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Journal of Lipid Research, Vol. 47, 215-227, January 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4

Yusuke Inoue^*, Ai-Ming Yu^*, Sun Hee Yim^*, Xiaochao Ma^*, Kristopher W. Krausz^*, Junko Inoue^*, Charlie C. Xiang, Michael J. Brownstein, Gösta Eggertsen, Ingemar Björkhem and Frank J. Gonzalez^1,*

^* Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD
Laboratory of Genetics, National Institute of Mental Health, National Institutes of Health, Bethesda, MD
Department of Medical Laboratory Sciences and Technology, Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden

Published, JLR Papers in Press, November 1, 2005.

¹ To whom correspondence should be addressed. e-mail: fjgonz{at}helix.nih.gov

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Hepatocyte nuclear factor 4 (HNF4) regulates many genes that are preferentially expressed in liver. Mice lacking hepatic expression of HNF4 (HNF4^L) exhibited markedly increased levels of serum bile acids (BAs) compared with HNF4-floxed (HNF4^F/F) mice. The expression of genes involved in the hydroxylation and side chain ß-oxidation of cholesterol, including oxysterol 7-hydroxylase, sterol 12-hydroxylase (CYP8B1), and sterol carrier protein x, was markedly decreased in HNF4^L mice. Cholesterol 7-hydroxylase mRNA and protein were diminished only during the dark cycle in HNF4^L mice, whereas expression in the light cycle was not different between HNF4^L and HNF4^F/F mice. Because CYP8B1 expression was reduced in HNF4^L mice, it was studied in more detail. In agreement with the mRNA levels, CYP8B1 enzyme activity was absent in HNF4^L mice. An HNF4 binding site was found in the mouse Cyp8b1 promoter that was able to direct HNF4-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 HNF4 plays a central role in BA homeostasis by regulation of genes involved in BA biosynthesis, including hydroxylation and side chain ß-oxidation of cholesterol in vivo.

Supplementary key words conditional knockout mice • sterol 12-hydroxylase • oxysterol 7-hydroxylase • sterol carrier protein x • cholic acid

Abbreviations: ACOX2, trihydroxycoprostancyl-coenzyme A oxidase 2; BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; CPF, cholesterol 7-hydroxylase promoter factor; CYP7A1, cholesterol 7-hydroxylase promoter factor; CYP7B1, oxysterol 7-hydroxylase; CYP8B1, sterol 12-hydroxylase; CYP27A1, sterol 27-hydroxylase; CYP39A1, oxysterol 7-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; HNF4, hepatocyte nuclear factor 4; HNF4^L, liver-specific hepatocyte nuclear factor 4-null; HNF4^F/F, hepatocyte nuclear factor 4-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; PPAR, peroxisome proliferator-activated receptor ; PXR, pregnane X receptor; RXR, retinoid X receptor ; 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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Hepatocyte nuclear factor 4 (HNF4; NR2A1), an orphan member of the nuclear receptor superfamily, regulates many genes that are preferentially expressed in the liver. HNF4 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). HNF4 is also involved in human genetic diseases; mutations in the HNF4 gene cause maturity-onset diabetes of the young 1 (3), and mutations in HNF4 binding sites were found in the promoter regions of the genes encoding blood coagulation factor IX and HNF1, causing hemophilia B Lyden and maturity-onset diabetes of the young-3, respectively (4–6).

Targeted disruption of the HNF4 gene was found to be embryo-lethal (7), indicating that HNF4 is an essential factor for mammalian development. To circumvent the problem of embryonic lethality, liver-specific HNF4-null mice, designated HNF4^L, were produced using the Cre-loxP 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, HNF4^L mice have increased bile acids (BAs), which could have a role in the high 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). HNF4^L 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 HNF4^L 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 HNF4 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 7-hydroxylase (CYP7A1), oxysterol 7-hydroxylase (CYP7B1), sterol 27-hydroxylase (CYP27A1), and sterol 12-hydroxylase (CYP8B1). The side chain ß-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 (PPAR) (19), small heterodimer protein (SHP) (20, 21), HNF1 and HNF1ß (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 HNF4 in BA biosynthesis was investigated. In vivo and in vitro data revealed that HNF4 is critical in the regulation of BA biosynthesis.

	MATERIALS AND METHODS

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Animals
HNF4^L mice were generated as described previously (8). All experiments were performed with 14, 28, and 45-day-old male HNF4-floxed (HNF4^F/F) mice and HNF4^L mice. Mice were housed in a pathogen-free animal facility under a standard 12 h light/12 h dark cycle with ad libitum water and chow. All experiments with mice were carried out under Association for Assessment and Accreditation of Laboratory Animal Care guidelines with the approval of the National Cancer Institute Animal Care and Use Committee.

Microarray analysis 1
cDNA fragments from selected mouse genes were obtained by PCR from cDNA clones inserted into the plasmid pBluescript II or pUC18. The majority of the cDNA fragments contained 400 and 1,000 bp. Before spotting, the PCR fragments were dissolved in 50% DMSO solution to a final concentration of 100 ng/µl. Microarrays were spotted on amino-silane-coated glass slides (Corning, London, UK) using the Affymetrix 417^TM Arrayer. Each set of PCR fragments was arranged in six grids. mRNA was prepared from total liver RNA of 45-day-old mice at 10 AM using magnetic beads (Dynal Biotech, Oslo, Norway), and 1–2 µg was subjected to reverse transcription and labeled with the fluorescent dye Cy3 (CyScribe First-Strand cDNA Labeling Kit; Amersham Pharmacia Biotech, Uppsala, Sweden). Prehybridization and hybridization of the slides were carried out in hybridization cassettes at 65°C essentially as described in the Amersham Pharmacia Biosciences manual. The slides were then analyzed with a ScanArray 4000XL (Perkin-Elmer, Foster City, CA).

Microarray analysis 2
Twenty micrograms of pooled total liver RNA from 45-day-old mice (n = 5 for each group) at 10 AM was reverse-transcribed using oligo(dT) primers with either Cy5 or Cy3 dye using the indirect labeling method (25). The raw data were obtained using a GenPix 4000A scanner (Axon, Union City, CA) and further analyzed with National Cancer Institute microarray analysis tools (http://nciarray.nci.nih.gov) for normalization of each individual array. Each experiment was replicated by reciprocally labeling with either Cy3 or Cy5. Final values for differentially expressed genes were obtained from average regression ratios of Cy5/Cy3 (HNF4^L/HNF4^F/F) and the reverse value of Cy5/Cy3 (HNF4^F/F/HNF4^L).

Northern blot analysis
Northern blot analysis was carried out as described previously (9). All probes were amplified from a mouse cDNA library using gene-specific primers and cloned into pCR II vector (Invitrogen, Carlsbad, CA). Sequences were verified using the CEQ 2000 Dye Terminator cycle sequencing kit (Beckman Coulter, Fullerton, CA) with a CEQ 2000XL DNA Analysis System (Beckman Coulter).

Real-time PCR
Real-time PCR was performed on an ABI PRISM 7900HT sequence detection system (Applied Biosystems, Foster City, CA). Mouse liver cDNAs encoding CYP7A1, CYP27A1, Oxysterol 7-hydroxylase (CYP39A1), ACOX2, D-PBE, and control 18S RNA were amplified using SYBR Green PCR Master Mix (Applied Biosystems) and 0.3 µM specific oligonucleotide primers (CYP7A1, 5'-GCTGTGGTAGTGAGCTGTTGCA-3' and 5'-CACAGCCCAGGTATGGAATCA-3'; CYP27A1, 5'-CTGCACTTCCTGCTGACCAAT-3' and 5'-TGTCAGTGTGTTGGATGTCGTG-3'; CYP39A1, 5'-CTCTTGCAGGCCATATTGGAA-3' and 5'-ATAGGAGGAGCATTGGCCAGA-3'; ACOX2, 5'-ATGCAAAGCTGGAAACCCAAG-3' and 5'-CCACCCCGTGCTTTAAATTCTT-3'; D-PBE, 5'-AGCTGTGAGGAAAATGGTGGC-3' and 5'-TGATTCCGCTTTCTGACGATG-3'; 18S, 5'-AACTTTCGATGGTAGTCGCCG-3' and 5'-CCTTGGATGTGGTAGCCGTTT-3'). The conditions were 95°C for 10 min followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The levels of each mRNA are expressed relative to 18S RNA.

Analysis of oxysterols in serum
The serum levels of the oxysterols 7-, 25-, and 27-hydroxycholestrol were determined by isotope dilution mass spectrometry and the use of deuterium-labeled internal standards as described previously (26).

Total BA analysis
Mice were anesthetized with 2.5% avertin and decapitated, and the trunk blood was collected in a serum separator tube (Becton Dickinson, Franklin Lakes, NJ). The serum was separated by centrifugation at 7,000 g for 5 min and stored at –20°C before analysis. The total BA pool was determined as described previously (24). Urine samples and gallbladder biles were diluted 10 times with water and 1,000 times with 10% methanol, respectively, before analysis. The BA content of all samples was measured by colorimetric analysis using a BA analysis kit (Sigma, St. Louis, MO).

Analysis of gallbladder bile
Bile was hydrolyzed and analyzed by GC-MS essentially as described (27) using a Hewlett-Packard 5973 mass-specific detection instrument equipped with a 0.33 µm phase HP-ultra 1 column. The samples were methylated with diazomethane and trimethylsilylated before analysis. The samples were first analyzed by repetitive scanning to identify different BAs. Cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and ursodeoxycholic acid (UDCA) were quantified by isotope dilution mass spectrometry with the use of deuterium-labeled internal standards and selected ion monitoring. Other BAs were quantified from the chromatogram/total ion current obtained in the analysis of material to which no internal standard had been added. The peak of trihydroxy BAs was compared with that of CA. The peak areas of the dihydroxy BAs were compared with that of DCA.

Analysis of serum BAs
Twenty microliters of a 50 µM dehydrocholic acid solution (in methanol, used as an internal standard) was added to 60 µl of the serum sample. The mixture was deproteinized by the addition of 200 µl of acetonitrile. The sample was vortexed for 10 s and centrifuged at 14,000 g at 4°C for 5 min. The supernatant was transferred to a new glass tube and extracted with 2 ml of a mixture of ethyl acetate and t-butyl methyl ether (2:1, v/v). After centrifugation at 3,000 g at 4°C for 15 min, the organic phase was transferred to a new glass tube and evaporated under a stream of nitrogen gas. The residue was reconstituted in 60 µl of 50% methanol solution containing 0.2% formic acid. Ten microliters of each reconstituted sample was injected for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.

Identification and quantitation of intact free BAs by LC-MS/MS
LC-MS/MS analysis was performed on a PE SCIEX API2000 ESI triple-quadrupole mass spectrometer (Perkin-Elmer) controlled by Analyst software. A Phenomenex Luna C18 3 µm, 100 mm x 2 mm inner diameter column (Torrance, CA) was used to separate free BAs. The flow rate through the column at ambient temperature was 0.2 ml/min, and optimal resolution was achieved by elution with a linear gradient of water containing 0.1% formic acid (45%0%) and methanol (55%100%) in 10 min at room temperature. The mass spectrometer was operated in the turbo ion spray mode with positive ion detection. The turbo ion spray temperature was maintained at 350°C, and a voltage of 4.8 kV was applied to the sprayer needle. Nitrogen was used as the turbo ion spray and nebulizing gas. The detection and quantification of free and conjugated BAs and the internal standard were accomplished by multiple reaction monitoring with the transition m/z 403.3/367.4 for dehydrocholic acid, 359.1/135.0 for lithocholic acid, 375.1/357.2 for free DCA, CDCA, hyodeoxycholic acid, UDCA, and murideoxycholic acid, 373.1/355.3 for free CA, hyocholic acid, and - and -muricholic acid (MCA), and 391.1/355.2 for ß-MCA. All raw data were processed using Analyst software. Calibration curves were linear for free DCA, CDCA, hyodeoxycholic acid, UDCA, and murideoxycholic acid concentrations ranging from 0.05 to 10 µM and for free CA, hyocholic acid, and -, ß-, and -MCA concentrations ranging from 0.2 to 20 µM.

Western blot analysis
Frozen livers were homogenized in a lysis buffer (9 M urea, 2% Triton X-100, 70 mM dithiothreitol, 10 µg/ml aprotinin, 10 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and allowed to sit on ice for 30 min. The homogenate was centrifuged at 12,000 g for 30 min at 4°C, and the supernatants were used as whole cell lysates. Total protein (100 µg), determined by the Bio-Rad assay, was diluted with 3x Laemmli sample buffer, incubated at 65°C for 15 min, fractionated by 10% SDS-PAGE, and blotted onto a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was incubated for 1 h with PBS containing 0.1% Tween 20, 5% dry milk, 6% glycine, and 3% fetal bovine serum and then for 1 h with a 1:1,000 dilution of primary antibodies against mouse CYP7A1 (a generous gift from Dr. David W. Russell, University of Texas Southwestern Medical Center) and ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA). After washing, the membrane was incubated for 1 h with a 1:10,000-diluted horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology), and the reaction product was visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

Determination of CYP8B1 enzyme activity
CYP8B1 enzyme activity was determined by a modification of a previous report (28). 7-Hydroxy-4-cholestene-3-one (Steraloids, Newport, RI) and 7,12-hydroxy-4-cholestene-3-one (a gift from Dr. Mizuho Une, Hiroshima University) were used as the substrate and its metabolite, respectively. Livers from CYP8B1-null and wild-type mice were used as negative and positive controls, respectively (29). Incubation reactions were carried out in 100 mM potassium phosphate (pH 7.4) containing 0.5 mg/ml microsomal protein, 2 mM NADPH, and 100 µM 7-hydroxy-4-cholestene-3-one at 37°C for 10 min with shaking. The reaction was terminated by the addition of 2 ml of ethyl acetate. 6ß-Hydroxytestosterone was used as an internal standard. The reaction mixture described above was centrifuged at 1,200 g for 5 min, and the upper layer was collected and dried at 40°C under nitrogen gas. The extracted residue was dissolved in 100 µl of methanol, and 10 µl was injected into an LC-MS/MS system for 7,12-hydroxy-4-cholestene-3-one detection. The LC-MS/MS system included a PE SCIEX API2000 ESI triple-quadrupole mass spectrometer (Perkin-Elmer) and a Synergi 4 µm MAX-RP, 50 x 2 mm inner diameter column. The flow rate was 0.2 ml/min with 70% methanol and 30% water containing 0.1% formic acid. The mass spectrometer was operated in the turbo ion spray mode with positive ion detection. The turbo ion spray temperature was maintained at 350°C, and a voltage of 5 kV was applied to the sprayer needle. Nitrogen was used as the turbo ion spray and nebulizing gas. The detection and quantitation of 7,12-hydroxy-4-cholestene-3-one and 6ß-hydroxytestosterone were accomplished by selected ion monitoring with the transition m/z 439.1 for 7,12-hydroxy-4-cholestene-3-one and 327.2 for 6ß-hydroxytestosterone. All raw data were processed using Analyst software. The detection limit for 7,12-hydroxy-4-cholestene-3-one is 0.5 pmol. The sensitivity of the CYP8B1 assay is 0.1 pmol/min/mg protein.

Construction of the mouse CYP8B1-luciferase reporter plasmids
The –707, –497, –235, –173, and –104/–1 fragments from the translation start site of the mouse Cyp8b1 promoter (30) containing BglII and KpnI sites were amplified by PCR and cloned into the luciferase reporter vector, pGL3/basic (Promega, Madison, WI). Mutations were introduced into the putative HNF4 binding site in the Cyp8b1-luciferase constructs by PCR-based site-directed mutagenesis using the following complementary primer pair: 5'-ccgagcctctgagcaCTGTCCAAGGGCAggaacct-3' and 5'-aggttccTGCCCTTGGACAGtgctcagaggctcgg-3' (Mut; mutations in the HNF4 binding site are underlined). Plasmid DNA sequences were verified with a CEQ 2000XL DNA Analysis System (Beckman Coulter).

Transient transfection assay
HepG2 and CV-1 cell lines were cultured at 37°C in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (HyClone, Logan, UT) and 100 U/ml penicillin/streptomycin (Invitrogen). Cells were seeded on 24-well tissue culture plates and grown to 90–95% confluence. Transfections were carried out as described previously (9). All transfections were performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. After 48 h, the cells were washed with phosphate-buffered saline and assayed for dual luciferase activity using commercial kits according to the manufacturer's instructions (Promega).

Gel shift analysis
Crude nuclear extracts were prepared and gel shift analysis was carried out as described previously (9). The following three double-stranded probes were used: HNF4 binding site for the mouse Cyp8b1 promoter (5'-CTGAGCAAAGTCCAAGGGCAGGAACCT-3' and 5'-AGGTTCCTGCCCTTGGACTTTGCTCAG-3'), consensus HNF4 binding site, and consensus PPAR response element (Santa Cruz Biotechnology). End-labeled double-stranded oligonucleotide (2 x 10⁵ cpm) was added, and the reaction mixture was incubated at room temperature for 30 min. For competition experiments, a 25-fold excess of unlabeled oligonucleotide was added to the reaction mixture and the mixture was incubated at room temperature for 20 min before the addition of a ³²P-labeled oligonucleotide probe. For supershift analysis, 1 µg of anti-HNF4, PPAR, and retinoid X receptor (RXR) antibody (Santa Cruz Biotechnology) was added to the reaction mixture, and the mixture was incubated at room temperature for 30 min after the addition of a ³²P-labeled oligonucleotide probe.

Statistical analysis
All values are expressed as means ± SEM or SD. All data were analyzed using the unpaired Student's t-test for significant differences between the mean values of each group.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Disruption of BA homeostasis in HNF4^L mice
HNF4^L mice showed age-dependent increases of serum BA levels; serum BAs in HNF4^L mice were increased as early as postnatal day 14 and were increased dramatically by 45 days of age compared with those of HNF4^F/F mice (Fig. 1A). CA, DCA, CDCA, -, ß-, and -MCA, and UDCA were markedly increased, ranging from 10- to 60-fold compared with HNF4^F/F mice, in 45-day-old HNF4^L mice (Table 1). Additionally, a large number of unidentified BAs were also observed in the serum of HNF4^L mice (data not shown). The expression of HNF4 was reduced 50% in the livers of HNF4^L mice on day 14 and was not detected in 28-day-old HNF4^L mice (Fig. 2), showing that the high serum BA levels in HNF4^L mice were correlated with the loss of HNF4 expression.

View larger version (25K): [in this window] [in a new window]   Fig. 1. Bile acid (BA) homeostasis in liver-specific hepatocyte nuclear factor 4-null (HNF4L) and hepatocyte nuclear factor 4-floxed (HNF4F/F) mice. A: Serum total BA levels in 14-, 28-, and 45-day-old mice. B: Total volume of biofluid in gallbladder. C: Total BA amount in gallbladder. D: Total BA pool. E, F: Fecal (E) and urinary (F) BA excretion rates. For B–D, all mice were 45 days old. Data are means ± SEM (HNF4F/F mice, n = 4–6; HNF4L mice, n = 4–8). Significant differences compared with HNF4F/F mice: * P < 0.05, ** P < 0.01, *** P < 0.001.

View this table: [in this window] [in a new window]   TABLE 1. Analysis of free bile acids in serum of HNF4L and HNF4F/F mice

View larger version (117K): [in this window] [in a new window]   Fig. 2. Expression of the genes involved in BA biosynthesis pathways. A: Northern blot analysis of liver RNA from 14, 28, and 45 day old mice euthanized at 10 AM. B–E: Real-time PCR of liver mRNA from 45 day old mice euthanized at 10 AM for sterol 27-hydroxylase (CYP27A1; B), CYP39A1 (C), acyl-coenzyme A oxidase 2 (ACOX2; D), and D-type peroxisomal bifunctional enzyme (D-PBE; E). Data are means ± SEM (n = 8 for each group). Significant difference compared with HNF4F/F mice: * P < 0.001.

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Expression of genes involved in BA biosynthesis in HNF4^L mice
To determine the differences in the expression of potential HNF4 target genes involved in BA biosynthesis, microarray analysis was carried out using liver RNA collected from mice killed at 10 AM (Table 2). A marked decrease in the expression of genes encoding CYP7B1, CYP8B1, OATP1, and NTCP was found in 45-day-old HNF4^L mice. Interestingly, there was no significant change in the expression of the Cyp7a1 gene, whereas expression of the apolipoprotein B gene was decreased and mRNAs encoding HMG-CoA synthase, scavenger receptor class B type I, PXR, RXR, and LCAT were increased in the HNF4^L mice. No increased expression of PXR and RXR mRNAs was observed, as revealed in earlier studies using Northern blot analysis (8). The differentially expressed genes between HNF4^L and HNF4^F/F mice by microarray analysis were further confirmed by Northern blot analysis and real-time PCR. By Northern analysis, the levels of CYP7A1 mRNA were not significantly different between HNF4^L and HNF4^F/F mice at the ages tested (Fig. 2A); however, serum levels of 7-hydroxycholesterol, which reflects the enzymatic activity of CYP7A1 (31), were significantly reduced in HNF4^L mice (10 ± 1 ng/ml) compared with HNF4^F/F mice (17 ± 1 ng/ml). CYP27A1 and CYP7B1 mRNA levels were reduced significantly in the livers of HNF4^L mice (Fig. 2A, B). However the serum levels of 27-hydroxycholesterol, the product of CYP27A1 and the substrate for CYP7B1 (32), were >2-fold higher in HNF4^L mice than in HNF4^F/F mice (118 vs. 49 ng/ml). This was also the case with serum levels of 25-hydroxycholesterol (17 vs. 6 ng/ml), a second substrate for CYP7B1 (32, 33). The levels of CYP8B1 mRNA were also markedly reduced in HNF4^L mice (Fig. 2A). The expression of another CYP7B1 (CYP39A1), with specificity for 24-hydroxycholesterol as a substrate (34), was much higher in adult HNF4^L mice (Fig. 2A, C).

View this table: [in this window] [in a new window]   TABLE 2. Gene expression changes in HNF4L mice-I

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View this table: [in this window] [in a new window]   TABLE 3. Gene expression changes in HNF4L mice-II

Expression of CYP7A1 is not induced during the dark cycle in HNF4^L mice

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View larger version (59K): [in this window] [in a new window]   Fig. 3. Circadian rhythm of the expression of cholesterol 7-hydroxylase (CYP7A1) and sterol 12-hydroxylase (CYP8B1) mRNAs in the livers of HNF4L and HNF4F/F mice. A, B: Northern blot analysis of mRNA in livers from 45-day-old mice at 10 AM (A) and 10 PM (B). C: Real-time PCR of liver mRNA for CYP7A1 at 10 AM (left) and 10 PM (right). Data are means ± SEM (n = 8 for each group). Significant differences compared with the other three groups: * P < 0.01. D, E: Western blots of total liver proteins (100 µg) at 10 AM (D) and 10 PM (E). CYP7A1 and ß-actin polyclonal antibodies were used to assess protein expression.

BA composition in HNF4^L and HNF4^F/F mice

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View larger version (38K): [in this window] [in a new window]   Fig. 4. Composition of BA in HNF4L and HNF4F/F mice. Amounts of individual BAs from 45-day-old mice were analyzed by GC-MS and are indicated as percentages of the entire pool. The position and stereochemistry of hydroxyl groups on the ring structure of each BA are indicated in parentheses.

Hepatic CYP8B1 enzyme activity is reduced in HNF4^L mice

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View larger version (13K): [in this window] [in a new window]   Fig. 5. Specific activities and expression of CYP8B1 in HNF4L and HNF4F/F mice. A: Specific activities of CYP8B1 were measured using liver microsomal protein from HNF4L and HNF4F/F mice. Data are means ± SEM (n = 5). Significant differences between HNF4L and HNF4F/F mice: * P < 0.001 and ** P < 0.005, respectively. B: Specific activities of CYP8B1 were measured using liver microsomal protein from Cyp8b1-null and wild-type (WT) mice. Data are means ± SEM (n = 2).

HNF4 directly binds to the promoter region of the mouse Cyp8b1 and positively regulates its expression

View larger version (59K): [in this window] [in a new window]   Fig. 6. Promoter analysis of the mouse Cyp8b1 gene. A: Luciferase reporter plasmids containing the mouse Cyp8b1 promoter were transfected into HepG2 cells. The normalized activity ± SEM (n = 4) of each construct is presented as arbitrary units. B: CV-1 cells were cotransfected with the HNF4 expression vector, as indicated. The normalized activity ± SEM (n = 4) of each construct is presented as arbitrary units. C: Nuclear extracts from liver of HNF4F/F mice (left panel) and HNF4L mice (right panel) were incubated with a labeled HNF4 binding derived from the mouse Cyp8b1 promoter in the absence (lanes 1 and 8) or presence of each unlabeled oligonucleotide for the HNF4 binding site of the mouse Cyp8b1 promoter (lanes 2 and 9), consensus HNF4 binding site (lanes 3 and 10), and consensus peroxisome proliferator-activated receptor (PPAR)-responsive element (lanes 4 and 11). For the supershift assay, nuclear extracts were incubated with labeled oligonucleotide probe in the presence of anti-HNF4 (lanes 5 and 12), anti-PPAR (lanes 6 and 13), and anti-retinoid X receptor (RXR) antibodies (lanes 7 and 14). The HNF4-DNA complex and its supershifted complex, caused by the HNF4-specific antibody, are indicated by the lower and upper arrows, respectively.

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To determine whether disruption of the HNF4 binding site in the Cyp8b1 gene affects promoter activity, mutations were introduced into the site in the (–330)-luciferase promoter construct (Fig. 7A, Mut). As shown in Fig. 7B, when HepG2 cells were used for transient transfections, promoter activity of the mutated –330 bp fragments (–330/Mut) was reduced to background levels compared with that of the wild-type promoter (–330/WT). Similarly, when CV-1 cells were used, the promoter activity of the mutated fragment was not induced by cotransfection of HNF4 expression vector (Fig. 7C). Together, the results shown in Figs. 6 and 7 suggest that HNF4 directly regulates the expression of the Cyp8b1 gene.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Effects of mutations of the HNF4 binding site in the mouse Cyp8b1 promoter. A: Scheme of the wild-type (WT) and mutated (Mut) HNF4 binding site of the mouse Cyp8b1 promoter. Mutations in the HNF4 binding site are represented in boldface type and underlined. B, C: Plasmids were transfected into HepG2 (B) and CV-1 (C) cells, and the normalized activity ± SEM (n = 4) of each construct is presented as arbitrary units.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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L

Studies with the HNF4^L mice indicate that the Cyp27a1 gene is directly regulated by HNF4 in vivo. This is in agreement with a recent report showing that HNF4 can bind to and transactivate the promoter region of the human Cyp27a1 gene (44, 45). Thus, both mouse Cyp27a1 and human Cyp27a1 are direct targets for HNF4. Interestingly, the levels of 27-hydroxycholesterol were significantly higher in HNF4^L mice than in HNF4^F/F mice. The circulating levels of this oxysterol are regulated by the relative activities of CYP27A1 and CYP7B1. Both Cyp27a1 and Cyp7b1 were downregulated in HNF4^L mice, but the downregulation of Cyp7b1 was almost complete. Therefore, it seems likely that the increased levels of 27-hydroxycholesterol in HNF4^L mice reflect a markedly reduced metabolism of oxysterols. This finding was also observed in the Cyp7b1-null mice that have higher serum levels of both 25- and 27-hydroxycholesterol (46).

HNF4 binds to the promoter region of human Cyp8b1 and activates transcription, as revealed by transactivation transcription assays (47). Other studies have suggested that BAs inhibit the expression of the rat Cyp8b1 by inducing CPF/LRH-1 and inhibiting HNF4 expression (48). However, repression of Cyp8b1 in HNF4^L mice might not be attributable to this negative feedback mechanism by BAs because CPF/LRH-1 is not induced in HNF4^L mice (8). The work described here indicates that HNF4 positively regulates the basal expression of Cyp8b1 in vivo; thus, lower HNF4 that results from high BA levels could potentially cause a decrease in Cyp8b1 transcription. It should also be noted that CYP8B1 is needed to synthesize CA, and Cyp8b1-null mice lack synthesis of CA and its metabolites (29). However, surprisingly, CA-derived metabolites (CA and DCA) in HNF4^L mice were not decreased compared with other BAs in the absence of CYP8B1 mRNA, and the concentration and/or total amount of CA in serum and gallbladder bile were increased along with other BAs. Furthermore, the enzyme activity of hepatic CYP8B1 was also markedly decreased, and no measurable levels of expression of CYP8B1 mRNA were detected in other tissues examined. We currently have no explanation for this discrepancy between CYP8B1 expression and CA levels.

The expression of CYP7B1 mRNA was almost extinguished in the HNF4^L mice. However, a functional HNF4 binding site could not be located in the Cyp7b1 promoter region. Because expression of the human Cyp7b1 gene is regulated by Sp1 (49), the mouse Cyp7b1 gene might also be regulated by Sp1. Another mechanism involving Sp1 could also contribute to the loss of Cyp7b1 gene expression in the HNF4^L mice. BA, found at high levels in HNF4^L mice, induces hepatic cytokine expression by activation of hepatic macrophage-like Kupffer cells (50). Tumor necrosis factor- produced by these cells represses growth hormone receptor expression by inhibiting Sp1 and Sp3 binding to their corresponding sites in the promoter (51). Thus, hepatic production of tumor necrosis factor- and other cytokines might be increased in HNF4^L mice, resulting in repression of the DNA binding activity of Sp1 family proteins to the Cyp7b1 promoter. As discussed above, the expression of the major genes involved in BA biosynthesis is reduced in HNF4^L mice, including that encoding Scpx. This gene also does not contain a functional HNF4 site within proximity to its transcription start site, but it does contain GC-rich sequences that have potential Sp1 binding (data not shown). Thus, a mechanism similar to that described for the Cyp7b1 gene may have a role in the loss of Scpx gene expression in HNF4^L mice. Finally, the presence of an HNF4 binding site at a remote distance from the Cyp7b1 and Scpx promoters cannot be ruled out.

Because side chain ß-oxidation of bile alcohols is catalyzed in peroxisomes, a deficiency in this pathway has been described as a secondary feature of various peroxisome disorders (52). The gene encoding SCPx also encodes SCP2, which contains the C-terminal sequence of SCPx (53), but the expression of SCP2 is unchanged (data not shown) because of the use of different promoters (32). Scp2/Scpx-null mice exhibit decreased catabolism of branched fatty acyl-CoA (54) and loss of side chain ß-oxidation of bile alcohol at the thiolytic step (55), indicating that SCPx is indeed critical for side chain ß-oxidation of bile alcohols. Scp2/Scpx-null mice also accumulate intermediate products of BA biosynthesis (55). These intermediate products might accumulate in HNF4^L mice as a result of the loss of SCPx expression, thus accounting for the additional unidentified peaks detected by LC-MS/MS in these mice.

After BA biosynthesis by hydroxylation of cholesterol and side chain ß-oxidation, BAs are conjugated with taurine to produce bile salts in mice. The two main enzymes involved in BA conjugation are VLACSR and BA CoA:amino acid N-acyltransferase in mice. The expression of these genes was reduced in HNF4^L mice and dependent on HNF4 binding sites and HNF4 expression (11). Thus, HNF4 regulates all steps of BA biosynthesis, transport, and conjugation.

In conclusion, characterization of liver-specific HNF4-null mice provided evidence for a critical role for hepatic HNF4 in the maintenance of BA homeostasis. Because other genes are also downregulated in the livers of HNF4^L mice in a mechanism that does not appear to involve HNF4, further investigation of these genes might be useful to understand the regulation of the complete pathway of BA biosynthesis and transport and possibly to establish novel therapeutic targets for diseases resulting from altered BA homeostasis.

Liver samples from Cyp8b1-null and control mice and antibody against mouse CYP7A1 were kindly provided by Dr. David W. Russell (University of Texas Southwestern Medical Center, Dallas, TX). The authors thank Anita Lövgren-Sandblom and Maria Olin for their technical assistance.

	ACKNOWLEDGMENTS

 
This work was partly supported by the Swedish Science Council and by the National Cancer Institute Intramural Research Program of the National Institutes of Health.

Manuscript received September 29, 2005 and in revised form October 26, 2005.

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