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

• Published in: Free radical biology & medicine 2002-04, Vol.32 (7), p.650-662
• Main Authors: Sekhar, Konjeti R, Spitz, Douglas R, Harris, Stephanie, Nguyen, Trung T, Meredith, Michael J, Holt, Jeffrey T, Guis, David, Marnett, Lawrence J, Summar, Marshall L, Freeman, Michael L
• Format: Article
• Language: English
• Subjects: Anti-Inflammatory Agents, Non-Steroidal - pharmacology; ARE; Blotting, Northern; Carcinoma, Hepatocellular - drug therapy; Carcinoma, Hepatocellular - enzymology; Carrier Proteins - metabolism; Chloramphenicol O-Acetyltransferase - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fluorescent Antibody Technique, Indirect; Free radicals; GCLC; GCLM; Glutamate-Cysteine Ligase - genetics; Glutamate-Cysteine Ligase - metabolism; Glutathione; Glutathione - metabolism; Glutathione disulfide; Humans; Ibuprofen - pharmacology; Indomethacin; Indomethacin - pharmacology; KIAA0132; Leucine Zippers; Liver Neoplasms - drug therapy; Liver Neoplasms - enzymology; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase Kinases - metabolism; N-acetylcysteine; NF-E2-Related Factor 2; Nrf2; NSAIDs; Oxidation-Reduction; Promoter Regions, Genetic; Protein-Serine-Threonine Kinases - metabolism; Resveratrol; RNA, Messenger - metabolism; Trans-Activators - genetics; Trans-Activators - metabolism; Transfection; Tumor Cells, Cultured - drug effects; Tumor Cells, Cultured - enzymology; Tumor Cells, Cultured - metabolism
• ISSN: 0891-5849; 1873-4596
• 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, γ-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 γ-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 γ-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.