title: Human constitutive androstane receptor mediates induction of CYP2B6 gene expression by phenytoin.
abstract: Compared with its rodent orthologs, little is known about the chemical specificity of human constitutive androstane receptor (hCAR) and its regulation of hepatic enzyme expression. Phenytoin (PHY), a widely used antiepileptic drug, is a potent inducer of CYP2B6 in primary human hepatocytes, but does not activate human pregnane X receptor (PXR) significantly in cell-based transfection assays at the same concentrations associated with potent induction of CYP2B6. Based on this observation, we hypothesized that PHY may be a selective activator of hCAR. In primary human hepatocytes, expression of CYP2B6 reporter genes containing phenobarbital-responsive enhancer module (PBREM) or PBREM/xenobiotic-responsive enhancer module (XREM) response elements were activated up to 14- and 28-fold, respectively, by 50 microm PHY. By contrast, parallel experiments in HepG2 cell lines co-transfected with an hPXR expression vector did not show increased reporter activity. These results indicated that a PXR-independent pathway, which is retained in primary hepatocytes, is responsible for PHY induction of CYP2B6. Further experiments revealed that PHY effectively translocates hCAR from the cytoplasm into the nucleus in both primary human hepatocytes and CAR(-/-) mice. Compared with vehicle controls, PHY administration significantly increased CYP2B6 reporter gene expression, when this reporter construct was delivered together with hCAR expression vector into CAR(-/-) mice. However, PHY did not increase reporter gene expression in CAR(-/-) mice in the absence of hCAR vector, implying that CAR is essential for mediating PHY induction of CYP2B6 gene expression. Taken together, these observations demonstrate that, in contrast to most of the known CYP2B6 inducers, PHY is a selective activator of CAR in humans.
authors.affiliation: Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina at Chapel Hill, North Carolina 27599, USA.
authors.forename: Hongbing
authors.initials: H
authors.lastname: Wang
authors.forename: Stephanie
authors.initials: S
authors.lastname: Faucette
authors.forename: Rick
authors.initials: R
authors.lastname: Moore
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sueyoshi
authors.forename: Masahiko
authors.initials: M
authors.lastname: Negishi
authors.forename: Edward
authors.initials: E
authors.lastname: LeCluyse
mesh_terms: D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D000090702:Constitutive Androstane Receptor; D065702:Cytochrome P-450 CYP2B6; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism*; D010672:Phenytoin / Q000494:pharmacology*; D000077297:Pregnane X Receptor; D011401:Promoter Regions, Genetic; D021381:Protein Transport / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000927:Anticonvulsants; D000090702:Constitutive Androstane Receptor; D000077297:Pregnane X Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D010672:Phenytoin; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating