Degradation of Transcription Factor Nrf2 via the
Ubiquitin-Proteasome Pathway and Stabilization by Cadmium*
Daniel
Stewart
§,
Erin
Killeen
,
Ryan
Naquin
,
Safdar
Alam
, and
Jawed
Alam
§¶
From the
Department of Molecular Genetics, Ochsner
Clinic Foundation, New Orleans, Louisiana 70121, the
§ Department of Biochemistry and Molecular Biology,
Louisiana State University Health Sciences Center, New Orleans,
Louisiana 70112, and the ¶ Department of Environmental Health
Sciences, Tulane University School of Medicine, New Orleans,
Louisiana 70112
Received for publication, September 9, 2002, and in revised form, November 12, 2002
 |
ABSTRACT |
Nrf2 mediates
inducer-dependent activation of the heme oxygenase-1 (HO-1)
gene (Alam, J., Stewart, D., Touchard, C., Boinapally, S., Choi,
A. M., and Cook, J. L. (1999) J. Biol. Chem.
274, 26071-26078), but the mechanism by which HO-1 inducers regulate
Nrf2 function is not known. Treatment of mouse hepatoma (Hepa)
cells with 50 µM CdCl2 increased the amount
of Nrf2 protein in a time-dependent manner;
induction was observed within 30 min, prior to the accumulation of HO-1
mRNA. Cadmium did not significantly affect the steady-state level
of Nrf2 mRNA or the initial rate of Nrf2 protein
synthesis but increased the half-life of Nrf2 from ~13 to 100 min. Proteasome inhibitors, but not other protease inhibitors, enhanced
the expression of Nrf2, and ubiquitinylated Nrf2 was
detected after proteasome inhibition. Cycloheximide inhibited
cadmium-stimulated Nrf2 expression and DNA binding activity and
attenuated HO-1 mRNA accumulation. Conversely, proteasome
inhibitors enhanced HO-1 mRNA and protein accumulation by a
Nrf2-dependent mechanism. Together, these results indicate that Nrf2 is targeted for rapid degradation by the
ubiquitin-proteasome pathway and that cadmium delays the rate of
Nrf2 degradation leading to ho-1 gene activation.
 |
INTRODUCTION |
Reactive oxygen species
(ROS),1 which are detrimental
to cellular structures and activities, are generated during the course of normal cellular metabolism and in response to noxious, exogenous stimuli such as heavy metals, UV irradiation, and bacterial toxins. Cells utilize both exogenous (e.g. vitamins and
plant-derived phenolic antioxidants) and endogenous (e.g.
catalase and superoxide dismutase) mechanisms to detoxify ROS. A
continuing imbalance between ROS production and ROS detoxification,
however, results in cellular oxidative stress. In an effort to maintain
cellular homeostasis, cells respond to this imbalance in part by
modulating the expression of a select set of genes that encode proteins
with antioxidant and cytoprotective activities. Central to this
response are the transcription factors that control the activation of
these stress-responsive genes. Examples of such stress-responsive
transcription factors include the well-characterized heat shock factors
(1) and members of the AP-1 (2, 3) and NF-
B (3) families of proteins.
Recent studies from several laboratories have identified another
potentially important stress-responsive transcription factor, Nrf2. Like the AP-1 constituents, Fos and Jun factors,
Nrf2 is a basic-leucine zipper (bZIP) protein that functions as
an obligate dimer (4, 5); the bZIP sequence of Nrf2 precludes
homodimerization, and it dimerizes most prominently with "small"
Maf proteins (5, 6) but also with other bZIP proteins, including Jun
family members (7) and ATF4 (8). Such dimers bind to
cis-elements with similar core sequences and are
alternatively known as MAREs (Maf recognition elements) (9), AREs
(antioxidant response elements) (10), and StREs (stress response
elements) (11). Multiple Nrf2 target genes, almost all of which
are inducible by various oxidants, electrophiles, or xenobiotics, have
been identified: among others these include genes that encode phase II
detoxification enzymes (12-17) such as NAD(P)H:quinone oxidoreductase,
-glutamylcysteine synthase, and glutathione
S-transferase; heme oxygenase-1 (18); and thioredoxin (19).
Individually and collectively, the select set of Nrf2-regulated
proteins function to detoxify xenobiotics, reduce oxidized proteins,
maintain cellular reducing equivalents, disrupt redox cycling
reactions, and inhibit the generation of, or counteract the effects of,
ROS. Given these activities of the target gene products, Nrf2
appears to be a key physiological regulator of the cellular adaptive
response to oxidants and xenobiotics. Consistent with this idea,
Nrf2-deficient mice are more prone to butylated
hydroxytoluene-mediated pulmonary dysfunction (20), are susceptible to
acetaminophen hepatotoxicity (21, 22), and exhibit a significantly
higher burden of benzo[a]pyrene-induced gastric neoplasia
and reduced chemoprotective efficacy (23). Moreover, macrophages
derived from such mice exhibit reduced resistance to toxic
electrophiles (24).
Although much is known about the regulation of Nrf2 target
genes, the mechanisms by which xenobiotics and oxidants regulate Nrf2 activity are not well characterized and are under active investigation. Accumulating evidence (25-30) suggests that such activity is regulated at least in part at the level of sub-cellular compartmentalization. According to this model, under normal conditions, Nrf2 exists in an inactive, cytoplasm-localized state, in part or fully as a consequence of binding to the cytoskeleton-associated protein Keap1 (25, 26). Upon cellular stimulation by stress agents, the
cytoplasmic retention mechanism is inactivated, and Nrf2 is
transported to the nucleus by one or more as yet uncharacterized mechanisms but one that, under certain circumstances, may involve protein kinase C-mediated phosphorylation of Nrf2 (16). In the nucleus, Nrf2 heterodimers bind to response elements to regulate target gene transcription. Another control point may be at the level of
DNA binding. Jaiswal and colleagues (7, 31) have shown that the
association and DNA-binding activity of Nrf2·Jun dimers
requires a cytosolic factor yet to be characterized. Whether such a
factor is responsive to oxidative stress and how it regulates an
ultimately nuclear activity are not known. Xenobiotics may also promote
Nrf2 activity by stimulating nrf2 gene
transcription. Kwak et al. (32) have recently described a
positive feedback mechanism in which Nrf2 autoregulates its own
expression in response to 3H-1,2-dithiole-3-thione via an
ARE-like sequence within the nrf2 gene.
In this report, we describe a different mechanism of Nrf2
regulation. We show that Nrf2 is a highly labile protein,
rapidly and specifically degraded by the ubiquitin-proteasome system, and that cadmium stimulates Nrf2 activity and subsequent
activation of the ho-1 target gene at least in part by
stabilization of the Nrf2 protein.
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EXPERIMENTAL PROCEDURES |
Materials--
Oligonucleotides were synthesized by IDT, Inc.,
and radiochemicals were obtained from PerkinElmer Life Sciences.
Antibodies against transcription factors (Nrf1, Nrf2, E2F1,
JunD, and ATF2) were obtained from Santa Cruz Biotechnology.
Inc., whereas anti-rat HO-1 was acquired from StressGen Biotech. Corp.
Protease inhibitors were purchased from Calbiochem. Reagents for
luciferase assays were acquired from Sigma Chemical Co. All other
chemicals were of reagent grade.
RNA and Protein Blot Analyses--
Mouse hepatoma (Hepa) cells
were cultured in a humidified atmosphere of 5% CO2 and
95% air at 37 °C in Dulbecco's modified Eagle's medium
(Invitrogen) containing 0.45% glucose, 10% fetal bovine serum
(Mediatech), and 50 µg/ml gentamicin. Cells were routinely passaged
every 3-4 days. Unless otherwise indicated, cells were typically
seeded (4 × 106 cells/100-mm plate or 1 × 106 cells/60-mm plate) and cultured for 40-48 h in
complete medium prior to treatment with agents in serum-free medium.
Total RNA was isolated by the procedure of Chomczynski and Sacchi (33), and Northern or dot blot analyses were carried out as previously described (34, 35). Western blot analyses were carried out using either
whole cell protein extracts or nuclear extracts (36). For detection of
Nrf2, extracts were electrophoresed on a 4-12% gradient
SDS-PAGE gel (Invitrogen), and proteins were transferred to a
polyvinylidene difluoride membrane. The membrane was blocked overnight
in Tris-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v)
nonfat dry milk and then incubated with the primary antibody (1:1000
dilution) for 3 h. Treatment with the secondary antibody and
antigen detection were carried out using the ECL system (Amersham
Biosciences) according to the manufacturer's recommendation. Detection
of HO-1, ATF2, JunD, and MafG was carried out as previously described
(8, 36). Additional details are provided in the figure legends.
Pulse Labeling and Pulse-Chase Analyses--
Hepa cells were
plated (5 × 105 cells/35-mm plate) and cultured until
~80% confluent. Cells were washed (2×) with pulse labeling media
(methionine-free, Hepes-buffered (pH 7.5) Dulbecco's modified Eagle's
medium containing 10% dialyzed fetal bovine serum) and incubated at
37 °C for 15 min. The media was removed and replaced with pulse
labeling media containing 0.1 mCi/ml [35S]methionine and
the appropriate inducing agent. After labeling for appropriate time
periods, the media was removed and the cells were washed (2×) with
cold phosphate-buffered saline and lysed by the addition of
radioimmune precipitation assay buffer (10 mM Tris-HCl
(pH 7.4), 0.5% sodium deoxycholate, 1% Nonidet P-40, 400 mM NaCl, 0.1% SDS) containing 50 mM NaF, 2 mM EGTA, and 0.1 mM PMSF. Samples were
collected by scraping, transferred to Eppendorf tubes, and sonicated
(3× for 30 s each). Lysates were collected after centrifugation
at 10,000 × g for 10 min at 4 °C. For pulse-chase experiments, labeling was carried out for 30 min and terminated by
direct addition of cold methionine (20-fold molar excess) to the
culture media. Cells were incubated for varying time periods prior to
lysis. For immunoprecipitation, cell lysates were pre-cleared using 1 µg of pre-immune rabbit IgG and 20 µl of a 50% slurry of
Sepharose-coupled Protein A. Pre-cleared lysates containing equivalent
amounts of trichloroacetic acid-precipitable counts (~150 µg
of protein) were incubated with 1 µg of anti-Nrf2 IgG at
4 °C with continuous rotation. After 18 h, a 20-µl slurry of Protein A-Sepharose was added, and incubation was continued for an
additional 2 h. Immunocomplexes were pelleted by centrifugation, washed in RIPA buffer (4×), and resuspended in 40 µl of Laemmli sample buffer containing 1%
-mercaptoethanol and 0.1 mM
PMSF. The samples were heated at 100 °C for 4 min, and 15-µl
aliquots were subjected to electrophoresis. Signals were detected and
quantified using a phosphorimaging device (Packard Instruments).
Ubiquitinylation Assay--
Ubiquitinylation of Nrf2 and
E2F1 was carried out by modification of the procedure of Treier
et al. (37). Human embryo kidney (HEK) 293 cells (1 × 106 cells/60-mm plate) were transfected with DNA mixtures
consisting of the following: empty vector or plasmids encoding
Nrf2 or E2F1 (500 ng), empty vector or plasmid encoding
His-tagged ubiquitin (500 ng), and a luciferase expression plasmid (25 ng) using FuGENE 6 transfection reagent (Roche Molecular Biochemicals)
according to the manufacturer's recommendations. Twenty-four hours
after transfection, the cells were treated with 10 µM
MG-132 for 3 h and collected in phosphate-buffered saline. A
portion (1/20) of the cells was lysed and assayed for luciferase
activity (38). The remainder of the cells was lysed in lysis binding
buffer (20 mM Tris-HCl (pH 7.9), 0.5 M NaCl, 8 M urea) containing 5 mM imidazole, and
luciferase-equivalent portions were adsorbed onto
Ni+2-charged agarose (25-µl packed volume). The resin was
washed (3×) with lysis binding buffer containing 5 mM
imidazole and then (3×) with lysis binding buffer containing 20 mM imidazole. Proteins were eluted within 30 µl of
elution buffer (20 mM Tris-HCl (pH 6.7), 0.5 M
NaCl, 8 M urea, and 200 mM imidazole) and
subjected to electrophoresis and Western blot analysis as described above.
Electrophoretic Mobility Shift Assay--
Cells were treated
with vehicle or 25 µM CdCl2 in the presence
or absence of 5 µg/ml cycloheximide in serum-free medium for 3 h
and then collected for preparation of whole cell extracts (36). A
double-stranded oligonucleotide containing the sequence 5'-TTTTATGCTGTGTCATGGTT-3' (core StRE sequence is
underlined) was used as probe in EMSA reactions using conditions
previously reported (36). In antibody supershift assays, 2 µg of
pre-immune IgG or anti-transcription factor IgG was added to the
reaction mixture and incubated for 20 min at room temperature prior to electrophoresis.
Transfection and Luciferase Assays--
The construction of
pE1-luc and pE1M789-luc has been described previously (35). Transient
transfections of Hepa cells, preparation of cell extract, and
measurement of reporter enzyme activities were carried out as
previously reported (38, 39). Additional details are provided in the
figure legends.
 |
RESULTS |
Cadmium Stimulates the Steady-state Level of Nrf2 Protein
but Not of Nrf2 mRNA--
We have previously
demonstrated the requirement for Nrf2 in
cadmium-dependent ho-1 gene activation in
several cell types, including Hepa cells (18, 35). Consistent with this
role, here we demonstrate that cadmium also regulates Nrf2
expression. Treatment of Hepa cells with 50 µM
CdCl2 increased the steady-state level of Nrf2 in a
time-dependent manner to greater than 20-fold above basal
values (Fig. 1). In our gel system, mouse
Nrf2 migrates as an 88-kDa protein, significantly larger than
the predicted size of 66 kDa. Accumulation of Nrf2 was detected
within 30 min after cell treatment, well before the observable increase
in HO-1 mRNA (typically between 1 and 2 h after exposure to
CdCl2) (Ref. 34 and Fig. 2)
or HO-1 protein (2 h) (Fig. 1). The induction of Nrf2 protein by
cadmium is a specific response, because the expression of ATF2 and of
other transcription factors such as JunD and CREB (8) is not affected
by this agent.

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Fig. 1.
Cadmium stimulates expression of Nrf2
and HO-1 protein in Hepa cells. Cells were exposed to 50 µM CdCl2 for the indicated times. Western
blot analyses were carried out as described under "Experimental
Procedures" using whole cell extracts (30 µg of protein/lane) and
antibodies directed against the indicated proteins. The Nrf2
protein band is marked by an arrow, and the migration of the
molecular mass (kDa) markers is indicated.
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Fig. 2.
Cadmium stimulates accumulation of HO-1
mRNA but not of Nrf2 RNA. Hepa cells were exposed to 50 µM CdCl2 for the indicated time and collected
for RNA isolation. Total RNA (10 µg/lane) was electrophoresed,
transferred to nylon membranes, and hybridized to the indicated
cDNA probes.
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In stark contrast to the effect on Nrf2 protein expression,
exposure of Hepa cells to 50 M cadmium (for up to 6 h)
did not significantly alter the steady-state level of Nrf2
mRNA (Fig. 2). As expected, cadmium treatment dramatically
increased the level of HO-1 mRNA in a time-dependent
manner. The level of ribosomal protein S3 mRNA, which was used as a
control for RNA loading, was not significantly changed during the
course of this treatment. Taken together, these results suggest that
cadmium regulates Nrf2 expression primarily by one or more
post-transcriptional mechanisms.
Effect of Cadmium on Nrf2 Synthesis and
Stability--
Cadmium may increase the level of Nrf2 by
regulating one or both of two general processes: the rate of
Nrf2 synthesis or the rate of Nrf2 degradation. The rate
of Nrf2 synthesis was monitored by pulse-labeling analysis, and
typical results are shown in Fig. 3.
[35S]Methionine-labeled Nrf2 steadily accumulated
over a period of 15 min in unstimulated cells, and the rate of
accumulation was not affected by cadmium (left panel). The
apparent stimulation of Nrf2 synthesis by cadmium was first
detected after 20 min of labeling and was more pronounced (~2.5-fold
over control) after 30 and 60 min (right panel).

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Fig. 3.
Cadmium does not affect the initial rate of
Nrf2 synthesis. Pulse labeling analyses was carried out as
described under "Experimental Procedures" in the absence ( ) or
presence (+) of 50 µM CdCl2 for the indicated
times. The electrophoresis gel was dried and exposed to the
phosphorimaging screen for 5 days. The migration of the molecular mass
(kDa) markers is indicated.
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To examine the role of protein stabilization in Nrf2 regulation
by cadmium, we monitored the decay of basal and cadmium-induced Nrf2 protein in Hepa cells after inhibition of protein synthesis by cycloheximide. In the experiment depicted in Fig. 1, whole cell
extracts were used to monitor both Nrf2 and the endoplasmic reticulum-localized HO-1 in the same sample. Subsequent studies using
cytoplasmic and nuclear fractions demonstrated that basal and induced Nrf2 are localized exclusively in the nucleus,
because the level of cytoplasmic Nrf2 is below the detection
limit of the Western blot assay (data not shown). Consequently, in the following experiment, nuclear extracts were used to obtain a stronger Nrf2 signal in unstimulated cells. Nrf2 in the nuclei of
untreated cells decayed rapidly after addition of cycloheximide and
could not be detected after 40 min even after longer exposure of the membrane to the phosphorimaging screen (Fig.
4A). In unstimulated Hepa
cells, the half-life of Nrf2 was calculated to be ~13 min. Cadmium-induced Nrf2, however, was more stable with an estimated t1/2 of nearly 100 min. The specificity of this
response was demonstrated by the fact that cadmium did not appreciably
affect the steady-state level or the rate of degradation of
transcription factor JunD (Fig. 4B). Similar results were
obtained by pulse-chase analysis (data not shown but see below). We
interpret these results to indicate that cadmium stimulates Nrf2
expression primarily by attenuating the rate of Nrf2
degradation. Although we cannot completely exclude an effect on
Nrf2 synthesis, it is likely that the apparent increase in the
synthetic rate observed at the later time points (30 to 60 min) is a
consequence of protein stabilization.

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Fig. 4.
Cadmium increases the stability of
Nrf2 protein. Hepa cells were incubated in serum-free
medium in the absence or presence of 50 µM
CdCl2 for 2 h. The culture medium was then replaced
with serum-free medium containing 100 µg/ml cycloheximide, and the
cells were harvested at the indicated times (t) after
addition of cycloheximide. Nuclear extracts (15 µg/lane) were
subjected to Western blot analyses for Nrf2 (A) or
JunD (B). The Nrf2 protein band is marked by an
arrow, and the migration of the molecular mass markers is
indicated. Whole cell extract (3.5 µg) from HEK293 cells transfected
with a mouse Nrf2 expression plasmid was used as a positive
control (+) for Nrf2. The Nrf2 signal was quantified by
densitometry and represents the average of two experiments.
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Proteasome Inhibitors Stimulate Nrf2 Expression--
Many
labile regulatory proteins, including signal-activated transcription
factors, are commonly degraded by the 26 S proteasome, a highly
conserved, multiprotein proteolytic system (40). If Nrf2 is
degraded by this pathway, then inhibition of the proteasome should
result in higher levels of Nrf2. As shown in Fig.
5A, with treatment of Hepa
cells with MG-132 (10 µM), lactacystin (20 µM), and PI 1 (20 µM), all selective
inhibitors of the 26 S proteasome significantly increased the
steady-state level of Nrf2. The most potent response was
observed with MG-132; lactacystin and PI 1 were less effective, but
unlike MG-132, further stimulated Nrf2 expression in the
presence of 25 µM CdCl2. Inhibitors of serine proteases (PMSF), calpain (PD150606), serine/cysteine proteases (leupeptin), and lysozomal proteases (chloroquine) were ineffective (Fig. 5A and data not shown). Control experiments showed
that none of the protease inhibitors stimulate expression of
-tubulin (Fig. 5A).

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Fig. 5.
A, proteasome inhibitors stimulate
Nrf2 expression. Hepa cells were treated with vehicle
(Me2SO, DMSO), MG-132 (10 µM),
lactacystin (20 µM), PI 1 (20 µM), PMSF
(100 µM), or PD150606 (20 µM) in the
presence or absence of 25 µM CdCl2 for the
indicated times. Western blot analyses for Nrf2 and -tubulin
(Tub) were carried out as described under "Experimental
Procedures" using whole cell extracts (30 µg of protein/lane). +,
positive control for Nrf2. B, MG-132 increases
Nrf2 stability. Hepa cells were pulse-labeled with
[35S]methionine for 30 min in the absence ( ) or
presence (+) of 10 µM MG-132 and "chased" with cold
methionine for the indicated time. Pulse-chase analysis was continued
as described under "Experimental Procedures." The electrophoresis
gel was dried and exposed to the phosphorimaging screen for 5 days. +,
immunoprecipitate of [35S]methionine-labeled Nrf2
in cell extract from HEK293 cells transfected with a mouse Nrf2
expression plasmid.
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The proteasome inhibitors did not affect the steady-state level of
Nrf2 mRNA (data not shown). To directly demonstrate that induction of Nrf2 by the proteasome inhibitors results from
inhibition of protein degradation, Nrf2 stability was assayed by
pulse-chase experiments. Under normal conditions,
[35S]methionine-labeled Nrf2 was rapidly degraded
and barely detected 30 min after termination of labeling (Fig.
5B, "
"). In the presence of MG-132, labeled
Nrf2 was maintained at constant levels up to the last time point
tested. Taken together, these results indicate that Nrf2 is
specifically degraded by the 26 S proteasome.
Ubiquitinylation of Nrf2--
Most, but not all, proteins
selectively degraded by the 26 S proteasome are marked by prior,
covalent ligation of ubiquitin molecules, a highly conserved ~8-kDa
polypeptide, to the
-amino group of lysine residues. The
ubiquitin-conjugating system usually generates substrate proteins
containing varying lengths of polyubiquitin chains resulting from
linkage of successive ubiquitin molecules to the previous moiety (40).
Identification of ubiquitinylated Nrf2 would suggest that this
transcription factor is regulated via the ubiquitin-proteasome system.
Because they are rapidly degraded by the proteasome, endogenous
ubiquitinylated species of a specific protein are generally difficult
to detect (37, 41). We, therefore, employed the strategy of Treier
et al. (37), which involves cellular co- and overexpression
of the target protein (e.g. Nrf2) and poly-His-tagged
ubiquitin. His-tagged ubiquitin-conjugated proteins are subsequently
purified and concentrated by affinity chromatography, and the
ubiquitinylated target protein is detected immunologically. In a
necessary modification, transfected cells were treated with MG-132 to
inhibit proteasome activity prior to isolation of ubiquitinylated
proteins. Transcription factor E2F1 was used as a positive control for
this experiment, because it is known to be ubiquitinylated (42). As
shown in Fig. 6, a ladder of mono- and
polyubiquitinylated Nrf2 was readily detected in cells
expressing Nrf2 and His-tagged ubiquitin (lane 4). No such species were detected when either of the corresponding expression plasmids was omitted (lanes 1-3). An analogous profile was
observed with E2F1 (lanes 5-8). In theory, unconjugated
proteins should not be detected in this assay as was the case for E2F1
(arrow, lane 8). Nonetheless, unconjugated
Nrf2 (arrow, lanes 3 and 4) was
observed, suggesting some affinity for the resin.

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Fig. 6.
Ubiquitinylation of Nrf2 and
E2F1. HEK293 cells were transfected with the indicated expression
plasmids, and ubiquitinylation assays were carried out as described
under "Experimental Procedures." The positions of the unconjugated
Nrf2 and E2F1 are marked by arrows, and the
mono-ubiquitinylated proteins are marked by asterisks. The
migration of the molecular mass (kDa) markers is indicated.
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Functional Consequence of Modulation of Nrf2
Levels--
That induction of Nrf2 is not simply an epigenetic
phenomenon but represents an effect with functional consequences is
suggested by data provided in Figs. 7 and
8. Consistent with the mode of regulation
described above, the basal and cadmium-stimulated expression of
Nrf2 was abrogated in the presence of the protein synthesis inhibitor cycloheximide (Fig. 7A). Under these conditions,
accumulation of MafG, an Nrf2 dimerization partner, was not
altered. Cycloheximide had a similar effect on Nrf2 at the level
of DNA binding activity. Electrophoretic mobility shift assays using an
oligonucleotide probe harboring an Nrf2 recognition site
(i.e. StRE from the ho-1 gene), and whole cell
extracts from untreated Hepa cells detected several StRE·protein
complexes of similar intensity (Fig. 7B, lane 2).
Treatment with cadmium substantially altered the electrophoretic profile resulting in one prominent complex (or several co-migrating complexes) (lane 4). Nrf2 appears to be a predominant
factor in this complex, because the complex was quantitatively shifted
with anti-Nrf2 antibodies (lane 7) but not with
pre-immunized IgG (lane 6) or an antibody to the related
StRE-binding protein, Nrf1 (lane 8). Consistent with
induction of Nrf2 protein by cadmium and the abrogation of this
response by cycloheximide, cycloheximide co-treatment abolished
formation of the Nrf2·StRE complex (lane 5).
Inhibition of cadmium-stimulated binding of Nrf2 to the StRE
correlated with the attenuation of cadmium-induced HO-1 mRNA
accumulation in response to cycloheximide (Fig. 7C).

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Fig. 7.
A, cycloheximide inhibits
cadmium-induced Nrf2 expression. Hepa cells were exposed to
vehicle ( ) or 50 µM CdCl2 (+Cd) in the
presence or absence of 5 µg/ml cycloheximide for 2 h. Western
blot analyses were carried out on nuclear extracts (15 µg/lane) using
anti-Nrf2 or anti-MafG antibodies. The migration of the
molecular mass markers is indicated. B, cycloheximide
inhibits cadmium-induced Nrf2·StRE complex formation. EMSA
reactions were carried out as described under "Experimental
Procedures" using extracts prepared from Hepa cells treated with
vehicle or 50 µM CdCl2 (Cd) in the
presence or absence of 5 µg/ml cycloheximide (CHX) for
3 h (lanes 2-5). Antibody-supershift EMSA reactions
(lanes 6-8) were carried out with protein extract from
+cadmium/ cycloheximide cells and with pre-immune IgG
("Ig") or antibodies directed against the indicated
transcription factors. No extract was used in the reaction in
lane 1. C, cycloheximide inhibits cadmium-induced
HO-1 mRNA accumulation. Hepa cells were exposed to 25 µM CdCl2 in the absence or presence of 5 µg/ml cycloheximide for the indicated time and harvested for RNA
isolation. Total RNA (5 µg/slot) was dot-blotted onto a nylon
membrane and hybridized consecutively to the HO-1 and S3 probes.
S3-normalized HO-1 mRNA levels (the average value from two
experiments) are presented.
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Fig. 8.
Proteasome inhibitors stimulate HO-1 mRNA
(A) and protein (B) accumulation. Hepa cells were
treated for 3 (A) or 4 (B) h with vehicle
(Me2SO, DMSO), MG-132 (10 µM),
lactacystin (20 µM), PI 1 (20 µM), PD150606
(20 µM), PMSF (100 µM), leupeptin (10 µg/ml), or chloroquine (100 µM) in the absence
( Cd) or presence (+) of 50 µM
CdCl2 as indicated. RNA and protein blot analyses were
carried out as described under "Experimental Procedures" and the
legends to Figs. 5 and 7. S3-normalized HO-1 mRNA levels
(S/H; average value from four experiments) are presented
(A). C, MG-132 stimulates Nrf2·StRE
complex formation. EMSA reactions were carried out as described under
"Experimental Procedures" using extracts prepared from Hepa cells
treated with vehicle ( ), 50 µM CdCl2
(Cd), or 10 µM MG-132 (MG) for
3 h (lanes 1-4). Antibody-supershift EMSA reactions
were carried out pre-immune IgG or anti-Nrf2 (lanes 5 and 6). D, proteasome inhibitors stimulate
ho-1 enhancer activity. Hepa cells were transfected with
plasmids pE1-luc (E1) or pE1M789-luc (E1M789) and
treated with vehicle (Me2SO, DMSO), 10 µM MG-132, 20 µM lactacystin, 20 µM PD150606, or 50 µM CdCl2 for
5 h. Luciferase activity normalized to that in vehicle-treated
cells is presented. Each data point represents the average ± S.E.
from three to four experiments.
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In contrast to cycloheximide, proteasome inhibitors, which positively
regulate Nrf2 expression (Fig. 5A), also induced HO-1 mRNA and protein accumulation (Fig. 8, A and
B). The highest level of HO-1 mRNA induction (17-fold)
was observed with MG-132, the most potent of the Nrf2
stimulatory proteasome inhibitors. PD150606, PMSF, leupeptin, and
chloroquine, which do not affect Nrf2 expression, also did not
induce HO-1 mRNA or protein levels (Fig. 7, A and B, and data not shown). The proteasome inhibitors did not
further stimulate cadmium-dependent HO-1 mRNA
accumulation, suggesting that these agents activate the ho-1
gene by similar mechanisms. Support for this supposition is
provided by the observation that MG-132, like cadmium, promotes
formation of the StRE·Nrf2 complex (Fig. 8C).
Additionally, both MG-132 and lactacystin stimulated expression of a
luciferase reporter gene under the control of the mouse ho-1
gene enhancer (E1) containing three StREs by ~3- and 2-fold,
respectively (Fig. 8D). By comparison, cadmium enhanced luciferase activity by ~13-fold and the calpain inhibitor PD150606 was without effect. Mutation of the StREs into sequences incapable of
binding Nrf2 (35) generates a variant E1 (E1M789) that was unresponsive to MG-132 or lactacystin and exhibited reduced sensitivity to cadmium. Taken together, the above results suggest that
stabilization of Nrf2 by cadmium (or proteasome inhibitors)
results in a functional transcription factor capable of binding to its
target sequences and promoting transcription of target genes such as
ho-1.
 |
DISCUSSION |
We have previously reported that cadmium does not regulate
Nrf2 protein expression in Hepa cells (8). This conclusion was based on artifactual results obtained using extract preparation, sample
electrophoresis, and Western blotting conditions that
generated a weak Nrf2 signal masked by a more
intense, co-migrating but nonspecific signal. The use of a lower
percentage polyacrylamide gel and polyvinylidene difluoride membranes
has allowed separation of the Nrf2 signal from the nonspecific
signal and also significantly improved the signal-to-noise ratio. The
new analysis reveals a robust induction of Nrf2 protein in
response to cadmium and demonstrates that such induction is regulated
primarily at the level of protein degradation/stabilization.
The present studies demonstrate that Nrf2 is a highly labile
protein (t1/2 ~ 13 min) specifically degraded by
the ubiquitin-proteasome system. This pathway is known to regulate the
activity of various transcription factors, such as c-Jun, c-Fos,
NF-
B, STAT1, and p53 (40), that are typically short-lived and often
activated in response to extracellular stimuli. Furthermore, Molinari
et al. (43) have shown that the rate of degradation of
transcription activators by the proteasome correlates with the potency
of one or more of the activation domains; Nrf2 contains a very
powerful activation domain (8, 26, 36). The detailed mechanism by which
transcription factors are marked and degraded by the
ubiquitin-proteasome pathway is generally unique for each protein, but
such regulation is often dependent on cis-acting sequences
(i.e. specific sequence domains within the target protein)
and may involve post-translational signals such as phosphorylation. One
structural signal commonly found in rapidly degraded proteins is the
PEST domain, which is defined as a hydrophilic stretch of
12 or greater amino acids, enriched in proline, glutamate, serine and
threonine residues and flanked by positively charged residues. PEST
sequences have been identified in many labile proteins, including
ornithine decraboxylase, the NF-
B inhibitor I
B
, and c-Fos (44,
45). Using the PESTfind algorithm (available at
Vienna.at.embnet.org/htbin/embnet/PESTfind), we have identified several
poor PEST candidates and one potential PEST sequence at position
350-380 (HSVESSIYGDPPPGFSDSEMEELDSAPGSVK) in mouse Nrf2. Serine
and threonine residues within PEST domains are potential
phosphorylation sites that may be necessary for degradation. For
instance, specific phosphorylation of Ser-32 and Ser-36 within
I
B
is required for ubiquitinylation and subsequent degradation of
I
B
leading to activation of NF-
B (46, 47). Studies to
determine the role of the putative Nrf2 PEST domain (or other
cis-elements) and of (de)phosphorylation reactions in degradation of Nrf2 are in progress.
How does cadmium promote Nrf2 stability? In the case
of hypoxia-mediated stabilization of hypoxia-inducible factor 1
, low oxygen tension reduces ubiquitinylation of hypoxia-inducible factor 1
(41). We have not, however, been able to detect a similar effect
of cadmium on Nrf2 (data not shown) suggesting that cadmium may
inhibit another step or steps within the degradation pathway. Whether
such interference involves the PEST domain identified above and
(de)phosphorylation reactions or other post-translational modifications
remains to be determined.
Finally, it is worth pointing out that, although proteasome inhibitors
are as or more effective than cadmium in stimulating Nrf2
levels, they are less potent inducers of HO-1 mRNA. One explanation for this discrepancy is that Nrf2 may not be the sole regulator of cadmium-dependent ho-1 gene activation and
that other transcription factors, which may not be regulated by the
proteasome, also contribute to this process. This possibility is
supported by the observation that mutation of the StREs within E1 does
not completely abolish cadmium-dependent transcription
activity of the enhancer (Fig. 8D). Additional support for a
non-StRE/Nrf2 mode of regulation is provided by Takeda et
al. (48) who identified a cadmium response element that is
distinct from, but in close proximity to, the StREs in the human
ho-1 gene. The protein that binds to this site and
presumably mediates the transcriptional response is distinct from
Nrf2. An alternative, or additional, possibility for the above
discrepancy is that the steady-state level of Nrf2 is not the
sole determinant of Nrf2 activity. Such activity is likely to
require post-translational modification, such as phosphorylation, which
may occur optimally in response to cadmium or other oxidants but not
during proteasome inhibition.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Stuart Orkin, Volker Blank, and
Erik Flemington for generously providing the Nrf2 expression
plasmid, the anti-MafG antibody, and plasmids encoding E2F1 and
His-tagged ubiquitin, respectively.
 |
FOOTNOTES |
*
This work was supported by the Department of Energy
cooperative agreement DE-FC26-00NT40843 and the Center for
Bioenvironmental Research, Tulane and Xavier Universities (to
B. S. B.).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: Dept. of Molecular
Genetics, Ochsner Clinic Foundation, 1516 Jefferson Highway, New
Orleans, LA 70121. Tel.: 504-842-3314; Fax: 504-842-3381; E-mail:
jalam@ochsner.org.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M209195200
 |
ABBREVIATIONS |
The abbreviations used are:
ROS, reactive oxygen
species;
HO-1, heme oxygenase-1;
Nrf, NF-E2-related factor;
AP-1, activator protein 1;
NF-
B, nuclear factor
B;
bZIP, basic
region/leucine zipper;
StRE, stress response element;
ARE, antioxidant
response element;
EMSA, electrophoretic mobility shift assay;
STAT1, signal transducers and activators of transcription 1;
PI 1, proteasome
inhibitor 1.
 |
REFERENCES |
1.
|
Morano, K. A.,
and Thiele, D. J.
(1999)
Gene Expr.
7,
271-282[Medline]
[Order article via Infotrieve]
|
2.
|
Karin, M.,
Liu, Z.,
and Zandi, E.
(1997)
Curr. Opin. Cell Biol.
9,
240-246[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Karin, M.,
Takahashi, T.,
Kapahi, P.,
Delhase, M.,
Chen, Y.,
Makris, C.,
Rothwarf, D.,
Baud, V.,
Natoli, G.,
Guido, F.,
and Li, N.
(2001)
Biofactors
15,
87-89[Medline]
[Order article via Infotrieve]
|
4.
|
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]
|
5.
|
Itoh, K.,
Igarashi, K.,
Hayashi, N.,
Nishizawa, M.,
and Yamamoto, M.
(1995)
Mol. Cell. Biol.
15,
4184-4193[Abstract]
|
6.
|
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]
|
7.
|
Venugopal, R.,
and Jaiswal, A. K.
(1998)
Oncogene
17,
3145-3156[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
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]
|
9.
|
Kataoka, K.,
Noda, M.,
and Nishizawa, M.
(1994)
Mol. Cell. Biol.
14,
700-712[Abstract]
|
10.
|
Rushmore, T. H.,
Morton, M. R.,
and Pickett, C. B.
(1991)
J. Biol. Chem.
266,
11632-11639[Abstract/Free Full Text]
|
11.
|
Choi, A. M.,
and Alam, J.
(1996)
Am. J. Respir. Cell Mol. Biol.
15,
9-19[Abstract]
|
12.
|
Venugopal, R.,
and Jaiswal, A. K.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14960-14965[Abstract/Free Full Text]
|
13.
|
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]
|
14.
|
Moinova, H. R.,
and Mulcahy, R. T.
(1999)
Biochem. Biophys. Res. Commun.
261,
661-668[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Wild, A. C.,
Moinova, H. R.,
and Mulcahy, R. T.
(1999)
J. Biol. Chem.
274,
33627-33636[Abstract/Free Full Text]
|
16.
|
Huang, H. C.,
Nguyen, T.,
and Pickett, C. B.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
12475-12480[Abstract/Free Full Text]
|
17.
|
Nguyen, T.,
Huang, H. C.,
and Pickett, C. B.
(2000)
J. Biol. Chem.
275,
15466-15473[Abstract/Free Full Text]
|
18.
|
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]
|
19.
|
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]
|
20.
|
Chan, K.,
and Kan, Y. W.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12731-12736[Abstract/Free Full Text]
|
21.
|
Chan, K.,
Han, X. D.,
and Kan, Y. W.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
4611-4616[Abstract/Free Full Text]
|
22.
|
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]
|
23.
|
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]
|
24.
|
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]
|
25.
|
Kobayashi, M.,
Itoh, K.,
Suzuki, T.,
Osanai, H.,
Nishikawa, K.,
Katoh, Y.,
Takagi, Y.,
and Yamamoto, M.
(2002)
Genes Cells
7,
807-820[Abstract/Free Full Text]
|
26.
|
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]
|
27.
|
Zipper, L. M.,
and Mulcahy, R. T.
(2002)
J. Biol. Chem.
277,
36544-36552[Abstract/Free Full Text]
|
28.
|
Sekhar, K. R.,
Spitz, D. R.,
Harris, S.,
Nguyen, T. T.,
Meredith, M. J.,
Holt, J. T.,
Guis, D.,
Marnett, L. J.,
Summar, M. L.,
and Freeman, M. L.
(2002)
Free Radic. Biol. Med.
32,
650-662[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Dinkova-Kostova, A. T.,
Holtzclaw, W. D.,
Cole, R. N.,
Itoh, K.,
Wakabayashi, N.,
Katoh, Y.,
Yamamoto, M.,
and Talalay, P.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
11908-11913[Abstract/Free Full Text]
|
30.
|
Dhakshinamoorthy, S.,
and Jaiswal, A. K.
(2001)
Oncogene
20,
3906-3917[CrossRef][Medline]
[Order article via Infotrieve]
|
31.
|
Jeyapaul, J.,
and Jaiswal, A. K.
(2000)
Biochem. Pharmacol.
59,
1433-1439[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Kwak, M. K.,
Itoh, K.,
Yamamoto, M.,
and Kensler, T. W.
(2002)
Mol. Cell. Biol.
22,
2883-2892[Abstract/Free Full Text]
|
33.
|
Chomczynski, P.,
and Sacchi, N.
(1987)
Anal. Biochem.
162,
156-159[CrossRef][Medline]
[Order article via Infotrieve]
|
34.
|
Alam, J.,
Shibahara, S.,
and Smith, A.
(1989)
J. Biol. Chem.
264,
6371-6375[Abstract/Free Full Text]
|
35.
|
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]
|
36.
|
Gong, P., Hu, B.,
Stewart, D.,
Ellerbe, M.,
Figueroa, Y. G.,
Blank, V.,
Beckman, B. S.,
and Alam, J.
(2001)
J. Biol. Chem.
276,
27018-27025[Abstract/Free Full Text]
|
37.
|
Treier, M.,
Staszewski, L. M.,
and Bohmann, D.
(1994)
Cell
78,
787-798[Medline]
[Order article via Infotrieve]
|
38.
|
Alam, J.
(2000)
in
Functional Analysis of the Heme Oxygenase-1 Gene Promoter: Vol. 1. Current Protocols in Toxicology
(Maines, M. D.
, Costa, L. G.
, Reed, D. J.
, Sassa, S.
, and Sipes, I. G., eds)
, pp. 9.7.1-9.7.21, John Wiley & Sons, Inc., New York
|
39.
|
Gong, P.,
Stewart, D., Hu, B., Li, N.,
Cook, J.,
Nel, A.,
and Alam, J.
(2002)
Antioxid. Redox Signal.
4,
249-257[CrossRef][Medline]
[Order article via Infotrieve]
|
40.
|
Hershko, A.,
and Ciechanover, A.
(1998)
Annu. Rev. Biochem.
67,
425-479[CrossRef][Medline]
[Order article via Infotrieve]
|
41.
|
Sutter, C. H.,
Laughner, E.,
and Semenza, G. L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4748-4753[Abstract/Free Full Text]
|
42.
|
Campanero, M. R.,
and Flemington, E. K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2221-2226[Abstract/Free Full Text]
|
43.
|
Molinari, E.,
Gilman, M.,
and Natesan, S.
(1999)
EMBO J.
18,
6439-6447[Abstract/Free Full Text]
|
44.
|
Rechsteiner, M.,
and Rogers, S. W.
(1996)
Trends Biochem. Sci.
21,
267-271[CrossRef][Medline]
[Order article via Infotrieve]
|
45.
|
Rogers, S.,
Wells, R.,
and Rechsteiner, M.
(1986)
Science
234,
364-368[Medline]
[Order article via Infotrieve]
|
46.
|
Alkalay, I.,
Yaron, A.,
Hatzubai, A.,
Orian, A.,
Ciechanover, A.,
and Ben-Neriah, Y.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
10599-10603[Abstract]
|
47.
|
Chen, Z.,
Hagler, J.,
Palombella, V. J.,
Melandri, F.,
Scherer, D.,
Ballard, D.,
and Maniatis, T.
(1995)
Genes Dev.
9,
1586-1597[Abstract]
|
48.
|
Takeda, K.,
Ishizawa, S.,
Sato, M.,
Yoshida, T.,
and Shibahara, S.
(1994)
J. Biol. Chem.
269,
22858-22867[Abstract/Free Full Text]
|
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