Effect Of Smad Ubiquitination Regulatory Factor 2 On TGF-β1-induced Activation In Lung Fibroblasts And Its Molecular Mechanism

BackgroundPulmonary fibrosis is a group of diffuse lung diseases which have common pathologic changes characterized by fibroblast proliferation and extracellular matrix(ECM) accumulation,and accompanied by tissue structure damage and function loss caused by inflammation and damage. Transforming growth factor β1(TGF-β1) is a kind of multifunctional cytokine and it’s also an important fibrogenic factor. The Smad signaling pathway acts as the primary and central signaling molecules when TGF-β superfamily transmit the cell membrane signals into intranuclear through the specific receptors on the cell membrane. TGF-β1/Smads signaling transduction pathway plays a crucial role in the development process of pulmonary fibrosis,including chemotaxis and activation of inflammatory cells, driving epithelial-to-mesenchymal transition(EMT), activating fibroblasts, and stimulating ECM synthesis and promoting its ECM deposition.The ubiquitin proteasome Pathway(UPP) regulates degree of proteins to maintain the dynamic balance of proteins. The UPP, Ubiquitous in cellular, is involved in the regulation of numerous physiological processes, such as apoptosis, the cell cycle progression, cell signal transduction and antigen presentation. If protein homeostasis is disrupted, the incidence of many important diseases will increase. The E3 ubiquitin ligases of UPP can promote the combination of ubiquitin-specific protein to make the target protein degradation,and determine the specificity and highly selective of substrate protein degradation, therefore it plays a decisive role in the UPP.Smad ubiquitination regulatory factor(Smurf)is a HECT-domain E3 ubiquitin ligases,including Smurf1 and Smurf2.Smurf2 plays an important role in adjusting the activity of the TGF-beta1 signal through the degradation of many components of Smad signal pathway. Once dysfunction, it may cause abnormal TGF-β1/Smads signal transduction pathway and then lead to a series of pathophysiological changes. Recent reports have implicated that Smurf2 can mediate the degradation of c-Ski, Sno N, Smad7, Smad2 and TβRI and other proteins involved in the regulation of TGF-β1/Smads signaling transduction pathway, and then participate in thedevelopment of fibrotic diseases, such as liver fibrosis, renal fibrosis and skin fibrosis diseases.However, little is known about the role of Smurfs in lung fibrosis.Objective To investigate the effect of 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(10μg·L–1) for 1, 2 and 6h in vitro, and control group(without TGF-β1) was set up. The expression levels of Smurf1 and Smurf2 m RNA 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 si RNA), TGF-β1 group(10 μg·L–1 TGF-β1), control si RNA group(10μg·L–1 TGF-β1+ Control si RNA), and Smurf2 si RNA group(10 μg·L–1 TGF-β1+ Smurf2 si RNA). The protein levels of α-smooth muscle actin(α-SMA) and collagen type I alpha1(COL1A1) in the fibroblasts in various groups were detected by Western blotting method. The expression levels of Smad7, Sno N(Ski-related novel gene N), TβRI, Smad2 and Smad3 m RNA and protein were detected by RT-PCR and Western blotting method, respectively.Results Compared with control group, the expression levels of Smurf2 m RNA 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 si RNA 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 si RNA group were decreased(P<0.05). Compared with control group, the expression levels of Smad7 and Sno N m RNA in TGF-β1 group and Smurf2 si RNA group were increased(P<0.05),and there were no significant differences between TGF-β1 group and Smurf2 si RNA group(P>0.05). Compared with control group, the expression levels of Smad7 and Sno N protein in TGF-β1 group were decreased(P<0.05). Compared with TGF-β1 group, the expression levels of Smad7 and Sno N protein in Smurf2 si RNA group were increased(P<0.05). Compared with control group, the expression levels of of TβRI m RNA and protein in TGF-β1 group and Smurf2 si RNA group were increased(P<0.05), and there were no significant differences between TGF-β1 group and Smurf2 si RNA group(P>0.05). There were no significant differences of the protein and m RNA levels of Smad2 and Smad3 among the four groups(P>0.05).Conclusion(1) TGF-β1 could promote the expression of Smurf2,which contribute to TGF-β1-induced activation in human lung fibroblasts, and had no effect on the expression of Smurf1;(2) Smurf2 could contribute to TGF-β1-induced activation in lung fibroblasts by enhancing TGF-β1 signaling through inducing the degradation of Smad7 and Sno N.