From the Steroid Hormones Section, NIDDK/LMCB,
National Institutes of Health, Bethesda, Maryland 20892 and
§ Université de Fribourg Suisse, CH-1700
Fribourg, Switzerland
Received for publication, March 22, 2001, and in revised form, April 9, 2001
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ABSTRACT |
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A major unanswered question of glucocorticoid and
progesterone action is how different whole cell responses arise when
both of the cognate receptors can bind to, and activate, the same
hormone response elements. We have documented previously that the
EC50 of agonist complexes, and the partial agonist
activity of antagonist complexes, of both glucocorticoid receptors
(GRs) and progesterone receptors (PRs) are modulated by increased
amounts of homologous receptor and of coregulators. We now ask whether
these components can differentially alter GR and PR transcriptional
properties. To remove possible cell-specific differences, we have
examined both receptors in the common environment of a line of mouse
mammary adenocarcinoma (1470.2) cells. In order to segregate the
responses that might be due to unequal nucleosome reorganization by the two receptors from those reflecting interactions with other components, we chose a transiently transfected reporter containing a simple glucocorticoid response element (i.e. GREtkLUC). No
significant differences are found with elevated levels of either
receptor. However, major, qualitative differences are seen with the
corepressors SMRT and NCoR, which afford opposite responses with GR and
PR. Studies with chimeric GR/PR receptors indicate that no one segment of PR or GR is responsible for these properties and that the composite response likely involves interactions with both the amino and carboxyl
termini of receptors. Collectively, the data suggest that GR and PR
induction of responsive genes in a given cell can be differentially
controlled, in part, by unequal interactions of multiple receptor
domains with assorted nuclear cofactors.
Among the longstanding conundrums of steroid hormone action is why
different whole cell responses are observed for androgen, glucocorticoid, mineralocorticoid, and progestin steroid hormones (1)
even though each receptor-steroid complex can bind to the same DNA
sequences to induce gene transcription (2, 3). Steroid
hormone-regulated gene transactivation requires ligand binding to the
cognate intracellular receptor. After binding to biologically active
DNA sequences, called hormone response elements (HREs),1 the activated
complexes are thought to recruit transcriptional coregulators and
components of the transcriptional complex prior to modifying the
transcription rates of target genes. Different ligands bind to the
various steroid receptors with different affinities. However, this
specificity for the different steroid hormones would seem to soon
disappear as the activated form of each complex of the above four
steroid receptors can bind to, and activate transcription from, the
same HREs.
The actions of glucocorticoid receptors (GRs) and progesterone
receptors (PRs) have been extensively studied in an effort to
understand how biological diversity can be maintained when the
activated receptor complexes act on a common HRE. Several explanations
have been proposed, including different levels of the receptor within a
given cell (4). HRE mutations have also been reported to affect
differentially GR versus PR transactivation (5), although
the differences seem to be less pronounced when receptor concentrations
are about equal (6). Nelson et al. (7) found that the
flanking and spacer DNA of the palindromic HRE can contribute to the
affinity and specificity of receptor binding. Similarly, DNA binding
specificity has been implicated in the androgen receptor (AR)
versus GR-specific induction of the probasin gene (8) and in
GR versus AR activation of the aspartate aminotransferase
gene (9). Thus, subtle differences in HRE sequence may regulate the
relative activities of GR and PR.
Chromatin architecture has recently emerged as another promising
modifier for receptor-regulated expression of some, but not all (10),
genes. Chromatin structure can repress gene expression (11, 12),
thereby increasing the fold induction by receptor-steroid complexes,
especially in cell-free systems (13, 14). Chromatin environment can
control gene inducibility by PR (15-17), and chromatin structure has
been proposed to be a determinant for PR induction (10, 15). However,
alterations in chromatin structure do not appear to be a prerequisite
for all steroid receptor-induced gene transactivation. In several
cases, chromatin reorganization appears to precede the actions of
receptor-steroid complexes in inducing gene expression (14, 18-20),
whereas in other cases chromatin disruption or remodeling is not
sufficient for transactivation, which requires a second step (21). In
T47D cells lacking both PR and GR, or expressing only GR, the
responsive B nucleosome of the MMTV enhancer is in a constitutively
"open" state, indicating that GR transcriptional activation is
independent of chromatin remodeling (20).
The low homology of the amino-terminal halves of GR and PR (<15%) has
been advanced as an additional possible cause for selective gene
expression (22). One mechanism by which this could be achieved is
through differential interactions with the recently discovered transcriptional coregulators such as coactivators and corepressors. Although the initially determined interactions of these coregulators were with the ligand binding domain (LBD) of receptors (23-25), several recent reports (26-31) describe interactions with the
amino-terminal domain of receptors. Unfortunately, receptor-specific
interactions with coregulators appear limited. ARA70 (compare
Ref. 32 with Refs. 33 and 34) and FHL2 (35) have been described to be specific coactivators for ARs, whereas a 68-kDa protein (p68) appears to be a coactivator for estrogen receptor (ER) More evidence exists for differential effects of coactivators and
corepressors on GR versus PR activities. The nuclear orphan receptor estrogen receptor-related receptor 2 is described to be
a repressor of GR transcriptional activity but does not affect PR
activity (39). Conversely, PIAS1 enhances GR transactivation but
represses PR transactivation in the same system (40). A 130-kDa
auxiliary protein increases the DNA binding of full-length GR but not
truncated PRs (41). Similar quantitative differences have been
noted for other steroid receptors. SRC-1 has a greater effect on ER
than AR action in the brain of developing rats (42), which may result
from SRC-1 interacting with the ER LBD via the LXXLL motifs
of SRC-1 but with the amino-terminal region of AR in a manner that does
not depend on the SRC-1 LXXLL motifs (34). SRC-1e interacts
with the fragments containing the DNA binding domain and LBD of ER but
not of AR (28). RIP140 represses GR (43) but increases AR (44)
transactivation. Whether Zac1 augments or represses the activity of
GRIP1 with AR versus ER depends on the target gene and cell
(45).
Recently we have found that varying concentrations of the homologous
receptor, coactivators, and corepressors can alter the EC50
of agonist complexes, and the partial agonist activity of antagonist
complexes, for both GRs (46-48) and PRs (49). Preliminary evidence
suggested that the responses of GR and PR to these factors might be
different (47-49). Therefore, the purpose of this study was to examine
whether the quantitative activities of those factors known to alter PR
and GR transactivation properties are the same or different for PR and
GR. To answer this question, we have performed multiple dose-response
curves to determine the EC50 of agonists and the partial
agonist activity of antagonists. It was important to conduct these
assays in the same cells so that cell-specific contributions to
transcriptional activities could be eliminated. Similarly, we needed to
use the same reporter so that effects of chromatin organization would
be minimized. In the context of such an assay system, we found two
instances in which the induction properties of PR and GR were
qualitatively different, with almost opposite effects being produced by
the same added component. Further studies with PR/GR chimeras indicated
that no one segment of PR or GR was responsible for these differences.
Collectively, the data suggest that the differences between GR and PR
induction in a selected cell can be controlled, in part, by unequal
responses from the combination of amino- and carboxyl-terminal domains
of each receptor to assorted nuclear components.
Unless otherwise indicated, all operations were performed at
0 °C.
Chemicals, Buffers, and
Plasmids--
[3H]Dexamethasone (Dex, 91 Ci/mmol) was
obtained from PerkinElmer Life Sciences and non-radioactive Dex from
Sigma. Dex-Ox (50) and Dex-Mes (51) were prepared as described.
Restriction enzymes and digestions were performed according to the
manufacturer's specifications (New England Biolabs, Beverly, MA).
The Renilla null luciferase reporter was purchased from
Promega (Madison, WI). GREtkLUC (52) has been described previously. The
cDNA plasmids of GR (pSVLGR from Keith Yamamoto, University of
California, San Francisco), MMTVLUC (pLTRLUC from Gordon Hager, National Institutes of Health, Bethesda), TIF2, and the B form of human
progesterone receptor (hPR-B) (Hinrich Gronemeyer, IGBMC, Strasbourg,
France), NCoR (Michael Rosenfeld, University of California, San Diego),
and s-SMRT (53) (Ron Evans, Salk Institute, La Jolla, CA) were each
received as gifts.
Construction of Chimeras--
The cDNA encoding GR or PR was
recombined through a compatible site (NspI/SphI)
that coincides with the position of cysteine 495 of the rat GR. No
amino acids were added or subtracted or changed at this junction. Both
expression vectors for the chimeric receptors start with the unrelated
sequence ASGSWP, which is due to the thymidine kinase promoter
AUG followed by a BamHI linker. The PR/GR chimera bears a
PR, which is lacking the first 24 amino acids of PR. This deletion has
no observable influence on the transactivation capacity of the
PR.2 The GR/PR chimera starts
with a rat GR that misses 3 amino acids of the amino terminus. Various
experiments since this construct was first employed (54) have confirmed
that there is no substantial difference between this GR and the wild
type GR.3
Cell Culture and Transfection--
Monolayer cultures of COS-7
and 1470.2 cells were grown at 37 °C with 5% CO2 in
Dulbecco's modified Eagle's medium (Life Technologies, Inc., and
Dulbecco's modified Eagle's medium with 4.5 g glucose/liter, Quality Biologicals, Inc., respectively) supplemented with 5 and 10%
of fetal bovine serum, respectively. We had previously used charcoal-stripped serum with 1470.2 cells to prevent any PR-mediated induction by endogenous progestins (49). However, we have confirmed the
observations of others (55) that this is not necessary (data not
shown). Therefore, charcoal-stripped serum was no longer used with
1470.2 cells. CV-1 cells were grown as described (46). Coregulator
plasmids were transiently cotransfected into 1470.2 cells using
LipofectAMINE (LIfe Technologies, Inc.) with hPR-B receptor-containing
plasmid, 1 µg of GREtkLUC, and 50 ng Renilla null
luciferase, with the total transfected DNA brought up to 3 µg/60-mm
dish with pBSK+ DNA (56). In experiments with varying
amounts of receptor or coregulator cDNA plasmids, equimolar amounts
of the same plasmid vector were cotransfected to control for artifacts
of the vector DNA. The cells were treated for 24 h with 1%
ethanol ± steroids in media containing 10% fetal bovine serum
and harvested in 1× Passive Lysis Buffer (0.5 ml/dish, Promega). Fifty
µl of the cell lysates were used to assay for luciferase activity
using the Dual-luciferase Assay System from Promega (Madison, WI)
according to the supplier. The data were then normalized for the
cotransfected Renilla activity.
Steroid Binding Assays--
Transient transfection of COS-7
cells with 1 µg/10-cm plate of GR or PR/GR plasmid DNA and 19 µg of
single-stranded DNA was performed as described (57). Cytosols of
transfected cells containing the steroid-free receptors were obtained
by the lysis of cells on dry ice and centrifugation at 15,000 × g (58). Thirty percent cytosol with 20 mM sodium
molybdate was added to varying concentrations of [3H]Dex
±100-fold excess of non-radioactive Dex and incubated at 0 °C for
18 h. Unbound [3H]Dex was removed by dextran-coated charcoal.
Whole cell steroid binding was performed by incubating suspensions of
cells (1.5-2 × 106) with increasing concentrations
of [3H]Dex (1.5 to 50 nM) in 200 µl of
serum-free medium in the presence or absence of a 100-fold molar excess
of unlabeled Dex (each with 1.2% ethanol) for 30-45 min at 37 °C.
The binding was terminated by the addition of 2 ml of
phosphate-buffered saline, followed by centrifugation for 15 s,
all at room temperature. Cells were washed three more times with
phosphate-buffered saline at room temperature. In both cases, the total
binding was determined by liquid scintillation counting. The specific
binding was calculated by subtracting the background
disintegrations/min (100-fold Dex) from the total [3H]Dex
binding. The binding capacity and affinity were determined by Scatchard
plot analysis by plotting the ratio of bound steroid/free steroid
versus bound steroid.
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 (30 nM R5020 or 1 µM Dex unless otherwise noted). The fold induction was
calculated as the luciferase activity (relative firefly light units/relative Renilla light units) with 30 nM
R5020, or 1 µM Dex, divided by the basal activity
obtained with ethanol. Individual values were generally within ±20%
of the average, which was plotted.
The dose-response curves were constructed from the theoretical
sigmoidal curve for the binding isotherm, which is described by the
equation of y = x/(x + k), where
y is the fractional response; x is the
concentration of free steroid, and k is an arbitrary value
for the binding affinity of steroid to receptor. This theoretical curve
was then aligned with the experimental data so as to give the best
visual fit.
Unless otherwise noted, all statistical analyses were by two-tailed
Student's t test using the program "InStat 2.03" for
Macintosh (GraphPad Software, San Diego, CA).
Selection of Assay System--
A bioassay with transiently
transfected receptors and reporters to analyze possible differences in
the biological properties of PR and GR was chosen for several reasons.
First, bioassays measure the cumulative effect of the proceeding steps
in the induction of protein synthesis. Bioassays are also often more
sensitive than other assays, like DNA binding (8). Furthermore, not all in vitro interactions are sufficiently strong to elicit an
effect in whole cell bioassays (59). A transiently transfected
template, in which nucleosome reorganization does not occur (60), was used in order to minimize the possible complications of differential chromosomal reorganization by receptors.
The ideal cells for this study would lack both GR and PR but would
display dynamic, induction responses over a range of transiently transfected receptors. CV-1 cells lack both GR and PR and respond well
to transfected GR (46). However, in our hands, CV-1 cells give a low
fold induction with transfected PR over a very narrow range of
transfected receptors (49).4
The 1470.2 mouse mammary adenocarcinoma cells do contain some GR but
possess excellent properties regarding gene induction by transfected
human PR-B (49). As both transiently and stably transfected PR induce
transactivation with transiently transfected MMTVLUC reporters (49, 55,
61), the use of transiently transfected PR should not pose a problem.
PR is limiting for transactivation and capable of displaying increasing
levels of gene induction over a range of transiently transfected
receptors. PR also responds to a variety of coactivators and
corepressors in these cells (49).
Induction Properties of PR and GR in 1470.2 Cells--
Increasing
concentrations of transiently transfected PR produce a progressive left
shift in the dose-response curve to lower EC50 values for
R5020 induction of transiently transfected MMTVLUC reporters in
1470.2 cells (49). Steroid hormone induction of MMTV is complicated,
due to the binding of NF1 and Oct1 to the MMTV promoter (62). In order
to avoid these additional complications, we elected to use the simpler
GREtkLUC reporter, which does not contain cis-acting binding
sequences for other transcription factors. We first determined that
higher concentrations of hPR-B plasmid afford increased total reporter
activity, indicating that PR is limiting in this range (Fig.
1A). Under these conditions,
the dose-response curve (or EC50) for R5020 induction of
the transiently transfected GREtkLUC reporter is shifted to lower
steroid concentrations and the partial agonist activity of the
antiprogestin dexamethasone mesylate (Dex-Mes) (49) is increased (Fig.
1B). Thus, we see the same responses to changing PR
concentrations with the GREtkLUC reporter as for the MMTVLUC reporter
(49). We conclude that the ability of added PR to reposition the
dose-response curve is independent of the GRE and promoter
sequences.
It should be noted that the endogenous GR of 1470.2 cells does not
interfere with the quantitation of PR induction. Not only does Dex
display negligible activity with PR but also Dex-Mes and R5020 have
little or no activity with the endogenous GR (49).
We reported previously that increasing amounts of transiently
transfected GR produce a left shift for the induction of a GREtkLUC reporter in HeLa and CV-1 cells (46). However, no further left shift
(or increased partial agonist activity of antiglucocorticoids) is seen
with high concentrations of GR (>1 µg of plasmid), indicating that
there is a limit to the effects of added GR (48). 1470.2 cells contain
some GR (49). Under conditions where added GR increases the total
transactivation of cotransfected GREtkLUC in 1470.2 cells, the higher
levels of GR also afford about a 2-fold left shift in the dose-response
curve with Dex (Fig. 1C). At the same time, the very low
amount of partial agonist activity of Dex-Mes and dexamethasone
oxetanone (Dex-Ox) with GR in 1470.2 cells (49) increases (Fig.
1C and data not shown). Thus, both the dose-response curve
for glucocorticoids and the partial agonist activity of
antiglucocorticoids are modulated by added GR in 1470.2 cells just as
has been observed in HeLa and CV-1 cells (46-48). These data indicate
that the transactivation properties of GR and PR are similarly altered
by elevated levels of receptor.
Modulation of PR and GR Activities by NCoR--
We recently
observed that added corepressor NCoR (63) had no effect on GR
transactivation of GREtkLUC in CV-1
cells5 but a significant
effect on PR transactivation properties in 1407.2 cells with the
MMTVLUC reporter (49). We therefore asked whether this behavior of the
two receptors was maintained with a common reporter in the same cells.
With the GREtkLUC reporter, NCoR decreases the maximal transactivation
of GREtkLUC by PR by 41 ± 8% (S.E., n = 4) while
slightly increasing the fold induction (18 ± 13%, S.E.,
n = 4). As indicated by the representative experiment of Fig. 2A, NCoR
simultaneously produces a 3.9 ± 0.8 (S.E., n = 4, p = 0.033)-fold left shift in the dose-response curve
and a 70 ± 17% (S.E., n = 3) increase in the
partial agonist activity of Dex-Mes. This is comparable to the effects
of NCoR on the transactivation properties of PR with MMTVLUC (49) and
shows that NCoR modulation of PR properties in 1470.2 cells is
independent of the enhancer and promoter sequences.
With the endogenous GR of 1470.2 cells, added NCoR also reduced the
total transactivation by 27 ± 11% (S.E., n = 4)
and increased the fold induction (54 ± 18%, S.E.,
n = 4), just as was observed above for PR. However, the
effect on the other GR transactivation properties was almost exactly
the reverse as seen with PR (Fig. 2B). NCoR afforded an
average of a 2.1 ± 0.2-fold (S.E., n = 4, p = 0.011) right shift in the dose-response curve. The
slight decrease in the low partial agonist activity of Dex-Ox was not statistically significant (to 84 ± 31%, n = 3, p = 0.47).
Modulation of PR and GR Activities by SMRT--
The transfection
of SMRT cDNA causes a right shift in the dose-response curve and
decreases the partial agonist activity of antisteroids, both for GR
induction of GREtkLUC in CV-1 cells (47) and for PR induction of
MMTVLUC in 1407.2 cells (49). When PR induction properties were
determined with a different reporter, GREtkLUC, SMRT still affords a
right shift (3.2 ± 0.6 fold, n = 2) in the
EC50 and a 54 ± 1% (n = 2) decrease
in the partial agonist activity of Dex-Mes (Fig.
3A). Interestingly, when GR
induction was examined, the addition of SMRT no longer yields a right
shift. Instead, a weak left shift (1.4 ± 0.2 fold, S.E.,
n = 5) is obtained (Fig. 3B) while causing a
33 ± 10% (S.E., n = 5, p = 0.032) decrease in the total transactivation. The magnitude of the left
shift of the GR dose-response curve with SMRT is not statistically
significant (p = 0.11). However, the observation that
SMRT causes a right shift with PR and little or no left shift with GR
is significant (p = 0.025). At the same time, the low partial agonist activity of Dex-Ox (8.0 ± 0.7%, S.E.,
n = 5) is increased by SMRT to 9.9 ± 0.6% (S.E.,
n = 5, p = 0.048 in paired Student
t test). This result further supports a left shift in the GR
dose-response curve by SMRT because a shift in the dose-response curve
to the left has always been accompanied by increased partial agonist
activity of an antisteroid (46-48, 64-66). The ability of added SMRT
to produce a decrease in Dex-Mes partial agonist activity with PR and
an increase in the partial agonist activity of Dex-Ox with GR is also
significant (p = 0.0014 by Alternate Welch t
test). These combined data indicate that the effects of SMRT on the
dose-response curve and partial agonist activity of antagonists for PR
complexes are the inverse of that seen with GR complexes.
Responses of Chimeric GR/PR Receptors--
In an effort to
understand the origins of the divergent responses of GR and PR
transactivation properties to NCoR and SMRT, we asked whether the
individual biological effects require specific domains of each
receptor. For this, we selected chimeras in which the amino-terminal
and DBD domains of one receptor were fused to the LBD of the other to
give the hybrid receptors that we call PR/GR and GR/PR (Fig.
4A). In both cases, the
junction is seamless so that no mutations have been introduced in the
body of the receptors. The construction of the chimeras did result in
changes at the amino termini (see Fig. 6A). However, these
changes have not been observed to affect any properties of the
receptors (data not shown). The experiments with PR/GR are not
compromised by the endogenous GR of 1470.2 cells as the total activity
and fold induction with PR/GR are 3.3 ± 0.6 and 2.8 ± 0.6 (S.E., n = 7) times greater, respectively, than the
values with the endogenous GR (data not shown).
With both chimeras, 3-30 ng of transfected receptor yielded increasing
total amounts of induced luciferase activity (data not shown). This
confirms that each receptor is limiting under our transfection
conditions, as is the case for all of the above experiments. The
dose-response curve for GR/PR is left-shifted by a factor of 2.36 ± 0.65 (range, n = 2) in going from 3 to 30 ng of
plasmid (Fig. 4B). There is no increase in the partial
agonist activity of Dex-Mes. Instead, there is a slight decrease of
21 ± 3% (range, n = 2).
A much larger response to changing concentrations is observed with the
PR/GR chimera. In a preliminary experiment in 1470.2 cells, the
dose-response curve of 30 ng of PR/GR plasmid is shifted to 6.8-fold
lower Dex concentrations than that with 3 ng of plasmid, whereas the
partial agonist activity of Dex-Mes and Dex-Ox each increased (data not
shown). At the same time, we noticed that the EC50 for
GREtkLUC induction by 30 ng of PR/GR plasmid was unexpectedly
5-10-fold lower than that for the endogenous GR (data not shown). To
be sure that these properties are not influenced by the endogenous GR
of 1470.2 cells, the PR/GR chimera was further examined in CV-1 cells,
which contain much lower levels of functional GR (46-48). Here, the
same two concentrations of PR/GR cause a 7.13 ± 0.07 (S.E.,
n = 3, p = 0.0001)-fold left shift in
the dose-response curve and a 31 ± 7% (S.E., n = 3, p < 0.050) increase in the partial agonist activity
of Dex-Mes (Fig. 4C). Interestingly, under conditions where
the total transactivation of GREtkLUC by 30 ng of PR/GR and 100 ng of
GR is the same (consistent with equivalent amounts of transcriptionally
active GR and PR/GR complexes), the dose-response curves and partial
agonist activities with the two receptors are dramatically different
(see also below).
Added SMRT plasmid causes a slight left shift with GR and a right shift
with PR (Fig. 3). With the chimeric receptors, SMRT produces a weak
right shift with PR/GR and a stronger right shift with GR/PR (Fig.
5, A and B). The
receptors can thus be ordered by decreasing ability of SMRT to afford a
left shift as follows, with the value for the fold left shift in
parentheses: GR (1.41 ± 0.2) > PR/GR (0.79 ± 0.11) > GR/PR (0.46 ± 0.09) > PR (0.33 ± 0.06)
(± S.E. with n = 5 for all samples except PR,
where ± range, n = 2). These differences are
significant with p = 0.027 for GR versus
PR/GR, p = 0.049 for PR/GR versus GR/PR, and
p = 0.0027 for GR versus GR/PR. This
ordering is maintained when the effect of SMRT on the partial agonist
activity of the various antisteroids is expressed in terms of increased
agonist activity, with 100% being the control value: GR (127 ± 10%) > PR/GR (75 ± 3%) > GR/PR (58 ± 8%) > PR (46 ± 1%) (p = 0.009 for GR
versus PR/GR and 0.08 for PR/GR versus GR/PR,
both using Welch's t test) (see Figs. 3 and 5, A
and B). We therefore conclude that not only can the action
of SMRT differ among receptors but also the eventual response results
from a combination of the amino- and carboxyl-terminal regions of PR
and GR as opposed to the actions of any one domain.
Similar conclusions were obtained from experiments on NCoR action with
the chimeric receptors. NCoR shifted the dose-response curve of PR/GR
to the left by a factor of 1.52 ± 0.14 (S.E., n = 3) (Fig. 5C) but had no significant effect on GR/PR (Fig.
5D). The rank order of altered dose-response curve by NCoR,
with the fold left shift in parentheses, was thus PR (3.9 ± 0.8, n = 4) > PR/GR (1.52 ± 0.14, n = 3) > GR/PR (1.19 ± 0.19, n = 4) > GR (0.48 ± 0.04, n = 4) (all values are ± S.E.) (see Figs. 2 and 5, C and
D). These values all differ from each other at the level of
p = 0.05 except for PR/GR versus GR/PR
(p = 0.24). The order for increased partial agonist
activity with added NCoR of PR Comparison of the PR/GR Chimera with Wild Type GR--
We noted
above (Fig. 4C) that the dose-response curve for PR/GR is
considerably left-shifted from that for GR. This was unexpected because
steroid binding to the receptor is thought to be the determining factor
for the dose-response curve and both GR and PR/GR contain the same LBD
(67). This question was therefore examined in greater detail. As shown
for 1470.2 cells in Fig. 6A,
the presence of 30 ng of transfected PR/GR affords a dose-response
curve for Dex that is left-shifted more than 10-fold from that for the
endogenous GR and more than 3-fold left-shifted from that for cells
containing an additional 200 ng of transfected GR. This difference in
dose-response curves could be due to unequal levels of expressed,
biologically active receptors because the total transactivation by 30 ng of PR/GR is about twice that by 200 ng of GR (data not shown). To examine this possibility, the experiments were repeated in CV-1 cells,
in which the ability of increasing amounts of transfected GR to cause a
progressive left shift in the dose-response curve, and higher amounts
of partial agonist activity with antisteroids, reach nearly plateau
values with 1000 ng of transfected GR plasmid (48). We therefore
compared the effects of near saturating amounts of GR to those obtained
with 3 and 30 ng of PR/GR plasmid. As seen in Fig. 6B, the
dose-response curve for 3 ng of PR/GR is the same as that with 300 times more GR plasmid, which yields close to the maximal left shift
seen with added GR (48). In contrast, 30 ng of PR/GR is able to further
shift the Dex dose-response curve another 7-fold to the left.
Similarly, marginal increases in the partial agonist activity of
Dex-Mes are seen with 100 versus 1000 ng of GR plasmid, and
substantial additional increases occur with 3 and 30 ng of PR/GR. This
suggests that the differences in transactivation properties of GR
versus PR/GR are not limited by the concentration of
expressed receptors but rather reflect intrinsic transactivation
properties of the wild type GR and the PR/GR chimeric receptors.
One trivial explanation for the different properties of PR/GR
versus GR is that the presence of the PR amino-terminal
domain and/or DBD somehow increases the affinity of Dex for the GR LBD. This explanation can be eliminated, however, because the cell-free affinity of Dex is the same for both receptors in a cell-free Scatchard
assay (Fig. 6C). Similarly, whole cell binding assays at
37 °C reveal that there is no appreciable difference in Dex binding
to PR/GR and GR under the conditions of the whole cell bioassay (Fig.
6D). Therefore, the lower EC50 of PR/GR,
compared with GR, is not the result of any difference in affinity
between the receptors for ligand at either 0 or 37 °C.
This study presents evidence that changes in the concentration of
the corepressors NCoR and SMRT can modify selected transcriptional properties of GR differently than those of PR. The most commonly examined transcriptional properties of steroid receptors are the total
transactivation and the fold induction. None of the species in this
study influence these properties of GR appreciably more or less than
those for PR. However, notable differences can be seen between GR and
PR with regard to changes in the dose-response curve, or
EC50, and the partial agonist activity of antisteroids. These two properties of receptor-steroid complexes have important physiological and pathological consequences. Physiological levels of
steroids are rarely sufficient for maximal induction and commonly correspond to the concentrations required for half-maximal induction or
the EC50. Therefore, changes in EC50 of a gene
will result in significant differences in the expressed activity under
physiological conditions. We do not know how similar the magnitude of
responses in intact animals will be to our results in transfected
cells. However, the effects could even be greater. In transgenic mice with ~50% higher level of GR protein, due to a 100% increase in the
GR gene dosage, the dose-response curve for glucocorticoid-induced DNA
binding-dependent apoptosis of primary thymocytes (68) is left-shifted to lower steroid concentrations by a factor of greater than 10 (69). Conversely, in cells from transgenic flies carrying the
human estrogen receptor and an estrogen receptor-responsive reporter
and no known coactivators, the EC50 for reporter induction by estradiol was right-shifted by a factor of about 100 (70). The other
transactivation parameter that we investigated, i.e. changes
in the partial agonist activity of antisteroids, is highly relevant for
endocrine therapies. An antisteroid that displays high levels of
partial agonist activity for the very gene that one wishes to suppress
would clearly be contraindicated. Alternatively, increased levels of
partial agonist activity, which is characteristic of selective receptor
modulators, is often desirable. To the extent that side effects of
antisteroid therapies result from the suppression of all responsive
genes, an antisteroid with partial agonist activity for as many genes
as possible, other than the one to be repressed, would be extremely useful.
In this study, we find that the corepressors NCoR and SMRT affect both
the EC50 of agonists and the partial agonist activity of
antisteroids complexed with GR and PR for induction of GREtkLUC-based reporters in 1470.2 cells. More significantly, these factors
differentially affect these responses for the two receptors. The
corepressor SMRT was previously found to shift the dose-response curve
to higher EC50 values and decrease the partial agonist
activity of antisteroids, both for GR induction of GREtkLUC in CV-1
cells (47) and for PR induction of MMTVLUC in 1470.2 cells (49). As
anticipated, the responses of PR to SMRT in 1470.2 cells are independent of the sequence of the glucocorticoid response element and
are the same for a GREtkLUC (Fig. 3A) and MMTV (49)
reporter. Unexpectedly, the response of GR to added SMRT for induction
of GREtkLUC in 1470.2 cells is the opposite of PR. The GR dose-response curve is repositioned to lower EC50 values, and the partial
agonist activity of antisteroids increases (Fig. 3B). This
argues not only that the responses of a given receptor can vary among
cell types (i.e. GR in CV-1 versus 1470.2 cells)
but also that the same cofactor can produce diametrically opposite
effects for the transactivation properties of two different steroid
receptors (GR and PR) acting on the same gene in the same cell.
Similarly, the effects of NCoR with GR and PR are cell- and
receptor-selective. The response of PR to added NCoR is the same for
GREtkLUC (Fig. 2A) and MMTVLUC (49) and thus is independent
of the reporter. In contrast, NCoR modulates GR the transactivation
properties for GREtkLUC induction in 1470.2 cells (Fig. 2B)
but not in CV-1 cells.5 Furthermore, NCoR shifts the
dose-response curves for GR and PR induction of GREtkLUC in 1470.2 cells in opposite directions and has opposite effects on the partial
agonist activity of antisteroids (Fig. 2). Earlier studies have also
documented specificity in receptor binding of coactivators and
corepressors (71, 72).
The net result is that two factors have been identified that cause
unequal modifications of GR versus PR induction properties of the same gene in the same cell. Furthermore, these changes are
biologically relevant. The normal variations of glucocorticoid and
progestin hormones are in the range of the EC50 for
induction of most genes, thus ensuring significant alterations in gene
expression. Within this context, the amount of corepressor in each cell
will inversely affect the response of GR and PR to physiological
concentrations of steroid, making the position of the agonist
dose-response curves and the partial agonist activity of antagonists
less similar. Thus, the presence of corepressors within a cell appears
to constitute a novel mechanism for imparting different transcription
properties to receptors that bind to the same HRE, such as GR and PR.
Many of the activities of steroid receptors can be localized to unique
domains of the proteins. By using chimeras containing the
amino-terminal plus DNA binding domain of one receptor and the LBD of
the other, it is apparent that no one domain of either receptor is
dominant for the differential responses of PR and GR to NCoR and SMRT.
Rather, the chimeras have properties that are intermediate between the
two wild type receptors. This result supports the notion that the
transcription properties of GR and PR are modified by the interactions,
either indirect or direct, of corepressors with the amino- and
carboxyl-terminal portions of both wild type and chimeric receptors.
Such inter-domain interactions and signaling appear to be common among
the steroid/nuclear receptors and have been observed for GR (73), PR
(74), androgen receptors (44, 75), estrogen receptors (76), and PPAR
receptors (77). Similarly, we propose that the combination of amino-
and carboxyl-terminal sequences forms a surface that differentially
interacts with cell-specific factors, thereby giving rise to the
unequal transcriptional responses regarding the EC50 and
partial agonist activity (Fig. 7). These cell-specific factors would be expected to include the corepressors, given the above consequences of added NCoR and SMRT. We do not yet have
any evidence for the corepressors directly contacting GR. Initially, it
was thought that NCoR and SMRT bound only to the nuclear receptors.
More recently, however, NCoR and SMRT have been shown to bind to
antagonist complexes of PR and ER (26, 78-80), and the amino-terminal
domain of receptors may be involved (31) in addition to the LBD
(81-83). Significantly, others (78, 79), in addition to ourselves (47,
49), have reported effects of corepressors on the total transactivation
of agonist-bound ER, PR, and GR complexes. These data support an
interaction of corepressors with agonist- and antagonist-bound
receptors. Further support for the importance of amino- and
carboxyl-terminal sequence interactions in determining the
transactivation properties of GR and PR comes from the PR/GR chimera.
Although the steroid binding affinity of PR/GR at 0 and 37 °C is, as
expected, the same as that of wild type GR (Fig. 6), the
EC50 values for Dex induction of GREtkLUC, and the partial
agonist activities of antisteroids, are quite different (Fig.
4C). The simplest explanation is that the combination of
domains alters steps in transactivation that follow steroid binding to
the receptor in a way that increases the sensitivity to circulating
steroids, as outlined in Fig. 7.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
but not for
ER
, AR, mineralocorticoid receptors, or retinoic acid receptor
(36). Repressor of estrogen receptor activity selectively interacts with ERs to decrease both the activity of agonists and the
concentration of antiestrogens required for half-maximal inhibition of
estrogens (37). NRIF3 is a ligand-dependent specific
coactivator that binds (and augments transactivation of) thyroid
and retinoid receptors but not retinoic acid, vitamin D, or
steroid receptors (38).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of steroid receptor concentration on
GREtkLUC induction properties. Triplicate cultures of 1470.2 cells
were transfected with GREtkLUC and Renilla null luciferase
reporters plus the indicated amounts of hPR-B (A and
B) or rat GR (C) cDNA plasmids, induced with
varying concentrations of agonist (R5020 (A and
B) or Dex (C)) or antagonist (Dex-Mes
(B) or Dex-Ox (C)), and assayed for luciferase
and Renilla activities as described under "Materials and
Methods." The average values, normalized for cotransfected
Renilla, were then plotted as either total luciferase
activity (A) or the percent of maximal induction above EtOH
controls (B and C). The error bars
represent the S.D. within a given experiment. Similar results were
obtained in one (A and B) and two (C)
additional experiments.
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Fig. 2.
Modulation by exogenous NCoR of steroid
receptor induction properties for GREtkLUC. Triplicate cultures of
1470.2 cells were transfected with GREtkLUC and Renilla null
luciferase reporters plus 30 ng of hPR-B cDNA plasmid
(A). The endogenous GR, with no transfected receptor
plasmids, was used in B to examine glucocorticoid-induced
responses. Both sets were cotransfected with either 1.2 µg of NCoR
plasmid or the molar equivalent of empty vector, induced with varying
concentrations of agonist (R5020 (A) or Dex (B)),
or antagonist (Dex-Mes in A and Dex-Ox in B), and
assayed for luciferase and Renilla activities as described
under "Materials and Methods." The average values, normalized for
cotransfected Renilla, were then plotted as percent of
maximal induction above EtOH controls. The error bars
represent the S.D. within a given experiment. Similar results were
obtained in three additional experiments with each receptor.
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Fig. 3.
Ability of SMRT to modify receptor-regulated
induction properties of GREtkLUC. The behavior of transfected
hPR-B (30 ng of plasmid DNA; A) or endogenous GR
(B) was assessed in triplicate cultures of 1470.2 cells that
were cotransfected with GREtkLUC and Renilla null luciferase
reporters plus 0.4 µg of SMRT plasmid or the molar equivalent of
empty vector, induced with varying concentrations of agonist (R5020
(A) or Dex (B)), or antagonist (Dex-Mes in
A and Dex-Ox in B), and assayed for luciferase
and Renilla activities as described under "Materials and
Methods." The average values, normalized for cotransfected
Renilla, were then plotted as percent of maximal induction
above EtOH controls. The error bars represent the S.D.
within a given experiment. Similar results were obtained in one
(A) and four (B) additional experiments.
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Fig. 4.
Induction properties of chimeric
receptors. A, schematic of the composition of
chimeric receptors. The linear structures of the wild type rat GR and
human PR-B are displayed on top. The individual domains of
the receptors are identified by the A-E on top
of the GR structure. The C and D domains, corresponding to the DNA
binding domain and the hinge region, respectively, are
differentially shaded. The chimeric receptors are joined at
the interface of the C and D domains, indicated by the vertical
line. The precise amino acids from each receptor are shown
above and below the two segments comprising each
chimera. B, effect of concentration of transfected GR/PR
on GREtkLUC induction properties in 1470.2 cells. C,
effect of steroid receptor concentration of GR versus PR/GR
on GREtkLUC induction properties in CV-1 cells. B and
C, triplicate cultures were cotransfected with the indicated
amounts of receptor plasmids and GREtkLUC plus Renilla null
luciferase reporters, induced with varying concentrations of agonist
(R5020 (B) or Dex (C)), or antagonist (Dex-Mes in
B and C), and assayed for luciferase and
Renilla activities as described under "Materials and
Methods." The average values, normalized for cotransfected
Renilla, were then plotted as percent of maximal induction
above EtOH controls. The error bars represent the S.D.
within a given experiment. Similar results were obtained in one
(B) and two (C) additional experiments.
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Fig. 5.
Effect of corepressors (SMRT and NCoR) on the
induction properties of GREtkLUC by chimeric receptors in 1470.2 cells. The consequences of cotransfected SMRT (0.4 µg;
A and B) and NCoR (1.2 µg; C and
D) on the properties of PR/GR (A and
C) and GR/PR (B and D) were examined.
Triplicate cultures were cotransfected with 30 ng of each chimeric
receptor plasmid, an equimolar amount of corepressor plasmid or vector,
and GREtkLUC plus Renilla null luciferase reporters, induced
with varying concentrations of agonist (Dex (A and
C) or R5020 (B and D)) or antagonist
(Dex-Ox (A and C) or Dex-Mes (B and
D)), and assayed for luciferase and Renilla
activities as described under "Materials and Methods." The average
values, normalized for cotransfected Renilla, were then
plotted as percent of maximal induction above EtOH controls. The
error bars represent the S.D. within a given experiment.
Similar results were obtained in four (with SMRT) and two or three
(with NCoR) additional experiments.
GR/PR > PR/GR > GR (see
Figs. 2 and 5, C and D) is slightly different from that for the left shift in the dose-response curve. Nevertheless, neither the LBD nor the amino-terminal half of the receptor (including the DBD) appears to be dominant in the responses to NCoR. Instead, the
final behavior of the chimeric receptor is an amalgam of the properties of each wild type protein with both halves of the receptor contributing to the final activity.
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Fig. 6.
Comparison of properties of PR/GR with those
of wild type GR. Induction properties of PR/GR versus
GR in 1470.2 (A) and CV-1 (B) cells. Triplicate
cultures were cotransfected with the indicated amounts of each receptor
plasmid and GREtkLUC plus Renilla null luciferase reporters,
induced with varying concentrations of agonist (Dex in A and
B) or antagonist (Dex-Ox (A) or Dex-Mes
(B)), and assayed for luciferase and Renilla
activities as described under "Materials and Methods." The average
values, normalized for cotransfected Renilla, were then
plotted as percent of maximal induction above EtOH controls. The
error bars represent the S.D. within a given experiment.
Similar results were obtained in two (A) and three
(B) additional experiments. Affinity of Dex binding to GR
and PR/GR in cell-free (C) and whole cell (D)
systems. Scatchard plots of Dex binding to GR or PR/GR in
cytosols from transfected COS-7 cells, or intact transfected COS-7
cells, were performed as described under "Materials and Methods."
The best fitting lines were determined by least squares analysis to
give the indicated Kd values. Similar results were
obtained in a second experiment.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Proposed model for role of receptor domains
in differential modulation of transactivation properties by various
cofactors. For simplicity, only one of the two receptor molecules
that bind to a single GRE of GREtkLUC are shown. Those factors that
interact with the structurally discrete amino- and carboxyl-terminal
domains of GR, PR, and the chimeric receptors (only PR/GR is shown) may
undergo a binding-induced conformational modification. These factors
may interact alone or as part of a multiprotein complex. The now
different tertiary structures of these interacting molecules or
complexes (indicated by the altered shape and shading of the species
contacting the amino- (N) and carboxyl
(C)-terminal domains of the receptors) will possess unequal
spatial relationships with, and/or will have altered affinities for,
different components of the transcriptional machinery (lightly
shaded species of PolII complex). These varied protein-protein
contacts, which may be mediated by additional nuclear proteins, would
result in the non-equal effects on the EC50 and partial
agonist activity that are seen for each of the illustrated receptors.
Pol, polymerase. See text for further description.
A differential effect of cofactors on steroid receptor regulated-gene
transcription is an attractive mechanism for conveying unequal
transactivation properties to receptors that bind to common DNA
targets. As is described in the present study with GR and PR, this
mechanism can help to account for the different responses between two
classes of steroid receptors within the same and different cells.
Another apparent example of differential responses to a common cofactor
has recently been reported for PIAS1. In CV-1 cells, PIAS1 increased
the fold induction by both androgen receptors and GR but decreased
progesterone receptor fold induction (40). In light of the variation of
cofactor levels among cell types (84-89), the model of Fig. 7 helps to
explain the cell-to-cell differences for a given receptor, as has been
seen here for GR in CV-1 versus 1470.2 cells. Finally, the
present modulation of EC50, and partial agonist activity of
antisteroids, in the same and different tissues offers yet another
method for achieving the differential control of gene expression that
is required during the differentiation, development, and homeostasis of
complex organisms. These results suggest that additional studies on the
differential consequences of cofactor binding to steroid receptors
represent a fertile area for future research.
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ACKNOWLEDGEMENTS |
---|
We thank Ron Evans (Salk Institute), Hinrich Gronemeyer (IGBMC, Strasbourg), Gordon Hager (National Institutes of Health), Michael Rosenfeld (University of California, San Diego), and Keith Yamamoto (University of California, San Francisco) for generously donating research materials, members of the Steroid Hormones Section for helpful discussions, and Cathy Smith (National Institutes of Health) for critical review of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Bldg. 8, Rm. B2A-07, NIDDK/LMCB, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-6796; Fax: 301-402-3572.
Published, JBC Papers in Press, May 1, 2001, DOI 10.1074/jbc.M102610200
2 B. Huse, unpublished data.
3 S. Rusconi, unpublished data.
4 D. Szapary and S. S. Simons, Jr., unpublished results.
5 Y. Huang and S. S. Simons, Jr., unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: HREs, hormone response elements; Dex, dexamethasone; GRs, glucocorticoid receptors; PRs, progesterone receptors; AR, androgen receptor; ER, estrogen receptor; LBD, ligand binding domain; DBD, DNA binding domain; Dex-Mes, dexamethasone mesylate; GRE, glucocorticoid response element; Dex-Ox, dexamethasone oxetanone; MMTV, mouse mammary tumor virus.
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