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

Jong-Min LeeDagger §, Janean M. Hanson, Waihei A. Chu, and Jeffrey A. JohnsonDagger §||**DaggerDagger

From the Dagger  School of Pharmacy, the § Environmental Toxicology Center, the || Waisman Center, and the ** Center for Neuroscience, University of Wisconsin, Madison, Wisconsin 53706 and the  Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160-7417

Received for publication, January 25, 2001, and in revised form, March 21, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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) by tert-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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 glutathione S-transferase (GST) (1, 2). 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 (3-7). 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 (8). Itho et al. (9) 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 that tert-butylhydroquinone (tBHQ) dramatically induces Nrf2 nuclear translocation in human neuroblastoma cells (10). 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 (11, 12). Erk1/2 plays an important role in the regulation of cell growth and differentiation (13-16). Activation of Erk1/2 culminates in the phosphorylation of downstream factors such as p90RSK, c-Myc, and Elk-1, which control various cellular processes (17-19). 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 (20, 21). 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 (21-25). Among the downstream targets of PI3-kinase are phospholipase C and the serine/threonine kinase Akt (22, 23, 26, 27). 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 (28, 29). 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 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 alpha /beta 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.

Plasmids and Expression Vectors-- The reporter gene fusion construct for human NQO1-ARE (hNQO1-ARE-luciferase; 5'-CTCAGCCTTCCAAATCGCAGTCACAGTGACTCAGCAGAATC-3') was made as described previously (30). The mammalian expression vector for dominant negative (DN) Nrf2 was described previously (10). Plasmids for constitutively active PI3-kinase p110* (CA PI3-kinase) and kinase-deficient PI3-kinase p110*Delta kin (KD PI3-kinase) were kindly provided by Dr. Anke Klippel (31).

Transient Transfection and Reporter Gene Activity Assay-- 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 (10). To investigate the role of PI3-kinase, IMR-32 cells were cotransfected with the hNQO1-ARE-luciferase reporter construct (80 ng/well), CMV-beta -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-beta -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 beta -galactosidase activity were determined as described previously (10, 30). Data are expressed as the ratio of luciferase to beta -galactosidase activity.

Preparation of Whole Cell and Nuclear Extracts-- 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 (10).

Immunocomplex Kinase Assay of Akt-- Akt enzymatic activity was measured using a commercially available Akt-kinase assay kit (New England Biolabs) using GSK-3 alpha /beta as a substrate.

Western Immunoblot Analysis-- For Western immunoblot analysis, a whole cell extract (for NQO1, phospho-Erk1/2, Erk1/2, and phospho-GSK-3 alpha /beta ) or nuclear fraction (for Nrf2) of IMR-32 cells was resolved by SDS-PAGE. The transferred membranes were probed with an hNQO1 (10), phospho-Erk1/2, Erk1/2, phospho-GSK-3 alpha /beta , or Nrf2 antibody. Chemiluminescence was produced using SuperSignal West Pico chemiluminescent substrate (Pierce). Representative Western blots are shown in the figures.

Cytotoxicity Assay-- Cytotoxicity was measured using an MTS cytotoxicity assay kit (Promega, Madison, WI) according to the protocol provided by the manufacturer.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

tBHQ Does Not Increase Erk1/2 Phosphorylation-- 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. 1A) or effect the level of Erk1/2 protein (Fig. 1B) up to 24 h. In contrast, endogenous NQO1 protein induction was significant by 12 h in tBHQ-treated groups (Fig. 1C). It should be noted that the phosphorylation status of Erk1/2 varied with time in both vehicle- and tBHQ-treated cells (Fig. 1A). 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.


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Fig. 1.   Effect of tBHQ on Erk1/2 phosphorylation and NQO1 induction. A, to investigate time-dependent activation of Erk1/2, IMR-32 human neuroblastoma cells were treated with the vehicle (-) or tBHQ (+, 10 µM) for the indicated times. After incubation, whole cell extracts were prepared, and 50 µg of protein was analyzed for phosphorylation of Erk1/2 using phospho-Erk1/2 antibody. B, the same samples described in A were analyzed with control the Erk1/2 antibody for loading control. C, the same samples described in A were used for the analysis of time-dependent NQO1 protein induction by tBHQ.

A Selective PI3-kinase Inhibitor, LY 294002, Inhibits ARE Activation-- 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 and B, 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).


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Fig. 2.   Effect of LY 294002 on ARE activation and induction of NQO1. A, IMR-32 cells were transfected with the hNQO1-ARE-luciferase reporter construct (80 ng/well) and CMV-beta -galactosidase (20 ng/well). After 24 h, cells were pretreated with increasing doses of LY 294002 (LY). After 30 min of pretreatment, cells were treated with the vehicle (black-square) or tBHQ (, 10 µM). 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 beta -galactosidase activity. Each data point represents the mean ± S.E. (n = 6). B, IMR-32 cells were pretreated with increasing doses of LY 294002, followed by vehicle (-) or tBHQ (+, 10 µM) treatment. After 24 h of treatment, whole cell extracts were prepared, and 50 µg of protein was resolved by SDS-PAGE. The transferred membrane was probed with the NQO1 antibody.

ARE Activation by tBHQ Is Not Inhibited by PD 98059-- 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. 3A). In contrast, pretreatment with PD 98059 (50 µM) did not inhibit ARE activation by tBHQ (Fig. 3A). Similarly, endogenous NQO1 protein induction by tBHQ (10 µM) was decreased by only LY 294002 pretreatment (Fig. 3B). tBHQ did not increase the level of Erk1/2 phosphorylation, and PD 98059 completely blocked the basal level of phospho-Erk1/2 (Fig. 3C), indicating that PD 98059 was functioning as expected.


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Fig. 3.   Effects of PD 98059 and LY 294002 on hNQO1-ARE activation in IMR-32 cells. A, IMR-32 cells were transfected with the hNQO1-ARE-luciferase reporter construct (80 ng/well) and CMV-beta -galactosidase (20 ng/well). After 24 h, the cells were pretreated for 30 min with the vehicle (V, ethanol 0.1%), LY 294002 (LY, 20 µM), or PD 98059 (PD, 50 µM) and then treated with vehicle or tBHQ (T, 10 µM). 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 beta -galactosidase activity. Each bar represents the mean ± S.E. (n = 6). B, IMR-32 cells were pretreated with vehicle, LY 294002 (20 µM), or PD 98059 (50 µM) for 30 min followed by vehicle or tBHQ (10 µM) treatment. 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." C, IMR-32 cells were pretreated with the vehicle or PD 98059 (50 µM) for 30 min followed by treatment with the vehicle or tBHQ (10 µM) for another 30 min. Whole cell extracts were prepared, and 50 µg of protein was used for Western immunoblot analysis of phospho-Erk1/2 as described under "Experimental Procedures."

Erk1/2 Activation Does Not Correlate with Inhibition of ARE Activation-- Interestingly, LY 294002 increased Erk1/2 phosphorylation (Fig. 4A), 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 (32, 33). NGF was very effective at increasing Erk1/2 phosphorylation in IMR-32 cells (Fig. 4B), but NGF treatment neither increased hNQO1-ARE reporter gene expression nor inhibited ARE activation by tBHQ up to 500 ng/ml (Fig. 4C). Similarly, treatment with LY 294002 strongly activated Erk1/2 (Fig. 4A), but LY 294002 significantly inhibited hNQO1-ARE-luciferase expression as well as NQO1 protein increase by tBHQ (Figs. 2 and 3).


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Fig. 4.   Erk1/2 activation does not correlate with inhibition of ARE activation. A, IMR-32 cells were pretreated with the vehicle (V) or LY 294002 (LY, 20 µM) for 30 min and treated with vehicle or tBHQ (10 µM). Whole cell extracts were prepared, and 50 µg of protein was resolved and probed with phospho-Erk1/2 antibody as described under "Experimental Procedures." B, IMR-32 cells were treated with vehicle, NGF (100 ng/ml), or tBHQ (10 µM). After 30 min, whole cell extracts were prepared, and 50 µg of protein was resolved and probed with phospho-Erk1/2 antibody as described under "Experimental Procedures." C, IMR-32 cells were transfected with the hNQO1-ARE-luciferase reporter construct (80 ng/well) and CMV-beta -galactosidase (20 ng/well). After 24 h of transfection, the cells were pretreated for 30 min with NGF (0-500 ng/ml) or LY 294002 (20 µM) and then treated with the vehicle (open bar) or 10 µM tBHQ (filled bar). 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 beta -galactosidase activity. Each bar represents the mean ± S.E. (n = 6).

tBHQ Does Not Increase Akt Activity or GSK-3 beta  Phosphorylation-- Recent findings linking PI3-kinase to Akt and subsequent phosphorylation of GSK-3 beta  (34-36) lead us to speculate that tBHQ may activate this same pathway. Using insulin as a positive control (37), the data show that tBHQ does not increase Akt activity (Fig. 5A) or lead to increased phosphorylation of GSK-3 beta  (Fig. 5B). As expected, the effect of insulin on the phosphorylation of GSK-3 beta  was blocked completely by inhibitors of PI3-kinase activity (Fig. 5C). Finally, insulin did not increase either hNQO1-ARE-luciferase expression (Fig. 6A) or hNQO1 protein (Fig. 6B), implying that not all activators of PI3-kinase lead to ARE activation.


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Fig. 5.   tBHQ does not activate Akt activity or increase GSK-3 beta  phosphorylation. A, IMR-32 cells were treated with the vehicle, tBHQ (10 µM), or insulin (2.5 µg/ml) for 30 min. Akt activity was measured using a commercially available Akt-kinase assay kit. B, IMR-32 cells were treated with the vehicle, tBHQ (10 µM), or insulin (2.5 µg/ml) for 30 min. Whole cell extracts were prepared, and 80 µg of protein was resolved by SDS-PAGE as described under "Experimental Procedures." The transferred membrane was probed with phospho-GSK-3 alpha /beta antibody. C, IMR-32 cells were pretreated with the vehicle (V), LY 294002 (LY, 20 µM), or wortmannin (Wort, 1 µM). After 30 min, the cells were treated with the vehicle or insulin (I, 2.5 µg/ml) for another 30 min. Whole cell extracts were prepared, and 80 µg of protein was resolved by SDS-PAGE as described under "Experimental Procedures." The transferred membrane was probed with phospho-GSK-3 alpha /beta antibody.


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Fig. 6.   Insulin does not activate ARE. A, IMR-32 cells were transfected with the hNQO1-ARE-luciferase reporter construct (80 ng/well) and CMV-beta -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 beta -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."

PI3-kinase Regulates ARE-- 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. 7A), and that induction was inhibited completely by treatment with LY 294002 (Fig. 7B). Second, we recently reported that tBHQ treatment effectively induces nuclear translocation of Nrf2 in IMR-32 cells (10). Pretreatment of IMR-32 cells with the PI3-kinase inhibitors LY 294002 or wortmannin significantly decreased Nrf2 nuclear translocation induced by tBHQ (Fig. 8A). Finally, ARE activation mediated by CA PI3-kinase was blocked completely by DN Nrf2 (Fig. 8B), 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).


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Fig. 7.   Constitutively active PI3-kinase activates the ARE. A, IMR-32 cells were cotransfected with the hNQO1-ARE-luciferase reporter construct (80 ng/well), CMV-beta -galactosidase (20 ng/well), and the indicated amounts of the PI3-kinase plasmid. After 24 h, the cells were harvested in lysis buffer. Luciferase and galactosidase activities were measured as described under "Experimental Procedures." Each data point represents the mean ± S.E. (n = 6). B, the cells were cotransfected with the hNQO1-ARE-luciferase reporter construct (80 ng/well), CMV-beta -galactosidase (20 ng/well), and 40 ng/well of the PI3-kinase plasmid. After 24 h, the cells were treated with the vehicle or LY 294002 (20 µM) for another 24 h. Luciferase and beta -galactosidase activities were determined as described under "Experimental Procedures." Each bar represents the mean ± S.E. (n = 6).


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Fig. 8.   PI3-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-beta -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 beta -galactosidase activities were determined as described under "Experimental Procedures." Each data bar represents the mean ± S.E. (n = 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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. (38) reported increased Erk2 activity by tBHQ and positive regulation of ARE by Erk1/2 in HepG2 cells. In contrast, Alam et al. (39) 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 (4) published evidence that phospho-Erk1/2 was increased by pyrrolidine dithiocarbamate and proposed a positive role for Erk1/2 in the regulation of the gamma -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 (4, 39, 40) or negatively (41). In our system, the inhibition of p38 MAP kinase by SB 203580 did not effect tBHQ-induced hNQO1-ARE-luciferase expression.2 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 (3, 4, 10, 39, 42, 43). Nrf2 has been hypothesized to be sequestered by its cytoplasmic binding partner Keap1 (9). We have shown that tBHQ treatment dramatically increased Nrf2 nuclear translocation (10), 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 (7, 9, 44). 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 (10). A second hypothesis is based on data from Huang et al. (42), 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 (30). 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 (45). Although the relevance of these putative phosphorylation sites has not been demonstrated, Yu et al. (46) 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. (40) 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 (37), 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 (21).

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 (47) 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 H2O2 toxicity.3 Furthermore, recently published studies using Nrf2 null mice show that these mice are more sensitive to butylated hydroxytoluene-induced pulmonary toxicity (48) and acetaminophen-induced hepatic toxicity (49). 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.

    ACKNOWLEDGEMENTS

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.

    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.

Dagger Dagger To whom correspondence should be addressed: School of Pharmacy, University of Wisconsin, 425 N. Charter St., Madison, WI 53706. Tel.: 608-262-2893; Fax: 608-262-3397; E-mail: jajohnson@pharmacy.wisc.edu.

Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M100734200

2 J. M. Lee and J. A. Johnson, unpublished observations.

3 J. Li and J. A. Johnson, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: 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*Delta kin; CMV, cytomegalovirus; PAGE, polyacrylamide gel electrophoresis; NGF, nerve growth factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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