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Originally published In Press as doi:10.1074/jbc.M311015200 on October 28, 2003

J. Biol. Chem., Vol. 279, Issue 4, 2480-2489, January 23, 2004
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Hepatocyte Nuclear Factor 4{alpha} Is a Central Regulator of Bile Acid Conjugation*

Yusuke Inoue, Ai-Ming Yu, Junko Inoue, and Frank J. Gonzalez{ddagger}

From the Laboratory of Metabolism, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, October 7, 2003 , and in revised form, October 24, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) has an important role in regulating the expression of liver-specific genes. Because bile acids are produced from cholesterol in liver and many enzymes involved in their biosynthesis are preferentially expressed in liver, the role of HNF4{alpha} in the regulation of bile acid production was examined. In mice, unconjugated bile acids are conjugated with taurine by the liver-specific enzymes, bile acid-CoA ligase and bile acid-CoA:amino acid N-acyltransferase (BAT). Mice lacking hepatic HNF4{alpha} expression exhibited markedly decreased expression of the very long chain acyl-CoA synthase-related gene (VLACSR), a mouse candidate for bile acid-CoA ligase, and BAT. This was associated with markedly elevated levels of unconjugated and glycine-conjugated bile acids in gallbladder. HNF4{alpha} was found to bind directly to the mouse VLACSR and BAT gene promoters, and the promoter activities were dependent on HNF4{alpha}-binding sites and HNF4{alpha} expression. In conclusion, HNF4{alpha} plays a central role in bile acid conjugation by direct regulation of VLACSR and BAT in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bile acids (BAs)1 are synthesized from cholesterol mainly in hepatocytes by a cascade of enzyme reactions. In humans, these reactions are initiated by steroid nucleus and side chain hydroxylation of cholesterol and followed by {beta}-oxidation of the side chain and resulting in the production of the primary BAs cholic acid and chenodeoxycholic acid (1, 2). In mice, cholic acid and {beta}-murichoric acid are the predominant primary BAs (3). Following BA biosynthesis, BAs are conjugated with glycine or taurine resulting in conjugates that are exported from hepatocytes via hepatic BA transporters such as the bile salt export pump located at the apical or canalicular membrane (1, 4–6). These BAs are stored in gallbladder and then transported into the small intestine. BAs are biological detergents that serve to solubilize fats, sterols, and fat-soluble vitamins in the intestine. Most BAs are recycled from the jejunum and ileum by transporters, such as apical sodium-dependent bile acid transporter (4, 7), and then transported back into hepatocytes from the portal circulation by transporters including sodium taurocholate cotransporter polypeptide (NTCP) and organic anion transporter polypeptide 1 (OATP1) located at the basolateral membrane (4).

Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is an orphan member of the nuclear receptor hormone superfamily that is mainly expressed in liver, intestine, kidney, and pancreas and regulates expression of target genes including several serum proteins such as apolipoproteins, blood coagulation factors, P450s, and enzymes involved in glucose, lipid, ammonia, steroid, and fatty acid metabolism by binding to direct repeat-1 elements in their promoter or enhancer regions (8, 9). Indeed, the expression of hepatic apolipoproteins, including A-II, A-IV, C-II, and C-III, ornithine transcarbamylase involved in ureagenesis, and CYP3A associated with xenobiotic metabolism, was significantly reduced in livers of liver-specific HNF4{alpha}-null mice (10–12). A second line of liver-specific HNF4{alpha}-null mice was established where Cre recombinase was placed under the control of the albumin promoter and the {alpha}-fetoprotein enhancer (13). These mice exhibited a marked reduction in the expression of hepatic glycogen synthase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase, involved in gluconeogenesis, and reduced levels of E-cadherin, ZO-1, and CEACAM1 proteins that are critical for normal liver morphology (13).

Liver-specific HNF4{alpha}-null mice in the adult exhibited increased serum BAs, and expression of the BA transporters, NTCP and OATP1, was also reduced in these mice (10). Whereas BA biosynthesis and transport are controlled to some degree by HNF4{alpha}, it remains unknown whether HNF4{alpha} regulates the BA conjugation pathway. Nuclear receptors are involved in the control of the BA biosynthesis and transport system (6, 14, 15), but the mechanism regulating BA conjugation pathways have not been analyzed. BA conjugation reactions are catalyzed by two liver-enriched enzymes, bile acid-CoA ligase (BAL) and bile acid-CoA:amino acid N-acyltransferase (BAT) (1, 14). BAL activities are associated with rat BAL protein (16) and human very long chain acyl-CoA synthase homolog 2 (VLACSR-H2) protein (17), but enzymatic analysis of BAL protein has not been performed in the mouse. Because the mouse very long chain acyl-CoA synthase-related (VLACSR) gene, which is highly expressed only in liver (18) as well as rat BAL and human VLACSR-H2 genes, has a high homology to these proteins (16), it was reasonable to assume that the VLACSR gene might be the mouse homolog for the BAL gene. On the other hand, BAT genes have been cloned from mouse (19), rat (20), and human (21). The human BAT protein has conjugation activity with both taurine and glycine as substrates (21), but the mouse BAT protein only uses taurine as a substrate (19).

In the present study, the biological function of HNF4{alpha} was investigated in vivo and in vitro using liver-specific HNF4{alpha}-null mice. These mice exhibit increased unconjugated and glycine-conjugated BAs in gallbladder bile and decreased expression of VLACSR and BAT. Expression of the VLACSR and BAT was shown to be regulated directly by HNF4{alpha} via HNF4{alpha}-binding sites in their promoter regions. These results indicate that HNF4{alpha} has an important role in the maintenance of BA homeostasis by regulating BA conjugation pathways.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatment—Liver-specific HNF4{alpha}-null mice were generated as described previously (10). All experiments were performed with 45-day-old HNF4{alpha}flox/flox x albumin-Cre+ (KO) and HNF4{alpha}flox/flox x albumin-Cre– (FLOX) mice. Mice were housed in a pathogen-free animal facility under standard 12-h light/12-h dark cycle with water and chow ad libitum. All experiments with mice were performed under Association for Assessment and Accreditation of Laboratory Animal Care guidelines with the approval of the NCI Animal Care and Use Committee.

Northern Blot Analysis—Total liver RNA (10 µg), extracted with Trizol reagent (Invitrogen), was fractionated by electrophoresis through a formaldehyde-agarose gel and transferred to GeneScreen Plus membranes (DuPont). Blots were hybridized at 68 °C in PerfectHyb plus solution (Sigma) with random primer-labeled cDNA probes and exposed to a PhosphorImager screen cassette followed by visualization using a Storm 860 PhosphorImager system (Amersham Biosciences). All probes were amplified from a mouse cDNA library using gene-specific primers and cloned into the pCR II vector (Invitrogen). Sequences were verified using CEQ 2000 Dye Terminator cycle sequencing kits (Beckman Coulter, Fullerton, CA) and a CEQ 2000XL DNA Analysis System (Beckman Coulter).

Extraction of Bile Acids from Bile—Three µl of bile or a 1/100 dilution (in methanol) was added into 200 µl of acetonitrile containing 20 µl of 50 µM dehydrocholic acid. The sample was vortexed for 10 s and then subsequently centrifuged at 14,000 x 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 x g at 4 °C for 15 min, the organic phase was evaporated under a stream of nitrogen gas. The residue was reconstituted in 60 µl of a 50% methanol solution containing 0.2% formic acid. Ten µl of each reconstituted sample was injected for liquid chromatography tandem mass spectrometry/mass spectrometry (LC-MS/MS) analysis.

Identification and Quantitation of Intact Free and Conjugated Bile Acids by LC-MS/MS—LC-MS/MS analysis was performed on a PE SCIEX API2000 ESI triple-quadrupole mass spectrometer (PerkinElmer Life Sciences) controlled by Analyst software. A Phenomenex Luna C18 3-µm 100 x 2 mm inner diameter column (Torrance, CA) was used to separate free bile acids. 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%) for 10 min at room temperature. Separation of glycine- and taurine-conjugated bile acids was performed on an Aquasil C18 5-µm 50 x 2 mm inner diameter column (Keystone Scientific Operations, Bellefonte, PA), and the high pressure liquid chromatography system was operated isocratically at a flow rate of 0.2 ml/min with the mobile phase (acetonitrile/water/methanol = 30/45/25, v/v/v) 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 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 bile acids and the internal standard were accomplished by multiple reactions monitoring with the transitions m/z 403.3/367.4 for dehydrocholic acid; 359.1/135.0 for lithocholic acid; 375.1/357.2 for deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, ursodeoxycholic acid, and murideoxycholic acid; 373.1/355.3 for cholic acid, hyocholic acid, {alpha}- and {omega}-muricholic acid (MCA); 391.1/355.2 for {beta}-MCA; 466.4/412.3 for glycocholic acid; 450.4/414.4 for glycodeoxycholic acid (GDCA) and glycochenodeoxycholic acid (GCDCA), 516.3/462.4 for taurocholic acid (TCA) and tauro-{beta}-muricholic acid (T-{beta}-MCA); and 500.4/464.4 for taurodeoxycholic acid (TDCA) and taurochenodeoxycholic (TCDCA). All raw data were processed using Analyst software. Calibration curves were linear for free deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, ursodeoxycholic acid, and murideoxycholic acid concentrations ranging from 0.05 to 10 µM; free cholic acid, hyocholic acid {alpha}-, {beta}-, and {omega}-MCA from 0.2 to 20 µM, GDCA and GCDCA from 0.1 to 10 µM; TDCA and TCDCA from 1.0 to 200 µM; and glycocholic acid, TCA, and T-{beta}-MCA from 2.5 to 500 µM.

Determination of the Transcription Start Sites of the Mouse VLACSR and BAT Genes—5'-Rapid amplification of cDNA ends was performed to determine the transcription start site of the mouse VLACSR and BAT genes using GeneRacer kit (Invitrogen). PCR products were cloned into pCR TOPO II, and 20 individual clones were sequenced. Based on these results, the major transcription start site was determined.

Construction of the Mouse VLACSR and BAT-Luciferase Reporter Plasmids—The mouse VLACSR (–2219, –1435, –1014, –256, –220, –194, and –146/–47 bp from translation start site) and BAT (–1972, –1567, –1447, –1322, –602, –505, –136, and –52/+178 bp from transcription start site) promoters were amplified by PCR using sequence data derived from the Celera data base (Celera Genomics, Rockville, MD) and cloned into KpnI and XhoI sites of the pGL3/basic vector (Promega, Madison, WI). Mutations were introduced into the putative HNF4{alpha}-binding site in the VLACSR-luciferase constructs by PCR-based site-directed mutagenesis using the following primer pair: 5'-acaaaCTGACAGAGTCCAtgggt-3' and 5'-acccaTGGACTCTGTCAGtttgt-3' (M2; mutations in the HNF4{alpha}-binding site are boldface and underlined). Similarly, mutations were introduced into an Sp1-binding site in the VLACSR-luciferase constructs by PCR-based site-directed mutagenesis using the following primer pair: 5'-tgtccaagTAAAGAAGTGag-3' and 5'-ctCACTTCTTTActtggaca-3' (mutations in the GC box are boldface and underlined). Similarly, mutations were introduced into an HNF4{alpha}-binding site in the BAT-luciferase constructs by PCR-based site-directed mutagenesis using the following primer pair: 5'-agtccaCTTTCCAAGGTCTtagtct-3' and 5'-agactaAGACCTTGGAAAGtggact-3' (mutations in the HNF4{alpha}-binding site are boldface and underlined). Sequences were verified using 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 units/ml penicillin/streptomycin (Invitrogen). Cells were seeded in 24-well tissue culture plates and grown to 90–95% confluency. For transfections, 500 ng/well pGL3 basic or the wild-type or mutated BAL and BAT promoters and 200 ng/well pRL/TK (Promega) were used with the total amount of DNA adjusted to 900 ng by the addition of pUC19 as a carrier DNA. For cotransfections, 200 ng/well of the rat HNF4{alpha} expression plasmid, pSG5/HNF4{alpha}, was used. 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 (Promega).

Gel Shift Analysis—Crude nuclear extracts were prepared, and gel shift analysis was carried out as described previously (11). The following three double-stranded probes were used: HNF4{alpha}-binding site for the mouse VLACSR promoter (wild-type), 5'-acaaaAGGACAGAGTCCAtgggt-3' and 5'-acccaTGGACTCTGTCCTtttgt-3', the mouse VLACSR promoter (mutant), 5'-acaaaCTGACAGAGTCCAtgggt-3' and 5'-acccaTGGACTCTGTCAGtttgt-3' (mutations in the HNF4{alpha}-binding site are boldface and underlined); GC box for the mouse VLACSR promoter (wild-type), 5'-tgtccaagGGGGCGGGGCag-3' and 5'-ctGCCCCGCCCCcttggacaC-3', HNF4{alpha}-binding site for the mouse VLACSR promoter (mutant), 5'-tgtccaagTAAAGAAGTGag-3' and 5'-ctCACTTCTTTActtggaca-3' (mutations in the GC box are boldface and underlined); the mouse BAT promoter (wild-type), 5'-agtccaAGTTCCAAGGTCTtagtct-3' and 5'-agactaAGACCTTGGAACTtggact-3', and the mouse BAT promoter (mutant), 5'-agtccaCTTTCCAAGGTCTtagtct-3' and 5'-agactaAGACCTTGGAAAGtggact-3' (mutations in the HNF4{alpha}-binding site are boldface and underlined). 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 prior to the addition of a 32P-labeled oligonucleotide probe. For supershift analysis, 1 µg of anti-HNF4{alpha} 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 the means ± S.E. (Figs. 3, 4, 6, and 7) or standard deviation (S.D., Tables I and II). All data were analyzed by the unpaired Student's t test for significant differences between the mean values of each group.



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  		FIG. 3.
Promoter analysis of the mouse VLACSR gene. A, luciferase reporter plasmids containing the mouse VLACSR promoter (–146, –194, –220, –256, –1014, –1435, and –2219/–47 bp from translation start site) were transfected into HepG2 cells. The normalized activity ± S.E. (n = 4) of each construct is presented as arbitrary units. B, CV-1 cells were cotransfected with the HNF4{alpha} expression vector, as indicated. The normalized activity ± S.E. (n = 4) of each construct is presented as arbitrary units. C, nuclear extracts (2 µg) from liver of H4Flox (left panel) and H4LivKO (right panel) mice were incubated with the labeled HNF4{alpha}-binding site oligonucleotide of the mouse VLACSR promoter in the absence (lanes 1 and 6) or presence of a 25-fold excess of each unlabeled oligonucleotide (lanes 2 and 7 for the wild-type of the HNF4{alpha}-binding site of the mouse VLACSR promoter, and lanes 3 and 8 for the mutated site). Similarly, the labeled mutated HNF4{alpha}-binding site oligonucleotide of the mouse VLACSR promoter was incubated with nuclear extracts from H4FLOX (lane 5) and H4LivKO (lane 10) mice. For the supershift assays, nuclear extracts were incubated with labeled probe in the presence of the anti-HNF4{alpha} antibody (lanes 4 and 9). HNF4{alpha}-DNA complex and the supershifted complex, caused by the HNF4{alpha}-specific antibody, are shown by the lower and upper arrow, respectively.

 



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  		FIG. 4.
Effects of mutations of the HNF4{alpha}-binding site and GC box in the mouse VLACSR promoter. A, schematic representation of the wild-type (WT) and mutated (Mut) HNF4{alpha}-binding site and GC box of the mouse VLACSR promoter. Mutations in the HNF4{alpha}-binding site and GC box are represented in boldface type. Plasmids were transfected into HepG2 (B) and CV-1 (C) cells, and the normalized activity ± S.E. (n = 4) of each construct is presented as relative activity.

 



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  		FIG. 6.
Promoter analysis of the mouse BAT gene. A, luciferase reporter plasmids containing the mouse BAT promoter were transfected into HepG2 cells. The normalized activity ± S.E. (n = 4) of each construct is presented as arbitrary units. B, CV-1 cells were cotransfected with the HNF4{alpha} expression vector, as indicated. The normalized activity ± S.E. (n = 4) of each construct is presented as arbitrary units. C, nuclear extracts from liver of H4Flox (left panel) and H4LivKO (right panel) mice were incubated with the labeled HNF4{alpha}-binding site oligonucleotide of the mouse BAT promoter in the absence (lanes 1 and 6) or presence of each unlabeled oligonucleotide (lanes 2 and 7 for the wild-type of the HNF4{alpha}-binding site of the mouse BAT promoter, and lanes 3 and 8 for the mutated site). Similarly, the labeled mutated HNF4{alpha}-binding site oligonucleotide of the mouse BAT promoter was incubated with nuclear extracts from H4Flox (lane 5) and H4LivKO (lane 10) mice. For the supershift assays, nuclear extracts were incubated with labeled probe in the presence of the anti-HNF4{alpha} (lanes 4 and 9). HNF4{alpha}-DNA complex and the supershifted complex, caused by the HNF4{alpha}-specific antibody, are shown by the lower and upper arrow, respectively.

 



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  		FIG. 7.
Effects of mutations of the HNF4{alpha}-binding site in the mouse BAT promoter. A, schematic representation of the wild-type (WT) and mutated (Mut) HNF4{alpha}-binding site of the mouse BAT promoter. Mutations in the HNF4{alpha}-binding site are represented in boldface type. Plasmids were transfected into HepG2 (B) and CV-1 (C) cells, and the normalized activity ± S.E. (n = 4) of each construct is presented as relative activity.

 


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  		TABLE I
Analysis of free bile acids in gallbladder bile of liver-specific HNF4{alpha}-null and control mice

Amounts of {alpha}- and {omega}-MCA were combined. Data are mean ± S.D. (FLOX (n = 7–8) and KO (n = 6–8)). N.D., not detectable. Significant differences compared with FLOX mice: *, p < 0.005; **, p < 0.001.

 


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  		TABLE II
Analysis of conjugated bile acids in gallbladder bile of liver-specific HNF4{alpha}-null and control mice

Data are mean ± S.D. (FLOX (n = 7–8) and KO (n = 6–8)). Significant differences compared with FLOX mice: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Genes Involved in BA Conjugation Is Reduced in Liver-specific HNF4a-null Mice—Free BAs are at first esterified with acetyl-CoA by BAL, and the esterified BAs are conjugated with glycine or taurine by BAT. The enzyme activity of BAL has not been analyzed in mouse, but the mouse VLACSR gene might be a candidate for a mouse BAL since VLACSR has high homology to both the rat BAL and human VLACSR-H2 that have BAL activities. Human BAT is able to conjugate both glycine and taurine (21), but mouse BAT has conjugation specificity toward taurine and not glycine (19). Northern blots were performed to determine whether the expression of VLACSR and BAT is significantly different between liver-specific HNF4{alpha}-null (HNF4{alpha}-floxed/floxed, albumin-Cre; H4LivKO) and control (HNF4{alpha}-floxed/floxed, without albumin-Cre; H4Flox) mice. Expression of VLACSR and BAT was markedly reduced in the livers of H4LivKO mice as compared with H4Flox mice (Fig. 1). It is also noteworthy that the expression of alanine:glyoxylate aminotransferase and taurine transporter, two enzymes that determine the levels of cytosolic glycine and taurine, were unchanged.



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  		FIG. 1.
Northern blot analysis of VLACSR and BAT. Total liver RNA was isolated, and 10 µg was subjected to electrophoresis on a 1% agarose gel, transferred to a nylon membrane, and hybridized with the indicated 32P-labeled cDNA probes. TAUT, taurine transporter; AGXT, alanine:glyoxylate aminotransferase.

 
Free BAs and Glycine-conjugated BAs Are Increased in Gallbladder Bile of Liver-specific HNF4{alpha}-null Mice—Because the expression of VLACSR and BAT was decreased in H4LivKO mice, it was expected that free BAs are increased and conjugation patterns are altered in H4LivKO mice. Most BAs in the H4Flox mice are also found as conjugated derivatives in gallbladder bile because only small amounts of free BAs were detected (Table I). However, free BAs were significantly increased in the gallbladders of H4LivKO mice (Table I), indicating that the conjugation enzymatic machinery of the BAs might be impaired by decreased expression of VLACSR in the livers of H4LivKO mice.

The conjugation pattern of BA in gallbladder bile was further determined by LC-MS/MS (Table II). In male H4Flox mice, taurocholic acid (TCA) and tauro-{beta}-muricholic acid (T-{beta}-MCA) were the predominant BAs. The concentrations of these BAs (Table II, upper) were decreased in H4LivKO mice, but the total amounts (Table II, lower) were almost unchanged (TCA) or increased (T-{beta}-MCA). Most mammals secrete BAs conjugated with taurine and/or glycine, but mouse gallbladder bile contains only the taurine-conjugated forms (18). However, small amounts of glycine-conjugated BAs including glycocholic acid, glycodeoxycholic acid (GDCA), and glycochenodeoxycholic acid (GCDCA) were detected in the H4Flox mice, and their levels were much lower as compared with their taurine-conjugated derivatives. In contrast to the results with control mice, taurodeoxycholic acid (TDCA), GDCA, taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA) were increased in H4LivKO mice with GDCA and GCDCA being markedly elevated. These results indicate that the conjugation pattern of BAs in H4LivKO mice is significantly different from H4Flox mice because of decreased expression of BAT. Because glycine-conjugated BAs are increased and taurine-conjugated BAs are maintained in H4LivKO mice, an unidentified BAT that can use both glycine and taurine as substrates might play an important role in H4LivKO mice.

Expression of the Mouse VLACSR Gene Is Directly Regulated by HNF4{alpha}—To investigate why expression of VLACSR was reduced in H4LivKO mice, the promoter region of the mouse VLACSR gene was cloned (Fig. 2). A transcription start site was determined by 5'-rapid amplification of cDNA ends, and five major sites were identified between –143 and –85 from translation start site (+1). A putative HNF4{alpha}-binding site and a GC box were found between –162 and –150 and –238 and –229, respectively (Fig. 2). To determine whether HNF4{alpha} has the potential to activate the mouse VLACSR promoter, several VLACSR promoter-luciferase reporter plasmids were constructed (Fig. 3A). When HepG2 cells, which express HNF4{alpha}, were used for transient transfections, the promoter activity of the –194-bp fragment containing a putative HNF4{alpha}-binding site was higher than that of the promoterless construct (pGL3/basic) and the –146-bp fragment (Fig. 3A). The promoter activities of the –220-bp fragment was higher than –194-bp fragment, indicating that the nucleotide sequence between –220 and –195 bp, which are GC-rich regions similar to the GC box between –238 and –229 bp, might be important for promoter activity. Furthermore, the promoter activities of the –256-, –1014-, –1435-, and –2219-bp fragments, which contain an HNF4{alpha}-binding site and GC-rich sequences, were much higher than that of –194- and –220-bp fragments. To determine whether HNF4{alpha} positively regulates the promoter activity, CV-1 cells, which do not express HNF4{alpha}, were used. The promoter activity of the –146-bp fragment was unchanged by cotransfection of the HNF4{alpha} expression vector, consistent with the absence of an HNF4{alpha}-binding site (Fig. 3B). However, the promoter activity of the –194-bp fragment was increased by cotransfection with an HNF4{alpha} expression vector. The same results were obtained using the longer fragments, indicating that HNF4{alpha} positively regulates expression of the VLACSR gene. Interestingly, the basal promoter activity of –220-bp fragment was increased in the absence of HNF4{alpha} expression, and the activity of –256-bp fragment was much higher. Because a GC-rich sequence was observed in the region between –256 and –195 bp, the Sp1 family proteins might be important in activating maximal promoter activity in the presence of HNF4{alpha}.



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  		FIG. 2.
Nucleotide sequence of the mouse VLACSR gene promoter. A, localization of HNF4{alpha}-binding site and GC box in the mouse VLACSR gene. B, numbering of nucleotides is relative to the translation start site (nucleotide +1, arrow). The asterisk marks the transcription start sites. The putative binding site for HNF4{alpha} at –162 and –150, and GC box at –238 and –229 are indicated by boldface and underlined, respectively.

 
To determine whether HNF4{alpha} can bind to the HNF4{alpha}-binding site in the mouse VLACSR promoter, gel shift analysis was performed (Fig. 3C). Liver nuclear extracts from H4Flox mice contained proteins that bound to the HNF4{alpha}-binding site (Fig. 3C, lane 1). Binding was diminished by the addition of excess amounts of unlabeled VLACSR probe (wild-type) but not by the mutated probe, indicating that a protein specifically bound to this site (Fig. 3C, lanes 2 and 3, the lower arrow). Furthermore, these bands were supershifted by the addition of anti-HNF4{alpha} antibody, indicating that the protein bound to this HNF4{alpha}-binding site was indeed HNF4{alpha} (Fig. 3C, lane 4, the upper arrow). However, supershifted bands were not detected using liver nuclear extracts from H4LivKO mice (Fig. 3C, lane 9) and no specific complex, indicated by the lower arrow, was detected using the labeled mutated probe (Fig. 3C, lanes 5 and 10). These results indicate that the HNF4{alpha}-binding site in the mouse VLACSR promoter is capable of binding HNF4{alpha}.

To determine whether disruption of the HNF4{alpha}-binding site and GC box decreases the promoter activity, mutations were introduced into the HNF4{alpha}-binding site and GC box in the mouse VLACSR (–1014/–47)-luciferase construct (Fig. 4A). As shown in Fig. 4B, when HepG2 cells were used for transient transfections, the promoter activity of the HNF4{alpha} mutant (–1014/HNF4{alpha} Mut) was decreased as compared with the wild-type construct (–1024/WT). The promoter activity of the GC box mutant (–1014/GC box Mut) was further decreased, and the double mutants for both the HNF4{alpha}-binding site and GC box (–1014/HNF4{alpha}/GC box Mut) had almost no promoter activity. When CV-1 cells were used for transient transfections, the promoter activity of the HNF4{alpha} mutant (–1014/HNF4{alpha} Mut) was decreased to 40% as compared with the wild-type fragment (–1014/WT) in the presence of HNF4{alpha} (Fig. 4C). The basal activity of the GC box mutant (–1014/GC box Mut) was decreased to the same level as the promoterless vector, but the activity was still increased by HNF4{alpha}. Furthermore, introduction of mutations into the binding sites of both factors (–1014/HNF4{alpha}/GC box Mut) caused a marked reduction of promoter activities even in the presence of HNF4{alpha}. These results indicate that the HNF4{alpha} and a GC box-binding protein can activate expression of the VLACSR gene, but both are needed for the maximal activation.

Expression of the Mouse BAT Gene Is Directly Regulated by HNF4{alpha}—In order to investigate why the expression of BAT was reduced in H4LivKO mice, the promoter region of the mouse BAT gene was cloned (Fig. 5B). An HNF4{alpha}-binding site was found between –68 and –56 from the transcription start site (Fig. 5, A and B). However, the promoter activity of the 3078-bp fragment between the beginning of intron 1 and translation start site was not induced in HepG2 and CV-1 cells by cotransfection with HNF4{alpha} (data not shown). To determine whether HNF4{alpha} has the potential to activate the mouse BAT promoter, several BAT promoter-luciferase reporter plasmids were constructed (Fig. 6A). When HepG2 cells were used for transient transfections, the promoter activity of the –136-bp fragment containing a putative HNF4{alpha}-binding site was higher than that of the promoterless construct (pGL3/basic) and the –52-bp fragment (Fig. 6A). The promoter activities of the longer fragments were much higher than that of the –136-bp fragment. To determine whether this effect was due to HNF4{alpha}, CV-1 cells were used. The promoter activity of the –52-bp fragment was unchanged by cotransfection of the HNF4{alpha}-expression vector, consistent with the absence of an HNF4{alpha}-binding site (Fig. 6B). However, the promoter activity of the –136-bp fragment was increased by HNF4{alpha}. The same results were obtained from experiments using the longer fragments.



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  		FIG. 5.
Nucleotide sequence of the mouse BAT gene promoter. A, localization of HNF4{alpha}-binding site in the mouse BAT gene. The asterisk marks the translation start site in exon 2. B, numbering of nucleotides is relative to the transcription start site (+1, arrow). The putative binding site for HNF4{alpha} at –68 and –56 is indicated by boldface and underlined.

 
To determine whether HNF4{alpha} can bind to the DR1-like elements in the mouse BAT promoter, gel shift analysis was performed (Fig. 6C). Liver nuclear extracts from H4Flox mice contained proteins that bound to the HNF4{alpha}-binding site (Fig. 6C, lane 1). Binding was diminished by the addition of excess amounts of unlabeled BAT probe (wild-type) but not by the mutated probe, indicating that a protein specifically bound to this site (Fig. 6C, lanes 2 and 3, the lower arrow). Furthermore, these bands were supershifted by the addition of anti-HNF4{alpha} antibody, indicating that the protein bound to this HNF4{alpha}-binding site was indeed HNF4{alpha} (Fig. 6C, lane 4, the upper arrow). Supershifted bands were not detected using liver nuclear extracts from H4LivKO mice (Fig. 6C, lane 9), and no specific complex, indicated by the lower arrow, was detected using the labeled mutated probe (Fig. 6C, lanes 5 and 10). These results indicate that the HNF4{alpha}-binding site in the mouse BAT promoter is capable of binding HNF4{alpha}.

To determine whether disruption of the HNF4{alpha}-binding site decreases the promoter activity, the same mutations used in Fig. 6C were introduced into the HNF4{alpha}-binding site in the mouse BAT (–602/+178)-luciferase construct (Fig. 7A). As shown in Fig. 7B, when HepG2 cells were used for transient transfections, the promoter activity of the mutated –602-bp fragment (–602/Mut) was decreased to 25% as compared with the wild-type fragment (–602/WT). When CV-1 cells were used, the promoter activity of the mutated fragment was decreased to 15% by cotransfection of HNF4{alpha} as compared with the wild-type fragment (Fig. 7C), indicating that the HNF4{alpha}-binding site is important for activation of the mouse BAT promoter.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
BAs are synthesized by steroid nucleus and side chain hydroxylation of cholesterol followed by {beta}-oxidation of the side chain. Because serum BA levels are significantly increased and expression of OATP1 and NTCP was decreased in liver-specific HNF4{alpha}-null mice (10), HNF4{alpha} is likely a critical transcription factor regulating genes involved in BA biosynthesis. In this study, HNF4{alpha} was also found to control the BA conjugation pathway by direct binding to the promoter regions of both the VLACSR and BAT genes and activation of their transcription.

Because the levels of unconjugated (free) BAs are very low in gallbladder bile (22), increased levels of unconjugated BAs in liver-specific HNF4{alpha}-null mice appear to be due to decreased expression of VLACSR and BAT. Because VLACSR and BAT are preferentially expressed in liver (18, 19) as well as HNF4{alpha}, HNF4{alpha} is a central factor in the regulation of VLACSR and BAT in vivo. An HNF4{alpha}-binding site and a GC-rich region were identified in the promoter region of the mouse VLACSR gene. Indeed, HNF4{alpha} binds to this HNF4{alpha}-binding site, and the promoter activities were dependent on this site and expression of HNF4{alpha}. Furthermore, a GC-rich sequence was also important for maximal activation of the VLACSR gene because the mutated GC-rich sequence reduced its basal promoter activity. It was reported that HNF4{alpha} and Sp1, a prototypical GC box-binding protein, binds to promoter regions of genes, including apolipoprotein C-III and CYP27, and increases their expression (23, 24). In the mouse VLACSR promoter, HNF4{alpha} can still activate promoter activity when mutations were introduced into the GC box, but the resultant activity was much lower when compared with that of the wild-type promoter. This result indicates that cooperative binding of HNF4{alpha} and Sp1 to the promoter might be required for maximal induction of VLACSR expression in the liver. Furthermore, the expression of sterol carrier protein x, which is involved in the side chain {beta}-oxidation of BA, was also reduced in liver-specific HNF4{alpha}-null mice, and Sp1 was identified as a critical factor regulating the sterol carrier protein x gene in an HNF4{alpha}-independent manner (data not shown). Because expression of Sp1 and Sp3, typical GC box-binding proteins, was unchanged and VLACSR expression is markedly reduced in liver-specific HNF4{alpha}-null mice (data not shown), post-transcriptional modification of Sp1 and/or Sp3 could reduce the expression of VLACSR in these null mice.

In addition to increased unconjugated BAs in serum, glycine-conjugated BAs such as GDCA and GCDCA were significantly increased in the gallbladder of liver-specific HNF4{alpha}-null mice. Because mouse BAT has no glycine conjugation activity (19), expression of an unidentified BAT gene might be activated in H4LivKO mice. Actually a few mouse genes that exhibit similarity to mouse BAT were cloned, but their function is still unknown. Expression of one of these genes (GenBankTM accession number NM_145368 [GenBank] ) was found to be increased in the livers of H4LivKO mice (data not shown). However, it remains unclear whether this gene encodes an enzyme with glycine conjugation activity. Furthermore, the physiological role of increased GDCA and GCDCA in H4LivKO mice also remains unclear, but these BAs in the intestinal lumen might change many aspects of intestinal and whole body cholesterol homeostasis.

Recently it was reported (25) that a mutation of the BAT coding sequence causes human familial hypercholanemia that is characterized by elevated serum BA, and serum of homozygous individuals for this mutation contains only unconjugated BAs. Because serum unconjugated BAs are increased, but most BAs in the gallbladder are conjugated forms in liver-specific HNF4{alpha}-null mice, protein and enzyme activity of VLACSR and BAT might still be active in these mice. In human disease, mutations in the HNF4{alpha}-binding sites were reported in HNF1{alpha}, blood coagulation factor VII, and IX (9). Because HNF4{alpha} regulates many liver-enriched genes, human diseases might be found to be caused by mutations of the HNF4{alpha}-binding sites in the VLACSR and BAT genes.

It was reported that the expression of BAL and BAT is induced by treatment with ligands for the farnesoid X receptor (FXR) in rat, and this induction was due to the binding of FXR to FXR-binding sites in their promoter regions (26). Because this phenomenon was not observed in mouse, regulation of both genes by FXR exhibits a species difference between rat and mouse (26). Actually, neither VLACSR nor BAT gene was induced in wild-type mice treated with cholic acid, a ligand for FXR (data not shown). Unlike other nuclear receptors including FXR, HNF4{alpha} is constitutively activated in vivo and in vitro without exogenous compounds (9). Thus, HNF4{alpha} positively regulates the basal levels of expression of the VLACSR and BAT genes. It may also be involved in the regulation of transporters such as OATP1 and NTCP that are required for hepatocyte uptake of conjugated BAs from the portal circulation (Fig. 8).



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  		FIG. 8.
Schematic representation of regulatory targets of HNF4{alpha} in bile acid transport and conjugation. Bile acids, conjugated by VLACSR and BAT, are exported from hepatocytes by transporters such as bile salt export pump located at the canalicular membrane. Most bile acids are recycled and transported from the portal circulation back into hepatocytes by transporters including NTCP and OATP1 located at the basolateral membrane. Expression of OATP1, NTCP, VLACSR, and BAT is reduced in liver-specific HNF4{alpha}-null mice. Note that HNF4{alpha} directly regulates the expression of VLACSR and BAT via HNF4{alpha}-binding sites in their promoters.

 
Many nuclear receptors including FXR are involved in regulating the BA biosynthesis pathway and transport system (6, 14, 15). Thus, these nuclear receptors also might have important roles in inducing or repressing the expression of VLACSR and BAT to regulate the BA conjugation pathway.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Laboratory of Metabolism, Center for Cancer Research, NCI, National Institutes of Health, 9000 Rockville Pike, Bldg. 37, Rm. 3106, Bethesda, MD 20892. Tel.: 301-496-9067; Fax: 301-496-8419; E-mail: fjgonz{at}helix.nih.gov.

1 The abbreviations used are: BAs, bile acids; HNF4{alpha}, hepatocyte nuclear factor 4{alpha}; BAL, bile acid-CoA ligase; BAT, bile acid-CoA:amino acid N-acyltransferase; VLACSR, very long chain acyl-CoA synthase-related; NTCP, sodium taurocholate cotransporter polypeptide; OATP1, organic anion transporter polypeptide 1; LC-MS/MS, liquid chromatography tandem mass spectrometry/mass spectrometry; MCA, muricholic acid; GDCA, glycodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; TCA, taurocholic acid; T-{beta}-MCA tauro-{beta}-muricholic acid; TDCA, taurodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; WT, wild-type; FXR, farnesoid X receptor. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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