Regulation of the Cyp2a5 gene involves an aryl hydrocarbon receptor-dependent pathway.
We have investigated the role of the aryl hydrocarbon receptor (AHR) in the regulation of the Cyp2a5 gene. The C57BL/6 and DBA/2 mouse strains with a genetically determined difference in AHR function were used to study the CYP2A5 induction by typical AHR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene. The CYP2A5 mRNA up-regulation in these mouse strains showed a difference in response, typical for AHR-regulated genes, both by TCDD in cultured primary hepatocytes and by 3-methylcholanthrene in vivo. In primary hepatocytes, TCDD caused a 3-fold elevation of the CYP2A5 protein level and a similar induction of the CYP2A5-catalyzed coumarin 7-hydroxylation activity. In reporter gene assays, the Cyp2a5 promoter region -3033 to +10 mediated a 2- to 5-fold induction of luciferase activity by TCDD treatment in primary hepatocytes and in Hepa-1 hepatoma cells with an intact AHR/AHR nuclear translocator (ARNT) complex. In Hepa-1 variant cell lines with deficiencies in the AHR/ARNT complex, the absence of ARNT abolished the induction. A putative AHR response element (XRE) was identified in the Cyp2a5 promoter at the position -2514 to -2492 and found to interact with the AHR/ARNT heterodimer. Transfection experiments combined with mutation of the XRE site indicated that the site partly mediates the TCDD induction of Cyp2a5. An additional AHR-dependent mechanism also regulates the proximal promoter of the Cyp2a5 gene. In conclusion, our studies showed that AHR ligands up-regulate Cyp2a5 transcriptionally by an AHR/ARNT-dependent mechanism and established Cyp2a5 as a novel AHR-regulated gene.
Arpiainen
Satu
S
Department of Pharmacology and Toxicology, P.O. Box 5000, University of Oulu, FIN-90014 Oulun yliopisto, Finland.
Raffalli-Mathieu
Françoise
F
Lang
Matti A
MA
Pelkonen
Olavi
O
Hakkola
Jukka
J
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D065637:Cytochrome P-450 CYP2A6; D000072467:Cytochrome P450 Family 2; D004268:DNA-Binding Proteins / Q000502:physiology; D004790:Enzyme Induction / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D006899:Mixed Function Oxygenases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D018336:Receptors, Aryl Hydrocarbon / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic
C497397:Arnt protein, mouse; D004268:DNA-Binding Proteins; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585520:Cyp2a5 protein, mouse; D065637:Cytochrome P-450 CYP2A6; D000072467:Cytochrome P450 Family 2