Direct Association with Thioredoxin Allows Redox Regulation of
Glucocorticoid Receptor Function*
Yuichi
Makino,
Noritada
Yoshikawa,
Kensaku
Okamoto,
Kiichi
Hirota
,
Junji
Yodoi§,
Isao
Makino, and
Hirotoshi
Tanaka¶
From the Second Department of Internal Medicine, Asahikawa Medical
College, 4-5-3 Nishikagura, Asahikawa 078-8510, the
Department of Anesthesia, Kyoto University Hospital,
Kyoto 606-8507, and the § Department of Biological
Responses, Institute for Virus Research, Kyoto University, Kyoto
606-8507, Japan
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ABSTRACT |
The glucocorticoid receptor (GR) is considered to
belong to a class of transcription factors, the functions of which are
exposed to redox regulation. We have recently demonstrated that
thioredoxin (TRX), a cellular reducing catalyst, plays an important
role in restoration of GR function in vivo under oxidative
conditions. Although both the ligand binding domain and other domains
of the GR have been suggested to be modulated by TRX, the molecular
mechanism of the interaction is largely unknown. In the present study,
we hypothesized that the DNA binding domain (DBD) of the GR, which is
highly conserved among the nuclear receptors, is also responsible for
communication with TRX in vivo. Mammalian two-hybrid assay and glutathione S-transferase pull-down assay revealed the
direct association between TRX and the GR DBD. Moreover, analysis of subcellular localization of TRX and the chimeric protein harboring herpes simplex viral protein 16 transactivation domain and the GR DBD
indicated that the interaction might take place in the nucleus under
oxidative conditions. Together these observations indicate that TRX,
via a direct association with the conserved DBD motif, may represent a
key mediator operating in interplay between cellular redox signaling
and nuclear receptor-mediated signal transduction.
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INTRODUCTION |
Gene expression is regulated via interactions between factors,
including DNA-binding proteins, coactivators/corepressors, histones,
and DNA, and it allows fine tuning of essential cellular processes;
e.g. proliferation, growth, differentiation, energy metabolism, and stress responses (1). Among others, redox regulation has now been considered to be one of the important determinants for
activity of transcription factors and subsequent gene expression; DNA
binding activity of a growing number of transcription factors, including AP-1 (2, 3), NF
B (4, 5), Sp-1 (6), p53 (7), c-Myb (8),
Egr-1 (9), PEBP2 (10), E2 (11), TTF-1 (12), and Ets (13), has been
shown to be regulated by thiol-redox controlling systems. Aside from
chemical oxidants/reductants, however, it largely remains to be
elucidated which endogenous factor might be involved in redox
regulation of transcription factors. A cellular reducing catalyst
thioredoxin (TRX)1 is a small
protein with a molecular mass of 13 kDa, and acts as a potent disulfide
reductase for a variety of target proteins (14, 15). Recently, TRX has
been shown to interact, directly or indirectly, with several
transcription factors. For example, TRX facilitates the DNA binding and
transcriptional activities of NF
B by reducing Cys62 in
the DNA binding loop of p50 subunit (4, 16). TRX has also been
suggested to participate in redox regulation of AP-1 via interaction
with another reducing catalyst, redox factor-1 (Ref-1) (17), which
reduces conserved cysteine residues within the DNA binding domains of
Fos and Jun (18-20). TRX has thus been suggested as a candidate
endogenous molecule operating in the redox-regulation of gene
expression via modulation of many transcription factors.
The glucocorticoid receptor (GR) is a ligand-inducible transcription
factor that belongs to the superfamily of the nuclear receptors,
comprising a central DNA binding domain (DBD), nuclear localization
signals (NLSs), a ligand binding domain (LBD), and several
transactivation functions (21-23). After binding hormone and
dissociation of heat shock proteins, the GR translocates into the
nucleus, thereby communicating with basal transcriptional machinery,
coactivators, other transcription factors, and DNA and modulating
target gene expression to produce pleiotropic glucocorticoid hormone
actions (24-26). Numerous biochemical studies have demonstrated that
GR function in vitro is subject to redox modulation, via reversible modification of functionally and structurally critical cysteine residues within the GR; oxidative treatment of the GR reduces
both ligand binding activity (27, 28) and binding to DNA cellulose (29,
30). We have previously demonstrated that metal ions that have high
affinity for thiols interfere GR functions in living cells, plausibly
via similar modification of cysteine thiols (31). Moreover, a recent
study has shown that cellular redox state is an important determinant
of GR function in vivo and that TRX is implicated in redox
regulation of GR function; GR-mediated gene expression is suppressed by
oxidative treatment of cells, which overexpression of TRX counteracts
(32). Suggested mechanisms are 1) that TRX may preserve ligand binding
activity of the GR, in accordance with previous biochemical
observations showing that ligand binding activity of cytosolic GR is
maintained by the presence of TRX systems (TRX and TRX reductase) (33, 34), and 2) that the nuclear translocation, DNA binding, and transactivation of the GR may also be influenced by TRX. The precise mechanisms of molecular interplay between the GR and TRX, however, are
not yet well understood.
To explore the molecular mechanism of redox regulation of GR function
with particular reference to its interaction with TRX, we here report
that the conserved DBD of the GR, independent of the LBD, is a target
for redox regulation by TRX. Mechanistically, direct association
between the DBD and TRX in the nucleus was shown by mammalian
two-hybrid and in vitro protein-protein interaction assays.
Thus, we suggest that TRX may play a critical role allowing cellular
redox potential to modulate steroid hormone receptor-mediated gene expression.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
COS7, CV-1, and HeLa cells were obtained from
RIKEN Cell Bank (Tsukuba Science City, Japan) and maintained in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.), pH 7.0, supplemented with 10% heat-inactivated fetal calf serum (Life
Technologies, Inc.) and antibiotics. The human GR overexpressing
(300,000-500,000 molecules/cell) Chinese hamster ovary-pMTGR cells
(35), were kindly provided by Dr. S. Nilsson (Karo Bio, Huddinge,
Sweden) and maintained in Ham's F-12 medium (Life Technologies, Inc.) supplemented with antibiotics and 10% heat-inactivated fetal calf serum in the presence of cadmium and zinc ions, each at a concentration of 40 µM. In all experiments, serum was stripped with
dextran-coated charcoal, and cells were cultured in a humidified
atmosphere at 37 °C with 5% CO2.
Reagents and Antibodies--
Diamide and dexamethasone were
purchased from Sigma. Other chemicals were from Wako Pure Chemical
(Osaka, Japan). Recombinant TRX was produced according to the method
described previously and kindly provided by Ajinomoto Co. Inc., Basic
Research Laboratory (Kawasaki, Japan) (36). Monoclonal antibody against
the carboxyl-terminal sequence of TRX was prepared as described
previously (37). Anti-GST polyclonal antibody was obtained from
Amersham Pharmacia Biotech. All enzymes were purchased from TaKaRa
Syuzo (Kyoto, Japan).
Plasmids--
The expression vectors for the wild-type and
mutant GR, RShGR
and I550, respectively, have been described
elsewhere (38) and were kindly supplied by Dr. R. M. Evans (Salk
Institute, La Jolla, CA). The expression plasmids for TRX and antisense
TRX, pcDSR
ADF and pASADF, respectively, have also previously been described (32). To construct expression plasmids for fusion protein
VP16 transactivation domain and the DBD of the human GR with or without
constitutive NLS, NL1, the DNA fragments encoding 129 amino acids
(serine 403 to leucine 532) or 87 amino acids (serine 403 to alanine
490) of the human GR were amplified by polymerase chain reaction with
appropriate flanking sequences for enzymatic cleavage and inserted into
the BamHI site of the parent pCMX-VP16 (39), resulting in
pCMX-VP16-GR DBD and pCMX-VP16-GR DBD
NL1, respectively. Construction
of the expression plasmid for the fusion protein of the DNA binding
domain of GAL4 (40) and TRX has been previously described (17). The
expression plasmids for the green fluorescent protein (GFP)-fused
chimeric protein, pCMX-GFP-VP16-GR DBD and pCMX-GFP-VP16-GR DBD
NL1,
were made by inserting a polymerase chain reaction-cloned DNA fragments
encoding VP16-GR DBD and VP16-GR DBD
NL1, respectively, into the
pCMX-GFP vector (41). The glucocorticoid-responsive reporter construct pGRE-Luc (31) and the GAL4-responsive luciferase reporter tk-GALpx3-Luc (17) were previously described. The
-galactosidase expression plasmid pCH110 (Amersham Pharmacia Biotech) was used as an internal control for transfection efficiency when appropriate.
Partial Purification of GR--
Partially purified GR was
prepared from Chinese hamster ovary-pMTGR whole cell extract
essentially as described by Cairns et al. (42). Briefly,
whole cell extracts were prepared in the presence of molybdate and
chromatographed through a phosphocellulose column. The flow-through
material was then applied to a DEAE-Sepharose column, and the absorbed
material was eluted with 200 mM NaCl. Salt and molybdate
were removed from the pooled, eluted material by chromatography on
Sephadex G-25. After transformation (25 °C for 60 min), the receptor
fraction was further purified by fast protein liquid anion-exchange
Mono Q chromatography (Amersham Pharmacia Biotech). Fractions
containing receptor were identified by ligand binding and specific DNA
binding assays. These fractions contained 10-20% pure receptor and
were used for protein-DNA interaction experiments.
Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assay was carried out as described previously (43).
Briefly, partially purified GR (usually 10 ng of protein per reaction)
was incubated with 0.2 ng of 32P-labeled GRE
oligonucleotide (5'-CGAGTAGCTAGAACAGACTGTTCTGAGG-3') probe in a 20-µl
reaction mixture containing 5 mM HEPES, pH 7.9, 60 mM KCl, 2.5 mM EDTA, 2.5 mM
MgCl2, 10 mM spermidine, 0.25 mM dithiothreitol, 10% glycerol, and 100 ng of poly(dI-dC) (Amersham Pharmacia Biotech) for 15 min on ice. The reaction mixture was loaded
onto a 4% nondenaturing polyacrylamide gel containing 0.25× TBE (1×
TBE is 89 mM Tris borate, 89 mM boric acid, and
2 mM EDTA). The gels were run at 350 V for 2 h and
dried. Results were visualized by autoradiography.
Immunocytochemical Analysis--
Cells grown on 8-chambered
sterile glass slides (Nippon Becton & Dickinson, Tokyo, Japan) were
fixed for immunostaining using a freshly prepared solution of 4%
paraformaldehyde (w/v) in phosphate-buffered saline (PBS) overnight at
4 °C. Immunocytochemistry was carried out as described previously
with minor modification (44). Briefly, after fixation, cells were
washed five times with PBS at room temperature and incubated with
anti-human TRX monoclonal mouse antibody at 1 µg/ml in PBS containing
0.1% Triton X-100 for 9 h at 4 °C. The cells were then again
washed five times with PBS and incubated with biotinylated rabbit
anti-mouse IgG antibody at a dilution of 1:200 in PBS containing 0.1%
Triton X-100 for 1 h at room temperature. The cells were then
washed a further five times with PBS and incubated with fluorescein
isothiocyanate-conjugated streptavidin at a dilution of 1:100 in PBS
containing 0.1% Triton X-100 for 1 h at room temperature. The
cells were then washed a final five times with PBS and mounted with
GEL/MOUNTTM (Biomeda Co. Ltd., Foster City, CA) for
examination on a laser scanning microscope (Zeiss LSM 510, Karl Zeiss
Jena GmbH, Jena, Germany).
Transfection and Luciferase Assay--
Transient transfection
was performed as described previously (31). Briefly, cells were plated
on plastic culture dishes (IWAKI Glass, Funabashi, Japan) to 30-50%
confluence and washed with PBS three times, and medium was replaced
with Opti-MEM (Life Technologies, Inc.). Plasmid mixture was mixed with
TransIT-LT1 transfection reagent (Pan Vera Corp., Madison, WI) and
added to the culture. After 6 h of incubation, the medium was
replaced with fresh Dulbecco's modified Eagle's medium supplemented
with 2% dextran-coated charcoal-stripped fetal calf serum, and the cells further cultured in the presence or absence of various ligands for 24 h. After normalization of transfection efficiency by
-galactosidase expression, luciferase enzyme activity was determined
in a luminometer (Berthold GmbH & Co. KG, Bad Wildbad, Germany)
essentially as described before (32).
Purification of GST Fusion Protein--
For the construction of
the expression plasmid for GST-GR DBD fusion protein, the DNA fragment
encoding the DNA binding domain (serine 403 to leucine 532) of the
human GR was amplified by polymerase chain reaction and ligated in
frame into the BamHI site of the pGEX4T-3 plasmid (Amersham
Pharmacia Biotech). GST fusion protein was expressed in
Escherichia coli BL21 (DE3) (Stratagene, La
Jolla, CA) by induction with 0.1 mM
isopropyl-
-D-thiogalactopyranoside. The cell pellets
were suspended in PBS containing 1 mM ZnCl2, 1% Triton X-100, and 5 mM dithiothreitol and subsequently
sonicated. Lysates were centrifuged at 12,000 × g for
10 min at 4 °C, and supernatants were incubated with 200 µg of
glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) at room
temperature for 30 min. Beads were washed three times with PBS, and
bound proteins were eluted with 1 ml of elution buffer (10 mM glutathione, 50 mM Tris, pH 8.0, 1 mM dithiothreitol). Eluted proteins were dialyzed against
PBS containing 1 mM dithiothreitol before storage at
80 °C. The protein was characterized by Western blot analysis.
In Vitro Protein-Protein Interaction Assays--
Bacterially
expressed GST-GR DBD fusion protein (10 µg) or GST (5 µg) was
incubated with 45 µg of glutathione-Sepharose 4B beads (Amersham
Pharmacia Biotech) in 250 µl of PBS at room temperature for 30 min.
After being washed three times, the beads were incubated with 10 µg
of recombinant TRX in PBS containing 3% bovine serum albumin and 0.1%
Triton X-100, with or without 5 mM diamide at room
temperature for 30 min. The beads were then washed five times, and
bound proteins were eluted by boiling in 30 µl of 2× SDS loading buffer (20% v/v glycerol, 4.6% w/v SDS, 0.125 M Tris-HCl,
pH 6.8, 4% 2-mercaptoethanol), and separated by polyacrylamide gel
electrophoresis. The proteins were then electrically transferred onto
polyvinylidine difluoride membrane (Bio-Rad) and probed with
appropriate antibodies. Antigen-antibody complexes were detected with
the ECL Western blot detection kit (Amersham Pharmacia Biotech)
according to the manufacturer's instructions.
 |
RESULTS |
Functional Interaction between the GR and TRX--
As described
above, we hypothesized that TRX may preserve GR-dependent
gene expression under oxidative conditions (32). To further explore
this hypothesis, we manipulated cellular TRX levels by means of
transfection of either sense or antisense TRX expression plasmids and
tested glucocorticoid-dependent reporter gene expression
under oxidative conditions. HeLa cells were transfected with the GR
expression plasmid, the glucocorticoid-responsive reporter plasmid, and
either TRX or antisense TRX expression plasmid, and cultured in the
presence of 100 nM dexamethasone and 1 mM H2O2 as indicated (Fig.
1). As reported previously (32),
treatment with H2O2 resulted in an
approximately 3-fold decrease in hormone induction response
(lanes 1-3). When TRX was overexpressed, the repression
effect of H2O2 was dose-dependently
abolished (lanes 4-6, hatched columns). In contrast,
reduction of cellular TRX levels, accomplished by expression of
antisense TRX (32, 45), resulted in increased sensitivity to
H2O2 and a further decrease in hormone
induction (lanes 4-6, filled columns). This indicates that
cellular TRX levels are an important determinant of
glucocorticoid-mediated gene expression in oxidative conditions.

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Fig. 1.
Effect of treatment with
H2O2 and overexpression of TRX or antisense TRX
on glucocorticoid-mediated gene expression. HeLa cells were
transfected with 10 ng of the GR expression plasmid pRShGR , 5 µg
of pGRE-Luc reporter plasmid, and various amounts of the TRX expression
plasmid pcDSR ADF or antisense TRX expression plasmid pASADF as
indicated. The cells were cultured in the presence or absence of 1 mM H2O2 and/or 100 nM
dexamethasone (DEX) for 24 h, and cellular luciferase
activity was determined as described under "Experimental
Procedures." All results are expressed as fold induction compared
with the cellular luciferase levels when the reporter and carrier
plasmids pGEM3Z were transfected (column 1). Three
independent experiments were performed, and means ± S.D. of the
results are shown.
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Subcellular Localization of TRX--
We next studied the
subcellular localization of TRX in HeLa cells using indirect
immunofluorescent analysis. HeLa cells were cultured in the presence or
absence of H2O2 for 2 h and then fixed for
immunodetection of TRX. In the absence of H2O2,
TRX is mainly found in the cytoplasm, with some cells showing partial
nuclear fluorescence (Fig. 2, left
panel). However, in the presence of H2O2,
the majority of the cells showed nuclear-predominant TRX fluorescence
(right panel), indicating that TRX translocates into the
nucleus under oxidative conditions.

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Fig. 2.
Effect of treatment with
H2O2 on subcellular distribution of TRX in HeLa
cells. HeLa cells were cultured in the absence or presence of 1 mM H2O2 for 2 h as indicated,
and then indirect immunofluorescence labeling using anti-human TRX
monoclonal antibody was carried out as described under "Experimental
Procedures."
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DNA Binding Domain of the GR Is a Target of Redox
Regulation--
If TRX translocates into the nucleus under oxidative
conditions, it is likely that TRX interacts with the GR in the nucleus to restore the transactivational function of the GR. The GR mutant I550
(which lacks the ligand binding domain, is constitutively present in
the nucleus even in the absence of hormone (46), and acts as a
ligand-independent transcriptional activator (47)) was shown to be
sensitive to oxidative stress (32). Indeed, suppression of
I550-mediated gene expression either by treatment with
H2O2 (Fig.
3A) or antisense TRX
expression (Fig. 3B) is similar to that seen for wild-type
GR (32), further evidence for the nuclear location of the GR-TRX
interaction.

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Fig. 3.
Regulation of ligand-independent
transactivation function of the LBD-truncated mutant GR by cellular TRX
levels. A, COS7 cells were grown in 100-mm-diameter culture
dishes and transfected with 5 µg of pGRE-Luc reporter plasmid and 10 ng of the carboxyl-terminally truncated GR expression plasmid I550 and
TRX expression plasmid pcDSR ADF as indicated. The cells were
cultured in the presence or absence of 1 mM
H2O2 for 24 h, and then cellular
luciferase activity was measured as described under "Experimental
Procedures." B, COS7 cells in 100-mm-diameter culture
dishes were transfected with 5 µg of pGRE-Luc, 10 ng of I550, and
various amounts of antisense TRX expression plasmid pASADF as
indicated. After further incubation for 24 h, cellular luciferase
activity was determined. Results are expressed as fold induction
compared with the luciferase activity in the cells without either
transfection of expression plasmids or treatment with reagents
(column 1 in A and B, respectively).
Means ± S.D. of three independent experiments are shown.
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Electrophoretic mobility shift assay using either partially purified
full-length GR (Fig. 4) or the
recombinant GR DBD (32) revealed that sequence-specific DNA binding
activity of the GR is abolished by addition of the oxidative reagent
diamide, and progressively restored by addition of recombinant TRX.
Based on these results and the fact that the GR DBD contains several
cysteine residues as a part of the zinc finger structures (22), we
speculated that the DBD of the GR could be one of the targets of TRX in
the nucleus. To further examine this possibility, we constructed
expression plasmids for fusion proteins of the GR DBD with or without
the constitutive NLS (NL1) plus the activation domain of the herpes simplex virus VP16 protein, VP16-GR DBD and VP16-GR DBD
NL1,
respectively (Fig. 5A). Note
that transactivation domain of VP16 does not contain cysteine residues
(48). When these chimeric proteins were expressed as a fusion protein
with GFP in COS7 cells, VP16-GR DBD was shown to be constitutively
localized in the nucleus, whereas VP16-GR DBD
NL1 was exclusively
cytoplasmic (Fig. 5B). Fig. 5C shows that VP16-GR
DBD but not VP16-GR DBD
NL1 acts as a GRE-specific constitutive transcriptional activator. Neither VP16-GR DBD
NL1, VP16, nor a
fusion protein of the VP16 activation domain plus GAL4 DBD (VP16-GAL4) transactivates the GRE-driven reporter plasmid (Fig.
5C).

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Fig. 4.
Restoration of the DNA binding activity of
the GR by TRX. 10 ng of partially purified GR were preincubated
with 5 mM diamide and various concentrations of recombinant
TRX (rTRX) for 30 min on ice as indicated and mixed with the
32P-labeled GRE oligonucleotide probe for another 15 min on
ice. Formation of protein-DNA complexes was monitored by
electrophoretic mobility shift assay and visualized by autoradiography.
Experiments were repeated three times with almost identical results and
a representative results are shown. C, protein-DNA complex;
F, free probe.
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Fig. 5.
A, schematic presentation of fusion
proteins VP16-GR DBD and VP16-GR DBD NL1. B, subcellular
localization of GFP-VP16-GR DBD and GFP-VP16-GR DBD NL1. COS7 cells
were transfected with 5 µg of GFP-VP16-GR DBD or GFP-VP16-GR
DBD NL1 expression plasmids. After 24 h of transfection,
subcellular localization of expressed chimeric proteins was examined by
fluorescence microscopy. C, VP16-GR DBD activates gene
expression through sequence-specific interaction with GRE in a
ligand-independent manner. COS7 cells were grown in 60-mm-diameter
culture dishes and transfected with 2 µg of reporter plasmids
pGRE-Luc or tk-GALpx3-Luc and 100 ng of the expression plasmids for
VP16-GR DBD, VP16-GR DBD NL1, VP16-GAL4, and VP16. The cells were
harvested at 24 h after transfection, and cellular luciferase activity was determined as described
under "Experimental Procedures." Expressed luciferase activities
when the cells were transfected with each reporter plasmid alone served
as controls (columns 1 and 6). Results are
plotted as mean ± S.D. of three experiments. D,
H2O2-mediated repression and TRX-mediated
restoration of the GR DBD-GRE interaction in living cells. COS7 cells
were transfected with 2 µg of pGRE-Luc reporter, 100 ng of VP16-GR
DBD expression plasmid, and various amounts of TRX expression plasmid
pcDSR ADF, as indicated. The cells were incubated with increasing
concentrations of H2O2 for 24 h, and then
cellular luciferase activity was measured as described under
"Experimental Procedures." Results are expressed as fold induction
compared with the cells without either cotransfection of any expression
plasmids or treatment with H2O2. Data represent
the mean ± S.D. of three independent experiments.
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VP16-GR DBD can thus be used in monitoring of the influence of TRX on
the GR DBD and its interaction with GRE in the nucleus. To this end,
VP16-GR DBD and TRX were coexpressed in COS7 cells, and the cells were
cultured in the absence or presence of H2O2 as
indicated in the legend for Fig. 5D. VP16-GR DBD induced
reporter gene expression by 105-fold in the absence of
H2O2 (Fig. 5D), indicating the
presence of a productive interaction between the GR DBD and GRE. As
expected, the transactivational function of VP16-GR DBD was lowered
15-fold after treatment with H2O2 (Fig. 5D) and restored by overexpression of TRX (Fig.
5D). On the other hand, when VP16-GR DBD
NL1 was used,
there were no effects of either H2O2 treatment
or TRX overexpression (data not shown). These results strongly indicate
that TRX, at least functionally, communicates with the GR DBD in the nucleus.
Demonstration of Physical Association between TRX and the GR DBD in
the Nucleus--
Because TRX has been shown to directly associate with
target proteins in exerting its reducing action (17, 49), we postulated that TRX may physically associate with the GR DBD. This possibility was
tested by using the mammalian two-hybrid assay, in which a cDNA of
the GR DBD or TRX was subcloned downstream of the GAL4 DBD (harboring
an NLS capable of taking fusion proteins to the nucleus (40)) or the
transactivation domain of VP16 in frame. These plasmids were then
cotransfected in CV-1 cells with a reporter plasmid driving luciferase
gene expression under the control of GAL4 binding sites. When GAL4-TRX,
which is constitutively in the nucleus (data not shown), was
coexpressed with either VP16 or VP16-GR DBD
NL1, luciferase activity
was not induced (Fig. 6). In contrast,
coexpression of increasing levels of GAL4-TRX and VP16-GR DBD
significantly induced luciferase expression in a
dose-dependent manner (Fig. 6). Because VP16-GR DBD
constitutively localizes in the nucleus, communication between
GR-mediated signal and TRX would appear to occur in the nucleus via
physical association between the GR DBD and TRX.

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Fig. 6.
Physical association of the GR DBD and TRX in
living cells. Mammalian two-hybrid assay in CV-1 cells. CV-1 cells
were grown in 60-mm-diameter culture dishes and transfected with 100 ng
of reporter plasmid tk-GALpx3-Luc and expression plasmids for
either GAL4-TRX and for either VP16, VP16-GR DBD, or VP16-GR DBD
NL1, as indicated. After further 24-h culture of the cells,
luciferase assay was performed as described under "Experimental
Procedures." The luciferase activity in the cells that were
transfected with reporter plasmid and each bait are served as controls
(column 1), and results are expressed as fold induction
compared with the controls. Means ± S.D. of three independent
transfections are shown.
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Finally, we analyzed direct protein-protein interaction between the GR
DBD and TRX using GST pull-down assay in combination with diamide
cross-linking (17). For this purpose, a cDNA encoding the GR DBD
was subcloned downstream of GST cDNA in frame, which resulted in
GST-GR DBD. Recombinant TRX was mixed with either Sepharose, GST-bound
Sepharose, or GST-GR DBD-bound Sepharose in the absence or presence of
diamide, and the formation of the complex consisting of TRX and GST-GR
DBD was analyzed by Western immunoblot assay as described under
"Experimental Procedures." When either Sepharose or GST-bound
Sepharose was added, TRX was not detected (Fig.
7, lanes 2-5), indicating
that neither Sepharose itself nor GST binds TRX. In contrast, when
GST-GR DBD-bound Sepharose was added, modest levels of TRX were
detected in the absence of diamide, and much higher levels in the
presence of diamide (lanes 6 and 7), strongly
suggesting direct protein-protein interaction between TRX and the GR
DBD under oxidative conditions in vitro.

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Fig. 7.
TRX directly binds to the GR DBD. 10 µg of recombinant TRX was incubated with glutathione-Sepharose beads
(lanes 2 and 3) or beads bound with GST
(lanes 4 and 5) or GST-GR DBD (lanes 6 and 7) in the presence or absence of 5 mM
diamide for 1 h at room temperature as indicated. The beads were
washed in PBS and collected by centrifugation. Bound proteins were
eluted in SDS sample buffer, run on a 15% SDS-polyacrylamide gel along
with an aliquot containing 1 µg of recombinant TRX, transferred to a
polyvinylidine difluoride membrane, and then immunoblotted with
anti-human TRX antibody (upper panel) or anti-GST antibody
(lower panel). For visualization, the ECL system was used.
Numbers at left represent molecular mass markers
run in parallel (not shown). The arrows denote the position
of TRX, GST-GR DBD, and GST as indicated.
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DISCUSSION |
We here demonstrate that TRX directly interacts with the GR in the
nucleus, allowing restoration of sequence-specific DNA binding and
subsequent transcriptional activation of GR under oxidative conditions.
In addition, we have shown that the DBD of the GR is the target of TRX.
Treatment of cells with H2O2 induced
translocation of TRX from the cytoplasm to the nucleus. Although TRX
has no authentic NLS and the mechanism of the translocation is to be
elucidated, UV irradiation also results in accumulation of TRX into the
nucleus in HSC-1 keratinocytes and HeLa cells (51). Similarly, in mouse Fe-NTA-induced renal tubular damage models, TRX has been largely recovered from nuclear fractions (52), suggesting that nuclear translocation of TRX may be a physiological cue in a variety of cellular stress responses and that nuclear TRX may play an important role in response to tissue damage, for example. TRX is also transported to the nucleus by treatment with phorbol 12-myristate 13-acetate, and
it potentiates AP-1 transcriptional activity via a
redox-dependent interaction with Ref-1 (17). Nuclear
accumulation of TRX might thus be an important process in the function
of certain transcription factors and regulation of gene expression. In
addition to the alteration of expression levels induced by variety
forms of cellular stress (51), such differential subcellular
localization in response to the environmental stimuli may constitute a
mechanism for the pleiotropic action of TRX.
Posttranslational modification of GR has been shown to be an important
determinant of the complexity to the response to glucocorticoid hormonal signals. Ligand binding or association with heat shock protein
90 has been suggested to be modulated by a variety of cellular factors
or stimuli, e.g. immunophilins (53), tyrosine kinases (54),
heat shock (55), metal ions (31), and geldanamycin (56). Redox
manipulation of the ligand binding function via modification of
critical thiols in the LBD of the GR by oxidizing reagents or reducing
reagents, including TRX systems (TRX and TRX reductase), has been
previously described (27, 28, 33, 34). In addition to these
observations, we have previously shown that ligand-independent
transactivation by the LBD-truncated mutant GR (termed I550, see Ref.
47) is also suppressed by oxidative treatment of cells and rescued by
overexpression of TRX (32), suggesting that the LBD of the GR is not a
unique domain conveying redox/TRX-mediated signals. The present study
further confirmed this issue by functional analysis of a chimeric
protein in which isolated GR DBD was fused with the heterologous
transactivation function of VP16 (VP16-GR DBD); VP 16-GR DBD-mediated
gene expression was shown to be markedly influenced by oxidative
conditions and TRX, indicating that the DBD of the GR also mediates the
effect of oxidative reagents and/or communicates with TRX, because
transactivation domain of VP16 does not contain any cysteine residues
(48). Consistently, the mammalian two-hybrid assay employing the GR DBD
and TRX as a bait and GST pull-down assay demonstrated the direct
protein-protein interaction between TRX and the GR DBD both in
vivo and in vitro. Redox signals including TRX systems, therefore, are suggested to communicate with the GR at two levels, the
LBD and DBD, which may contribute to fine tuning of receptor function
and/or glucocorticoid hormonal signal reception.
In contrast with the LBD, the DBD is the most highly conserved region
between members of the steroid hormone receptor family (21). TRX may
thus interact with other members via the DBD as well. We have
previously demonstrated that TRX also augments estrogen receptor
function, which is negatively modulated under oxidative conditions
(57), indicating that TRX may be a general factor that allows
cross-talk between redox signal and steroid hormone actions. Recently,
a small nuclear RING finger protein SNURF, which directly binds to the
DBD of the androgen receptor and can coactivate receptor function, was
identified (58). In addition to a panel of coactivators or corepressors
for steroid receptors that have been shown to associate with the LBD or
the transactivation domains (59, 60), DBD-associating proteins, such as
TRX or SNURF, might play an important role in regulation of steroid
receptor-mediated gene expression.
In addition to the steroid hormone receptors, the DBD structure is
widely shared as a DNA binding motif by the other nuclear receptors
(25). Some nuclear receptors are considered to be ligand-independent
transcription factors, activity of which is modulated by
posttranslational modification or cross-coupling with the other protein
factors (25). Such ligand-independent receptors, compared with the
receptors for known ligands, have been shown to be ancient in
evolutionary terms (61) and thus have been suggested as potential
regulators of development, differentiation, and other fundamental
physiological processes. TRX, via its interaction with the conserved
DBD, might play a role in regulation of such nuclear receptors or their
target gene expression and thus influence those biological processes.
Correspondingly, TRX is a ubiquitous protein and widely conserved from
prokaryote to eukaryote (14), and targeted disruption of the mouse TRX
gene causes early embryonic lethality (62).
Recent studies on the redox regulation of gene expression suggest that
in some but not all cases, direct or indirect association between the
catalysts of cellular reduction and the transcription factors is
essential. For example, in addition to the present case of interaction
between TRX and the GR, direct physical association of TRX and
oligopeptides from the DNA binding loop of p50 subunit of NF
B has
been demonstrated by nuclear magnetic resonance (49). Ref-1 has also
been shown to be directly targeted by TRX (17), which in turn restores
the DNA binding activity of transcription factor AP-1 (19, 20).
Similarly, Ref-1 has been shown to augment DNA binding and
transcriptional activities of p53 (7). Besides the transcription
factors, direct interaction between apoptosis signal-regulating kinase
and TRX has been suggested to be a possible mechanism for the
redox-dependent regulation of apoptosis (63). Probably on
structural grounds, there seems to be distinctions in the interplay
between transcription factors and reducing catalysts; for example,
whereas Ref-1 acts on the DNA binding activity of AP-1 but not on that
of the GR, AP-1 is not itself a direct substrate of TRX (19). Therefore
redox signals initially generated as broad intracellular reactive
oxygen species might converge onto or be directed toward target
molecules via specific interactions with reducing catalysts, such as
TRX and Ref-1. Elucidation of the coupling partner catalysts of the
transcription factors, therefore, may be extremely important for
understanding the mechanism of redox regulation of gene expression.
In the present study, we also showed that the GR can communicate with
redox/TRX-mediated signals in the nucleus independent from a
cytoplasmic interaction; the biological meaning of this two-compartment
interaction between GR and TRX remains to be explored. In the case of
the redox regulation of the NF
B function, the action of TRX in the
cytoplasm and in the nucleus has been suggested to be distinct, with
TRX inhibiting prooxidant-mediated activation of NF
B in the
cytoplasm (64) and potentiating DNA binding in the nucleus (4).
Moreover, some reducing factors vary in their subcellular localization;
TRX and glutathione can translocate from the cytoplasm to the nucleus,
whereas Ref-1 and nucleoredoxin are resident in the nucleus (19, 50).
Consideration of redox regulation of the transcription factors with
respect to cellular compartments, therefore, might be an important
issue, especially in attempting to modulate transcription factor
function in a redox-dependent manner.
In summary, we have shown that a direct interaction between the GR DBD
and TRX in the nucleus, distinct from the GR LBD-TRX interaction in the
cytoplasm, might participate in redox regulation of GR function. Such
an interaction affects the complexity of redox-mediated modification of
GR function and, moreover, may provide a possible model for the redox
regulation of nuclear receptor function and target gene expression.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Stefan Nilsson for Chinese
hamster ovary-pMTGR cells, Dr. R. M. Evans for plasmids, Dr. K. Umesono for plasmids and valuable comments, and Drs. S. Hayashi and H. Eguchi for technical advice.
 |
FOOTNOTES |
*
This work was supported in part by grants from the Ministry
of Health and Welfare and the Ministry of Education, Science, Sports
and Culture of Japan (to H. T.), the Japan Rheumatism Association (to
Y. M. and K. O.), and the Akiyama Foundation (to Y. M.).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.:
81-166-68-2451; Fax: 81-166-68-2459; E-mail:
hirotnk{at}asahikawa-med.ac.jp.
The abbreviations used are:
GR, glucocorticoid
receptor; TRX, thioredoxin; DBD, DNA binding domain; GST, glutathione
S-transferase; VP16, viral protein 16; Ref-1, redox
factor-1; NLS, nuclear localization signal; LBD, ligand binding domain; GFP, green fluorescent protein; GRE, glucocorticoid response element; PBS, phosphate-buffered saline; NF
B, nuclear factor
B.
 |
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