Hepatocyte nuclear factor-4α is a central transactivator of the mouse Ntcp gene

Sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake system for conjugated bile acids. Deletions of hepatocyte nuclear factor (HNF)-1 alpha and retinoid X receptor-alpha: retinoic acid receptor-alpha binding sites in the mouse 5'-flanking region corresponding to putatively central regulatory elements of rat Ntcp do not significantly reduce promoter activity. We hypothesized that HNF-4 alpha, which is increasingly recognized as a central regulator of hepatocyte function, may directly transactivate mouse (mNtcp). A 1.1-kb 5'-upstream region including the mouse Ntcp promoter was cloned and compared with the rat promoter. In contrast to a moderate 3.5-fold activation of mNtcp by HNF-1 alpha, HNF-4 alpha cotransfection led to a robust 20-fold activation. Deletion analysis of mouse and rat Ntcp promoters mapped a conserved HNF-4 alpha consensus site at -345/-326 and -335/-316 bp, respectively. p-475bpmNtcpLUC is not transactivated by HNF-1 alpha but shows a 50-fold enhanced activity upon cotransfection with HNF-4 alpha. Gel mobility shift assays demonstrated a complex of the HNF-4 alpha-element formed with liver nuclear extracts that was blocked by an HNF-4 alpha specific antibody. HNF-4 alpha binding was confirmed by chromatin immunoprecipitation. Using Hepa 1-6 cells, HNF-4 alpha-knockdown resulted in a significant 95% reduction in NTCP mRNA. In conclusion, mouse Ntcp is regulated by HNF-4 alpha via a conserved distal cis-element independently of HNF-1 alpha.