Smad泛素化调节因子2在转化生长因子β1诱导人肺成纤维细胞活化中的作用及其分子机制

目的:观察Smad泛素化调节因子2(Smurf2)在转化生长因子β1(TGF-β1)诱导人肺成纤维细胞活化中的作用,并探讨其可能的分子机制。方法:体外培养人胚肺成纤维细胞MRC-5,10 μg·L^-1 TGF-β1作用1、2和6 h(分别为TGF-β1 1 h组、TGF-β1 2 h组和TGF-β1 6 h组),并设对照组(不加TGF-β1), RT-PCR和Western blotting法分别检测各组细胞中Smurf1和Smurf2 mRNA和蛋白的表达水平。MRC-5细胞随机分为对照组(未加入TGF-β1或siRNA)、TGF-β1组 (10 μg·L^-1 TGF-β1)、Control siRNA转染组(10 μg·L^-1TG F-β1+Control siRNA)和Smurf2 siRNA转染组(10 μg·L^-1 TGF-β1+Smurf2 siRNA)。Western blotting法检测各组细胞中α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原α1(COL1A1)的表达水平;RT-PCR和Western blotting法分别检测各组细胞中Smad7、核转录共抑制因子SnoN、TGF-β Ⅰ型受体(TβRⅠ)、Smad2和Smad3 mRNA和蛋白的表达水平。结果:与对照组比较,各TGF-β1组细胞中Smurf2 mRNA和蛋白表达水平均明显升高(P<0.05),且随着TGF-β1作用时间的延长,其表达水平呈逐渐增加的趋势;与对照组比较,各TGF-β1组细胞中Smurf1表达水平无明显变化(P>0.05)。与TGF-β1组比较,Smurf2 siRNA组细胞中Smurf2蛋白表达水平下降(P<0.05)。与对照组比较,TGF-β1组细胞中α-SMA和COL1A1蛋白表达水平均明显升高(P<0.05);与TGF-β1组比较,Smurf2 siRNA组细胞中α-SMA和COL1A1蛋白表达水平均下降(P<0.05)。与对照组比较,TGF-β1组和Smurf2 siRNA组细胞中Smad7和SnoN mRNA表达水平均升高(P<0.05),而Smurf2 siRNA组和TGF-β1组细胞中Smad7和SnoN mRNA表达水平无明显变化(P>0.05)。与对照组比较,TGF-β1组细胞中Smad7和SnoN蛋白表达水平均明显下降(P<0.05);与TGF-β1组比较,Smurf2 siRNA组细胞中Smad7和SnoN蛋白表达水平明显升高(P<0.05)。与对照组比较,TGF-β1组和Smurf2 siRNA组细胞中TβRⅠ mRNA和蛋白表达水平均明显升高(P<0.05),而Smurf2 siRNA组和TGF-β1组细胞中TβRⅠ mRNA和蛋白表达水平比较差异无统计学意义(P>0.05)。各组细胞中Smad2和Smad3 mRNA和蛋白表达水平比较差异无统计学意义(P>0.05)。 结论: Smurf2可能通过泛素化降解Smad7和SnoN,参与调控TGF-β1/Smads信号通路,从而发挥其促进TGF-β1活化肺成纤维细胞的作用。

Effect of Smad ubiquitination regulatory factor 2 on TGF-β1-induced activation in lung fibroblasts and its molecular mechanism

Objective To investigate the effect of Smad ubiquitination regulatory factor 2(Smurf2) on the TGF-β1-induced activation in lung fibroblasts,and to explore the possible molecular mechanism. Methods The human embryonic lung fibroblasts (MRC-5) were treated with TGFβ1(10 μg·L^-1) for 1,2 and 6 h in vitro(used as TGF-β1 1 h group,TGF-β1 2 h group,and TGF-β1 6 h group),while control group(without TGF-β1) was set up.The expression levels of Smurf1 and Smurf2 mRNA and protein were detected by RT-PCR and Western blotting method,respectively.The MRC-5 cells were randomly divided into control group(without TGF-β1 or siRNA),TGF-β1 group(10 μg·L^-1 TGF-β1),Control siRNA group(10 μg·L^-1 TGF-β1+ Control siRNA),and Smurf2 siRNA group(10 μg·L^-1 TGF-β1+ Smurf2 siRNA).The protein levels of α-smooth muscle actin(α-SMA) and collagen type Ⅰ alpha 1(COL1A1) in the fibroblasts in various groups were detected by Western blotting method.The expression levels of Smad7,SnoN(Ski-related novel gene N),TβRⅠ,Smad2 and Smad3 mRNA and protein were detected by RT-PCR and Western blotting method,respectively. Results Compared with control group,the expression levels of Smurf2 mRNA and protein in TGF-β1 groups were significantly increased (P<0.05),and the expression levels of Smurf1 had no significant change (P>0.05).Compared with TGF-β1 group,the expression level of Smurf2 protein in Smurf2 siRNA group was decreased (P<0.05).Compared with control group,the expression levels of α-SMA and COL1A1 protein in TGF-β1 group were increased (P<0.05).Compared with TGF-β1 group,the expression levels of α-SMA and COL1A1 protein in Smurf2 siRNA group were decreased (P<0.05).Compared with control group,the expression levels of Smad7 and SnoN mRNA in TGF-β1 group and Smurf2 siRNA group were increased (P<0.05),and there were no significant differences between TGF-β1 group and Smurf2 siRNA group (P>0.05).Compared with control group,the expression levels of Smad7 and SnoN protein in TGF-β1 group were decreased (P<0.05).Compared with TGF-β1 group,the expression levels of Smad7 and SnoN protein in Smurf2 siRNA group were increased (P<0.05).Compared with control group,the expression levels of of TβRⅠ mRNA and protein in TGF-β1 group and Smurf2 siRNA group were increased (P<0.05),and there were no significant differences between TGF-β1 group and Smurf2 siRNA group (P>0.05).There were no significant differences of the mRNA and protein levels of Smad2 and Smad3 among the four groups (P>0.05). Conclusion Smurf2 could contribute to TGF-β1-induced activation in lung fibroblasts by enhancing TGF-β1 signaling through inducing the degradation of Smad7 and SnoN in vitro.