泛素系统在人近端小管上皮细胞Smad信号传导通路中的作用

目的:观察Smad7在TGF-β致近端小管上皮细胞转分化中的变化,泛素系统对该过程中Smad信号的调节及对TGF-β所致的小管上皮细胞转分化的作用。方法:体外培养的人近端小管上皮细胞(HK-2),随机分为对照组、TGF-β1(5μg/L)组和TGF-β1(5μg/L)+MG-132(5 mmol/L)组。Western blot检测细胞中Smad2/3磷酸化水平的改变和Smad7蛋白水平的改变;半定量RT-PCR方法观察细胞中Smad7 mRNA表达的改变。结果:West-ern blot的结果显示:①MG-132+TGF-β1组中,Smad2/3磷酸化蛋白的表达减少90%(P<0.01)。②TGF-β1作用于细胞后,Smad7蛋白表达进行性减少,30 min较0 min降低20%(P<0.05),24 h时几乎无表达(P<0.01);MG-132+TGF-β1组中Smad7蛋白表达与对照组无明显差异,与TGF-β1组相比,Smad7蛋白水平上调。③半定量RT-PCR显示:TGF-β1(5μg/L)作用于细胞后,与Smad7蛋白表达不同,Smad7 mRNA迅速升高,24 h其表达增高近1倍(与0 min比较,P<0.01);MG-132+TGF-β1组中Smad7 mRNA的表达与对照组无明显差异。结论:TGF-β可诱导Smad7 mRNA表达上调,但Smad7蛋白的表达显著减少,其机制与泛素系统活化导致Smad7蛋白的降解增加有关。

Role of Ubiquitinproteasome Pathway in TGF-beta/Smad Signaling Pathway in Human Proximal Tubular Epithelial Cells

Objective: To identify how Smad7 changing and Smad signaling regulated by ubiquitinproteasome pathway(UPP) in tubulointerstitial on human proximal epithelial cells.Methods: Human kidney proxima tubular cells HK2 were divided into three groups according to the differnet treatment: control group,TGF-β1(5 μg/L) group,TGF-β1(5 μg/L)+MG-132(5 mmol/L) group.Western blot were performed to determine the changes of Smad7 protein and Smad2/3 phosphorylation,and semi-quantitive RT-PCR were performed to test diversify of Smad7 mRNA.Results: Compared with that in TGF-β1 group,the expression of phospho-Smad2/3 decreased by 90% in MG-132+TGF-β1 group(P<0.01).The level of Smad7 protein decreased progressively in tubular cells after TGF-β1 inducing,Smad7 protein expression decreased by 20% at 30 min(P<0.05) and almost showed no expression at 24 h(P<0.01).Compared with that in TGF-β1 group(P<0.01),Smad2/3 phosphorylation and significantly decreasing of Smad7 protein was hindered by addition of MG-132 in MG-132+TGF-β group.Semi-quantitative RT-PCR showed in contrast to expression of Smad7 protein,Smad7 mRNA was up-regulated rapidly by TGF-β1 inducion and no difference was found between MG-132+TGF-β1 group and control group.Conclusion: Different expression between Smad7 protein and Smad7 mRNA after TGF-β1 stimulation is discovered in this study.Up-regulation of Smad7 mRNA and UPP degradating Smad7 protein are both involved in this change.