Redox-dependent Regulation of Nuclear Import of the
Glucocorticoid Receptor*
Kensaku
Okamoto
,
Hirotoshi
Tanaka
§,
Hidesato
Ogawa¶,
Yuichi
Makino
,
Hidetaka
Eguchi
,
Shin-ichi
Hayashi
,
Noritada
Yoshikawa
,
Lorenz
Poellinger**,
Kazuhiko
Umesono¶, and
Isao
Makino
From the
Second Department of Internal Medicine,
Asahikawa Medical College, Nishikagura, Asahikawa 078-8510, the
¶ Department of Genetics and Molecular Biology, Institute for
Virus Research, Kyoto University, Shogoin, Kawahara-cho, Kyoto
606-8507, the
Department of Biochemistry, Saitama Cancer Center
Research Institute, Ina, Saitama 362-0800, Japan, and the ** Department
of Cell and Molecular Biology, Medical Nobel Institute, Karolinska
Institutet, Stockholm S-171 77, Sweden
 |
ABSTRACT |
A number of transcription factors including the
glucocorticoid receptor (GR) are regulated in a
redox-dependent fashion. We have previously reported that
the functional activity of the GR is suppressed under oxidative
conditions and restored in the presence of reducing reagents. In the
present study, we have used a chimeric human GR fused to the
Aequorea green fluorescent protein and demonstrated that
both ligand-dependent and -independent nuclear
translocation of the GR is impaired under oxidative conditions in
living cells. Substitution of Cys-481 for Ser within NL1 of the human
GR resulted in reduction of sensitivity to oxidative treatment,
strongly indicating that Cys-481 is one of the target amino acids for
redox regulation of the receptor. Taken together, we may conclude that
redox-dependent regulation of nuclear translocation
of the GR constitutes an important mechanism for modulation of
glucocorticoid-dependent signal transduction.
 |
INTRODUCTION |
Glucocorticoids are indispensable not only for maintenance of
metabolic homeostasis but also for treatment of a wide variety of human
disorders including inflammatory diseases (1-4). Glucocorticoids exert
hormone action in target tissues via binding to the glucocorticoid receptor (GR).1 The GR, as a
ligand-inducible transcription factor belonging to the nuclear receptor
superfamily, is docked in the cytoplasm in the absence of hormonal
ligands. Upon hormone binding, the GR dissociates from hsp90 and
translocates into the nucleus to regulate target gene expression
(5-8). Previous biochemical studies have proven that GR function is
sensitive to redox state in vitro, most possibly via
reversible modification of cysteine residues in the GR. For instance,
oxidative modification of the GR decreases ligand binding and
nonspecific DNA binding activities of the GR in vitro
(9-13). Moreover, we have recently presented evidence demonstrating
that glucocorticoid hormone action in vivo is strictly controlled by cellular redox state and cysteine-affinitive metal ions
(14-19). Although a number of transcription factors have been shown to
be regulated in a redox-dependent fashion (reviewed in Refs. 20-22), the molecular mechanism for redox regulation of cellular GR remains largely unknown.
From a signal transduction point of view, the glucocorticoid signal
that finally influences gene expression must be transmitted to the
nucleus via receptor translocation. Therefore, nuclear import of the GR
is one of the key control points in regulation of glucocorticoid
hormone action. In general, protein transport from the cytoplasm to the
nucleus involves the nuclear localization signal (NLS), i.e.
short peptide sequences that are necessary and sufficient for nuclear
localization of their respective proteins (23). One of the best
characterized NLS motifs is that of SV40 large tumor antigen (T-ag)
(23). Nuclear import of the GR is mediated by NL1, a stretch of basic
amino acids at the immediate C-terminal end of the receptor DNA binding
domain, and a second significantly less characterized NLS in the ligand
binding domain, NL2 (24). Whereas the NLS of SV40 T-ag consists of a
short domain of basic amino acids, NL1 of the GR is a bipartite domain
and confers constitutive nuclear localization of the receptor (24). In
contrast, NL2 acts as a dominant negative NLS in the absence of ligands
(24). A number of studies have proven that the GR shuttles between the
cytoplasm and the nucleus, and subcellular localization of the GR is
determined by an equilibrium of both nuclear import and export. The GR
translocates to the nucleus in a ligand- and
energy-dependent manner, and nuclear export of the GR also
requires ATP (25-34). On the other hand, subcellular localization of
certain transcription factors is conditionally regulated to confer
extracellular stimulus-dependent gene expression. For
example, cellular treatment with tumor growth factor-
causes nuclear
translocation of the transcription factors Smad3/Smad4 (35). Moreover,
oxygen also variably modulates intracellular compartmentalization of
several transcription factors including nuclear factor-
B (36). In
the case of the GR, it is well known that glucocorticoid ligands are a
unique molecular switch to trigger nuclear translocation of the
receptor; however, it has not yet been documented whether the cellular
redox state modulates subcellular localization of the receptor.
In the present study, we have used a fusion protein of human GR and
Aequorea green fluorescent protein (GFP) to study nuclear translocation of the GR in living cells (26). Using this model system,
we demonstrate that not only ligand-dependent but also ligand-independent nuclear import of the GR is negatively modulated under oxidative conditions, illustrating the critical importance of the
cellular redox state in modulation of GR-mediated signal transduction.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Dexamethasone was purchased from Sigma, and other
chemicals were from Wako Pure Chemical (Osaka, Japan) unless otherwise specified.
Plasmids--
The expression plasmid for the chimeric protein of
GFP and the human GR, pGFP-hGR, was described previously (referred to
as pCMX-GFP-hGR in Ref. 26). The construction of pCMX-GFP-VP16-GR DBD,
which encodes fusion protein for GFP, VP16 transactivation domain, and
the DNA binding domain (DBD) of the human GR (serine 403 to leucine
532), is described elsewhere (15). pGFP-hGR/C481S, which carries a TGT
to TCT point mutation to generate a cysteine for serine substitution at
position 481 in the human GR, was constructed using pGFP-hGR as a
template with the QuickChangeTM site-directed mutagenesis
kit (Stratagene, La Jolla, CA) according to the manufacturer's
protocol. The oligonucleotides used were 5'-GCATGCCGCTATCGAAAATCTCTTCAGGCTGGAATGAACC-3' and
5'-GGTTCATTCCAGCCTGAAGAGATTTTCGATAGCGGCATGC-3'. pCMX-GFP-VP16-GR
DBD/C481S was constructed in a similar fashion using pCMX-GFP-VP16-GR
DBD as a template with the identical pair of primers. The resulting
plasmid constructs were confirmed by DNA sequencing. The
glucocorticoid-responsive reporter plasmid pGRE-Luc has been described
elsewhere, in which the firefly luciferase gene expression is driven
under the control of a tandem repeat of glucocorticoid response
elements (17). The
-galactosidase expression plasmid pCH110
(Amersham Pharmacia Biotech, Uppsala, Sweden) was used as an internal
control for transfection efficiency when appropriate.
Cell Culture--
The human GR-expressing CHO cells, CHOpMTGR,
were originally developed and kindly provided by Dr. Stefan Nilsson
(Karo Bio, Huddinge, Sweden) and cultured in Ham's F-12 medium (Life
Technologies, Inc.) (37). COS7 cells and HeLa cells were obtained from
the RIKEN Cell Bank (Tsukuba Science City, Japan) and maintained in Dulbecco's modified Eagle's medium (DMEM, Iwaki Glass Inc., Chiba, Japan). CHO-K1 cells were obtained from the RIKEN Cell Bank and maintained in Ham's F-12 medium. All media used in this study were
phenol red-free and supplemented with 10% fetal calf serum (FCS) and
antibiotics. Serum steroids were stripped with dextran-coated charcoal
(DCC), and cells were cultured in a humidified atmosphere at 37 °C
with 5% CO2 unless otherwise specified.
Immunocytochemical Analysis of Subcellular Localization of the
GR--
Cells grown on eight-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 (38) with a
small modification. Briefly, cells were washed five times with PBS at
room temperature and incubated with anti-human GR polyclonal rabbit
antibody PA1-512 (Affinity Bioreagents, Neshanic Station, NJ) at 2 µg/ml in PBS containing 0.1% Triton X-100 for 9 h at 4 °C.
The cells were washed five times with PBS and incubated with
biotinylated anti-rabbit Ig species-specific whole antibody from donkey
at a dilution of 1:200 in PBS containing 0.1% Triton X-100 for 1 h at room temperature, and then the cells were washed five times with
PBS and incubated with fluorescein isothiocyanate-conjugated
streptoavidin at a dilution of 1:100 in PBS containing 0.1% Triton
X-100 for 1 h at room temperature. Finally, the cells were washed
five times with PBS and mounted with GEL/MOUNTTM (Biomeda
Co. Ltd., Foster city, CA) and then examined by a Zeiss Axiovert 135 microscope equipped with a fluorescein isothiocyanate filter set.
Transfection and Reporter Gene Assay--
Before transfection,
cell culture medium was replaced with Opti-MEM medium lacking phenol
red (Life Technologies). A plasmid mixture containing pGRE-Luc in the
presence or absence of pGFP-human GR expression plasmids was mixed with
TransIT-LT1 reagent (Panvera Corp., Madison, WI) and added to the
culture. The total amount of the plasmids was kept constant by adding
an irrelevant plasmid (pGEM3Z was used unless otherwise specified).
After 6 h of incubation, the medium was replaced with fresh DMEM
supplemented with 2% DCC-treated FCS, and the cells were further
cultured in the presence or absence of various ligands for 24 h at
30 °C to increase transactivation function of expressed GFP-hGR
(26). Luciferase enzyme activity was determined using a luminometer
(Berthold GmbH & Co. KG, Bad Wildbad, Germany) essentially as described
before (17).
Visualization of Intracellular Trafficking of GFP Fusion Proteins
in Living Cells--
For analysis of nuclear translocation of the GR
in living cells, we transiently expressed GFP-tagged human GR or its
mutants in COS7 cells. The cells were cultured on the silane-coated
coverslips in 6-cm diameter plastic dishes, and the medium was changed
to Opti-MEM medium lacking phenol red before transfection. A plasmid mixture containing 6 µg of expression plasmids for various GFP-tagged proteins was mixed with 12 µl of TransIT-LT1 reagent and added to the
culture. After 6 h of incubation, the medium was replaced with
DMEM supplemented with 2% DCC-treated FCS, and the cells were cultured
at 37 °C for 24 h, at 30 °C for at least 4 h, and then
at 37 °C thereafter. GFP was expressed at detectable levels between
24 and 72 h after transfection. Routinely, cells were used for
further experiments 48 h after transfection. After various treatments, cells were examined using a Zeiss Axiovert 135 microscope enclosed by an incubator and equipped with a heating stage, a fluorescein isothiocyanate filter set, and epifluorescence with illumination from a Gixenon burner (26). Quantitative assessment of the
subcellular localization of expressed GFP fusion proteins was performed
according to methods described elsewhere (39). In brief, subcellular
localization analysis of GFP-tagged proteins was performed by blinded
observers, counting approximately 200 cells in which GFP fluorescence
was detected. The GFP fluorescence-positive cells were classified into
four different categories: N < C for cytoplasmic dominant
fluorescence; N = C, cells having equal distribution of
fluorescence in the cytoplasmic and nuclear compartments; N > C
for nuclear-dominant fluorescence; and N for exclusive nuclear fluorescence.
Immunoprecipitation and Western Immunoblot Assays--
Whole
cell extract was prepared by lysing cells treated with dexamethasone
and/or H2O2 in 25 mM
N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH
8.2), 1 mM EDTA, 50 mM NaCl, 2.5 mM
molybdate, and 10% glycerol. Immunoprecipitation experiments, with
either the anti-hsp90 IgM antibody 3G3 (Affinity Bioreagents) or
control mouse IgM antibody TEPC 183 (Sigma) was carried out as
described previously (40). Briefly, goat anti-mouse IgM (Sigma) was
coupled to CNBr- activated Sepharose 4B (Amersham Pharmacia Biotech) by
incubating in the coupling buffer (0.1 M
NaHCO3, 0.5 M NaCl, pH 8.3) overnight at 4 °C. Thirty-five micrograms of either the monoclonal anti-hsp90 IgM
antibody or control mouse IgM antibody was then incubated with 80 µl
of a 1:1 suspension of the goat anti-mouse IgM antibody coupled to
Sepharose in MENG buffer (25 mM Mops (pH 7.5), 1 mM EDTA, 0.02% NaN3, 10% glycerol) on ice for
90 min. This Sepharose-adsorbed material was then pelleted and washed
successively once with 1 ml of MENG buffer containing 0.5 M
NaCl and twice with MENG buffer containing 20 mM sodium
molybdate. After brief centrifugation, the pellet was resuspended in 80 µl of MENG buffer containing 20 mM sodium molybdate, 2 mM dithiothreitol, 0.25 M NaCl, and 2.5% (w/v)
bovine serum albumin. In immunoprecipitation experiments, 66 µg of
cellular protein was added to the resuspension. The reaction mixtures
were incubated on ice for 90 min, after which Sepharose beads were
pelleted by centrifugation and washed three times with MENG buffer
containing 20 mM sodium molybdate and 2 mM
dithiothreitol. Immunoprecipitated proteins were eluted by boiling in
SDS sample buffer and analyzed by SDS-polyacrylamide gel
electrophoresis and electrically transferred to an Immobilon-NC Pure
(Millipore Corp., Bedford, MA). Subsequently, immunoblotting was
performed with a monoclonal anti-GFP antibody
(CLONTECH Laboratories, Palo Alto, CA) diluted at
1:500 followed by horseradish peroxidase-conjugated sheep anti-mouse Ig
(Amersham Pharmacia Biotech) diluted at 1:750. In parallel, 20 µg of
whole cell extracts was independently used for immunodetection of
GFP-hGR and hsp90. Western immunoblot analysis for detection of hsp90
was performed in the same membrane after stripping off the immune
complex for the detection of GFP-hGR, using monoclonal mouse anti-hsp90
IgG antibody 3B6 (Affinity Bioreagents) diluted at 1:500 followed by
horseradish peroxidase-conjugated sheep anti-mouse Ig diluted at 1:750.
Antibody-protein complexes were visualized using the enhanced
chemiluminescence method according to the manufacturer's protocol
(Amersham Pharmacia Biotech). After autoradiography, intensities of the
appropriate bands for GFP-hGR and hsp90 were quantified using a densitometer.
Preparation and Microinjection of Recombinant GST-NLSc-GFP
Protein--
Recombinant GST-NLSc-GFP, encoding the NLS of SV40 T-ag,
glutathione S-transferase (GST), and GFP, was prepared as
described previously (41). Glass capillary-mediated transfer of the
recombinant protein was carried out using a Leitz micromanipulator as
also described previously (41). In brief, COS7 cells were plated on the
silane-coated coverslips in 35-mm diameter dishes in DMEM supplemented
with 2% DCC-stripped FCS and treated with or without 2 mM
H2O2 for 2 h. The medium was then changed
to H2O2-free medium just prior to the
injection. Microinjection of the recombinant protein was performed
within the ensuing 5 min (approximately 30 cells were injected per
dish) and the medium was immediately changed to DMEM containing 2%
DCC-treated FCS with or without 2 mM of
H2O2. Subcellular localization of the protein
was monitored by fluorescent microscopy, and photographs were taken at
intervals of 1 min for 20 min.
 |
RESULTS |
Inhibition of Hormone-dependent Nuclear Import of the
GR by Treatment with H2O2--
We recently
reported that the functional activities of the GR, including ligand
binding, DNA binding, and transactivation, are strictly controlled by
the cellular redox state. Since, as described in the Introduction,
nuclear translocation is a prerequisite for the GR to mediate hormone
signaling, we wanted to examine whether subcellular localization of the
GR is modulated via redox-dependent mechanisms. For this
purpose, first, we assessed whether treatment with
H2O2 influenced subcellular localization of the
GR by indirect immunofluorescent analysis using human GR-expressing
CHOpMTGR cells. As shown in Fig. 1, we
found that ligand-dependent nuclear targeting of the human
GR is markedly inhibited after treatment with 1 mM
H2O2, whereas pretreatment with
N-acetyl-L-cysteine (NAC) effectively titrates
this negative effect of H2O2. Based on these
results, we decided to transiently express GFP-hGR chimeric protein
(26) to perform a kinetic analysis of subcellular localization of the
GR in living cells with special reference to the regulatory role of
cellular redox state. In these experiments, we also examined whether
the nuclear translocation process was individually controlled via a
redox-dependent mechanism.

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Fig. 1.
Oxidative treatment suppressed
ligand-dependent nuclear translocation of the GR. The
human GR-expressing CHOpMTGR cells were treated with 100 nM
dexamethasone (Dex), 1 mM
H2O2, and 10 mM NAC for 1 h as
indicated. The subcellular distribution of the GR was
immunocytochemically analyzed as described under "Experimental
Procedures." Experiments were repeated for three times with almost
identical results, and representative photographs are shown.
Bar, 10 µm.
|
|
To this end, we tested whether GFP-hGR mimics endogenous GR in terms of
ligand-dependent nuclear translocation and transactivation using COS7 cells, since those cells show relatively high transfection efficiency. After transient transfection of GFP-hGR into COS7 cells,
20-30% of the cells showed cytoplasmic green fluorescence, indicating
expression of GFP-hGR fusion protein in those cells. In the absence of
hormone, expressed GFP-hGR chimera localized exclusively in the
cytoplasm, whereas it translocated into the nucleus in a
time-dependent manner after hormone treatment (Fig. 2, and data not shown). Quantitative
analysis (see "Experimental Procedures") also revealed a strict
ligand concentration dependence of nuclear translocation of GFP-hGR
(Fig. 2). This hormone-dependent nuclear localization of
GFP-hGR correlated with reporter gene expression (data not shown). We
also performed similar experiments using CHO-K1 cells and HeLa cells
and found that, despite significantly lower transfection efficiency,
GFP-hGR again mimics native GR in these cells as well (data not shown).
Therefore, although GFP was present in the N-terminal end of the human
GR, we concluded that GFP-hGR mimics the native human GR, at least with
reference to ligand-dependent translocation and
transactivation properties (26, 28, 33).

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Fig. 2.
Effect of ligand on subcellular localization
of GFP-hGR. pGFP-hGR was transiently transfected into COS7 cells,
and various concentrations of dexamethasone were added to the culture
medium: 0.1 nM (open squares), 1 nM (open circles), 10 nM
(filled squares), 100 nM
(filled circles). Photographs were taken using a
fluorescent microscope at the indicated time intervals after the
addition of dexamethasone. For quantitative analysis, cells were
classified according to the methods described under "Experimental
Procedures," and the percentages of the cells belonging to the
category N are shown. Experiments were repeated 3-5 times with almost
identical results, and a representative graph is shown.
|
|
We next examined the effect of oxidative treatment on nuclear
translocation of GFP-hGR in COS7 cells. As shown in Fig.
3, the rate of
ligand-dependent nuclear translocation of GFP-hGR was
markedly delayed upon the addition of H2O2.
This suppressive effect of H2O2 was
dose-dependent (Fig. 3). Indeed, in the presence of 2 mM H2O2, nuclear translocation was
severely compromised even after treatment with 100 nM
dexamethasone (Fig. 3). Neither distribution nor intensity of GFP
fluorescence was significantly influenced after treatment with
H2O2 when GFP alone was expressed in COS7 cells
(data not shown and Fig. 5). To test the reversibility of H2O2-dependent suppression of
nuclear translocation, GFP-hGR-expressing COS7 cells were treated with
10 mM NAC after 1-h treatment with 2 mM
H2O2 and 100 nM dexamethasone. As
shown in Fig. 3, NAC partially but efficiently reversed the negative
effects of 2 mM H2O2, resulting in
the nuclear translocation of GFP-hGR in the presence of 100 nM dexamethasone. These results strongly indicate that the
suppressive effects of H2O2 can be inhibited by
the addition of reducing reagents and do not appear to be related to a
decrease in cell viability.

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Fig. 3.
Redox regulation of nuclear translocation of
GFP-hGR. GFP-hGR-expressing COS7 cells were incubated for 2 h
in the absence (open circles) or presence of
various concentrations of H2O2: 0.5 mM (filled triangles), 1 mM (open squares), and 2 mM H2O2 (filled and
shaded circles). Then 100 nM dexamethasone was
added. As indicated by the arrow, NAC was added after a 1-h
incubation with dexamethasone (shaded circles).
At the indicated time points after the addition of dexamethasone, the
subcellular localization of GFP-hGR was quantitatively assessed, and
the percentages of the category N are shown. Experiments were repeated
3-5 times with almost identical results, and a representative graph is
shown.
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|
We also compared the sensitivity to H2O2 of
nuclear translocation of GFP-hGR in COS7 cells, CHO-K1, and HeLa cells,
all of which revealed almost complete nuclear translocation of GFP-hGR after treatment with 100 nM dexamethasone in 2 h (data
not shown). When concentration-dependent curves were
compared, the different cells showed the following rank order of
sensitivity to oxidative treatment: HeLa, CHO-K1, and COS7 cells.
IC50 values for inhibition of nuclear translocation of
GFP-hGR were 0.49, 0.78, and 1.00 mM for HeLa, CHO-K1, and
COS7 cells, respectively. These data indicate that, despite variation
in sensitivity to oxidative treatment, oxidative inhibition of
nuclear translocation of the GR may be a general phenomenon.
Oxidative Stress Inhibits Ligand-dependent Dissociation
of hsp90 from the GR--
To assess the mechanism of
oxidation-mediated repression of GR nuclear translocation, we
investigated whether treatment with H2O2
influenced the ligand-dependent dissociation between the GR
and hsp90, since we previously have reported that oxidative treatment
decreases ligand binding activity of the GR (16, 17) and
ligand-dependent dissociation of hsp90 has been
demonstrated to correlate with initiation of nuclear translocation
(29). First, to demonstrate interaction between the GR and hsp90, we employed in coimmunoprecipitation experiments a monoclonal IgM antibody
capable of recognizing both free hsp90 and hsp90 complexed with other
proteins (40). These experiments revealed that GFP-hGR coprecipitated
with hsp90 using whole cell extracts from pGFP-hGR-transfected COS7
cells (Fig. 4A). In contrast,
only background levels of GFP-hGR were coprecipitated by control IgM
antibodies (Fig. 4A), indicating that GFP-hGR as well as
native human GR forms a stable complex with hsp90 in solution in the
absence of ligands. The IgM anti-hsp90 antibody did not react with GFP
itself (data not shown). We then examined the effect of
H2O2 on GFP-hGR-hsp90 interaction. After transfection of pGFP-hGR to COS7 cells, cells were cultured in the
absence or presence of 1 and 2 mM
H2O2 for 2 h, exposed to 100 nM dexamethasone for 30 min and harvested, and then whole cell extracts were prepared. Fig. 4B clearly demonstrates
that dissociation of hsp90 from GFP-hGR requires ligand (top
part, compare lanes 2 and
3). Moreover, treatment with H2O2
appeared to partially suppress this ligand-dependent
dissociation of hsp90. The intensities of the bands in lanes
4 and 5 were 22 and 48% of that of
lane 2 in Fig. 4B, respectively, when
analyzed densitometrically (top part). On the
other hand, treatment with H2O2 did not
significantly affect complex formation between GFP-hGR and hsp90 in the
absence of ligand (data not shown). Western immunoblot analysis of
whole cell extracts using either the anti-GFP or anti-hsp90 antibodies revealed that total amounts of expressed GFP-hGR and cellular hsp90
were not significantly affected under these experimental conditions
(Fig. 4B, middle and bottom). Thus,
these data suggest that oxidative treatment, most possibly via
interference with ligand binding, suppresses subsequent dissociation of
hsp90 from the receptor. However, note that oxidative inhibition of
ligand-dependent dissociation of hsp90 is partial.

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Fig. 4.
Inhibition of ligand-dependent
dissociation of hsp90 from the GR under oxidative condition.
A, 66 µg of whole cell extracts from GFP-hGR-expressing
COS7 cells were coimmunoprecipitated with hsp90-specific antibodies
(anti-hsp90 Ab) or control antibodies (control Ig), as described under
"Experimental Procedures," and analyzed by SDS-polyacrylamide gel
electrophoresis and subsequent Western immunoblotting using anti-GFP
antibodies. B, after transfection of pGFP-hGR to COS7 cells,
cells were cultured in the absence or presence of 1 and 2 mM H2O2 for 2 h and then
exposed to 100 nM dexamethasone (Dex) for 30 min
and harvested. Whole cell extracts were prepared as described under
"Experimental Procedures," and 66 µg of protein samples were
coimmunoprecipitated with hsp90-specific antibodies. Immunoprecipitated
proteins (top) or 20 µg of whole cell extracts
(middle and bottom) were run on 7.5%
SDS-polyacrylamide gel electrophoresis. Western immunoblotting was
performed as described under "Experimental Procedures" using
anti-GFP antibodies (top and middle) or
anti-hsp90 antibodies (bottom). Mr
was calculated according to the protein samples run in parallel (not
shown). Filled triangles depict the position of
GFP-hGR (approximate Mr is 120,000), and an
open triangle shows the position of hsp90
(approximate Mr is 90,000). Experiments were
repeated twice with identical results, and representative results are
shown.
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Hormone-independent Nuclear Translocation Is Also Negatively
Modulated under Oxidative Conditions--
The segregation between the
effects of oxidative treatment on hsp90 release from the GR and those
on nuclear translocation of GR prompted us to consider that the effects
of oxidative treatment on these two phenomena are separable, although
both of these events occur subsequent to ligand binding. It has already
been shown that the truncation of the ligand binding domain of the GR
results in constitutive nuclear localization of the mutant receptor
(41). Therefore, to eliminate the involvement of the effect on
ligand-receptor interaction, we constructed the expression plasmid for
the fusion protein of GFP, VP16 transactivation domain, and the DNA
binding domain of the GR (15). We have already shown that this plasmid contains NL1 of the GR and constitutively localizes in the nucleus (15). After transient expression of GFP-VP16-GR DBD in COS7 cells, the
cells were cultured in the presence or absence of
H2O2 for 12 h, and subcellular
localization of this fusion protein was analyzed using a fluorescent
microscope. In the absence of H2O2, GFP-VP16-GR
DBD was constitutively localized in the nucleus (Fig.
5). However, in the presence of 1 mM H2O2, part of the cells showed
cytoplasmic retention of GFP fluorescence (Fig. 5). Moreover, almost
all cells having GFP fluorescence revealed significant cytoplasmic
fluorescent signal at 2 mM H2O2,
indicating that the expressed fusion protein between GFP, VP16, and GR
DBD, at least in part, docks in the cytoplasm under oxidative
conditions (Fig. 5). We thus may conclude that the GR nuclear
translocation process is negatively modulated in a
redox-dependent mechanism even after ligand-dependent dissociation of hsp90.

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Fig. 5.
Effect of oxidative treatment on
ligand-independent nuclear translocation of the GR. COS7
cells were transfected with the expression plasmid for GFP-VP16-GR DBD
and cultured for 12 h in the absence or presence of
H2O2. Then photographs were taken as described
under "Experimental Procedures." Bar, 10 µm.
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Export of the GR from the Nucleus Is Not Affected under Oxidative
Conditions--
Because the trafficking of the GR between the
cytoplasm and the nucleus is dynamic and bidirectional (31), the
equilibrium of distribution of the GR is determined by the relationship
between nuclear import and export rates. Compared with nuclear protein import, it is generally believed that the export kinetics is relatively slow. Therefore, nuclear protein transport appears to be strictly unidirectional in the short term (23). Predominantly cytoplasmic localization of the GR in the absence of hormone strongly suggests that
nuclear import is a rate-limiting step. Moreover, as shown in Fig.
6, export of GFP-hGR from the nucleus to
the cytoplasm was not yet completed 24 h after withdrawal of
dexamethasone and was not significantly affected by the addition of
0.5-2 mM H2O2. Thus, these results
strongly suggest that treatment with H2O2 largely affects nuclear import of GFP-hGR.

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Fig. 6.
Effect of oxidative treatment on nuclear
export of the GR. GFP-hGR-expressing COS7 cells were treated with
100 nM dexamethasone (Dex) for 1 h to allow
cells to show almost complete nuclear translocation of GFP-hGR. After
withdrawal of hormone by intensive washing with PBS three times, the
medium was replaced with Opti-MEM medium lacking phenol red. Then the
cells were incubated in the absence (open
circles) or presence of various concentrations of
H2O2 (0.5 mM (open
triangles), 1 mM (open
squares), 2 mM (filled
squares)), and subcellular localization of GFP-hGR was
assessed at indicated time points. Filled circles
represent the percentages of the cells belonging to category N when the
cells were further cultured in the presence of 100 nM
dexamethasone. Quantitative analysis was performed as described under
"Experimental Procedures." Experiments were repeated three times
with almost identical results, and a representative graph is
shown.
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Effect of Oxidative Conditions on Nuclear Import of SV40 T-ag
NLS--
Next, we examined whether treatment with
H2O2 affected the nuclear transport of the well
characterized SV40 T-ag NLS. We microinjected a recombinant chimeric
protein, GST-NLSc-GFP, which consists of GST, SV40 T-ag NLS, and GFP,
into the cytoplasm of COS7 cells (42). Regardless of the presence or
absence of 2 mM H2O2, all cells so
far examined revealed nuclear translocation of GST-NLSc-GFP within 15 min (Fig. 7), indicating that the
cytoplasmic-nuclear transport machinery is not generally affected under
oxidative conditions. Considering that NL1 but not the SV40 T-ag NLS
contains a cysteine residue that is well conserved among nuclear
receptors (Fig. 8), we supposed that NLS
of the GR, especially NL1, could be a target of
redox-dependent modulation.

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Fig. 7.
Nuclear translocation of GST-SV40 T-ag
NLSc-GFP was not affected under oxidative conditions. Recombinant
GST-SV40 T-ag NLSc-GFP was prepared as described under "Experimental
Procedures." After a 2-h incubation of COS7 cells in the absence or
presence of 2 mM H2O2, the
recombinant GST-SV40 T-ag NLSc-GFP was microinjected into the
cytoplasm, and then subcellular localization of GST-SV40 T-ag NLSc-GFP
was examined at intervals of 1 min for 20 min (at least 20 cells were
photographed per dish). Representative photographs are shown.
Bar, 10 µm.
|
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Fig. 8.
Amino acid sequence comparison of the
immediate C-terminal regions of the second zinc finger motifs of
nuclear receptors. The amino acid sequences of the immediate
C-terminal regions of the second zinc finger motifs of nuclear
receptors are shown. hGR , human glucocorticoid
receptor- (43); hGR , human glucocorticoid receptor-
(43); hMR, human mineralocorticoid receptor (44);
hPR, human progesterone receptor (45); hAR, human
androgen receptor (46); hER , human estrogen receptor-
(47); hER , human estrogen receptor- (48);
hVDR, human vitamin D receptor (49); hTR- ,
human thyroid hormone receptor- (50); hTR- , human
thyroid hormone receptor- (51); hRAR- , human retinoic
acid receptor- (52); hRAR- , human retinoic acid
receptor- (53); hRAR- , human retinoic acid
receptor- (54); hPPAR- , human peroxisome
proliferator-activated receptor- (55); hPPAR- , human
peroxisome proliferator-activated receptor- (56);
hPPAR- , human peroxisome proliferator-activated
receptor- (57); hNOR-1, human neuron-derived orphan
receptor-1 (58); hTINUR, human transcriptionally inducible
nuclear receptor (59); hLXR- , human liver X receptor
(60); hERR- , human estrogen-related receptor- (61);
hERR- , human estrogen-related receptor- (62);
hERR- , human estrogen-related receptor- (62);
hAd4BP, human adrenal 4 binding protein (63);
hROR- , human retinoic acid receptor-related orphan
receptor- (64); hROR- , human retinoic acid
receptor-related orphan receptor- (65); hGCNF, human germ
cell nuclear factor (66); hRXR- , human retinoid X
receptor- (67); hRXR- , human retinoid X receptor-
(68); hRXR- , human retinoid X receptor- (69);
hTAK1/hTR4 (70); hTR2 (71); hARP-1, human apolipoprotein AI
regulatory protein-1 (72); hEAR-3, human v-Erb-A-related
protein (73); hHNF-4, human hepatocyte nuclear factor-4
(74). Amino acids are represented in a single letter code. Conserved
amino acids are shown in boldface type, and the
box represents the conserved cysteine residue.
|
|
Reduction of Redox Sensitivity of the Human GR by Substitution of
Cys-481 for Ser--
To test the hypothesis that this conserved
cysteine residue is a target of redox regulation, we substituted Cys
for Ser at position 481 in the human GR to generate GFP-hGR/C481S and
GFP-VP16-GR DBD/C481S and examined nuclear import of these fusion
proteins. As shown in Fig. 9A,
GFP-hGR/C481S translocated into the nucleus in a
ligand-dependent fashion similar to that of wild type
GFP-hGR (compare with Fig. 2). In contrast, the sensitivity to
H2O2 of GFP-hGR/C481S was significantly reduced
when compared with that of GFP-hGR. Treatment with 1 mM
H2O2 did not significantly suppress nuclear
import of GFP-hGR/C481S, and 60% of cells showed complete nuclear
localization of this receptor mutant even in the presence of 2 mM H2O2 (Fig. 9A,
compare with Fig. 3). Moreover, even in the presence of 1 or 2 mM H2O2, constitutive nuclear
localization was observed when this conserved cysteine residue was
mutated to serine in GFP-VP16-GR DBD (Fig. 9B). These
results strongly indicate that NL1 of the GR is a direct target of
redox regulation.

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Fig. 9.
Substitution of Cys-481 for Ser reduced
sensitivity of the human GR to oxidative treatment. A,
GFP-hGR/C481S-expressing COS7 cells were incubated for 2 h in the
absence (open circles) or presence of various
concentrations of H2O2: 0.5 mM
(filled triangles), 1 mM
(open squares), and 2 mM
H2O2 (filled circles).
Then 100 nM dexamethasone was added. At the indicated time
points after the addition of dexamethasone, subcellular localization of
GFP-hGR/C481S was quantitatively assessed, and the percentages of
the category N are shown. Experiments were repeated 3-5 times
with almost identical results, and a representative graph is shown.
B, COS7 cells were transfected with the expression plasmid
for GFP-VP16-GR DBD/C481S fusion protein and cultured for 12 h in
the absence or presence of H2O2. Then
photographs were taken as described under "Experimental
Procedures."
|
|
 |
DISCUSSION |
GFP has been developed as a protein tag for in situ and
real time visualization of target proteins (75). We (26) and others (28, 33) have recently studied subcellular localization of the GR in
living cells using GFP-hGR chimera. Here we again confirm that GFP-hGR
exclusively docks in the cytoplasm in the absence of hormonal ligand
and that nuclear translocation of GFP-hGR is strictly controlled in a
ligand-dependent fashion. The kinetics of GFP-hGR
translocation appears to be similar to those reported for native GR;
the time for the half-maximum translocation (t1/2) is 5-10 min (Fig. 2, also compare with Refs. 24 and 27). Since not
only ligand-dependent release of hsp90 and nuclear
transport but also transactivational function of GFP-hGR is maintained, this translocation assay system using GFP-hGR appears to be suitable for pharmacological screening of various reagents affecting the glucocorticoid signal transduction pathway. Indeed, nonsteroidal reagents have been reported to translocate the GR to the nucleus by us
(38) and others (76, 77).
In the present study, we observed that subcellular localization of both
wild-type and GFP-tagged human GR is dynamically controlled by
redox-dependent mechanisms. Treatment of cells with
H2O2 inhibits ligand-dependent
nuclear translocation of the human GR, and treatment with NAC reverses
this inhibitory effect of H2O2. We have also shown that oxidative treatment impairs ligand-dependent
release of hsp90 from GFP-hGR. Given the putative importance of this
chaperone protein for folding of the ligand binding domain (78), this effect may be closely associated with the reduction in ligand-binding activity of the GR in cultured cells under oxidative conditions (16,
17). Moreover, we showed that ligand-independent nuclear translocation
of the GR is also inhibited under oxidative conditions. Thus, oxidative
stress-mediated repression of GR nuclear import may not only be related
to decreased ligand binding activity and subsequent impairment of
release of hsp90, but may also be related to dysfunction of the nuclear
translocation process itself. Under severe oxidative conditions,
general dysfunction of subcellular organs may occur, and the nuclear
transport machinery itself may be generally damaged. Although GR NL1 is
a bipartite motif and structurally distinct from canonical NLS motif
such as that of SV40 T-ag (23), both NLS categories use, at least in
part, similar transportation apparatus involving importins/karyopherins
and Ran GTPase (23, 28, 79). Considering that nuclear localization of
GFP-tagged SV40 T-ag NLS was not affected by treatment with H2O2, oxidative stress does not appear to
generally repress the cytoplasmic-nuclear transport machinery but may
rather preferentially target the GR. In addition, while SV40 T-ag NLS
does not contain a cysteine residue, the N-terminal part of NL1
contains a cysteine residue that is extremely well conserved among
nuclear receptors (Refs. 5 and 43-74; Fig. 8). Our present data argue
that this conserved Cys-481 might confer redox dependence of nuclear
import of the GR. The fact that Cys-481 for Ser substitution almost
completely abolished redox sensitivity of NL1 function strongly
indicates that Cys-481 is one of the regulatory amino acids involved in redox-dependent intramolecular disulfide bond formation in
the GR. For example, disulfide bond formation involving this cysteine residue may result in a protein conformation that hampers efficient interaction of the GR with NLS receptor proteins, e.g.
importin-
/karyopherin-
even in the presence of ligands. Under
reducing conditions, NL1 may interact with NLS receptor proteins in a
ligand-dependent fashion, or alternatively, this may occur
after liberation from hsp90, resulting in nuclear translocation of the
GR. Although this cysteine residue is well conserved among nuclear
receptors (Fig. 8), the functional significance has not yet been
characterized for other receptor proteins. It has previously been shown
that amino acid substitution of Cys-481 to Ser, contrary to amino acid substitution of Cys-481 to Arg (80), does not affect either DNA binding
or transactivation functions of the GR (81). Therefore, it will be
interesting to test whether this cysteine residue generally defines
redox-dependent subcellular localization of nuclear receptors.
Although several reports have already documented that treatment with
H2O2 of rat liver cytosol results in a decrease
in ligand binding activity of the GR in vitro, we cannot
compare the concentrations of H2O2 used in
those studies (i.e. 20-100 mM in Refs. 12 and 82) with those used in the present in vivo study
(i.e. 0.5-2 mM). We have also observed that
sensitivity to H2O2 of nuclear translocation of
GFP-hGR is extremely variable among distinct cell types (see also Ref.
83 for NF-
B activation), and steroid hormone-inducible gene
expression is suppressed at lower concentrations of
H2O2 in certain cells (16, 17, 84). Among the
cells that we have studied, human mammary tumor cells ZR-75-1 were most
sensitive to treatment with H2O2, and
expression of reporter genes for either the estrogen receptor or the GR
was affected under physiological concentrations of
H2O2 (Ref. 84, and data not shown). Considering that reactive oxygen species including H2O2 are
known to be generated as a consequence of, for example, stimulation
with cytokines (85) and phagocytosis (86, 87), repression of cellular
glucocorticoid action under oxidative conditions may be physiologically
important in inflammatory processes. To further confirm this model,
redox sensitivity of the GR should be studied in a variety of cell
types with reference to their physiological processes.
Together with our previous observations, we conclude that GR-mediated
signals communicate with redox signals at multiple regulatory levels
including ligand binding, nuclear translocation, DNA binding, and
transcriptional activation. Although involvement of the cysteine residues is suggested in both cases, nuclear translocation of the
transcription factor yAP-1, in clear contrast to the GR, is rather
promoted in response to oxidative stress (88). Moreover, the multicopy
suppressor of SNF1 protein 2, Msn2p, which contains two zinc finger
motifs, translocates into the nucleus in response to a broad variety of
stresses, e.g. exposure to heat shock, oxidative stress,
ethanol, sorbate, and osmostress (89). Thus,
redox-dependent modification of cysteine residues is
considered to be one of the key regulatory mechanisms of protein
localization within the cells. From a mechanistic point of view, it
should be emphasized that not only oxidative stress but hypoxic
conditions influence subcellular compartmentalization of transcription
factors. Notably, nuclear translocation of the hypoxia-inducible factor
1-
is promoted under hypoxic conditions (39). Therefore, cells may
respond to alteration in oxygen tension via variable mechanisms
including segregation of distinct transcription factors. In summary,
the present results suggest that the dynamic cellular responses to redox state could play an important role of the glucocorticoid signal
transduction mechanism. Further analysis may be necessary to clarify
not only underlying molecular mechanisms but physiological significance
as well.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Stefan Nilsson for the CHOpMTGR
cells, Dr. Katarina Gradin for sharing experimental materials and
excellent technical advice, and Dr. Pekka J. Kallio for critical
reading of the manuscript and valuable comments.
 |
FOOTNOTES |
*
This work was supported in part by grants from the Ministry
of Health and Welfare of Japan; the Ministry of Education, Science, Sports, and Culture of Japan; Japan Rheumatism Association; Uehara Memorial Foundation; Akiyama Foundation; Japan Research Foundation for
Clinical Pharmacology; and Ito Foundation.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: Second Dept. of
Internal Medicine, Asahikawa Medical College, Nishikagura, Asahikawa 078-8510, Japan. Tel.: 81-166-68-2451; Fax: 81-166-68-2459; E-mail: hirotnk{at}asahikawa-med.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
GR, glucocorticoid
receptor;
hGR, human GR;
hsp90, heat shock protein 90;
NLS, nuclear
localization signal;
T-ag, large T-antigen;
GFP, green fluorescent
protein;
CHO, Chinese hamster ovary;
DMEM, Dulbecco's modified
essential medium;
DCC, dextran-coated charcoal;
FCS, fetal calf serum;
PBS, phosphate-buffered saline;
GST, glutathione
S-transferase;
NAC, N-acetyl-L-cysteine;
DBD, DNA binding domain;
Mops, 4-morpholinepropanesulfonic acid.
 |
REFERENCES |
-
Boumpas, D. T.,
Chrousos, G. P.,
Wilder, R. L.,
Cupps, T. R.,
and Balow, J. E.
(1993)
Ann. Intern. Med.
119,
1198-1208[Abstract/Free Full Text]
-
Tyrrell, J. B.
(1995)
Endocrinology and Metabolism, 3rd Ed., pp. 855-882, McGraw-Hill, Inc., New York
-
Cole, T. J.,
Blendy, J. A.,
Monaghan, A. P.,
Krieglstein, K.,
Schmid, W.,
Aguzzi, A.,
Fantuzzi, G.,
Hummler, E.,
Unsicker, K.,
and Schütz, G.
(1995)
Genes Dev.
9,
1608-1621[Abstract]
-
Reichardt, H. M.,
Kaestner, K. H.,
Tuckermann, J.,
Kretz, O.,
Wessely, O.,
Bock, R.,
Gass, P.,
Schmid, W.,
Herrlich, P.,
Angel, P.,
and Schütz, G.
(1998)
Cell
93,
531-541[Medline]
[Order article via Infotrieve]
-
Evans, R. M.
(1988)
Science
240,
889-895[Medline]
[Order article via Infotrieve]
-
Kamei, Y.,
Xu, L.,
Heinzel, T.,
Torchia, J.,
Kurokawa, R.,
Gloss, B.,
Lin, S.-C.,
Heyman, R. A.,
Rose, D. W.,
Glass, C. K.,
and Rosenfeld, M. G.
(1996)
Cell
85,
403-414[Medline]
[Order article via Infotrieve]
-
Glass, C. K.,
Rose, D. W.,
and Rosenfeld, M. G.
(1997)
Curr. Opin. Cell Biol.
9,
222-232[CrossRef][Medline]
[Order article via Infotrieve]
-
Beato, M.,
Herrlich, P.,
and Schütz, G.
(1995)
Cell
83,
851-857[Medline]
[Order article via Infotrieve]
-
Simons, S. S., Jr.,
and Pratt, W. B.
(1995)
Methods Enzymol.
251,
406-422[Medline]
[Order article via Infotrieve]
-
Opoku, J.,
and Simons, S. S., Jr.
(1994)
J. Biol. Chem.
269,
503-510[Abstract/Free Full Text]
-
Bodwell, J. E.,
Holbrook, N. J.,
and Munck, A.
(1984)
Biochemistry
23,
1392-1398[Medline]
[Order article via Infotrieve]
-
Tienrungroj, W.,
Meshinchi, S.,
Sanchez, E. R.,
Pratt, S. E.,
Grippo, J. F.,
Holmgren, A.,
and Pratt, W. B.
(1987)
J. Biol. Chem.
262,
6992-7000[Abstract/Free Full Text]
-
Hutchison, K. A.,
Matic, G.,
Meshinchi, S.,
Bresnick, E. H.,
and Pratt, W. B.
(1991)
J. Biol. Chem.
266,
10505-10509[Abstract/Free Full Text]
-
Tanaka, H.,
Makino, Y.,
and Okamoto, K.
(1998)
Vitamins Hormones
57,
153-175
-
Makino, Y.,
Yoshikawa, N.,
Okamoto, K.,
Hirota, K.,
Yodoi, J.,
Makino, I.,
and Tanaka, H.
(1999)
J. Biol. Chem.
274,
3182-3188[Abstract/Free Full Text]
-
Okamoto, K.,
Tanaka, H.,
Makino, Y.,
and Makino, I.
(1998)
Biochem. Pharmacol.
56,
79-86[CrossRef][Medline]
[Order article via Infotrieve]
-
Makino, Y.,
Okamoto, K.,
Yoshikawa, N.,
Aoshima, M.,
Hirota, K.,
Yodoi, J.,
Umesono, K.,
Makino, I.,
and Tanaka, H.
(1996)
J. Clin. Invest.
98,
2469-2477[Abstract/Free Full Text]
-
Makino, Y.,
Tanaka, H.,
Dahlman-Wright, K.,
and Makino, I.
(1996)
Mol. Pharmacol.
49,
612-620[Abstract]
-
Tanaka, H.,
Makino, Y.,
Dahlman-Wright, K.,
Gustafsson, J.-Å.,
Okamoto, K.,
and Makino, I.
(1995)
Mol. Pharmacol.
48,
938-945[Abstract]
-
Sen, C. K.
(1998)
Biochem. Pharmacol.
55,
1747-1758[CrossRef][Medline]
[Order article via Infotrieve]
-
Sen, C. K.,
and Packer, L.
(1996)
FASEB J.
10,
709-720[Abstract/Free Full Text]
-
Sun, Y.,
and Oberley, L. W.
(1996)
Free Radical Biol. Med.
21,
335-348[CrossRef][Medline]
[Order article via Infotrieve]
-
Nigg, E. A.
(1997)
Nature
386,
779-787[CrossRef][Medline]
[Order article via Infotrieve]
-
Picard, D.,
and Yamamoto, K. R.
(1987)
EMBO J.
6,
3333-3340[Abstract]
-
Hsu, S.,
Qi, M.,
and DeFranco, D. B.
(1992)
EMBO J.
11,
3457-3468[Abstract]
-
Ogawa, H.,
Inouye, S.,
Tsuji, F. I.,
Yasuda, K.,
and Umesono, K.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
11899-11903[Abstract]
-
Sackey, F. N. A.,
Haché, R. J. G.,
Reich, T.,
Kwast-Welfeld, J.,
and Lefebvre, Y. A.
(1996)
Mol. Endocrinol.
10,
1191-1205[Abstract]
-
Carey, K. L.,
Richards, S. A.,
Lounsbury, K. M.,
and Macara, I. G.
(1996)
J. Cell Biol.
133,
985-996[Abstract]
-
Yang, J.,
and DeFranco, D. B.
(1996)
Mol. Endocrinol.
10,
3-13[Abstract]
-
Tang, Y.,
Ramakrishnan, C.,
Thomas, J.,
and DeFranco, D. B.
(1997)
Mol. Biol. Cell
8,
795-809[Abstract]
-
Madan, A. P.,
and DeFranco, D. B.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
3588-3592[Abstract]
-
Tang, Y.,
and DeFranco, D. B.
(1996)
Mol. Cell. Biol.
16,
1989-2001[Abstract]
-
Htun, H.,
Barsony, J.,
Renyi, I.,
Gould, D. L.,
and Hager, G. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4845-4850[Abstract/Free Full Text]
-
Yang, J.,
Liu, J.,
and DeFranco, D. B.
(1997)
J. Cell Biol.
137,
523-538[Abstract/Free Full Text]
-
Zawel, L.,
Dai, J. L.,
Buckhaults, P.,
Zhou, S.,
Kinzler, K. W.,
Vogelstein, B.,
and Kern, S. E.
(1998)
Mol. Cell
1,
611-617[Medline]
[Order article via Infotrieve]
-
Baeuerle, P. A.,
and Baltimore, D.
(1996)
Cell
87,
13-20[Medline]
[Order article via Infotrieve]
-
Alksnis, M.,
Barkhem, T.,
Strömstedt, P.-E.,
Ahola, H.,
Kutoh, E.,
Gustafsson, J.-Å.,
Poellinger, L.,
and Nilsson, S.
(1991)
J. Biol. Chem.
266,
10078-10085[Abstract/Free Full Text]
-
Tanaka, H.,
Makino, Y.,
Miura, T.,
Hirano, F.,
Okamoto, K.,
Komura, K.,
Sato, Y.,
and Makino, I.
(1996)
J. Immunol.
156,
1601-1608[Abstract]
-
Kallio, P. J.,
Okamoto, K.,
O'Brien, S.,
Carrero, P.,
Makino, Y.,
Tanaka, H.,
and Poellinger, L.
(1998)
EMBO J.
17,
6573-6586[Abstract/Free Full Text]
-
McGuire, J.,
Whitelaw, M. L.,
Pongratz, I.,
Gustafsson, J.-Å.,
and Poellinger, L.
(1994)
Mol. Cell. Biol.
14,
2438-2446[Abstract]
-
Jewell, C. M.,
Webster, J. C.,
Burnstein, K. L.,
Sar, M.,
Bodwell, J. E.,
and Cidlowski, J. A.
(1995)
J. Steroid Biochem. Mol. Biol.
55,
135-146[CrossRef][Medline]
[Order article via Infotrieve]
-
Eguchi, H.,
Ikuta, T.,
Tachibana, T.,
Yoneda, Y.,
and Kawajiri, K.
(1997)
J. Biol. Chem.
272,
17640-17647[Abstract/Free Full Text]
-
Hollenberg, S. M.,
Weinberger, C.,
Ong, E. S.,
Cerelli, G.,
Oro, A.,
Lebo, R.,
Thompson, E. B.,
Rosenfeld, M. G.,
and Evans, R. M.
(1985)
Nature
318,
635-641[Medline]
[Order article via Infotrieve]
-
Arriza, J. L.,
Weinberger, C.,
Cerelli, G.,
Glaser, T. M.,
Handelin, B. L.,
Housman, D. E.,
and Evans, R. M.
(1987)
Scinece
237,
268-275[Medline]
[Order article via Infotrieve]
-
Misrahi, M.,
Atger, M.,
d'Auriol, L.,
Loosfelt, H.,
Meriel, C.,
Fridlansky, F.,
Guiochon-Mantel, A.,
Galibert, F.,
and Milgrom, E.
(1987)
Biochem. Biophys. Res. Commun.
143,
740-748[Medline]
[Order article via Infotrieve]
-
Lubahn, D. B.,
Joseph, D. R.,
Sar, M.,
Tan, J.,
Higgs, H. N.,
Larson, R. E.,
French, F. S.,
and Wilson, E. M.
(1988)
Mol. Endocrinol.
2,
1265-1275[Abstract]
-
Greene, G. L.,
Gilna, P.,
Waterfield, M.,
Baker, A.,
Hort, Y.,
and Shine, J.
(1986)
Science
231,
1150-1154[Medline]
[Order article via Infotrieve]
-
Ogawa, S.,
Inoue, S.,
Watanabe, T.,
Hiroi, H.,
Orimo, A.,
Hosoi, T.,
Ouchi, Y.,
and Muramatsu, M.
(1998)
Biochem. Biophys. Res. Commun.
243,
122-126[CrossRef][Medline]
[Order article via Infotrieve]
-
Baker, A. R.,
McDonnell, D. P.,
Hughes, M.,
Crisp, T. M.,
Mangelsdorf, D. J.,
Haussler, M. R.,
Pike, J. W.,
Shine, J.,
and O'Malley, B. W.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
3294-3298[Abstract]
-
Pfahl, M.,
and Benbrook, D.
(1987)
Nucleic Acids Res.
15,
9613[Medline]
[Order article via Infotrieve]
-
Weinberger, C.,
Thompson, C. C.,
Ong, E. S.,
Lebo, R.,
Gruol, D. J.,
and Evans, R. M.
(1986)
Nature
324,
641-646[Medline]
[Order article via Infotrieve]
-
Petkovich, M.,
Brand, N. J.,
Krust, A.,
and Chambon, P.
(1987)
Nature
330,
444-450[CrossRef][Medline]
[Order article via Infotrieve]
-
Giguère, V.,
Ong, E. S.,
Segui, P.,
and Evans, R. M.
(1987)
Nature
330,
624-629[CrossRef][Medline]
[Order article via Infotrieve]
-
Lehmann, J. M.,
Hoffmann, B.,
and Pfahl, M.
(1991)
Nucleic Acids Res.
19,
573-578[Abstract]
-
Sher, T.,
Yi, H.-F.,
McBride, O. W.,
and Gonzalez, F. J.
(1993)
Biochemistry
32,
5598-5604[Medline]
[Order article via Infotrieve]
-
Schmidt, A.,
Endo, N.,
Rutledge, S. J.,
Vogel, R.,
Shinar, D.,
and Rodan, G. A.
(1992)
Mol. Endocrinol.
6,
1634-1641[Abstract]
-
Lambe, K. G.,
and Tugwood, J. D.
(1996)
Eur. J. Biochem.
239,
1-7[Abstract]
-
Ohkura, N.,
Ito, M.,
Tsukada, T.,
Sasaki, K.,
Yamaguchi, K.,
and Miki, K.
(1996)
Biochim. Biophys. Acta
1308,
205-214[Medline]
[Order article via Infotrieve]
-
Okabe, T.,
Takayanagi, R.,
Imasaki, K.,
Haji, M.,
Nawata, H.,
and Watanabe, T.
(1995)
J. Immunol.
154,
3871-3879[Abstract/Free Full Text]
-
Willy, P. J.,
Umesono, K.,
Ong, E. S.,
Evans, R. M.,
Heyman, R. A.,
and Mangelsdorf, D. J.
(1995)
Genes Dev.
9,
1033-1045[Abstract]
-
Giguère, V.,
Yang, N.,
Segui, P.,
and Evans, R. M.
(1988)
Nature
331,
91-94[CrossRef][Medline]
[Order article via Infotrieve]
-
Eudy, J. D.,
Yao, S.,
Weston, M. D.,
Ma-Edmonds, M.,
Talmadge, C. B.,
Cheng, J. J.,
Kimberling, W. J.,
and Sumegi, J.
(1998)
Genomics
50,
382-384[CrossRef][Medline]
[Order article via Infotrieve]
-
Oba, K.,
Yanase, T.,
Nomura, M.,
Morohashi, K.,
Takayanagi, R.,
and Nawata, H.
(1996)
Biochem. Biophys. Res. Commun.
226,
261-267[CrossRef][Medline]
[Order article via Infotrieve]
-
Giguère, V.,
Tini, M.,
Flock, G.,
Ong, E.,
Evans, R. M.,
and Otulakowski, G.
(1994)
Genes Dev.
8,
538-553[Abstract]
-
Hirose, T.,
Smith, R. J.,
and Jetten, A. M.
(1994)
Biochem. Biophys. Res. Commun.
205,
1976-1983[CrossRef][Medline]
[Order article via Infotrieve]
-
Kapelle, M.,
Krätzschmar, J.,
Husemann, M.,
and Schleuning, W.-D.
(1997)
Biochim. Biophys. Acta
1352,
13-17[Medline]
[Order article via Infotrieve]
-
Mangelsdorf, D. J.,
Ong, E. S.,
Dyck, J. A.,
and Evans, R. M.
(1990)
Nature
345,
224-229[CrossRef][Medline]
[Order article via Infotrieve]
-
Leid, M.,
Kastner, P.,
Lyons, R.,
Nakshatri, H.,
Saunders, M.,
Zacharewski, T.,
Chen, J.-Y.,
Staub, A.,
Garnier, J.-M.,
Mader, S.,
and Chambon, P.
(1992)
Cell
68,
377-395[Medline]
[Order article via Infotrieve]
-
Mangelsdorf, D. J.,
Borgmeyer, U.,
Heyman, R. A.,
Zhou, J. Y.,
Ong, E. S.,
Oro, A. E.,
Kakizuka, A.,
and Evans, R. M.
(1992)
Genes Dev.
6,
329-344[Abstract]
-
Hirose, T.,
Fujimoto, W.,
Tamaai, T.,
Kim, K. H.,
Matsuura, H.,
and Jetten, A. M.
(1994)
Mol. Endocrinol.
8,
1667-1680[Abstract]
-
Chang, C.,
and Kokontis, J.
(1988)
Biochem. Biophys. Res. Commun.
155,
971-977[Medline]
[Order article via Infotrieve]
-
Ladias, J. A. A.,
and Karathanasis, S. K.
(1991)
Science
251,
561-565[Medline]
[Order article via Infotrieve]
-
Wang, L.-H.,
Tsai, S. Y.,
Cook, R. G.,
Beattie, W. G.,
Tsai, M.-J.,
and O'Malley, B. W.
(1989)
Nature
340,
163-166[CrossRef][Medline]
[Order article via Infotrieve]
-
Chartier, F. L.,
Bossu, J. P.,
Laudet, V.,
Fruchart, J. C.,
and Laine, B.
(1994)
Gene (Amst.)
147,
269-272[CrossRef][Medline]
[Order article via Infotrieve]
-
Chalfie, M.
(1995)
Photochem. Photobiol.
62,
651-656[Medline]
[Order article via Infotrieve]
-
Pariante, C. M.,
Pearce, B. D.,
Pisell, T. L.,
Owens, M. J.,
and Miller, A. H.
(1997)
Mol. Pharmacol.
52,
571-581[Abstract/Free Full Text]
-
Calleja, C.,
Pascussi, J. M.,
Mani, J. C.,
Maurel, P.,
and Vilarem, M. J.
(1998)
Nat. Med.
4,
92-96[Medline]
[Order article via Infotrieve]
-
Pratt, W. B.
(1993)
J. Biol. Chem.
268,
21455-21458[Free Full Text]
-
Jans, D. A.,
and Hübner, S.
(1996)
Physiol. Rev.
76,
651-685[Abstract/Free Full Text]
-
Zilliacus, J.,
Dahlman-Wright, K.,
Carlstedt-Duke, J.,
and Gustafsson, J-Å
(1992)
J. Steroid Biochem. Mol. Biol.
42,
131-139[Medline]
[Order article via Infotrieve]
-
Schena, M.,
Freedman, L. P.,
and Yamamoto, K. R.
(1989)
Genes Dev.
3,
1590-1601[Abstract]
-
Bresnick, E. H.,
Sanchez, E. R.,
Harrison, R. W.,
and Pratt, W. B.
(1988)
Biochemistry
27,
2866-2872[Medline]
[Order article via Infotrieve]
-
Schreck, R.,
and Baeuerle, P. A.
(1994)
Methods Enzymol.
234,
151-163[Medline]
[Order article via Infotrieve]
-
Hayashi, S-I.,
Hajiro-Nakanishi, K.,
Makino, Y.,
Eguchi, H.,
Yodoi, J.,
and Tanaka, H.
(1997)
Nucleic Acids Res.
25,
4035-4040[Abstract/Free Full Text]
-
Klebanoff, S. J.,
Vadas, M. A.,
Harlan, J. M.,
Sparks, L. H.,
Gamble, J. R.,
Agosti, J. M.,
and Waltersdorph, A. M.
(1986)
J. Immunol.
136,
4220-4225[Abstract/Free Full Text]
-
Tsan, M.-F.,
Douglass, K. H.,
and Mclntyre, P. A.
(1977)
Blood
49,
437-444[Abstract]
-
Shepherd, V. L.
(1986)
Semin. Respir. Infect.
1,
99-106[Medline]
[Order article via Infotrieve]
-
Kuge, S.,
Jones, N.,
and Nomoto, A.
(1997)
EMBO J.
16,
1710-1720[Abstract/Free Full Text]
-
Görner, W.,
Durchschlag, E.,
Martinez-Pastor, M. T.,
Estruch, F.,
Ammere, G.,
Hamilton, B.,
Ruis, H.,
and Schüller, C.
(1998)
Genes Dev.
12,
586-597[Abstract/Free Full Text]
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