Sp1 dependent transcriptional regulation of G-protein coupled bile acid receptor Gpbar-1: role of Krüppel like factor 5

Abstract

TGR5 (Gpbar-1) is a G-protein coupled bile acid receptor, which is highly expressed in cholangiocytes, liver sinusoidal endothelial cells, liver macrophages and CD14 positive monocytes of peripheral blood. TGR5-/- mice show impaired liver regeneration after partial hepatectomy and the increased inflammation in liver disease models, thus emphasizing a protective role of TGR5 in liver. While TGR5 functions are being studied extensively, little is known about the transcriptional regulation of this receptor. Aim: To investigate the mechanism and transcriptional regulation of TGR5. Methods: The potential TGR5 promoter (-154/-79) was cloned into a pGL3 Luciferase expression vector. Luciferase gene expression was used to evaluate the effect of specificity protein 1 (Sp1) and Krüppel like factor 5 (KLF5) on TGR5 expression by co-transfection. Binding of the two transcription factors to the promoter was verified by Chromatin Immunoprecipitation (ChIP). Phosphorylation of Sp1 was shown by immunoprecipitation. Results: KLF5 increased the luciferase gene expression in a concentration dependent manner. The effect was lost when the binding site for KLF5 was mutated. High expression of Sp1 mediated a downregulation of luciferase activity, which was absent when mutated Sp1 with no DNA binding activity was co-transfected. KLF5 enhanced the phosphorylation of Sp1. ChIP analysis confirmed the binding of both transcription factors to the promoter region. Conclusion: This study demonstrates that the transcription factor Sp1 downregulates whereas KLF5 upregulates the gene expression of TGR5 in vitro. KLF5 increases the transcription of TGR5 either directly through interaction with the promoter and/or by decreasing the DNA binding activity of Sp1 via increased Sp1 phosphorylation.