Differential regulation of G-protein coupled bile acid receptor (Gpbar-1) by Sp1/KLF5 family transcription factors

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 Sp1 and KLF5 on TGR5 expression by co-transfection. Binding of the two transcription factors to the promoter was verified by Chromatin Immunoprecipitation (ChIP). Immunoprecipitation was used to confirm the interaction of HDAC1 and 2 with Sp1. Transfection of KLF5 siRNA was used to verify the TGR5 transcriptional regulation. 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 TGR5 promoter luciferase activity, which was absent when mutated Sp1 with a defective DNA binding domain was co-transfected. ChIP analysis confirmed the binding of both transcription factors to the predicted TGR5 promoter region. Sp1 forms an inhibitory complex with HDAC1 and 2 at the site of binding there by downregulating the transcription of TGR5. Inhibition of casein kinase 2 mediated phosphorylation of HDAC proteins by apigenin resulted in a decreased complex formation with Sp1 there by loss of Sp1 mediated downregulation. TGR5 is differentially regulated by Sp1 in different carcinoma cell lines (colon carcinoma and cholangiocyte carcinoma). KLF5 siRNA decreased the mRNA expression of TGR5 in colon and cholangiocyte carcinoma cell lines. Conclusion: This study demonstrates that the transcription factor Sp1 downregulates whereas KLF5 upregulates the gene expression of TGR5 in vitro. Sp1 decreases the transcription of TGR5 by binding to the promoter region and recruiting HDAC1 and 2 to forming an inhibitory complex thereby leading to the deacetylation of histones. Phosphorylation of HDAC proteins by casein kinase 2 is important in the formation of inhibitory complex. Differential regulation of TGR5 by Sp1 was confirmed in different carcinoma cell lines.

Corresponding author: Chintalapati, Chakravarthi

E-Mail: chintalapaticv@gmail.com