Overexpression of Smad ubiquitin regulatory factor 2 suppresses transforming growth factor-ß mediated liver fibrosis

OBJECTIVE: To determine the function of Smad ubiquitin regulatory factor 2 (Smurf2) on the development of liver fibrosis and cirrhosis. METHODS: In vivo Smurf2 expression in fibrotic and cirrhotic rat and human liver tissues were measured using reverse transcription-polymerase chain reaction, Western blot (WB) and immunohistochemistry. In vitro Smurf2 levels were determined in LX-2 cell line with or without transforming growth factor (TGF)-beta 1 treatment; I, III, IV collagen and laminin levels were determined by ELISA. The recombinant plasmid pcDNA3.1-Smurf2 was transfected into LX-2 cells, and WB and ELISA were utilized to analyze the expression of TGF-beta receptor type I (T beta RI), Smad7, collagens and laminin with or without proteasome inhibitor MG-132. Coimmunoprecipitation was utilized to characterize the interactions among these factors and the ubiquitination levels. pcDNA3.1-Smad7 vector was transfected and subsequent examinations were conducted just as Smurf2. RESULTS: Smurf2 levels were elevated in the early period of fibrotic rat liver and TGF-beta 1-treated LX-2 cells but were reduced in the cirrhotic livers. Smurf2 overexpression in LX-2 cells reduced T beta RI and Smad7 levels, which was accompanied by decreased collagen and laminin levels. Coimmunoprecipitation demonstrated that Smurf2 interacted with T beta RI and Smad7, which increased T beta RI and Smad7 ubiquitin levels. Smad7 overexpression reduced the T beta RI level and was accompanied by decreased collagen and laminin levels. MG-132 could antagonize these effects. CONCLUSION: Smurf2 interacts with Smad7 to suppress TGF-beta-mediated liver fibrosis through the ubiquitin-dependent degradation of T beta RI during the early period of liver fibrosis.