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INTRODUCTION |
The ability of both natural and synthetic glucocorticoids to act
on a target tissue and elicit specific biological responses is
dependent on the presence of the 
isoform of the glucocorticoid receptor (GR).1 GR
belongs to the superfamily of steroid/thyroid/retinoic acid receptor
proteins that function as ligand-dependent transcription factors (1). This family also includes receptors for vitamin D and a
large group of proteins termed orphan receptors whose ligands and/or
functions are unknown. Like other members of this receptor superfamily,
GR is composed of an amino-terminal domain that is involved in gene
activation, a central DNA binding domain, and a carboxyl-terminal
hormone binding domain (HBD).
GR is expressed in almost all tissues and cells, and in the absence
of hormone it resides in the cytoplasm of cells as part of a large
multiprotein complex. This complex consists of the receptor
polypeptide, two molecules of the heat shock protein hsp90, and several
additional proteins (2, 3). When hormone binds the receptor, a
conformational change ensues, resulting in the dissociation of hsp90
and the other associated proteins. In its new conformation, GR
translocates into the nucleus, where it binds as a homodimer to
glucocorticoid-responsive elements (GREs) located in the regulatory
regions of target genes. GR then communicates with the basal
transcription machinery and, depending on the GRE sequence and promoter
context, either positively or negatively regulates expression of the
linked gene. The receptor can also modulate gene expression apart from
DNA binding by physically interacting with other transcription factors
such as AP-1 and NF-
B (4-7).
Sensitivity to glucocorticoids varies between tissues and even within
the same tissue during different stages of development (8, 9). In
addition, the beneficial effects of glucocorticoids in the treatment of
many immune and inflammatory diseases is often limited by the
development of glucocorticoid resistance in the diseased tissue (10).
The molecular basis for these variations in glucocorticoid
responsiveness is poorly understood. As the sole effector molecule in
the glucocorticoid signaling cascade, GR is the primary target for
regulatory events that modulate target cell sensitivity to
glucocorticoids. Changes in GR expression and/or the potency with
which GR functions as a ligand-dependent transcription
factor will elicit corresponding changes in glucocorticoid responsiveness. Because glucocorticoids profoundly influence all aspects of biological function and are extensively used as therapeutic agents in the treatment of many diseases and disorders, understanding the factors that regulate GR expression and/or activity, and hence
glucocorticoid responsiveness, is an important goal of current research.
Alternative splicing of the human GR (hGR) gene produces a splice
variant termed hGR
. hGR differs from the wild-type receptor (hGR) only at the carboxyl terminus (11-13). The two isoforms are
identical through amino acid 727 but then diverge, with hGR having
an additional 50 amino acids and hGR an additional, nonhomologous 15 amino acids. hGR has been detected in many different tissues (13-17). Within tissues, hGR is most abundant in certain epithelial cells (16). In contrast to hGR, hGR resides in the nucleus of
cells independent of glucocorticoid treatment and does not bind
glucocorticoids or activate glucocorticoid-responsive genes (11, 13,
16, 18). We and others have shown that hGR functions as a dominant
negative inhibitor of hGR on both complex and simple glucocorticoid-responsive promoters (13, 14).
The ability of hGR to antagonize the function of hGR suggests
that hGR will play a critical role in the regulation of target cell
sensitivity to glucocorticoids. Indeed, recent studies have demonstrated that hGR expression is elevated in patients suffering from generalized and tissue-specific glucocorticoid resistance (19,
20). In the present study, we investigate the ability of hGR to
repress hGR in multiple cell types and evaluate how much hGR
(relative to hGR) is necessary for the observed repression. hGR's dominant negative activity is further explored on other closely related steroid hormone receptors and on genes negatively regulated by glucocorticoids. We also assess the ability of hGR to
associate with hsp90, bind GRE-containing DNA, and heterodimerize with
hGR in order to understand the mechanism responsible for the
dominant negative activity of hGR. Finally, we investigate whether
the hGR-specific amino acids mediate this repression of hGR function.
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EXPERIMENTAL PROCEDURES |
Materials--
Dexamethasone (DEX) and progesterone were
purchased from Steraloids (Wilton, NH), and the synthetic androgen
R1881 was obtained from NEN Life Science Products.
[14C]Chloramphenicol (40-60 mCi/mmol) was obtained from
NEN Life Science Products, and [-32P]UTP and dCTP
(3000 Ci/mmol) were supplied by ICN Radiochemicals (Irvine, CA). Acetyl
coenzyme A and protease inhibitors were obtained from Roche Molecular
Biochemicals. The peroxidase-labeled anti-rabbit and anti-mouse
secondary antibodies and the chemiluminescent detection reagents were
purchased from Amersham Pharmacia Biotech. Dithiobis(succinimidyl propionate) (DSP) was from Pierce. The TNT Coupled Reticulocyte Lysate
Translation System was from Promega (Madison, WI).
Recombinant Plasmids--
Construction of pCMVhGR and
pCMVhGR expression vectors as well as the human cytomegalovirus
major intermediate early gene promoter expression vector backbone pCMV5
have been described previously (13). Plasmids pGST-hGRHBD and
pGST-hGRHBD were generated by polymerase chain reaction cloning and
express the hGR HBD (amino acids 523-777) and hGR HBD (amino
acids 523-742), respectively, fused to the glutathione
S-transferase (GST) protein. Site-directed mutagenesis by
polymerase chain reaction was employed to generate the truncated
receptor pCMVhGR728T. Nucleotides GTG, encoding valine at amino acid
728 of hGR, were replaced with TAG, which mutates the valine to a
stop codon. The expressed hGR728T protein was recognized by the
antipeptide hGR antibody 57 but not by the hGR-specific antibody
AShGR nor the hGR-specific antibody BShGR (16, 21, 22). All
constructs were confirmed by DNA sequencing. The expression vector
CMV3.1AR (pCMVhAR) encodes the human androgen receptor and was obtained
from Dr. M. J. McPhaul (University of Texas Southwestern) (23).
The expression vector phPR-B, kindly provided by Dr. D. P. McDonnell (Duke University), encodes the B form of the human
progesterone receptor and is driven by the SV40 enhancer (24). The
expression vector for the transcriptionally active p65 subunit of
NF-B (pCMVp65) and the NF-B-responsive reporter 3XMHCCAT
(MHC-CAT) were obtained from Dr. A. S. Baldwin (University of
North Carolina, Chapel Hill, NC) (7). Plasmids pHHluc and pGMCS contain
the mouse mammary tumor virus (MMTV) promoter cloned upstream of the
luciferase and CAT genes, respectively (25, 26). Plasmids pT7/T3-hGR
and pT7/T3-hGR, used for in vitro synthesis of the hGR
and hGR proteins, have been described previously (16, 27).
Cell Culture and Transfections--
COS-1, CV-1, and COS-7 cells
were grown in Dulbecco's minimum essential medium supplemented with 2 mM glutamine and 10% (v/v) of a mixture (1:1) of
heat-inactivated fetal calf and calf serum. All cultures were
maintained in a 5% CO2 humidified atmosphere at 37 °C
and passaged every 3-4 days. For transfection of cells, medium was
removed from subconfluent cells and replaced with fresh Dulbecco's
minimum essential medium containing 3% serum. Plasmid DNA was prepared
as a calcium phosphate precipitate, and the total amount of DNA in each
transfection was kept constant by the addition of empty vector (pCMV5)
and salmon sperm DNA. The precipitates were incubated with cells for
5 h. The medium was then removed, and the cells were shocked for
30 s with 15% glycerol and then refed with fully supplemented
medium stripped of endogenous steroids.
Luciferase Assays--
Cells were transfected as described above
and in the appropriate figure legends. Immediately after the
transfection, DEX or vehicle was added to the cells, which were then
incubated an additional 18 h. Cells were harvested, lysates were
prepared, and luciferase assays were performed according to the
manufacturer's instructions (Analytical Luminescence Laboratory, San
Diego, CA). Luciferase activity was calculated per µg of protein for
each sample.
CAT Assays--
Cells were transfected as described above and in
the appropriate figure legend. Immediately after the transfection, the
appropriate steroid (DEX, progesterone, or R1881) or vehicle was added
to the cells. After an 18-h incubation, cells were harvested, and CAT
assays were performed as described previously (13). Briefly, a lysate
was prepared and inactivated by heating at 68 °C for 6 min.
Equivalent amounts of protein were adjusted to 156 mM Tris, 1 mM acetyl coenzyme A, and 0.1 µCi of
[14C]chloramphenicol in a final volume of 150 µl. The
CAT reaction was then allowed to proceed for 16 h at 37 °C.
Samples were applied to a thin layer chromatography plate and
chromatographed in chloroform/methanol (95:5). After autoradiography,
CAT activity was quantitated by excising the appropriate area from the
thin layer chromatography plate and counting the
[14C]chloramphenicol and acetylated derivatives in a
Beckman LS 7000 scintillation counter.
Western Blotting--
Transfected cells were harvested and lysed
in TENT buffer (20 mM Tris-HCl, pH 7.5, 2 mM
EDTA, 150 mM NaCl, 0.5% Triton X-100) containing protease
inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
aprotinin, 1 µM pepstatin, 1 µM leupeptin).
Proteins were resolved on SDS-polyacrylamide gels and transferred to
nitrocellulose. Membranes were stained with Ponceau S to evaluate
loading equivalency and transfer efficiency and were blocked in
Tris-buffered saline (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) containing 10% nonfat dry milk.
Blots was then washed in Tris-buffered saline containing 1% nonfat dry
milk (TBS-1%) and incubated for 1 h at room temperature with the
appropriate dilution of primary antibody in TBS-1%. The antipeptide
hGR antibody 57, the hGR-specific antibody AShGR, and the
hGR-specific antibody BShGR were each used at a 1:1000 dilution (16,
21, 22). The hsp90 monoclonal antibody AC88 was used at 6 µg/ml and
was kindly provided by Dr. G. R. Pearson (28). Membranes were
subsequently washed in TBS-1%, reacted for 1 h at room
temperature with a horseradish peroxidase-labeled anti-rabbit or
anti-mouse secondary antibody in TBS-1%, washed in TBS-1%, reacted
with chemiluminescent reagents, and then processed for autoradiography.
Blots were stripped by incubating at 55 °C for 45 min in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, 100 mM
2-mercaptoethanol. hGR, hGR, and hGR728T signals were quantitated
densitometrically using NIH Image analysis software.
Northern Blotting--
Poly(A)+ RNA (5 µg) from
transfected cells was isolated, electrophoresed, and transferred as
described previously (29). After transfer, the RNA was UV-cross-linked
to the membrane and subsequently stained with methylene blue (0.04%
methylene blue, 0.5 M NaOAc, pH 5.2) to examine both the
integrity of the RNA and the completeness of the transfer. Membranes
were prehybridized (3 h at 65 °C) and hybridized (18 h at 65 °C)
in 50% formamide, 5× saline/sodium phosphate/EDTA, 5× Denhardt's
solution, 2% SDS, 200 µg/ml yeast RNA, and 200 µg/ml denatured,
sheared salmon sperm DNA. The [-32P]UTP-labeled hGR
or hGR-specific cRNA probes, which have been described previously
(13), were included in the hybridization fluid (1 × 106 cpm/ml). Following hybridization, blots were washed
once at room temperature and four times at 65 °C in 0.1×
saline/sodium phosphate/EDTA, 0.1% SDS. To control for loading
differences in RNA, membranes were stripped (30-min incubation in 0.1×
saline/sodium phosphate/EDTA, 0.1% SDS heated to 100 °C) and
reprobed with a -actin cRNA probe. The hGR and hGR mRNA
signals were quantitated densitometrically using NIH Image analysis
software and normalized to -actin mRNA levels.
Immunoprecipitation of Receptor-hsp90 Complexes--
For
immunoprecipitation of receptor-hsp90 complexes from whole cells,
transfected cells were lysed using a Dounce homogenizer in HEPES buffer
(10 mM HEPES, pH 7.4, 1 mM EDTA, 20 mM sodium molybdate) containing protease inhibitors. The
homogenate was centrifuged at 165,000 × g in a Beckman
50 Ti rotor for 1 h at 4 °C, and the supernatant was collected.
Proteins (200 µg) were precleared with preimmune serum and protein
A-Sepharose for 1 h at 4 °C. After centrifugation, the
supernatant was removed and transferred to a new tube. The antipeptide
hGR antibody 59 (1:50 dilution) or preimmune serum was added and each
tube incubated for 2 h at 4 °C with rotation (21). For binding
immune complexes, 80 µl of protein A-Sepharose beads (Sigma) were
added, and the incubation continued an additional 30 min at 4 °C
with rotation. Protein A-Sepharose was prepared in TEGM buffer (10 mM TES, pH 7.6, 4 mM EDTA, 50 mM
NaCl, 10% glycerol, 20 mM sodium molybdate). The protein
A-Sepharose immune complexes were washed four times with 1 ml of TEGM
buffer and then resuspended in sample buffer containing 10% glycerol,
2% SDS, 0.2 mg/ml bromphenol blue, 62.5 mM Tris-HCl, pH
6.8, and 5% 2-mercaptoethanol. Immunoprecipitated proteins were eluted
from the protein A-Sepharose by boiling 5 min and resolved on
SDS-polyacrylamide gels. After electrophoretically transferring the
proteins to nitrocellulose, immunoblotting was carried out as described
above. Immunoprecipitation of receptor-hsp90 complexes from
reticulocyte lysates expressing equivalent amounts of
35S-labeled hGR or 35S-labeled hGR was
performed using the anti-hsp90 monoclonal antibody 3G3 (Affinity
BioReagents, Golden, CO) essentially as described (30).
DNA Binding Analysis of in Vitro Translated hGR and
hGR--
DNA binding analysis was carried out essentially as
described previously (27). The hGR and hGR proteins were
synthesized in vitro using reticulocyte lysates. Translation
products (5 fmol) were incubated with an equal volume of DNA binding
buffer (20 mM NaHPO4, pH 7, 10% glycerol, 50 mM NaCl, 1 mM EDTA, and 2 mM 2-mercaptoethanol) for 2 h at 0 °C with or without 200 nM DEX. Lysates were then heat-activated for 30 min at
25 °C, after which they were transferred to tubes coated with 1%
BSA. An 868-base pair ClaI/SphI fragment
containing the MMTV long terminal repeat from plasmid pLTR190 or a
777-bp PstI/ClaI fragment from the plasmid pBR322
was labeled on one end with [-32P]dCTP using Klenow to
fill in the ClaI-generated 5' overhang. Labeled DNA
fragments (10-fold molar excess) and nonspecific DNA (poly(dI-dC) at a
50-fold excess and Escherichia coli DNA at a 50-fold excess)
were added to the lysates, and the incubation continued at 0 °C for
2 h. For some experiments, unlabeled competitor DNA was added at a
50-fold excess. Antipeptide hGR antibody 57 (1:50 dilution) was then
added to the lysates, and the incubation continued for 2 h at
0 °C. Protein A-Sepharose (133 mg/ml DNA binding buffer) was then
added to the lysates for an additional 1-h incubation, and the pellets
were subsequently washed four times with 1 ml of DNA binding buffer.
DNA fragments were recovered from the pellets by phenol chloroform
extraction and analyzed on 8 M urea, 4% polyacrylamide
gels. Gels were fixed in 30% methanol, 10% acetic acid for 15 min,
dried for 1 h under vacuum at 60 °C, and processed for autoradiography.
Immunoprecipitation of hGR-hGR Heterodimers--
The
hGR and hGR proteins were synthesized in vitro using
reticulocyte lysates. Thirty-five µl of 35S-labeled
hGR translation mix were added to 35 µl of unlabeled hGR
translation mix or 35 µl of unprogrammed translation mix and
incubated for 30 min at room temperature in 20 mM HEPES, pH 7.9, 50 mM KCl, 2.5 mM MgCl2, 1.0 mM dithiothreitol, 200 nM DEX, and protease
inhibitors. An aliquot was then removed from each tube to verify that
equivalent amounts of hGR were present in each mixture. Proteins
were cross-linked by the addition of 2.5 mM DSP for 10 min
at room temperature. Reactions were then terminated by the addition of
0.1 M ethanolamine. The cross-linked proteins were diluted
in immunoprecipitation buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% sodium deoxycholate,
0.5% Nonidet P-40, 0.1 M ethanolamine, and protease
inhibitors), precleared, and immunoprecipitated with the
hGR-specific antibody BShGR (16). Immune complexes were recovered,
washed extensively in the immunoprecipitation buffer, and resuspended
in Laemmli buffer with or without -mercaptoethanol. After proteins
were resolved on SDS-polyacrylamide gels, gels were fixed in 30%
methanol-10% acetic acid for 30 min, dried for 1 h under vacuum
at 60 °C, and processed for autoradiography using liquid
EN3HANCE.
GST Pull-down Assays--
GST pull-down assays were carried out
essentially as described (31). In brief, the GST-hGRHBD and
GST-hGRHBD fusion proteins were induced in DH5 E. coli
by the addition of 0.1 mM isopropyl -D-thiogalactoside to log phase cells. Following a 4-h
incubation, the cells were harvested and lysed by sonication in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 10% glycerol, 0.2 mg/ml lysozyme, and protease
inhibitors. Following centrifugation, the soluble protein extract was
incubated with phosphate-buffered saline-washed glutathione agarose in
the presence of 1% Triton X-100 and 1 mM dithiothreitol.
The immobilized fusion proteins were then incubated with full-length,
in vitro translated 35S-labeled hGR or
35S-labeled hGR treated with or without 1 µM DEX. Assays were carried out at 4 °C with constant
rotation in binding buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 0.01% Nonidet P-40, and protease
inhibitors). Recovered proteins were resolved on SDS-polyacrylamide
gels and processed as described above.
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RESULTS |
The Dominant Negative Activity of hGR Occurs in Multiple Cell
Types When hGR Is More Abundant than hGR--
We have
demonstrated previously in HeLa S3 cells that hGR can
inhibit the transcriptional activity of hGR in a dominant negative
fashion (13). This early study, however, did not investigate whether
the dominant negative activity occurred in other cell types; nor did it
determine how much hGR (relative to hGR) was needed for the
observed repression. To address these issues, we initially analyzed the
ability of hGR to repress hGR-mediated activation of the
glucocorticoid-responsive MMTV promoter in transfected COS-1 cells.
COS-1 cells (which are devoid of endogenous GR) were transfected with a
fixed amount of the MMTV-luciferase reporter pHHluc, a fixed amount of
the hGR expression vector pCMVhGR, and increasing amounts of the
hGR expression vector pCMVhGR corresponding to a 5- and 10-fold
molar excess over the amount of transfected pCMVhGR. The cells were
then treated with or without hormone for 18 h and analyzed for
luciferase activity. As increasing amounts of pCMVhGR were
transfected into the cells, the glucocorticoid-induced, hGR-mediated
activation of the MMTV promoter was diminished in a
dose-dependent manner (Fig.
1A). Transfection of a 5- and
10-fold molar excess of pCMVhGR resulted in a 36.7 and 63.7%
decrease, respectively, in hGR activity. Similar reductions in
hGR activity were also observed following a 48-h hormone treatment
(data not shown).
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Fig. 1.
The dominant negative activity of
hGR occurs in multiple cell types.
A, COS-1 cells were transfected with 0.1 µg of the
MMTV-luciferase reporter pHHluc, 0.1 µg of pCMVhGR, and various
amounts of pCMV5 and/or pCMVhGR. Molar ratios of transfected
plasmids are indicated. Following an 18-h incubation with vehicle
(CON) or 100 nM DEX, cells were harvested, and
luciferase activity was determined. Data are plotted as -fold change
from basal activation and represent the mean ± S.E. of three or
four independent experiments (*, p < 0.05 versus -fold induction in the absence of pCMVhGR;
t test). B, COS-1 cells transfected with
equimolar amounts of pCMVhGR (lane 1) or pCMVhGR
(lane 2) were analyzed on a Western blot with the
antipeptide hGR antibody 57. COS-1 cells transfected with 1.0 µg of
pCMVhGR and various amounts of pCMVhGR corresponding to a 1-, 5-, and 10-fold molar excess (lanes 3-5, respectively) were
analyzed on a Western blot with the hGR-specific antibody AShGR and
the hGR-specific antibody BShGR. C, COS-7 and CV-1 cells
were transfected with 0.1 µg of pHHluc, 0.1 µg of pCMVhGR, and
various amounts of pCMV5 and/or pCMVhGR. Molar ratios of transfected
plasmids are indicated. Cells were processed as described in
A. Data are plotted as -fold change from basal activation
and represent the mean ± S.E. of four independent experiments (*,
p < 0.05 versus -fold induction in absence
of pCMVhGR; t test).
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Measuring the amount of hGR and hGR expressed in these cells with
an antibody that recognizes both receptor isoforms is problematic
because the 90-kDa hGR protein comigrates with a 90-kDa hGR
degradation product (16). Therefore, the relative levels of the hGR
and hGR proteins were assessed in the following manner. First, COS-1
cells were separately transfected with an equimolar amount of
pCMVhGR or pCMVhGR. A Western blot was performed on these cells
with the anti-peptide hGR antibody 57, which recognizes the same
epitope (amino acids 346-367) in both receptor isoforms (Fig.
1B, lanes 1-2). hGR was expressed at
approximately 80% the level of hGR. Second, COS-1 cells were
co-transfected with a fixed amount of pCMVhGR and various amounts of
pCMVhGR corresponding to a 1-, 5-, or 10-fold molar excess. Western
blots were performed on these cells with isoform-specific antibodies
(Fig. 1B, lanes 3-5). hGR levels remained
constant in these cells, and hGR levels increased the expected 5- and 10-fold when a 5- and 10-fold molar excess of pCMVhGR was
included in the transfection mixture. Taken together, these blots
indicate that the dose-dependent reduction in hGR
activity observed for COS-1 cells in Fig. 1A occurred when
hGR was expressed at levels 4- and 8-fold greater than hGR.
The ability of hGR to function as a dominant negative inhibitor of
hGR was evaluated in two additional cell lines devoid of endogenous
GR. COS-7 and CV-1 cells were each transfected with a fixed amount of
pHHluc, a fixed amount of pCMVhGR, and a 5- or 10-fold molar excess
of pCMVhGR. The cells were then treated with our without
glucocorticoids for 18 h and analyzed for luciferase activity.
Expression of hGR inhibited hGR-mediated activation of the MMTV
promoter in both cell types (Fig. 1C). The transcriptional activity of hGR was reduced 55.6 and 50.4% in COS-7 cells and 25.6 and 69.0% in CV-1 cells when a 5- and 10-fold molar excess, respectively, of pCMVhGR was included in the transfection mixture. Similar reductions in hGR activity were also observed following a
48-h hormone treatment (data not shown). Western blots, performed exactly as described above for COS-1 cells, again showed that hGR
was expressed at levels approximately 4- and 8-fold greater than hGR
(data not shown). Thus, the dominant negative activity of hGR
appears to be a general phenomenon that occurs in multiple cell types
when hGR is more abundant than hGR.
The Dominant Negative Activity of hGR Is Selective for
hGR--
hGR is a member of a subfamily of receptors that
includes the mineralocorticoid receptor (MR), progesterone receptor
(PR), and androgen receptor (AR). The DNA-binding domains of these
receptors are highly conserved. As a consequence, each receptor
activates the MMTV promoter by binding the same hormone response
element (HRE) (32). Our observation that hGR represses the
transcriptional activity of hGR on the MMTV promoter raised an
important question. Does hGR also inhibit the ability of these other
closely related steroid hormone receptors to activate the MMTV
promoter? To answer this question, COS-1 cells were transfected with a
fixed amount of the MMTV-CAT reporter pGMCS; a fixed amount of the
expression vector for hGR, the B-form of the human PR (hPRB), or the
human AR (hAR); and a 10-fold molar excess of pCMV5 or pCMVhGR. The cells were then treated with or without the appropriate hormone for
18 h. Expression of hGR inhibited the activity of hGR by 60% in cells transfected with pCMVhGR (Fig.
2A, left panel). In
cells transfected with the more weakly expressing pRSVhGR, expression of hGR produced an even greater 75% reduction in the transcriptional activity of hGR (Fig. 2A, right
panel). However, hGR had no effect on the transcriptional
activity of hPRB, and only a small inhibition (27%) was observed for
hAR (Fig. 2, B and C). These results suggest that
the dominant negative activity of hGR is selective for hGR.
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Fig. 2.
Effect of hGR on
hPRB- or hAR-mediated activation of the MMTV promoter in transfected
COS-1 cells. A, cells were transfected with 2.5 µg of
the MMTV-CAT reporter pGMCS, 2.5 µg of pCMVhGR (left
panel) or pRSVhGR (right panel), and a 10-fold molar
excess of pCMV5 or pCMVhGR. B, cells were transfected
with 2.5 µg of pGMCS, 2.5 µg of phPR-B, and a 10-fold molar excess
of pCMV5 or pCMVhGR. C, cells were transfected with 2.5 µg of pGMCS, 2.5 µg of pCMVhAR, and a 10-fold molar excess of pCMV5
or pCMVhGR. Molar ratios of the transfected plasmids are indicated.
Following an 18-h incubation with vehicle (CON) or the
appropriate ligand (100 nM DEX, 100 nM
progesterone (PROG), or 1.0 µM R1881), cells
were harvested, and CAT activity was determined. Data are plotted as
-fold change from basal activation and represent the mean ± S.E.
(n = 2 for hGR, n = 4-5 for hPRB
and hAR; *, p < 0.05 versus -fold induction
in the absence of pCMVhGR; t test).
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The Dominant Negative Activity of hGR Occurs on Some Genes
Negatively Regulated by hGR--
hGR enhances the expression of
some genes, and it represses the expression of others. However, it is
not known whether the dominant negative activity of hGR occurs on
genes negatively regulated by hGR. One gene that is repressed by
hGR in almost all cell types is the hGR gene itself by a process
termed homologous down-regulation (33). In COS-1 cells expressing only
hGR, the hGR mRNA was down-regulated to approximately 20% of
control levels following a 24-h glucocorticoid treatment (Fig.
3A). In contrast, no reduction
in hGR mRNA was observed in cells expressing only hGR (Fig.
3A). The ability of hGR to antagonize hGR-mediated down-regulation was assessed in COS-1 cells transfected with a fixed
amount of pCMVhGR and increasing amounts of pCMVhGR corresponding to a 5- and 10-fold molar excess. After treating the cells with or
without glucocorticoids for 24 h, Western blots were performed with isoform-specific antibodies. Expression of hGR (at levels approximately 4- and 8-fold higher than hGR) had no effect on the
magnitude of hGR down-regulation (Fig. 3B). hGR not
only fully down-regulated its own expression but also partially
down-regulated the expression of hGR (Fig. 3B). This
partial down-regulation of hGR was also observed at the mRNA
level in cells expressing equivalent amounts of the two receptor
isoforms (Fig. 3A).
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Fig. 3.
Effect of hGR on
hGR-mediated down-regulation of the
transfected hGR gene in COS-1 cells.
A, cells were transfected with pCMVhGR, pCMVhGR, or
pCMVhGR and pCMVhGR together. Molar ratios of the transfected
plasmids are indicated. Following a 24-h incubation with vehicle
(lane 1) or 100 nM DEX (lane 2),
poly(A)+ mRNA was isolated and analyzed on a Northern
blot using hGR- or hGR-specific cRNA probes. hGR and hGR
mRNA were quantitated, normalized to actin mRNA, and plotted as
a percentage of control. Representative autoradiographs are shown
above the appropriate bar graph.
B, cells were transfected with 5 µg of pCMVhGR and
various amounts of pCMV5 or pCMVhGR. Molar ratios of the transfected
plasmids are indicated. Following a 24-h incubation with vehicle
(lane 1) or 100 nM DEX (lane 2),
cells were harvested, and whole cell lysates were prepared. Western
blots were performed with the hGR-specific antibody AShGR or the
hGR-specific antibody BShGR. hGR and hGR protein levels were
quantitated and plotted as a percentage of control. Representative
immunoblots are shown above the appropriate bar graph. Data represent the mean ± S.D. of two
independent experiments.
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hGR also represses gene expression apart from DNA binding by
physically associating with other transcription factors. For example,
hGR physically associates with NF-B, an activator of a broad
class of immune system genes, and prevents NF-B from activating
target genes (7). This is illustrated in Fig.
4A, where ligand-bound hGR
blocked the constitutively active p65 subunit of NF-B from
activating the NF-B-responsive reporter MHC-CAT in COS-1 cells. The
DNA-binding domain of hGR is required for this repression (34).
hGR has an intact DNA-binding domain, but this receptor isoform did
not repress the transcriptional activity of NF-B following hormone
treatment (Fig. 4A). In addition, hGR did not
constitutively inhibit NF-B, since similar absolute amounts of CAT
activity were measured in the pCMV5- and pCMVhGR-transfected cells
(data not shown). We next investigated whether hGR could inhibit
hGR's antagonism of NF-B. hGR repressed the transcriptional activity of NF-B to approximately 15% of control levels in the absence of co-expressed hGR (Fig. 4B). This response was
reduced to 33% of control levels in cells transfected with a 5-fold
molar excess of pCMVhGR (Fig. 4B). However, no further
inhibition was observed with greater amounts of hGR (up to 10-fold),
suggesting that the efficacy of hGR as a dominant negative inhibitor
is greater on genes activated by hGR than on genes repressed by hGR.
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Fig. 4.
Effect of hGR on
hGR-mediated repression of an
NF-B-responsive promoter in transfected COS-1
cells. A, cells were transfected with 2.5 µg of the
MHC-CAT reporter, 2.5 µg of the p65 subunit of NF-B, and equimolar
amounts of pCMV5, pCMVhGR, or pCMVhGR. After an 18-h incubation
with vehicle (CON) or 100 nM DEX, cells were
harvested, and CAT activity was determined. CAT activity is plotted as
a percentage of control and represents the mean ± S.E. of 4-7
independent experiments (*, p < 0.05 versus
control; t test). B, cells were transfected with
2.5 µg of the MHC-CAT reporter, 2.5 µg of the p65 subunit of
NF-B, 2.5 µg of pCMVhGR, and various amounts of pCMV5 and/or
pCMVhGR. Molar ratios of transfected plasmids are indicated. Cells
were processed as described in A. CAT activity is plotted as
a percentage of control and represents the mean ± S.E. of five
independent experiments (*, p < 0.05 versus
percentage repression in absence of pCMVhGR; t
test).
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hGR Associates with hsp90--
The ability of hGR to repress
the transcriptional activity of hGR requires hGR to be expressed
at levels 4-8-fold greater than hGR. This requirement may indicate
that a portion of the expressed hGR molecules are held in an
inactive state. In the absence of hormone, hGR is held in an
inactive state that cannot dimerize nor bind DNA by its association
with the heat shock protein hsp90 (2, 3). To test whether hGR
associates with hsp90, we transfected COS-1 cells with equimolar
amounts of pCMVhGR, pCMVhGR, or the expression vector backbone
pCMV5 (mock). Immunoprecipitations were performed with preimmune serum
or the antipeptide hGR antibody 59, which recognizes an epitope common
to both receptor isoforms. The immunoprecipitated proteins were then
analyzed on a Western blot with the hsp90 antibody AC88. AC88 detected
the 90-kDa hsp90 protein in the immunoprecipitates prepared from the
hGR- and hGR-transfected cells but not the mock-transfected cells
(Fig. 5, upper panel). The
blot was then stripped and reprobed with the antipeptide hGR antibody
57 to verify that hGR and hGR were immunoprecipitated at
comparable levels (Fig. 5, lower panel). These results
indicate that hGR can associate with hsp90 and suggest that a
portion of the expressed hGR molecules may be unavailable for
repressing ligand-bound hGR.
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Fig. 5.
Association of hGR with hsp90 in transfected COS-1 cells. Whole cell lysates
were prepared from COS-1 cells transfected with equimolar amounts of
pCMVhGR, pCMVhGR, or pCMV5 (mock). Proteins were
immunoprecipitated with preimmune serum (lanes 1,
3, and 5) or the antipeptide hGR antibody 59 (lanes 2, 4, and 6). The
immunoprecipitated proteins were analyzed on a Western blot using the
hsp90 antibody AC88 (upper panel). The blot was then
stripped and reprobed with the antipeptide hGR antibody 57 (lower
panel). Molecular mass standards are indicated on the
left.
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hGR Binds GRE-containing DNA--
The molecular mechanisms
responsible for hGR's antagonism of hGR are currently unknown.
Cellular sensitivity to glucocorticoids is regulated by changes in
receptor expression and/or alterations in the efficacy with which the
receptor functions as a ligand-dependent transcription
factor. Because overexpression of hGR had no effect on the
expression of hGR (Fig. 1B), hGR may interfere with
the ability of hGR to function as a ligand-dependent
transcription factor. hGR has an intact DNA-binding domain and is
located in the nucleus of cells independent of hormone treatment (13,
16). Therefore, hGR might compete with hGR for GRE binding. To
test whether hGR can bind a GRE-containing piece of DNA, equivalent amounts of in vitro translated hGR or hGR (Fig.
6D) were heat-activated in the
presence of glucocorticoids and incubated with a 10-fold molar excess
of a radiolabeled DNA fragment. The DNA fragment utilized was a
GRE-containing MMTV promoter fragment or a similar-sized piece of DNA
from pBR322 that does not contain a GRE. Receptor-DNA complexes were
immunoprecipitated using the antipeptide hGR antibody 57, and the DNA
recovered from the immune complexes was analyzed on a denaturing gel.
Recovery of radiolabeled DNA is indicative of receptor binding. As
shown in Fig. 6A, both hGR and hGR bound the MMTV
fragment but not the pBR322 fragment. hGR bound the MMTV fragment
with a greater capacity than hGR as results from four independent
experiments revealed a 3-4-fold difference in DNA binding between the
two receptor isoforms. Binding of hGR and hGR to the MMTV
fragment was also specific (Fig. 6B). In the presence of a
50-fold excess of unlabeled MMTV fragment, binding above background was
eliminated for both receptor isoforms. In contrast, no reduction in
binding was observed for either isoform in the presence of a 50-fold
excess of the pBR322 fragment (Fig. 6B). In addition, a 50%
reduction in DNA binding was observed for both hGR and hGR when a
100-fold excess of a GRE oligonucleotide was included in the reaction,
whereas a non-GRE oligonucleotide had no effect (data not shown).
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Fig. 6.
Binding of hGR to
the GRE-containing MMTV promoter. A, in
vitro translated hGR, hGR, or unprogrammed lysates
(mock) were heat-activated in the presence of 200 nM DEX and incubated with a 10-fold molar excess of a
radiolabeled MMTV promoter fragment (MMTV) or a radiolabeled
DNA fragment that does not contain a GRE (pBR322).
Receptor-DNA complexes were immunoprecipitated with antibody 57. DNA
recovered from the immune complexes was then analyzed on a 4%
denaturing urea-polyacrylamide gel. B, in vitro
translated hGR or hGR were heat-activated in the presence of 200 nM DEX and incubated with a 10-fold molar excess of the
radiolabeled MMTV promoter fragment in the absence (None) or
presence of a 50-fold excess of unlabeled MMTV or pBR322 and processed
as described above. C, in vitro translated
hGR, hGR, or unprogrammed lysates (mock) were
heat-activated in the absence or presence of 200 nM DEX,
incubated with a 10-fold molar excess of the radiolabeled MMTV promoter
fragment, and processed as described above. D, Western blot
analysis with antibody 57 of in vitro translated hGR,
hGR, or unprogrammed lysates (mock) utilized in the
reactions above. E, association of in vitro
translated hGR and hGR with hsp90. Immunoprecipitations were
performed with the anti-hsp90 antibody 3G3 on heat-activated
reticulocyte lysates expressing equivalent amounts of
35S-labeled hGR or 35S-labeled hGR in the
absence of glucocorticoids. Left panel, autoradiograph of
receptor input (10% of total); right panel, autoradiograph
of co-immunoprecipitated receptor.
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The 3-4-fold difference in the capacity of hGR and hGR to bind
the MMTV fragment (Fig. 6A) might reflect differences in the
affinity of the two receptor isoforms for GREs and/or differences in
the number of hGR and hGR molecules free from hsp90 and available to bind DNA. Removal of hsp90 is necessary for DNA binding to occur,
and glucocorticoid binding triggers the dissociation of hGR-hsp90
complexes (2, 3). However, hGR does not bind glucocorticoids (11,
13). Therefore, we next compared the binding of hGR and hGR to
the GRE-containing MMTV fragment after heat activation in the absence
of glucocorticoids. Surprisingly, the capacity for hGR to bind the
MMTV fragment under these conditions was greater than that for hGR
(Fig. 6C, left panel). Four independent experiments revealed
a 2.5-3.5-fold difference in DNA binding. Lysates prepared in parallel
and activated by heat in the presence of glucocorticoids revealed no
significant change in the capacity of hGR to bind the MMTV fragment
but a marked increase in hGR binding, consistent with
hormone-mediated dissociation of the hGR-hsp90 complexes but not
hGR-hsp90 complexes (Fig. 6C, right panel).
These results suggest that in the absence of glucocorticoids the
hGR-hsp90 interaction may be weaker than the hGR-hsp90
interaction; consequently, more hGR molecules are free of hsp90 and
available to bind DNA. To test this hypothesis, receptor-hsp90
complexes were immunoprecipitated from heat-activated lysates in the
absence of glucocorticoids using the anti-hsp90 antibody 3G3. The
amount of recovered hGR was approximately 40% less than the amount
of recovered hGR (Fig. 6E), consistent with hGR-hsp90
complexes being less stable than hGR-hsp90 complexes.
hGR Heterodimerizes with hGR--
Homodimerization of hGR
and the presence of ligand on each receptor monomer is required for
glucocorticoid induction of gene expression (35, 36). Therefore, an
hGR-hGR heterodimer, in which ligand is bound to one partner
(hGR) but not the other (hGR), would be transcriptionally
impaired. Co-immunoprecipitation experiments were performed with
in vitro translated hGR and hGR to test whether the
two proteins could physically associate with each other. Equivalent
amounts of 35S-labeled hGR were incubated with lysates
containing unlabeled hGR or lysates containing no receptor (mock) in
the presence of hormone (Fig.
7A, left panel,
lanes 1 and 2). After cross-linking with DSP,
proteins were immunoprecipitated with the hGR-specific antibody
BShGR (16). 35S-Labeled hGR was recovered by BShGR from
the hGR/hGR mixture (Fig. 7A, middle panel,
lane 1). Moreover, the 35S-labeled hGR
monomers were shifted to a size consistent with 35S-labeled
hGR-hGR heterodimers when the cross-linker was not broken (Fig.
7A, right panel, lane 1). No shift was
observed in the small amount of 35S-labeled hGR
recovered nonspecifically from the hGR/mock mixture (Fig.
7A, compare middle and right panels,
lane 2). To further investigate the ability of hGR and
hGR to physically associate as a heterodimer, GST fusion proteins
were constructed with the HBD of hGR (amino acids 523-777) and the
HBD of hGR (amino acids 523-742). In addition to binding
glucocorticoids, this region of hGR is thought to contain a
dimerization interface (37). Equivalent amounts of in vitro
translated 35S-labeled hGR or 35S-labeled
hGR, treated with or without glucocorticoids, were incubated with
immobilized GST, GST-hGRHBD, and GST-hGRHBD. Full-length
hGR interacted specifically with both the HBD of hGR and the HBD
of hGR independent of ligand (Fig. 7B, left panel). Full-length hGR also interacted specifically with the both the HBD of hGR and the HBD of hGR independent of ligand (Fig. 7B, right panel). Thus, results from both
co-immunoprecipitation experiments and GST pull-down assays indicate
that hGR and hGR can physically associate with each other as a
heterodimer.
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Fig. 7.
Association of hGR with hGR. A,
reticulocyte lysates containing 35S-labeled hGR were
incubated with unlabeled lysates containing hGR (lane 1,
each panel) or unlabeled lysates not containing receptor (lane
2, each panel) in the presence of 200 nM DEX. Proteins
were cross-linked with DSP, and immunoprecipitations were performed
with the hGR-specific antibody BShGR. Left panel,
autoradiograph showing the amount of 35S-labeled hGR in
each mixture. Middle and right panels,
autoradiographs showing 35S-labeled hGR
immunoprecipitated by BShGR. Proteins immunoprecipitated by BShGR were
resuspended in sample buffer containing -mercaptoethanol
(BShGR + ME) or not containing -mercaptoethanol
(BShGR  ME) and resolved on an
SDS-polyacrylamide gel. -Mercaptoethanol cleaves the DSP cross
linker. Exposure times for the autoradiographs were 18 h
(BShGR + ME) and 72 h (BShGR ME). B, unmodified GST and the GST-hGRHBD
and GST-hGRHBD fusion proteins were immobilized on glutathione
agarose. The immobilized proteins were then incubated with reticulocyte
lysates containing 35S-labeled hGR (left
panel) or 35S-labeled hGR (right panel)
treated with or without DEX. Recovered proteins were resolved on
SDS-polyacrylamide gels. Input represents 25% of total protein.
Positions of hGR monomers, hGR monomers, and hGR-hGR
heterodimers are indicated on the left and
right.
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The Dominant Negative Activity of hGR Resides within the Unique
15 Amino Acids at the Carboxyl Terminus of hGR--
The hGR and
hGR proteins are identical through amino acid 727 but then diverge,
with hGR having an additional 50 amino acids and hGR having an
additional, nonhomologous 15 amino acids. What role, if any, these
unique 15 amino acids play in the dominant negative activity of hGR
has never been explored. We investigated this issue by truncating the
receptor after amino acid 727 (hGR728T). The hGR728T protein, like
hGR, did not bind DEX.2
COS-1 cells were transfected with a fixed amount of the MMTV luciferase
reporter pHHluc, a fixed amount of pCMVhGR, and increasing amounts
of pCMVhGR728T corresponding to a 5- or 10-fold molar excess over
the amount of transfected pCMVhGR. The cells were then treated with
or without glucocorticoids for 18 h and analyzed for
luciferase activity. In contrast to our findings for hGR (see
Fig. 1A), transfection of increasing amounts of pCMVhGR728T did not diminish the transcriptional activity of hGR.
Western blots were then performed to examine the relative levels of the
hGR728T and hGR proteins. hGR728T was expressed at approximately
70% the level of hGR in COS-1 cells separately transfected with an
equimolar amount of pCMVhGR or pCMVhGR728T (Fig.
8B, lanes 1 and
2). In COS-1 cells co-transfected with a fixed amount of
pCMVhGR and various amounts of pCMVhGR728T corresponding to a 1, 5, or 10-fold molar excess, hGR levels did not change, but hGR728T
levels increased the expected 5- and 10-fold (Fig. 8B,
lanes 3-5). Together, these blots indicate that hGR728T was expressed at levels 3.5- and 7-fold greater than hGR in the
experiments presented in Fig. 8A. We showed earlier that
hGR functioned as a dominant negative inhibitor when expressed at
levels 4- and 8-fold greater than hGR (see Fig. 1, A and
B). Therefore, the inability of hGR728T to repress
hGR-mediated activation of the MMTV promoter was not due to
insufficient expression of the truncated receptor.
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Fig. 8.
Removal of the
hGR-specific amino acids produces a truncated
receptor (hGR728T) that does not function as a dominant negative
inhibitor of hGR. A, COS-1
cells were transfected with 0.1 µg of pHHluc, 0.1 µg of pCMVhGR,
and various amounts of pCMV5 and/or pCMVhGR728T. Molar ratios of
transfected plasmids are indicated. Following an 18-h incubation with
vehicle (CON) or 100 nM DEX, cells were
harvested, and luciferase activity was determined. Data are plotted as
-fold change from basal activation and represent the mean ± S.E.
of three independent experiments. B, COS-1 cells transfected
with equimolar amounts of pCMVhGR (lane 1) or pCMVhGR728T
(lane 2) were analyzed on a Western blot with antibody 57. COS-1 cells transfected with 1.0 µg of pCMVhGR and various amounts
of pCMVhGR728T corresponding to a 1-, 5-, and 10-fold molar excess
(lanes 3-5, respectively) were analyzed on a Western blot
with the hGR-specific antibody AShGR and antibody 57.
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DISCUSSION |
In the present study, we have characterized the dominant negative
activity of hGR. We show that hGR inhibits the transcriptional activity of hGR in multiple cell types. This antagonism is selective for hGR, since other closely related steroid hormone receptors are
only weakly inhibited by hGR. In addition, we show that the dominant
negative activity of hGR occurs on some, but not all, genes
negatively regulated by glucocorticoids. We demonstrate that hGR can
associate with hsp90 and provide evidence that hGR-hsp90 complexes
are less stable than hGR-hsp90 complexes in the absence of hormone.
We also show that hGR can bind GRE-containing DNA and heterodimerize
w
Isoform
§,
TOP
ABSTRACT
INTRODUCTION
