P0358 FUNCTIONAL ANALYSIS OF THE TRANSCRIPTIONAL REGULATION OF THE HUMAN BILE SALT EXPORT PUMP GENE

Abstract

Introduction: The bile salt export pump, BSEP, encoded by ABCB11, is the major bile salt transporter of human liver. ABCB11 is mutated in a form of low gamma-GT progressive familial intrahepatic cholestasis (PFIC), known as BSEP deficiency. It has been documented that transcription of ABCB11 is positively regulated by bile salts. However, little is known about which other transcription factors are involved in controlling the expression of the human gene, as well as the effect of exogenous compounds on its transcription. This study was designed to investigate these questions. Methods: 5′ and 3′ Rapid Amplification of cDNA Ends (RACE) were performed using total RNA from normal liver and various cholestatic livers as a template. Various ABCB11 promoter fragments were PCR amplified from genomic DNA and cloned into pGEM-Teasy by T-A cloning for sequencing purposes. They were then sub-cloned to the Firefly luciferase vector pGL3-basic (Promega). 2.7 mug of each promoter construct was transfected into 9 ×105 HepG2, HEK 293, and CaCo2 cells in triplicate using Tfx-20 (Promega), with a lipid:DNA ratio of 3:1. 0.3 μg of the Renilla luciferase vector pRL-TK was co-transfected with each sample to serve as a transfection efficiency control. In the case of HepG2 cells, 50 muM oestrogen or progesterone was added to the culture medium. Cells were harvested after 48 hours and the Dual-Luciferase Reporter Assay System (Promega) was used to determine the values of Firefly and Renilla luciferase in each sample. Protein concentrations were determined using a Bradford assay (Bio-Rad). Results were presented as Firefly luciferase units corrected to Renilla luciferase units per mg of protein. Results: 5′ RACE identified 3 transcription start sites, two of which are novel. 3′ RACE yielded products which on sequencing determined that the ABCB11 polyadenylation site is 1.365 kb downstream from the published mRNA sequence. The ABCB11 promoter fragment/Firefly luciferase constructs identified a putative silencer in the −845 to 670 bp region in HepG2 cells. The addition of oestrogen and progesterone caused no decrease in promoter activity in these cells. However, differential promoter activity was detected in CaCo2 and HEK293 cells. Conclusion: These preliminary findings are in keeping with both enhancer and silencer elements in the ABCB11 promoter. DNA footprinting and EMSA are underway to identify transcriptionally active molecules binding to the promoter. The effect of drugs on promoter activity is also being assessed. This work provides an insight into the transcriptional regulation of ABCB11 under normal and cholestatic conditions and an experimental system to assess the effect of ABCB11 promoter sequence variants found in cholestatic patients.