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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.

SUBMITTER: Liu Y

PROVIDER: S-EPMC1829287 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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A genomic screen for activators of the antioxidant response element.

Liu Yanxia Y Kern Jonathan T JT Walker John R JR Johnson Jeffrey A JA Schultz Peter G PG Luesch Hendrik H

Proceedings of the National Academy of Sciences of the United States of America 20070312 12

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 pre ...[more]

PMID: 17360324

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Project description:The transcription factor NFE2L2 (Nuclear Factor, Erythroid 2-Like 2, or NRF2) plays a key role in maintaining the redox state within cells. Characterization of this pathway has extended to fish, most notably zebrafish (Danio rerio), in which two paralogs of the transcription factor exist: Nrf2a, an activator, and Nrf2b, a negative regulator during embryogenesis. Only one ARE target has been thoroughly delineated in zebrafish, and this deviated from the canonical sequence derived from studies in mammals. In general, the mechanistic pathway has not been characterized in non-model aquatic organisms that are commonly exposed to environmental pollutants. The current study compares the zebrafish paralogs to those found in a non-model teleost, the ecologically important salmonid, Oncorhnychus kisutch (coho salmon). Two salmon paralogs, Nrf2A and -2B, described here were found to possess only slightly greater identity between one another (84% of amino acids) than to the singleton ortholog of the esocid Esox lucius (80-82%), the nearest non-salmonid outgroup. Unlike one of the zebrafish forms, each is a strong activating factor based on sequence homology and in vitro testing. To uncover functional target AREs in coho, promoter flanking sequences were isolated for five genes that protect cells against oxidative stress: heme oxygenase 1, peroxiredoxin 1, glutamate-cysteine ligase, and the glutathione S-transferases pi and rho (hmox1, prdx1, gclc, gstp, and gstr). All except gstr had functional elements and all fit the standard mammalian-derived canonical sequence, unlike the motif found in zebrafish gstp. Expression studies demonstrate the presence of both Nrf2 paralogs in multiple organs, although in differing ratios. Collectively, our findings extend the conservation of Nrf2 and the ARE to salmonids, and should help inform future work in teleosts on mechanisms of redox control, as well as responsiveness of this pathway and its downstream antioxidant gene targets to chemical exposures in the environment.

Project description:Oxidative stress after burn injury induces inflammatory and hypermetabolic responses associated with adverse outcomes. We propose that antioxidant and trace element supplementation may reduce oxidative stress and subsequently alleviate inflammation and hypermetabolism, thus improving clinical outcomes. We conducted a cohort study of adult patients with an acute burn injury admitted to our provincial burn center. Patients in the antioxidant group received an intravenous infusion of multivitamins and trace elements for the first 14 days after admission. The inflammatory profile was assessed at early time points, < 14 days postburn, and later time points, ≥ 15 days postburn, and included interleukin (IL)-1β, interferon-γ, IL-1 receptor antagonist, IL-6, granulocyte-macrophage colony-stimulating factor, and FMS-like tyrosine kinase 3 ligand. Hypermetabolism was assessed by resting energy expenditure. Clinical outcomes included mortality, morbidities, hospital length of stay, and infections including days to the last positive culture after injury. We studied 172 patients, mean age 49 ± 17 years and 33 ± 13% TBSA burned, with 91 controls and 81 patients in the antioxidant group. Patients in the antioxidant group had significantly lower levels of inflammatory markers at both early and late time points, P < .05. Antioxidant treatment was associated with decreased measure of hypermetabolism, P < .05. Morbidity and mortality were not significantly different between groups. Length of hospital stay was significantly shorter in the antioxidant group when adjusted for patient demographics and injury characteristics (risk ratio (RR), 0.78; 95% confidence interval (CI), 0.66-0.92). In the antioxidant group, while infections were not different, the last positive culture post-injury was documented at median 19 days (Interquartile range (IQR), 11-43 days) compared with controls at 35 days (IQR, 15-59 days), P = .012. Patients receiving antioxidant and trace element supplementation had reduced markers of burn stress-induced inflammation; they were also associated with a decreased hypermetabolic response, shorter length of stay, and improved bacterial clearance.

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A genomic screen for activators of the antioxidant response element.

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A genomic screen for activators of the antioxidant response element.

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