Redox-sensitive interaction between KIAA0132 and Nrf2 mediates indomethacin-induced expression of gamma-glutamylcysteine synthetase.

Sekhar KR, Spitz DR, Harris S, Nguyen TT, Meredith MJ, Holt JT, Gius D, Marnett LJ, Summar ML, Freeman ML, Guis D
Free Radic Biol Med. 2002 32 (7): 650-62

PMID: 11909699 · DOI:10.1016/s0891-5849(02)00755-4

Exposure of HepG2 cells to nonsteroidal anti-inflammatory drugs (i.e., indomethacin and ibuprofen; NSAIDs) as well as resveratrol, caused increased expression of the mRNAs coding for the catalytic (Gclc) and modifier (Gclm) subunits of the glutathione synthetic enzyme, gamma-glutamylcysteine synthetase. In addition, indomethacin exposure increased intracellular glutathione content as well as inhibited glutathione depletion and cytotoxicity caused by diethyl maleate. Indomethacin-induced increases in the expression of gamma-glutamylcysteine synthetase mRNA were preceded by increases in steady state levels of intracellular pro-oxidants and glutathione disulfide accumulation. Simultaneous incubation with the thiol antioxidant N-acetylcysteine (NAC) inhibited indomethacin-mediated increases in GCLC mRNA, suggesting that increases in GCLC message were triggered by changes in intracellular oxidation/reduction (redox) reactions. Indirect immunofluorescence using intact cells demonstrated that indomethacin induced the nuclear translocation of Nrf2, a transcription factor believed to regulate GCLC expression. Immunoprecipitation studies showed that indomethacin treatment also inhibited Nrf2 tethering to KIAA0132 (the human homolog of Keap1 accession #D50922), which is believed to be a negative regulator of Nrf2. Consistent with this idea, over-expression of Nrf2 increased GCLC reporter gene expression and over-expression of KIAA0132 inhibited GCLC reporter gene activity as well as inhibited indomethacin-induced increases in the expression of GCLC. Finally, simultaneous treatment with NAC inhibited both indomethacin-induced release of Nrf2 from KIAA0132 and indomethacin-induced nuclear translocation of Nrf2. These results demonstrate that NSAIDs and resveratrol cause increases in the expression of gamma-glutamylcysteine synthetase mRNA and identify these agents as being capable of stimulating glutathione metabolism. These results also support the hypothesis that indomethacin-induced transcriptional activation of GCLC involves the redox-dependent release of KIAA0132 from Nrf2 followed by the nuclear translocation of Nrf2.

MeSH Terms (24)

Anti-Inflammatory Agents, Non-Steroidal Blotting, Northern Carcinoma, Hepatocellular Carrier Proteins Chloramphenicol O-Acetyltransferase DNA-Binding Proteins Fluorescent Antibody Technique, Indirect Glutamate-Cysteine Ligase Glutathione Humans Ibuprofen Indomethacin Leucine Zippers Liver Neoplasms MAP Kinase Kinase 1 Mitogen-Activated Protein Kinase Kinases NF-E2-Related Factor 2 Oxidation-Reduction Promoter Regions, Genetic Protein-Serine-Threonine Kinases RNA, Messenger Trans-Activators Transfection Tumor Cells, Cultured

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