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INTRODUCTION |
The antioxidant response element
(ARE)1 is a
cis-acting enhancer that mediates the transcriptional
activation of genes encoding antioxidant and Phase II drug-metabolizing
enzymes in response to electrophilic compounds and phenolic
antioxidants. Among genes that contain a functional ARE, which has a
core sequence 5'-TGACNNNGC-3' (1), are those encoding the mouse and rat
glutathione S-transferase (GST) A1 and A2 subunits,
respectively (2-4), the rat and human NAD(P)H:quinone reductases
(NQO1) (5-7), the human
-glutamylcysteine synthetase heavy
(
-GCSh) and light (
-GCSl) subunits
(8, 9), heme oxygenase 1 (HO-1) (10), and others.
Transcriptional activation through the ARE is largely dependent upon
the transcription factor NF-E2-related factor 2 (Nrf2) (11), a
member of the Cap'n'Collar (CNC) family of bZIP proteins, as
demonstrated by in vitro electrophoretic mobility shift
assays and transient transfection experiments (12-15). These data are
further supported by in vivo studies using
Nrf2-deficient mice. The expression of GstA1 and
Nqo1 has been shown to be markedly reduced in
Nrf2 (
/
) mice as compared with wild-type animals (16). Both the basal and/or the inducible expression of these enzymes
and the
-GCS subunits by known ARE inducers were also found to be
impaired in these null mice (16, 17). Furthermore, the decreased
expression of many antioxidant and Phase II enzymes in
Nrf2 (
/
) mice has been linked to the increased
sensitivity of these animals to the toxic effects of acetaminophen and
carcinogens (18-20). These data provide strong evidence that
Nrf2 is critical in the regulation of ARE-responsive genes.
A number of recent studies have focused on identifying the signaling
pathways that may be involved in transducing the ARE-mediated response.
These include the mitogen-activated protein kinase cascades (21-23),
the phosphatidylinositol 3-kinase (PI3K)-dependent pathways (24-26), and the protein kinase C (PKC)-dependent pathway
(27). We have previously reported that activation of Nrf2 in
HepG2 cells treated with tBHQ,
-NF, or
12-O-tetradecanoylphorbol-13-acetate involves in part
a phosphorylation-dependent mechanism mediated by PKC,
which appears to promote translocation of the transcription factor into
the nucleus (27). These findings support the notion that Nrf2
may be activated by a process that involves a disruption of its
interaction with the cytoskeleton-associated protein Keap1 that retains
Nrf2 in the cytoplasm (28). Indeed, we have recently obtained
evidence showing that phosphorylation of Nrf2 at Ser-40 by PKC
interferes with the association between the two proteins (29).
In the present study, we have further explored the
mechanisms involved in the activation of this transcription factor.
Nrf2 was found to undergo rapid degradation mediated by the
26 S proteasome but became stabilized in cells exposed to tBHQ
and
-NF, leading to its accumulation and enhanced transcriptional
activity. The stabilization of Nrf2 appears to depend upon its
phosphorylation by a protein kinase(s) associated with the MAPK/ERK
signaling cascade. Thus regulation of Nrf2 stability may
represent an important mechanism in the activation of
ARE-dependent gene expression in response to oxidative stress.
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EXPERIMENTAL PROCEDURES |
Chemicals and Antibodies--
Cycloheximide (CHX), tBHQ, and
-NF were obtained from Sigma. Lactacystin (LC), MG-132, okadaic acid
(OA), staurosporine (stauro), PD 98059, and U 0126 were obtained from
Calbiochem. Antibodies against ubiquitin, and a C-terminal peptide of
Nrf2 were obtained from Santa Cruz Biotechnology. Anti-GAPDH
antibodies were obtained from Research Diagnostics Inc. (Flanders, NJ).
In addition, antibodies against a peptide derived from the N-terminal
region of Nrf2 were raised in rabbits by
BIOSOURCE International (Camarillo, CA) for the present study. This antibody specifically recognizes both human and
rat Nrf2.
Cell Culture--
HepG2 and H4IIEC3 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, non-essential amino acids, penicillin, and streptomycin,
all of which were obtained from Invitrogen. Cells were seeded in
6-well plates at 40-50% confluency and incubated for 20 h prior
to treatment with chemicals or for use in transfection experiments. For
time-course experiments, cells were treated with chemicals following a
schedule such that they would all be harvested simultaneously.
Transient transfections were performed using LipofectAMINE Plus
reagents according to the manufacturer's instructions (Invitrogen).
Transfection procedures, plasmid DNA, CAT activity assays, and
quantitation methods have been described previously (15).
Immunoblot Analysis and Immunoprecipitation--
HepG2 or
H4IIEC3 cells were cultured and treated with chemicals as described
above. In the CHX experiments, cells were treated with CHX (5 µg/ml)
or pretreated with tBHQ (50 µM) for 2 h before addition of CHX in the time-course assays. In experiments where cells
were exposed to OA (20 nM) and tBHQ, both reagents were added simultaneously. Following the treatment period, cells were washed
twice with ice-cold phosphate-buffered saline buffer and lyzed directly
with SDS sample buffer. Total cell lysates were fractionated by
SDS-PAGE on 10% or 4-12% polyacrylamide gels, transferred onto
polyvinylidene difluoride membranes and probed with specific
antibodies. Immunoreactive polypeptides were detected by
chemiluminescence using ECL reagent purchased from Amersham Biosciences
and subsequent autoradiography. Quantitation of the results was
performed by exposing the immunoblots directly to a Fuji
phosphorimaging device or by analyzing the autoradiogram with a
densitometer. For immunoprecipitation, cells were treated with tBHQ (50 µM), LC (5 µM), and MG-132 (10 µM) for 4 h, washed twice with ice-cold
phosphate-buffered saline buffer, and lyzed in
immunoprecipitation buffer as described previously (27). Lysates
were incubated with anti-Nrf2 antibodies at 4 °C for 16 h, and the immune complexes were precipitated with protein A-Sepharose beads at 4 °C for an additional 2 h. The precipitates were then washed extensively with immunoprecipitation buffer before fractionation by SDS-PAGE and subsequent immunoblotting with an anti-ubiquitin antibody.
Determination of Human Nrf2 and
-GCSh
mRNA levels in HepG2 Cells--
The relative levels of mRNA
encoding Nrf2 and
-GCSh in HepG2 cells were
compared by TaqMan® real-time PCR. Total RNA was isolated using
Trizol reagent (Invitrogen) and then treated with RQ1 DNase (Promega).
The cDNA was synthesized from 500 ng of total RNA using
Superscript-RT (Invitrogen) and subsequently analyzed by TaqMan®
using an ABI Prism 7700 Sequence Detection System (Applied Biosystems).
Primers and probes were designed for human Nrf2 and
-GCSh using the Primer Express Software and
were obtained from Applied Biosystems.
For analysis of human Nrf2, the forward
5'-TACTCCCAGGTTGCCCACA-3' and reverse 5'-CATCTACAAACGGGAATGTCTGC-3'
primers were used along with the TaqMan® probe
5'-[FAM]-TCAGATGCTTTGTACTTTGATGACTGCATGC-[TAMARA]-3'. For
human
-GCSh, the forward 5'-TTGCAGGAAGGCATTGATCA-3' and
reverse 5'-GCATCATCCAGGTGTATTTTCTCTT-3' primers were used along with
the TaqMan® probe
5'-[FAM]-TGGCCCAGCATGTTGCTCATCTCTTTAT-[TAMARA]-3'. In both cases,
the forward primer was designed over the first intron/exon boundary of
the respective gene and, along with the reverse primer, gave rise to a
91-bp (Nrf2) or 101-bp (
-GCSh) amplicon. A
primer/probe set for human GAPDH was also obtained from Applied Biosystems.
TaqMan® reactions included 200 nM of each primer with 100 nM of each probe and 1× final concentration of TaqMan®
Universal Mastermix (Applied Biosystems). All reactions were carried
out with duplicate pipefitting. TaqMan® software was used to
calculate a Ct value. The fold increase or decrease in
individual mRNAs was calculated as follows:
CtNrf2 or
Ct
GCSh
CtGAPDH =
Ct; then the
240
Ct value for treated samples was divided
by the same calculated value for control samples.
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RESULTS |
Increased Levels of Nrf2 Protein in Cells Treated with tBHQ
or
-NF--
Nrf2 has been demonstrated to regulate both the
basal and inducible expression of many ARE-responsive genes (17). These observations indicate that Nrf2 is constitutively expressed and that its transactivation activity increases in response to an inducing
signal. We sought to determine whether the level of the Nrf2
protein is affected during induction. HepG2 cells were treated with
either tBHQ or
-NF in a time-course experiment, and the level of
Nrf2 protein in cell lysates was determined by immunoblot analysis. The blots were first probed with anti-Nrf2 antibodies and followed with anti-GAPDH antibodies. The level of GAPDH was used to
normalize for sample loading. As shown in Fig.
1, A and C, the
amount of Nrf2 was found to increase within 15 min of treatment with tBHQ, and this continued up to the 8-h time point, where an
8.5-fold increase was observed. By 24 h post-treatment, the level
of Nrf2 protein decreased to ~5-fold over the basal level. A
similar increase of Nrf2 was also observed in the tBHQ-treated rat hepatoma H4IIEC3 cells (Fig. 1D). To determine whether
such effects could be produced by a different ARE inducer, we analyzed lysates from HepG2 cells treated with
-NF. In this case, the Nrf2 protein level was also found to increase during the
time-course experiment, becoming apparent at the 4-h time point with a
3-fold increase over the basal level. The maximal level was attained at
8 h post-treatment (5-fold) and remained at this plateau up to the
24-h time point (Fig. 1, B and C).

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Fig. 1.
Expression of Nrf2 is increased in
cells exposed to tBHQ or -NF. Total
lysates prepared from HepG2 cells treated with 50 µM tBHQ
(A) or -NF (B) or from H4IIEC3 cells treated
with tBHQ (D) in a time-course assay over a 24-h period were
analyzed by immunoblotting with anti-Nrf2 and anti-GAPDH
antibodies. The exposure times are indicated at the top of
each panel. The positions of protein bands representing Nrf2 and
GAPDH are indicated. Nrf2 translated in vitro from an
Nrf2-expressing plasmid (15) using a wheat-germ TNT translation
system (Promega) was included as control (TNT-Nrf2).
The blots shown are representatives of three independent experiments
with similar results. C, the results in A and
B were quantitated by densitometry, and the Nrf2
values were plotted after normalization with those of GAPDH.
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Increased Nrf2 Expression Is Mediated by a
Post-transcriptional Mechanism--
To determine whether the increased
Nrf2 expression induced by tBHQ in HepG2 cells was the result of
an increase in Nrf2 transcription or by a
post-transcriptional mechanism, we measured Nrf2
mRNA levels in these cells. Total RNA was isolated from HepG2 cells following exposure to tBHQ for 0, 0.5, 1, 2, 4, 6, and 24 h. The RNA was subject to analysis by a quantitative PCR-based approach using
oligonucleotide primers specific for Nrf2. As a
control, the GAPDH mRNA levels were similarly quantified
and used to normalize the data. Fig. 2
shows the results of these experiments. The levels of
Nrf2 mRNA were found to be unaffected by tBHQ. To
ensure that the tBHQ treatment was effective, we also measured the
levels of mRNA for the
-GCSh subunit
gene. The transcriptional regulation of this gene by tBHQ has been
shown to be Nrf2-dependent (13, 17). As shown in
Fig. 2, the level of
-GCSh mRNA was found to increase gradually starting at 2 h of post-treatment and was highest at the 6-h time point with an ~4-fold increase. Thus tBHQ was
effective in eliciting an ARE response in these cells. Together these
data indicate that the elevated level of Nrf2 in tBHQ-treated HepG2 cells was not due to an increase in Nrf2
transcription.

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Fig. 2.
Nrf2 mRNA level is unaffected in
HepG2 cells exposed to tBHQ. HepG2 cells were treated with tBHQ
(50 µM) in a time-course assay over a 24-h period, and
total RNA was isolated. The RNA was subject to analysis by a
quantitative, PCR-based approach (TaqMan®) using the ABI Prism 7700 Sequence Detection System (Applied Biosystems). The PCR was performed
using three sets of primers that are specific for
Nrf2, -GCSh, and
GAPDH, respectively. The values obtained for
GAPDH mRNA were used to normalize those of
Nrf2 (light shade bars) and
-GCSh (dark shade bars)
mRNA.
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High Rate of Intracellular Nrf2
Turnover--
The elevated Nrf2 level in the absence of an
increased rate of transcription implies that the turnover rate for
Nrf2 may decrease in response to tBHQ, therefore increasing its
stability. To examine this possibility, HepG2 cells were treated with
the protein synthesis inhibitor CHX in a time-course experiment (for 0 min, 5 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, and 2 h)
and cell lysates were analyzed by immunoblotting. The results of this
experiment show that the Nrf2 protein level decreased to ~50%
within 15 min of treatment with CHX. A trace amount of Nrf2 was
detected after 30 min of exposure to CHX (Fig.
3, A and C). To
determine the effects of tBHQ on Nrf2 stability, HepG2 cells
were pretreated with tBHQ for 2 h before exposure to CHX in a
similar time-course experiment as described above. The results depicted
in Fig. 3, B and C show that the level of
Nrf2 decreased to ~50% after 30 min of CHX exposure and 5%
at the 1-h time point.

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Fig. 3.
Stabilization of Nrf2 in HepG2 cells
exposed to tBHQ. HepG2 cells were treated with 5 µg/ml CHX
(A) or pretreated with tBHQ (50 µM) for 2 h followed by CHX (B) over a 2-h time-course period. Cells
were lyzed, and total lysates were immunoblotted with anti-Nrf2
antibodies followed by autoradiography. The times indicated above each
panel refers to CHX exposure times. The asterisk (*) indicates a
nonspecific, cross-reacting protein band. C, the immunoblots
were exposed and scanned using a Fuji phosphorimaging device, and the
intensity of the protein band was quantitated and plotted on a semi-log
graph with the value obtained for cells not treated with CHX set as
100%. The values were normalized with those of GAPDH. The blots shown
are representatives of four independent experiments.
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Nrf2 Degradation by the Proteasome--
The decreased
turnover rate in tBHQ-treated HepG2 cells implies that Nrf2 may
become more resistant to degradation. Proteins with regulatory
functions, such as the cyclins and transcription activators (30, 31),
are commonly degraded by the proteasome through the
ubiquitin-dependent pathway. Therefore we sought to determine if Nrf2 might be proteolytically degraded by this
pathway. In a time-course experiment, HepG2 cells were treated with the proteasome inhibitor LC for 0 min, 30 min, 1 h, 1.5 h,
2 h, 4 h, 8 h, and 24 h. The cells were
lyzed, and total cell extracts were analyzed by immunoblotting with
anti-Nrf2 and anti-GAPDH antibodies. The results of these
experiments show that intracellular Nrf2 protein started to
accumulate within 1.5 h of incubation with LC (a 2-fold increase),
and this continued with a 5.5-fold increase by 8 h. By 24 h,
the Nrf2 protein decreased to a lower level than that of
untreated cells (basal level), presumably because LC was no longer
effective in the cells or became toxic (Fig. 4A). To support these data a
parallel experiment using a structurally different proteasome
inhibitor, MG-132, was performed. As with LC, MG-132 caused a similar
pattern of Nrf2 accumulation in the cells (data not shown; Fig.
5A, lane
5). To further confirm the involvement of the 26 S proteasome,
immunoprecipitation experiments were performed to detect ubiquitinated
forms of Nrf2. Following treatment with LC or MG-132 in the
presence or absence of tBHQ, HepG2 cell lysates were immunoprecipitated
with anti-Nrf2 antibodies and then analyzed by immunoblotting
with an anti-ubiquitin monoclonal antibody. As shown in Fig.
4B, protein bands with slower mobility than Nrf2 were
detected only in the lysates of cells treated with either LC or MG-132
(Fig. 4C, lanes 3 and 5). Treatment
with tBHQ did not have any apparent effect on the accumulation of these slow-migrating protein bands (Fig. 4C, lanes 2,
4, and 6). Similar observations have been made in
recent studies on the ubiquitination of the aryl hydrocarbon receptor
(32) and the
2-adrenergic receptor and
-arrestin
protein (33).

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Fig. 4.
Accumulation of Nrf2 induced by
inhibitors of the ubiquitin-dependent proteasome.
A, HepG2 cells were exposed to the proteasome inhibitor LC
(5 µM) over a 24-h time-course period (as indicated), and
total lysates were analyzed by immunoblotting with anti-Nrf2 and
anti-GAPDH antibodies. The results were quantitated using a
densitometer, and the Nrf2 values were plotted after
normalization with those of GAPDH. The blot shown is a representative
of three independent experiments with similar results. B,
HepG2 cells were exposed to Me2SO (lane 1), tBHQ
(50 µM, lane 2), LC (5 µM,
lane 3), LC and tBHQ (lane 4), MG-132 (10 µM, lane 5), or MG-132 and tBHQ (lane
6) for 4 h, and total cell lysates were subject to
immunoprecipitation with anti-Nrf2 ( -Nrf2) antibodies.
The immunoprecipitated complexes were fractionated by SDS-PAGE and
immunoblotted with anti-ubiquitin ( -Ub) antibodies. The
blot shown is a representative of two independent experiments.
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Fig. 5.
Phosphorylation by MAPK/ERK pathway increases
Nrf2 stability. A, HepG2 cells were exposed to
solvent (Me2SO, lane 2), 50 µM
tBHQ (lane 3), LC (lane 4), 10 µM
MG-132 (lane 5), 20 nM OA (lane 6),
OA and tBHQ (lane 7), 10 nM staurosporine
(Stauro, lane 8), Stauro and tBHQ (lane
9) for 4 h, and total cell lysates were prepared and analyzed
by immunoblotting with anti-Nrf2 antibodies. B, HepG2
cells were treated with Me2SO (lane 2), or 50 µM tBHQ (lane 3) for 4 h, 50 µM PD 98059 (lane 4) or 10 µM U
0126 (lane 5) for 1 h, and followed by tBHQ for 4 h, and total cell lysates were analyzed by immunoblotting with
anti-Nrf2 and anti-GAPDH antibodies. TNT-Nrf2, in
vitro translated Nrf2 as positive control. The asterisk (*)
indicates a nonspecific, cross-reacting band. The results in
A and B were quantitated using a densitometer, and the Nrf2 values were plotted after
normalization with those of GAPDH. The blots shown are representatives
of three independent experiments with similar results.
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Transient transfection experiments were also performed to
determine if the proteasome inhibitors can mimic ARE inducers such as
tBHQ to elicit a transcriptional response through the ARE. As shown in
Fig. 6, LC or MG-132 treatment of HepG2
cells transfected with the GSTA2 ARE-CAT reporter construct
induced CAT activity ~3.5-fold over the basal level. Further
treatment with tBHQ did not lead to higher CAT activity.

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Fig. 6.
Activation of ARE-mediated reporter gene by
the proteasome inhibitors and by okadaic acid. HepG2 cells
transfected with the GSTA2 ARE-CAT reporter construct were
treated with LC (5 µM), MG-132 (10 µM), OA
(20 nM), or staurosporine (Stauro, 10 nM) in
the presence or absence of tBHQ (50 µM) for 20 h.
Cell lysates were prepared, and CAT assays were performed as described
previously (15). The CAT activity from the lysate of cells exposed to
only Me2SO was arbitrarily set at 1. The data represent the
average of three independent transfection experiments.
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Stabilization of Nrf2 Is Dependent on
Phosphorylation by the MAPK Pathway--
Phosphorylation of
Nrf2 by PKC caused by ARE inducers appears to promote its
nuclear translocation (27). To determine if phosphorylation of
Nrf2 also contributes to its stability, we employed OA, a
protein phosphatase inhibitor that induces intracellular hyperphosphorylation (34). Treatment of HepG2 cells with OA resulted in
an increase in the Nrf2 protein by ~2-fold as determined by
immunoblot analysis (Fig. 5A) as well as a 2.5-fold
induction of CAT activity from the GSTA2 ARE-CAT construct
(Fig. 6). The level of Nrf2 (9.5-fold) as well as the CAT
activity (4.5-fold) was increased further in cells co-treated with OA
and tBHQ (Fig. 5A, and Fig. 6) as compared with those
treated with tBHQ alone (6.2-fold and 3-fold, respectively). In
contrast, treatment with the broad spectrum PKC inhibitor staurosporine
did not affect the level of Nrf2 (Fig. 5A), although
this inhibitor did attenuate the induction of CAT activity by tBHQ
(Fig. 6). These data indicate that phosphorylation of Nrf2 by
PKC may not be essential for its stability. However, cellular
hyperphosphorylation induced by OA appears to have a positive effect on
Nrf2 stability and transactivation activity.
It has been previously reported that the MAPK/ERK signaling pathway may
be involved in the regulation of the ARE response (21, 22). We sought
to determine if phosphorylation events mediated by this pathway may
have a role in promoting Nrf2 stability. In this experiment,
HepG2 cells were pretreated with PD 98059 or U 0126 for 1 h
followed by tBHQ treatment for 4 h. The lysates were analyzed by
immunoblotting with both anti-Nrf2 and anti-GAPDH antibodies.
The results of this experiment show that both of these compounds
attenuated the inducing effects of tBHQ on the Nrf2 protein
level, with PD 98059 and U 0126 causing a reduction of ~60 and 50%,
respectively (Fig. 5B). These data suggest a link between
phosphorylation and Nrf2 stability and a positive effect from
the MAPK/ERK pathway.
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DISCUSSION |
The expression of many antioxidant and Phase II
drug-metabolizing enzymes is increased in cells during oxidative stress
(35), and the induction process is primarily mediated by the
transcription factor Nrf2 acting on the ARE present in the
promoter of genes encoding these enzymes. The established mechanism of
activation of the ARE-mediated pathway has been demonstrated to lead to
the dissociation of Nrf2 from the cytoskeleton-anchored Keap1
protein, which results in its translocation into the nucleus to
increase gene transcription (28, 36). Several recent studies have
examined the association between these two proteins at the molecular
levels, including potential sites involved in the interaction (28), the
necessity for dimerization of Keap1 to associate with Nrf2 (37),
and critical cysteine residues of Keap1 that become modified by
electrophilic agents to induce the release of Nrf2 (38). Separately, phosphorylation of a serine residue in Nrf2 by PKC has been shown to interfere with its binding to Keap1 (29).
In the present study, we have examined other mechanisms involving
Nrf2 that are important for ARE-driven transcription. We observed that the level of cellular Nrf2 increased in HepG2
cells treated with either tBHQ or
-NF, two structurally diverse
compounds that activate gene expression via the ARE (1, 2). The
increase of cellular Nrf2 by
-NF occurred at a later time
point as compared with tBHQ. This delay is consistent with the
observation that
-NF must first be metabolized to a reactive
intermediate by CYP1A1 to become an active inducer of the ARE response
(2).
Our results suggest that the increase in the level of Nrf2 in
response to tBHQ is mediated by a post-transcriptional mechanism, rather than an increase in Nrf2 mRNA levels.
These findings are consistent with other studies that have demonstrated
that Nrf2 mRNA levels are unaffected by various
inducers of ARE activity (13, 39). A more recent study, however,
reported that the expression of Nrf2 was increased in murine
keratinocytes by 3H-1,2-dithiole-3-thione (D3T), but
this effect was attributed to an increase in the rate of
Nrf2 transcription (40). These results are
contradictory to those observed in this and other studies and are
possibly due to differences in the experimental procedures, cell types,
and compounds being used. Our data also suggest that increased
Nrf2 stability, rather than an increase in the rate of protein
translation, is responsible for the higher level of Nrf2
observed in tBHQ- or
-NF-treated cells. We have also obtained
evidence demonstrating that the 26 S proteasome plays an important role
in the regulation of the Nrf2 protein. Thus it appears that the
steady-state level of Nrf2 is maintained by a precise balance
between the rates of its synthesis and its degradation by the
proteasome. We hypothesize that in response to oxidative stress
Nrf2 would continue to be synthesized at a normal rate, but the
rate of its degradation decreases such that the balance would now tip
toward accumulation of the protein, ultimately leading to an enhanced
transcriptional activity.
These data raise an important question as to the mechanism(s) that
regulate Nrf2 stability in the cell. Since degradation by the 26 S proteasome requires prior ubiquitination of the substrate molecule,
recognition and targeting of the Nrf2 protein by the ubiquitin
ligases may represent the critical, rate-limiting step. As
phosphorylation of Nrf2 was found to be promoted in cells
treated with ARE inducers (27), it is possible that phosphorylation may
also contribute to its stabilization, perhaps by rendering the protein inaccessible to the ubiquitin ligases. This type of mechanism is exemplified by the p53 transcription factor whose phosphorylation has been shown to reduce its affinity for the Mdm2
protein and therefore increase its stability (41-43). Indeed, our
findings that both an accumulation of Nrf2 as well as an
increase in ARE-dependent reporter gene activity were
induced by OA in HepG2 cells support the hypothesis that
phosphorylation does have an important role in the stability and
transcriptional activity of Nrf2. Furthermore, using inhibitors
of the MAPK/ERK pathway, it appears that this pathway may lead to
phosphorylation of Nrf2 and its increased stability. These
findings confirm previous reports implicating the MAPK/ERK pathway in
the regulation of ARE-responsive genes (21, 22). Although PKC can
phosphorylate Nrf2, these kinases did not seem to affect its
stability. The treatment of cells with staurosporine does attenuate the
induction of ARE-dependent reporter gene activity by tBHQ
but had no effects on the Nrf2 protein level. Together, our data
indicate that regulation of the Nrf2 activity may involve
phosphorylation at multiple sites, each of which is associated with a
particular function. Finally, it remains to be seen what role, if any,
the PI3K/Akt pathway, which has also been implicated in regulation of
ARE-responsive genes (24-26), may have on processes controlling the
activation of Nrf2 activity. Inhibition of the PI3-kinase enzyme
interfered with the inducing effects of tBHQ on a number of genes in
IMR-32 neuroblastoma cells, one of which encodes Keap1 (44). The
significance of the induction of Keap1 by tBHQ is not known but was
suggested to possibly constitute a feedback mechanism to repress the
activation of ARE-dependent genes and return their
expression to basal level (44).
Thus the induction of ARE-dependent enzymes appears to be
regulated by a coordinated process that controls the activation of
Nrf2 activity at multiple levels, including its nuclear
translocation, protein stability, and possibly feedback repression.
Recent studies have begun to elucidate mechanisms governing its
interaction with Keap1 and thus its movement across the nuclear envelop
(29, 37, 38). Elucidation of mechanisms that mediate the increased stability of Nrf2 will require the identification of the
specific protein kinase(s) involved as well as the site(s) of
phosphorylation. Equally important is the identification of other
components, such as those that mediate the interaction of Nrf2
with the proteasome proteolytic pathway. Together, such mechanistic
data will provide insights into the various regulatory mechanisms
involved in the induction of antioxidant and drug-metabolizing enzymes
important for the protection of cells against oxidative damage caused
by reactive oxygen species.