Original Contribution REDOX-SENSITIVE INTERACTION BETWEEN KIAA0132 AND Nrf2 MEDIATES INDOMETHACIN-INDUCED EXPRESSION OF -GLUTAMYLCYSTEINE SYNTHETASE KONJETI R. SEKHAR,* DOUGLAS R. SPITZ, † STEPHANIE HARRIS,* TRUNG T. NGUYEN,* MICHAEL J. MEREDITH, ‡ JEFFREY T. HOLT, § DAVID GUIS,  LAWRENCE J. MARNETT, ¶ MARSHALL L. SUMMAR,* and MICHAEL L. FREEMAN* *Dept of Radiation Oncology, Vanderbilt University School of Medicine, Nashville, TN, USA; † Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa, Iowa City, IA, USA; ‡ Oregon Health Science University School of Dentistry, Portland OR, USA; § Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN, USA;  Washington University School of Medicine, St. Louis, MO, USA; and ¶ Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA (Received 10 October 2001; Accepted 8 January 2002) Abstract—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, -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 -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 -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. © 2002 Elsevier Science Inc. Keywords—GCLC, GCLM, NSAIDs, Indomethacin, Resveratrol, Nrf2, KIAA0132, ARE, Glutathione, Glutathione disulfide, N-acetylcysteine, Free radicals INTRODUCTION Mammalian cells contain between 1 and 10 mM of the tripeptide L-glutamyl-L-cysteinylglycine (glutathione) [1]. The majority resides in the cytosol and the nucleus in the reduced form (GSH), with a small percentage exist- ing in an oxidized state as glutathione disulfide (GSSG). Under normal steady state conditions the ratio of intra- cellular GSH to GSSG is usually greater than 10:1, although this value can vary substantially between dif- ferent cell types [1]. About 10% of total cellular gluta- thione is located in the mitochondria. In the endoplasmic reticulum the ratio of GSH to GSSG is 3:1 [2]. The maintenance of high intracellular concentrations and the Address correspondence to: Michael L. Freeman, Ph.D., B 902 TVC Radiation Oncology, Vanderbilt University School of Medicine, Nash- ville, TN 37232, USA; Tel: (615) 322-3606; Fax: (615) 343-3061; E-Mail: michael.freeman@mcmail.vanderbilt.edu. Free Radical Biology & Medicine, Vol. 32, No. 7, pp. 650 – 662, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter PII S0891-5849(02)00755-4 650