Abstract
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.
Footnotes
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This article is partly based on A'edah Abu-Bakar's doctoral research that was funded by the University of Queensland International Postgraduate Research Scholarship (UQIPRS) and the International Postgraduate Research Scholarship (IPRS). The National Research Centre for Environmental Toxicology is funded by Queensland Health, the University of Queensland, Queensland University of Technology, and Griffith University, Australia. Jukka Hakkola was supported by the Academy of Finland (contract 110591).
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A.A.-B. and V.L. contributed equally to this work.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.106.014423.
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ABBREVIATIONS: P450, cytochrome P450; AHR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; XRE, xenobiotic response element; Nrf2, nuclear factor (erythroid-derived 2)-like 2; HO-1, haem oxygense-1; StRE, stress response element(s); CdCl2, cadmium chloride; PMSF, phenylmethylsulfonyl fluoride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; COH, coumarin 7-hydroxylase; PCR, polymerase chain reaction; ChIP, chromatin immunoprecipitation; bp, base pair; ANOVA, analysis of variance; EMSA, electrophoretic mobility shift assay(s); BR, bilirubin.
- Received December 19, 2006.
- Accepted February 12, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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