Redox-sensitive interaction between KIAA0132 and Nrf2 mediates indomethacin-induced expression of gamma-glutamylcysteine synthetase.
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.
Sekhar
Konjeti R
KR
Dept of Radiation Oncology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Spitz
Douglas R
DR
Harris
Stephanie
S
Nguyen
Trung T
TT
Meredith
Michael J
MJ
Holt
Jeffrey T
JT
Gius
David
D
Marnett
Lawrence J
LJ
Summar
Marshall L
ML
Freeman
Michael L
ML
Guis
David
D
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D015152:Blotting, Northern; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000201:enzymology; D002352:Carrier Proteins / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007052:Ibuprofen / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology*; D016350:Leucine Zippers; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000201:enzymology; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D051267:NF-E2-Related Factor 2; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D012333:RNA, Messenger; D015534:Trans-Activators; D015500:Chloramphenicol O-Acetyltransferase; D017346:Protein Serine-Threonine Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D007052:Ibuprofen; D007213:Indomethacin