Hepatocyte nuclear factor HNF-6 regulates hepatic cholesterol 7 alpha-hydroxylase (CYP7A1) during liver injury from biliary obstruction

Abstract

Introduction: The liver enriched transcription factor HNF-6 (OC-1) is expressed in the embryonic and mature hepatocytes and bile duct epithelia, and has potential DNA binding sites for numerous hepatic target genes involved in critical liver function. The relevance of HNF-6 in liver development and function is illustrated in the HNF-6 knock out mice where the extra and intrahepatic bile ducts are abnormally differentiated and animals developed non obstructive cholestatic injury. Hypothesis: HNF-6 regulates in vivo hepatic genes involved in bile acid metabolism. Methods: CD1 mice underwent common bile duct ligation (BDL) (n = 4) or sham operation (SH) (n = 2) for 24 hours and the effects of biliary obstruction on the expression of hepatic HNF-6 and its in vivo target genes involved in bile acid transport and synthesis are examined. Whole liver total RNA was extracted for RNAse protection analyses. Data were collected on the Phosphoimager and analyzed by the ImageQuant program. Differences in mRNA signals between groups were evaluated by the student t test. Results: A 4 fold reduction in HNF-6 mRNA expression in BDL samples relative to SH samples (p = 0.005) is associated with a 3 fold (p = 0.001) and a 10 fold (p = 0.005) downregulation in the sodium dependent taurocholate bile acid transporter NTCP and the bile acid synthesis enzyme Cholesterol 7α hydroxylase (CYP7A1) respectively, but a 7 fold upregulation in the apical bile acid transporter protein (ABAT). Overexpression of HNF-6 mRNA and protein in liver following 48 hours of recombinant adenovirus expressing HNF-6 cDNA (AdH6 n = 3) or control LacZ (n = 2) by tail vein injection showed a 4 fold upregulation of Cyp7A1 (p = 0.03) but no effect on NTCP, or ASBT expression in AdH6 infected liver. Electromobility shift assay using liver nuclear protein extracts, HNF-6 antibody and oligonucleotide of the CYP7A1 promoter bearing the DNA consensus sequences for HNF-6 binding confirmed that the mouse Cyp7A1 promoter at nt -1442/–1429 contains a binding site for HNF-6. Conclusions: CYP7A1 is a likely in vivo target gene for HNF-6. The downregulation of HNF-6 transcription factor expression in association with a reduction in Cyp7A1 during biliary obstruction is a likely protective mechanism to further limit the production and therefore the excess of intracellular toxic bile acids accumulated during cholestasis.