PKC Downstream of PI3-Kinase Regulates Peroxynitrite Formation for Nrf2-Mediated GSTA2 Induction

  • Kim, Sang-Geon (College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University) ;
  • Kim, Sun-Ok (College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University)
  • Published : 2004.07.01


The protective adaptive response to electrophiles and reactive oxygen species is mediated by the induction of phase II detoxifying genes including glutathione S-transferases (GSTs). NF-E2-related factor-2 (Nrf2) phosphorylation by protein kinase C (PKC) is a critical event for its nuclear translocation in response to oxidative stress. Previously, we have shown that peroxynitrite plays a role in activation of Nrf2 and Nrf2 binding to the antioxidant response element (ARE) via the pathway of phosphatidylinositol 3-kinase (PI3-kinase) and that nitric oxide synthase in hepatocytes is required for GSTA2 induction. In view of the importance of PKC and Pl3-kinase in Nrf2-mediated GST induction, we investigated the role of these kinases in peroxynitrite formation for GSTA2 induction by oxidative stress and determined the relationship between PKC and PI3-kinase. Although PKC activation by phorbol 12-myristate-13-acetate (PMA) did not increase the extents of constitutive and inducible GSTA2 expression, either PKC depletion by PMA or PKC inhibition by staurosporine significantly inhibited GSTA2 induction by tert-butylhydroquinone (t-SHa) a prooxidant chemical. Therefore, the basal PKC activity is req- uisite for GSTA2 induction. 3-Morpholinosydnonimine (SIN-1), which decomposes and yields peroxynitrite, induced GSTA2, which was not inhibited by PKC depletion, but slightly enhanced by PKC activation, suggesting that PKC promotes peroxynitrite formation for Nrf2-mediated GSTA2 induction. Treatment of cells with S-nitroso-N-acetyl-penicillamine (SNAP), an exogenous NO donor, in combination with t-BHQ may produce peroxynitrite. GSTA2 induction by SNAP + t-BHQ was not decreased by PKC depletion, but rather enhanced by PKC activation, showing that the activity of PKC might be required for peroxynitrite formation. LY294002 a P13-kinase inhibitor blocked GSTA2 induction by t-BHQ, which was reversed by PMA-induced PKC activation. These results provide evidence that PKC may playa role in formation of peroxynitrite that activates Nrf2 for GSTA2 induction and that PKC may serve an activator for GSTA2 induction downstream of PI3-kinase.


  1. Cho, M. K., Kim, Y. G., Lee, M. G., and Kim, S. G., The effect of cysteine on the altered expression of class alpha and mu glutathione S-transferase genes in the rat liver during proteincalorie malnutrition. Biochim. Biophys. Acta, 1502, 235-246 (2000)
  2. Huang, H. C., Nguyen T., and Pickett, C. B, Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor-2. Proc. Natl. Acad. Sci. U.S.A., 97, 12475-12480 (2000)
  3. Huang, H. C., Nguyen, T., and Pickett, C. B., Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription. J. Biol. Chem., 277, 42769-42774 (2002)
  4. Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, T, O'Connor, T, and Yamamoto, M., Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of Nrf2 in response to electrophiles. Genes Cells, 8, 379-391 (2003)
  5. Kang, K. W., Ryu, J. H., and Kim, S. G., The essential role of phosphatidylinositol 3-kinase and of p38 mitogen-activated protein kinase activation in the antioxidant response elementmediatedrGSTA2 induction by decreased glutathione in H4IIE hepatoma cells. Mol. Pharmacol., 58, 1017-1025 (2000)
  6. Kang, K. W, Cho, M. K., Lee, C. H., and Kim, S. G., Activation of phosphatidylinositol 3-kinase and Akt by ten-butylhydroquinone is responsible for antioxidant response element-mediated rGSTA2 induction in H4IIE cells. Mol. Pharmacol., 59, 1147-1156 (2001)
  7. Kang, K. W., Choi, S. H., and Kim, S. G., Peroxynitrite activates NF-E2-related factor 2/antioxidant response element through the pathway of phosphatidylinositol 3-kinase: the role of nitric oxide synthase in rat glutathione S-transferase A2 induction. Nitric Oxide, 7, 244-253 (2002)
  8. Kang, K. W., Choi, S. Y., Cho, M. K., Lee, C. H., and Kim, S. G., Thrombin induces nitric-oxide synthase via G alpha 12/13coupled protein kinase C-dependent I-kapa B alpha phos-phorylation and JNK-mediated I-kappa B alpha degradation. J. Biol. Chem., 278, 17368-17378 (2003)
  9. Kang, K. W., Cho, I. J., Lee, C. H., and Kim, S. G., Essential role of phosphatidylinositol 3-kinase-dependent CCAAT/enhancer binding protein beta activation in the induction of glutathione S-transferase by oltipraz. J. Natl. Cancer. Inst., 95, 53-66 (2003)
  10. Kim, S. G., Nam, S. Y., Kim, J. H., Cho, C. K., and Yoo, S. Y., Enhancement of radiation-inducible hepatic glutathione $transferase Ya1, Yb1, Yb2, Yc1, and Yc2 expression by oltipraz: Possible role in radioprotection. Mol. Pharmecol., 51, 225-233 (1997)
  11. Klotz, L. O., Schieke, S. M., Sies, H., and Holbrook, N. J., Peroxynitrite activates the phosphoinositide 3-kinase/Akt pathway in human skin primary fibroblasts. Biochem. J., 352, 219-225 (2000)
  12. Liu, S. and Pickett, C. B., The rat liver glutathione S-transferase Ya subunit gene: Characterization of the binding properties of a nuclear protein from HepG2 cells that has high affinity for the antioxidant response element. Biochemistry, 35, 11517-11521 (1996)
  13. Marshall, H. E., Merchant, K., and Stamler, J. S., Nitrosation and oxidation in the regulation of gene expression. FASEB J., 14, 1889-1900 (2000)
  14. McMahon, M., Itoh, K., Yamamoto, M., Chanas, S. A., Henderson, C. J., McLellan, L. I., Wolf, C. R., Cavin, C., and Hayes, J. D., The Cap'n'Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. Cancer Res., 61, 3299-3307 (2001)
  15. Nguyen, T. and Pickett, C. B., Regulation of rat glutathione S-transferase Ya subunit gene expression. DNA-protein interaction at the antioxidant responsive element. J. Biol. Chem., 267,13535-13539 (1992)
  16. Numazawa, S., Ishikawa, M., Yoshida, A., Tanaka, S., and Yoshida, T, Atypical protein kinase C mediates activation of NF-E2-related factor 2 in response to oxidative stress. Am. J. Physiol. Cell Physiol., 285, C334-342 (2003)
  17. Park, H. S., Huh, S. H., Kim, M. S., Lee S. H., and Choi, E. J., Nitric oxide negatively regulates $\sigma$-Jun N-terminal kinase/stress-activated protein kinase by means of S-nitrosylation. Proc. Natl. Acad. Sci. U.S.A .., 97, 14382-14387 (2000)
  18. Shah, B. H. and Catt, K. J., Calcium-independent activation of extracellularly regulated kinases 1 and 2 by angiotensin II in hepatic C9 cells: Role of protein kinase C$\delta$, Src/proline-rich tyrosine kinase 2, and epidermal growth factor receptor trans-activation. Mol. Pharmacol., 61, 343-351 (2002)
  19. Vaughan, R. A., Huff, R. A., Uhi, G. R., and Kuhar, M. J., Protein kinase C-mediated phosphorylation and functional regulation of dopamine transporters in striatal synaptosomes. J. Biol. Chem., 272, 15541-15546 (1997)