Opposing Effects of Corepressor and Coactivators in Determining the Dose-Response Curve of Agonists, and Residual Agonist Activity of Antagonists, for Glucocorticoid Receptor-Regulated Gene Expression
Daniele Szapary,
Ying Huang and
S. Stoney Simons, Jr.
Steroid Hormones Section National Institute of Diabetes and
Digestive and Kidney Diseases Laboratory of Molecular and Cellular
Biology National Institutes of Health Bethesda, Maryland
20892-0805
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ABSTRACT
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A distinguishing, but unexplained, characteristic
of steroid hormone action is the dose-response curve for the regulation
of gene expression. We have previously reported that the dose-response
curve for glucocorticoid induction of a transfected reporter gene in
CV-1 and HeLa cells is repositioned in the presence of increasing
concentrations of glucocorticoid receptors (GRs). This behavior is now
shown to be independent of the reporter, promoter, or enhancer,
consistent with our proposal that a transacting factor(s) was being
titrated by added receptors. Candidate factors have been identified by
the observation that changes in glucocorticoid induction parameters in
CV-1 cells could be reproduced by varying the cellular levels of
coactivators [transcriptional intermediary factor 2 (TIF2), steroid
receptor coactivator 1 (SRC-1), and amplified in breast cancer 1
(AIB1)], comodulator [CREB-binding protein (CBP)], or corepressor
[silencing mediator for retinoid and thyroid-hormone receptors
(SMRT)] without concomitant increases in GR. Significantly, the
effects of TIF2 and SMRT were mutually antagonistic. Similarly,
additional SMRT could reverse the action of increased levels of GRs in
HeLa cells, thus indicating that the effects of cofactors on
transcription may be general for GR in a variety of cells. These data
further indicate that GRs are yet an additional target of the
corepressor SMRT. At the same time, these cofactors were found to be
capable of regulating the level of residual agonist activity displayed
by antiglucocorticoids. Finally, these observations suggest that a
novel role for cofactors is to participate in processes that determine
the dose-response curve, and partial agonist activity, of GR-steroid
complexes. This new activity of cofactors is disconnected from their
ability to increase or decrease GR transactivation. An equilibrium
model is proposed in which the ratio of coactivator-corepressor bound
to either receptor-agonist or -antagonist complexes regulates the final
transcriptional properties.
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INTRODUCTION
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The dose-response curve, showing the fold induction of a
biological response with increasing concentration of steroid,
summarizes a major physiological consequence of steroid hormones in a
responsive cell. Many of the events leading to steroid-regulated gene
induction have been elucidated and include steroid binding to the
cognate receptor, activation or transformation of the receptor-steroid
complex, complex binding to the hormone response element, recruitment
of coactivators vs. corepressors, and interaction with
auxiliary transcription factors followed by a series of events starting
with transcription initiation and culminating in the mature, induced
protein (1, 2, 3, 4). Steroid binding to receptors is an obligatory step in
such steroid-induced responses. The observance of a dose-response curve
that could be superimposed on the curve for steroid binding to a
receptor was early evidence for the requirement of that receptor
protein in steroid hormone action (5). Generally, the concentrations of
circulating steroid in humans and animals are below those required for
saturation of the receptor. Therefore, a physiologically relevant
action of steroids is to specify the percentage of maximal induction
that is elicited from a given responsive gene.
The close correlations that were often noted between steroid binding to
receptors and steroid induction of a biological response led to the
conclusion that the dose-response curve was dictated by the affinity of
steroid for the receptor (6, 7, 8). Thus, all genes responding to a
particular steroid were predicted to display the same dose-response
curves, at least in the same cell. It was not uncommon for a given
steroid to afford different dose-response curves in different cells or
organisms, possibly due to phenomena such as unequal amounts of
metabolism, serum binding proteins, and nonspecific binding to cells.
However, reports of dissimilar dose-response curves for the regulation
of two genes by the same receptor-steroid complex in the same cell
caused the model to be seriously questioned (9, 10, 11, 12). Additional
discrepancies included the findings that the dose-response curve of
some, but not all, glucocorticoid-responsive genes could be modified by
altering the density of the cells in culture (13), by the presence of a
21-bp cis-acting element of the rat tyrosine
aminotransferase (TAT) gene (14, 15, 16, 17), and finally by elevated levels of
glucocorticoid receptor (GR) (18). In the face of these unexpected
results, no model has yet been able to explain the molecular
determinants of dose-response curves for steroid induction, or
repression, of gene expression.
Another aspect of steroid hormone action that has resisted descriptions
at a molecular level pertains to the residual agonist activity
displayed by antisteroids. While it has long been thought that the
amount of residual agonist activity of each antisteroid is independent
of the gene examined, many exceptions have been noted (9, 10, 12, 14, 18, 19, 20, 21, 22, 23, 24). A prominent feature of the current model is that corepressors
are bound to the ligand-free, or antagonist-bound, receptors. Agonist
binding is believed to result in a conformational change of the
protein, thereby releasing the corepressor and/or recruiting
coactivators. However, this has been envisaged as an all-or-none
process with corepressor or coactivator binding mediating gene
repression or activation, respectively (2, 3, 25, 26, 27, 28, 29) and, therefore,
cannot readily account for changes in residual agonist activity. The
activity of antisteroids can also be altered by trans-acting
factors, such as dopamine (30), EGF (31), and protein kinase A inducers
(32, 33). Thus, general models are presently lacking for the molecular
determinants of both dose-response curves and the residual agonist
activity of antisteroids.
We have recently described a system in which both the dose-response
curve of glucocorticoids and the residual agonist activity of
antiglucocorticoids were modified by increased levels of GRs (18). The
parallel behavior of the two responses intimated that they might be
closely linked at the molecular level. These effects were seen when
receptors were both limiting and nonlimiting for gene induction,
thereby suggesting that another component of the transcriptional
machinery could be titrated by added receptors. The purpose of the
current study was to further define those components that are proposed
to affect the dose-response curve and the partial agonist activity of
antisteroids. We now report that the effect of transfected receptors
was independent of reporter, promoter, and enhancer. Surprisingly,
coactivator and corepressor molecules exerted opposing effects that
could modify the induction properties in a manner that was independent
of changes in the fold induction or GR protein. A model is advanced in
which the ratio of coactivator to corepressor is a defining ingredient
for both the dose-response curve for agonist steroids and for the
amount of residual agonist activity displayed by antisteroids.
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RESULTS
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Modified Induction Parameters with Increasing Receptor Are
Independent of Reporter, Promoter, and Enhancer
We previously reported that the dose-response curve for
GR-regulated transactivation of a transiently transfected reporter
construct is progressively shifted to lower concentrations of steroid
hormone when the GR concentrations are raised (18). Furthermore, we
showed that these changes in the dose-response curve under different
conditions can be more easily detected by comparing the induction by a
subsaturating, physiological level of agonist (
1 nM Dex)
after expressing this value as the percent of maximal induction with 1
µM Dex to normalize for interexperimental variations in
absolute response. Any increase in the percent agonist activity for
approximately 1 nM Dex is then due to a left shift in the
dose-response curve (14, 15, 18). Similarly, an increase in the
residual agonist activity of an antiglucocorticoid can be directly
determined. In this manner, increased GR in CV-1 and HeLa cells caused
both a left shift in the dose-response curve with Dex and increased
amounts of agonist activity with antiglucocorticoids from a
chloramphenicol acetyltransferase reporter (18). This same behavior was
obtained with a luciferase reporter in both CV-1 (Fig. 1A
) and HeLa cells (data not shown).
Thus, the effect of transfected GR is independent of the reporter gene.
The absence of a statistically significant increase in the agonist
activity of RU 486 in CV-1 cells (P = 0.27, n =
5), compared with HeLa cells (18), presumably reflects the lack of a
tissue-specific component and is reminiscent of the report that RU 486
is a better agonist for the B form progesterone receptors (PR) than is
R5020 in HeLa cells but not in CV-1 cells (24). Also, RU 486 displayed
agonist activity with stably transfected PR (and transiently
transfected PRE2tkLUC) in HeLa but not CV-1 cells (34).

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Figure 1. Effect of Reporter, Promoter, and Enhancer on
Activity of Agonists and Antagonists with Increased Levels of GR
Triplicate dishes of CV-1 cells were transfected with the indicated
amounts of pSVLGR and 2 µg of GREtkLuc (panel A) or 1 µg of MMTVLuc
reporter (panel B) (18 ). The fold induction by 1 µM Dex
at low and high concentrations of GR was typically 7- and 20-fold for
GREtkLuc and 200- and 450-fold for MMTVLuc. The activity of each
steroid was expressed as percent of maximal induction as described in
Materials and Methods. The data are averages ±
SEM for n = 45 experiments.
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The role of the promoter was examined in HeLa cells using the
chloramphenicol acetyltransferase (CAT) reporter under the control of
either the minimum thymidine kinase promoter (tkmin) or the TAT gene
promoter (tat). While the absolute activities were different with the
two promoters (Table 1
), both promoters
were equally effective in mediating the effect of increased GR. The
mouse mammary tumor virus (MMTV) promoter/enhancer, which contains a
complex glucocorticoid response element (GRE) (35), gave results in
CV-1 cells (Fig. 1B
) that were very similar to those with the different
promoters in CV-1 and HeLa cells (Table 1
and Ref. 18). Interestingly,
the MMTV promoter/enhancer permitted a significant increase in the
agonist activity of RU 486 (P = 0.049, n = 4) that
was not apparent with the simple GRE enhancer (Fig. 1A
;
P = 0.27, n = 5). This may be due to a heightened
capacity of the MMTV promoter/enhancer to mediate increased levels of
agonist activity of antisteroids, as witnessed by the very high levels
of agonist activity for the antiglucocorticoid dexamethasone
21-mesylate (Dex-Mes) (Fig. 1B
vs. A and Table 1
).
Nevertheless, the ability of the increased levels of GR to modify the
induction parameters was relatively independent of enhancer, promoter,
and reporter.
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Table 1. Effect of Promoter on Ability of Transfected
GR to Increase Steroid Activity as Percent of Maximal Induction by 1
µM Dex in HeLa Cells
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We had previously found that the effects of transfected GR were
specific in HeLa cells in that no change was seen with the transfected
A form of human PR (PR-A) (18). Similarly, the addition in CV-1 cells
of 0.36 µg of PR-A cDNA plasmid with 0.04 µg of GR cDNA had no
effect compared with the further addition of 0.36 µg of GR cDNA
(percent agonist activity of 0.5 nM Dex: 7 ± 11% for
0.04 µg GR; 2.5 ± 9.1% for 0.04 µg GR plus 0.36 µg PR-A;
68 ± 2% for 0.4 µg GR [± range; n = 2]). PR-A was
being expressed as seen by the squelching of GR induction when 10
nM R5020 was added along with Dex (data not shown).
Therefore, the above effects of transfected GR are not an artifact of
transfection but are selective for GR.
Coactivators Mimic Transfected GR
Given the ubiquitous role of coactivators in steroid hormone
action, we inquired whether they might participate in determining the
GR dose-response curve. Transcriptional intermediary factor 2
(TIF2)/glucocortiocoid interacting protein 1 (GRIP-1) caused a moderate
increase in the total transactivation by saturating concentrations of
Dex (100 nM) in the presence of low levels of transiently
transfected GR in CV-1 cells (Fig. 2A
).
This is consistent with TIF2 being a coactivator. At the same time, the
activity of 1 µM concentrations of Dex-Mes increased
about 4-fold. To determine whether or not the dose-response curve of
Dex, or the percent residual agonist activity of Dex-Mes, had been
altered by the added TIF2, the data were replotted as percent of
maximal induction by 100 nM Dex. As shown in Fig. 2B
, the
added TIF2 produced both a clear left shift in the Dex dose-response
curve and a marked increase in the percent agonist activity of the
antagonist Dex-Mes. Further experiments were then conducted with the
simpler, three-point dose-response curve (14, 15, 18), in which an
increase in the percent of maximal activity by a subsaturating
concentration of Dex, such as with 1 or 3 nM in Fig. 2B
, was diagnostic of a left shift in the dose-response curve. Using this
abbreviated assay, we confirmed the ability of added TIF2 to shift the
Dex dose-response curve to the left (Fig. 3A
). This shift was accompanied by a 2-
to 3-fold increase in the fold induction by saturating concentrations
of Dex (1 µM), as in Fig. 2A
, which contrasts with the
reports that TIF2 has little effect on the fold induction by GR in HeLa
cells (36, 37). At the same time, the residual agonist activity of the
antagonists Dex-Mes and progesterone was significantly augmented. The
lack of appreciable change in RU 486 activity parallels the inability
of transfected GR to alter RU 486 activity (Figs. 1
and 3A
), suggesting
that increased levels of either GR or TIF2 are acting via the same
mechanism. These responses require the C-terminal sequences of TIF2 as
a truncated protein (TIF2.0 = amino acids 1627) lacking
the receptor interacting and transactivation domains (4) was inactive
(data not shown).

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Figure 2. Dose-Response Curves for Dex Induction by
Transfected GR ± Cotransfected TIF2
Triplicate dishes of CV-1 cells were transfected with 1 µg of
GREtkLuc reporter and low (0.04 µg) amounts of pSVLGR plus 0.2 µg
of TIF2, followed by the indicated concentrations of Dex (18 ). A, Total
luciferase activity (relative). The total activity of induced
luciferase activity was plotted for each series
(circles). At the same time, the total activity seen
with 1 µM Dex-Mes (DM) was included at the edge of the
graph (bars). B, Normalized dose-response curves. To
directly determine whether or not the Dex dose-response curves for
GR ± TIF2 were the same, the activity at each point in the
curves, or bars, of panel A was expressed as percent of maximal
induction by 100 nM Dex in similarly treated cells. In both
graphs, the responses with GR alone are indicated by the
open symbols while the data for GR + TIF2 are designated
by the solid symbols.
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Figure 3. Ability of Added Coactivator/Comodulator to
Reproduce the Enhanced Activity of Agonists and Antagonists with
Increased Levels of GR
Triplicate dishes of CV-1 cells were transfected with 12 µg of
GREtkLuc reporter and low (0.020.04 µg) or high (0.20.4 µg)
amounts of pSVLGR plus 0.2 µg of TIF2 (panel A), 0.9 µg of SRC-1
(panel B), 0.9 µg of AIB1 (panel C), or 0.8 µg of CBP plasmid
(panel D) as in Fig. 2 . The activity of each steroid was expressed as
percent of maximal induction by 1 µM Dex as described in
Materials and Methods. The data are averages ±
SEM for n = 34 experiments, except for the high GR
data with AIB1 where the average ± range of two experiments is
given.
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TIF2/GRIP-1 is related to a group of approximately 160-kDa coactivators
(p160) that includes steroid receptor coactivator 1 (SRC-1) and
amplified in breast cancer 1 (AIB1)/activator of thyroid and retinoic
receptors (ACTR)/p300/CBP interacting protein
(pCIP)/receptor-associated coactivator 3 (RAC3) (3). Almost all of
these factors have been shown to interact with GR (37, 38). We
therefore examined the effects of these related factors. SRC-1, which
is reported to produce only marginal increases in the total activation
by GRs with saturating concentrations of Dex in HeLa cells (39), caused
a 1.2- to 3-fold increase in GR transactivation in CV-1 cells (data not
shown). This small enhancement of the fold induction could reflect the
presence of appreciable quantities of endogenous SRC-1 in CV-1 cells
(40). AIB1 (41) caused between no change and a 2-fold increase (data
not shown). In both cases, however, added cofactor mimicked the ability
of additional GR to cause a left shift in the Dex dose-response curve
(i.e. increased activity of 1 nM Dex) and to
augment the residual agonist activity of antiglucocorticoids (Fig. 3
, B
and C). Again, the increased activity of the antagonists Dex-Mes and
progesterone, but not RU 486, with added SRC-1 (Fig. 2B
) argues that
added coactivator, or GR, are each mediating the same responses.
The ability of increased concentrations of GR, SRC-1, TIF2, and AIB1 to
cause the same net result suggested that a common factor is being
titrated. One candidate was the adapter protein CREB-binding protein
(CBP) (42), which is known to interact with all of the above proteins
(2, 3, 4, 43, 44, 45, 46). Therefore, if each coactivator acted by sequestering
the available CBP, added CBP should reverse the responses and cause a
right shift in the Dex dose-response curve and a decrease in the
partial agonist activity of antisteroids. However, increased CBP
resulted in the same behavior as added GR instead of counteracting the
GR (Fig. 3D
and data not shown). Thus, the effect of increased GR or
coactivator cannot be due to titration of endogenous CBP.
Interestingly, the increased activity with added CBP occurred under
conditions where the fold induction by saturating concentrations of Dex
was reduced by 25 ± 8% (±SEM, n = 4).
TIF2 Effects Occur without Raising GR Levels
Given the ability of each of the coactivators, and CBP, to mimic
the effects of high levels of transfected GR in causing increased
activities for both subsaturating concentrations of Dex (due to a left
shift in the dose-response curve, as in Fig. 2
) and saturating
concentrations of antagonists (Fig. 3
, AD), we asked whether the
coactivators were acting simply by increasing the amount of GR obtained
after transfection with low levels of GR cDNA to those seen with high
concentrations of transfected GR cDNA. Detection of transfected GR in
CV-1 cells was complicated by the presence of endogenous, biologically
inactive GR that specifically bound [3H]Dex but was
unable to transactivate the GREtkLUC reporter (data not shown). In
fact, the specific Dex binding in CV-1 cells transiently transfected
with 0.2 µg of pSVLGR was indistinguishable from that in
untransfected cells. However, the ratio of transfected GR to
endogenous, biologically inactive GR could be dramatically increased by
using magnetic isolation procedures to select for those cells that had
taken up the exogenous plasmid DNAs. Western blots of the cytosols from
those magnetically isolated cells, which had been transfected with 0.4
µg of pSVLGR, revealed the overexpressed GR as the usual 98-kDa
species (Fig. 4
, species labeled as
a) plus two lower molecular mass species (labeled as
b and c) arising from alternative translational start sites (47) (lane
9 vs. lanes 2 and 4). Cells transfected with 0.04 µg of GR
did not contain noticeably higher levels of GR (lanes 7 vs.
6). Most significant, though, was that the transfection of 0.04 µg of
GR plus 0.2 µg of TIF2, conditions affording the same increased
activities of 1 nM Dex and 1 µM Dex-Mes as
obtained with 0.4 µg of GR (see Fig. 3A
), did not result in any more
GR than that without TIF2 (lanes 8 vs. 7) and much less than
that seen with 0.4 µg of GR (lanes 8 vs. 9). Therefore,
the presence of added TIF2 with low concentrations of GR protein
affords the same biological responses as seen with higher amounts of GR
in the absence of TIF2. Thus, the ability of coactivators to modulate
GR transactivation properties occurs via a mechanism that is different
from simply increasing the GR concentrations.

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Figure 4. Effect of TIF2 on GR Concentration in Transiently
Transfected CV-1 Cells
CV-1 cells were transfected with 0.1 µg of IL2R cDNA plus the
following DNAs (amounts are listed per 60-mm dish but were scaled up
for the larger volumes of cells used): carrier and vector DNAs only
(lane 6), 40 ng pSVLGR (lane 7), 40 ng pSVLGR plus 200 ng TIF2 (lane
8), or 400 ng pSVLGR (lane 9). The transfected cells were enriched by
adding mouse monoclonal antihuman IL2R antibody, followed by M-450
Dynabeads coated with goat antimouse IgG. The magnetically tagged cells
were immobilized by magnetic plates on the walls of the flask and
washed to remove untransfected cells. The cytosols from the washed
pellets were prepared and the receptor was visualized by ECL as
described in Materials and Methods. Lanes 4, 5, and 10
contain the indicated units of GR that was overexpressed from pSVLGR in
Cos-7 cells. The heterogeneity of receptors overexpressed in Cos
(labeled as a, b, and c, with a being the full length GR) and CV-1
cells is due to downstream translational start sites (47 ). Lanes 1 and
2 are a shorter exposure of the lanes 3 and 4 to show that the smaller
translation product of pSVLGR is the most intense, consistent with the
pattern seen in lane 9 for 400 ng of pSVLGR in CV-1 cells.
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Corepressor Antagonizes the Effect of Coactivators
Corepressors have inverse effects from coactivators with the
nuclear receptors (48, 49) and are present, along with coactivators, in
CV-1 and HeLa cells (40). Therefore, we examined the response in our
system of added corepressor, even though they have not been described
to interact with GR or to alter their transactivation (49). Transfected
silencing mediator for retinoid and thyroid hormone receptors
(SMRT) repressed the activity of subsaturating concentrations of
Dex (i.e. caused a right shift of the dose-response curve)
and decreased the residual agonist activity of the antiglucocorticoid
Dex-Mes (Fig. 5
). Thus, the corepressor
SMRT acted in the opposite direction of GR, coactivators, or CBP. At
the same time, the added SMRT resulted in a 52 ± 12%
(±SEM, n = 3) reduction in the fold induction by
saturating concentrations of Dex. Importantly, when SMRT and TIF2 were
both added, an intermediate response was obtained (Fig. 5
). The
activity of SMRT plus TIF2 in the presence of low concentrations of GR
was significantly less than that with TIF2 only (P
0.007) and more than that with SMRT only (P
0.05;
both P values are for the paired Students t test,
n = 5). Therefore, added coactivator and corepressor counteract, or
antagonize, the effects of each other in establishing the position of
the GR dose-response curve with agonists and the partial agonist
activity of antiglucocorticoids.

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Figure 5. Activity of Agonists and Antagonists Responds to
the Opposing Effects of the Corepressor SMRT and the Coactivator TIF2
Triplicate dishes of CV-1 cells were transfected with 0.04 µg (low
GR) and 0.2 µg (high GR) of pSVLGR and 2 µg of GREtkLuc reporter
without or with 0.2 µg of SMRT, 0.2 µg of TIF2, or both as in Fig. 2 . The activity of each steroid was expressed as percent of maximal
induction as described in Materials and Methods. The
data are averages ± SEM for n = 5 experiments.
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Effects of Cofactors in HeLa Cells
Cotransfection of TIF2 into HeLa cells produced little change in
the fold induction by 1 µM Dex (1.3- to 1.6-fold) and no
significant difference in the Dex dose-response curve or residual
agonist activity of Dex-Mes (data not shown). Assuming that this could
result from high levels of endogenous TIF2, which might be
counterbalanced by added SMRT, we treated HeLa cells with SMRT both
without and with additional GR. Little if any effect was seen when SMRT
was added to the endogenous GR. However, the changes produced by
increased GR could be prevented by the added SMRT (Fig. 6
). Thus, SMRT could repress the
increases in activity caused by both added TIF2 in CV-1 cells and extra
GR in HeLa cells. It should be noted that the fold induction by 1
µM Dex in HeLa cells was not decreased, but increased by
up to 2-fold, in the presence of added SMRT.

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Figure 6. Reversal of Effects of Added GR by SMRT in HeLa
Cells
Triplicate dishes of HeLa cells were transfected with 0.8 µg of SMRT
with or without and 0.4 µg of pSVLGR and 1 µg of GREtkLuc reporter
as in Fig. 2 . The activity of each steroid was expressed as percent of
maximal induction (± SD of the triplicate values) as
described in Materials and Methods. Similar results were
obtained in two other experiments.
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DISCUSSION
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The GR dose-response curve can be modified in a manner that is
independent of the gene, the promoter, the enhancer (Fig. 1
and Table 1
), and the cell (18). We further find that coactivators (TIF2/GRIP-1,
SRC-1, AIB1/ACTR/pCIP/RAC3, and CBP) and corepressors (SMRT) display a
previously unsuspected property in being able to oppose each other, and
added receptor, in the regulation of the dose-response curve for GR.
Thus, when the endogenous ratio of coactivator-corepressor in CV-1
cells was perturbed by the overexpression of one factor, the
dose-response curve was repositioned. The effects of
coactivators/corepressor were specific as neither a TIF2 mutant lacking
all GR-interacting domains nor PR was able to produce the same
responses. At the same time, this balance of coactivators and
corepressors was seen to modify the residual agonist activity of
antiglucocorticoids. These are new activities of coactivators and
corepressors, which have been thought to affect only the fold induction
of receptors bound with saturating concentrations of ligand (36, 39, 41, 43, 45, 48, 49). Thus, coactivators and corepressors are equally
important for the activity of both physiological and pharmacological
levels of glucocorticoid steroids.
Given the effect of variations in GR levels on both the dose-response
curve and the partial agonist activity of antisteroids, the response to
increased coactivators and corepressor could theoretically be indirect
by modifying the level of GR. However, Western blots of cytosols of
transfected CV-1 cells that had been enriched by magnetic isolation of
those cells expressing the transfected DNA revealed that biologically
active concentrations of TIF2 (Figs. 2
and 3
) caused no discernible
increase in GR protein levels (Fig. 4
). Thus, the modification of GR
activity by coactivators occurs via mechanisms other than simple
elevation of the level of GR protein. This inability of coactivators to
increase steroid receptor levels has also been noted for AIB1 with
estrogen receptors (ERs) in tumor cell lines or breast tumor samples
(41), for CBP and SRC-1 with GR in COS-7 cells (44), and for a fragment
of GRIP-1/TIF2 with truncated mouse GR in yeast (27). Even stronger
support for this conclusion derives from an analysis of the
biologically active GRs ± cofactors in our experiments. We had
previously demonstrated that the total induced reporter activity is
directly proportional to GR concentration in CV-1 cells (18) (Y. Huang
and S. Simons, submitted). Because the level of reporter activity with
low levels of GR did not increase in the presence of additional AIB1 or
CBP (±10%, data not shown), we conclude that the cotransfection of
coactivators did not uniformly increase the levels of either GR protein
or functional GR. Also, there was no consistent relationship between
the fold induction by 1 µM Dex ± cofactor in CV-1
cells. The fold induction went down with CBP and up with AIB1, SRC-1,
and TIF2. Added CBP and SMRT each decreased the fold induction with
saturating concentrations of agonist steroid but had opposite effects
on the position of the dose-response curve and the residual agonist
activity of Dex-Mes (Figs. 3
and 5
). Finally, SMRT exhibited some
cell-specific effects in that it decreased the fold induction by 1
µM Dex in CV-1 cells but caused an increase in HeLa
cells. Nevertheless, SMRT caused a right shift in the Dex dose-response
curve and decreased agonist activity for Dex-Mes in both cell lines
(Figs. 5
and 6
). Thus, the consequences of added SMRT in our assays
were unchanged even in HeLa cells where SMRT acted as a coactivator for
GR. These results also indicate that the influences of cofactors on the
GR induction parameters of the present study can be dissociated from
the more extensively studied effects on total transactivation by
saturating concentrations of ligand. Therefore, as no consistent
relationship could be found between the overexpression of coactivators
(or corepressors) and the absolute activity, or fold induction, of the
reporter construct, we conclude that the modulation of GR activity in
Figs. 2
, 3
, 5
, and 6
cannot result simply from changes in the
concentration of either GR protein or functional GR that might be
caused by overexpressed cofactors. It should also be noted that others
have observed dramatic increases in the fold induction by progesterone
receptors without any change in the dose-response curve (50).
The alteration of glucocorticoid dose-response curves appears to be a
novel property of corepressors and the p160 coactivators in mammalian
cells. GRIP-1 causes a left shift in the dose-response curve of
full-length GR (and mineralocorticoid and ERs) in yeast (27), but no
reports have appeared in mammalian cells, where the effect of
TIF2/GRIP-1 has varied from squelching (38) to inactivity (36) to a
2-fold increase in fold induction (37). In our hands, overexpression
of TIF2/GRIP-1 in mammalian cells had multiple effects, i.e.
of shifting the dose-response curve to the left and enhancing the
residual agonist activity of antagonists in addition to magnifying the
fold induction by saturating concentrations of agonist by a factor of
23. L7/SPA, which does not appear to be related to the p160
coactivators, enhances the residual agonist activity of RU 486 bound to
GR but has no effect on agonist-bound receptors (51). It was initially
thought that corepressors did not bind to the steroid receptors, like
GR and ER (49). More recently, though, corepressors have been found to
decrease the residual agonist activity of antisteroids bound to PR and
ER (29, 51, 52, 53). In most cases, the corepressor was also seen to
interact with the receptor (29, 51, 52, 54). We therefore suspect that
the effects of SMRT on GR action (Figs. 3
, 5
, and 6
), which have not
been previously reported, also derive from an interaction of SMRT with
GR.
TIF2 counteracted the effects of SMRT and vice versa (Fig. 5
). This
contrasts with the report with ERs that added coactivator (SRC-1) could
not reverse the effects of the corepressor SMRT (52). Thus, the
interactions of coactivators and corepressors with GR may be more
closely balanced than with other receptors, such as ERs.
Previous observations that only full-length, functionally active, GR
were able to alter the Dex dose-response curve, and the residual
agonist activity of antiglucocorticoids (18) suggested that both amino
and carboxyl termini of GR are required for productive interactions
with at least the coactivators, as has been observed by others
(54, 55, 56, 57, 58, 59). The fact that the induction properties of reporters regulated
by either TAT or MMTV enhancers responded to increased GR (Fig. 1
)
lends further support to this conclusion since receptors lacking the
amino-terminal AF-1 domain were unable to induce MMTV-regulated
reporters (60).
Histone acetylation and release of the repressive effects of DNA-bound
nucleosomes has emerged as an attractive control mechanism for
steroid-regulated gene expression (61, 62, 63). Several, but not all (4),
cofactors have been found to possess histone acetylase or deacetylase
activity (2, 26, 28, 64, 65). However, histone acetylation has been
reported to cause both increased and decreased induction by receptors
(66). Also, histone acetylation does not appear to be required in all
cases of steroid-regulated transcription, especially in transiently
transfected cells (67), and nucleosome reorganization may precede
receptor-induced transactivation (68, 69). Nucleosome formation and
phasing are highly ordered over the MMTV GREs (70) and have been found
to be greatly reduced, or absent, in transiently transfected reporters
(71, 72), which is the usual substrate in studies of coactivators and
corepressors. Because we see both a high-fold induction of MMTVLuc
expression (200450 fold) and the typical dose-response curve left
shift, and increased residual agonist activity of antisteroids, with
elevated GR levels (Fig. 1B
), we suspect that these effects of added GR
and cofactors do not require histone acetylation or deacetylation.
The ability of coactivators and corepressors to alter glucocorticoid
dose-response curves, and residual agonist activity of antagonists,
cannot be explained by the current model in which one or the other
group of cofactors is associated exclusively with a single GR species.
Such behavior would be expected to lead to an all-or-nothing response
as opposed to the gradual increases that are seen with added GR (18) or
cofactors (data not shown). Furthermore, if antagonist-bound GR
associated only with corepressors, it is not obvious how added
coactivator could exert any effect. We propose that the effects
described in this study reflect changes in the equilibrium mixture of
oligomeric species containing corepressors, or coactivators, bound to
GR complexed with either agonist or antagonists (Fig. 7
). This is similar to the proposal of an
equilibrium population of cofactor complexes with ligand-free nuclear
receptors (73). However, we now extend this concept to ligand-bound GR.
From our previous work with mutant GRs (18, 74), we conclude that
effective interactions of coactivators and corepressors occur
predominantly, if not exclusively, with steroid-bound GR. Thus, for
agonist-bound GR, the ratio of coactivator-bound GR to
corepressor-bound GR would determine the position of the dose-response
curve without any change in the dissociation constant (Kd)
of steroid binding to GR. Addition of more coactivator or CBP would
force the equilibrium to the side of coactivator-bound GR by the
principle of mass action, thereby causing a further left shift in the
dose-response curve. Similarly, the amount of residual agonist activity
displayed when an antiglucocorticoid bound to GR would be controlled by
the ratio of the two sets of receptor/cofactor complexes (Fig. 7
).
Support for the generality of this model derives from the recent
finding that SRC-3 (= AIB1) associated with ERs bound by either
agonists or antagonists (75) and that the antiestrogen ICI 164,384
stimulated the binding of the coactivator RIP140 to ER ligand-binding
domain (76). Furthermore, coactivators (TIF2, AIB1, and SRC-1) and
comodulators (CBP and p300) increased the percent agonist activity of
tamoxifen bound to mutant ERs (G440V) (77). A comparable situation
appears to exist with the androgen receptor: the binding of
antiandrogens has recently been reported to promote the interaction of
receptors with the coactivator ARA70 (78). Finally, this determination
of biological activity by a balance of interactions with coactivators
vs. corepressors may not be unique to mammalian systems. The
activity of the Drosophila transcription factor Dorsal
appears to be modulated by the association of coactivators (CBP) and
corepressors (Groucho) (79).

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|
Figure 7. Model for Control of Dose-Response Curve and
Residual Agonist Activity by Ratio of GR-Steroid Complexes That Are
Associated with Coactivators vs. Corepressors
Cartoon depicts some of the species proposed to be associated with
DNA-bound GR-steroid complexes (shaded and cross-hatched symbolsrepresent unspecified, probable additional components).
However, not all components may be simultaneously associated with the
same molecule of GR. The two shapes representing GR depict presumed
conformational differences resulting from the association of either
coactivators or corepressors. Either agonist or antagonist steroids can
be bound to each GR conformer. Extreme ratios of
coactivators/corepressors would afford almost exclusively the higher
order complexes on the left or right while intermediate ratios would
give a mixture of higher order complexes. See text for further details.
|
|
A mathematical analysis of our model was performed to understand how
variations in GR concentration could affect the final ratio of
coactivator- vs. corepressor bound to GR-steroid complexes.
We assumed that GR bound to either coactivators (CoA) or corepressors
(CoR) as dictated by the following simple equations: GR + CoA
GR-CoA, with [GR-CoA] = K1[GR][CoA], and GR + CoR
GR-CoR, with [GR-CoR] = K2[GR][CoR]. Solving
these two equations revealed that, for any cellular concentration of
coactivators and corepressors, increasing amounts of receptor would
yield higher ratios of coactivator-/corepressor-bound GR
(i.e. GR-CoA/GR-CoR) only when the affinity of coactivators
for steroid-bound GR (K1) was less than that for
corepressors (K2). We therefore predict that GR complexes,
regardless of the nature of the bound steroid, will have an
intrinsically higher affinity for corepressors than for coactivators
and/or CBP. However, the magnitude of preferential association of GR
with corepressors vs. coactivators might further be modified
by steroid-induced changes in receptor tertiary structure (80, 81, 82, 83) such
that more coactivators associate with agonist-bound GR. This balance
could be additionally modified by other factors binding to
cis-acting elements (14, 15, 74).
Our model of an equilibrium interaction of cofactors with GR suggests
that the ratio of cofactors plays a pivotal role in both the
dose-response curve of agonists and the residual agonist activity of
antagonists. Thus, in addition to the absolute level of cofactors (54),
the ratio of cofactors is important. Our model is based predominantly
on data from the overexpression of GR and cofactors in CV-1 cells.
However, the similar results with added GR (18) and SMRT (Fig. 6
) in
HeLa cells suggest that this effect is not cell-specific. Furthermore,
the recent reports that transfected GRIP-1/TIF2 caused a left shift in
the dose-response curve of ER-mediated induction of an
estrogen-responsive reporter gene in yeast (84), that all three classes
of coactivators (and two comodulators) increased the agonist activity
of the antiestrogen tamoxifen in HeLa cells (77), and that the
coactivator ARA70 associates with antagonist-bound androgen receptors
in human prostate cancer cells (78) argue that our model may apply to
other steroid receptors in numerous cellular environments. These
considerations, coupled with the recent findings of variations in
levels of cofactors in different (40, 75, 85) or the same (53) cells
provide an attractive mechanism for explaining cellular variations
in the dose-response curve and residual agonist activity of
specific receptor-steroid complexes. These results further suggest
novel controls of receptor activity during differentiation,
development, and homeostasis.
The model of Fig. 7
deviates from that which has evolved for the
nuclear receptors, where agonist binding causes a dissociation of
corepressor and is required for the association of coactivators (2, 3, 25, 26, 27, 28, 29). However, the interactions of cofactors with GR may be much
more loosely regulated than those seen for the nuclear receptors. This
is consistent with variations in other properties of the nuclear
receptors, such as a predominantly cytoplasmic localization of the
steroid-free GR, the requirement of bound hsp90 for steroid binding of
GR, the absence of either DNA binding or transcriptional repression by
steroid-free GR, the loss of steroid binding activity upon deletion of
amino acids beyond the proposed helix 12, different tryptic digestion
sites in receptor ligand-binding domains (86), and the lack of
heterodimerization of GR with retinoid X receptor (RXR). Conversely, an
equilibrium nature of cofactor association with receptors may be
more prevalent than previously recognized. For example, the presence of
a sequence in retinoic acid receptors that interacts with coactivators
prevents gene repression by the ligand-free receptor in CV-1 cells
(87). Given the recent report of SRC-1 interaction with nuclear
factor-
B (NF-
B) (88), it will be interesting to determine whether
SRC-1 can also modify the dose-response curve of various NF-
B
inducers in addition to other steroid receptors. Finally, our model
suggests that an added consequence of titration/squelching, in which
one transcription factor competitively binds a cofactor needed by a
second transcription factor (11, 18, 44, 89) and thus alters the ratio
of interacting factors, can be to modify the dose-response curve and
residual agonist activity of a given receptor-steroid complex.
 |
MATERIALS AND METHODS
|
---|
Unless otherwise indicated, all operations were performed at 0
C.
Chemicals, Buffers, and Plasmids
Most of the chemicals, buffers, and plasmids, including rat GR
(pSVLGR from Keith Yamamoto, UCSF, San Francisco, CA) are the same as
described previously (18). GREtkminCAT and GREtatCAT were described
elsewhere (14). MMTVLuc (pLTRLuc) was obtained from Gordon Hager
(National Institutes of Health, Bethesda, MD). The cDNA plasmids of
SRC-1a (B. W. OMalley, Baylor College of Medicine, Houston, TX),
TIF2 and and the truncated TIF2 (TIF2.0) plus the A-form of human
progesterone receptor (Hinrich Gronemeyer, IGBMC, Strasbourg, France),
AIB1 (Paul Meltzer, National Institutes of Health, Bethesda, MD), CBP
(Richard Goodman, Vollum Institute, Portland, OR), and SMRT (Ron Evans,
Salk Institute, La Jolla, CA) were each graciously sent as gifts.
Cell Culture and Transfection
The conditions of both growth and transient transfection (in
triplicate) of CV-1 and HeLa cells, with the total transfected DNA
brought up to 3 µg/60-mm dish with pBSK+ DNA have been
described (18). CAT activity was determined as described previously
(18). The luciferase activity was assayed using the Luciferase Assay
System from Promega Corp. (Madison, WI) and the methods
recommended by the supplier, including one cycle of freeze-thaw lysis
of the cells in dry ice followed by centrifugation (15,000 rpm) for 15
min. In experiments with cofactor cDNA plasmid, all cells not
transfected with cofactor were cotransfected with an equimolar amount
of the same plasmid vector to control for artifacts of the vector
DNA.
Western Blotting of Cytosols from Magnetically Isolated,
Transfected Cells
CV-1 cells (three semiconfluent T150 flasks per treatment) were
transfected by the usual calcium phosphate method (18), with the
amounts of DNA (pSVLGR, pSG5, and TIF2 in the pSG5 vector, and
pBSK+ as carrier DNA) being scaled up from that used for
60-mm dishes. Those cells that had taken up the added DNA were
separated for analysis by the magnetic isolation procedure of Smith
et al. (90). The initial transfection included 2.1 µg/T150
flask of pCMVIL2R (from Cathy Smith, National Cancer Institute/National
Institutes of Health) (90) for the expression of interleukin 2
receptor (IL2R). The transfected cells were harvested by adding
mouse monoclonal antihuman IL2R antibody (50 µg per 1 ml of
Dynabeads; Upstate Biotechnology, Inc., Lake Placid, NY)
followed by M-450 Dynabeads coated with goat antimouse IgG (Perspective
Biosystems, Framingham, MA). Magnetic plates (BioMag Separators;
Perspective Biosystems) were used to attract the magnetically tagged
cells to the walls of the flask. The cytosols from the washed pellets
were prepared as usual (83) and separated on an 8% SDS-PAGE gel. The
receptors were visualized by Western blotting with BUGR-2 monoclonal
antirat GR antibody, followed by enhanced chemiluminescence (ECL) as
described (83).
Analysis of Data
The activity for subsaturating concentrations of agonist, or
saturating concentrations of antagonist, was expressed as percent of
maximal activity with saturating concentrations of agonist [1
µM dexamethasone (Dex) unless otherwise noted]. The fold
induction with 1 µM Dex was calculated as the luciferase
activity [relative light units/(mg protein)(sec)] with 1
µM Dex divided by the basal activity obtained with
ethanol. Individual values were generally within ± 20% of the
average, which was plotted. Unless otherwise noted, all statistical
analyses were by two-tailed Students t test using the
program InStat 2.03 for Macintosh (GraphPad Software, Inc., San Diego, CA).
 |
ACKNOWLEDGMENTS
|
---|
We thank Ron Evans, Richard Goodman, Hinrich Gronemeyer, Gordon
Hager, Paul Meltzer, Bert OMalley, Cathy Smith, and Keith Yamamoto
for their generous donation of reagents; Cathy Smith for help with the
magnetic isolation of transiently transfected cells; Allen Minton (NIH)
for the mathematical analysis of our model; and Keiko Ozato (NIH) and
Paul Meltzer (NIH) for their critical review of the paper.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. S. Stoney Simons, Jr., Building 8, Room B2A-07, National Institute of Diabetes and Digestive and Kidney Diseases/LMCB, National Institutes of Health, Bethesda, Maryland 20892.
Received for publication May 21, 1999.
Revision received July 27, 1999.
Accepted for publication August 17, 1999.
 |
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