Identification of the NF-E2-related Factor-2-dependent Genes Conferring Protection against Oxidative Stress in Primary Cortical Astrocytes Using Oligonucleotide Microarray Analysis*

Jong-Min LeeDagger §, Marcus J. CalkinsDagger §, Kaimin Chan, Yuet Wai Kan||**Dagger Dagger , and Jeffrey A. JohnsonDagger §§§¶¶||||

From the Dagger  School of Pharmacy, the § Molecular and Environmental Toxicology Center, the §§ Waisman Center, and the ¶¶ Center for Neuroscience, University of Wisconsin, Madison, Wisconsin 53705, the  Department of Medicine, University of Hong Kong, Pokfulam Road, Hong Kong, and the || Cardiovascular Research Institute, the ** Department of Laboratory Medicine, and the Dagger Dagger  Howard Hughes Medical Institute, University of California, San Francisco, California 94143

Received for publication, November 13, 2002, and in revised form, January 14, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The antioxidant responsive element (ARE) mediates transcriptional regulation of phase II detoxification enzymes and antioxidant proteins such as NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferases, and glutamate-cysteine ligase. In this study, we demonstrate that NF-E2-related factor-2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes and identify Nrf2-dependent genes by oligonucleotide microarray analysis using primary cortical astrocytes from Nrf2+/+ and Nrf2-/- mice. Nrf2-/- astrocytes had decreased basal NQO1 activity and no induction by tert-butylhydroquinone compared with Nrf2+/+ astrocytes. Similarly, both basal and induced levels of human NQO1-ARE-luciferase expression in Nrf2-/- astrocytes were significantly lower than in Nrf2+/+ astrocytes. Furthermore, human NQO1-ARE-luciferase expression in Nrf2-/- astrocytes was restored by overexpression of Nrf2, whereas ARE activation in Nrf2+/+ astrocytes was completely blocked by dominant-negative Nrf2. In addition, we observed that Nrf2-dependent genes protected primary astrocytes from H2O2- or platelet-activating factor-induced apoptosis. In support of these observations, we identified Nrf2-dependent genes encoding detoxification enzymes, glutathione-related proteins, antioxidant proteins, NADPH-producing enzymes, and anti-inflammatory genes using oligonucleotide microarrays. Proteins within these functional categories are vital to the maintenance and responsiveness of a cell defense system, suggesting that an orchestrated change in gene expression via Nrf2 and the ARE gives a synergistic protective effect against oxidative stress.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The antioxidant responsive element (ARE)1 is a cis-acting regulatory element in promoter regions of several genes encoding phase II detoxification enzymes and antioxidant proteins (1). The ARE plays an important role in transcriptional activation of downstream genes such as NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferases (GSTs), UDP-glycosyltransferase 1A6, glutamate-cysteine ligase (previously known as gamma -glutamylcysteine synthetase), heme oxygenase-1 (HO-1), thioredoxin reductase-1 (TXNRD1), thioredoxin, and ferritin (2-12).

NF-E2-related factor-2 (Nrf2) is a basic leucine zipper transcription factor that can bind to an NF-E2/AP-1 repeat sequence in the promoter of the beta -globin gene (13). Nrf2 has two kinds of binding partners, a cytoplasmic repressor and multiple nuclear binding partners. Itoh et al. (14, 15) have demonstrated that Nrf2 is sequestered in the cytoplasm by its repressor Keap1 (mouse), released under conditions of oxidative stress, and translocated into the nucleus. This cytoplasmic repressor of Nrf2 was also identified in human and rat (14, 16, 17). The suggested binding partners that have been demonstrated to bind with Nrf2 consist of other basic leucine zipper proteins such as small Maf (18, 19), Jun (20), activating transcription factor-4 (21), and cAMP-responsive element-binding protein-binding protein (22).

The DNA binding sequence of Nrf2 (5'-TGA(C/G)TCA-3') (23) is very similar to the ARE core sequence (5'-TGACnnnGC-3') (1). Several lines of evidence suggest that Nrf2 binds to the ARE sequence, leading to transcriptional activation of downstream genes encoding GSTs (24-27), glutamate-cysteine ligase (28), HO-1 (26, 29), and thioredoxin (7). Previously, our laboratory demonstrated that Nrf2 is a critical transcription factor for both basal and induced levels of NQO1 expression in IMR-32 human neuroblastoma cells (2, 3). In contrast to the clear evidences for a role of Nrf2 in ARE activation, the upstream signaling pathway is controversial. For example, mitogen-activated protein kinase (30), protein kinase C (31), and phosphatidylinositol 3-kinase (3, 32-35) have been suggested to play an important role in ARE activation.

The function of Nrf2 and its downstream proteins has been shown to be important for protection against oxidative stress- or chemical-induced cellular damage in liver (36, 37) and lung (38) as well as for prevention of cancer formation in the gastrointestinal tract (39, 40) and promotion of the wound-healing process (41). In addition, many chronic neurodegenerative diseases (i.e. Parkinson's disease and Alzheimer's disease) are thought to involve oxidative stress as a component contributing to the progression of the disease. The regulation and cell-specific expression of these genes in cells derived from brain could therefore be important for understanding how to protect neural cells from oxidative stress. One of the Nrf2-dependent ARE-driven genes, NQO1, has been demonstrated to play an important role in protecting cells against oxidative stress (42-44). Interestingly, overexpression of NQO1 and one GST isoenzyme does not protect N18-RE-105 rodent neuroblastoma cells from free radical-mediated toxicity (45), although tert-butylhydroquinone (tBHQ) treatment, which up-regulates a battery of ARE-driven genes, protects N18-RE-105 cells from glutamate toxicity (43). These observations imply that the coordinate up-regulation of ARE-driven genes, not one or two genes, is more efficient in protecting cells from oxidative damage. A recent study identified the ARE-driven genes including NQO1 that are responsible for protecting IMR-32 human neuroblastoma cells from H2O2-induced apoptosis (32, 33). Therefore, Nrf2, which mediates transcription of ARE-driven genes, is presumably the driving force behind increasing a cluster of protective genes that play an important role in cellular defense against oxidative stress.

In the central nervous system, astrocytes have been shown to express many of these protective ARE-driven genes and ARE-driven human placental alkaline phosphatase in primary cortical neuronal cultures derived from transgenic reporter mice (34). To further understand how Nrf2 contributes to the regulation of ARE-driven genes in astrocytes and how expression of these genes affects the sensitivity of astrocytes to oxidative stress, we compared primary cortical astrocyte cultures derived from Nrf2+/+ and Nrf2-/- mice. Astrocytes were treated with tBHQ to induce nuclear translocation of Nrf2 leading to ARE activation and H2O2 or platelet-activating factor (PAF) (46) to determine differential sensitivity. To understand how Nrf2-dependent genes are associated with this differential sensitivity, we performed oligonucleotide microarray analysis.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nrf2 Knockout Mice-- Nrf2 knockout mice were generated by replacing the basic leucine zipper domain with the lacZ reporter construct as described previously (47).

Primary Cortical Astrocyte Culture-- Nrf2+/- mice were bred with Nrf2+/- mice, and primary cortical astrocyte cultures were prepared individually. Cerebral cortices from newborn pup littermates were removed, placed in ice-cold Hanks' balance salt solution (3 ml/pup; Invitrogen), centrifuged at 300 × g for 2 min, and digested individually in 0.5 mg/ml trypsin (Invitrogen) in Hanks' balance salt solution at 37 °C for 25 min. Tissues were washed twice with Hanks' balance salt solution and resuspended in minimal essential medium with Earle's salt (Mediatech) containing heat-inactivated (55 °C, 30 min) fetal bovine serum (10%) and horse serum (10%) (both from Atlanta Biologicals, Inc.). Cell suspensions were sieved through cell strainers (70 µm; Falcon) and plated at a density of 5 × 104 cells/ml. The medium was changed after 24 h of initial plating and every 3 days thereafter. Cultures were maintained at 37 °C in a humidified three-gas incubator (5% O2, 90% N2, and 5% CO2; Forma Scientific, Inc.). The Nrf2 genotype of each culture was determined by a PCR-based method (3'-primer, 5'-GGAATGGAAAATAGCTCCTGCC-3'; 5'-primer, 5'-GCCTGAGAGCTGTAGGCCC-3'; and lacZ primer, 5'-GGGTTTTCCCAGTCACGAC-3') from genomic DNA (DNeasy DNA isolation kit, QIAGEN Inc.). Cells were used for experiments between 5 and 10 days in vitro. Typically, >95% of the cells in the cultures (both Nrf2-/- and Nrf2+/+) were astrocytes as determined by immunostaining of the astrocyte-specific marker glial fibrillary acidic protein (1:1000 dilution; Dako Corp.) (data not shown).

Transient Transfection and Reporter Gene Activity Assay-- Astrocytes in 96-well plates were transfected with human NQO1 (hNQO1)-ARE-luciferase (80 ng/well) and cytomegalovirus (CMV)-beta -galactosidase reporter constructs (20 ng/well) by the calcium phosphate transfection method as described previously (2, 3). For overexpression, pEF (control vector), pEF-wild-type Nrf2, and pEF-dominant-negative Nrf2 were transfected together with hNQO1-ARE-luciferase and CMV-beta -galactosidase. After 24 h of transfection, cells were treated with chemicals for another 24 h, and luciferase and beta -galactosidase activities were determined (2, 3). Reporter gene expression is presented as the ratio of luciferase to beta -galactosidase activity (for transfection efficiency correction).

NQO1 Activity-- Endogenous NQO1 enzymatic activity was determined by a colorimetric method for whole cell extracts (with menadione as a substrate) (48) and histochemistry for fixed cultures (LY 83583 as a substrate) (34) as described previously.

Western Blotting-- For glutamate-cysteine ligase modifier subunit (GCLM) and glutamate-cysteine ligase catalytic subunit (GCLC) Western blotting, 50 µg of whole cell extracts (2, 3) were used. Representative Western blots are shown in the figures.

GSH Levels-- Total glutathione (GSH + GSSG) levels were measured as described previously (34).

Cytotoxicity-- Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt assay (Promega), and apoptotic cell death was determined by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining (Roche Molecular Biochemicals). Primary astrocytes in 96-well plates were pretreated with vehicle (0.01% Me2SO) or tBHQ (50 µM). After 48 h, cells were treated with H2O2 (0-300 µM, 4 h) or PAF (0-50 µM, 24 h). For PAF treatment, the medium was changed with serum-free Dulbecco's modified Eagle's medium. The media were changed with fresh media, and 3-(4,5-dimethylthiazol-2-yl)-5-3-carboxymethoxyphenyl)tetrazolium salt substrate was added. After a 2-h incubation, the absorbance at 490 nm was measured. Percent cell viability was calculated by A490(treatment)/A490(control) × 100%. For TUNEL staining, astrocytes in eight-chamber slides were pretreated (0.01% Me2SO or 50 µM tBHQ, 48 h), treated (phosphate-buffered saline; 150 µM H2O2, 4 h; or 20 µM PAF, 24 h), and stained according to the manufacturer's protocol.

Oligonucleotide Microarray Analysis-- Nrf2-/- and Nrf2+/+ primary astrocytes were treated with vehicle (0.01% Me2SO) or tBHQ (50 µM) for 24 h. Biotinylated cRNA was prepared from total RNA, and fragmented cRNA was hybridized to MG U74 Av2 arrays (Affymetrix) (32, 33). Affymetrix Microarray Suite 5.0 was used to scan and analyze the relative abundance of each gene (scaling target signal 2500 and default analysis parameters). Data were analyzed by rank analysis as previously described (32, 33). Briefly, the definition of increase, decrease, or no change of expression for individual genes was based on ranking the difference call from two intergroup comparisons (2 × 2 matrix), viz. no change = 0, marginal increase = 1, marginal decrease = -1, increase = 2, and decrease = -2. The final rank reflects the sum of the four values (2 × 2 matrix) corresponding to the difference calls. The cutoff values for increase/decrease were set as +4/-4 (2 × 2 matrix). The reproducibility of paired comparisons was based on the coefficient of variation (S.D./mean) for the fold change of the ranked genes. A distribution curve of the coefficient of variation (CV) was used to determine its cutoff value. The cutoff values were CV < 1.0 and >= 1.2-fold for increased genes and CV > -1.0 and <= -1.2-fold for decreased genes. This method of analysis is critical in generating an accurate list of genes associated with Nrf2 and tBHQ treatment. Because these littermate cultures were derived from mice of mixed background, there is the possibility that some changes in expression may be associated with differences in genetic background. However, this type of matrix analysis selects for consistent reproducible changes associated with the presence of Nrf2 and tBHQ treatment in lieu of random changes due to genetic background (32, 33). Gene categorization was based on the NetAffx Database.2

Reverse Transcription-PCR-- Total RNA was isolated using TRIzol reagent (Invitrogen), and cDNA was synthesized (reverse transcription system, Promega) according to the manufacturer's protocol. Aliquots of cDNA were used for PCR amplification using Taq DNA polymerase (Promega). PCR primers specific to each gene are as follows: NQO1, 5'-CATTCTGAAAGGCTGGTTTGA-3 and 5'-CTAGCTTTGATCTGGTTGTCAG-3'; GST Mu1, 5'-CTCCCGACTTTGACAGAAGC-3' and 5'-CAGGAAGTCCCTCAGGTTTG-3'; GST A4, 5'-GCCAAGTACCCTTGGTTGAA-3' and 5'-CAATCCTGACCACCTCAACA-3'; UDP-glycosyltransferase 1A6, 5'-TAGTGCTTTGGGCCTCAGTT-3' and 5'-CCAAGCATGTGTTCCAGAGA-3'; GCLM, 5'-ACCTGGCCTCCTGCTGTGTG-3' and 5'-GGTCGGTGAGCTGTGGGTGT-3'; GCLC, 5'-ACAAGCACCCCCGCTTCGGT-3' and 5'-CTCCAGGCCTCTCTCCTCCC-3'; TXNRD1, 5'-GGGAGAAAAAGGTCGTCTA-3' and 5'-ACATTGGTCTGCTCTTCATC-3'; HO-1, 5'-TACACATCCAAGCCGAGAAT-3' and 5'-GTTCCTCTGTCAGCATCACC-3'; protamine-1, 5'-CAGCAAAAGCAGGAGCAG-3' and 5'-GACAGGTGGCATTGTTCCTT-3'; and beta -actin, 5'-AGAGCATAGCCCTCGTAGAT-3' and 5'-CCCAGAGCAAGAGAGGTATC-3'.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

tBHQ Selectively Activates the ARE in Nrf2+/+ Astrocytes-- Initially, to choose an ARE activator for this study, we tested several known ARE activators in other cell types such as tBHQ in IMR-32 human neuroblastoma cells (2, 3), and H2O2 (1) and phorbol 12-myristate 13-acetate in HepG2 human hepatoma cells (31). Nrf2-/- and Nrf2+/+ astrocytes were transfected with hNQO1-ARE-luciferase and treated with vehicle, tBHQ, H2O2, or phorbol 12-myristate 13-acetate. First, the basal level of hNQO1-ARE-luciferase expression in Nrf2+/+ astrocytes (3086.5 ± 320.7) (V in Fig. 1B) was significantly higher than in Nrf2-/- astrocytes (657 ± 91.6) (V in Fig. 1A). Second, none of the tested chemicals activated the ARE in Nrf2-/- astrocytes (Fig. 2A). Third, only tBHQ increased reporter gene expression in Nrf2+/+ astrocytes (Fig. 2B), suggesting that a tBHQ-specific signaling pathway mediates Nrf2-dependent ARE activation in primary astrocytes.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   ARE activation by tBHQ in Nrf2+/+ astrocytes. Nrf2-/- (A) and Nrf2+/+ (B) astrocytes were transfected with hNQO1-ARE-luciferase (80 ng/well) and CMV-beta -galactosidase (20 ng/well). After 24 h of transfection, cells were treated with vehicle (V; 0.01% Me2SO), tBHQ, H2O2, and phorbol 12-myristate 13-acetate (PMA) for 24 h. Luciferase and galactosidase activities were measured, and ARE-luciferase gene expression was calculated by the ratio of luciferase to galactosidase activity. Each data bar represents the mean ± S.E. (n = 6). *, significantly different from the vehicle-treated group by Student's t test (p < 0.05).


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 2.   Nrf2-dependent ARE activation and NQO1 expression. A, primary astrocytes were transfected with hNQO1-ARE-luciferase (80 ng/well) and CMV-beta -galactosidase (20 ng/well). After 24 h of transfection, cells were treated with tBHQ (0-20 µM) for 24 h. Luciferase and galactosidase activities were measured, and ARE-luciferase gene expression was calculated by the ratio of luciferase to galactosidase activity. Each data bar represents the mean ± S.E. (n = 6). B, primary astrocytes were treated with tBHQ (0-50 µM) for 72 h, and NQO1 activity was determined from cell lysates. Each data bar represents the mean ± S.E. (n = 6). C, primary astrocytes were treated with vehicle (0.01% Me2SO) or tBHQ (50 µM) for 72 h, and NQO1 activity was determined by histochemistry using LY 83583 as a substrate. Magnification is ×200.

Nrf2-dependent ARE Activation-- hNQO1-ARE-luciferase gene expression and endogenous NQO1 activity were determined in tBHQ-treated Nrf2-/- and Nrf2+/+ astrocytes. In Nrf2-/- astrocytes, basal ARE-luciferase reporter gene expression was markedly decreased, and there was no induction of reporter gene expression by tBHQ compared with Nrf2+/- and Nrf2+/+ astrocytes (Fig. 2A). Similarly, both basal and induced levels of endogenous NQO1 activity in Nrf2-/- astrocytes were significantly lower than in Nrf2+/- and Nrf2+/+ astrocytes (Fig. 2B), implying that Nrf2 plays an important role in both basal and induced ARE-driven gene expression in mouse primary cortical astrocytes. In addition, histochemical detection of NQO1 activity confirmed the Nrf2-dependent NQO1 gene expression. The NQO1 staining of vehicle-treated Nrf2+/+ astrocytes was significantly higher than that of vehicle-treated Nrf2-/- cells (Fig. 2C, upper left panel versus lower left panel), and tBHQ increased NQO1 staining intensity only in Nrf2+/+ astrocytes (lower left panel versus lower right panel). To further investigate the role of Nrf2 in ARE activation, we transfected Nrf2-/- astrocytes with an Nrf2 overexpression vector to restore ARE activation and Nrf2+/+ astrocytes with dominant-negative Nrf2 to inhibit ARE activation. Dominant-negative Nrf2 (N-terminally truncated Nrf2) inhibits endogenous Nrf2 function by occupying and limiting its binding partners and DNA-binding sites (5). Indeed, overexpression of Nrf2 led to dramatic ARE activation in Nrf2-/- astrocytes (Fig. 3A). tBHQ did not activate the ARE in pEF-transfected Nrf2-/- astrocytes. However, tBHQ did activate the ARE in Nrf2-overexpressing Nrf2-/- astrocytes in a dose-dependent manner (Fig. 3A). Finally, dominant-negative Nrf2 blocked both basal and induced ARE activation by tBHQ in Nrf2+/+ astrocytes (Fig. 3B).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3.   Restoration by Nrf2 and inhibition by dominant-negative Nrf2. A, Nrf2-/- astrocytes were cotransfected with hNQO1-ARE-luciferase (80 ng/well), CMV-beta -galactosidase (20 ng/well), and pEF (control vector; 10 ng/well) or pEF-wild-type Nrf2 (Nrf2; 10 ng/well). B, Nrf2+/+ astrocytes were cotransfected with hNQO1-ARE-luciferase (80 ng/well), CMV-beta -galactosidase (20 ng/well), and pEF (20 ng/well) or pEF-dominant-negative Nrf2 (DN Nrf2). After 24 h of transfection, cells were treated with tBHQ (0-20 µM) for 24 h. Luciferase and galactosidase activities were measured, and ARE-luciferase gene expression was calculated by the ratio of luciferase to galactosidase activity. Each data bar represents the mean ± S.E. (n = 6).

Differential Sensitivity to H2O2- and PAF-induced Cytotoxicity-- Nrf2 regulates ARE-driven genes involved in detoxification and antioxidant potential. Therefore, we hypothesized that Nrf2-/- astrocytes would be more sensitive to oxidative stress compared with Nrf2+/+ astrocytes due to reduced levels of detoxification and antioxidant potential. To investigate this differential sensitivity, we pretreated Nrf2-/- and Nrf2+/+ astrocytes with tBHQ (50 µM, 48 h) to increase ARE-driven gene expression and then with H2O2 to investigate differential sensitivity. Also, we used the potent inflammatory agent PAF (46) to investigate the anti-inflammatory effect of Nrf2. As shown in Fig. 4A, vehicle-pretreated Nrf2-/- astrocytes were more sensitive to H2O2-induced cytotoxicity compared with vehicle-pretreated Nrf2+/+ astrocytes. Furthermore, tBHQ pretreatment significantly increased cell viability in Nrf2+/+ (but not Nrf2-/-) astrocytes (Fig. 4A). Similarly, Nrf2-/- astrocytes were more sensitive to PAF compared with Nrf2+/+ astrocytes, and tBHQ pretreatment protected only Nrf2+/+ astrocytes (Fig. 4B). TUNEL staining and the corresponding phase-contrast microscope pictures confirmed this differential sensitivity. As shown in Fig. 4C, the numbers of TUNEL-positive cells in H2O2- or PAF-treated Nrf2-/- astrocytes were greater than in the corresponding Nrf2+/+ astrocytes. Although tBHQ did not decrease the number of TUNEL-positive cells in Nrf2-/- astrocytes, tBHQ pretreatment decreased TUNEL-positive cells in both H2O2- and PAF-treated Nrf2+/+ astrocytes (data not shown). Consistent with the TUNEL data, H2O2 and PAF induced more caspase-3 activation in Nrf2-/- astrocytes than in Nrf2+/+ astrocytes (data not shown). These observations suggest that Nrf2-/- astrocytes are more sensitive to oxidative stress and inflammation compared with Nrf2+/+ astrocytes and that coordinate up-regulation of ARE-driven genes by tBHQ further protects Nrf2+/+ cells from H2O2- and PAF-induced cytotoxicity.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 4.   Protective role of Nrf2-dependent genes. Nrf2-/- (knockout (KO)) and Nrf2+/+ (wild-type (WT)) primary astrocytes were pretreated with vehicle (V; 0.01% Me2SO) or tBHQ (T; 50 µM, 48 h), followed by H2O2 (4 h) or PAF (24 h). To measure cell viability, 3-(4,5-dimethylthiazol-2-yl)-5-3-carboxymethoxyphenyl)tetrazolium salt assay was used (A and B), and to measure apoptotic nuclei, TUNEL staining was performed (C) according to the manufacturers' protocols. Scale bars = 20 µm. Treatment with tBHQ alone (tBHQ - vehicle) did not induce cytotoxicity in either Nrf2+/+ or Nrf2-/- astrocytes (data not shown).

Identification of the Nrf2-dependent Genes-- To identify the Nrf2-dependent genes that play an important role in protecting astrocytes from H2O2- and PAF-induced apoptosis, we performed oligonucleotide microarray analysis. The genes changed by Nrf2 and/or tBHQ were identified by four comparisons, as depicted in Fig. 5A. tBHQ increased 16 genes (stromal cell-derived factor, Induced in fatty liver dystrophy-2, histones 1H2B and H2A, histone H1, TG-interacting factor, Thy-1.2 glycoprotein, Lumican, cysteine- and histidine-rich-1, ectonucleotide pyrophosphatase/phosphodiesterase-2, proteasome 26 S subunit, and six expressed sequence tags) and decreased 27 genes in Nrf2-/- astrocytes (comparison I in Fig. 5A), suggesting that the changes in expression of these genes are Nrf2-independent. Genes changed by Nrf2 in the absence of tBHQ (comparison II) are listed in Table I, and genes changed by tBHQ in the presence (comparison III) or absence (comparison I) of Nrf2 are listed in Table II. Interestingly, the majority of the genes increased by tBHQ in Nrf2+/+ astrocytes (97.6%) were not changed by tBHQ in Nrf2-/- astrocytes (Fig. 5B and Table II), suggesting that most of the tBHQ-induced genes are Nrf2-dependent. Only five genes (Induced in fatty liver dystrophy-2, ectonucleotide pyrophosphatase/phosphodiesterase-2, TG-interacting factor, Thy-1.2 glycoprotein, and expressed sequence tag AW124185) were increased by tBHQ in both Nrf2-/- and Nrf2+/+ astrocytes. The gene list in comparison IV includes most of the genes changed in comparisons I and III. The major functional categories of Nrf2-dependent genes are 1) detoxification, 2) antioxidant/reducing potential, 3) growth, and 4) defense/immune/inflammation (Tables I and II). Clearly, the oligonucleotide microarray data verify that Nrf2 is important for the expression of NQO1 and other ARE-driven genes such as GSTs. Interestingly, cytochrome P450 1B1 was the only member of the cytochrome P450 family that appeared to be Nrf2-dependent in primary astrocytes (Tables I and II). Another evident Nrf2-dependent gene category is antioxidant/reducing potential. As shown in Tables I and II, many glutathione-related proteins (GCLM, GCLC, GSTs, glutathione reductase, and glutathione peroxidase), antioxidant proteins (TXNRD1, thioredoxin, peroxiredoxin, HO-1, ferritin, catalase, and superoxide dismutase), and genes involved in NADPH production (glucose-6-phosphate dehydrogenase, malic enzyme, transaldolase, and transketolase) were identified as Nrf2-dependent genes. Furthermore, oligonucleotide microarray analysis revealed that many defense/immune/inflammation-related genes (i.e. cathepsin, complements, lipopolysaccharide-binding protein, and PAF acetylhydrolase), metabolic enzymes (i.e. lipoprotein lipase and esterase), growth factors (i.e. platelet-derived growth factor and nerve growth factor), and signaling proteins (i.e. protein kinase C) were regulated in an Nrf2-dependent manner (Tables I and II). Clearly, oligonucleotide microarray data showed that Nrf2-dependant antioxidant and anti-inflammatory genes play an important role in protecting primary astrocytes from the H2O2- and PAF-induced apoptosis observed in this study.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5.   Changes in gene expression revealed by oligonucleotide microarray analysis. A, Nrf2-dependent genes were identified by oligonucleotide microarray analysis using four comparisons (I-IV). The numbers of genes altered in each comparison are presented in boxes. B, shown are Venn diagrams of the number of genes altered in each comparison (comparisons I and III and comparisons II and IV).


                              
View this table:
[in this window]
[in a new window]
 
Table I
Identification of Nrf2-dependent genes in primary cortical astrocytes
The genes changed by Nrf2 were functionally categorized. R, rank; CV, coefficient of variation of -fold; G6PD, glucose-6-phosphate dehydrogenase; MMTV, murine mammary tumor virus. For decreased genes, genes with fold lower than -1.4 are listed.


                              
View this table:
[in this window]
[in a new window]
 
Table II
Identification of inducible Nrf2-dependent genes in primary cortical astrocytes
The genes changed by tBHQ in the presence or absence of Nrf2 were functionally categorized. R, rank; CV, coefficient of variation of fold; NR, not ranked; G6PD, glucose-6-phosphate dehydrogenase; EST, expressed sequence tag.

Verification of Microarray Data-- To verify the oligonucleotide microarray data, we performed reverse transcription-PCR and Western blot analysis for selected genes. As shown in Fig. 6 (A and B), the expression levels of the selected genes observed by reverse transcription-PCR were consistent with the oligonucleotide microarray analysis results, verifying the change in Nrf2-dependent genes identified by the oligonucleotide microarray. Also, Western blot analysis (Fig. 6C) and GSH quantification data (Fig. 6D) showed that Nrf2 plays an important role in both GCLM/GCLC expression and GSH synthesis, as expected from the reverse transcription-PCR and oligonucleotide microarray data.


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 6.   Verification of microarray data. A, primary astrocytes were treated with vehicle (V; 0.01% Me2SO) or tBHQ (T; 50 µM) for 24 h. Total RNA was isolated, and cDNA was synthesized for PCR amplification. PCR cycle numbers were as follows: NQO1, 30; GST A4, 20; GST Mu1, 25; UDP-glycosyltransferase 1A6 (UGT 1.6), 30; GCLM, 25; GCLC, 25; TXNRD1, 30; HO-1, 30; protamine-1, 30; and beta -actin, 30. B, the averages of the signal values from oligonucleotide microarray analysis are listed. a Increase in comparison II; b increase in comparison III; c increase in comparison IV; d decrease in either comparison II or IV (see Fig. 5A). The genes shown here were not changed by tBHQ in the absence of Nrf2 (comparison I in Fig. 5A). Primary astrocytes were treated with vehicle (0.01% Me2SO) or tBHQ (50 µM) for 48 h. C, whole cell extracts were prepared, and Western blot analysis for GCLC and GCLC was performed as described under "Experimental Procedures." D, total glutathione levels (GSH + GSSG) were measured 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 demonstrated that the basic leucine zipper transcription factor Nrf2 plays a critical role in both basal and induced gene expression of NQO1 in primary cortical astrocytes. Overexpression of wild-type Nrf2 restored the basal expression and activation of ARE by tBHQ in Nrf2-/- cells, and dominant-negative Nrf2 significantly decreased both basal expression and activation of ARE by tBHQ in Nrf2+/+ astrocytes. The reduced expression and lack of ARE activation in Nrf2-/- astrocytes directly correlate with an increased sensitivity to H2O2- and PAF-induced cytotoxicity compared with Nrf2+/+ astrocytes. Finally, the genes responsible for conferring protection against oxidative stress or inflammation were identified by oligonucleotide microarray analysis. The major functional categories are detoxification enzymes, antioxidant proteins, NADPH-producing proteins, growth factors, defense/immune/inflammation-related proteins, and signaling proteins. Proteins within these functional categories are vital to the maintenance and responsiveness of a cell's defense system, suggesting that an orchestrated change in expression via Nrf2 and the ARE would give a synergistic protective effect.

Recently, Kwak et al. (49) observed 3H-1,2-dithiole-3-thione-increased Nrf2 gene expression and demonstrated that Nrf2 autoregulates its own expression through an ARE-like element. In the present study, tBHQ did not increase Nrf2 expression levels, but induced nuclear translocation of Nrf2 (data not shown), suggesting that ARE activation by tBHQ is mediated by nuclear translocation of Nrf2, not by induction of Nrf2 gene expression in primary astrocytes. tBHQ did, however, increase expression of binding partners of Nrf2 (i.e. MafG and activating transcription factor-4) in Nrf2+/+ astrocytes (Table II). Maf proteins have been shown to regulate ARE activation negatively or positively depending on cell types and genes (27, 28, 50, 51), and activating transcription factor-4 has been demonstrated to bind to Nrf2, leading to HO-1 gene expression (21). In addition, CCAAT/enhancer-binding protein-beta was increased by Nrf2 and tBHQ in Nrf2+/+ astrocytes. CCAAT/enhancer-binding protein-beta has been shown to mediate negative regulation of rat GST-Ya expression (52). Finally, in contrast to the increased expression of KIAA0132 (human homolog of Keap1) by tBHQ in IMR-32 cells (32), Keap1 was not changed by either Nrf2 or tBHQ in mouse primary astrocytes (Tables I and II). These observations suggest a possible balancing mechanism between positive and negative regulation in Nrf2-mediated gene expression and that the role and regulation of other binding partners of Nrf2 are dependent on the cell type and/or genes being studied.

A recent study reported Nrf2-regulated genes induced by sulforaphane in the small intestine (53). Interestingly, only nine genes were commonly increased by sulforaphane (small intestine) (53) and by tBHQ (primary astrocytes) (this study) in Nrf2+/+ cells (NQO1, epoxide hydrolase, GST A4, GST Mu1, GST Mu3, transaldolase, transketolase, GCLM, and GCLC). In the small intestine, genes coding GSTs and drug-metabolizing enzymes were induced by sulforaphane (53). In primary astrocytes, however, tBHQ increased the expression of many antioxidant and anti-inflammatory genes (i.e. HO-1, TXNRD1, thioredoxin, ferritin, peroxiredoxin, glucose-6-phosphate dehydrogenase, superoxide dismutase, catalase, malic enzyme, and PAF acetylhydrolase), suggesting cell type-specific gene expression.

The function of a number of Nrf2-dependent genes is dependent on GSH. GSTs catalyze the nucleophilic addition of GSH to an electrophilic group of a broad spectrum of xenobiotic compounds (54). Other GSH-dependent enzymes (i.e. glutathione peroxidase, peroxiredoxin, and glutathione reductase) were also increased in an Nrf2-dependent manner. Glutathione peroxidase and peroxiredoxin metabolize H2O2, generating H2O and oxidized GSH (GSSG), and glutathione reductase regenerates reduced GSH. Ideally, in association with an increased utilization of GSH, there would also be an increased production of GSH. The rate-limiting step in the GSH biosynthesis is mediated by GCLM/GCLC. In this study, solute carrier family-1, glycine transporter, GCLM, and GCLC were shown to be Nrf2-dependent genes. The coordinate regulation of these genes can have a synergistic effect in the maintenance of GSH levels as well as detoxification of reactive intermediates (Fig. 7A).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7.   Orchestrated regulation of Nrf2-dependent genes. Nrf2-dependent genes identified by oligonucleotide microarray analysis are depicted in shaded boxes. Genes are categorized based on function and metabolic pathway. Genes are related to glutathione homeostasis (A), detoxification of H2O2 and iron homeostasis (B), and NADPH homeostasis (C). GR, glutathione reductase; GPX, glutathione peroxidase; PRX, peroxiredoxin; SOD, superoxide dismutase; TRX, thioredoxin; G6PD, glucose-6-phosphate dehydrogenase.

Another cluster of genes including superoxide dismutase and HO-1 are very important for cellular defense against oxidative stress. Superoxide dismutase detoxifies superoxide, resulting in H2O2, and HO-1 generates a potent radical scavenger, bilirubin. However, superoxide dismutase and HO-1 can induce more oxidative stress because they increase the cellular concentrations of H2O2 and free iron, which together can generate hydroxyl radical through the Fenton reaction. For complete detoxification of superoxide, H2O2 should be further metabolized to H2O by glutathione peroxidase, catalase, or peroxiredoxin. Catalase directly detoxifies H2O2, whereas peroxiredoxin uses GSH (Fig. 7A) and/or thioredoxin as an electron donor for peroxidation of H2O2, resulting in generation of GSSG and oxidized thioredoxin, respectively (Fig. 7B). GSSG and oxidized thioredoxin are converted to their reduced forms by glutathione reductase and TXNRD1. Oligonucleotide microarray data showed that superoxide dismutase, catalase, peroxiredoxin, thioredoxin, and TXNRD1 are transcriptionally regulated through an Nrf2-dependent mechanism. In addition, proper management of free iron is also important for minimizing oxidative stress, and this can be best achieved by ferritin. Ferritin converts Fe2+ to Fe3+ (ferroxidase activity) and sequesters it, thereby preventing Fe2+ from participating in the Fenton reaction. Thus, up-regulation of HO-1 together with ferritin is a way to increase antioxidant potential while minimizing hydroxyl radical formation. Based on these observations, we speculate that increased expression of these genes can dramatically increase the efficiency of detoxification of reactive oxygen species. Also, the genes depicted in Fig. 7B provide a molecular mechanism by which tBHQ-treated Nrf2+/+ astrocytes are resistant to H2O2-induced apoptosis.

Finally, NQO1, glutathione reductase, and TXNRD1 are important in detoxifying quinones and maintaining the cellular redox balance. One common feature of these proteins is that they use NADPH as an electron donor. So, for efficient detoxification and maintenance of cellular redox status, it would be beneficial to up-regulate these proteins together with the appropriate reducing potential (NADPH) to support enzymatic reactions. Glucose-6-phosphate dehydrogenase/malic enzyme can directly generate NADPH, and transketolase/transaldolase can increase NADPH production by regenerating substrates for glucose-6-phosphate dehydrogenase. Oligonucleotide microarray data showed that NQO1, glutathione reductase, TXNRD1, glucose-6-phosphate dehydrogenase, malic enzyme, transketolase, and transaldolase are Nrf2-dependent genes (Fig. 7C). These Nrf2-dependent genes would also contribute significantly to a cell's detoxification potential and cellular redox balance. Together, these coordinately regulated gene clusters presented in Fig. 7 strongly support the hypothesis that Nrf2-dependent gene expression is central to efficient detoxification of reactive metabolites and reactive oxygen species as well as a cell's ability to deal with stress such as inflammation.

Can these changes in astrocytes protect neurons from oxidative stress-induced apoptosis? Astrocytes have been suggested to interact with neurons and to confer neuronal protection. It has been demonstrated in numerous neuronal culture systems that the survival of neurons is significantly enhanced by astrocytes (55-57). They can promote neuronal survival by removing excitotoxins (i.e. glutamate) from the synapse, modulating antioxidant levels (i.e. GSH), and secreting trophic factors (i.e. glial-derived neurotrophic factor) (58-60). In support of this idea, Nrf2-dependent detoxification and antioxidant proteins in astrocytes can play a role in protecting neurons. However, genetic changes in neurons associated with increased expression of ARE-driven genes in astrocytes could also contribute to an overall protective mechanism. The extent to which this intercellular communication is required and the specific genetic remodeling in the neurons versus the astrocytes in a co-culture system remain to be determined. Preliminary data from our laboratory suggest that there are unique changes in both astrocytes and neurons that, when combined, may be responsible for protecting neurons from oxidative stress.3

In summary, oxidative stress and reactive metabolites can induce apoptosis or programmed cell death. Programmed cell death can be prevented in many ways, such as addition of external growth factors, antioxidant supplementation, and inhibition of apoptotic signaling pathways. Here we present an alternative in that the coordinate up-regulation of Nrf2-dependent genes provides a way to protect cells through genetic remodeling, a process referred to as programmed cell life. We hypothesize that increased activation of programmed cell life pathways can balance programmed cell death and that, in combination with other techniques known to prevent programmed cell death, may be a powerful tool in controlling progressive neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Currently, we are focused on evaluating the neuroprotective role of Nrf2-dependent genes in vivo by crossing Nrf2 knockout mice with established transgenic models representing human neurological disorders.

    ACKNOWLEDGEMENTS

We thank Matthew Slattery and the Molecular Biology Core Facility of the University of Wisconsin Environmental Health Science Center for conducting the gene array hybridizations and Dr. Terrance Kavanagh for providing anti-GCLM and anti-GCLC antibodies. We also thank Delinda Johnson, Jiang Li, Thor Stein, and Andrew Kraft for helpful suggestions.

    FOOTNOTES

* This work was supported by Grants ES08089 and ES10042 (to J. A. J.) and Grant ES09090 (to the Environmental Health Sciences Center) from the NIEHS, National Institutes of Health, by the Burroughs Wellcome New Investigator in Toxicological Sciences award (to J. A. J.), and by Grant DK16666 from the National Institutes of Health (to Y. W. K.).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.

|||| To whom correspondence should be addressed: School of Pharmacy, University of Wisconsin, 6125 Rennebohm Hall, 777 Highland Ave., Madison, WI 53705-2222. Tel.: 608-262-2893; Fax: 608-262-5345; E-mail: jajohnson@pharmacy.wisc.edu.

Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M211558200

2 Available at www.NetAffx.com.

3 J.-M. Lee and J. A. Johnson, unpublished data.

    ABBREVIATIONS

The abbreviations used are: ARE, antioxidant responsive element; NQO1, NAD(P)H:quinone oxidoreductase-1; hNQO1, human NAD(P)H:quinone oxidoreductase; GST, glutathione S-transferase; HO-1, heme oxygenase-1; TXNRD1, thioredoxin reductase-1; Nrf2, NF-E2-related factor-2; tBHQ, tert-butylhydroquinone; PAF, platelet-activating factor; CMV, cytomegalovirus; GCLM, glutamate-cysteine ligase modifier subunit; GCLC, glutamate-cysteine ligase catalytic subunit; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Rushmore, T. H., Morton, M. R., and Pickett, C. B. (1991) J. Biol. Chem. 266, 11632-11639[Abstract/Free Full Text]
2. Lee, J.-M., Moehlenkamp, J. D., Hanson, J. M., and Johnson, J. A. (2001) Biochem. Biophys. Res. Commun. 280, 286-292[CrossRef][Medline] [Order article via Infotrieve]
3. Lee, J.-M., Hanson, J. M., Chu, W. A., and Johnson, J. A. (2001) J. Biol. Chem. 276, 20011-20016[Abstract/Free Full Text]
4. Moehlenkamp, J. D., and Johnson, J. A. (1999) Arch. Biochem. Biophys. 363, 98-106[CrossRef][Medline] [Order article via Infotrieve]
5. Alam, J., Wicks, C., Stewart, D., Gong, P., Touchard, C., Otterbein, S., Choi, A. M., Burow, M. E., and Tou, J. (2000) J. Biol. Chem. 275, 27694-27702[Abstract/Free Full Text]
6. Favreau, L. V., and Pickett, C. B. (1995) J. Biol. Chem. 270, 24468-24474[Abstract/Free Full Text]
7. Kim, Y. C., Masutani, H., Yamaguchi, Y., Itoh, K., Yamamoto, M., and Yodoi, J. (2001) J. Biol. Chem. 276, 18399-18406[Abstract/Free Full Text]
8. Mulcahy, R. T., Wartman, M. A., Bailey, H. H., and Gipp, J. J. (1997) J. Biol. Chem. 272, 7445-7454[Abstract/Free Full Text]
9. Nguyen, T., and Pickett, C. B. (1992) J. Biol. Chem. 267, 13535-13539[Abstract/Free Full Text]
10. Orino, K., Lehman, L., Tsuji, Y., Ayaki, H., Torti, S. V., and Torti, F. M. (2001) Biochem. J. 357, 241-247[CrossRef][Medline] [Order article via Infotrieve]
11. Rushmore, T. H., King, R. G., Paulson, K. E., and Pickett, C. B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3826-3830[Abstract]
12. Venugopal, R., and Jaiswal, A. K. (1998) Oncogene 17, 3145-3156[CrossRef][Medline] [Order article via Infotrieve]
13. Moi, P., Chan, K., Asunis, I., Cao, A., and Kan, Y. W. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9926-9930[Abstract/Free Full Text]
14. Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, T., Igarashi, K., Engel, J. D., and Yamamoto, M. (1999) Genes Dev. 13, 76-86[Abstract/Free Full Text]
15. Itoh, K., Ishii, T., Wakabayashi, N., and Yamamoto, M. (1999) Free Radic. Res. 31, 319-324[Medline] [Order article via Infotrieve]
16. Bloom, D., Dhakshinamoorthy, S., and Jaiswal, A. K. (2002) Oncogene 21, 2191-2200[CrossRef][Medline] [Order article via Infotrieve]
17. Dhakshinamoorthy, S., and Jaiswal, A. K. (2001) Oncogene 20, 3906-3917[CrossRef][Medline] [Order article via Infotrieve]
18. Itoh, K., Chiba, T., Takahashi, S., Ishii, T., Igarashi, K., Katoh, Y., Oyake, T., Hayashi, N., Satoh, K., Hatayama, I., Yamamoto, M., and Nabeshima, Y. (1997) Biochem. Biophys. Res. Commun. 236, 313-322[CrossRef][Medline] [Order article via Infotrieve]
19. Marini, M. G., Chan, K., Casula, L., Kan, Y. W., Cao, A., and Moi, P. (1997) J. Biol. Chem. 272, 16490-16497[Abstract/Free Full Text]
20. Venugopal, R., and Jaiswal, A. K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14960-14965[Abstract/Free Full Text]
21. He, C. H., Gong, P., Hu, B., Stewart, D., Choi, M. E., Choi, A. M., and Alam, J. (2001) J. Biol. Chem. 276, 20858-20865[Abstract/Free Full Text]
22. Katoh, Y., Itoh, K., Yoshida, E., Miyagishi, M., Fukamizu, A., and Yamamoto, M. (2001) Genes Cells 6, 857-868[Abstract/Free Full Text]
23. Motohashi, H., Shavit, J. A., Igarashi, K., Yamamoto, M., and Engel, J. D. (1997) Nucleic Acids Res. 25, 2953-2959[Abstract/Free Full Text]
24. Chanas, S. A., Jiang, Q., McMahon, M., McWalter, G. K., McLellan, L. I., Elcombe, C. R., Henderson, C. J., Wolf, C. R., Moffat, G. J., Itoh, K., Yamamoto, M., and Hayes, J. D. (2002) Biochem. J. 365, 405-416[CrossRef][Medline] [Order article via Infotrieve]
25. Hayes, J. D., Chanas, S. A., Henderson, C. J., McMahon, M., Sun, C., Moffat, G. J., Wolf, C. R., and Yamamoto, M. (2000) Biochem. Soc. Trans. 28, 33-41[Medline] [Order article via Infotrieve]
26. Ishii, T., Itoh, K., Takahashi, S., Sato, H., Yanagawa, T., Katoh, Y., Bannai, S., and Yamamoto, M. (2000) J. Biol. Chem. 275, 16023-16029[Abstract/Free Full Text]
27. Nguyen, T., Huang, H. C., and Pickett, C. B. (2000) J. Biol. Chem. 275, 15466-15473[Abstract/Free Full Text]
28. Wild, A. C., Moinova, H. R., and Mulcahy, R. T. (1999) J. Biol. Chem. 274, 33627-33636[Abstract/Free Full Text]
29. Alam, J., Stewart, D., Touchard, C., Boinapally, S., Choi, A. M., and Cook, J. L. (1999) J. Biol. Chem. 274, 26071-26078[Abstract/Free Full Text]
30. Yu, R., Lei, W., Mandlekar, S., Weber, M. J., Der, C. J., Wu, J., and Kong, A. T. (1999) J. Biol. Chem. 274, 27545-27552[Abstract/Free Full Text]
31. Huang, H. C., Nguyen, T., and Pickett, C. B. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 12475-12480[Abstract/Free Full Text]
32. Li, J., Lee, J.-M., and Johnson, J. A. (2002) J. Biol. Chem. 277, 388-394[Abstract/Free Full Text]
33. Li, J., and Johnson, J. A. (2002) Physiol. Genomics 9, 137-144[Abstract/Free Full Text]
34. Johnson, D. A., Andrews, G. K., Xu, W., and Johnson, J. A. (2002) J. Neurochem. 81, 1233-1241[CrossRef][Medline] [Order article via Infotrieve]
35. Kang, K. W., Cho, M. K., Lee, C. H., and Kim, S. G. (2001) Mol. Pharmacol. 59, 1147-1156[Abstract/Free Full Text]
36. Chan, K., Han, X. D., and Kan, Y. W. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4611-4616[Abstract/Free Full Text]
37. Enomoto, A., Itoh, K., Nagayoshi, E., Haruta, J., Kimura, T., O'Connor, T., Harada, T., and Yamamoto, M. (2001) Toxicol. Sci. 59, 169-177[Abstract/Free Full Text]
38. Chan, K., and Kan, Y. W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12731-12736[Abstract/Free Full Text]
39. Fahey, J. W., Haristoy, X., Dolan, P. M., Kensler, T. W., Scholtus, I., Stephenson, K. K., Talalay, P., and Lozniewski, A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7610-7615[Abstract/Free Full Text]
40. Ramos-Gomez, M., Kwak, M. K., Dolan, P. M., Itoh, K., Yamamoto, M., Talalay, P., and Kensler, T. W. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3410-3415[Abstract/Free Full Text]
41. Braun, S., Hanselmann, C., Gassmann, M. G., auf dem Keller, U., Born-Berclaz, C., Chan, K., Kan, Y. W., and Werner, S. (2002) Mol. Cell. Biol. 22, 5492-5505[Abstract/Free Full Text]
42. Dinkova-Kostova, A. T., and Talalay, P. (2000) Free Radic. Biol. Med. 29, 231-240[CrossRef][Medline] [Order article via Infotrieve]
43. Murphy, T. H., De Long, M. J., and Coyle, J. T. (1991) J. Neurochem. 56, 990-995[Medline] [Order article via Infotrieve]
44. Radjendirane, V., Joseph, P., Lee, Y. H., Kimura, S., Klein-Szanto, A. J., Gonzalez, F. J., and Jaiswal, A. K. (1998) J. Biol. Chem. 273, 7382-7389[Abstract/Free Full Text]
45. Duffy, S., So, A., and Murphy, T. H. (1998) J. Neurochem. 71, 69-77[Medline] [Order article via Infotrieve]
46. Hostettler, M. E., Knapp, P. E., and Carlson, S. L. (2002) Glia 38, 228-239[CrossRef][Medline] [Order article via Infotrieve]
47. Chan, K., Lu, R., Chang, J. C., and Kan, Y. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13943-13948[Abstract/Free Full Text]
48. Prochaska, H. J., and Santamaria, A. B. (1988) Anal. Biochem. 169, 328-336[Medline] [Order article via Infotrieve]
49. Kwak, M. K., Itoh, K., Yamamoto, M., and Kensler, T. W. (2002) Mol. Cell. Biol. 22, 2883-2892[Abstract/Free Full Text]
50. Dhakshinamoorthy, S., and Jaiswal, A. K. (2000) J. Biol. Chem. 275, 40134-40141[Abstract/Free Full Text]
51. Dhakshinamoorthy, S., and Jaiswal, A. K. (2002) Oncogene 21, 5301-5312[CrossRef][Medline] [Order article via Infotrieve]
52. Chen, Y. H., and Ramos, K. S. (2000) J. Biol. Chem. 275, 27366-27376[Abstract/Free Full Text]
53. Thimmulappa, R. K., Mai, K. H., Srisuma, S., Kensler, T. W., Yamamoto, M., and Biswal, S. (2002) Cancer Res. 62, 5196-5203[Abstract/Free Full Text]
54. Pickett, C. B., and Lu, A. Y. (1989) Annu. Rev. Biochem. 58, 743-764[CrossRef][Medline] [Order article via Infotrieve]
55. Chen, Y., Vartiainen, N. E., Ying, W., Chan, P. H., Koistinaho, J., and Swanson, R. A. (2001) J. Neurochem. 77, 1601-1610[CrossRef][Medline] [Order article via Infotrieve]
56. Drukarch, B., Schepens, E., Stoof, J. C., Langeveld, C. H., and Van Muiswinkel, F. L. (1998) Free Radic. Biol. Med. 25, 217-220[CrossRef][Medline] [Order article via Infotrieve]
57. Tanaka, J., Toku, K., Zhang, B., Ishihara, K., Sakanaka, M., and Maeda, N. (1999) Glia 28, 85-96[CrossRef][Medline] [Order article via Infotrieve]
58. Aschner, M. (2000) Neurotoxicology 21, 1101-1107[Medline] [Order article via Infotrieve]
59. Mount, H. T., Dean, D. O., Alberch, J., Dreyfus, C. F., and Black, I. B. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9092-9096[Abstract]
60. Sonnewald, U., Qu, H., and Aschner, M. (2002) J. Pharmacol. Exp. Ther. 301, 1-6[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.