Mouse NTCP is directly transactivated via a conserved HNF-4α element in the proximal promoter region, with further co-activation by PGC-1α

Background & Aim: Sodium taurocholate co-transporting polypeptide (Ntcp), a 50 kDa basolaterally localized membrane transporter, is the major transport system for conjugated bile acids. Induction of cholestasis in rats leads to down-regulation of Ntcp involving decreased HNF–1α binding to a proximal element within the minimal promoter. Mouse Ntcp promoter is missing this proximal HNF–1α element and is activated by a distal HNF–1α cis element (Geier et al. AJP 2005, in press). In addition, other potential cis elements not previously identified are also likely to regulate the mouse gene. We therefore functionally characterized the mouse Ntcp promoter to identify further potential regulators. Methods: A 1.1 kb 5’-upstream region including the mouse promoter (Genbank AF190697) was cloned upstream of firefly luciferase gene in pXP2 delta vector. Nested deletion constructs of the mouse Ntcp promoter were generated by PCR. HepG2 cells were transfected with the 1.1 kb mouse promoter using FuGene6 and were also cotransfected with plasmids encoding HNF–1α, HNF–4α and PGC–1α. Gel mobility shift (EMSA) assays and supershift assays were done using ³²P-labeled HNF–4 consensus site and antibodies to HNF–4α. Site-directed mutagenesis and siRNA mediated blockade was used to verify the specific HNF–4α effect on the promoter. Results: In contrast to a moderate 3.5-fold increase of mouse Ntcp promoter activity by HNF–1α cotransfection, HNF–4α cotransfection led to a robust 17–fold increase over basal promoter activity which has not been reported previously. Deletion analysis of the mouse Ntcp promoter mapped the HNF4α binding site to –352/–326, representing a consensus element which is also present in the rat Ntcp promoter. EMSA assays demonstrated that this consensus element for HNF–4 formed a retarded complex with liver nuclear extracts which was shifted by a HNF–4α specific antibody. Mutations in this element and co-transfection with HNF–4α siRNA only halved promoter activity indicating additional HNF–4 binding sites and incomplete inhibition efficiency by siRNA. In contrast, co-transfection with PGC–1α together with HNF–4α increased promoter activity by a further 50%. Conclusion: Mouse Ntcp is directly regulated by HNF–4α via a conserved proximal cis-element, conferring a much higher promoter activation than HNF–1α. This activating effect is increased by a considerable co-activation upon co-transfection with PGC–1α.

Key words

Bile acid transport - cholestasis - nuclear factors - regulation