合物的组成部分。(4)将Smad2和Smad3分别与p-1500RGCluc共同转染NRK-52E细胞,Smad3可以显著增加RGC-32启动子活性,表明Smad3是TGF-β诱导的转录复合物的组成部分,而不是Smad2。结论:在TGF-β诱导肾小管EMT过程中,Smad3通过与RGC-32启动子区SBE结合,从而调控肾小管EMT过程。Objective： To research the molecular mechanisms of Smad3 regulation RGC-32 transcription in TGF-β1 induced Epithelial-mesenchymal Transition on NRK-52 E Cells. Methodology： The NRK-52 E cells were cultured and transiently transfected in vitro. Luciferase assays were performed to determine whether the SBE plays roles in TGF-β1-induced RGC-32 promoter activation. Electrophoretic mobility shift assays（ EMSA） were selected to determine which proteins are the formations of TGF-β1-induced nuclear complex. Results：（ 1） The Smad binding element（ SBE） was important for TGF-β1-induced RGC-32 transcriptional activation. The SBE mutation structure significantly inhibited RGC-32 promoter activity.（ 2） The SBE was essential for the interaction of TGF-β1-induced nuclear proteins with RGC-32 promoter.It was found that the TGF-β-induced interaction between nuclear factors and RGC-32 promoter in EMSA,and the wild-type DNA containing SBE site as competitors abolished the formation of TGF-β-induced complex.（ 3） Smad2 / 3 was the composition of TGF-β1-inducible transcription factor complex bound to RGC-32 promoter as recognized by their specific antibodies in EMSA.（ 4） Smad2 and Smad3 expression plasmids were co-transfected individually into NRK-52 E cells,and then Smad3 significantly increased the RGC-32 promoter activity. This suggests that Smad3,but not Smad2,be presented in the TGF-β1-inducible complex formed by nuclear proteins with RGC-32 promoter. Conclusion： These results illuminated that Smad3 may be combined with RGC-32 promoter region SBE and play an important role in transcriptional regulation in TGF-β-induced renal tubular EMT of NRK-52 E cells.

关 键 词：补体应答基因32 上皮-间充质转化 转化生长因子Β 转录调控 

分 类 号：R692[医药卫生—泌尿科学]