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α1-Fetoprotein Transcription Factor Is Required for the Expression of Sterol 12α-Hydroxylase, the Specific Enzyme for Cholic Acid Synthesis

2000; Elsevier BV; Volume: 275; Issue: 23 Linguagem: Inglês

10.1074/jbc.m000996200

1083-351X

Antonio del Castillo‐Olivares, Gregorio Gil,

Pediatric Hepatobiliary Diseases and Treatments

Cholesterol conversion to bile acids occurs via the “classic” (neutral) or the “alternative” (acidic) bile acid biosynthesis pathways. Sterol 12α-hydroxylase/CYP8b1 is the specific enzyme required for cholic acid synthesis. The levels of this enzyme determine the ratio of cholic acid to chenodeoxycholic acid and thus the hydrophobicity of the circulating bile acid pool. Expression of the 12α-hydroxylase gene is tightly down-regulated by hydrophobic bile acids. In this study, we report the characterization of two DNA elements that are required for both the 12α-hydroxylase promoter activity and bile acid-mediated regulation. Mutation of these elements suppresses 12α-hydroxylase promoter activity. Mutations of any other part of the promoter do not alter substantially the promoter activity or alter regulation by bile acids relative to the wild type promoter. These two DNA elements bind α1-fetoprotein transcription factor (FTF), a member of the nuclear receptor family. We also show that overexpression of FTF in a non-liver cell line activates the sterol 12α-hydroxylase promoter. These studies demonstrate the crucial role of FTF for the expression and regulation of a critical gene in the bile acid biosynthetic pathways. Cholesterol conversion to bile acids occurs via the “classic” (neutral) or the “alternative” (acidic) bile acid biosynthesis pathways. Sterol 12α-hydroxylase/CYP8b1 is the specific enzyme required for cholic acid synthesis. The levels of this enzyme determine the ratio of cholic acid to chenodeoxycholic acid and thus the hydrophobicity of the circulating bile acid pool. Expression of the 12α-hydroxylase gene is tightly down-regulated by hydrophobic bile acids. In this study, we report the characterization of two DNA elements that are required for both the 12α-hydroxylase promoter activity and bile acid-mediated regulation. Mutation of these elements suppresses 12α-hydroxylase promoter activity. Mutations of any other part of the promoter do not alter substantially the promoter activity or alter regulation by bile acids relative to the wild type promoter. These two DNA elements bind α1-fetoprotein transcription factor (FTF), a member of the nuclear receptor family. We also show that overexpression of FTF in a non-liver cell line activates the sterol 12α-hydroxylase promoter. These studies demonstrate the crucial role of FTF for the expression and regulation of a critical gene in the bile acid biosynthetic pathways. chenodeoxycholic acid cholesterol 7α-hydroxylase sterol 12α-hydroxylase cytochrome P450 7α-hydroxylase gene cytochrome P450 7α-hydroxylase CYP7A promoter-binding factor A1-fetoprotein transcription factor sterol regulatory element growth hormone receptor hepatocyte nuclear factor Proper control of intracellular and circulating cholesterol levels is essential for maintaining cholesterol homeostasis, since metabolic disarrangement can lead to many degenerative conditions with a probable genetic component, such as atherosclerosis, cholestasis, and cholesterol gallstone disease. Because nearly 50% of the body cholesterol is catabolized to bile acids, this pathway plays an important role in the cholesterol homeostasis of mammals. Current evidence suggests a decreased bile acid output as the major contributing factor in the production of lithogenic bile. This leads to an abnormal ratio of cholesterol to bile acids and lecithin, which is a major risk factor for cholesterol gallstone formation (1.Turley S.D. Dietschy J.M. Arias I. Popper H. Schachter D. Shafritz D.A. The Liver: Biology and Pathology. Raven Press, New York1982: 467-492Google Scholar). Cholesterol conversion to bile acids occurs via the “classic” (neutral) or the “alternative” (acidic) bile acid biosynthesis pathways (2.Javitt N.B. FASEB J. 1994; 8: 1308-1311Crossref PubMed Scopus (147) Google Scholar). Cholic acid and chenodeoxycholic acid (CDCA)1 are the end products of these pathways (Fig. 1) and the major primary bile acids found in most vertebrates. Cholic acid is hydroxylated at position 12α, whereas CDCA is not. There are three enzymes that play major regulatory roles in these two pathways. Cholesterol 7α-hydroxylase/CYP7a1 (7α-hydroxylase) is the rate-limiting enzyme in the classic pathway. Sterol 27-hydroxylase/CYP27 is the first enzyme in the alternative pathway. Sterol 12α-hydroxylase/CYP8b1 (12α-hydroxylase) is the specific enzyme for cholic acid synthesis and determines the ratio of cholic acid to chenodeoxycholic acid and thus the hydrophobicity of the circulating pool. The altered ratio of cholic to CDCA has been postulated to play a role in cholesterol gallstone formation (3.Shaw R. Elliot W.H. J. Biol. Chem. 1979; 254: 7177-7182Abstract Full Text PDF PubMed Google Scholar). Suppression of 12α-hydroxylase by specific inhibitors has been suggested as a possible therapeutic strategy for dissolution of cholesterol gallstone (3.Shaw R. Elliot W.H. J. Biol. Chem. 1979; 254: 7177-7182Abstract Full Text PDF PubMed Google Scholar). Because chenodeoxycholic acid is a more potent suppressor of HMG-CoA reductase and 7α-hydroxylase than cholic acid (3.Shaw R. Elliot W.H. J. Biol. Chem. 1979; 254: 7177-7182Abstract Full Text PDF PubMed Google Scholar, 4.Heuman D.M. Hylemon P.B. Vlahcevic Z.R. J. Lipid. Res. 1989; 30: 1161-1171Abstract Full Text PDF PubMed Google Scholar), the relative activity of 12α-hydroxylase may play an important role in the regulation of hepatic cholesterol homeostasis. The alteration of cholic/CDCA ratio affects biliary cholesterol and phospholipid secretion, thus altering intestinal cholesterol absorption and receptor-mediated lipoprotein uptake by the hepatocyte (4.Heuman D.M. Hylemon P.B. Vlahcevic Z.R. J. Lipid. Res. 1989; 30: 1161-1171Abstract Full Text PDF PubMed Google Scholar). It is well documented that bile acids exert negative feedback regulation on their own synthesis (5.Carey M.C. Cahalene M.J. Arias I.M. Jakoby W.B. Popper H. Schachter D. Shafritz D.A. The Liver: Biology and Pathobiology. Raven Press, New York1988: 573-616Google Scholar). Interruption of the enterohepatic circulation, by biliary diversion or by feeding bile acid-binding resins (cholestyramine), enhances cholesterol and bile acid synthesis (6.Danielsson J. Einarsson K. Johansson G. Eur. J. Biochem. 1967; 2: 44-49Crossref PubMed Scopus (183) Google Scholar). Conversely, feeding bile acids suppresses bile acid and cholesterol synthesis (7.Dietschy J.M. Wilson J. N. Engl. J. Med. 1970; 282: 1128-1138Crossref PubMed Scopus (203) Google Scholar). Bile acids negatively regulate the transcription of the 7α-hydroxylase gene, which controls output from the classic pathway (8.Ramirez M.I. Karaoglu D. Haro D. Barillas C. Bashirzadeh R. Gil G. Mol. Cell. Biol. 1994; 14: 2809-2821Crossref PubMed Google Scholar). Two bile acid response elements have been localized within the 5′-flanking region of the rat gene (9.Stroup D. Crestani M. Chiang J.Y.L. Am. J. Physiol. 1997; 273: G508-G517PubMed Google Scholar, 10.Chiang J.Y.L. Stroup D. J. Biol. Chem. 1994; 269: 17502-17507Abstract Full Text PDF PubMed Google Scholar), but the factors that mediate regulation have not been characterized. It remains to be demonstrated whether these elements are indeed involved in this regulation. Similarly, bile acids also down-regulate transcription of the sterol 27-hydroxylase gene (11.Vlahcevic Z.R. Jairath S.K. Heuman D.M. Stravitz R.T. Hylemon P.B. Avadhani N.G. Pandak W.M. Am. J. Physiol. 1996; 270: G646-G652PubMed Google Scholar), and it has been reported that hepatocyte nuclear factor 1 (HNF-1) is involved in this regulation (12.Rao Y.P. Vlahcevic Z.R. Stravitz R.T. Mallonee D.H. Mullick J. Avadhani N.G. Hylemon P.B. J. Steroid Biochem. Mol. Biol. 1999; 70: 1-14Crossref PubMed Scopus (32) Google Scholar). However, the molecular mechanisms involved in that regulation are not well defined. More recently, it has been shown that a transcriptional factor, named the CYP7A promoter-binding factor (CPF), is required for the expression of the 7α-hydroxylase gene (13.Nitta M. Ku S. Brown C. Okamoto A.Y. Shan B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6660-6665Crossref PubMed Scopus (249) Google Scholar). This factor is a member of the Ftz-F1 family of the class IV orphan nuclear receptor superfamily (14.Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. et al.Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6110) Google Scholar). CPF binds to the region of the 7α-hydroxylase promoter previously characterized as a bile acid response element (10.Chiang J.Y.L. Stroup D. J. Biol. Chem. 1994; 269: 17502-17507Abstract Full Text PDF PubMed Google Scholar), which suggests that CPF might play a role in the bile acid-mediated down-regulation of 7α-hydroxylase transcription. The cDNAs and genes encoding the rabbit, rat, and human 12α-hydroxylase enzyme have been cloned (15.Andersson U. Yang Y.Z. Björkhem I. Einarsson C. Eggertsen G. Gafvels M. Biochim. Biophys. Acta. 1999; 1438: 167-174Crossref PubMed Scopus (37) Google Scholar, 16.Eggertsen G. Olin M. Andersson U. Ishida H. Kubota S. Hellman U. Okuda K.-I. Björkhem I. J. Biol. Chem. 1996; 271: 32269-32275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 17.Gafvels M. Olin M. Chowdhary B.P. Raudsepp T. Andersson U. Persson B. Jansson M. Björkhem I. Eggertsen G. Genomics. 1999; 56: 184-196Crossref PubMed Scopus (62) Google Scholar), and studies of the molecular basis of its regulation are now feasible. It is expressed exclusively in the liver, since it corresponds to a liver-specific process. Recently, Vlahcevic et al. (18.Vlahcevic Z.R. Eggertsen G. Björkhem I. Hylemon P.B. Redford K. Pandak W.M. Gastroenterology. 2000; 118: 599-607Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) showed that expression of the 12α-hydroxylase gene is tightly regulated in a similar fashion to the 7α-hydroxylase gene. Cholestyramine-fed rats contained approximately 2-fold more 12α-hydroxylase mRNA than control rats, whereas deoxycholate- or cholate-fed rats had undetectable levels of 12α-hydroxylase mRNA (at least 10-fold less than controls). Interestingly, cholesterol feeding decreased the amount of 12α-hydroxylase mRNA by about 2-fold. Similar regulation was observed for both enzymatic and transcriptional activity, which indicates that both bile acids and cholesterol regulate the expression of the 12α-hydroxylase gene mainly at the transcriptional level. In this study, we have characterized the 12α-hydroxylase promoter. We have localized two DNA elements that are required for both expression of the 12α-hydroxylase promoter and bile acid-mediated regulation. These elements bind α1-fetoprotein transcription factor (FTF) (19.Galarneau L. Pare J.F. Allard D. Hamel D. Levesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar), a member of the Ftz-F1 family of receptors. Rat FTF is the ortholog of mouse liver receptor homolog 1 2J. D. Tugwood, I. Issemann, and S. Green, GenBankTM accession number M81385. and human PHR-1 (21.Becker-Andre M. Andre E. DeLamarter J.F. Biochem. Biophys. Res. Commun. 1993; 194: 1371-1379Crossref PubMed Scopus (232) Google Scholar). We have also shown that when FTF is expressed in a non-liver cell line, the 12α-hydroxylase promoter becomes active, demonstrating the critical role of FTF for 12α-hydroxylase promoter activity. These studies demonstrate the crucial role of FTF in the bile acid biosynthetic pathway. Reagents used in DNA cloning and sequencing were from New England Biolabs, Roche Molecular Biochemicals, U.S. Biochemical Corp., or Life Technologies, Inc. Common laboratory chemicals were from Fisher, Sigma, or Bio-Rad. The luciferase promoterless vector, pGL3-Basic, was purchased from Promega. Oligonucleotides were prepared in the Medical College of Virginia DNA Synthesis Facility by the phosphoramidite method on an automated DNA synthesizer. pCI-FTF, an expression plasmid that contains the human FTF cDNA in the expression vector pCI (Promega), was a generous gift from Dr. Bélanger (19.Galarneau L. Pare J.F. Allard D. Hamel D. Levesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar). pCMX, a plasmid for expression in mammalian cells and in vitro, contains the cytomegalovirus and the T7 promoters and was a gift from Dr. Ronald M. Evans. Anti-FTF antibodies were a gift from Dr. David W. Russell and were raised against a peptide corresponding to amino acids 180–1197 of the DNA binding domain. Standard recombinant DNA procedures were carried out essentially as described (8.Ramirez M.I. Karaoglu D. Haro D. Barillas C. Bashirzadeh R. Gil G. Mol. Cell. Biol. 1994; 14: 2809-2821Crossref PubMed Google Scholar). DNA sequencing was done by the dideoxy chain termination method using DNA fragments subcloned into M13 vectors or with double-stranded clones and the universal primer or sequence-specific primers with reagents from U.S. Biochemical Corp. A rat genomic library was obtained from CLONTECH. The library was plated and screened with a doubled-stranded probe made by reverse transcriptase-polymerase chain reaction and contained a 200-nt fragment from the 5′-end of the rabbit 12α-hydroxylase cDNA. The polymerase chain reaction primers were synthesized based on published DNA sequence (22.Teixeira J. Gil G. J. Biol. Chem. 1991; 266: 21030-21036Abstract Full Text PDF PubMed Google Scholar). Southern blot analysis was done to characterize the λ clone. About 3 kilobases was sequenced by the dideoxy method. PGL3-R12α-865 was prepared by placing a 903-nucleotide SacI–SacI fragment containing nucleotides −865 to +37 into the SacI site of pGL3-Basic (Promega). The three 5′-deletion constructs PGL3-R12α-289, PGL3-R12α-163, and PGL3-R12α-106 were prepared by polymerase chain reaction using a specific 5′ primer and a common 3′ primer corresponding to nucleotides 20–37. Mutation constructs were generated by oligonucleotide-directed mutagenesis in M13 (23.Subramanian A. Teixeira J. Wang J. Gil G. Mol. Cell. Biol. 1995; 15: 4672-4682Crossref PubMed Google Scholar) or by using the QuikChange site-directed mutagenesis kit (Stratagene). All constructs were confirmed by DNA sequencing. HepG2 and CV-1 cells were obtained from American Type Culture Collection. Both cell types were transfected with Lipofectin (Life Technologies, Inc.), using 1.5 μg of total DNA in 35-mm plates. HepG2 were transfected with 25 ng of test plasmid and 5 ng of pCMV-Gal (a plasmid containing the human cytomegalovirus promoter in from of the bacteria galactosidase gene) to normalize for transfection efficiencies. CV-1 cells were transfected with 200 ng of test plasmid, 50 ng of pCMV-Gal, and the indicated amounts of pCI-FTF, an expression plasmid that contains the FTF cDNA driven by the cytomegalovirus promoter. After 16 h, the DNA was removed, and where indicated, 100 μm CDCA was added. Cells were harvested 48 h later, and luciferase and β-galactosidase assays were performed with a kit from Tropix (Bedford, MA), according to the manufacturer's protocol. Average values are for the number of experiments indicated. Transcription/translation of cDNAs encoding FTF or of the growth hormone receptor (GHR) as a control was performed using the TNT T7-coupled rabbit reticulocyte lysate system according to the manufacturer's protocol (Promega). HepG2 nuclear extracts were prepared as indicated (24.Subramanian A. Wang J. Gil G. Nucleic Acids Res. 1998; 29: 2173-2178Crossref Scopus (40) Google Scholar). DNA binding reactions were set up in 50 mm KCl, 20 mmTris-HCl (pH 8.0), 0.2 mm EDTA, 4% Ficoll, 1.0 μg of poly (dI-dC), 4 μl of the translated protein, and a 1500-fold molar excess of an irrelevant single-stranded DNA, in a final volume of 20 μl on ice. After a 15-min incubation, 320 fmol of the indicated32P-labeled DNA probes (∼2 × 105 cpm) were added. All probes were adjusted to the same specific radioactivity. After incubation for 20 min on ice, samples were loaded onto a 4.5% polyacrylamide gel and subjected to electrophoresis at 4 °C. Gels were dried and exposed to XAR-5 (Eastman Kodak Co.). For supershift experiments, 1 μl of the crude serum was used. The DNA sequence of the 5′-flanking region of the 12α-hydroxylase gene is shown in Fig.2. The transcriptional initiation site was located by primer extension techniques (data not shown) and is numbered +1. This sequence contains a TATA box-like element, two consensus binding sites for HNF-3, a liver-specific DNA-binding protein (16.Eggertsen G. Olin M. Andersson U. Ishida H. Kubota S. Hellman U. Okuda K.-I. Björkhem I. J. Biol. Chem. 1996; 271: 32269-32275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and two sterol regulatory elements (SREs) that are implicated in cholesterol-mediated regulation of the transcription of several genes (17.Gafvels M. Olin M. Chowdhary B.P. Raudsepp T. Andersson U. Persson B. Jansson M. Björkhem I. Eggertsen G. Genomics. 1999; 56: 184-196Crossref PubMed Scopus (62) Google Scholar). One stretch of DNA located between nucleotides −63 and −48 contains two potential sites for the liver-specific nuclear receptor FTF (19.Galarneau L. Pare J.F. Allard D. Hamel D. Levesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar). To functionally characterize the 12α-hydroxylase promoter, we prepared a chimeric gene (pGL3-R12α-865, as shown in Fig.3) by fusing theSacI–SacI fragment (Fig. 2), which contains 865 base pairs of the 5′-flanking region and 33 base pairs of 5′-untranslated region, to the coding region of the luciferase gene. We then used HepG2 cells as recipient cells to transfect this chimeric gene. After transfection, cells were treated with or without 100 μm CDCA, which was shown to suppress expression of the endogenous gene in primary hepatocytes (data not shown), mimicking the bile acid-mediated regulation that has been described in vivo (18.Vlahcevic Z.R. Eggertsen G. Björkhem I. Hylemon P.B. Redford K. Pandak W.M. Gastroenterology. 2000; 118: 599-607Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Cells were harvested for luciferase and galactosidase activities as explained under “Experimental Procedures.” As shown in Fig. 3, pGL3-R12α-865 had promoter activity (100-fold above background levels) that was regulated approximately 5-fold upon the addition of CDCA. To narrow down the promoter region involved in both transcriptional activity and bile acid-mediated regulation, we made the three 5′-deletion constructs shown in Fig. 3. All three constructs showed regulated activity, indicating that all of the DNA elements required for both promoter activity and bile acid-mediated regulation are located in the first 106 nucleotides of the 12α-hydroxylase promoter. To further characterize the 12α-hydroxylase promoter, we mutated the first 106 nucleotides in blocks of approximately 20 nucleotides each as shown in Fig. 2 (brackets A–G). The results of these experiments are shown in Fig.4 A, and the actual mutations introduced in mutants D, E, and F are shown in Fig. 4 B. All mutants exhibited promoter activity and bile acid-mediated regulation, except when nucleotides −62 to −49 were mutated. This region contains two putative elements with homology to the rat FTF site, a member of the Drosophila orphan receptor fushi tarazu F1 (Ftz-F1) (25.Lavorgna G. Ueda H. Clos J. Wu C. Science. 1991; 252: 848-851Crossref PubMed Scopus (247) Google Scholar), and we named them FTF sites. These elements are also homologous to the recently described CPF site for the 7α-hydroxylase gene (26.Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Mol. Cell. 1999; 3: 543-553Abstract Full Text Full Text PDF PubMed Scopus (1304) Google Scholar), another nuclear receptor of the same family. These homologies are shown in Fig. 5.Figure 5Homology between the 12α-hydroxylase promoter FTF sites and other homologous binding sites. Nucleotides −63 to −48 from the rat 12α-hydroxylase promoter are shown at the top, with the two FTF sites boxed. These two sites are aligned with the rat FTF site (19.Galarneau L. Pare J.F. Allard D. Hamel D. Levesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar); the Drosophila Ftz-F1 consensus binding site (25.Lavorgna G. Ueda H. Clos J. Wu C. Science. 1991; 252: 848-851Crossref PubMed Scopus (247) Google Scholar); and the human, rat, and hamster 7α-hydroxylase CPF sites (13.Nitta M. Ku S. Brown C. Okamoto A.Y. Shan B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6660-6665Crossref PubMed Scopus (249) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Based on these homologies, we hypothesized that there are two FTF elements within the 12α-hydroxylase promoter located at positions −63 to −55 and −56 to −48. The data in Fig. 4 A show that both mutants D and F, which have the two potential FTF sites independently mutated, and mutant E, which has both sites mutated, had very low, if detectable, activities. This suggests that both elements are required for activity. Fig. 6 shows that in vitrosynthesized FTF binds to the 12α-hydroxylase promoter (lanes 2 and 3). When in vitro made FTF was preincubated with antibodies raised against a peptide corresponding to the DNA binding domain of FTF, binding was mostly abolished (lane 5), demonstrating that the protein binding to the 12α-hydroxylase probe is in fact FTF. Preimmune serum did not produce any effect (lane 4). Mutation of 5 nucleotides (−62 to −58) within the first site diminished binding about 5-fold (lanes 7 and 8). Mutation of 4 nucleotides (−52 to −49) within the second putative site also diminished binding by about 5-fold (Fig. 6 A, lanes 13 and14). Mutation of 8 nucleotides (−56 to −49) that modifies both sites abolished binding completely (lanes 10and 11). As a negative control, we used in vitrosynthesized GHR (lanes 1, 6,9, and 12). To determine the specificity of the binding and if the 7α-hydroxylase CPF site binds to the same protein, we performed the competition experiment shown in Fig. 6 B. Wild type probe competed as expected (lanes 3–5). A probe made from the rat 7α-hydroxylase promoter sequence (−150 to −131) containing the described CPF site (13.Nitta M. Ku S. Brown C. Okamoto A.Y. Shan B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6660-6665Crossref PubMed Scopus (249) Google Scholar) competed even better than the 12α-hydroxylase probe itself (lanes 6–8), suggesting that the 7α-hydroxylase CPF site has higher affinity for FTF than the 12α-hydroxylase sites. A mutated 12α-hydroxylase fragment (mutant E), with nucleotides mutated in both FTF sites, did not compete for binding (lanes 9–11), confirming the specificity of the binding. To demonstrate that the protein binding to the 12α-hydroxylase FTF sites exists in liver cells, HepG2 nuclear extracts were used for binding experiments (Fig. 6 C, lanes 7–9). The binding activity found in HepG2 cells was also inhibited by incubation with a specific antibody against FTF (lane 9), and the DNA-protein complexes formed exhibited mobility identical to that of the complex formed by in vitro made FTF (lanes 4–6). As a control, we used a probe from the rat 7α-hydroxylase promoter known to bind CPF, a highly homologous protein to FTF (13.Nitta M. Ku S. Brown C. Okamoto A.Y. Shan B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6660-6665Crossref PubMed Scopus (249) Google Scholar). Both in vitromade FTF and HepG2 nuclear extracts bind the 7α-hydroxylase probe, and that binding was specific as indicated by the fact that anti-FTF antibodies also prevented binding (Fig. 6 C, lanes 10–18). The 7α-hydroxylase probe binds FTF at higher affinity that the 12α-hydroxylase probe, in agreement with the competition experiments shown in Fig. 6 B. To further demonstrate the key role of FTF for 12α-hydroxylase promoter activity, we overexpressed FTF in the kidney cell line CV-1 (Fig. 7). Since 12α-hydroxylase is expressed only in the liver, pGL3-R12α-865 was inactive in CV-1 cells as expected. However, when we cotransfected pCI-FTF, pGL3-R12α-865 became active. The level of activation was nearly 13-fold when 200 ng of pCI-FTF was used. As a control, we used pGL3-Basic and pGL3-R12α-865 mutant E, which had no promoter activity in HepG2 cells (Fig. 4). The activities from these two plasmids were the same as pGL3-R12α-865 when no pCI-FTF was included in the transfection. Overexpression of FTF produced only a slight activation (2–3-fold) of both the promoterless plasmid pGL3-Basic and the pGL3-R12α-865 mutant E. For comparison, a construct containing the rat 7α-hydroxylase promoter was activated only 8-fold when 2-fold more pCI-FTF (400 ng) was cotransfected (data not shown), suggesting a greater activity of FTF for the 12α-hydroxylase promoter than the 7α-hydroxylase promoter. The data presented in this study demonstrate that FTF is a factor required for 12α-hydroxylase expression. Several lines of evidence support this conclusion. First, mutagenesis of either of its two binding sites abolished promoter activity completely in HepG2 cells (Fig. 4); therefore, both sites are required for promoter activity. Second, in vitro-made FTF binds specifically to these sites (Fig. 6), and the same binding activity was observed in nuclear extracts prepared from HepG2 cells. Third, expression of FTF in a non-liver cell line activates the 12α-hydroxylase promoter, which is otherwise inactive (Fig. 7). Additionally, this study also demonstrates that all of the DNA elements required for the bile acid-mediated regulation of 12α-hydroxylase promoter activity are located in the first 106 nucleotides of the 5′-flanking region (Fig. 3). The 12α-hydroxylase promoter sequence also has two other potential regulatory elements. Two putative HNF-3 sites are located at approximately nucleotides −455 and −385 (Fig. 2). HNF-3 sites are found in the promoter of some liver-specific genes and are required for the expression of those genes (27.Jackson D.A. Rowader K.E. Stevens K. Jiang C. Milos P. Zaret K. Mol. Cell. Biol. 1993; 13: 2401-2410Crossref PubMed Scopus (134) Google Scholar). Additionally, two SRE sites are located at approximately nucleotides −315 and −328. SREs are regulatory elements found in cholesterol and fatty acid-regulated genes (28.Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1110) Google Scholar). However, neither the HNF-3 sites nor the SRE sites seem to be required for either promoter activity or regulation by bile acids, since deletion of these sites has very little effect, if any (Fig. 3). It is conceivable that the SRE sites are involved in the cholesterol-mediated regulation of 12α-hydroxylase transcription that has been observed in rats (18.Vlahcevic Z.R. Eggertsen G. Björkhem I. Hylemon P.B. Redford K. Pandak W.M. Gastroenterology. 2000; 118: 599-607Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), although this was not the objective of the present studies. Sterol 12α-hydroxylase is the second gene involved in bile acid biosynthesis that has been shown to require a member of the Ftz-F1 family of the class IV orphan nuclear receptor superfamily for its expression. Recently, it has been shown that another member of the same family, CPF, is required for 7α-hydroxylase expression (13.Nitta M. Ku S. Brown C. Okamoto A.Y. Shan B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6660-6665Crossref PubMed Scopus (249) Google Scholar). CPF also activates the 12α-hydroxylase promoter. In transactivation experiments similar to these shown in Fig. 6, CPF also activated 12α-hydroxylase promoter activity, although to a lesser extent (data not shown). It is possible that several members of the Ftz-F1 family have similar activity on genes involved in bile acid biosynthesis. In fact, it has been shown that at least both CPF and CPF variant 1 (another member of the same family) were active on the 7α-hydroxylase promoter (13.Nitta M. Ku S. Brown C. Okamoto A.Y. Shan B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6660-6665Crossref PubMed Scopus (249) Google Scholar). This suggests a crucial role of this family of factors in the control of bile acid biosynthesis. An interesting issue is the apparent existence of two FTF binding sites within the 12α-hydroxylase promoter. These two FTF sites were located based on homology with other FTF sites, and they overlap by the last 2 nucleotides of the first site based on published consensus sequences (13.Nitta M. Ku S. Brown C. Okamoto A.Y. Shan B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6660-6665Crossref PubMed Scopus (249) Google Scholar, 19.Galarneau L. Pare J.F. Allard D. Hamel D. Levesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar, 25.Lavorgna G. Ueda H. Clos J. Wu C. Science. 1991; 252: 848-851Crossref PubMed Scopus (247) Google Scholar). Although DNA probes containing an individual mutation of either site still bind FTF weakly (Fig. 6 A), both sites are required for an active promoter (Fig. 4 A). Nuclear receptors of the Ftz-F1 family are known to bind as monomers (19.Galarneau L. Pare J.F. Allard D. Hamel D. Levesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar), and given the migration pattern of the protein-probe complex, only one protein molecule seemed to bind per probe molecule. Thus, mutant D, which has only the first site mutated (Fig. 4 B), has some binding capability (Fig. 6 A) but has no promoter activity (Fig.4 A). Mutant F, which has 4 nucleotides mutated within the second site, has a binding capability similar to that of mutant D (Fig.6 A) and has some promoter activity (Fig. 4 A), but much lower than wild type. Mutant E, which has 8 nucleotides mutated across both binding sites, lost all binding (Fig. 6 A) and promoter activity. The most likely explanation to this issue is that binding of FTF to the 12α-hydroxylase promoter site requires more nucleotides than the binding of FTF or homologous factors to other promoters, and in fact, there may be only one extended binding site. Whether the activity of FTF is regulated by small molecules in a way similar to most nuclear receptors is still unknown. Attempts in our laboratory to identify a bile acid molecule or derivative that could act as ligand and/or regulator of FTF have not been successful. It is possible that there is another unidentified factor of the same family that is specific for genes of the bile acid biosynthetic pathways and that this factor has a ligand binding activity for bile acids or a derivative. Besides the similarity between the 12α-hydroxylase and 7α-hydroxylase promoters in the requirement for FTF or a homologous factor such as CPF for its expression, these two genes seem to differ in the factors required for bile acid regulation of their transcription. In experiments using HepG2 cells and with CDCA, the 7α-hydroxylase promoter required exogenous farnesol X receptor in order to show bile acid-mediated regulation of its activity (20.Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2182) Google Scholar), whereas the 12α-hydroxylase promoter did not show this requirement (Figs. 3 and 4). Although this study was not specifically directed to study the factors involved in the bile acid-mediated regulation of 12α-hydroxylase expression, our data strongly suggest that FTF is also implicated in that regulation. This is based on the fact that only mutations within the FTF binding sites abolished regulation. All deletion promoter constructs (Fig. 3) and all block mutant constructs (Fig. 4) are regulated by bile acids except for the two constructs that abolished FTF binding. It could be argued that elimination of FTF binding renders the promoter inactive, and no conclusion could be drawn about its regulation. However, mutation of the rest of the promoter did not alter bile acid-mediated regulation, and therefore the FTF sites should be involved in the regulation either directly or indirectly. Whether the FTF binding sites or FTF itself is capable of interacting with a bile acid-regulated factor is still unknown. In conclusion, this study demonstrates the key role of FTF or its homologues in the regulation of bile acid biosynthesis and should help to elucidate the molecular mechanisms involved in the bile acid-mediated down-regulation of gene transcription, a process poorly understood to date. Lesley D. Johnson provided invaluable technical help. We thank Dr. Luc Bélanger for plasmid pCI-FTF and anti-FTF antibodies, Dr. David W. Russell for anti-FTF antibodies, and Dr. Ronald M. Evans for pCMX. We are grateful to Drs. Hylemon and Vlahcevic for sharing data before publication, critical comments, and support. We also thank Dr. Wells for discussions and critical review of the manuscript.