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

Free Radic Biol Med. 2002 Apr 1;32(7):650-62. 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Anti-Inflammatory Agents, Non-Steroidal / pharmacology*
  • 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
  • Glutamate-Cysteine Ligase / genetics
  • Glutamate-Cysteine Ligase / metabolism*
  • Glutathione / metabolism
  • Humans
  • Ibuprofen / pharmacology
  • Indomethacin / pharmacology*
  • Leucine Zippers
  • Liver Neoplasms / drug therapy*
  • Liver Neoplasms / enzymology
  • MAP Kinase Kinase 1
  • Mitogen-Activated Protein Kinase Kinases / metabolism
  • NF-E2-Related Factor 2
  • Oxidation-Reduction
  • Promoter Regions, Genetic
  • Protein Serine-Threonine Kinases / metabolism
  • RNA, Messenger / metabolism
  • Trans-Activators / genetics
  • Trans-Activators / metabolism*
  • Transfection
  • Tumor Cells, Cultured / drug effects
  • Tumor Cells, Cultured / enzymology
  • Tumor Cells, Cultured / metabolism


  • Anti-Inflammatory Agents, Non-Steroidal
  • Carrier Proteins
  • DNA-Binding Proteins
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • RNA, Messenger
  • Trans-Activators
  • Chloramphenicol O-Acetyltransferase
  • Protein Serine-Threonine Kinases
  • MAP Kinase Kinase 1
  • MAP2K1 protein, human
  • Map2k1 protein, mouse
  • Mitogen-Activated Protein Kinase Kinases
  • Glutamate-Cysteine Ligase
  • Glutathione
  • Ibuprofen
  • Indomethacin