Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4a

Hepatocyte nuclear factor 4a (HNF4a) regulates many genes that are preferentially expressed in liver. Mice lacking hepatic expression of HNF4a (HNF4a) exhibited markedly increased levels of serum bile acids (BAs) compared with HNF4a-floxed (HNF4a) mice. The expression of genes involved in the hydroxylation and side chain b-oxidation of cholesterol, including oxysterol 7ahydroxylase, sterol 12a-hydroxylase (CYP8B1), and sterol carrier protein x, wasmarkedly decreased inHNF4amice. Cholesterol 7a-hydroxylase mRNA and protein were diminished only during the dark cycle in HNF4a mice, whereas expression in the light cycle was not different between HNF4a and HNF4a mice. Because CYP8B1 expression was reduced in HNF4a mice, it was studied in more detail. In agreement with the mRNA levels, CYP8B1 enzyme activity was absent in HNF4a mice. An HNF4a binding site was found in the mouse Cyp8b1 promoter that was able to direct HNF4a-dependent transcription. Surprisingly, cholic acid-derived BAs, produced as a result of CYP8B1 activity, were still observed in the serum and gallbladder of these mice. These studies reveal that HNF4a plays a central role in BA homeostasis by regulation of genes involved in BA biosynthesis, including hydroxylation and side chain b-oxidation of cholesterol in vivo.— Inoue, Y., A-M. Yu, S. H. Yim, X. Ma, K. W. Krausz, J. Inoue, C. C. Xiang, M. J. Brownstein, G. Eggertsen, I. Björkhem, and F. J. Gonzalez. Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4a. J. Lipid Res. 2006. 47: 215–227. Supplementary key words conditional knockout mice . sterol 12ahydroxylase . oxysterol 7a-hydroxylase . sterol carrier protein x . cholic acid Hepatocyte nuclear factor 4a (HNF4a; NR2A1), an orphan member of the nuclear receptor superfamily, regulates many genes that are preferentially expressed in the liver. HNF4a target genes include several serum proteins such as apolipoproteins, blood coagulation factors, cytochromes P450, and enzymes involved in glucose, ammonia, lipid, steroid, and fatty acid metabolism (1, 2). HNF4a is also involved in human genetic diseases; mutations in the HNF4a gene cause maturity-onset diabetes of the young 1 (3), and mutations in HNF4a binding sites were found in the promoter regions of the genes encoding blood coagulation factor IX and HNF1a, causing hemophilia B Lyden and maturity-onset diabetes of the young-3, respectively (4–6). Targeted disruption of the HNF4a gene was found to be embryo-lethal (7), indicating that HNF4a is an essential factor for mammalian development. To circumvent the problem of embryonic lethality, liver-specific HNF4a-null mice, designated HNF4a, were produced using the CreloxP system (8). These mice exhibited impaired lipid homeostasis and increased serum ammonia levels as a result of decreased expression of hepatic ornithine transcarbamylase (8, 9). In addition, HNF4a mice have increased bile acids (BAs), which could have a role in the high Manuscript received 29 September 2005 and in revised form 26 October 2005. Published, JLR Papers in Press, November 1, 2005. DOI 10.1194/jlr.M500430-JLR200 Abbreviations: ACOX2, trihydroxycoprostancyl-coenzyme A oxidase 2; BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; CPF, cholesterol 7a-hydroxylase promoter factor; CYP7A1, cholesterol 7a-hydroxylase promoter factor; CYP7B1, oxysterol 7a-hydroxylase; CYP8B1, sterol 12a-hydroxylase; CYP27A1, sterol 27-hydroxylase; CYP39A1, oxysterol 7a-hydroxylase; DBP, albumin D site-binding protein; DCA, deoxycholic acid; D-PBE, D-type peroxisomal bifunctional enzyme; DR1, direct repeat 1; FXR, farnesoid X receptor; HNF4a, hepatocyte nuclear factor 4a; HNF4a, liver-specific hepatocyte nuclear factor 4a-null; HNF4a, hepatocyte nuclear factor 4a-floxed; LC-MS/MS, liquid chromatography tandem mass spectrometry; LRH-1, liver receptor homologue-1; MCA, muricholic acid; NTCP, sodium taurocholate cotransporter polypeptide; OATP1, organic anion transporter polypeptide 1; PPARa, peroxisome proliferator-activated receptor a; PXR, pregnane X receptor; RXRa, retinoid X receptor a; SCPx, sterol carrier protein x; SCP2, sterol carrier protein 2; SHP, small heterodimer partner; UDCA, ursodeoxycholic acid; VLACSR, very long chain acyl-coenzyme A synthase-related gene. 1 To whom correspondence should be addressed. e-mail: [email protected] This article is available online at http://www.jlr.org Journal of Lipid Research Volume 47, 2006 215 by gest, on O cber 1, 2017 w w w .j.org D ow nladed fom mortality of these mice as they age (8). BAs derived from cholesterol are preferentially produced in the liver, and the resulting BA pool is maintained by enterohepatic circulation. BAs secreted from liver form micelles with hydrophobic compounds, including fatty acids, sterols, and vitamins, and are absorbed from the jejunum and ileum and recycled via the portal venous system. As a result, only a small portion of BA is excreted into urine and feces, and the constant BA pool is maintained by newly synthesized BAs (10). HNF4a mice exhibited greatly increased serum BA levels and reduced expression of BA transporters, such as sodium taurocholate cotransporter polypeptide (NTCP) and organic anion transporter polypeptide 1 (OATP1) (8). Furthermore, increased levels of unconjugated and glycine-conjugated BAs in gallbladder were observed in HNF4a mice, which were associated with the reduced expression of the very long chain acyl-CoA synthase-related gene (VLACSR) and BA CoA:amino acid N-acyltransferase involved in BA conjugation (11). These results indicate that HNF4a plays an important role in the regulation of enterohepatic circulation of BA. BA production is attributable mainly to the neutral and acidic pathways of BA biosynthesis via a cascade of enzymatic reactions (12, 13). Hydroxylation of the steroid nucleus and side chain of cholesterol is catalyzed by enzymes such as cholesterol 7a-hydroxylase (CYP7A1), oxysterol 7a-hydroxylase (CYP7B1), sterol 27-hydroxylase (CYP27A1), and sterol 12a-hydroxylase (CYP8B1). The side chain b-oxidations are catalyzed by the enzymes trihydroxycoprostanoyl-CoA oxidase [acyl-coenzyme A oxidase 2 (ACOX2)], D-type peroxisomal bifunctional enzyme (D-PBE), and sterol carrier protein x (SCPx). Using gene knockout mice and transient transfection analysis, several transcription factors and/or nuclear receptors, such as the farnesoid X receptor (FXR) (14), liver X receptor (15), CYP7A1 promoter transcription factor (CPF) (16), pregnane X receptor (PXR) (17, 18), peroxisome proliferator-activated receptor a (PPARa) (19), small heterodimer protein (SHP) (20, 21), HNF1a and HNF1b (22, 23), and fibroblast growth factor receptor 4 (24), were shown to regulate genes encoding BA biosynthesis enzymes. In this study, the role of hepatic HNF4a in BA biosynthesis was investigated. In vivo and in vitro data revealed that HNF4a is critical in the regulation of