Transcriptional Regulation of the Human Sterol 12α-Hydroxylase Gene (CYP8B1)

Sterol 12α-hydroxylase catalyzes the synthesis of cholic acid and controls the ratio of cholic acid over chenodeoxycholic acid in the bile. Transcription of CYP8B1is inhibited by bile acids, cholesterol, and insulin. To study the mechanism of CYP8B1 transcription by bile acids, we have cloned and determined 3389 base pairs of the 5′-upstream nucleotide sequences of the human CYP8B1. Deletion analysis of CYP8B1/luciferase reporter activity in HepG2 cells revealed that the sequences from −57 to +300 were important for basal and liver-specific promoter activities. Hepatocyte nuclear factor 4α (HNF4α) strongly activated human CYP8B1 promoter activities, whereas cholesterol 7α-hydroxylase promoter factor (CPF), an NR5A2 family of nuclear receptors, had much less effect. Electrophoretic mobility shift assay identified an overlapping HNF4α- and CPF-binding site in the +198/+227 region. The human CYP8B1 promoter activities were strongly repressed by bile acids, and the bile acid response element was localized between +137 and +220. Site-directed mutagenesis of the HNF4α-binding site markedly reduced promoter activity and its response to bile acid repression. On the other hand, mutation of the CPF-binding site had little effect on promoter activity and bile acid inhibition. A negative nuclear receptor, small heterodimer partner markedly inhibited transactivation of CYP8B1 by HNF4α. Mammalian two-hybrid assay confirmed that HNF4α interacted with small heterodimer partner. Furthermore, bile acids and farnesoid X receptor reduced the expression of nuclear HNF4α in HepG2 cells and rat livers and its binding to DNA. Bile acids and farnesoid X receptor also inhibited mouse HNF4α gene transcription. In summary, our data revealed the critical roles HNF4α play on CYP8B1transcription and its repression by bile acids. Bile acids repress human CYP8B1 transcription by reducing the transactivation activity of HNF4α through interaction of HNF4α with SHP and reduction of HNF4α expression in the liver. Sterol 12α-hydroxylase catalyzes the synthesis of cholic acid and controls the ratio of cholic acid over chenodeoxycholic acid in the bile. Transcription of CYP8B1is inhibited by bile acids, cholesterol, and insulin. To study the mechanism of CYP8B1 transcription by bile acids, we have cloned and determined 3389 base pairs of the 5′-upstream nucleotide sequences of the human CYP8B1. Deletion analysis of CYP8B1/luciferase reporter activity in HepG2 cells revealed that the sequences from −57 to +300 were important for basal and liver-specific promoter activities. Hepatocyte nuclear factor 4α (HNF4α) strongly activated human CYP8B1 promoter activities, whereas cholesterol 7α-hydroxylase promoter factor (CPF), an NR5A2 family of nuclear receptors, had much less effect. Electrophoretic mobility shift assay identified an overlapping HNF4α- and CPF-binding site in the +198/+227 region. The human CYP8B1 promoter activities were strongly repressed by bile acids, and the bile acid response element was localized between +137 and +220. Site-directed mutagenesis of the HNF4α-binding site markedly reduced promoter activity and its response to bile acid repression. On the other hand, mutation of the CPF-binding site had little effect on promoter activity and bile acid inhibition. A negative nuclear receptor, small heterodimer partner markedly inhibited transactivation of CYP8B1 by HNF4α. Mammalian two-hybrid assay confirmed that HNF4α interacted with small heterodimer partner. Furthermore, bile acids and farnesoid X receptor reduced the expression of nuclear HNF4α in HepG2 cells and rat livers and its binding to DNA. Bile acids and farnesoid X receptor also inhibited mouse HNF4α gene transcription. In summary, our data revealed the critical roles HNF4α play on CYP8B1transcription and its repression by bile acids. Bile acids repress human CYP8B1 transcription by reducing the transactivation activity of HNF4α through interaction of HNF4α with SHP and reduction of HNF4α expression in the liver. cholesterol 7α-hydroxylase gene sterol 12α-hydroxylase cholic acid chenodeoxycholic acid cholesterol 7α-hydroxylase promoter factor deoxycholic acid direct repeat electrophoretic mobility shift assay α-fetoprotein transcription factor farnesoid X receptor hepatocyte nuclear factor 4α hormone response element liver X receptor peroxisome proliferator-activated receptor α small heterodimer partner sterol response element-binding protein ursodeoxycholic acid bile acid response elements liver-related homologue kilobase pair Chinese hamster ovary Na+ taurocholate cotransport peptide phosphate-buffered saline retinoid X receptor High serum cholesterol contributes to atherosclerosis and cardiovascular diseases (1Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Crossref PubMed Scopus (4331) Google Scholar). The conversion of cholesterol to bile acids is the most significant pathway for cholesterol disposal and occurs exclusively in the liver (2Russell D.W. Setchell K.D. Biochemistry. 1992; 31: 4737-4749Crossref PubMed Scopus (653) Google Scholar, 3Russell D.W. Cardiovasc. Drugs Ther. 1992; 6: 103-110Crossref PubMed Scopus (149) Google Scholar, 4Chiang J.Y.L. Front. Biosci. 1998; 3: D176-D193Crossref PubMed Scopus (259) Google Scholar). The neutral pathway of bile acid synthesis is subjected to bile acid feedback inhibition of the rate-limiting step catalyzed by cholesterol 7α-hydroxylase (CYP7A1)1 (5Crestani M. Sadeghpour A. Stroup D. Galli G. Chiang J.Y. J. Lipid Res. 1998; 39: 2192-2200Abstract Full Text Full Text PDF PubMed Google Scholar). CYP8B1 catalyzes the synthesis of cholic acid and controls the ratio of cholic acid (CA) over chenodeoxycholic acid (CDCA) that determines the hydrophobicity of the bile acid pool (6Bjorkhem I. Eriksson M. Einarsson K. J. Lipid Res. 1983; 24: 1451-1456Abstract Full Text PDF PubMed Google Scholar). CYP8B1 was purified in 1992 (7Ishida H. Noshiro M. Okuda K. Coon M.J. J. Biol. Chem. 1992; 267: 21319-21323Abstract Full Text PDF PubMed Google Scholar) from rabbit livers. Recently, the cDNA and the gene encoding CYP8B1 were cloned in the rabbit, rat, mouse, and human (8Andersson U. Yang Y.Z. Bjorkhem I. Einarsson C. Eggertsen G. Gafvels M. Biochim. Biophys. Acta. 1999; 1438: 167-174Crossref PubMed Scopus (37) Google Scholar, 9Gafvels M. Olin M. Chowdhary B.P. Raudsepp T. Andersson U. Persson B. Jansson M. Bjorkhem I. Eggertsen G. Genomics. 1999; 56: 184-196Crossref PubMed Scopus (62) Google Scholar, 10Eggertsen G. Olin M. Andersson U. Ishida H. Kubota S. Hellman U. Okuda K.I. Bjorkhem I. J. Biol. Chem. 1996; 271: 32269-32275Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Interestingly, the CYP8B1 has no intron. 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Eggertsen G. Bjorkhem I. Hylemon P.B. Redford K. Pandak W.M. Gastroenterology. 2000; 118: 599-607Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). is also that be in bile acid repression of of to the bile acid repression. Deletion from to −57 the bile acid repression we the sequences from the between +137 and bile acid promoter activities were reduced HNF4α- and CPF-binding the bile acid effect on in HepG2 in the promoter activities of and which had the site the HNF4α site were suppressed by in the HNF4α site was site was in the the reporter activities. that HNF4α binding is and for bile acid repression of human CYP8B1 and be in bile acid repression of the Bile acids repress transcription by of SHP which with and transcription M. K. J. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, B. C. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). HNF4α was to play and had much effect on we the effect of SHP on HNF4α or of CYP8B1 transcription. A that of HNF4α of strongly human reporter activity by and of SHP have effect on CYP8B1 reporter with HNF4α and reporter activity was the the which was with SHP repressed the reporter activity by HNF4α. that HNF4α human reporter and with of SHP strongly repressed reporter activity in reporter activity by to with of the receptor over reporter SHP repressed the reporter activity by by much less its effect on reporter activity by HNF4α. two-hybrid assay to study the interaction between HNF4α and SHP in HepG2 cells of two-hybrid with and in of reporter with and in of reporter activity by over of with or also two-hybrid with and A of reporter activity by was and were for of reporter activity in HepG2 cells that HNF4α and SHP by two-hybrid is that bile acid repression of human CYP8B1 transcription be also to inhibition of HNF4α binding to CYP8B1 or inhibition of HNF4α expression in the effect of on HNF4α and binding to the to HepG2 cells were with with or with were from HepG2 cells and for nuclear of HepG2 cells with were for the was and the was Interestingly, nuclear of HepG2 cells with the shift nuclear from nuclear from HepG2 cells with and with were the were the HNF4α was for the nuclear were that and reduced HNF4α binding to DNA. to the expression of HNF4α was for the HNF4α binding to DNA. the nuclear HNF4α protein in HepG2 cells by assay The HNF4α protein in HepG2 cells was by the of and with HNF4α protein also with with or cholesterol for were from rat livers for that and markedly reduced the nuclear HNF4α protein and cholesterol the HNF4α protein the effect of on mouse reporter activity in assay in HepG2 cells repressed HNF4α reporter activity by with reduced reporter activity by and of repressed reporter activity by data revealed that bile acid repression of HNF4α transcription also to the inhibition of CYP8B1 transcription by bile acids, in to the repression by has revealed that is bile acid receptor that is activated by bile acids to the transcription of the in bile acid and cholesterol transcription J.Y. C. Stroup D. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M. K. J. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, B. C. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). play in cholesterol by the cholesterol from the to the liver for its conversion to bile acids. mechanism the bile acid pool in the liver cells from effect of bile acids. that the bile negative nuclear receptor SHP which with and transcription M. K. J. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, B. C. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, Yang Stroup D. Chiang J.Y.L. J. Lipid Res. Full Text Full Text PDF PubMed Google Scholar). was that the mechanism also CYP8B1 transcription and bile acid M. K. J. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, B. 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Full Text Full Text PDF PubMed Google Scholar). is important in human CYP8B1 transcription by mutagenesis analysis In study we revealed that bile acids were to repress nuclear HNF4α protein expression in HepG2 cells with and with and also in bile rat reporter activity was strongly repressed by and The rat HNF4α gene is by S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). interaction of SHP with repress transcription. M. Chiang J.Y. J. Lipid Res. 2000; Full Text Full Text PDF PubMed Google Scholar) that HNF4α protein expression was reduced by and to the inhibition of transcription by A. Galli G. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that bile acids could transcription by reducing the transactivation activity of HNF4α through protein our that HNF4α in bile acid synthesis and SHP nuclear with expression with other and gene expression liver and is that bile acid receptor receptor SHP that with other nuclear to repress gene transcription. The expression of nuclear in be to liver gene expression and and CYP8B1 transcription is strongly inhibited by bile acids, cholesterol, and insulin. The expression of CYP8B1 activity the bile acid hydrophobicity in the bile that the of bile acid synthesis by feedback inhibition of transcription. has that cholic acid and cholesterol by the of cholesterol in the B. J. 1999; PubMed Google Scholar). A cholesterol is to CYP8B1 transcription and in the hydrophobicity of the bile. that and transcription in J.Y. Stroup D. PubMed Scopus Google Scholar). cholesterol to the bile acid hydrophobicity and pool that the negative effect of over the effect of and repress transcription. On the other hand, the effect of over the negative effect of and in the of transcription in cholesterol strongly and CYP8B1 transcription and in the conversion of cholesterol to bile acids. is to the synthesis of cholesterol and by sterol response element-binding protein transcription and to in the with I. S. M.S. Goldstein J.L. U. S. A. 1999; PubMed Scopus Google Scholar). of the HNF4α gene have identified in with of the K. H. S. S. M. 1996; PubMed Scopus Google Scholar). Interestingly, of the SHP gene have identified in with H. H. M. S. K. I. K. I. T. K. Yang T. S. J. U. S. A. PubMed Scopus Google Scholar). that SHP is gene that activity in and and In bile acid synthesis and pool in with an in CYP8B1 activity H. C. Noshiro M. J. 2000; PubMed Scopus Google Scholar, H. C. Noshiro M. 1999; PubMed Scopus Google Scholar). is with our that SHP and HNF4α play important roles in human CYP8B1 transcription. the of CYP8B1transcription by bile acids, cholesterol, and insulin is important for the of and of In summary, we have mechanism of bile acid repression of human CYP8B1 transcription. Bile acids that in to nuclear and HNF4α the transcription of the in bile acid and and to the reduction of serum cholesterol levels and the of and other liver The of and Stroup is