From the 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
Howard Hughes Medical Institute, University
of California, San Francisco, California 94143
Received for publication, November 13, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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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 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
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 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 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+/ 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)- 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 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 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 Nrf2-dependent ARE
Activation--
hNQO1-ARE-luciferase gene expression and endogenous
NQO1 activity were determined in tBHQ-treated
Nrf2 Differential Sensitivity to H2O2- and
PAF-induced Cytotoxicity--
Nrf2 regulates ARE-driven genes
involved in detoxification and antioxidant potential. Therefore, we
hypothesized that Nrf2 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 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.
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 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- 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).
/
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
-glutamylcysteine synthetase), heme oxygenase-1 (HO-1), thioredoxin
reductase-1 (TXNRD1), thioredoxin, and ferritin (2-12).
-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).
/
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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
-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-
-galactosidase. After 24 h of
transfection, cells were treated with chemicals for another 24 h,
and luciferase and
-galactosidase activities were determined (2, 3).
Reporter gene expression is presented as the ratio of luciferase to
-galactosidase activity (for transfection efficiency correction).
/
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
-actin,
5'-AGAGCATAGCCCTCGTAGAT-3' and 5'-CCCAGAGCAAGAGAGGTATC-3'.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
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.
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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-
-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).
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Fig. 2.
Nrf2-dependent ARE
activation and NQO1 expression. A, primary astrocytes
were transfected with hNQO1-ARE-luciferase (80 ng/well) and
CMV- -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.
/
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).
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Fig. 3.
Restoration by Nrf2 and inhibition by
dominant-negative Nrf2. A,
Nrf2 /
astrocytes were cotransfected with
hNQO1-ARE-luciferase (80 ng/well), CMV-
-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-
-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).
/
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.
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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).
/
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.
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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).
Identification of Nrf2-dependent genes in primary
cortical astrocytes
1.4 are listed.
Identification of inducible Nrf2-dependent genes in
primary cortical astrocytes
View larger version (48K):
[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 -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
/
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.
was increased by Nrf2 and
tBHQ in Nrf2+/+ astrocytes. CCAAT/enhancer-binding
protein-
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.
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[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.
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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.
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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.
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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.
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