title: Hepatocyte nuclear factor 4{alpha} regulates rifampicin-mediated induction of CYP2C genes in primary cultures of human hepatocytes.
abstract: CYP2C enzymes are expressed constitutively and comprise approximately 20% of the total cytochrome P450 in human liver. However, the factors influencing the transcriptional regulation of the CYP2C subfamily have only been addressed recently. In the present study, we used primary cultures of human hepatocytes to investigate the role of HNF4alpha in the pregnane X receptor (PXR)/rifampicin-mediated up-regulation of CYP2C8, CYP2C9, and CYP2C19 gene expression. We first identified new proximal cis-acting HNF4alpha sites in the proximal CYP2C8 promoter [at -181 base pairs (bp) from the translation start site] and the CYP2C9 promoter (at -211 bp). Both sites bound HNF4alpha in gel shift assays. Thus, these and recent studies identified a total of three HNF4alpha sites in the CYP2C9 promoter and two in the CYP2C8 promoter. Mutational studies showed that the HNF4alpha sites are needed for up-regulation of the CYP2C8 and CYP2C9 promoters by rifampicin. Furthermore, silencing of HNF4alpha abolished transactivation of the CYP2C8 and CYP2C9 promoters by rifampicin. Constitutive promoter activity was also decreased. Quantitative polymerase chain reaction analysis demonstrated that silencing HNF4alpha reduced the constitutive expression of CYP2C8 (53%), CYP2C9 (55%), and CYP2C19 (43%) mRNAs and significantly decreased the magnitude of the rifampicin-mediated induction of CYP2C8 (6.6- versus 2.7-fold), CYP2C9 (3- versus 1.5-fold), and CYP2C19 (1.8- versus 1.1-fold). These results provide clear evidence that HNF4alpha contributes to the constitutive expression of the human CYP2C genes and is also important for up-regulation by the PXR agonist rifampicin.
authors.affiliation: Human Metabolism Section, Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Ritu
authors.initials: R
authors.lastname: Rana
authors.forename: Yuping
authors.initials: Y
authors.lastname: Chen
authors.forename: Stephen S
authors.initials: SS
authors.lastname: Ferguson
authors.forename: Grace E
authors.initials: GE
authors.lastname: Kissling
authors.forename: Sailesh
authors.initials: S
authors.lastname: Surapureddi
authors.forename: Joyce A
authors.initials: JA
authors.lastname: Goldstein
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000328:Adult; D000368:Aged; D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D002478:Cells, Cultured; D000090702:Constitutive Androstane Receptor; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D051557:Hepatocyte Nuclear Factor 4 / Q000502:physiology*; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D000077297:Pregnane X Receptor; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects; D011987:Receptors, Steroid / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012293:Rifampin / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection
chemical_list: D000904:Antibiotics, Antitubercular; D000090702:Constitutive Androstane Receptor; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; C400322:cytochrome P-450 CYP2C subfamily; D003577:Cytochrome P-450 Enzyme System; D012293:Rifampin