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J. Biol. Chem., Vol. 281, Issue 36, 26245-26252, September 8, 2006

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Nrf2-mediated Induction of Cytoprotective Enzymes by 15-Deoxy-^12,14-Prostaglandin J₂ Is Attenuated by Alkenal/one Oxidoreductase^*

Xiang Yu, Patricia A. Egner, Junko Wakabayashi, Nobunao Wakabayashi, Masayuki Yamamoto^¶, and Thomas W. Kensler¹

From the Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, the Division of Toxicology, Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205 and the ^¶Center for Tsukuba Advanced Research Alliance and Institute for Basic Medical Sciences, University of Tsukuba, Tsukuba, 305-8577 Japan

NADPH-dependent alkenal/one oxidoreductase (Aor) was discovered to be highly inducible in rat liver following treatment with the cancer chemopreventive agent 3H-1, 2-dithiole-3-thione. Aor was further characterized as an Nrf2-regulated antioxidative enzyme that reduces carbon-carbon double bonds in a variety of , -unsaturated aldehydes and ketones. 15-Deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂) is a reactive membrane lipid metabolite that activates multiple pathways, including Nrf2-mediated induction of cytoprotective enzymes. Physiologically, it is postulated that 15d-PGJ₂ alkylates key regulatory proteins via the electrophilic carbon centers found in two , -unsaturated ketone moieties. This current study addresses the metabolism of 15d-PGJ₂ by rat Aor (rAor) and subsequent deactivation of the Nrf2 signaling pathway by both rat and human AOR. We demonstrate that induction of NADPH-dependent quinone oxidoreductase activity by 15d-PGJ₂ is markedly attenuated in mouse embryonic fibroblasts that overexpress rAor. Luciferase reporter assay and quantitative real-time PCR confirmed these findings. Concentrations required for doubling the NADPH-dependent quinone oxidoreductase response are increased from 1.8µM in wild-type to >10µM in rat Aor transgenic fibroblasts. 15d-PGJ₂ is metabolized by recombinant rAor with a K_m of 9.6 µM and k_cat of 18.5 min^-1. The major product is 12,13-dihydro-15-deoxy-^12,14-prostaglandin J₂ (dihydro-15d-PGJ₂). The reduction of C=C by Aor yielding dihydro-15d-PGJ₂ abolishes the inducibility in an antioxidant response element-driven luciferase assay. Collectively, these results demonstrate that 15d-PGJ₂ can be catabolized by Aor, thereby attenuating subsequent Nrf2 signaling and possibly inflammatory and apoptotic processes also influenced by 15d-PGJ₂.

Received for publication, May 15, 2006 , and in revised form, June 30, 2006.

^* This work was supported by National Institutes of Health Grants CA39416 and ES06052 and Center Grant ES03819. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-4712; Fax: 410-955-0116; E-mail: tkensler{at}jhsph.edu.