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

J Dig Dis. 2012 Jun;13(6):327-34. doi: 10.1111/j.1751-2980.2012.00592.x.

Abstract

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)-β1 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-β receptor type I (TβRI), 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-β1-treated LX-2 cells but were reduced in the cirrhotic livers. Smurf2 overexpression in LX-2 cells reduced TβRI and Smad7 levels, which was accompanied by decreased collagen and laminin levels. Coimmunoprecipitation demonstrated that Smurf2 interacted with TβRI and Smad7, which increased TβRI and Smad7 ubiquitin levels. Smad7 overexpression reduced the TβRI level and was accompanied by decreased collagen and laminin levels. MG-132 could antagonize these effects.

Conclusion: Smurf2 interacts with Smad7 to suppress TGF-β-mediated liver fibrosis through the ubiquitin-dependent degradation of TβRI during the early period of liver fibrosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carbon Tetrachloride
  • Cells, Cultured
  • Collagen / biosynthesis
  • Disease Progression
  • Gene Expression Regulation / physiology
  • Humans
  • Laminin / biosynthesis
  • Liver / drug effects
  • Liver / metabolism
  • Liver Cirrhosis / metabolism*
  • Liver Cirrhosis, Experimental / chemically induced
  • Liver Cirrhosis, Experimental / metabolism
  • Male
  • Protein-Serine-Threonine Kinases / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Receptor, Transforming Growth Factor-beta Type I
  • Receptors, Transforming Growth Factor beta / metabolism
  • Signal Transduction / physiology
  • Smad7 Protein / metabolism
  • Transfection
  • Transforming Growth Factor beta / pharmacology*
  • Ubiquitin / physiology
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitin-Protein Ligases / physiology*

Substances

  • Laminin
  • Receptors, Transforming Growth Factor beta
  • SMAD7 protein, human
  • Smad7 Protein
  • Smad7 protein, rat
  • Transforming Growth Factor beta
  • Ubiquitin
  • Collagen
  • Carbon Tetrachloride
  • SMURF2 protein, human
  • Smurf2 protein, rat
  • Ubiquitin-Protein Ligases
  • Protein-Serine-Threonine Kinases
  • Receptor, Transforming Growth Factor-beta Type I
  • Tgfbr1 protein, rat