title: A genomic screen for activators of the antioxidant response element.
abstract: The antioxidant response element (ARE) is a cis-acting regulatory enhancer element found in the 5' flanking region of many phase II detoxification enzymes. Up-regulation of ARE-dependent target genes is known to have neuroprotective effects; yet, the mechanism of activation is largely unknown. By screening an arrayed collection of approximately 15,000 full-length expression cDNAs in the human neuroblastoma cell line IMR-32 with an ARE-luciferase reporter, we have identified several cDNAs not previously associated with ARE activation. A subset of cDNAs, encoding sequestosome 1 (SQSTM1) and dipeptidylpeptidase 3 (DPP3), activated the ARE in primary mouse-derived cortical neurons. Overexpression of SQSTM1 and DPP3 in IMR-32 cells stimulated NF-E2-related factor 2 (NRF2) nuclear translocation and led to increased levels of NAD(P)H:quinone oxidoreductase 1, a protein which is transcriptionally regulated by the ARE. When transfected into IMR-32 neuroblastoma cells that were depleted of transcription factor NRF2 by RNA interference, SQSTM1 and DPP3 were unable to activate the ARE or induce NAD(P)H:quinone oxidoreductase 1 expression, indicating that the ARE activation upon ectopic expression of these cDNAs is mediated by NRF2. Studies with pharmacological inhibitors indicated that 1-phosphatidylinositol 3-kinase and protein kinase C signaling are essential for activity. Overexpression of these cDNAs conferred partial resistance to hydrogen peroxide or rotenone-induced toxicity, consistent with the induction of antioxidant and phase II detoxification enzymes, which can protect from oxidative stress. This work and other such studies may provide mechanisms for activating the ARE in the absence of general oxidative stress and a yet-unexploited therapeutic approach to degenerative diseases and aging.
authors.affiliation: Department of Medicinal Chemistry, University of Florida, 1600 Southwest Archer Road, Gainesville, FL 32610, USA.
authors.forename: Yanxia
authors.initials: Y
authors.lastname: Liu
authors.forename: Jonathan T
authors.initials: JT
authors.lastname: Kern
authors.forename: John R
authors.initials: JR
authors.lastname: Walker
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Johnson
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
authors.forename: Hendrik
authors.initials: H
authors.lastname: Luesch
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis; D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects; D018076:DNA, Complementary / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D015723:Gene Library; D016678:Genome / Q000187:drug effects; D023281:Genomics / Q000379:methods*; D006360:Heat-Shock Proteins / Q000096:biosynthesis; D051379:Mice; D016660:NAD(P)H Dehydrogenase (Quinone); D009252:NADPH Dehydrogenase / Q000096:biosynthesis; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D020218:Response Elements / Q000187:drug effects / Q000235:genetics*; D000071456:Sequestosome-1 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000975:Antioxidants; D018076:DNA, Complementary; D006360:Heat-Shock Proteins; D051267:NF-E2-Related Factor 2; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D000071456:Sequestosome-1 Protein; C101343:Sqstm1 protein, mouse; D015534:Trans-Activators; D016660:NAD(P)H Dehydrogenase (Quinone); C491732:Nqo1 protein, mouse; D009252:NADPH Dehydrogenase