title: AMP-activated protein kinase mediates phenobarbital induction of CYP2B gene expression in hepatocytes and a newly derived human hepatoma cell line.
abstract: Phenobarbital (PB) administration is known to trigger pleiotropic responses, including liver hypertrophy, tumor promotion, and induction of genes encoding drug-metabolizing enzymes. The induction of human CYP2B6 and the rat (CYP2B1) and mouse (Cyp2b10) homologues by PB is mediated by the nuclear receptor constitutive androstane receptor (CAR). The study of CYP2B gene regulation and CAR activity by PB has been difficult due to the lack of a cellular model. In this study, we describe a novel differentiated human hepatoma cell line (WGA), derived from HepG2, which expresses CYP2B6 and CAR. WGA cells represent a powerful system to study the regulation of CYP2B6 gene expression by PB. There is evidence that CAR activity is regulated by phosphorylation and that regulation of some CYP genes depends on the nutritional status of cells. The AMP-activated protein kinase (AMPK) functions as an energy sensor and is activated when cells experience energy-depleting stresses. In this report, we show that addition of 5-amino-imidazole carboxamide riboside, an AMPK activator, to WGA and human hepatocytes induces CYP2B6 gene expression. Expression of a constitutively active form of AMPK mimics the PB induction of CYP2B6 and CYP2B1 gene expression. Conversely, the expression of a dominant negative form of AMPK inhibits the induction of these genes by PB. Finally, we demonstrate, for the first time, that AMPK activity increases in cells cultured with PB. Our data strongly support a role for AMPK in the PB induction of CYP2B gene expression and provide new insights into the regulation of gene expression by barbiturate drugs.
authors.affiliation: Biomedical Research Centre and Cancer Research UK Molecular Pharmacology Unit, Level 5, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.
authors.forename: Franck
authors.initials: F
authors.lastname: Rencurel
authors.forename: Alasdair
authors.initials: A
authors.lastname: Stenhouse
authors.forename: Simon A
authors.initials: SA
authors.lastname: Hawley
authors.forename: Thomas
authors.initials: T
authors.lastname: Friedberg
authors.forename: D Grahame
authors.initials: DG
authors.lastname: Hardie
authors.forename: Calum
authors.initials: C
authors.lastname: Sutherland
authors.forename: C Roland
authors.initials: CR
authors.lastname: Wolf
mesh_terms: D055372:AMP-Activated Protein Kinases; D000255:Adenosine Triphosphate / Q000737:chemistry; D000256:Adenoviridae; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis* / Q000737:chemistry; D001463:Barbiturates / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D000090702:Constitutive Androstane Receptor; D003470:Culture Media; D019362:Cytochrome P-450 CYP2B1 / Q000096:biosynthesis; D065702:Cytochrome P-450 CYP2B6; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D005799:Genes, Dominant; D017930:Genes, Reporter; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D009097:Multienzyme Complexes / Q000502:physiology*; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis* / Q000737:chemistry; D010634:Phenobarbital / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012263:Ribonucleosides / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D001463:Barbiturates; D000090702:Constitutive Androstane Receptor; D003470:Culture Media; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D012263:Ribonucleosides; D014157:Transcription Factors; D000620:Aminoimidazole Carboxamide; C011651:acadesine; D012313:RNA; D000255:Adenosine Triphosphate; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D019362:Cytochrome P-450 CYP2B1; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D001616:beta-Galactosidase; D010634:Phenobarbital