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

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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