From the Institute for Virus Research, Kyoto University, 53 Shogoin, Kawahara-cho, Sakyo, Kyoto 606-8507, Japan and the § Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8577, Japan
Received for publication, January 5, 2001, and in revised form, February 28, 2001
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ABSTRACT |
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Thioredoxin plays an important role in various
cellular processes through redox regulation. Here, we have demonstrated
that thioredoxin expression is transcriptionally induced in K562 cells by hemin (ferriprotoporphyrin IX) through activation of a regulatory region positioned from Thioredoxin was originally identified in Escherichia
coli and is known to be a dithiol hydrogen donor for a variety of
target proteins. The two cysteine residues of thioredoxin undergo
reversible oxidation-reduction reactions catalyzed by an
NADPH-dependent enzyme, thioredoxin reductase. The thioredoxin
and glutathione systems constitute the major cellular reducing systems
(1). Human thioredoxin was cloned as an adult T cell leukemia-derived factor (2). Several cytokine-like factors such as 3B6-IL-1 (3, 4) are
identical to thioredoxin, indicating that thioredoxin plays a
multifunctional role in the eukaryotic system. Thioredoxin also
modulates the activity of various transcription factors, including
nuclear factor- Previous studies of the isolation and characterization of the human and
mouse thioredoxin gene (14-17) showed that there are conserved SP-1
binding motifs in the gene regulatory region of the thioredoxin gene.
In an erythroleukemic cell line K562, thioredoxin was reported to be
induced transcriptionally by ferriprotoporphyrin IX
(hemin)1 (18). Although heat
shock factor (HSF)-2 was indicated to be responsible for this
activation, the responsive element to hemin in the thioredoxin promoter
has not yet been determined. There is accumulating evidence that heme
(or more accurately, hemin, which is the oxidized form) itself acts as
an intracellular regulator of a wide variety of metabolic pathways
(19). The mechanisms of transmission of the signals induced by hemin
appear heterogeneous and remain to be elucidated. A report showed that
the heme responsive element of the mouse heme oxygenase-1 gene is an
extended AP-1 binding site, which resembles the recognition sequences
for MAF and NF-E2 transcription factors (20). The optimal recognition sequence of v-Maf (21) and NF-E2p45 (22) is similar to the antioxidant
responsive element (ARE) (23)/electrophile-responsive element (EpRE)
(24). The CNC-bZIP transcription factors including NF-E2p45 (22, 25),
Nrf1, and Nrf2 (26-28) form heterodimers with small Maf
proteins, binding to the ARE (29). Although the liberation of
Nrf2 from Keap1 and subsequent nuclear translocation of
Nrf2 is reported to be an important mechanism of activation through the ARE (30), the regulation of factor binding and activation of ARE remains to be elucidated.
These previous studies prompted us to investigate the mechanism of the
regulation of thioredoxin gene expression by hemin. We report here that
hemin activates the thioredoxin gene through the ARE. We also showed
that the thioredoxin gene is regulated through the ARE by the binding
of NF-E2p45/small Maf under unstimulated conditions, that of
Nrf2/small Maf with hemin stimulation, and that of the Jun/Fos
proteins with PMA stimulation. The binding of these factors to the ARE
correlated well with their nuclear expression pattern. We also present
evidence that Nrf2 plays a role in the hemin-induced activation
of the thioredoxin gene. We here propose a novel mechanism of the
regulation of the ARE by a switch of binding factors including
CNC-bZIP/small Maf transcription factors and the Jun/Fos proteins,
depending on different stimuli.
Materials, Cell Lines, and Cell Culture--
Hemin and PMA were
purchased from Sigma (St. Louis, MO). Hemin stocks were prepared as
reported previously (31). PMA was dissolved in dimethyl sulfoxide
(Me2SO). The final concentration of Me2SO was
kept to 0.1%. K562 (erythroleukemic cell line) cells were cultured in
RPMI 1640 medium, supplemented with 10% heat-inactivated fetal calf
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in 5%
CO2 at 37 °C.
Plasmids--
The pTrxCAT plasmids were constructed as described
previously (14). The HindIII-BamHI inserts from
the pTrxCAT vectors were subcloned into pBluescriptII KS (+) (pTRXblue
vectors). The pTRX(
pEFNrf2, pEFMafK, pEFBOS have been previously described (32).
The dominant negative mutant of Nrf2 (Nrf2-M) was
generated by polymerase chain reaction as described previously (33).
The amplification product was cloned into pcDNA3 (Invitrogen) to
produce pcDNA3-Nrf2-M.
Transfection and Luciferase Assay--
K562 cells were
transfected with luciferase reporter expression vectors using DMRIE-C
(Life Technologies, Inc.) according to the manufacturer's instruction.
After a 4-h incubation, various doses of hemin or control were added.
For controlling the efficiency of transfection, Renilla
luciferase gene expression was monitored using pRL-TK, pRL-SV40, or
pRL-CMV (Promega). Luciferase gene expression, normalized by
Renilla luciferase activity was analyzed 24 h later
using an assay kit (Promega). Assays were performed in duplicate.
Magnetic cell separation of transiently transfected cells was performed
according to the instruction of manufacturer (MACS, Miltenyi Biotec, Germany).
Antibodies, Immunoblotting, and Immunofluorescent
Analysis--
Anti-thioredoxin monoclonal antibody was provided by
Fuji-Rebio (TRX11Ab). Anti-NF-E2p45 and anti-Nrf2 rabbit
polyclonal antibody are specific for NF-E2p45 and Nrf2,
respectively. Anti-c-Jun polyclonal antibody recognizes c-Jun, JunB,
and JunD (data not shown). Anti-c-Fos polyclonal antibody (K-25)
recognizes c-Fos, FosB, Fra-1, and Fra-2 (data not shown). These
antibodies, anti-heat shock factor (HSF)-1, and anti-HSF-2 polyclonal
antibodies were purchased from Santa-Cruz Biotechnology. Anti-MafK
antibodies have been described previously (34). Immunoblotting was
performed as previously described (6). Whole cell lysates were prepared
in the sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS,
10% glycerol, 50 mM DTT). Immunofluorescent study was
performed as described previously (6). Rabbit polyclonal antibody
against human thioredoxin was prepared and used for the
immunofluorescent study.
Electrophoretic Mobility Shift Assay--
EMSA was performed as
described previously (35). Nuclear extracts were prepared from
exponentially growing K562 cells incubated with various amounts of
hemin, PMA, or control. Aliquots of 10 µg of nuclear extract was
incubated with 32P-end-labeled double-stranded
oligonucleotides in a binding reaction buffer containing 20 mM HEPES, pH 7.9, 0.02 mM EDTA, 14% glycerol, 1 µg of poly(dI-dC), 100 mM KCl, 1.5 mM
MgCl2, 1.2 mM DTT for 20 min at 25 °C. For
specificity analyses, 100-fold molar excesses of unlabeled
oligonucleotide competitors were added and were preincubated for 15 min. When indicated, reaction mixtures were incubated with antibodies
for 20 min on ice before labeled oligonucleotides were added.
Affinity Purification Assay--
Affinity purification assay
using biotinylated oligonucleotides conjugated to magnetic beads was
performed as described with modification (36). Briefly, 5'-biotinylated
double-stranded oligonucleotides (AREW and AREM4) were immobilized to
streptavidin-conjugated magnetic beads (DynabeadsTM M-280
streptavidin, Dynal) as per manufacturer's instructions. Aliquots of
200 µg of nuclear extract (200 µl) were incubated with the beads in
a reaction buffer (final concentration: 100 mM KCl,
20 mM HEPES, pH 7.9, 10% glycerol, 1 mM DTT,
0.01% Triton X-100) for 30 min at 25 °C. The beads were washed
three times with 100 mM KCl buffer C (20 mM
HEPES, pH 7.9, 10% glycerol, 100 mM KCl, 1 mM
DTT, 0.01% Triton X-100). The beads were eluted with 50 µl of 400 mM KCl buffer C (20 mM HEPES, pH 7.9, 10%
glycerol, 400 mM KCl, 1 mM DTT, 0.01% Triton
X-100). Immunoblotting was performed as previously described (6).
Identification of the Hemin Responsive Region in the Thioredoxin
Promoter--
To investigate the mechanism of activation by hemin, we
first tested the response of the thioredoxin gene regulatory region to
hemin treatment by a luciferase reporter assay. An activation of the
thioredoxin gene was observed in a luciferase reporter construct
containing whole thioredoxin promoter (pTRX( Marked Reduction in Responsiveness to Hemin by the Insertion of a
Mutation in the Sequence Similar to the ARE Overlapping with the AP-1
Site--
In the hemin responsive region, the typical consensus
sequence for binding of HSFs is not included. However, the region
contains a sequence TGCTGAGTAAC, which resembles the optimal
recognition sequence, TGCTGA(C/G)TCAGCA (21) and
(T/C)GCTGA(G/C)TCA(C/T) (22) of v-Maf and NF-E2p45,
respectively. The sequence is also similar to the ARE,
(A/G)GTGACNNNGC (23) and the AP-1 consensus binding
sequence, TGA(C/G)TCA. We therefore tested the involvement of the
sequence in hemin responsiveness. Activation of the thioredoxin gene by
hemin was markedly reduced in a vector (pTRX( Involvement of NF-E2p45 and Nrf2 in Binding Complexes to the
ARE in the Thioredoxin Promoter--
We next analyzed ARE-binding
proteins by the EMSA. A constitutively bound complex (complex I),
hemin-induced complex (complex II), and PMA-induced complex (complex
III) to the ARE were detected in nuclear extracts of K562 cells. The
binding of these complexes appeared to be specific because the binding
of complex I, II, and III was abrogated by the addition of an excess
amount of the wild-type oligonucleotides (Fig.
3) but not by the M4 mutant of the ARE
(data not shown). Faster migrating complexes seemed nonspecific because
they were not competed by wild-type oligonucleotides. Complex I and II
binding showed no competition by an excess of oligonucleotides
encompassing the heat shock element (HSE), and no supershift was
induced by either anti-HSF-1 antibody or anti-HSF-2 antibody. In
contrast, hemin-induced specific binding to the HSE was abolished by an
excess amount of oligonucleotide encoding the HSE and was supershifted
by these antibodies (Fig. 3A). We next tested whether the
complexes include proteins known to bind to the ARE or the AP-1 site.
Complex I was supershifted by the addition of anti-NF-E2p45 or
anti-MafK antibodies, but not by antibodies against Nrf2 (Fig.
3B), Jun, Fos (Fig. 3D), Nrf1, or v-Maf (data not
shown), suggesting that complex I includes NF-E2p45 and small Maf
proteins. Binding of complex II increased after treatment with hemin
(Fig. 3, A and C). The hemin-induced complex (complex II) was completely abrogated or supershifted by the addition of anti-Nrf2 or anti-MafK antibodies but not by antibodies
against NF-E2p45, Jun, Fos (Fig. 3C), Nrf1, or v-Maf (data
not shown), suggesting that complex II includes Nrf2 and small
Maf proteins. We also observed a PMA-induced binding complex to the
ARE. The PMA-induced complex (complex III) was abrogated or
supershifted by the addition of anti-Jun or anti-Fos antibodies but not
by anti-Nrf2 antibodies (Fig. 3D), suggesting that
complex III includes the Jun/Fos proteins.
Analysis of ARE-binding Proteins Using DNA Affinity Purification
Assay--
We performed the DNA affinity purification assay to further
analyze binding factors to the ARE. NF-E2p45 was detected in eluates from affinity beads conjugated with wild type ARE (AREW), but not from
those with mutated ARE (AREM4), using nuclear extracts from untreated
or hemin-treated cells. Nrf2 was only detected in eluates from
AREW affinity beads using nuclear extracts from hemin-treated cells but
not from untreated cells. We observed MafK in eluates from AREW beads
using nuclear extracts from untreated or hemin-treated cells. In
contrast, Jun and Fos proteins were only detected in eluates using
nuclear extracts from PMA-treated cells (Fig.
4).
Nuclear Expression of ARE and AP-1 Binding Factors--
As nuclear
translocation is considered to be an important mechanism of activation
of transcription factors including Nrf2, we tested nuclear
expression of Nrf2, NF-E2p45, small MafK, and the Jun/Fos
proteins. The Jun and Fos proteins were detected in nuclei only after
PMA stimulation. Staining with anti-NF-E2p45 or anti-MafK antibodies
was shown in nuclei before and after hemin treatment. In contrast,
Nrf2 proteins were stained in nuclei only after hemin treatment
(Fig. 5A). Because thioredoxin
is translocated into nucleus by PMA (37), we also tested nuclear
expression of thioredoxin in hemin treatment. Thioredoxin translocated
into nuclei after PMA or hemin stimulation in K562 cells (Fig.
5A). We then examined nuclear expression of these factors by
Western blotting. NF-E2p45 expression was detected in nuclear extracts without stimulation. However, after hemin or PMA treatment, the expression decreased. Nrf2 expression was augmented by hemin. In
contrast, expression of the Jun/Fos proteins was augmented by PMA, not
by hemin (Fig. 5B). MafK remained in the nuclei before and
after stimulation. Either hemin or PMA treatment augmented thioredoxin
nuclear expression (Fig. 5B).
Activation of the Thioredoxin Promoter by Overexpression of
Nrf2 and MafK--
To further test whether Nrf2 and MafK
was involved in the hemin-induced activation of the thioredoxin gene,
we used transient transfection experiments in K562 cells using
pTrxAREWT-Luc. Overexpression of both Nrf2 and MafK augmented
hemin-induced activation of the thioredoxin promoter in K562 cells,
dependent on the dose of Nrf2 (Fig.
6A). In addition,
overexpression of the dominant negative mutant of Nrf2
suppressed hemin-induced activation of the thioredoxin gene (Fig.
6B). These data collectively showed that Nrf2 and the small Maf proteins mediate the hemin-induced activation of the thioredoxin gene.
In the present study, we have shown that a region from In the EMSA and DNA affinity binding assays, we showed that
NF-E2p45/small Maf complex constitutively binds to the ARE and that
Nrf2/small Maf complex is induced to bind to the ARE by hemin (Figs. 3, B and C; and 4). Other members of small
Maf proteins may also contribute to these complexes, although the level
of MafK is higher than MafG in K562 cells (39). The Jun and Fos proteins have been reported to be involved in the binding complex to
the ARE (21, 29, 40-42). In our experiment, we could not detect
members of the Jun/Fos proteins in constitutively bound or
hemin-induced binding complexes to the ARE (Figs. 3, C and D; and 4). In contrast, the binding of the Jun/Fos proteins
to the ARE was detected only after PMA stimulation (Figs. 3,
C and D; and 4). Taken together, we here propose
a model that the ARE of the thioredoxin gene is regulated by a
switch of its binding proteins (Fig.
7).
452 to
420 bp of the thioredoxin gene. Insertion of a mutation in the antioxidant responsive element (ARE)/AP-1 consensus binding sequence in this region abolished the response to hemin. With electrophoretic mobility shift and DNA
affinity assays, we have shown that the NF-E2p45/small Maf complex
constitutively binds to the ARE. The binding of the Nrf2/small Maf complex to ARE was induced by hemin, whereas the binding of Jun/Fos
proteins to ARE was induced by phorbol 12-myristate 13-acetate, but not hemin. Hemin induced nuclear translocation of Nrf2 but did not affect nuclear expression of Jun/Fos proteins. Overexpression of Nrf2 augmented the response to hemin in a
dose-dependent manner. In contrast, overexpression of the
dominant negative mutant of Nrf2 suppressed hemin-induced
activation through the ARE. We show here hemin-induced
activation of the thioredoxin gene by Nrf2 through the ARE and
propose a novel mechanism of the regulation of the ARE through a switch
of its binding factors.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (NF-
B), activator protein-1 (AP-1), hypoxia
inducing factor 1 (HIF-1), and p53 (5, 6) by the regulation of
reduction and oxidation (redox regulation). Thioredoxin has radical
scavenging activity (7) and can protect cells from tumor necrosis
factor (8), hydrogen peroxide (9), and ischemic reperfusion injury
(10). Overexpression of thioredoxin in transgenic mice attenuates focal
ischemic brain damage (11). Thioredoxin expression is induced in
vivo in ischemia followed by reperfusion (12). These observations
indicated that thioredoxin physiologically protects cells against
oxidative stress-related conditions (13). Therefore, it is important to
elucidate the molecular mechanism of the regulation of thioredoxin gene
expression by oxidative stress.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1148)-Luc, the pTRX(
980)-Luc, the
pTRX(
874)-Luc, the pTRX(
463)-Luc, and the pTRX(
352)-Luc vectors
were constructed by ligating the KpnI-BamHI fragments of the pTRXblue vectors to the
KpnI-BglII sites of the pGL3 basic vector
(Promega, WI). The pTRX(
463)-Luc vector was excised by
HindIII, filled in, and then excised by XhoI. The
insert was ligated to the SmaI/XhoI site of the
pGL3 promoter vector to produce the pTRX(
463,
352)-Luc vector. The
BstEII-XhoI or NheI-BstEII
insert of the pTRX (
463,
352)-Luc vector was excised, filled in,
and self-ligated to produce the pTRX (
463,
447)-Luc or the pTRX
(
452,
352)-Luc vector, respectively. The pTRX(
468,
435)-Luc,
the pTRX (
452,
420)-WT-Luc, the pTRX(
452,
420)-M-Luc, the
pTrxAREWT-Luc, the pTrxAREM1-Luc, the pTrxAREM2-Luc, the pTrxAREM3-Luc, and the pTrxAREM4-Luc vectors were constructed by inserting
oligonucleotides OLIGO1, AREwt, AREm, AREW, AREM1, AREM2, AREM3, and
AREM4 into the KpnI-NheI site of the pGL3
promoter vectors, respectively. All constructs were controlled by
direct nucleotide sequencing using a Thermo Sequenase II dye terminator
cycle sequencing kit (Amersham Pharmacia Biotech). The pRL-CMV, the
pRL-SV40, and the pRL-TK vectors were purchased from Promega. The
oligonucleotides used for construction of vectors and electrophoretic
mobility shift assay (EMSA) were as follows: OLIGO1: FW,
5'-cGAGATACTTCCCGGTCACCGTTACTCAGCACTg-3' and Rev,
5'-ctagcAGTGCTGAGTAACGGTGACCGGGAAGTATCTCggtac-3'; AREwt: FW,
5'-cGGTCACCGTTACTCAGCACTTTGTGGGGTTCACg-3' and Rev,
5'-ctagcGTGAACCCCACAAAGTGCTGAGTAACGGTGACCggtac-3'; AREm: FW,
5'-cGGTCACCGTTACCTTGCACTTTGTGGGGTTCACg-3' and Rev,
5'-ctagcGTGAACCCCACAAAGTGCAAGGTAACGGTGACCggtac-3'; AREW: FW,
5'-cGGTCACCGTTACTCAGCACTTTG-3' and Rev,
5'-ctagCAAAGTGCTGAGTAACGGTGACCggtac-3'; AREM1: FW,
5'-cGGCTTCCGTTACTCAGCACTTTG-3' and Rev,
5'-ctagCAAAGTGCTGAGTAACGGAAGCCggtac-3'; AREM2: FW,
5'-cGGTCACCACCACTCAGCACTTTG-3' and Rev,
5'-ctagCAAAGTGCTGAGTGGTGGTGACCggtac-3'; AREM3: FW,
5'-cGGTCACCGTTACCTTGCACTTTG-3' and Rev,
5'-ctagCAAAGTGCAAGGTAACGGTGACCggtac-3'; AREM4: FW,
5'-cGGTCACCACCACCTTGCACTTTG-3' and Rev,
5'-ctagCAAAGTGCAAGGTGGTGGTGACCggtac-3'; HSE: FW,
5'-CTAGAAGCTTCTAGAAGCTTCTAG-3' and Rev,
5'-CTAGAAGCTTCTAGAAGCTTCTAG-3'.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1148)-Luc). To analyze
the region responsible for the response to hemin, we used luciferase
reporter genes containing a series of deletions of the thioredoxin
promoter region. As shown in Fig.
1A, 3.6, 5.5, 2.9, or 3.5-fold
induction of relative luciferase activity was observed using reporter
genes (pTRX(
1148)-Luc, pTRX(
980)-Luc, pTRX(
874)-Luc, or
pTRX(
463)-Luc) in response to hemin, respectively. The response was
dose-dependent (data not shown). In contrast, no
significant induction was observed using a reporter gene
(pTRX(
352)-Luc), suggesting that
463 to
352 of the thioredoxin
upstream sequence is important for the hemin response (Fig.
1A). To further determine the region required for hemin
responsiveness, we used reporter vectors containing various lengths of
nucleotides derived from
463 to
352 of the thioredoxin upstream
sequence. Hemin activated both pTRX(
463,
352)-Luc and pTRX(
452,
352)-Luc vectors. In addition, hemin activated pTRX (
468,
435)-Luc, but not pTRX (
463,
447)-Luc. These findings showed that
the
452 to
435 region is important for hemin response. Indeed,
experiments using pTRX (
452,
420)-Luc showed a 25-fold response to
hemin treatment, revealing that the
452 to
420 sequence is
necessary for the hemin response (Fig. 1B).
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Fig. 1.
Determination of the thioredoxin promoter
region required for hemin response. A, hemin-induced
activation of the thioredoxin gene. K562 cells were transfected with
the pTRX-Luc vectors indicated on the left, together with
pRL-CMV, then treated with 30 µM hemin. B,
identification of the region responsible for the response to hemin in
the thioredoxin promoter. K562 cells were transfected with the pTRX-Luc
vectors as indicated in the upper panel, together with
pRL-CMV. Values shown represent the ratio of luciferase activity of 30 µM hemin-treated cells to that of untreated cells. The
result is representative of three independent experiments.
452,
420)-M-Luc), which has a mutation in the region (Fig.
2A). As the effect of the
mutation was partial, we further analyzed the hemin response, using
constructs with various mutations in the hemin responsive region. A
10-fold activation was observed with a reporter vector containing
wild-type ARE (pTrxAREWT-Luc) in response to hemin. The response was
reduced in the pTrxAREM1-Luc, pTrxAREM2-Luc, or pTrxAREM3-Luc vector
and was almost abolished in the pTrxAREM4-Luc vector (Fig.
2B). Collectively, these results showed the involvement of
the ARE sequence overlapping with the AP-1 site in the response to
hemin. In addition, the deletion of the ARE resulted in decrease of the
basal activity of the thioredoxin promoter, showing that the ARE also
contribute to the basal activity of the thioredoxin gene (data not
shown).
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Fig. 2.
Mutational analysis of the ARE of the
thioredoxin gene. A, decrease of hemin responsiveness
in a vector harboring a mutation in the ARE/AP-1-like sequence. The
wild-type and mutated sequences are indicated in the left
panel. An x below the sequence indicates mutated bases.
K562 cells were transfected with the indicated plasmids together with
pRL-CMV. The values shown represent the ratio of luciferase activity of
30 µM hemin-treated cells to that of untreated cells.
Similar results were obtained in a total of three independent
experiments. B, abrogation of the hemin responsiveness by
the insertion of a mutation in the ARE. Oligonucleotides used for the
construction of reporter genes are indicated in the left
panel. An x below the sequence shows a mutated base.
K562 cells were transfected with the indicated plasmids with pRL-CMV
and then treated with 30 µM hemin. Results shown are the
-fold activation of luciferase activity, compared with that of
untreated cells.
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Fig. 3.
Identification of binding proteins to the ARE
of the thioredoxin promoter. A, failure of detection of
HSFs in the binding complexes to the ARE of the thioredoxin promoter.
Nuclear extracts from K562 cells in the absence or presence of 100 µM hemin for 6 h was analyzed by the EMSA as
described under "Experimental Procedures." Unlabeled
oligonucleotide encompassing either ARE (lanes 3 and
10) or the heat shock element (HSE) (lanes 4 and
9), aliquots of 100 ng of anti-HSF-1 (lanes 5 and
11) or anti-HSF-2 (lanes 6 and 12)
were added prior to the addition of radiolabeled probes. AREW
(lanes 1-6) or HSE (lanes 7-12)
oligonucleotides were used as probes. B, binding proteins to
the ARE using unstimulated K562 nuclear extract. Unlabeled
oligonucleotide encompassing either ARE (lane 2) or HSE
(lane 3), aliquots of 100 ng of anti-NF-E2p45 (lane
4), anti-MafK (lane 5), or anti-Nrf2 (lane
6) were incubated with reaction mixture prior to the addition of
radiolabeled probes, respectively. The AREW oligonucleotides were used
as a probe. C, hemin-induced binding complex in the EMSA.
Nuclear extracts from cells without treatment (lane 1) or
treated for 6 h with 100 µM hemin (lanes
2-8) were used. AREW oligonucleotide (lane 3) or
aliquots of 100 ng of anti-NF-E2p45 (lane 4), anti-MafK
(lane 5), anti-Nrf2 (lane 6), anti-Jun
(lane 7), and anti-Fos (lane 8) antibodies were
incubated with reaction mixture prior to the addition of radiolabeled
probes. AREW was used as a probe. D, PMA-induced binding
complex in the EMSA. Nuclear extracts from cells without treatment
(lanes 1-4) or treated for 6 h with 50 ng/ml PMA
(lanes 5-9) were used. Oligonucleotides encompassing AREW
(lanes 2 and 6) or aliquots of 100 ng of anti-Jun
(lanes 3 and 8), anti-Fos (lanes 4 and
9), or anti-Nrf2 (lane 7) antibodies were
incubated with reaction mixture prior to the addition of radiolabeled
probes. AREW was used as a probe.
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Fig. 4.
Analysis of ARE binding proteins using the
DNA affinity purification assay. K562 nuclear extracts without
stimulation (Control), 100 µM hemin treatment
for 6 h (Hemin), or 30 ng/ml PMA treatment for 6 h
(PMA) were incubated with DNA affinity beads conjugated with
either wild-type ARE (AREW) (WT) or mutated ARE (AREM4)
(M4). Eluates from these beads were analyzed by Western
blotting using antibodies indicated.
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Fig. 5.
Nuclear expression of ARE and AP-1 binding
factors. A, nuclear expression of Nrf2,
NF-E2p45, small MafK, and the Jun/Fos proteins. K562 cells, without
treatment, treated with 100 µM hemin for 6 h, or
treated with 50 ng/ml PMA for 6 h were immunostained with the
antibodies indicated and analyzed by confocal microscopy. Thioredoxin
expression was analyzed 3 h after stimulation. B,
nuclear expression of ARE or AP-1 binding factors by Western blotting.
Nuclear extracts were prepared from untreated, 100 µM
hemin-treated, or 50 ng/ml PMA-treated K562 cells and analyzed by
Western blotting using the antibodies indicated.
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Fig. 6.
Effect of overexpression of Nrf2, and
MafK on the transactivation of the thioredoxin promoter.
A, augmentation of hemin-induced activation by Nrf2
and MafK. K562 cells were transfected with the indicated plasmids,
together with the pTrxAREWT-Luc and the pRL-TK vector. The amount of
vector was normalized using pEFBOS. Transfected cells were cultured in
the presence and absence of 5 µM hemin. The results are
representative of two independent experiments. Values shown represent
the relative luciferase activity normalized with Renilla
luciferase activity. B, suppression of hemin-induced
activation by a dominant negative mutant of Nrf2. Magnetic
separated cells from transiently transfected K562 cells with either
pcDNA3 or pcDNA3-Nrf2-M
(pcDNA3-dnNrf2) were again transfected with the
pTrxAREWT-Luc and the pRL-TK vector. Transfected cells were cultured in
the presence and absence of 5 or 30 µM hemin. The result
is representative of two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
452 to
420 bp of the gene regulatory region of the thioredoxin gene is
required for hemin responsiveness and is also important for basal
expression of the thioredoxin gene. The sequence from position
452 to
420 bp of the human thioredoxin promoter is highly homologous to that
from position
607 to
575 bp of the mouse thioredoxin gene (15),
indicating that the sequence in the thioredoxin promoter is conserved
in both the human and mouse thioredoxin genes. We previously reported
an oxidative responsive element (ORE), which is the sequence from
953
to
930 bp and mediates the stress response of hydrogen peroxide (14).
The possible contribution of ORE to the hemin response should be
further examined. Sistonen and co-workers (18) suggested that HSF-2
mediates hemin-induced activation of the thioredoxin gene. However, as
observed in EMSA, specific binding complexes to the hemin responsive
sequence were not affected by the addition of either unlabeled
oligonucleotides encompassing HSE or antibodies against HSF-1 or HSF-2
(Fig. 3A). These observations showed that HSFs are not
included among proteins that bind to the sequence. Indeed, no typical
consensus sequence for HSFs was identified in the thioredoxin gene. The
possible involvement of HSFs in hemin-induced activation of the
thioredoxin gene should be further tested. In contrast, we found the
TGCTGAGTAAC sequence, which resembles the ARE and AP-1 binding site, in
the hemin responsive region of the thioredoxin promoter. Mutation in
the ARE core sequence abolished the hemin response (M4 mutant in Fig.
2B), showing that the ARE is important for hemin-induced thioredoxin gene activation. We also observed a decrease of the hemin
response with the M1 mutant (Fig. 2B), suggesting that the extended ARE sequence is necessary to be fully functional. This observation is consistent with a previous report (38), further indicating that hemin-induced thioredoxin gene activation is mediated by the ARE.
View larger version (25K):
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Fig. 7.
Schematic model of the regulation of the ARE
of the thioredoxin promoter. The thioredoxin gene is regulated
through the ARE in K562 cells by the binding of NF-E2p45/small Maf
under unstimulated conditions, of Nrf2/small Maf in hemin
stimulation, and of the Jun/Fos proteins in PMA stimulation.
This change of binding proteins seemed to be regulated by the control
of nuclear expression of ARE and AP-1 binding factors, because their
nuclear expression pattern (Fig. 5) correlated well with the binding to
the ARE (Figs. 3 and 4). Nuclear expression of these factors may be
regulated by several mechanisms. Cycloheximide treatment abolished
hemin-induced binding to the
ARE,2 suggesting an
involvement of protein synthesis. Induction of small Maf protein
synthesis is reported in -naphthoflavone-induced activation of the
-glutamylcysteine synthetase subunit gene (43). In our data, the
nuclear expression of MafK was unchanged or slightly augmented by hemin
treatment. In contrast, Nrf2 nuclear expression was
significantly augmented by hemin. The hemin-induced binding of
Nrf2 to the ARE of thioredoxin gene was not preceded by
augmentation of Nrf2 mRNA level (data not shown). Our
results using confocal microscopy showed hemin-induced up-regulation of
Nrf2 expression in nuclei. These data are consistent with a
previous study that reports exposure to electrophilic agents does not
change the Nrf2 steady-state mRNA level and liberates
Nrf2 from Keap1, leading to Nrf2 nuclear translocation
(30). Thus, the control of translocation and turnover of Nrf2
protein seems to be an important mechanism for hemin-induced
augmentation of nuclear Nrf2 expression. Meanwhile, the reason
for the decrease of nuclear expression of NF-E2p45 after hemin or PMA
treatment (Fig. 5) is currently unclear. Regulation of nuclear export
of Bach2 by oxidative stress has been reported (44). Nuclear expression
of NF-E2p45 may also be regulated at the level of nuclear export.
Overexpression of both Nrf2 and small Maf protein augmented hemin-induced thioredoxin gene activation, which was dependent on the dose of Nrf2. In contrast, overexpression of a dominant negative mutant of Nrf2 suppressed hemin-induced activation of the thioredoxin gene (Fig. 6). Therefore, the major stimulatory effect on the thioredoxin gene by hemin seemed to be mediated by Nrf2 and small Maf proteins. Meanwhile, the role of the Jun/Fos proteins in the regulation of the ARE remains to be clarified. PMA stimulation did not change the luciferase activity using the pTrxAREWT-Luc vector (data not shown), suggesting that the PMA-induced switch from NF-E2p45/small Maf to the Jun/Fos proteins does not change the activation status of the thioredoxin gene in K562 cells. In other tissues or developmental stages, however, the Jun/Fos proteins might be important for basal and inducible thioredoxin gene activation. In addition, we can also speculate that in a pathological condition, perturbation of signal to the ARE by dysregulated activation of the AP-1 system causes dysregulation of differentiation process, resulting in oncogenesis.
It is important to note that the phase II enzyme genes such as the
human and rat NAD(P)H: quinone oxidoreductase genes, the rat and murine
glutathione S-transferase Ya genes, and the human -glutamylcysteine synthetase subunit gene all contain the ARE. Nrf2 has been shown to be a regulator of the phase II enzyme
genes (30, 43, 45). Thus, thioredoxin and these redox enzymes all have
a common regulatory mechanism and may have co-ordinated roles against
oxidative stress. Moreover, thioredoxin may be involved in
cytoprotection against heme- and iron-related oxidative stress. Hemin,
iron, and hemoglobin are promoters of free radical formation (46, 47).
Heme release from hemoglobin has been implicated in the pathogenesis of
reperfusion injury (48). Previously, we showed that thioredoxin
expression is induced in ischemic reperfusion (13). Thioredoxin is also
reported to facilitate the induction of the heme oxygenase-1 (49).
Collectively, study of the regulation of hemin-induced thioredoxin gene
activation may lead to the clarification of the molecular basis of host
defense against heme-induced oxidative stress-associated conditions
such as reperfusion injury. In addition, because thioredoxin has a
potent reducing activity, the current study indicates a role of
thioredoxin in the protection against oxidation of hemoglobin in
erythroid differentiation. Furthermore, our results show nuclear
translocation of thioredoxin after PMA or hemin stimulation (Fig. 5).
CNC-bZIP transcription factors and the Jun/Fos proteins have conserved
redox-sensitive cysteine residues (40). Because the thioredoxin system
has an important role in the redox regulation of transcription factors,
ARE-mediated thioredoxin gene activation may contribute to the
regulation of ARE binding factors.
Finally, we have previously reported that thioredoxin negatively
regulates p38 MAP kinase activation (50). We observed suppression of
hemin-induced activation of the thioredoxin gene by p38 MAP kinase
inhibitors in K562 cells, suggesting the involvement of the p38 MAP
kinase system in the activation of the thioredoxin gene.2
It is possible to speculate that ARE-mediated thioredoxin gene activation is a negative feedback mechanism. Meanwhile, a recent report
showed that the heme oxygenase-1 gene is activated through the ARE by
MEKK1, TAK1, and ASK-1, but not by p38 MAP kinase (51). In addition,
another report showed that protein kinase C-mediated Nrf2
phosphorylation is involved in the PMA-induced activation of the ARE
(52), although we observed only slight augmentation of nuclear
expression of Nrf2 after PMA treatment (Fig. 5). Our results and
these studies indicate that several distinct signaling pathways lead to
the ARE, depending on individual stimulus and cell types. Further work
is in progress to elucidate the upstream pathway to the ARE of the
thioredoxin gene.
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ACKNOWLEDGEMENTS |
---|
We thank Y. Kanekiyo for secretarial help, Dr. F. Hosoi for preparation of antibody, and Drs. H. Nakamura, Y. Ishii, and A. Nishiyama for discussion.
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FOOTNOTES |
---|
* This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan and by a grant-in-aid for research for the future from the Japan Society for the Promotion of Science.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. Tel.:
81-75-751-4026; Fax: 81-75-761-5766; E-mail:
hmasutan@virus1.virus.kyoto-u.ac.jp.
Published, JBC Papers in Press, March 1, 2001, DOI 10.1074/jbc.M100103200
2 H. Masutani, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: hemin, ferriprotoporphyrin IX; ARE, antioxidant responsive element; EpRE, electrophile-responsive element; CNC-bZIP, Cap `n' Collar/basic leucine zipper; Nrf2, NF-E2-related factor 2; Me2SO, dimethyl sulfoxide; EMSA, electrophoretic mobility shift assay; HSF, heat shock factor; HSE, heat shock element; DTT, dithiothreitol; bp, base pair(s); PMA, phorbol 12-myristate 13-acetate; Jun/Fos proteins, Jun/Fos families of proteins.
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