Regulation of CYP2A5 gene by the transcription factor nuclear factor (erythroid-derived 2)-like 2.
We have previously shown that cadmium, a metal that alters cellular redox status, induces CYP2A5 expression in nuclear factor (erythroid-derived 2)-like 2 wild-type (Nrf2+/+) mice but not in the knockout (Nrf2-/-) mice. In the present studies, the potential role of Nrf2 in cadmium-mediated regulation of Cyp2a5 gene was investigated in mouse primary hepatocytes. Cadmium chloride (CdCl2) caused a time-dependent induction of the CYP2A5 at mRNA, protein, and activity levels, with a substantial increase observed within 3 h of exposure. Immunoblotting showed cadmium-dependent nuclear accumulation of Nrf2 within 1 h of exposure. Cotransfection of mouse primary hepatocytes with Cyp2a5 promoter-luciferase reporter plasmids and Nrf2 expression plasmid resulted in a 3-fold activation of Cyp2a5 promoter-mediated transcription relative to the control. Deletion analysis of the promoter localized the Nrf2 responsive region to an area from -2656 to -2339 base pair. Computer-based sequence analysis identified two putative stress response elements (StRE) within the region at positions -2514 to -2505 and -2386 to -2377. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that interaction of the more proximal StRE with Nrf2 was stimulated by CdCl2. Finally, site-directed mutagenesis of the proximal StRE in Cyp2a5 promoter-luciferase reporter plasmids abolished Nrf2-mediated induction. Collectively, the results indicate that Nrf2 activates Cyp2a5 transcription by directly binding to the StRE in the 5'-flanking region of the gene. This acknowledges Cyp2a5 as the first phase I xenobiotic-metabolizing gene identified under the control of the StRE-Nrf2 pathway with a potential role in adaptive response to cellular stress.
Abu-Bakar
A'edah
A
National Research Centre for Environmental Toxicology, University of Queensland, Brisbane, QLD, Australia. a.abubakar@uq.edu.au
Lämsä
Virpi
V
Arpiainen
Satu
S
Moore
Michael R
MR
Lang
Matti A
MA
Hakkola
Jukka
J
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects; D015153:Blotting, Western; D019256:Cadmium Chloride / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation / Q000379:methods; D065637:Cytochrome P-450 CYP2A6; D000072467:Cytochrome P450 Family 2; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008811:Mice, Inbred DBA; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D024363:Transcription Initiation Site
D008565:Membrane Proteins; D051267:NF-E2-Related Factor 2; D012333:RNA, Messenger; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585520:Cyp2a5 protein, mouse; D065637:Cytochrome P-450 CYP2A6; D000072467:Cytochrome P450 Family 2; D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse; D019256:Cadmium Chloride