Microarray analysis reveals an antioxidant responsive element-driven gene set involved in conferring protection from an oxidative stress-induced apoptosis in IMR-32 cells.

The present study was designed to investigate how tert-butylhydroquinone (tBHQ) prevents hydrogen peroxide-induced apoptosis in IMR-32 cells. tBHQ pretreatment (10 microm) attenuated hydrogen peroxide-induced cell death and reduced the number of TUNEL (terminal deoxynucleotidyltransferase-mediated, dUTP-incorporated nick end labeling)-positive cells. We hypothesize that tBHQ-mediated activation of the antioxidant responsive element is critical for generating this protective response. Addition of LY294002, a selective inhibitor of phosphatidylinositol 3-kinase (PI3K), 30 min prior to tBHQ treatment completely reversed the protective effect of tBHQ. Oligonucleotide microarrays were used to analyze the gene expression profile associated with tBHQ treatment in the absence and presence of LY294002. Ranking analysis using Affymetrix's difference call indicated that the expression of 137 genes changed with tBHQ treatment. Further analysis using the coefficient of variation for -fold change or average difference change reduced the list to 63 increased and 0 decreased genes. Reverse transcription-PCR for selected genes also confirmed the gene expression pattern. Many of these genes function to combat oxidative stress and increase the detoxification potential of the cells. Inhibition of PI3K significantly blocked the enhanced expression of 49 of the 63 genes induced by tBHQ. These data are the first to show a set of programmed cell life genes involved in conferring protection from an oxidative stress-induced apoptosis.

Accumulated evidence strongly suggests that apoptosis contributes to neuronal cell death in a variety of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease (1). Central to the apoptotic response is a family of aspartate-directed cysteine proteases termed the caspases. Caspases function both dependently and independently in the disruption of the mitochondria and its release of proapoptotic factors that are known to serve as signal transducers and positive effectors in the apoptotic pathway (2,3). Activation of the cysteine protease caspase-3 appears to be a key event in the execution of apoptosis in the central nervous system (CNS). 1 The CNS is particularly vulnerable to oxidative stress because of a high rate of oxidative metabolism, which results in high rates of strong oxidant formation. In addition, the CNS contains an abundance of polyunsaturated fatty acids that are susceptible to lipid peroxidation.
The cellular toxicity of hydrogen peroxide (H 2 O 2 ) is initiated by oxidative stress resulting in the rapid modification of cytoplasmic constituents, the depletion of intracellular glutathione (GSH) and ATP, a decrease in NAD ϩ level, an increase in free cytosolic Ca 2ϩ , and lipid peroxidation (4). H 2 O 2 also activates the mitochondria permeability transition pore and the release of cytochrome c (5). In the cytoplasm, cytochrome c, in combination with Apaf-1, activates caspase-9 leading to the activation of caspase-3 and subsequent apoptosis (6 -8). Because oxidative stress is involved in H 2 O 2 -induced cell death, modulation of antioxidant defenses, such as the increased concentration of the intracellular GSH, may protect cells from programmed cell death (PCD).
Others have shown that treating cells with tert-butylhydroquinone (tBHQ), a strong inducer of phase II detoxification enzymes via activation of the antioxidant responsive element (ARE), can protect cells from oxidative stress (9 -11). Induction of NAD(P)H:quinone oxidoreductase (NQO1) in N18-RE-105 neuronal cells by tBHQ prior to glutamate treatment was correlated with a significant decrease in glutamate toxicity (11). Glutamate toxicity in these cells is not due to N-methyl-Daspartate receptor activation and calcium influx. Rather, it is a result of competitive inhibition of cysteine uptake, depletion of GSH, increased oxidative stress, and apoptosis (9). Subsequent studies by Murphy and colleagues (10) using H 2 O 2 and dopamine to induce oxidative stress, however, demonstrated that N18-RE-105 cells overexpressing NQO1 were not resistant to cytotoxicity. These data suggest that the protective effect conferred by tBHQ may not simply be due to an increase in one gene but the coordinate up-regulation of many genes. We hypothesize that tBHQ-mediated activation of the ARE is a prin-cipal component generating this protective response.
Based on these observations and recent work in our laboratory characterizing a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism of ARE activation for tBHQ in IMR-32 human neuroblastoma cells (12,13), we were interested in identifying the ARE-driven gene set increased by tBHQ and correlating this information with the antiapoptotic effect of tBHQ (11). The present investigation was designed to: 1) determine if tBHQ pretreatment protects human neuroblastoma cells from H 2 O 2 -induced apoptosis; 2) evaluate how the inhibition of PI3K activity modulates any protective effect manifested by treatment with tBHQ; and 3) identify the set of genes conferring this protection using oligonucleotide microarray technology.
Cell Viability-Cell viability was determined using MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2terazolium, inner salt) as a substrate. Viability of remaining IMR-32 cells was determined as described in the protocol supplied with the CellTiter 96 nonRadioactive Cell Proliferation Assay kit, commercially available from Promega Corp. (Madison, WI). Early-passage IMR-32 cells seeded at a density of 4 ϫ 10 4 /well in a 96-well dish were pretreated with tBHQ for 24 h and then treated for 30 min or 1 h with H 2 O 2 at various concentrations from 100 to 800 M. Fresh medium was placed on the cells for 5 h followed by an MTS assay that was allowed to develop for another 4 h. In brief, a mixture of MTS and phenazine ethosulfate was added to the treated neuroblastoma cells cultured in 96-well plates. Following a 4-h incubation in at 37°C in a humidified 10% CO 2 atmosphere, aliquots of medium were collected and absorbance was read at 490 nm. Cell survival rate was calculated by A treatment / A control ϫ 100% (A represents the absorbance recorded at 490 nm).
Apoptosis Detection-Cells were harvested for TUNEL (terminal deoxynucleotidyltransferase-mediated, dUTP-incorporated nick end labeling) staining. The proportion of cells showing DNA fragmentation was measured by incorporation of fluorescein isothiocyanate (FITC)-12-dUTP into DNA by using terminal deoxynucleotidyltransferase (In Situ Cell Death Detection kit, FITC, Roche Molecular Biochemicals). Briefly, after 2-min incubation in permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate), slides were covered with the TUNEL mix (calf thymus terminal deoxynucleotidyltransferase, FITC-12-dUTP, and cobalt chloride) for 1.5 h at 37°C. Negative control was performed by omitting terminal deoxynucleotidyltransferase. DNase I-treated slides were used as positive control. The morphologic feature was visualized by fluorescence phase-contrast microscopy. Images were randomly collected, and cell counting was conducted under five bright and fluorescence fields (10ϫ), respectively. The total of cells numbered 1000. Quantitative analysis of apoptosis was represented by Apoptotic Index (AI), which is the number of FITC-positive cells (those with condensed chromosomes and fragmented nuclei) per 1000 cells counted (14).
Western Blot-Attached cells were lifted with virsene (containing 0.5 mM EDTA), and the detached cells in the supernatant were centrifuged 300 ϫ g for 3 min before resuspension in the lysis buffer (Tris-HCl, pH 7.4, containing 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mg/ml benzamidine, 0.1 mg/ml leupeptin, 0.1 mg/ml aprotinin, 1 mM sodium orthovanadate, and 2 mM dithiothreitol). The soluble faction was harvested by centrifugation at 300 ϫ g for 3 min after incubation on ice for 10 min. Equal amounts of protein were loaded, based on protein concentrations determined by the bicinchoninic acid (BCA) method according to the manufacture's instruction (Pierce, Rockford, IL). Before SDS-PAGE, SDS-reducing buffer (containing 0.5% Tris, 10% glycerol, 2% SDS, 5% (v/v) 2-mercaptoethanol, 0.01% bromphenol blue, pH 6.8) was added to cell lysates, and samples were boiled for 10 min at 95°C. SDS-PAGE and blotting were performed. The polyvinylidene difluoride membrane was incubated with PARP or cleaved caspase-3 antibody (1:1000) overnight at 4°C. Anti-rabbit IgG labeled with horseradish peroxidase was used as a secondary antibody (1:2000). The chemiluminescence emitted from luminal oxidize by horseradish peroxidase was detected by using the enhanced chemiluminescence Western blotting detection system (Amersham Biosciences, Inc., Piscataway, NJ).
Microarray Analysis-Cells were harvested, and total RNA was extracted by RNeasy Mini Kit (Qiagen). cDNA was synthesized from the total RNA by using Superscript choice kit (Invitrogen) with a T7-(dT) 24 primer incorporating a T7 RNA polymerase promoter. The cRNA was prepared and biotin-labeled by in vitro transcription (Enzo Biochemical). Labeled cRNA was fragmented by incubation at 94°C for 35 min in the presence of 40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate. Fifteen g of fragmented cRNA was hybridized 16 h at 45°C to an HG U95a array (Affymetrix, Santa Clara, CA). After hybridization, the gene chips were automatically washed and stained with streptavidin-phycoerythrin by using a fluidics station. Finally, probe arrays were scanned at 3-m resolution using the Genechip System confocal scanner made for Affymetrix by Aligent. Affymetrix Microarray Suite 4.1 was used to scan and analyze the relative abundance of each gene from the average difference of intensities. Analysis parameters used by the software were set to values corresponding to moderate stringency (SDT ϭ 30, SRT ϭ 1.5). Output from the microarray analysis was merged with the Unigene or GenBank TM descriptor and stored as an Excel data spreadsheet. The definition of increase, decrease, or no change of expression for individual genes was based on ranking the Difference Call from three inter-group comparisons (3 ϫ 3), namely No Change ϭ 0, Marginal Increase/Decrease ϭ 1/Ϫ1, Increase/Decrease ϭ 2/Ϫ2. The final rank referred to summing up the nine values corresponding to the Difference Calls, and the value varied from Ϫ18 to 18. The cutoff value for the final determination of Increase/Decrease was set as 9/Ϫ9.
RT-PCR-Validation of up-regulated expression was done by RT-PCR for seven selected genes. PCR primers specific to the genes of interest were used for cDNA synthesis and amplification as follows. We designed unique oligonucleotide primer pairs by using software PRIMER3 (available at www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and using sequence data from the NCBI data base. Primers for seven genes of interest were designed (prepared by IDT, Coralville, IA) for human NQO1, CATTCTGAAAGGCTGGTTTGA and TTTCTTCCATCCTTCCAGGAT; hemo oxygenase 1 (HO1), ACATCTATGTGGCCCTGGAG and GCGGTA-GAGCTGCTTGAACT; glutathione reductase (GR), GATCCCAAGCCCA-CAATAGA and AACCATGCTGACTTCCAAGC; ␥-glutamylcysteine ligase regulatory subunit (GCLR), TTTGGTCAGGGAGTTTCCAG and ACACAG-CAGGAGGCAAGATT; ␥-glutamylcysteine ligase catalytic subunit (GCLC), TGAGATTTAAGCCCCCTCCT and TTGGGATCAGTCCAGGAAAC; thioredoxin reductase (TR), ACAAGCCCTGCAAGACTCTC and CCTCT-GAGCCAGCAATCTTC; and ␤-actin, ATGGATGATGATATCGCCGC and GGGGTGTTGAAGGTCTCA. Total RNA, purified from cell pellets with TRIzol reagent (Invitrogen), was subjected to RT-PCR with the Promega Reverse Transcription System (Madison, WI). The reaction mix (20 l) contained 200 M dNTP, 0.45 M of each primer, 1 g of total RNA, and 15 units of avian myeloblastosis virus reverse transcriptase. RNA was reversetranscribed at 42°C for 30 min. DNA was amplified by an initial incubation at 94°C for 4 min followed by 25-35 cycles of 94°C for 0.5 min, 55-58°C for 0.6 min, 72°C for 0.5 min, and a final extension at 72°C for 7 min. The PCR products were then separated by electrophoresis in a 1.2% agarose gel and visualized by ethidium bromide staining. The number of cycles and melting temperature was adjusted depending on the genes amplified.
Statistical Analysis-All the experimental data shown were repeated at least three times, unless otherwise indicated. Results are presented as mean Ϯ S.E. Experimental groups were compared by a one-tailed, unequal S.D., t test. A statistical probability of p Ͻ 0.05 was considered significant.

H 2 O 2 -induced
Apoptosis in IMR-32 Cells-As described in current literature, the toxic dose of H 2 O 2 varies with both the density of cells and the cell types studied. In addition, its cytotoxicity is dependent on the concentration expressed as picomolar/cell rather than the absolute concentration in micromolar (15). This information led us to carefully evaluate the effects of cell density, dose, and time of exposure on H 2 O 2mediated cytotoxicity (Fig. 1). Pretreatment with 10 M tBHQ (24 h) significantly increased cell viability following treatment with H 2 O 2 for 30 min beginning at 200 M and continuing to at least 800 M (Fig. 1A). There was also a time-dependent loss of cell viability at a dose of 200 M H 2 O 2 (Fig. 1, B and C). Density did not change the protective effect of tBHQ even though there were dramatic differences in the slopes of the death curves at low (40 -50%; Fig. 1B) and high (80 -90%; Fig. 1C) density.
Activation of caspase-3 and poly(A)DP-ribose polymerase (PARP) cleavage are hallmarks of apoptosis in most biological systems. In apoptotic cells, active caspase-3 can proteolytically cleave numerous cellular targets, including PARP. We therefore wanted to verify whether exposure of IMR-32 cells to H 2 O 2 resulted in PARP cleavage and caspase-3 activation. H 2 O 2 treatment did indeed lead to activation of caspase-3 and PARP cleavage (Fig. 2).
Protection by ARE Activation-TUNEL staining was used to evaluate the apoptotic rate under different experimental conditions and is expressed as an apoptotic index (AI) as described under "Experimental Procedures." The nuclei of control cells showed no, or very weak, staining without H 2 O 2 treatment, indicating that cells were healthy and their nuclei were intact (Fig. 3A). Spontaneous apoptosis varied around 5 to 10 per 1000 cells in untreated or vehicle-treated IMR-32 cells, and tBHQ treatment did not significantly change these basal values (Fig. 3D). After 8 and 12 h of treatment with 200 M H 2 O 2 , a fraction of the IMR-32 cells exhibited the typical strong yellow-green TUNEL staining. Further observation of these TUNEL-positive cells demonstrated typical characteristics of apoptosis, such as condensed and shrunken nuclei (crescent nuclei) that were clearly distinguishable from normal nuclei. The AIs were significantly increased by H 2 O 2 at both 8 and 12 h (Fig. 3D). Pretreatment of the cells with tBHQ (10 M) for 8 or 24 h significantly reduced the AIs (Fig. 3D).
Reversal by Inhibition of PI3K-We hypothesize that tBHQmediated activation of ARE is critical for generating this protective response. Our laboratory has recently shown that a selective inhibitor of PI3K, LY294002, blocks ARE activation by tBHQ (13). Using the MTS cell viability assay, it was determined that addition of LY294002 (20 M) 30 min prior to tBHQ treatment completely reversed the protective effect of tBHQ (Table I). These data suggest that PI3K-dependent, AREdriven genes are responsible for the cytoprotective effect manifested by tBHQ.
Antiapoptotic Gene Set Revealed by Oligonucleotide Microarry Analysis-Oligonucleotide microarrays were used to analyze the gene expression profile associated with tBHQ treatment in the absence or presence of LY294002. The Af- fymetrix HG U95a arrays were used for these studies and contain 12,386 probe sets corresponding to 9670 full-length human cDNAs. Total RNA was isolated from IMR-32 cells treated with 10 M tBHQ or vehicle (0.01% EtOH) for 8 h. Biotinylated cRNA was prepared from cDNA synthesized from total RNA.
The Affymetrix algorithm for detecting differential expression considers several parameters from the raw data. The algorithms are presented in the Supplemental Appendix of the Affymetrix Microarray Suite 4.0 User Guide. By using the rank analysis described under "Experimental Procedures," we have identified 137 genes (1.2% of total genes) whose expression was consistently increased or decreased by tBHQ treatment. Interestingly, the number of increased genes (111 genes) was much larger than the number of decreased genes (26 genes). Transcriptional activation in IMR-32 cells after treatment with tBHQ, therefore, was unexpectedly frequent. In our analysis, evaluation of the reproducibility of paired experiments was based on calculation of the coefficient of variation (CV) (S.D./ mean) for fold change (FC) or average difference change (ADC) on the 137 genes mentioned above. According to the distribution of CV, a majority of genes have their CV of FC below 0.7 or CV of ADC below 1.0 (data not shown). In addition, only genes with a FC over 1.5 were considered significant. These cutoff values gave a conservative estimate of the numbers of genes whose expression level changed after treatment with tBHQ. Only those changes complying with the criteria were considered further. After excluding genes with large CV and/or small FC, 63 of the original 137 genes remained. All 63 genes were increased genes because no decreased genes met the criteria. These genes were categorized by function, as shown in Table II. The transcriptional changes were also confirmed by RT-PCR for seven selected genes (Fig. 4).
Gene Expression Profiles-A major cluster of genes increased in IMR-32 cells are the Phase-II detoxification enzymes (NQO1, HO1, GR, GSTM3, GCLR, TR) and other antioxidant systems such as hepatic dihydrodiol dehydrogenase (HDD) and malate NADP oxidoreductase, soluble form (Table II). These genes ranked higher compared with other genes on the list, suggesting their importance in the protective effect of tBHQ. In addition, most have been shown to be, or are proposed to be, members of the ARE-driven gene family.
Interestingly, there were a number of genes increased that were associated with neuronal growth and differentiation, including neuronal olfactomedin-related ER-localized protein, neurofilament heavy subunit (NF-H), CB1 cannabinoid receptor, secretogranin II, and axin. The expression level of certain nuclear proteins and transcriptional factors were also increased, such as kruppel-related zinc finger protein, ERF-2 (butyrate response factor 2), and Forkhead/winged helix-like transcription factor 7. Of particular note, KIAA0132, the human homolog of rat Inrf (16) and mouse Keap1 (21), was increased by 3.0-fold.
Inhibition of PI3K by LY294002 Significantly Attenuates the Increased Gene Expression Associated with tBHQ Treatment-Previous work in our laboratory has shown that ARE activation by tBHQ is blocked by inhibition of PI3K (13). Furthermore, inhibition of PI3K completely reversed the cytoprotective effect of tBHQ (Table I). To aid in determining which of these 63 genes are important for conferring the antiapoptotic effects of tBHQ treatment, microarrays were run on cells treated with tBHQ in the presence of LY294002. The data are presented in Table II. A multiple analysis on the inter-group comparison was performed based on the ADC using a one-tailed, unequal S.D. t test. As shown in Table II, the induction of Phase II detoxification enzymes and antioxidant genes such as NQO1, HO1, GR, GSTM3, cytosolic malic enzyme, and HDD and its homologue, KIAA0119, were significantly attenuated by LY294002 pretreatment. Surprisingly, up-regulation of GCLR and TR were unaffected by the PI3K inhibitor. Some other blocked genes were Wnt-5a, NF-H, MAPK kinase 4 (MAPKK4), and ERF-2 (butyrate response factor 2). Of the 63 genes increased by tBHQ, 46 genes were significantly reduced by inhibition of PI3K (Table II). RT-PCR confirmed the microarray data for NQO1 and GCLR (Fig. 5).

DISCUSSION
In this study, H 2 O 2 was used to generate oxidative injury and subsequent apoptosis in cultured IMR-32 cells. Our results clearly show that H 2 O 2 induced IMR-32 cell apoptosis through a caspase-3-dependent pathway. The apoptotic process and development of cell injury in H 2 O 2 -treated IMR-32 cells was prevented and/or delayed by strengthening the antioxidant capacity of these cells through the transcriptional activation of ARE-driven genes in response to treatment with tBHQ. Oligonucleotide microarray analysis identified the genes that changed with the treatment. The protective effects of tBHQ pretreatment were reversed by inhibition of PI3K and 46 of the 63 genes increased by tBHQ were significantly blocked. Although we have not screened the entire human genome, the

TABLE II Effect of LY294002 on the transcriptional up-regulation of genes induced by tBHQ
The genes up-regulated by tBHQ were functionally categorized. Genes with FC over 1.5 were listed. R represents the values calculated by ranking analysis based on Different Call, and the value ranging from 9 to 18 is considered as a significantly increased gene expression. Sig represents significance by one-tailed, unequal variance t test based on ADC gathered from 3 ϫ 3 groups without LY294002 compared to the corresponding value in the presence of LY294002. S means significant (p Ͻ 0.05), HS, highly significant (p Ͻ 0.01); NS, not significant. The names of genes whose up-regulation was blocked by LY294002 are shown in boldface. genes identified here represent the first set of antiapoptotic genes encoding proteins that can directly or indirectly attenuate the apoptotic process. The fact that H 2 O 2 toxicity was not totally prevented by tBHQ pretreatment is probably due to the mechanism by which H 2 O 2 causes toxicity. It has been reported that H 2 O 2 depletes NADPH and GSH within 10 -30 min after administration (18). The genes induced by tBHQ can increase the steady-state concentration of these essential molecules in the cell and delay but not completely prevent their depletion or the eventual cell death induced by H 2 O 2 depending on the time and dose of exposure. Similarly, Murphy and colleagues (11,19) have demonstrated that pretreatment of rodent neuroblastoma cells with compounds that activated the ARE and increased NQO1 partially protects cells from H 2 O 2 -and dopamine-induced cytotoxicity. They also demonstrated that 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 (10). Microarray analysis revealed a cluster of phase II detoxification enzymes and other genes, which can coordinately combat oxidative stress and prevent apoptosis. Among these genes, HO1, an enzyme that catalyzes the ratelimiting reaction in heme degradation, is involved in a catabolic pathway that leads to the production of bilirubin, a potent antioxidant. NQO1 catalyzes two-electron reduction of quinones and prevents the participation of such compounds in redox cycling and oxidative stress. GCLR catalyzes the rate-limiting reaction in glutathione biosynthesis. GSTM3 conjugates hydrophobic electrophiles and reactive oxygen species with glutathione. GR catalyzes the reduction of oxidized glutathione to reduced GSH and maintains adequate levels of reduced cellular GSH. TR is part of a family of selenium-containing pyridine nucleotide-disulfide oxidoreductase, which utilize NADPH to catalyze the conversion of oxidized thioredoxin into reduced thioredoxin and to reduce the oxidized forms of ascorbate into reduced ascorbate. Hepatic dihydrodiol dehydrogenase and KIAA0119 are aldo-keto reductases and detoxify reactive carbinyl-containing compounds, which are widely distributed in nature and pose a serious threat to living organisms because of their ability to react with cellular macromolecules. Finally, cytosolic NADP ϩ -dependent malic enzyme, an NADPH-producing cytosolic enzyme, catalyzes both oxidative decarboxylation of malate and reductive carboxylation of pyruvate.
Other categories of genes include those that enhance the growth and differentiation of neuron (neuronal olfactomedinrelated ER localized protein, axin, NF-H), function as chaperone proteins or respond to heat shock (hsp40 homolog, Bip protein), participate in various signaling pathways (Wnt-5a, MAPKK4, phosphotyrosine-independent ligand p62 for the Lck SH2 domain), and modulate transcription (KIAA0132 and ERF-2). All these genes, in addition to the detoxification and antioxidant genes, may contribute to the antiapoptotic effects of tBHQ treatment. The mechanisms by which they contribute are open to speculation and remain to be determined.
tBHQ-mediated activation of ARE is critical to generating the protective response against H 2 O 2 -induced apoptosis. We and others (13,17) have demonstrated that exposure to tBHQ triggers nuclear accumulation of the transcription factor, Nrf2, which binds to the ARE. These data provide evidence for a novel pathway of signal transduction in response to oxidative stress via the ARE reminiscent of the well-characterized AP-1-mediated or NF-B-mediated pathway. A search for potential Nrf2 binding sites within the 5Ј-flanking regions of a selected gene set was performed using MatInspector V2.2 based on TRANSFAC 4.0 (available at http://transfac.gbf.de) to determine whether the expression profile could be predicted by the presence or absence of Nrf2 binding sites. Indeed, the gene containing no Nrf2 binding site(s) such as GCLC was not increased by tBHQ whereas the promoter regions for the upregulated genes (e.g. NQO1, GCLR, Wnt5a, TR, and malate NADP oxidoreductase) contained Nrf2 binding site(s). In contrast, AP-1 and NF-B are distributed abundantly in the 5Јflanking region of all genes, including GCLC. They are not supposed to play an important role in transcriptional activation by tBHQ. Nrf2 binding sites appear to be in direct correlation with up-regulation of these genes.
Recent reports indicate that Nrf2 is normally localized in the cytoplasm bound to a chaperone, Keap1. Exposure of cells to inducers/stressors disrupts the Keap1⅐Nrf2 complex, and Nrf2 migrates to the nucleus where it binds (in heterodimeric forms with other transcription factors) to the ARE and stimulates transcription. Keap1, a 624-amino acid protein, contains 25 cysteine residues, 9 of which are expected to have highly reactive sulfhydryl groups because they are flanked by one or more basic amino acid residues (20 -22). Because many activators of the ARE can react with sulfhydryl groups, the Keap1⅐Nrf2 complex is a plausible candidate for the cytoplasmic sensor system that recognizes and reacts with inducers of ARE-driven genes (23). A recent study describes the identification of Inrf2, a rat homolog of Keap1, as part of a pathway mediating the response to oxidative stress through the ARE (16). Of particular interest, KIAA0132, a human homolog of Keap1, was upregulated by tBHQ, whereas other Nrf2 binding proteins, such as the small Maf protein family members (MafK and MafF) present on the U95a array, were unchanged. MatInspector also found putative Nrf2 binding sites in the 5Ј-flanking region of KIAA0132 indicating that transcription of KIAA0132 can be increased by the transcription factor that it sequesters. We suppose that this feedback effect aims to keep in balance the expression of ARE-driven genes, however, little is understood as to the mechanism by which the Keap1⅐Nrf2 complex functions as a cytoplasmic sensor for oxidative stress.
The signal transduction pathway leading to the activation of gene expression via the Nrf2⅐ARE interaction has recently been shown to require activation of the PI3K pathway in IMR-32 cells (13). We exploited this PI3K dependence in the present study to address the question of tBHQ-mediated cytoprotection and identify the associated changes in gene expression. Inhibition of PI3K completely blocked the protective effect seen by tBHQ treatment and significantly inhibited the increased expression of 46 out of the 63 genes increased by tBHQ. Although the data strongly support our hypothesis for the existence of a PI3K-Nrf2⅐ARE pathway leading to protection following treat- RT-PCR with specific primers for NQO1, GCLC, and GCLR was performed. RT-PCR for ␤-actin was used as equal loading control. ment with tBHQ, microarray analysis revealed two important phase II detoxification enzymes, GCLR and TR, that were induced by tBHQ but not inhibited by LY294002. This suggests an alternative pathway for GCLR and TR induction that is dependent on the Nrf2⅐ARE interaction but independent of PI3K. Others have suggested that ERK 1/2 and/or p38 MAPK are positive modulators of the ARE in several cell lines (24 -27). Because inhibition of ERK1/2 or p38 MAPK does not block tBHQ-mediated activation of the ARE or NQO1 induction, this does not appear to be the case in IMR-32 cells (12). These data imply that, in addition to cell-specific mechanisms for ARE activation, a new level of complexity exists for ARE activation within the same cell.
In conclusion, oxidative stress can be defined as an imbalance in which free radicals and their products exceed the capacity of cellular antioxidant defense mechanisms. A gain in product formation or a loss in protective mechanisms can disturb this equilibrium, leading to PCD. Numerous publications have demonstrated a correlation between direct supplementation of medium with chemical antioxidants and a decreased apoptotic rate in cell lines (28,29). Alternatively, induction of multiple antioxidant genes also may provide a way to increase an antioxidative potential and protect cells from apoptosis, as demonstrated here. We refer to this process as programmed cell life or PCL. An equilibrium exists such that any increase in the forces that drive PCD, therefore, must be balanced by increasing the forces that drive PCL. The set of ARE-driven survival genes identified here represents the first set of genes shown to modulate PCD and confirms that the PI3K-Nrf2⅐ARE pathway is crucial for the transcription of these PCL genes.