Phosphatidylinositol 3-Kinase, Not Extracellular Signal-regulated Kinase, Regulates Activation of the Antioxidant-Responsive Element in IMR-32 Human Neuroblastoma Cells

The antioxidant-responsive element (ARE) plays an important role in the induction of phase II detoxifying enzymes including NADPH:quinone oxidoreductase (NQO1). We report herein that activation of the human NQO1-ARE (hNQO1-ARE) bytert-butylhydroquinone (tBHQ) is mediated by phosphatidylinositol 3-kinase (PI3-kinase), not extracellular signal-regulated kinase (Erk1/2), in IMR-32 human neuroblastoma cells. Treatment with tBHQ significantly increased NQO1 protein without activation of Erk1/2. In addition, PD 98059 (a selective mitogen-activated kinase/Erk kinase inhibitor) did not inhibit hNQO1-ARE-luciferase expression or NQO1 protein induction by tBHQ. Pretreatment with LY 294002 (a selective PI3-kinase inhibitor), however, inhibited both hNQO1-ARE-luciferase expression and endogenous NQO1 protein induction. In support of a role for PI3-kinase in ARE activation we show that: 1) transfection of IMR-32 cells with constitutively active PI3-kinase selectively activated the ARE in a dose-dependent manner that was completely inhibited by treatment with LY 294002; 2) pretreatment of cells with the PI3-kinase inhibitors, LY 294002 and wortmannin, significantly decreased NF-E2-related factor 2 (Nrf2) nuclear translocation induced by tBHQ; and 3) ARE activation by constitutively active PI3-kinase was blocked completely by dominant negative Nrf2. Taken together, these data clearly show that ARE activation by tBHQ depends on PI3-kinase, which lies upstream of Nrf2. The antioxidant-responsive element (ARE) plays an important role in the induction of phase II detoxifying enzymes including NADPH:quinone oxidoreductase (NQO1). We report herein that activation of the human NQO1-ARE (hNQO1-ARE) bytert-butylhydroquinone (tBHQ) is mediated by phosphatidylinositol 3-kinase (PI3-kinase), not extracellular signal-regulated kinase (Erk1/2), in IMR-32 human neuroblastoma cells. Treatment with tBHQ significantly increased NQO1 protein without activation of Erk1/2. In addition, PD 98059 (a selective mitogen-activated kinase/Erk kinase inhibitor) did not inhibit hNQO1-ARE-luciferase expression or NQO1 protein induction by tBHQ. Pretreatment with LY 294002 (a selective PI3-kinase inhibitor), however, inhibited both hNQO1-ARE-luciferase expression and endogenous NQO1 protein induction. In support of a role for PI3-kinase in ARE activation we show that: 1) transfection of IMR-32 cells with constitutively active PI3-kinase selectively activated the ARE in a dose-dependent manner that was completely inhibited by treatment with LY 294002; 2) pretreatment of cells with the PI3-kinase inhibitors, LY 294002 and wortmannin, significantly decreased NF-E2-related factor 2 (Nrf2) nuclear translocation induced by tBHQ; and 3) ARE activation by constitutively active PI3-kinase was blocked completely by dominant negative Nrf2. Taken together, these data clearly show that ARE activation by tBHQ depends on PI3-kinase, which lies upstream of Nrf2. antioxidant responsive element NADPH:quinone oxidoreductase glutathione S-transferase NF-E2-related factor 2 extracellular signal-regulated kinase mitogen-activated protein phosphatidylinositol 3-kinase tert-butylhydroquinone glycogen synthase kinase human dominant negative constitutively active PI3-kinase p110* kinase-deficient PI3-kinase p110*Δkin cytomegalovirus polyacrylamide gel electrophoresis nerve growth factor The antioxidant-responsive element (ARE)1 plays an important role in transcriptional activation of several phase II detoxifying enzymes such as NADPH:quinone oxidoreductase (NQO1) and glutathioneS-transferase (GST) (1Rushmore T.H. Pickett C.B. J. Biol. Chem. 1990; 265: 14648-14653Abstract Full Text PDF PubMed Google Scholar, 2Prestera T. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8965-8969Crossref PubMed Scopus (219) Google Scholar). The consensus ARE core sequence in the human NQO1 gene (5′-TGACTCAGC-3′) is very similar to the DNA binding sequence for NF-E2-related factor 2 (Nrf2, 5′-TGAGTCA-3′). Several lines of evidence suggest that Nrf2 binds to the ARE sequence (3Wild A.C. Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1999; 274: 33627-33636Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 4Zipper L.M. Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; 278: 484-492Crossref PubMed Scopus (217) Google Scholar, 5Venugopal R. Jaiswal A.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14960-14965Crossref PubMed Scopus (929) Google Scholar, 6Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (486) Google Scholar, 7Itho K. Chiba T. Takahasi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Comm. 1997; 236: 313-322Crossref PubMed Scopus (3189) Google Scholar). Nrf2 was originally cloned using an AP1-NF-E2 tandem repeat as a recognition site probe and belongs to the basic leucine zipper family of transcription factors (8Moi P. Chan K. Asunis I. Cao A. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9926-9930Crossref PubMed Scopus (1236) Google Scholar). Itho et al. (9Itho K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2787) Google Scholar) suggest that Nrf2 is sequestered in the cytoplasm by Keap1 protein and that oxidative stress releases Nrf2 from the Nrf2-Keap1 complex, resulting in nuclear translocation of Nrf2. Recently our laboratory showed that activation of the human NQO1-ARE depends on Nrf2 and thattert-butylhydroquinone (tBHQ) dramatically induces Nrf2 nuclear translocation in human neuroblastoma cells (10Lee J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google Scholar). Although the role of Nrf2 in ARE activation seems evident, the upstream regulatory mechanisms by which ARE-activating signals are linked to Nrf2 and how this transcription factor is released from the Nrf2-Keap1 complex remain to be elucidated. Extracellular signal-regulated kinase (Erk1/2) is a member of the mitogen-activated protein (MAP) kinases, a serine/threonine kinase family (11Crews C.M. Alessandrini A.A. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8845-8849Crossref PubMed Scopus (71) Google Scholar, 12Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3205) Google Scholar). Erk1/2 plays an important role in the regulation of cell growth and differentiation (13Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1197) Google Scholar, 14Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2600) Google Scholar, 15Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4235) Google Scholar, 16Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar). Activation of Erk1/2 culminates in the phosphorylation of downstream factors such as p90RSK, c-Myc, and Elk-1, which control various cellular processes (17Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 18Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 74: 589-607Crossref PubMed Scopus (1389) Google Scholar, 19Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). Although there are several reports attempting to address the relationship between MAP kinases and ARE activation, the role of MAP kinases in ARE activation remains controversial, and the mechanism by which MAP kinases drive ARE activation through Nrf2 is unresolved. Phosphatidylinositol 3-kinase (PI3-kinase) phosphorylates phosphatidylinositol at the D-3 position of the inositol ring and has been shown to form a heterodimer consisting of a 85 kDa (adapter protein) and 110 kDa (catalytic) subunit (20Klippel A. Escobedo J.A. Hu Q. Williams L.T. Mol. Cell. Biol. 1993; 13: 5560-5566Crossref PubMed Scopus (87) Google Scholar, 21Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (838) Google Scholar). The role of PI3-kinase in intracellular signaling has been underscored by its implication in a plethora of biological responses such as cell growth, differentiation, apoptosis, calcium signaling, and insulin signaling (21Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (838) Google Scholar, 22Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1522) Google Scholar, 23Rameh L.E. Rhee S.G. Spokes K. Kazlauskas A. Cantley L.C. Cantley L.G. J. Biol. Chem. 1998; 273: 23750-23757Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 24Sabbatini P. McCormick F. J. Biol. Chem. 1999; 274: 24263-24269Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 25Jiang B.H. Aoki M. Zheng J.Z. Li J. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2077-2081Crossref PubMed Scopus (227) Google Scholar). Among the downstream targets of PI3-kinase are phospholipase C and the serine/threonine kinase Akt (22Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1522) Google Scholar, 23Rameh L.E. Rhee S.G. Spokes K. Kazlauskas A. Cantley L.C. Cantley L.G. J. Biol. Chem. 1998; 273: 23750-23757Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 26Falasca M. Logan S.K. Lehto V.P. Baccante G. Lemmon M.A. Schlessinger J. EMBO J. 1998; 17: 414-422Crossref PubMed Scopus (484) Google Scholar, 27Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (972) Google Scholar). Akt (protein kinase B), one of the most well known downstream targets of PI3-kinases, protects cells from apoptosis by the phosphorylation and inhibition of the Bad protein (28Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4946) Google Scholar, 29Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2218) Google Scholar). Based on these diverse effects of PI3-kinase (especially protective effects) and because the induction of phase II enzymes is thought to be a protective response in cells, we were interested in determining whether PI3-kinase is involved in ARE regulation. The present investigation was designed, therefore, to distinguish between the roles of Erk1/2 and PI3-kinase in ARE regulation using IMR-32 human neuroblastoma cells. tert-butylhydroquinone (tBHQ) was obtained from Acros Organics (St. Louis, MO). PD 98059, LY 294002, wortmannin, and insulin were purchased from Calbiochem. Antibodies for phospho-Erk1/2, Erk1/2, and phospho-GSK-3 α/β were obtained from New England Biolabs, Inc. (Beverly, MA). The Nrf2 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Tissue culture supplies were purchased from Atlanta Biologics (Norcross, GA), Life Technologies, Inc., and Midwest Scientific (St. Louis, MO). All other reagents were purchased from Fisher. The reporter gene fusion construct for human NQO1-ARE (hNQO1-ARE-luciferase; 5′-CTCAGCCTTCCAAATCGCAGTCACAGTGACTCAGCAGAATC-3′) was made as described previously (30Moehlenkamp J.D. Johnson J.A. Arch. Biochem. Biophys. 1999; 363: 98-106Crossref PubMed Scopus (70) Google Scholar). The mammalian expression vector for dominant negative (DN) Nrf2 was described previously (10Lee J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google Scholar). Plasmids for constitutively active PI3-kinase p110* (CA PI3-kinase) and kinase-deficient PI3-kinase p110*Δkin (KD PI3-kinase) were kindly provided by Dr. Anke Klippel (31Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (517) Google Scholar). IMR-32 human neuroblastoma cells (ATCC, CCL-127) were plated at a density of 2.5 × 104 cells/well in 96-well plates and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transient transfections were performed using the calcium phosphate methods as described previously (10Lee J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google Scholar). To investigate the role of PI3-kinase, IMR-32 cells were cotransfected with the hNQO1-ARE-luciferase reporter construct (80 ng/well), CMV-β-galactosidase (20 ng/well), and the CA PI3-kinase or KD PI3-kinase plasmid. To investigate the effect of DN Nrf2 on ARE activation by constitutively active PI3-kinase, IMR-32 cells were cotransfected with the hNQO1-ARE-luciferase (80 ng/well), CMV-β-galactosidase (20 ng/well), CA PI3-kinase (40 ng/well), and DN Nrf2 (5 ng/well). After 24 h of transfection, the cells were treated for another 24 h and harvested. Luciferase and β-galactosidase activity were determined as described previously (10Lee J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google Scholar, 30Moehlenkamp J.D. Johnson J.A. Arch. Biochem. Biophys. 1999; 363: 98-106Crossref PubMed Scopus (70) Google Scholar). Data are expressed as the ratio of luciferase to β-galactosidase activity. IMR-32 human neuroblastoma cells were plated at a density of 2.0 × 106 cells/10-cm dish and grown in 10 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were treated with various chemicals as described in each figure legend. After washing two times with cold phosphate-buffered saline, whole cell extracts and nuclear extracts were prepared as described previously (10Lee J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google Scholar). Akt enzymatic activity was measured using a commercially available Akt-kinase assay kit (New England Biolabs) using GSK-3 α/β as a substrate. For Western immunoblot a whole cell phospho-Erk1/2, Erk1/2, and phospho-GSK-3 or nuclear of IMR-32 cells was by The were with an (10Lee J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google phospho-Erk1/2, Erk1/2, phospho-GSK-3 or Nrf2 was using Western are shown in the was measured using an assay kit to the provided by the To investigate the relationship between Erk1/2 activation and ARE activation, we treated IMR-32 cells with a in phosphate-buffered or tBHQ and Western immunoblot for the phosphorylation of Erk1/2 as well as for the induction of shown in and tBHQ did not phosphorylation of Erk1/2 with or effect the of Erk1/2 protein to 24 In endogenous NQO1 protein induction was by h in be that the phosphorylation of Erk1/2 with in both and cells We the of the of be by a cell effect or a of of this tBHQ increased with the cells in not data suggest that tBHQ treatment endogenous NQO1 protein without Erk1/2 activity. to the of PI3-kinase in ARE activation, we a selective PI3-kinase LY shown in pretreatment with LY 294002 inhibited both expression and endogenous NQO1 in a dose-dependent a role for PI3-kinase in ARE of LY 294002 effect on cell as determined by the assay not To the of Erk1/2 in ARE regulation we IMR-32 cells with hNQO1-ARE-luciferase and treated with PD 98059, a selective of tBHQ treatment in a in hNQO1-ARE-luciferase expression that was significantly inhibited by pretreatment with LY 294002 In pretreatment with PD 98059 did not inhibit ARE activation by tBHQ endogenous NQO1 protein induction by tBHQ was decreased by LY 294002 pretreatment tBHQ did not the of Erk1/2 and PD 98059 completely blocked the of that PD 98059 was as LY 294002 increased Erk1/2 phosphorylation the that increased Erk1/2 activity be to the effect of LY To this IMR-32 cells were treated with nerve growth factor a of Erk1/2 J.M. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, Yao H. T. P.J. 1998; PubMed Scopus Google Scholar). was very at Erk1/2 phosphorylation in IMR-32 cells B), treatment increased reporter gene expression inhibited ARE activation by tBHQ to treatment with LY 294002 activated Erk1/2 LY 294002 significantly inhibited hNQO1-ARE-luciferase expression as well as NQO1 protein by tBHQ 2 and PI3-kinase to Akt and phosphorylation of GSK-3 Alessi D.R. Cohen P. M. 1995; PubMed Scopus Google Scholar, S.K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M. T. Y. S. K. A. A.C. N. M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) to that tBHQ this insulin as a control R. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google the data show that tBHQ not Akt activity or to increased phosphorylation of GSK-3 the effect of insulin on the phosphorylation of GSK-3 was blocked completely by of PI3-kinase activity insulin did not hNQO1-ARE-luciferase expression or protein B), that not of PI3-kinase to ARE not IMR-32 cells were with the hNQO1-ARE-luciferase reporter construct (80 and CMV-β-galactosidase (20 ng/well). After 24 h of transfection, the cells were treated with the tBHQ or insulin for After 24 h of the cells were and luciferase and were determined as described Data are expressed as the ratio of luciferase to β-galactosidase activity. the IMR-32 cells were treated with the tBHQ or insulin After 24 whole cell extracts were and of protein was for Western immunoblot of NQO1 as described evidence that PI3-kinase is involved in ARE activation is in and in IMR-32 cells with CA PI3-kinase or KD PI3-kinase, the CA PI3-kinase increased reporter gene expression and that induction was inhibited completely by treatment with LY 294002 we that tBHQ treatment induces nuclear translocation of Nrf2 in IMR-32 cells (10Lee J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google Scholar). Pretreatment of IMR-32 cells with the PI3-kinase LY 294002 or significantly decreased Nrf2 nuclear translocation induced by tBHQ ARE activation mediated by CA PI3-kinase was blocked completely by DN Nrf2 B), that Nrf2 is downstream of PI3-kinase in IMR-32 cells. KD PI3-kinase did not show a dominant negative effect on endogenous PI3-kinase, and ARE activation by tBHQ was not inhibited by KD PI3-kinase not is linked to IMR-32 cells were with the LY 294002 or for and treated with the or tBHQ 10 After nuclear extracts were and by The was with the Nrf2 the cells were cotransfected with the hNQO1-ARE-luciferase reporter construct (80 ng/well), CMV-β-galactosidase (20 ng/well), CA PI3-kinase (40 ng/well), and DN Nrf2 (5 ng/well). After 24 the cells were and Luciferase and β-galactosidase were determined as described data the In this we clearly showed that activation of the by tBHQ is mediated by PI3-kinase, not Erk1/2, in IMR-32 human neuroblastoma cells. tBHQ treatment increased protein without phospho-Erk1/2, and inhibition of Erk1/2 phosphorylation did not effect hNQO1-ARE-luciferase expression or protein induction. PI3-kinase inhibitors, however, significantly decreased both ARE activation and nuclear translocation of Nrf2 by tBHQ. In addition, ARE activation by constitutively active PI3-kinase was blocked completely by dominant negative the important role for PI3-kinase in ARE at the relationship between MAP kinases and the regulation of the et al. R. S. M.J. C.J. J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) increased activity by tBHQ and regulation of ARE by Erk1/2 in cells. In et al. J. P. S. M.E. J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) that increased inhibition of Erk1/2 did not effect induction in cells. and Mulcahy L.M. Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; 278: 484-492Crossref PubMed Scopus (217) Google Scholar) evidence that was increased by and a role for Erk1/2 in the regulation of the gene and its ARE in cells. in the present with IMR-32 cells, of Erk1/2 activity did not effect ARE MAP MAP has been to the ARE L.M. Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; 278: 484-492Crossref PubMed Scopus (217) Google Scholar, J. P. S. M.E. J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, S.G. Mol. 2000; PubMed Scopus Google Scholar) or R. S. T.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In our the inhibition of MAP kinase by did not effect hNQO1-ARE-luciferase M. and J. A. The data show that Erk1/2 such as LY 294002 and not ARE activation in IMR-32 cells. In LY 294002 significantly inhibited ARE activation in these neuroblastoma cells. Taken together, these suggest that activation of Erk1/2 not to activation of the ARE in cell and that the role of MAP kinases in gene expression is cell the the MAP kinases, is that Nrf2 and its translocation to the are to ARE activation in cell (3Wild A.C. Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1999; 274: 33627-33636Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 4Zipper L.M. Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; 278: 484-492Crossref PubMed Scopus (217) Google Scholar, J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google Scholar, J. P. S. M.E. J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, J. S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Nrf2 has been to be sequestered by its binding Keap1 (9Itho K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2787) Google Scholar). We shown that tBHQ treatment dramatically increased Nrf2 nuclear translocation (10Lee J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google that Nrf2 is released from Keap1 by treatment with tBHQ in IMR-32 cells. the mechanism by which Nrf2 is released from the Nrf2-Keap1 complex is not is that protein as at by oxidative stress releases Nrf2 from the Nrf2-Keap1 complex K. Chiba T. Takahasi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Comm. 1997; 236: 313-322Crossref PubMed Scopus (3189) Google Scholar, K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2787) Google Scholar, T. Itho K. Takahasi S. H. T. Katoh Y. S. Yamamoto M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). we that pretreatment of or antioxidant enzymes did not inhibit activation by tBHQ in IMR-32 cells, that activation by tBHQ not oxidative stress (10Lee J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google Scholar). is on data from et al. T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google that tBHQ or treatment induced the phosphorylation of Nrf2 through a protein kinase mechanism the of Nrf2. data from our laboratory suggest that protein kinase C is not involved in ARE activation in IMR-32 cells (30Moehlenkamp J.D. Johnson J.A. Arch. Biochem. Biophys. 1999; 363: 98-106Crossref PubMed Scopus (70) Google Scholar). to the MAP kinases and the phosphorylation of Nrf2 at several MAP kinase phosphorylation consensus the Nrf2 protein J.D. D.J. to U. Google Scholar). Although the of these phosphorylation has not been al. R. T.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) shown that dominant negative Nrf2 kinase kinase induction of and activation of the in cells. In this we PI3-kinase, not MAP as a regulatory protein to Nrf2 nuclear translocation and ARE activation in IMR-32 human neuroblastoma cells. et al. S.G. Mol. 2000; PubMed Scopus Google Scholar) that selective PI3-kinase decreased induction by in cells. the an these data are with our that NQO1 induction by tBHQ is blocked by LY In addition, we show that of PI3-kinase reporter gene activation and nuclear translocation of Nrf2 induced by tBHQ. the data that dominant negative Nrf2 completely blocked the increased hNQO1-ARE-luciferase expression by constitutively active a well known of PI3-kinase R. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google however, did not the that not of PI3-kinase in ARE we that the PI3-kinase for ARE activation is an PI3-kinase or phosphatidylinositol kinase (21Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (838) Google Scholar). laboratory and that increased expression of to the of cells to a of and S. A. T.H. J. 1998; PubMed Scopus Google Scholar) that pretreatment of neuroblastoma cells with that the ARE and NQO1 protects cells from and In addition, these that the of NQO1 in this cell did not to that the regulation of is for data from our laboratory that pretreatment of IMR-32 human neuroblastoma cells with tBHQ protects cells from Li and J. A. using Nrf2 show that these are to K. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar) and A. K. J. T. T. T. Yamamoto M. Sci. 2001; PubMed Scopus Google Scholar). The increased to these chemicals was with a expression of in the data the of how or cell control the expression of and the of expression on cell We Dr. for the DN Nrf2 expression vector and Dr. Anke Klippel for the PI3-kinase expression