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Objective@#To investigate the effect and regulatory mechanism of Smurf2 on the negative regulator Smad7 of TGF-β1/Smad signaling pathway in hypertrophic scar fibroblasts.@*Methods@#From January to October 2014, 8 patients with hypertrophic scar after burn were admitted. The age of patients ranged from 1 year 8 months to 7 years, and the time of scar hyperplasia ranged from 2 to 12 months. The residual normal skin of the same patient was used as the control. The fibroblasts were isolated from hypertrophic scar and normal skin respectively and cultured. The third to fifth passage cells were used for the experiments. ① The protein expression of Smad7 in the two groups was detected by Western Blot. ② Hypertrophic scar fibroblasts and normal skin fibroblasts were treated with exogenous TGF-β1 at concentration of 10 ng/ml for 0 min, 5 min, 15 min, 30 min, 1 h, 2 h and 12 h. The expression of Smad7 protein and mRNA were detected by Western Blot and RT-PCR, respectively. ③ The cell lysates of the two groups were collected and incubated with the ubiquitin mixture for 1 h, 2 h and 6 h at 37℃, respectively. The degradation level of Smad7 protein was detected by Western Blot. ④ The cell lysate of hypertrophic scar fibroblasts was collected and incubated with ubiquitin mixture with or without proteasome inhibitor (MG132: MG115=1: 1) to study its inhibitory effect on the degradation of Smad7 in vitro. ⑤ Immunoprecipitation (IP) technique was used to detect the interaction between Smad 7 and E3 ubiquitin ligase Smurf2 in hypertrophic scar fibroblasts. ⑥ The expression of Smad7 protein in hypertrophic scar fibroblasts stimulated by TGF-β1 after Smurf2 silencing by small interference RNA (siRNA) technique were detected by Western blot.@*Results@#① There was no significant difference in the expression level of Smad7 protein between hypertrophic scar and normal skin fibroblasts(t=0.76, P=0.46). ② Expressions of Smad7 mRNA and protein in normal skin fibroblasts stimulated by exogenous TGF-β1 gradually increased in a time-dependent manner(P<0.05). The expression of Smad7 mRNA in the hypertrophic scar fibroblasts increased at all-time points except at 5min , (P<0.05), while there was no significant difference in the expression level of Smad7 protein at all-time points with or without TGF-β1 stimulation(P>0.05). ③ Degradation of Smad7 protein was enhanced in hypertrophic scar fibroblasts (the expression level of Smad 7 protein at each time point was compared with that of the control group and the last time point, P<0.05), while there was no significant difference in Smad7 protein degradation in normal skin fibroblasts(P=0.162). ④ Enhanced degradation of Smad7 in hypertrophic scar fibroblasts was blocked by the addition of the proteasome inhibitors MG132/MG115. ⑤ In hypertrophic scar fibroblasts, the Smurf2-Smad7 complex was detected, which indicated the interaction between Smurf2 and Smad7 in hypertrophic scar fibroblasts. ⑥ The expression of Smad7 protein was not increased in the hypertrophic scar fibroblasts stimulated by TGF-β1, whereas the stimulation of TGF-β1 increased the expression of Smad7 protein after silencing of Smurf2 gene expression.@*Conclusions@#In the hypertrophic scar fibroblasts, Smurf 2 attenuates the inhibitory effect of Smad 7 on TGF-β1 signaling pathway through the degradation of Smad7 by ubiquitination, which may be involved in the formation of hypertrophic scar.
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Objective To investigate the mechanism of Smad ubiquitination-related factor 2 (Smurf2)neddylation. Methods The Smurf2 protein level was tested by overexpression of Nedd8,while the method of immunoprecipitation(IP) and Western blotting were used to analyz Smurf2-Nedd8 modification.The GST-pulldown experiment was conducted to prove protein interactions.The protein was obtained by grinding mouse tissue and Western blotting was used to test the protein expression level.Results Over expression of Nedd8 could lead to the down regulation of the Smurf2′s protein level.Smurf1 and Smurf2 could interact in the GST-pulldown experiment. Smurf1 could enhance Smurf2-Nedd8 modification.The Smurf2′s protein level was up-regulated in mouse tissue of Smurf1 C426A.Conclusion There is some relationship and also difference between Smurf1 and Smurf2.Smurf1 can enhance Smurf2-Nedd8 modification.
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Objective To investigate the possible mechanism that hepatocyte growth factor (HGF) inhibits renal tubular epithelial-mesenchymal transition (EMT),and to determine whether Smurf2 expression induced by TGF-β1 can be reversed by HGF in normal rat kidney epithelial cells (NRK-52E).Methods Using rat NRK-52E cell line as an in vitro system,NRK-52E cells were incubated with 5 μg/L TGF-β1 for 0-24 h.Part of cells were pretreated with 20 μg/L HGF for 30 min or not,then incubated with or without 5 μg/L TGF-β1 for 1 h or 48 h.The other cells were transfected with pFlag-Smurf2 or Smurf2 siRNA for 24 h,then treated with or without 20 μg/L HGF for 24 h.The expressions of Smurf2,SnoN,E-cadherin,alpha-smooth muscle actin (α-SMA) and fibronectin (FN) were detected by Western blotting and indirect immunofluorescence staining assays.Results Compared to normal control,TGF-β1 could rapidly induce Smurf2 protein expression in a short time (P＜0.01).Meanwhile,the expressions of FN and α-SMA were significantly induced,and the expression of E-cadherin was reduced in NRK-52E cells by TGF-β1.In contrast,in the NRK-52E cells pretreated with HGF,HGF could obviously inhibit Smurf2 expression induced by TGF-β1,and reversed the down-regulation of SnoN (P＜0.01) and E-cadherin (P＜0.05),the up-regulation of α-SMA (P＜0.01) and FN (P＜0.01) induced by TGF-β1.Moreover,overexpression of Smurf2 in NRK-52E cells could partly inhibit the up-regulation of SnoN protein by HGF,while down-regulation of Smurf2 could up-regulate the expression of SnoN induced by HGF.Conclusions HGF can abolish EMT induced by TGF-β1 in renal tubular epithelial cells through down-regulating Smurf2 expression and suppressing ubiquitin-proteasome dependent degradation of SnoN.
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Objective To investigate the expression and significance of Smad 7,Smurf 1 and Smurf 2 in basal cell carcinoma and squamous cell carcinoma.Methods Biopsy specimens were resected from 14 patients with basal cell carcinoma,19 patients with squamous cell carcinoma and 30 normal controls.Quanti- tative real-time PCR and immunohistochemical techniques were utilized to assess the expression of Smad 7, Smurf 1 and Smurf 2 in these specimens.Results The gray scale for staining of Smad 7,Smurf 1 and Smurf 2 was 166.61?7.11,166.08?8.71,and 166.25?8.15 respectively in basal cell carcinoma,161.66?5.52,166.84?9.27,and 169.98?9.48 respectively in squamous cell carcinoma.The expression levels of Smad 7,Smurf 1 and Smurf 2 were all significantly increased in basal cell carcinoma and squamous cell car- cinoma in comparison with normal controls.Conclusions The over-expression of Smad 7,Smurf 1 and Smurf 2 may interfere with transforming growth factor?signaling transduction pathway through several links,therefore prevent the inhibitory effect of transforming growth factor?on epidermal proliferation,and accelerate the abnormal proliferation in above epidermal tumors.