Hydroxynonenal induces glutathione synthesis through JNK

University Of California, Merced, Merced CA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): This competing renewal application focuses on induction of glutamate cysteine ligase (GCL), the rate limiting enzyme in glutathione (GSH) synthesis, by the lipid peroxidation product, 4-hydroxy-2-nonenal (HNE) through activation of EpRE and TRE cis elements and the JNK signaling pathway. A common response to sublethal exposure to oxidants and electrophiles is an increase in GSH concentration, which is critical to adaptation to greater stress. This increase is dependent on the increased expression of the catalytic and modulatory subunits of glutamate cysteine ligase (GCL, also called (-glutamylcysteine synthetase), the first enzyme in de novo GSH synthesis. HNE in healthy human plasma is 0.3-0.7 micromolar, but increases more than tenfold in inflammatory diseases and exposure to air pollutants. Although HNE can be toxic, at concentrations found in tissues under oxidative stress, it is a potent inducer of GSH, both GCL subunit genes, and other Phase II enzymes. We recently found that transcription factor switching appears to regulate GCL and that inhibition of JNK by a permeable dominant negative peptide completely inhibited GCL induction by HNE while neither ERK or p38MAPK inhibitors were effective. Our hypothesis is that induction of both human GCL genes by HNE occurs through alteration of the compositions of the EpRE and AP-1 binding complexes from those that repress to those that activate transcription. We also hypothesize that the alteration of composition of the EpRE and AP-1 binding complexes is mediated through HNE activation of the JNK signaling pathway. Using human bronchial epithelial cells (HBE1) for all studies, the specific aims of this proposal are to: 1- determine the composition of the complexes binding to the EpRE that regulate GCLC, GCLM, NQO1, and GST4A4 in response to HNE; 2- determine the mechanism of HNE by which HNE causes changing composition of the EpRE binding complexes; 3- determine how the changes in AP-1 complexes in response to HNE relate to the increase of GCL gene transcription; and 4- determine how HNE activates the JNK pathway. The approaches include DNA affinity chromatography, HPLC tandem mass spectrometry, immunodepletion EMSA, chromatin immunoprecipitation assays, Western blotting, protein translocation, protein phosphorylation measurements. Understanding the molecular basis of HNE induction of GCL to assist development of rational design of a drug with a less potential toxicity is the long range goal. [unreadable] [unreadable]