Hepatocyte nuclear factor-4alpha 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)-1alpha 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-4alpha, 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-1alpha, HNF-4alpha cotransfection led to a robust 20-fold activation. Deletion analysis of mouse and rat Ntcp promoters mapped a conserved HNF-4alpha consensus site at -345/-326 and -335/-316 bp, respectively. p-475bpmNtcpLUC is not transactivated by HNF-1alpha but shows a 50-fold enhanced activity upon cotransfection with HNF-4alpha. Gel mobility shift assays demonstrated a complex of the HNF-4alpha-element formed with liver nuclear extracts that was blocked by an HNF-4alpha specific antibody. HNF-4alpha binding was confirmed by chromatin immunoprecipitation. Using Hepa 1-6 cells, HNF-4alpha-knockdown resulted in a significant 95% reduction in NTCP mRNA. In conclusion, mouse Ntcp is regulated by HNF-4alpha via a conserved distal cis-element independently of HNF-1alpha.
Geier
Andreas
A
Dept. of Internal Medicine, Div. of Gastroenterology & Hepatology, Univ. Hospital Zurich (USZ) Rämistrasse 100, CH-8091 Zurich, Switzerland. andreas.geier@usz.ch
Martin
Ina V
IV
Dietrich
Christoph G
CG
Balasubramaniyan
Natarajan
N
Strauch
Sonja
S
Suchy
Frederick J
FJ
Gartung
Carsten
C
Trautwein
Christian
C
Ananthanarayanan
Meenakshisundaram
M
D000818:Animals; D015536:Down-Regulation; D051557:Hepatocyte Nuclear Factor 4 / Q000502:physiology*; D051379:Mice; D029363:Organic Anion Transporters, Sodium-Dependent / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D027981:Symporters / Q000235:genetics*; D015533:Transcriptional Activation / Q000187:drug effects*
D051557:Hepatocyte Nuclear Factor 4; D029363:Organic Anion Transporters, Sodium-Dependent; D034741:RNA, Small Interfering; D027981:Symporters; C071908:sodium-bile acid cotransporter