Cytochrome P450 2C11 5'-flanking region and promoter: regulation by aromatic hydrocarbons in vitro.
Aromatic hydrocarbons elicit toxic and adaptive responses via the aryl hydrocarbon receptor (AHR). Aromatic hydrocarbons suppress the transcription of the growth hormone-regulated, male-specific rat hepatic cytochrome P450 2C11 gene (CYP2C11) in vivo via an unknown mechanism. We hypothesize that the suppression of CYP2C11 by aromatic hydrocarbons is mediated by the gene's promoter and 5'-flanking region. Following bioinformatic analysis of putative transcription factor (TF) binding sites, we cloned extended lengths of the CYP2C11 5'-flanking region into a promoterless luciferase plasmid. Suppression of CYP2C11 constructs was not observed upon treatment of transfected rat 5L, BP8 or mouse Hepa-1 cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylcholanthrene. In human HepG2 cells, the 10.1-kb construct displayed a pronounced 6- to 8-fold induction by TCDD. Deletion analysis localized the paradoxical induction response to a region between -1.8 kb and -1.3 kb, which contains a dioxin-responsive element (DRE) previously shown by us to be capable of binding activated AHR. This was confirmed by site-directed mutagenesis of the DRE. Induction of the 10.1-kb construct by TCDD in HepG2 cells was blocked by alpha-naphthoflavone, an AHR antagonist/partial agonist. The AHR is likely involved in the induction of CYP2C11-luciferase activity by TCDD in HepG2 cells and this response is at least partly DRE-mediated. Although CYP2C11 is suppressed by aromatic hydrocarbons in vivo, CYP2C11-luciferase constructs display a potentially misleading paradoxical induction in vitro that is cell-specific. Regulation of CYP2C11-luciferase plasmids is being studied in vivo in rat liver, where an intact endocrine system and the full complement of TFs needed for CYP2C11 suppression are present.
Sawaya
Rana M
RM
Department of Pharmacology & Toxicology, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Riddick
David S
DS
D024506:5' Flanking Region / Q000502:physiology*; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D001571:Benzoflavones / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor / Q000187:drug effects; D003001:Cloning, Molecular; D000072467:Cytochrome P450 Family 2; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008748:Methylcholanthrene / Q000633:toxicity*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000037:antagonists & inhibitors; D039181:Steroid 16-alpha-Hydroxylase / Q000235:genetics* / Q000378:metabolism
D001571:Benzoflavones; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D008748:Methylcholanthrene; C011512:alpha-naphthoflavone; D008156:Luciferases; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase