β-连环素互作蛋白1参与PPARγ转录活性调控及在高糖诱导系膜细胞表型转化中的作用

Roles of ICAT in regulation of PPARγ transcriptional activity and phenotypic transition of human mesangial cells induced by high glucose

目的探讨β-连环素互作蛋白1(β-catenin-interacting protein 1,ICAT)在过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)转录活性调控中的作用及在高糖诱导系膜细胞表型转化中的意义。方法将人肾小球系膜细胞分别用常规葡萄糖浓度(5.5 mmol/L,对照组)和高糖(30 mmol/L,高糖组)培养48 h,检测PPARγ、ICAT和β-catenin的mRNA及蛋白质表达变化。观察过表达ICAT和敲减ICAT及β-catenin对上述培养条件下系膜细胞PPARγ转录活性的影响。利用实时荧光定量PCR和蛋白质免疫印迹技术检测不同分组中细胞表型转化标志物平滑肌动蛋白α(α-smooth muscular actin,α-SMA)表达水平。利用CCK-8试剂盒检测细胞增殖水平。结果与对照组比较,高糖培养可诱导系膜细胞表型标志蛋白α-SMA的表达上调以及系膜细胞增殖;高糖培养虽对系膜细胞PPARγ蛋白表达水平无影响(P>0.05),但可使PPARγ转录活性明显降低(P<0.01),同时,可抑制系膜细胞ICAT表达和促进β-catenin表达;过表达ICAT可部分升高高糖培养的系膜细胞PPARγ的转录活性(P<0.01);在常规糖浓度条件下,敲减ICAT可降低PPARγ的转录活性(P<0.01),敲减β-catenin表达,同样可降低PPARγ的转录活性(P<0.01);高糖培养条件下,过表达ICAT可抑制系膜细胞α-SMA的表达与细胞增殖。结论 ICAT可能是PPARγ转录活性的重要调控分子之一,并参与介导了高糖诱导系膜细胞表型转化。

Objective To explore the roles of β-catenin-interacting protein 1（ ICAT） in regulating peroxisome proliferator-activated receptor gamma（ PPARγ） transcriptional activity and inducing cell phenotypic transition in human mesangial cells（ HMCs） cultured in high glucose（ HG）. Methods HMCs were cultured in either 5. 5（ NC） or 30（ HG） mmol/L glucose medium. RT-PCR was used to detect the expression of PPARγ,ICAT and β-catenin in the cells. The protein levels of PPARγ,ICAT and β-catenin were detected by Western blotting. Cell phenotype transition of HMCs was detected by the expression levels ofα-smooth muscular actin（ α-SMA）. And CCK-8 assay was used to measure cell proliferation. Results Compared with NC,HG induced the expression of α-SMA and promoted the proliferation of HMCs. Though HG showed no significant influence on PPARγ expression in HMCs（ P〉 0. 05）,it lowered the transcriptional activity of PPARγ（ P 〈0. 05）,inhibited the expression of ICAT but enhanced β-catenin. Over-expression of ICAT partially promoted the transcriptional activity of PPARγ（ P〈 0. 05）. Under normal glucose condition,knockdown of ICAT reduced the transcriptional activity of PPARγ（ P 〈0. 01）,so was β-catenin knockdown（ P〈 0. 01）. However, under high glucose condition, over-expression of ICAT resulted in decreased expression of α-SMA and reduced proliferation of HMCs. Conclusion ICAT might be one of potential molecules in regulation of PPARγ transcriptional activity,and it also participates in mediating the cell phenotypic transition of HMCs induced by high glucose.