Abstract:

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.

Key words: Smad ubiquitination regulatory factor 2, lung fibroblast, transforming growth factor-β1, signal transduction