Redox-sensitive interaction between KIAA0132 and Nrf2 mediates indomethacin-induced expression of γ-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, Michael L. Freeman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

65 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)650-662
Number of pages13
JournalFree Radical Biology and Medicine
Issue number7
StatePublished - Apr 1 2002


  • ARE
  • Free radicals
  • GCLC
  • GCLM
  • Glutathione
  • Glutathione disulfide
  • Indomethacin
  • KIAA0132
  • N-acetylcysteine
  • NSAIDs
  • Nrf2
  • Resveratrol

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)


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