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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*

  • Jong-Min Lee
    Affiliations
    School of Pharmacy, the

    Environmental Toxicology Center, the
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  • Janean M. Hanson
    Affiliations
    Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160-7417
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  • Waihei A. Chu
    Affiliations
    Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160-7417
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  • Jeffrey A. Johnson
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    School of Pharmacy, the

    Environmental Toxicology Center, the

    Waisman Center, and the

    Center for Neuroscience, University of Wisconsin, Madison, Wisconsin 53706 and the
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  • Author Footnotes
    * This work was supported by National Institutes of Environmental Health Sciences Grant ES08089 and the Burroughs Wellcome New Investigators in Toxicology Award.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:January 01, 2001DOI:https://doi.org/10.1074/jbc.M100734200
      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.
      ARE
      antioxidant responsive element
      NQO1
      NADPH:quinone oxidoreductase
      GST
      glutathione S-transferase
      Nrf2
      NF-E2-related factor 2
      Erk1/2
      extracellular signal-regulated kinase
      MAP
      mitogen-activated protein
      PI3-kinase
      phosphatidylinositol 3-kinase
      tBHQ
      tert-butylhydroquinone
      GSK
      glycogen synthase kinase
      h
      human
      DN
      dominant negative
      CA PI3-kinase
      constitutively active PI3-kinase p110*
      KD PI3-kinase
      kinase-deficient PI3-kinase p110*Δkin
      CMV
      cytomegalovirus
      PAGE
      polyacrylamide gel electrophoresis
      NGF
      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) (
      • Rushmore T.H.
      • Pickett C.B.
      ,
      • Prestera T.
      • Talalay P.
      ). 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 (
      • Wild A.C.
      • Moinova H.R.
      • Mulcahy R.T.
      ,
      • Zipper L.M.
      • Mulcahy R.T.
      ,
      • Venugopal R.
      • Jaiswal A.K.
      ,
      • Venugopal R.
      • Jaiswal A.K.
      ,
      • Itho K.
      • Chiba T.
      • Takahasi S.
      • Ishii T.
      • Igarashi K.
      • Katoh Y.
      • Oyake T.
      • Hayashi N.
      • Satoh K.
      • Hatayama I.
      • Yamamoto M.
      • Nabeshima Y.
      ). 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 (
      • Moi P.
      • Chan K.
      • Asunis I.
      • Cao A.
      • Kan Y.W.
      ). Itho et al. (
      • Itho K.
      • Wakabayashi N.
      • Katoh Y.
      • Ishii T.
      • Igarashi K.
      • Engel J.D.
      • Yamamoto M.
      ) 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 (
      • Lee J.M.
      • Moehlenkamp J.D.
      • Hanson J.M.
      • Johnson A.J.
      ). 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 (
      • Crews C.M.
      • Alessandrini A.A.
      • Erikson R.L.
      ,
      • Seger R.
      • Krebs E.G.
      ). Erk1/2 plays an important role in the regulation of cell growth and differentiation (
      • Hill C.S.
      • Treisman R.
      , ,
      • Marshall C.J.
      ,
      • Pang L.
      • Sawada T.
      • Decker S.J.
      • Saltiel A.R.
      ). Activation of Erk1/2 culminates in the phosphorylation of downstream factors such as p90RSK, c-Myc, and Elk-1, which control various cellular processes (
      • Davis R.J.
      ,
      • Whitmarsh A.J.
      • Davis R.J.
      ,
      • Gutkind J.S.
      ). 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 (
      • Klippel A.
      • Escobedo J.A.
      • Hu Q.
      • Williams L.T.
      ,
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ). 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 (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ,
      • Rameh L.E.
      • Rhee S.G.
      • Spokes K.
      • Kazlauskas A.
      • Cantley L.C.
      • Cantley L.G.
      ,
      • Sabbatini P.
      • McCormick F.
      ,
      • Jiang B.H.
      • Aoki M.
      • Zheng J.Z.
      • Li J.
      • Vogt P.K.
      ). Among the downstream targets of PI3-kinase are phospholipase C and the serine/threonine kinase Akt (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ,
      • Rameh L.E.
      • Rhee S.G.
      • Spokes K.
      • Kazlauskas A.
      • Cantley L.C.
      • Cantley L.G.
      ,
      • Falasca M.
      • Logan S.K.
      • Lehto V.P.
      • Baccante G.
      • Lemmon M.A.
      • Schlessinger J.
      ,
      • Le Good J.A.
      • Ziegler W.H.
      • Parekh D.B.
      • Alessi D.R.
      • Cohen P.
      • Parker P.J.
      ). 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 (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ). 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.

      DISCUSSION

      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. (
      • Yu R.
      • Lei W.
      • Mandlekar S.
      • Weber M.J.
      • Der C.J.
      • Wu J.
      • Kong A.T.
      ) reported increased Erk2 activity by tBHQ and positive regulation of ARE by Erk1/2 in HepG2 cells. In contrast, Alam et al. (
      • Alam J.
      • Wicks C.
      • Stewart D.
      • Gong P.
      • Touchard C.
      • Otterbein S.
      • Choi A.M.K.
      • Burow M.E.
      • Tou J.
      ) 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 (
      • Zipper L.M.
      • Mulcahy R.T.
      ) 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 (
      • Zipper L.M.
      • Mulcahy R.T.
      ,
      • Alam J.
      • Wicks C.
      • Stewart D.
      • Gong P.
      • Touchard C.
      • Otterbein S.
      • Choi A.M.K.
      • Burow M.E.
      • Tou J.
      ,
      • Kang K.W.
      • Ryu J.H.
      • Kim S.G.
      ) or negatively (
      • Yu R.
      • Mandlekar S.
      • Lei W.
      • Fahl W.E.
      • Tan T.H.
      • Kong A.T.
      ). In our system, the inhibition of p38 MAP kinase by SB 203580 did not effect tBHQ-induced hNQO1-ARE-luciferase expression.
      J. 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 (
      • Wild A.C.
      • Moinova H.R.
      • Mulcahy R.T.
      ,
      • Zipper L.M.
      • Mulcahy R.T.
      ,
      • Lee J.M.
      • Moehlenkamp J.D.
      • Hanson J.M.
      • Johnson A.J.
      ,
      • Alam J.
      • Wicks C.
      • Stewart D.
      • Gong P.
      • Touchard C.
      • Otterbein S.
      • Choi A.M.K.
      • Burow M.E.
      • Tou J.
      ,
      • Huang H.C.
      • Nguyen T.
      • Pickett C.B.
      ,
      • Alam J.
      • Stewart D.
      • Touchard C.
      • Boinapally S.
      • Choi A.M.
      • Cook J.L.
      ). Nrf2 has been hypothesized to be sequestered by its cytoplasmic binding partner Keap1 (
      • Itho K.
      • Wakabayashi N.
      • Katoh Y.
      • Ishii T.
      • Igarashi K.
      • Engel J.D.
      • Yamamoto M.
      ). We have shown that tBHQ treatment dramatically increased Nrf2 nuclear translocation (
      • Lee J.M.
      • Moehlenkamp J.D.
      • Hanson J.M.
      • Johnson A.J.
      ), 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 (
      • Itho K.
      • Chiba T.
      • Takahasi S.
      • Ishii T.
      • Igarashi K.
      • Katoh Y.
      • Oyake T.
      • Hayashi N.
      • Satoh K.
      • Hatayama I.
      • Yamamoto M.
      • Nabeshima Y.
      ,
      • Itho K.
      • Wakabayashi N.
      • Katoh Y.
      • Ishii T.
      • Igarashi K.
      • Engel J.D.
      • Yamamoto M.
      ,
      • Ishii T.
      • Itho K.
      • Takahasi S.
      • Sato H.
      • Yanagawa T.
      • Katoh Y.
      • Bannai S.
      • Yamamoto M.
      ). 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 (
      • Lee J.M.
      • Moehlenkamp J.D.
      • Hanson J.M.
      • Johnson A.J.
      ). A second hypothesis is based on data from Huang et al. (
      • Huang H.C.
      • Nguyen T.
      • Pickett C.B.
      ), 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 (
      • Moehlenkamp J.D.
      • Johnson J.A.
      ). 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 (
      • Hayes J.D.
      • Ellis E.M.
      • Neal G.E.
      • Harrison D.J.
      • Manson M.M.
      ). Although the relevance of these putative phosphorylation sites has not been demonstrated, Yuet al. (
      • Yu R.
      • Chen C.
      • Mo Y.Y.
      • Hebbar V.
      • Owuor E.D.
      • Tan T.H.
      • Kong A.T.
      ) 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. (
      • Kang K.W.
      • Ryu J.H.
      • Kim S.G.
      ) 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 (
      • Ruderman N.B.
      • Kapeller R.
      • White M.F.
      • Cantley L.C.
      ), 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 (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ).
      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 (
      • Duffy S.
      • So A.
      • Murphy T.H.
      ) 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.
      J. 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 (
      • Chan K.
      • Kan Y.W.
      ) and acetaminophen-induced hepatic toxicity (
      • Enomoto A.
      • Itoh K.
      • Nagayoshi E.
      • Haruta J.
      • Kimura T.
      • O'Connor T.
      • Harada T.
      • Yamamoto M.
      ). 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.

      Acknowledgments

      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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