Overexpression of Smad ubiquitin regulatory factor 2 suppresses transforming growth factor-ß mediated liver fibrosis
Overexpression of Smad ubiquitin regulatory factor 2 suppresses transforming growth factor-ß mediated liver fibrosis
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DOI：
10.1111/j.1751-2980.2012.00592.x
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发表时间：
2012-06-01
影响因子：
3.5
通讯作者：
Shen, Xi Zhong
中科院分区：
文献类型：
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作者：
Cai, Yu;Zhou, Chao Hui;Shen, Xi Zhong
目的：探讨Smadubiquitinregulationfactor 2（Smurf 2）在肝纤维化和肝硬化发生发展中的作用。方法：使用逆转录-聚合酶链反应、蛋白质印迹（WB）和免疫组织化学方法测定纤维化和硬化大鼠和人肝组织中Smurf 2的体内表达。在有或没有转化生长因子（TGF）-β 1处理的LX-2细胞系中测定体外Smurf 2水平;通过ELISA测定I、III、IV胶原和层粘连蛋白水平。将重组质粒pcDNA3.1-Smurf 2转染LX-2细胞，用WB和ELISA法检测TGF-β I型受体（T β RI）、Smad 7、胶原和层粘连蛋白在蛋白酶体抑制剂MG-132作用下的表达。利用免疫共沉淀来表征这些因子之间的相互作用和泛素化水平。转染pcDNA3.1-Smad 7载体，并与Smurf 2一样进行后续检查。研究结果：Smurf 2水平在纤维化大鼠肝脏和TGF-β 1处理的LX-2细胞的早期升高，但在纤维化肝脏中降低。LX-2细胞中Smurf 2过表达降低了T β RI和Smad 7水平，同时伴有胶原和层粘连蛋白水平的降低。免疫共沉淀证实Smurf 2与T β RI和Smad 7相互作用，从而增加T β RI和Smad 7泛素水平。Smad 7过表达降低了T β RI水平，并伴有胶原和层粘连蛋白水平的降低。MG-132可拮抗上述作用。结论：Smurf 2与Smad 7相互作用，在肝纤维化早期通过泛素依赖性的T β RI降解来抑制TGF-β介导的肝纤维化。
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.