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J. Biol. Chem., Vol. 275, Issue 21, 16023-16029, May 26, 2000
,§,From the Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan
Received for publication, September 10, 1999, and in revised form, January 19, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Electrophiles and reactive oxygen species have
been implicated in the pathogenesis of many diseases. Transcription
factor Nrf2 was recently identified as a general regulator of
one defense mechanism against such havoc. Nrf2 regulates the
inducible expression of a group of detoxication enzymes, such as
glutathione S-transferase and NAD(P)H:quinone
oxidoreductase, via antioxidant response elements. Using peritoneal
macrophages from Nrf2-deficient mice, we show here that
Nrf2 also controls the expression of a group of electrophile- and oxidative stress-inducible proteins and activities, which includes
heme oxygenase-1, A170, peroxiredoxin MSP23, and cystine membrane
transport (system xc Oxidative stress conditions or enhanced production of reactive
oxygen species (ROS)1 result
from a variety of stimuli including ionizing radiation, exposure to
xenobiotics, inflammation, and phagocytosis (1). Treatment of mammalian
cells with electrophilic agents usually provokes cellular responses,
including transcriptional activation of genes encoding proteins that
partake in the defense against oxidative stress. This process is
referred to as the electrophile counterattack response (2). Through
analyses of mouse and rat glutathione S-transferase (GST) Ya
subunit genes and the rat NAD(P)H:quinone oxidoreductase (NQO1) subunit
gene, the cis-acting element responsible for the induction
by electrophiles was independently identified as an
electrophile-responsive element (EpRE) (3) or antioxidant-responsive element (ARE) (4). The consensus ARE sequence has been extensively characterized (5).
The consensus binding sequence of erythroid transcription factor NF-E2
shows high similarity to the ARE/EpRE sequence. Also, the expression
profile of Nrf2, one of the NF-E2 subunit factors, overlaps with
those of drug-metabolizing enzymes such as GST and NQO1. Based on these
facts, we recently demonstrated that transcription factor Nrf2
(6-8) is essential for the coordinated transcriptional activation of
genes encoding the drug-metabolizing enzymes, such as GST and NQO1, via
AREs/EpREs (9). Nrf2-deficient mice fed with butylated
hydroxyanisole, which normally leads to a pronounced up-regulation of
Alpha, Pi, and Mu classes of GSTs and NQO1, failed to induce either of
these detoxication enzymes in the liver or intestine (9). Since these
detoxication enzymes decrease the level of oxidative stress by removing
compounds capable of generating ROS or other highly reactive
substances, they thereby constitute part of the defense mechanism
against oxidative stress (10). Because ARE-type cis-acting
sequences are frequently found in the regulatory regions of a number of
other oxidative stress-inducible genes (5, 11-13), we hypothesized
that Nrf2 might also serve as the key transcription factor
activating these genes.
A number of defense proteins and activities in murine peritoneal
macrophages are markedly induced upon exposure to electrophilic agents
or other oxidative stresses. These proteins include heme oxygenase-1
(HO-1) (14-16), peroxiredoxin MSP23 (17), the cystine membrane
transporter (system xc Culture of Macrophages--
Female wild type ICR and
nrf2 mutant mice (9) weighing 20-25 g received an
intraperitoneal injection of 2 ml of 4% thioglycollate broth. Four
days later, macrophages were collected by peritoneal lavage (17). The
cells were resuspended at 7.5 × 105 cells/ml and
cultured in RPMI 1640 medium containing 10% (v/v) fetal bovine serum
as described previously (17). For the 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (27)
assay, the cells were resuspended at 2 × 105 cells/ml
and cultured in medium without fetal bovine serum. After 1 h of
culture, stress agents were added to the medium. Final concentrations
of the agents in the medium were 100 µM for DEM, paraquat, and hydrogen peroxide (H2O2); 80 µM for catechol; 20 milliunits/ml for glucose oxidase
(GO); 5 µM for CdCl2 and menadione (2-methyl-1,4-naphthoquinone); 10 µM for
1-chloro-2,4-dinitrobenzene (CDNB); 20 µM for iodoacetic
acid; 2.5 µM for t-butylhydroquinone (t-BHQ) and sodium arsenite (NaAsO2); and 1 ng/ml for lipopolysaccharide (LPS). The macrophages were harvested at
the times indicated in the figure legends. Cell viability was measured
by the MTT assay (27) and the trypan blue dye exclusion test.
RNA Blot Hybridization Analysis--
Total cellular RNA was
extracted from macrophages by RNAzolTM B (TEL-TEST, Inc.,
Friendswood, TX). The RNA samples (10 µg) were electrophoresed and
transferred to Zeta-Probe GT membranes (Bio-Rad). The membranes were
probed with 32P-labeled cDNA probes as indicated in the
figure legends. Immunoblotting--
Macrophages were solubilized with SDS-sample
buffer (without dye or 2-mercaptoethanol), and protein concentrations
were estimated by the BCA protein assay (Pierce). The proteins were
separated by SDS-polyacrylamide gel electrophoresis in the presence of
2-mercaptoethanol and electrotransferred onto Immobilon membrane
(Millipore Corp., Bedford, MA). To detect immunoreactive proteins, we
used horseradish peroxidase-conjugated anti-rabbit IgG and ECL blotting
reagents (Amersham Pharmacia Biotech). Polyclonal rabbit antisera
raised against rat HO-1, rat MSP23, and recombinant murine A170 were used as described previously (19, 21). Specific antibody was raised
against Nrf2 by immunizing rabbits with recombinant Nrf2 protein (amino acids 140-318 fused with E. coli
maltose-binding protein). An anti-actin antiserum was purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Densitometric analysis
was performed using NIH Image software.
Determination of Cystine Uptake and GSH Level--
Cystine
transport activity was measured using 14C-labeled cystine
in phosphate-buffered saline containing 0.1% glucose as described previously (20). Total (i.e. reduced plus oxidized) GSH was extracted from macrophages with 5% trichloroacetic acid solution, and
GSH content was measured as described previously (20).
Transient Transfection Assay--
The quail fibroblast cell line
QT6 (28) was maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and seeded in 24-well dishes
24 h before transfection. The cells were transfected with reporter
(pHO-1-Luc (see "Results") and pRBGP2 (7)) plasmids and an effector
plasmid (cNrf2; Ref. 7) using calcium phosphate precipitation as
described previously (29). The Luciferase assay was performed by
utilizing the Luciferase Assay System (Promega, Madison, WI) following
the supplier's protocol and measured in a Biolumat Luminometer
(Berthold, Germany). Transfection efficiencies were routinely
normalized to the activity of a co-transfected Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared from macrophages as described previously (7). An
oligonucleotide containing the stress-responsive element of the
ho-1 AB1 enhancer (5'-TCTGTTTTCGCTGAGTCATGGTTCCCGTTG-3') was
labeled with [ Impaired Induction of Antioxidative Stress Proteins in
Nrf2-deficient Macrophages--
A number of proteins or
activities are induced by electrophilic agents in murine peritoneal
macrophages. To test whether the electrophilic induction of this group
of genes shares a common regulatory mechanism with that of the
drug-metabolizing enzymes, we examined their expression in
Nrf2-deficient macrophages. Peritoneal macrophages were
harvested from nrf2-homozygous and -heterozygous mutant (or wild type) female ICR siblings. The macrophages were then
independently challenged with DEM (an electrophilic agent), paraquat
(an O
We first measured the levels of HO-1, MSP23, and A170 proteins 1 h
after the start of in vitro culture and found that the basal
levels of these proteins in the heterozygous macrophages were similar
to those of nrf2-homozygous mutant macrophages (Fig. 1A, lanes
1 and 7). After transfer of the
nrf2-heterozygous cells to in vitro
culture, these stress-inducible proteins were gradually induced by
unknown mechanisms (compare lanes 1 and
2). The gradual induction of HO-1 and MSP23 expression was
not seen in Nrf2-deficient cells (lanes 2 and 8). The important finding here was that whereas all of
the stress agents tested induced HO-1, MSP23, and A170 in
nrf2-heterozygous cells, induction was largely
canceled in Nrf2-deficient cells. Closer examination revealed
that in nrf2-null mutant cells (lanes
9-12) induction of HO-1 and A170 by DEM and GO was severely
affected, but induction by paraquat and CdCl2 was less
impaired. In contrast, while MSP23 was markedly induced by these agents
in nrf2-heterozygous cells, induction was largely absent in nrf2-null mutant cells.
Quantitative Analysis of Antioxidative Stress Protein Induction by
Various Stress Agents--
We also examined induction of these
proteins by other stress agents: catechol, CDNB,
H2O2, iodoacetic acid, sodium arsenite, menadione, and t-BHQ. Quantitative analysis by densitometry
of the stained bands is shown in Table I.
Menadione and catechol induced HO-1 in the Nrf2-deficient cells
at levels comparable with those in nrf2-heterozygous
mutant cells, suggesting the involvement of signal-transducing
pathway(s) other than the Nrf2 system. Apparently, induction of
HO-1 by sodium arsenite, t-BHQ, CDNB, and iodoacetic acid is
largely, if not exclusively, dependent on the presence of Nrf2.
A significant increase of MSP23 by all stress agents except sodium
arsenite, H2O2, and iodoacetic acid was also
observed in the heterozygous mutant cells but not in homozygous mutant cells (Table I). These results thus indicate that inducible expression of MSP23 is regulated mainly through the ARE/Nrf2 system.
To assess the induction process at the level of transcription, RNA was
extracted from macrophages that had been treated with various stress
agents and analyzed by the RNA blot analysis. Treatment with stress
agents significantly increased the levels of HO-1 and MSP23 mRNA in
nrf2-heterozygous cells (Fig. 1B,
lanes 1-6), but the induction was markedly
impaired in nrf2-null mutant cells (lanes
7-12). In contrast, whereas the lack of the induction of A170 mRNA in nrf2-null mutant cells was evident
when DEM was used as the stress agent, the induction was only partially
affected in Nrf2-deficient cells when paraquat, GO, or
CdCl2 was used as an inducer. Marked induction of the genes
was also observed in the nrf2-heterozygous cells
treated with menadione, t-BHQ, catechol, or CDNB (data not
shown). The induction of HO-1 mRNA by menadione and catechol was
diminished in Nrf2-null mutant cells, while that by
t-BHQ and CDNB was largely abolished. Induction of MSP23
mRNA by all of these agents was markedly impaired in
nrf2-null mutant cells. The three oxidative
stress-inducible genes we addressed here showed roughly comparable
variation in the mRNA and protein levels in response to various
stress agents (Fig. 1, compare A and B, and data
not shown). These results thus indicate that Nrf2 regulates the
stress agent-mediated induction of HO-1, MSP23, and A170 gene
expression. The results also clearly show that the contribution of
Nrf2 to the transcriptional activation of these genes differed
based upon the stress-inducing agent.
Induction of the Cystine Transporter
xc
It should be noted that system xc Overexpression of Nrf2 Up-regulates the Activities of the
Distal Enhancer of the ho-1 Gene--
The AB1 and SX2 enhancers of the
ho-1 gene (31) contain three and two copies, respectively,
of the Maf recognition element (MARE), which largely overlaps with ARE
(32). Due to their responsiveness to a wide variety of stress agents
including oxidative stress, the MAREs were also named stress-responsive
elements (StREs) (31-34). To ask whether Nrf2 can regulate the
expression of this antioxidative stress gene via the ho-1
AB1 enhancer, we co-transfected a Nrf2 expression plasmid and a
ho-1 AB1 enhancer-luciferase (pHO-1-Luc) reporter or
Stress Agents Post-transcriptionally Induce DNA Binding Activity of
Nrf2--
Given the clear lack of inductive response in a
number of electrophile-inducible genes, it was of interest to determine
how oxidative stress agents activate Nrf2 and thereby induce a
group of genes to counteract oxidative stress. To this end, we first examined the level of Nrf2 mRNA in macrophages under various
oxidative stresses and found that the mRNA level was not changed
significantly by any of the oxidative stress agents tested (Fig.
4A). In contrast, we found
that these same stress agents significantly enhanced the DNA binding
activity of Nrf2 to the StRE of HO-1 AB1 enhancer.
StRE binding activity in nuclear extracts was strongly induced by DEM
treatment of macrophages, as revealed by the retarded band
(arrow in Fig. 4B, lanes 1 and 2). The induction of binding activity was not observed
in nuclear extracts prepared from nrf2-deficient cells (Fig. 4B, lanes 3 and
4). The complex was effectively competed by the addition of
an excess of unlabeled StRE, mouse GST Ya gene ARE, or chicken
Lack of Nrf2 Renders Macrophages Sensitive to Oxidative
Stress--
The analysis thus far clearly indicates that macrophages
invoke an electrophile-inducible response upon exposure to oxidative stress agents and that the response is mediated by Nrf2. To ask whether this response has a major impact on cell viability, we incubated both nrf2-null and heterozygous control
macrophages with 5-20 µM CDNB for 12 h and measured
cell viability by the colorimetric MTT assay. While cell viability was
decreased in both nrf2-null mutant and control
macrophages treated with 20 µM CDNB, with 10 µM CDNB, nrf2-null mutant cells were
more sensitive to the CDNB treatment than the heterozygous control
cells (Table II). Notably, 10 µM CDNB treatment resulted in an approximately 2-fold
difference in the MTT assay, and this difference is statistically significant (p < 0.05). The difference in the
sensitivity to CDNB between the nrf2-null and
-heterozygous cells was much clear when we measured the cell viability
by the trypan blue dye exclusion test after 12 h of the CDNB
treatment. The viability of the cells was 77 and 16% (mean of two
independent experiments) for nrf2-heterozygous and
nrf2-null mutant cells, respectively. Nrf2
thus appears to contribute significantly to cellular defense mechanisms
against toxic electrophiles.
To highlight the Nrf2 contribution to cellular defense
mechanisms, we pretreated macrophages for 36 h with 100 µM DEM, a concentration that potently induces the
antioxidative stress response but with low cytotoxicity. The
macrophages were subsequently treated with 5 or 10 µM
CDNB for an additional 12 h. After incubation, we measured cell
viability by trypan blue dye exclusion. The Nrf2-deficient macrophages were more sensitive to treatment with CDNB than the heterozygous control cells. After CDNB treatment, less than 20% of the
Nrf2-deficient macrophages were viable (Fig.
5B), whereas more than 95% of
the heterozygous cells were viable (Fig. 5A). These results
unequivocally demonstrate that there are electrophile-inducible responses mediated by ARE/EpRE and Nrf2 and that the response machinery protects cells against toxic electrophiles and ROS
stresses.
We demonstrate in this study that, in addition to the drug
metabolizing enzymes that have already been shown to be regulated by
the Nrf2 pathway (9), a group of oxidative stress-inducible genes is also under the immediate transcriptional influence of Nrf2-small Maf heterodimer regulatory proteins. The fact that Nrf2 regulates a group of stress-inducible protein genes via
ARE/EpRE, as schematically illustrated in Fig.
6, is intriguing in the context of the
physiological origin of these defense mechanisms. In an evolutionary
sense, the acquisition of the ARE regulatory mechanism by genes that
protect against oxidative stress seems to confer a significant
advantage on the survival of living creatures. It should also be noted
that, whereas AREs are acquired by different categories of enzymes
(i.e. detoxication enzymes and antioxidant proteins) during
evolution, the transcription factors regulating the gene expression of
these two categories have remained the same. Thus, an array of
electrophile-responsive genes can be induced to act cooperatively upon
exposure to toxic xenobiotics.
) activity. The
response to electrophilic and reactive oxygen species-producing agents
was profoundly impaired in Nrf2-deficient cells. The lack of
induction of system xc activity
resulted in the minimum level of intracellular glutathione, and
Nrf2-deficient cells were more sensitive to toxic electrophiles. Several stress agents induced the DNA binding activity of Nrf2 in the nucleus without increasing its mRNA level. Thus Nrf2
regulates a wide-ranging metabolic response to oxidative stress.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) (18) and
60-kDa stress protein A170 (19). HO-1 is prominently induced under
various oxidative stress conditions in many different cell types (14).
HO-1-deficient embryonic fibroblasts are hypersensitive to the
cytotoxicity of both hemin and hydrogen peroxide (15). Induction of
system xc activity increases the
intracellular cysteine pool, which consequently augments the synthesis
of GSH (20), a potent antioxidant with a short half-life. MSP23 is the
murine peroxiredoxin I with antioxidative activity (21). It was
recently shown that a mammalian peroxiredoxin isoform reduces the
intracellular hydrogen peroxide level utilizing thioredoxin as an
immediate electron donor (22) and protects cells from apoptosis by
oxidative stress (23). A170 has a structural domain that interacts with
ubiquitin (24) and PKC-
(25). Electrophilic agents, such as
diethylmaleate (DEM), and other oxidative stress agents have been
reported to induce the proteins HO-1, A170, MSP23, and system
xc activity in peritoneal macrophages
(20) and fibroblasts (26). To determine whether these antioxidant
stress proteins are also under the regulation of Nrf2, we
examined in this study the electrophilic induction of this group of
genes in peritoneal macrophages from the nrf2-null
mutant mouse.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Actin cDNA was used as a positive control.
-galactosidase
expression plasmid, pENL. Normally, three independent experiments, each
carried out in duplicate, were performed, and the results were
averaged. The cells were washed 12 h after transfection, and then
DEM (Wako, Tokyo) was immediately added to the culture medium.
-32P]ATP by T4 polynucleotide kinase.
EMSA was performed as described previously (7). Where indicated,
antibodies were included in the binding reaction at 1:10 to 1:100
dilutions. The anti-chicken MafK antibody was described previously (9),
and that against human Nrf2 was purchased from Santa Cruz
Biotechnology. In competition experiments, a 100-fold excess of
unlabeled double-stranded oligonucleotides was included in the reaction
(7).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 generator), GO (an H2O2
generator), or CdCl2 (heavy metal). After challenging
macrophages with these agents, we examined expression levels of three
oxidative stress-inducible proteins (below) by immunoblotting and RNA
blotting analyses. Since wild type and heterozygous mutant mice did not
show large differences in induction of the antioxidative proteins, we
used both types of macrophages as positive controls.
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Fig. 1.
Stress agent-dependent loss of
HO-1, MSP23, and A170 gene induction in Nrf2-deficient
macrophages. A, immunoblot analysis of the effects of
oxidative stress agents on the expression levels of HO-1 (34 kDa),
MSP23 (23 kDa), and A170 (60 kDa). Macrophages from
nrf2-heterozygous (+/ 
) (lanes
1-6) and homozygous (/) (lanes
7-12) mutant mice were cultured for 1 h
(lanes 1 and 7) or 9 h
(lanes 2 and 8). Stress agents were
added to the culture medium after a 1-h culture, and the macrophages
were incubated with the agents for another 8 h (lanes
3-6 and 9-12). The stress agents added were DEM
(lanes 3 and 9), paraquat
(lanes 4 and 10), GO (lanes
5 and 11), and CdCl2
(lanes 6 and 12). B, RNA
blot analysis of the effects of oxidative stress agents on the levels
of mRNAs for HO-1, MSP23, and A170. Total RNAs were prepared from
macrophages cultured for 1 h (lanes 1 and
7) or 5 h (other lanes). Stress
agents were added at 1 h after the start of the culture.
Lane arrangements are as in A.
Quantitative analysis of the induction of HO-1 and MSP23 proteins by
various stress agents
System Is Defective in
Nrf2-deficient Cells--
Because oxidative stress agents
transcriptionally induce system xc
activity in macrophages (20, 26), the stress induction of system
xc activity in
nrf2-null mutant cells was examined next (Fig.
2A). Whereas
nrf2-heterozygous cells show system
xc activity comparable with that of
wild type cells under both basal and induced conditions (data not
shown), the oxidative stress agents DEM, paraquat, GO, and
CdCl2 barely induced system
xc activity in
nrf2-null mutant cells (Fig. 2A). In
contrast, LPS, a well known inducer of system
xc activity (30), significantly
induced the system xc activity even in
nrf2-null mutant cells, indicating that LPS induction
is mediated through an alternative regulatory pathway rather than the
Nrf2 pathway. These results argue that the transcription of the
cystine transporter gene may be under the regulatory influence of
Nrf2.
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Fig. 2.
Oxidative stress agents do not induce system
xc or GSH in
Nrf2-deficient macrophages. A, inability to
induce the system xc cystine transport
activity in the nrf2-deficient macrophages.
Macrophages were incubated for 12 h with DEM, paraquat, GO,
CdCl2, and LPS as described in the legend to Fig.
1A. B, decreased cellular GSH level in
nrf2-deficient macrophages treated with 100 µM DEM. The values in A and B
represent mean ± S.E. of three independent experiments, each
carried out in duplicate.
activity is necessary to maintain a high GSH level in cultured
macrophages, since cysteine is easily oxidized to cystine upon exposure
to air (18). While the addition of 500 µM to 1 mM of DEM to the culture medium depletes the intracellular
stores of GSH significantly, the addition of 100 µM DEM
only diminishes the GSH level minimally, and then its level increases
as a result of induced system xc
activity (26). As expected, the defect in inducible expression of
system xc activity in
nrf2-null mutant cells resulted in a decrease in cellular GSH content; after a 24-h incubation with DEM, the GSH level
dropped to less than half its original level (Fig. 2B).
-globin enhancer-luciferase (pRBGP2) reporter into QT6 fibroblasts
(Fig. 3A). DEM (Fig.
3B) and Nrf2 overexpression (Fig. 3C) both
activated HO-1-Luc reporter gene expression, with the highest
concentrations of these agents generating more than 10-fold activation.
This increase was strictly dependent on the presence of the AB1
enhancer (data not shown). We also tested a pRBGP2 reporter that
contains three tandem copies of the NF-E2 binding sequence of the
chicken -globin enhancer (7), a sequence that is very similar to the
AB1 enhancer sequence. Interestingly, the pRBGP2 reporter responded
more efficiently than the pHO-1-Luc reporter did to both DEM and the
overexpression of Nrf2 (Fig. 3, B and C).
These results indicate that StREs in the AB1 enhancer are actually
responsible for the activation of ho-1 gene expression and
that Nrf2 can activate the ho-1 gene expression
through StREs.
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Fig. 3.
Overexpression of Nrf2 is sufficient
to activate reporter gene expression driven by the AB1 enhancer of the
ho-1 gene. A, schematic representation
of the luciferase reporter constructs. In the HO-1-Luc reporter
construct, a 161-base pair fragment of the AB1 enhancer of the
ho-1 gene is placed upstream of the rabbit -globin TATA
box and luciferase reporter gene. The pRBGP2 construct is similar, but
three copies of the NF-E2 binding sequence from the chicken -globin
enhancer precede the TATA box and luciferase gene (7). B,
pRBGP2 or pHO-1-Luc reporter construct was transfected into QT6
fibroblasts in the presence or absence of the electrophilic agent DEM.
Luciferase activity in the absence of DEM was set at 100%, and results
of three independent experiments each carried out in duplicate are
shown with S.E. C, an increasing amount of chicken
Nrf2 was transfected with pRBGP2 or pHO-1-Luc. Experiments were
performed as in B.
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Fig. 4.
Increase in DNA binding activity of
Nrf2-small Maf heterodimers upon exposure to stress agents.
A, RNA blot analysis of Nrf2 mRNA in macrophages
treated with various stress agents. The
nrf2-heterozygous mutant cells were incubated for
4 h with the stress agents as in Fig. 1B. Total RNAs
extracted from the macrophages were analyzed with Nrf2 and
-actin cDNA probes. B, EMSA analysis of
Nrf2-small Maf DNA binding activity in macrophage nuclear
extracts. Nuclear extracts were prepared from wild type
(lanes 1 and 2) or
nrf2-deficient peritoneal macrophages
(lanes 3 and 4). These macrophages
were either treated with DEM for 4 h (lanes
2 and 4) or not treated (lanes
1 and 3). A double-stranded oligonucleotide of
the StRE sequence in the ho-1 gene AB1 enhancer
(ho-1 probe) was used as a probe. The complex containing
Nrf2 is indicated by an arrow. C,
Nrf2-small Maf heterodimer binds to StRE. The ho-1
probe was incubated with the nuclear extract from control macrophages
(lane 1) or the nuclear extract of macrophages
treated with DEM (lane 2). Anti-Nrf2
(lane 3), anti-MafK (lane
4), or normal rabbit IgG (lane 5) was
included in the reaction. The complex containing Nrf2 is
indicated by an arrow. D, the binding specificity
of the Nrf2-small Maf heterodimer was confirmed by an EMSA
competition experiment. The ho-1 probe was incubated with
the nuclear extract of macrophages treated with DEM (lane
1). An excess of unlabeled probe oligonucleotide
(lane 2) or double-stranded oligonucleotides from
mouse GST Ya gene ARE (lane 3), chicken
-globin enhancer NF-E2 binding site (lane 4),
or yeast transcription factor Gal4 cognate site (lane
5) was included in the binding reaction. The complex containing Nrf2 is indicated by an
arrow. E, increase in the DNA binding activity of
Nrf2-small Maf in stress agent-treated macrophages. Nuclear
extracts were prepared from the peritoneal macrophages treated with DEM
(lane 2), GO (lane 3),
paraquat (lane 4), or CdCl2
(lane 5) and examined by EMSA. An
arrow indicates the complex containing Nrf2.
F, increase of Nrf2 protein in the nucleus of the
stress agent-treated macrophages. Immunoblotting analysis was performed
with macrophage nuclear extracts.
-globin enhancer NF-E2 binding site but not by a yeast Gal4 binding
consensus sequence (Fig. 4D). This DNA-protein complex was
markedly diminished by treatment with antibodies against mouse
Nrf2 (Fig. 4C, lane 3) and
chicken MafK (lane 4), which are partner
molecules together comprising the heterodimeric transcription factor
complex (34), but the decrease was not obvious using a normal rabbit
IgG (lane 5). These results indicate that the DNA-protein complex contains both Nrf2 and small Maf proteins. The same binding activity was also induced in nuclear extracts of
macrophages treated with GO, paraquat, or CdCl2 (Fig.
4E). The Nrf2 expression level was also examined by
immunoblotting analyses of nuclear extracts prepared from the
macrophages treated with these stress agents. We found that DEM, GO,
paraquat, CdCl2, and CDNB all increased the Nrf2
level 1.5-3-fold (Fig. 4F). We therefore concluded that
post-transcriptional regulation might be involved in the activation of
Nrf2 by electrophilic agents and ROS.
CDNB sensitivity of Nrf2-null mutant macrophages
) and -homozygous (/)
mutant macrophages were incubated with 0, 5, 10, and 20 µM CDNB for 12 h. The cell viability was examined
with an MTT assay. Values are mean ± S.E. of relative absorbance
of three independent experiments each carried out in duplicate. Both
heterozygous and homozygous mutant cells showed similar absorbance in
the MTT assay without CDNB. * p < 0.05.
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Fig. 5.
Nrf2-null mutant macrophages are
sensitive to stress agents. Both
nrf2-heterozygous (+/ ) (A) and
homozygous (/) (B) mutant macrophages were first
incubated with DEM for 36 h. After changing the medium, the cells
were incubated with 10 µM CDNB for 12 h. Cell
viability was examined with the trypan blue dye exclusion test.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Schematic presentation of the antioxidative
stress response through Nrf2 and ARE/EpRE. The oxidative
stress agents liberate Nrf2 from the cytoskeleton into the
nucleus (40). Nrf2 then forms a heterodimer with a small Maf
protein, interacts with ARE, and induces transcription of a set of
genes that encode antioxidant functions. Nrf2 is a general
regulator of the defense genes against oxidative stress, which include
HO-1, MSP23, and system xc , and also
of a group of detoxication enzyme genes including GSTs and NQO1
(9).
Three levels of regulation should be important for ARE/EpRE-mediated regulation of gene expression. First, the ARE/EpRE cognate sequence shares a high degree of similarity to the consensus MARE sequence (32, 35), so that AREs are competitively bound by a number of bZip transcription factors in the Maf, Jun, Fos, and Cap'n'Collar families. Second, heterodimer formation between these bZip factors creates another level of diversity of regulation (for a review, see Ref. 32). Third, transactivation through an ARE may be regulated coordinately during oxidative stress conditions. For example, the DNA binding activity of transcription factor AP-1 was inhibited when AP-1 was exposed to oxidative stress in vitro (36). This inhibition of AP-1 binding activity may allow the binding of other activating transcription factors, such as an Nrf2-small Maf heterodimer, to ARE and hence induce genes with protective properties against oxidative stress. An interesting extension of this speculation on the physiological roles of the bZip transcription factor network and its perturbation is that the oncogenic property of the bZip (i.e. Maf, Jun, and Fos family) factors may be in part due to the lack of a proper response to electrophiles or ROS, leaving the cells in a rather prooxidative state.
The AB1 and SX2 enhancers of the ho-1 gene mediate the inducible expression of HO-1 by various stimuli (31, 33, 34). Multiple StREs reside in both ho-1 enhancers and have been shown to play important roles in the regulation of ho-1 gene expression by various electrophiles, ROS, heavy metals, and LPS (31, 33, 34). The StRE consensus sequence shares a high degree of similarity to those of ARE/EpRE and MARE (for a review, see Ref. 32), suggesting that StRE is also competitively bound by various bZip transcription factors. In this regard, we previously showed through gene targeting analysis that Nrf2 plays a central role in the regulation of GST and NQO1 gene expression through ARE (9), and the present study has extended the finding. We found that the inducible expression of the ho-1 gene by electrophiles and ROS was severely affected in the Nrf2-null mutant macrophages. We also showed that Nrf2 could activate transcription of the reporter gene through the AB1 enhancer of the ho-1 gene in a transfection assay. These results thus demonstrate that Nrf2 is one of the essential regulators of antioxidative stress genes acting through ARE/EpRE and StRE.
It should also be noted that Nrf2-dependence of stress-inducible
gene expression appeared to differ from gene to gene. For instance,
none of the electrophilic agents tested induced the expression of MSP23
in the nrf2-null mutant macrophages, whereas three of
the agents, menadione, CdCl2, and catechol, induced
ho-1 gene expression to a substantial level even in the
nrf2-null mutant macrophages. We envisage that this
variation in Nrf2 dependence reflects differences in the
structures of the enhancers that mediate the stress signals to gene
expression, since integration of signals from several stress-sensing
pathways should be executed at the level of enhancer sequence. Indeed,
accumulating evidence suggests that in addition to Nrf2,
transcription factors NF-
B, AP-1, and heat shock factor are also
activated in response to various oxidative stresses. Three lines of
evidence further support this hypothesis. First, CAAT boxes,
metal-responsive elements and NF-B binding sequences are found in
the ho-1 enhancers (33, 37). Second, menadione is known to
effectively activate NF-B, perhaps by generating ROS (38). Third,
NF-B is also known to mediate signals from LPS (39). Based on these
lines of evidence, we speculate that menadione most likely utilized the
NF-B pathway for ho-1 gene induction in
nrf2-null mutant cells. LPS induction of system
xc activity in Nrf2-null mutant
cells might also be mediated by the NF-B pathway.
Our present and previous studies uncovered the importance of Nrf2 in cellular protection against oxidative stress. This thesis was supported by the fact that induction of antioxidative stress genes by electrophilic agents was practically absent in the Nrf2-deficient macrophages. An important observation here is that the activation of Nrf2 by electrophilic agents and ROS did not accompany transcriptional induction of the nrf2 gene (Fig. 4A). Based on this observation, we recently identified Keap1, a new factor that binds to the N-terminal Neh2 domain of Nrf2 and negatively regulates Nrf2 activity (40). Electrophilic agents liberate Nrf2 from Keap1 repression. The results shown in Fig. 4 suggest a possibility that the stress agents facilitate translocation of Nrf2 from the cytosol to the nucleus. We are now investigating the precise molecular mechanism(s) whereby electrophilic agents and ROS affect interaction between the Neh2 domain of Nrf2 and Keap1.
Elevation of the cellular GSH level is one of the most important events
in the electrophile-inducible defense response (2). The increase in GSH
has been shown to be achieved by induction of a highly specific,
sodium-independent, system xc
transport system for anionic cystine in exchange for glutamate. Interestingly, hepatic -glutamylcysteine synthetase, which catalyzes the rate-limiting step of de novo GSH synthesis, is also
induced in mice fed on butylated hydroxyanisole (41) and in HepG2 cells treated with t-BHQ (42). Recently, multiple AREs in the
distal enhancer of the -glutamylcysteine synthetase heavy subunit
were reported to mediate inducible expression of the gene by
-naphthoflavone, an inducer of both phase I and II drug detoxifying
enzymes (43). AREs in the murine ferritin L promoter (12) and
metallothionein-I promoter (13) were also shown to be responsive to
H2O2. Based on these broad observations, we
speculate that many genes that function against oxidative stress are
also regulated by Nrf2 via AREs. Thus, the Nrf2-centered
gene expression regulatory system creates a coordinated cellular
defense against a wide range of electrophilic compounds and ROS.
Coordinated and inducible expression of these defense proteins should
be important in preventing various free radical-related diseases, such
as carcinogenesis, atherosclerosis, ischemia, and neurodegenerative disorders.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Shigeru Taketani for providing polyclonal antibody against rat HO-1 and Drs. Kim-Chew Lim, Kazuhiko Umesono, Priscilla Wilkins Stevens, and Ruth T. Yu for productive discussions and assistance.
| |
FOOTNOTES |
|---|
* This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture, the Japanese Society for Promotion of Sciences (JSPS), and CREST.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.
These two authors contributed equally to this work.
§ Research Fellow of the JSPS.
¶ To whom correspondence should be addressed: Center for TARA, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan. Tel.: 81-298-53-6158; Fax: 81-298-53-7318; E-mail: masi@tara. tsukuba.ac.jp.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: ROS, reactive oxygen species; GST, glutathione S-transferase; NQO1, NAD(P)H:quinone oxidoreductase; EpRE, electrophile-responsive element; ARE, antioxidant-responsive element; HO-1, heme oxygenase-1; DEM, diethylmaleate; MTT, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; CDNB, 1-chloro-2,4-dinitrobenzene; t-BHQ, t-butylhydroquinone; LPS, lipopolysaccharide; EMSA, electrophoretic mobility shift assay; MARE, Maf recognition element; StRE, stress-responsive element.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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