泛素特异性修饰酶2-69对大鼠系膜细胞内饰胶蛋白聚糖表达和泛素化降解的调节

目的 研究泛素特异性修饰酶2-69（USP2-69）对系膜细胞（MC）内饰胶蛋白聚糖（DCN）表达和泛素化的调节作用。方法 ①培养MC细胞，采用Western blot法检测大鼠肾MC内USP2-69的表达；②采用免疫共沉淀、激光共聚焦和免疫荧光法，检测MC内USP2-69是否与DCN结合，并观察两者在MC内的定位；③将pRK5-USP2-69-HA质粒瞬时转染MC后，用Western blot法检测HA、USP2-69和DCN的蛋白表达，用免疫共沉淀法检测DCN的泛素化水平；④采用USP2-69 siRNA处理MC，用PCR检测USP2-69和DCN的mRNA表达，用time-course Western blot法检测DCN的半衰期， Western blot法检测DCN的下游效应分子（TGF-β1和Col Ⅳ）的蛋白表达。结果 ① USP2-69在MC、肝细胞（BRL-3A）和足细胞（GEC）内均有表达，其在MC内的基础蛋白表达水平显著高于BRL-3A和GEC［MCs：(0.27±0.05)，BRL-3A：(0.035±0.009)，GEC：(0.012±0.004)， P<0.01］。② MC总蛋白经DCN抗体免疫沉淀后，可检测到USP2-69的表达；经USP2-69抗体免疫沉淀后，可检测到DCN表达；且在MC胞质内，代表USP2-69蛋白的荧光与代表DCN蛋白的荧光存在共定位。③ 转染pRK5-USP2-69-HA质粒的MC内可检测到代表外源性USP2-69表达的HA蛋白，USP2-69和DCN蛋白表达的升高，同时DCN的泛素化水平明显降低。④ 经USP2-69 RNA干扰的MC内DCN的mRNA水平没有明显改变，但其半衰期明显缩短（3 h），且TGF-β1和Col Ⅳ的蛋白表达均明显升高。结论 大鼠肾MC内USP2-69可与DCN相结合，及两者在胞质内共定位，USP2-69能够降低MC内DCN的泛素化水平及提高DCN蛋白总量并促进其功能。

Effect of USP2-69 on regulating the expression and ubiquitous degradation of intracellular decorin in mesangial cells in rats

Objective To investigate the effects of ubiquitin-specific processing protease 2-69 (USP2-69) on regulating the expression and ubiquitous degradation of decorin (DCN) in mesangial cells (MC). Methods Western blot was used to examine the protein expression of USP2-69 in rat MC. Co-IP, confocal and immunofluorescence were used to analyze the interaction between USP2-69 and DCN, and their location in MC. After pRK5-USP2-69-HA eukaryon expression plasmid was transfected into MC, Western blot was used to examine protein expression of HA, USP2-69 and DCN. Co-IP was used to analyze the ubiquitination of DCN. After USP2-69 was knocked down by USP2-69 RNA interference, PCR was used to analyze the mRNA expression of USP2-69 and DCN, time-course Western blot was used to analyze the half-life of DCN and Western blot was used to examine the protein levels of TGF-β1 and Col Ⅳ, two downstream effectors of DCN.Results ① USP2-69 was expressed in MC, BRL-3A and GEC, the basal protein expression was higher in MC than in hepatocytes and podocytes ［MCs：(0.27±0.05) vs BRL-3A：(0.035±0.009), GEC：(0.012±0.004)，P<0.01］. ② After the total protein of MC was immunoprecipitated by anti-DCN antibody, USP2-69 could be detected. Conversely, DCN could be detected after the total protein of MC was immunoprecipitated by anti-USP2-69 antibody. Moreover, there was a co-localization between fluorescence of USP2-69 and DCN in cytoplasm of MC. ③ Protein expressions of HA which represented extrinsic USP2-69, elevated protein level of USP2-69 and DCN, and an obvious decrease of ubiquitinated DCN was detected in MCs transfected with pRK5-USP2-69-HA plasmid. ④ RNA interference of USP2-69 in MC didn′t change the mRNA level of DCN, but markedly shortened the half-life of DCN from 6 h to 3 h and increased the protein expression of TGF-β1 and Col Ⅳ.ConclusionIn cultured rat MC, a physical interaction and a co-localization in cytoplasm existed between USP2-69 and DCN. USP2-69 could decrease the ubiquitinated form of DCN and increase its protein expression and biological function.