Abstract
Nf-E2 related factor-2 (Nrf2) is a basic leucine zipper transcription factor that binds and activates the antioxidant response element (ARE) in the promoters of many antioxidant and detoxification genes. We found that H2O2 treatment caused a rapid increase in endogenous Nrf2 protein level in rat cardiomyocytes. Semiquantitative or real-time reverse transcription-polymerase chain reaction failed to show an increase of Nrf2 mRNA level by H2O2 treatment. Measurements of Nrf2 protein stability excluded the possibility of Nrf2 protein stabilization. Although inhibiting protein synthesis with cycloheximide prevented H2O2 from elevating Nrf2 protein level, RNA synthesis inhibition with actinomycin D failed to do so. Measurements of new protein synthesis with [35S]methionine incorporation confirmed that H2O2 increased the translation of Nrf2 protein. Inhibitors of phosphoinositide 3-kinase were able to abolish the induction of Nrf2 protein by H2O2. Although H2O2 increased phosphorylation of p70 S6 kinase, rapamycin failed to inhibit H2O2 from elevating Nrf2 protein. H2O2 also induced phosphorylation of eukaryotic translation initiation factor (eIF) 4E and eIF2α within 30 and 10 min, respectively. Inhibiting eIF4E with small interfering siRNA or increasing eIF2α phosphorylation with salubrinal did not affect Nrf2 elevation by H2O2. Our data present a novel phenomenon of quick onset of the antioxidant/detoxification response via increased translation of Nrf2 by oxidants. The mechanism underlying such stress-induced de novo protein translation may involve multiple components of translational machinery.
Footnotes
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This work has been supported by the Burroughs Wellcome Foundation, American Heart Association, American Federation for Aging Research, Arizona Disease Control Research Commission, and National Institutes of Health (NIH) grants R01-ES010826 and NIH R01-HL076530-01 (Q.M.C.). S.E.P.-D. was supported for 1 year by NIH grant T32-ES007091. The Genomics Core facility of Southwest Environmental Health Sciences Center is supported by P30-ES006694.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.035360.
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ABBREVIATIONS: Nrf2, NF-E2 related factor-2; ARE, antioxidant response element; PI3, phosphatidylinositol 3; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; siRNA, small interfering RNA; eIF, eukaryotic translation initiation factor; PAGE, polyacrylamide gel electrophoresis; RT, reverse transcription; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; PCR, polymerase chain reaction; CXM, cycloheximide; UTR, untranslated region; IRES, internal ribosomal entry site; ER, endoplasmic reticulum.
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↵1 Current affiliation: Arizona Cancer Center, University of Arizona Health Sciences Center, Tucson, Arizona.
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↵2 Current affiliation: Department of Anesthesiology, Division of Molecular Medicine, University of California Los Angeles, Los Angeles, California.
- Received February 20, 2007.
- Accepted July 23, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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