Regulation of insulin gene expression by small G protein related protein, Rho-kinase.

本研究的目的是探讨Rho激酶在胰岛β细胞胰岛素合成中的作用。首先，我们用含有人胰岛素基因启动子和Rho激酶特异性抑制剂Y-27632的荧光素酶载体，对胰岛β细胞株WIN 6进行荧光素酶检测，以评价Rho激酶对胰岛素基因启动子活性的影响。荧光素酶活性随着Y-27632的处理而显著增加，表明胰岛素启动子活性通过Rho激酶的抑制而增加。接下来，为了鉴定与Rho激酶介导的胰岛素基因表达机制相关的顺式作用元件，我们使用包含各种缺失的胰岛素基因启动子的荧光素酶载体进行荧光素酶测定。用Y-27632处理所增加的荧光素酶活性被转染的荧光素酶载体显著降低，所述荧光素酶载体包括缺乏A3元件的胰岛素基因启动子。这一结果表明， ...更多信息 人胰岛素基因启动子中的eA 3元件在Rho激酶介导的胰岛素基因表达中具有重要作用。胰腺和十二指肠同源盒基因-1（PDX-1）是在胰岛β细胞特异性和葡萄糖诱导的胰岛素基因转录中起重要作用的反式作用因子，PDX-1可与胰岛素基因启动子中的A3元件结合。然后，为了评估Rho激酶对PDX-1的DNA结合活性的影响，我们使用A3元件DNA探针和用/不用Y-27632处理的MIN 6细胞的核提取物进行电泳迁移率变动分析（EMSA）。经Y-27632处理后，PDX-1的DNA结合活性增加，表明Rho激酶可调节PDX-1的DNA结合活性。综上所述，提示胰岛素基因启动子活性通过PDX-1介导的途径通过抑制Rho激酶活性而增加。Rho激酶介导PDX-1活性的机制有待进一步研究。少

The purpose of this study is to identify the role of Rho-kinase for the insulin synthesis in pancreatic β-cell At first, to evaluate the effect of Rho-kinase on the insulin gene promoter activity, we performed luciferase assay using pancreatic β-cell line WIN6, luciferase vector which include human insulin gene promoter and Rho-kinase specific inhibitor, Y-27632. The luciferase activity was significantly increased with the treatment of Y-27632, suggesting that insulin promoter activity was increased by the inhibition of Rho-kinase. Next, to identify the cis-acting element which correlate with the Rho-kinase mediated insulin gene expression mechanism, we performed luciferase assay using luciferase vector which include various deleted insulin gene promoter. The increased luciferase activity by the treatment with Y-27632 was significantly decreased by the transfection of luciferase vector which include insulin gene promoter lacking A3-element. This result suggested the possibility that th … More e A3-element in human insulin gene promoter had important role of the Rho-kinase mediated insulin gene expression. It is known that the pancreatic and duodenal homeobox gene-1(PDX-1) is the trans-acting factor which play a important role in pancreatic β-cell specific and glucose induced insulin gene transcription and PDX-1 can bind to A3-element in insulin gene promoter. Then, to evaluate the effect of Rho-kinase on DNA binding activity of PDX-1, we performed electropholetic mobility shift assay(EMSA)using A3-element DNA probe and the nuclear extract of MIN6 cells which were treated with/without Y-27632. DNA binding activity of PDX-1 was increased by the treatment of Y-27632, indicating that the Rho-kinase can modulate PDX-1 DNA binding activity. Taken together, It is suggested that insulin gene promoter activity is increased by the inhibition of Rho-kinase activity via PDX-1 mediated pathway. Further investigation is needed for identifying the mechanism how Rho-kinase can mediate PDX-1 activity. Less