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

2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês

10.1074/jbc.m100734200

ISSN

1083-351X

Autores

Jong‐Min Lee, Janean M. Hanson, Waihei A. Chu, Jeffrey A. Johnson,

Tópico(s)

Aldose Reductase and Taurine

Resumo

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. 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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. 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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 analysis, a whole cell extract (for NQO1, phospho-Erk1/2, Erk1/2, and phospho-GSK-3 α/β) or nuclear fraction (for Nrf2) of IMR-32 cells was resolved by SDS-PAGE. The transferred membranes were probed with an hNQO1 (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), phospho-Erk1/2, Erk1/2, phospho-GSK-3 α/β, or Nrf2 antibody. Chemiluminescence was produced using SuperSignal West Pico chemiluminescent substrate (Pierce). Representative Western blots are shown in the figures. Cytotoxicity was measured using an MTS cytotoxicity assay kit (Promega, Madison, WI) according to the protocol provided by the manufacturer. To investigate the relationship between Erk1/2 activation and ARE activation, we treated IMR-32 cells with a vehicle (0.01% Me2SO in phosphate-buffered saline) or tBHQ (10 μm) and carried out time-course Western immunoblot analysis for the phosphorylation of Erk1/2 as well as for the induction of NQO1. As shown in Fig.1, A and B, tBHQ did not increase phosphorylation of Erk1/2 compared with vehicle-treated controls (Fig. 1 A) or effect the level of Erk1/2 protein (Fig. 1 B) up to 24 h. In contrast, endogenous NQO1 protein induction was significant by 12 h in tBHQ-treated groups (Fig. 1 C). It should be noted that the phosphorylation status of Erk1/2 varied with time in both vehicle- and tBHQ-treated cells (Fig. 1 A). We think the change of the basal level of phospho-Erk1/2 might be caused by a cell cycle effect or a challenge of temperature change during treatment. Irrespective of this observation, tBHQ never consistently increased phospho-Erk1/2 when compared with the appropriate vehicle-treated cells in repeated experiments (data not shown). These data suggest that tBHQ treatment increase endogenous NQO1 protein without changing Erk1/2 activity. Initially, to evaluate the involvement of PI3-kinase in ARE activation, we used a selective PI3-kinase inhibitor, LY 294002. As shown in Fig. 2, A andB, pretreatment with LY 294002 inhibited both tBHQ-mediated NQO1-ARE-luciferase expression and endogenous NQO1 increase in a dose-dependent manner, suggesting a positive role for PI3-kinase in ARE activation. These concentrations of LY 294002 had no effect on cell viability as determined by the MTS cytotoxicity assay (data not shown). To evaluate the involvement of Erk1/2 in ARE regulation further, we transiently transfected IMR-32 cells with hNQO1-ARE-luciferase and treated them with PD 98059, a selective inhibitor of MAP/Erk kinase. tBHQ (10 μm) treatment resulted in a 57-fold increase in hNQO1-ARE-luciferase expression that was significantly inhibited by pretreatment with LY 294002 (Fig.3 A). In contrast, pretreatment with PD 98059 (50 μm) did not inhibit ARE activation by tBHQ (Fig. 3 A). Similarly, endogenous NQO1 protein induction by tBHQ (10 μm) was decreased by only LY 294002 pretreatment (Fig. 3 B). tBHQ did not increase the level of Erk1/2 phosphorylation, and PD 98059 completely blocked the basal level of phospho-Erk1/2 (Fig. 3 C), indicating that PD 98059 was functioning as expected. Interestingly, LY 294002 increased Erk1/2 phosphorylation (Fig. 4 A), raising the possibility that increased Erk1/2 activity may actually be contributing to the inhibitory effect of LY 294002. To test this hypothesis, IMR-32 cells were treated with nerve growth factor (NGF), a potent activator of Erk1/2 (32Volente C. Angelastro J.M. Greene L.A. J. Biol. Chem. 1993; 268: 21410-21415Abstract Full Text PDF PubMed Google Scholar, 33York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (757) Google Scholar). NGF was very effective at increasing Erk1/2 phosphorylation in IMR-32 cells (Fig. 4 B), but NGF treatment neither increased hNQO1-ARE reporter gene expression nor inhibited ARE activation by tBHQ up to 500 ng/ml (Fig.4 C). Similarly, treatment with LY 294002 strongly activated Erk1/2 (Fig. 4 A), but LY 294002 significantly inhibited hNQO1-ARE-luciferase expression as well as NQO1 protein increase by tBHQ (Figs. 2 and 3). Recent findings linking PI3-kinase to Akt and subsequent phosphorylation of GSK-3 β (34Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4375) Google Scholar, 35Moule S.K. Welsh G.I. Edgell N.J. Foulstone E.J. Proud C.G. Denton R.M. J. Biol. Chem. 1997; 272: 7713-7719Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 36Takata M. Ogawa W. Kitamura T. Hino Y. Kuroda S. Kotani K. Klip A. Gingras A.C. Sonenberg N. Kasuga M. J. Biol. Chem. 1999; 274: 20611-20618Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) lead us to speculate that tBHQ may activate this same pathway. Using insulin as a positive control (37Ruderman N.B. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (395) Google Scholar), the data show that tBHQ does not increase Akt activity (Fig. 5 A) or lead to increased phosphorylation of GSK-3 β (Fig. 5 B). As expected, the effect of insulin on the phosphorylation of GSK-3 β was blocked completely by inhibitors of PI3-kinase activity (Fig. 5 C). Finally, insulin did not increase either hNQO1-ARE-luciferase expression (Fig. 6 A) or hNQO1 protein (Fig. 6 B), implying that not all activators of PI3-kinase lead to ARE activation.Figure 6Insulin does not activate ARE.A, IMR-32 cells were transfected with the hNQO1-ARE-luciferase reporter construct (80 ng/well) and CMV-β-galactosidase (20 ng/well). After 24 h of transfection, the cells were treated with the vehicle, tBHQ (10 μm), or insulin (2.5 μg/ml) for 30 min. After 24 h of treatment, the cells were lysed, and luciferase and galactosidase activities were determined as described under “Experimental Procedures.” Data are expressed as the ratio of luciferase to β-galactosidase activity. Each bar represents the mean ± S.E. (n= 6). B, IMR-32 cells were treated with the vehicle, tBHQ (10 μm), or insulin (2.5 μg/ml). After 24 h, whole cell extracts were prepared, and 50 μg of protein was used for Western immunoblot analysis of NQO1 as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) More direct evidence that PI3-kinase is involved in ARE activation is presented in Figs.7 and 8. First, in IMR-32 cells transfected with CA PI3-kinase or KD PI3-kinase, only the CA PI3-kinase increased hNQO1-ARE reporter gene expression (Fig. 7 A), and that induction was inhibited completely by treatment with LY 294002 (Fig.7 B). Second, we recently reported that tBHQ treatment effectively 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 inhibitors LY 294002 or wortmannin significantly decreased Nrf2 nuclear translocation induced by tBHQ (Fig. 8 A). Finally, ARE activation mediated by CA PI3-kinase was blocked completely by DN Nrf2 (Fig. 8 B), suggesting 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 (data not shown).Figure 8PI3-kinase is linked to Nrf2.A, IMR-32 cells were pretreated with the vehicle (V), LY 294002 (LY, 20 μm) or wortmannin (Wort, 1 μm) for 30 min and treated with the vehicle or tBHQ (T, 10 μm). After 6 h, nuclear extracts were isolated and resolved by SDS-PAGE. The transferred membrane was probed with the Nrf2 antibody.B, 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 h, the cells were harvested, and Luciferase and β-galactosidase activities were determined as described under “Experimental Procedures.” Each data barrepresents the mean ± S.E. (n = 6).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this study, we clearly showed that activation of the hNQO1-ARE by tBHQ is mediated by PI3-kinase, not Erk1/2, in IMR-32 human neuroblastoma cells. tBHQ treatment increased hNQO1 protein without changing phospho-Erk1/2, and inhibition of Erk1/2 phosphorylation did not effect hNQO1-ARE-luciferase expression or hNQO1 protein induction. Selective 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 Nrf2, demonstrating the critically important role for PI3-kinase in Nrf2-dependent ARE activation. Recent publications have looked at the relationship between MAP kinases and the regulation of the ARE. Yu et al. (38Yu R. Lei W. Mandlekar S. Weber M.J. Der C.J. Wu J. Kong A.T. J. Biol. Chem. 1999; 274: 27545-27552Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) reported increased Erk2 activity by tBHQ and positive regulation of ARE by Erk1/2 in HepG2 cells. In contrast, Alam et al. (39Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M.K. Burow M.E. Tou J. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) reported that cadmium increased phospo-Erk1/2, but inhibition of Erk1/2 did not effect heme oxygenase-1 induction in MCF-7 cells. Recently, Zipper and Mulcahy (4Zipper L.M. Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; 278: 484-492Crossref PubMed Scopus (217) Google Scholar) published evidence that phospho-Erk1/2 was increased by pyrrolidine dithiocarbamate and proposed a positive role for Erk1/2 in the regulation of the γ-glutamylcysteine synthetase gene and its ARE in HepG2 cells. However, in the present study with IMR-32 cells, modulation of Erk1/2 activity did not effect Nrf2-dependent ARE activation. Another MAP kinase, p38 MAP kinase, also has been suggested to regulate the ARE positively (4Zipper L.M. Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; 278: 484-492Crossref PubMed Scopus (217) Google Scholar, 39Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M.K. Burow M.E. Tou J. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 40Kang K.W. Ryu J.H. Kim S.G. Mol. Pharmacol. 2000; 58: 1017-1025Crossref PubMed Scopus (105) Google Scholar) or negatively (41Yu R. Mandlekar S. Lei W. Fahl W.E. Tan T.H. Kong A.T. J. Biol. Chem. 2000; 275: 2322-2327Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). In our system, the inhibition of p38 MAP kinase by SB 203580 did not effect tBHQ-induced hNQO1-ARE-luciferase expression. 2J. M. Lee and J. A. Johnson, unpublished observations. The data show that strong Erk1/2 activators such as LY 294002 and NGF do not induce ARE activation in IMR-32 cells. In fact, LY 294002 significantly inhibited ARE activation in these neuroblastoma cells. Taken together, these observations suggest that activation of Erk1/2 does not necessarily lead to activation of the ARE in all cell types studied and that the role of MAP kinases in regulating ARE-driven gene expression is probably cell type-specific. Despite the controversy surrounding the MAP kinases, it is quite clear that Nrf2 and its translocation to the nucleus are principal to ARE activation in all cell types (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, 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, 39Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M.K. Burow M.E. Tou J. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 42Huang H.C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (440) Google Scholar, 43Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar). Nrf2 has been hypothesized to be sequestered by its cytoplasmic binding partner 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 have 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 Scholar), suggesting that Nrf2 is released from Keap1 by treatment with tBHQ in IMR-32 cells. However, the mechanism by which Nrf2 is released from the Nrf2-Keap1 complex is not characterized fully. One hypothesis is that protein modification (such as oxidation at cysteine residues) by oxidative stress releases Nrf2 from the Nrf2-Keap1 complex (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, 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, 44Ishii T. Itho K. Takahasi S. Sato H. Yanagawa T. Katoh Y. Bannai S. Yamamoto M. J. Biol. Chem. 2000; 275: 16023-16029Abstract Full Text Full Text PDF PubMed Scopus (1234) Google Scholar). Previously, we demonstrated that pretreatment of antioxidants or antioxidant enzymes did not inhibit hNQO1-ARE activation by tBHQ in IMR-32 cells, implying that hNQO1-ARE activation by tBHQ does not involve 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). A second hypothesis is based on data from Huang et al. (42Huang H.C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (440) Google Scholar), who reported that tBHQ or phorbol 12-myristate 13-acetate treatment induced the phosphorylation of Nrf2 through a protein kinase C-dependent mechanism leading the release of Nrf2. Again, 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). A third hypothesis brings us back to the MAP kinases and the potential phosphorylation of Nrf2 at several identified MAP kinase phosphorylation consensus sites throughout the Nrf2 protein (45Hayes J.D. Ellis E.M. Neal G.E. Harrison D.J. Manson M.M. Downes C.P. Wolf C.R. Lane D.P. Cellular Responses to Stress. Portland Press, London, U. K.1999: 141-168Crossref Google Scholar). Although the relevance of these putative phosphorylation sites has not been demonstrated, Yuet al. (46Yu R. Chen C. Mo Y.Y. Hebbar V. Owuor E.D. Tan T.H. Kong A.T. J. Biol. Chem. 2000; 275: 39907-39913Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar) have shown that dominant negative Nrf2 blocks MAP/Erk kinase kinase 1-mediated induction of heme oxygenase-1 and activation of the mouse GSTA1-ARE in HepG2 cells. In this study, we implicate PI3-kinase, not MAP kinase, as a key regulatory protein leading to Nrf2 nuclear translocation and subsequent ARE activation in IMR-32 human neuroblastoma cells. Recently, Kang et al. (40Kang K.W. Ryu J.H. Kim S.G. Mol. Pharmacol. 2000; 58: 1017-1025Crossref PubMed Scopus (105) Google Scholar) reported that selective PI3-kinase inhibitors decreased rat GSTA2 mRNA induction by sulfur amino acid deprivation in H4IIE rat hepatoma cells. Because the rat GSTA2 promoter contains an ARE, these data are consistent with our finding that NQO1 induction by tBHQ is blocked by LY 294002. In addition, we show that inhibitors of PI3-kinase block hNQO1-ARE reporter gene activation and nuclear translocation of Nrf2 induced by tBHQ. Furthermore, the data indicate that dominant negative Nrf2 completely blocked the increased hNQO1-ARE-luciferase expression by constitutively active PI3-kinase. Insulin, a well known activator of PI3-kinase (37Ruderman N.B. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (395) Google Scholar), however, did not activate the ARE, suggesting that not all activators of PI3-kinase result in ARE activation. Therefore, we speculate that the PI3-kinase responsible for ARE activation is an insulin-independent PI3-kinase or phosphatidylinositol kinase-related kinase (21Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (838) Google Scholar). Our laboratory and others propose that increased expression of ARE-driven genes contribute to the ability of cells to tolerate a variety of chemical-induced stresses. Murphy and co-workers (47Duffy S. So A. Murphy T.H. J. Neurochem. 1998; 71: 69-77Crossref PubMed Scopus (121) Google Scholar) have demonstrated that pretreatment (24 h) of rodent neuroblastoma cells with compounds that activate the ARE and induce NQO1 protects cells from H2O2 and dopamine-induced cytotoxicity. In addition, these investigators demonstrated that the stable overexpression of NQO1 in this cell line did not confer resistance to cytotoxicity, suggesting that the regulation of multiple genes is required for protection. Preliminary data from our laboratory also indicate that pretreatment (24 h) of IMR-32 human neuroblastoma cells with tBHQ protects cells from H2O2toxicity. 3J. Li and J. A. Johnson, unpublished observations. Furthermore, recently published studies using Nrf2 null mice show that these mice are more sensitive to butylated hydroxytoluene-induced pulmonary toxicity (48Chan K. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12731-12736Crossref PubMed Scopus (524) Google Scholar) and acetaminophen-induced hepatic toxicity (49Enomoto A. Itoh K. Nagayoshi E. Haruta J. Kimura T. O'Connor T. Harada T. Yamamoto M. Toxicol. Sci. 2001; 59: 169-177Crossref PubMed Scopus (637) Google Scholar). The increased sensitivity to these chemicals was correlated with a lower expression of ARE-driven genes in the respective tissues. These data demonstrate the importance of understanding how different tissues or cell types control the expression of ARE-driven genes and the potential impact of modulating their expression on cell survival. We thank Dr. Jawd Alam (Alton Ochsner Medical Foundation) for the DN Nrf2 expression vector and Dr. Anke Klippel (Chiron Corporation) for the PI3-kinase expression vector.

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