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Department of Pathology (E.K.G., S.A.L., S.K.N., D.P.E.) and Molecular Biology Program S.K.N. D.P.E.), University of Colorado Health Sciences Center, Denver, Colorado 80262
Address all correspondence and requests for reprints to: Dr. Dean P. Edwards, Department of Pathology and Molecular Biology Program, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262. E-mail: edwards_d{at}defiance.uchsc.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The biological actions of progesterone are mediated by progesterone receptor (PR), which is a member of the nuclear receptor superfamily of transcriptional activators (5). In the absence of progesterone, PR is associated with several heat shock proteins (hsp90, hsp70, and hsp56) and possibly other proteins to form an inactive oligomeric complex (6). Upon hormone binding, the receptor becomes activated, resulting in dissociation from the oligomeric complex, dimerization, and binding to specific hormone response elements (HREs) of target genes (7). Consensus HREs consist of palindromic hexanucleotide motifs separated by three-nucleotide spacers (8). Binding of PR to progesterone response elements (PREs) promotes the formation of a stable initiation complex, resulting in gene transcription. How PR or other steroid receptors couple with the general transcriptional machinery is not well understood. It appears that this occurs by protein-protein interactions with general transcription factors (9, 10, 11) or with specific coactivators/corepressors (12, 13, 14).
Several steroid antagonists have been developed that effectively compete for progesterone binding to PR and render the receptor transcriptionally inactive or substantially reduce its trans-activation potential (15). A central property of progestin antagonists is the induction of a conformational change in PR distinct from that induced by hormone agonists. Effects on PR structure have been detected by several methods, including an altered electrophoretic mobility of PR-DNA complexes on native polyacrylamide gels (3, 16, 17), differential recognition by an antibody directed to the extreme C-terminus of PR (18), and limited proteolytic digestion patterns (19). Additionally, progestin agonist and antagonists appear to contact noncoincident, but overlapping, amino acids in the ligand-binding domain (LBD), as suggested by results with a monoclonal antibody (mAb) to the C-terminal tail of PR that blocks binding of progesterone, but not RU486 (18); a point mutation at Gly722 that affects only RU486, but not progesterone, binding (20); and a C-terminal deletion mutant that fails to bind agonist, but binds RU486 (21). How an altered conformation induced by antagonists leads to inactivation of PR is not well understood. Recent studies suggest that the altered conformation in the LBD of PR induced by antagonists impairs the ability of receptors to interact with coactivators (22). Alternatively, antagonists may cause PR to recruit corepressors, suggesting that the relative amounts of coactivators and corepressors may contribute to the partial agonist or antagonist activity of a ligand (23, 24).
Earlier studies based on in vitro DNA binding assays by electrophoretic gel mobility shift assay (EMSA) suggested that there are at least two mechanistic classes of antiprogestins (16). Onapristone (ZK98299) was proposed to be a type I compound that failed to promote binding of PR to PREs. In contrast, mifepristone (RU486) and other related compounds increase the binding of PR to PREs and thus have been considered type II antagonists (3, 16, 25, 26). Based on these criteria, ZK98299 was predicted to be a more pure antagonist than type II compounds. Indeed, the pharmacology of ZK98299 indicates that it is a more complete antagonist than RU486. Further, we and others have shown that cross-talk with cAMP signaling pathways potentiates the agonist activity of RU486, but has no effect on the biological activity of ZK98299 (26, 27).
This initial classification of antagonists based upon EMSA binding has become controversial as a result of experiments designed to detect PR binding to PREs in the intact cell. In cotransfection studies with wild-type PR and a constitutively active truncated PR that lacks the LBD, RU486 and ZK98299 were both observed to inhibit the activity of the mutant PR, suggesting that both compounds stimulate binding of wild-type PR to PREs in vivo (28). In contrast, genomic footprint assays with a chromosomally integrated copy of mouse mammary tumor virus (MMTV) failed to detect RU486 or ZK98299 protection of specific sites in the MMTV promoter. However, the agonist R5020 induced a strong in vivo footprint (29). From these results, it was concluded that neither type of antagonist is able to induce PR binding in vivo when the target DNA sites are integrated into chromatin.
More recent results suggest by different criteria that ZK98299 does, in fact, represent a separate class of antagonist that affects the structure of PR differently from RU486. Phosphopeptide mapping of PR revealed that site-specific phosphorylation of PR in whole cells is affected differently by RU486 and ZK98299. Treatment of cells with RU486 stimulated phosphorylation of the same hormone-dependent sites as the agonist R5020, whereas ZK98299 failed to induce the hormone-dependent sites (30). Additionally, partial protease digestion assays have indicated that ZK98299 promotes a conformational change in PR distinct from that induced by RU486 or agonist (31, 32).
In the present study we have used three different transfection protocols to determine the ability of progesterone antagonists to stimulate binding of human PR to PREs in mammalian cells. This included a promoter interference assay, competition between PR antagonist and glucocorticoid receptor (GR)-agonist complexes for interaction with a common glucocorticoid response element (GRE)/PRE-controlled reporter construct, and activation of a PR-VP16 chimeric receptor. By all three approaches, each antiprogestin evaluated increased binding of PR to PREs in whole cells, including the previously classified type I compound ZK98299. Additionally, EMSA conditions were found that allowed detection of ZK98299 induction of PR-PRE complexes in vitro. Under these conditions, the receptor-DNA complex was less stable than the complex induced by the agonist R5020, and it exhibited an electrophoretic mobility distinct from that induced by RU486. We conclude from these results that RU486 and ZK98299 represent two different mechanistic classes of progestin antagonists based more upon their ability to induce different conformational changes in PR than on their promoting or failing to promote binding to target DNA.
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Materials and Methods |
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-methyl-3H]17,21-dimethyl-19-norpregna-4,9-diene-3,20-one;
87 Ci/mmol) and unlabeled R5020 were obtained from DuPont-New England
Nuclear Research Products (Boston, MA). The progestin antagonist
(mifepristone) RU486
(17-hydroxy-11[4-dimethyl-aminophenyl)17-propenyl-estra-4,5-diene-3-one)
was a gift from Roussel-UCLAF (Romainville, France), and ZK98299
(onapristone), ZK112993, and ZK98734 were provided by David Henderson
(Schering, Berlin, Germany). AB-52 is a mouse IgG1 mAb produced against
purified human PR that recognizes both the A and B isoforms of PR (33),
and 1294/H9 is an unpublished mAb to PR that also recognizes A and B
forms of human PR.
Promoter interference reporter and PR plasmids
A cytomegalovirus enhancer-driven chloramphenicol
acetyltransferase reporter plasmid (CMV-TATA-CAT) and promoter
interference plasmids (CMV-ERE2-CAT and
CMV-EREm-CAT) containing, respectively, two estrogen
response elements (EREs; AGGTCAcagTGACCT) or two mutant EREs
(AGATCAcagTGGCCT) inserted into the unique SacI site of
CMV-TATA-CAT were provided by B. Katazenellenbogen (University of
Illinois, Urbana, IL) and have been described previously (34). Promoter
interference plasmids containing one, two, or three consensus PREs
(CMV-PRE1–3-CAT) were constructed by ligating the
following double stranded synthetic oligonucleotides into the
SacI site of CMV-TATA-CAT: PRE1,
5-'-AGAACAAACTGTTCTTa-3'; PRE2, 5'-AGAACAAACTGTTCTtaaag
AGAACAAACTGTTCTt-3'; and PRE3,
5'-AGAACAAACTGTTCTtaaagAGAACAAACCTGT TCTtaaagAGAACAAACTGTTCT-3'.
The 15-bp core palindromic PRE-binding sites are capitalized. Each
oligonucleotide was synthesized to contain 5' single stranded
AGTC extensions for cloning into the unique SacI site of
CMV-TATA-CAT by the linker tailing method as previously described (35).
A reporter plasmid, pDHRE-E1b-CAT, that contains two optimal hormone
response elements linked to the TATA box of E1b (36) and CAT was used
for the detection of PR-mediated induction of transcription (Thackray,
U. G., B. A. Lieberman, and S. K. Nordeen, unpublished
data). The mammalian cell expression plasmid, phPR-B, containing the
complementary DNA (cDNA) for human PR-B under the control of the simian
virus 40 enhancer and the human metallothionein IIa promoter was
provided by Donald P. McDonnell at Duke Medical Center and has been
previously described (37). The phPR-B plasmid was partially digested
with BamHI to generate three PR cDNA fragments of 0.24, 2.8,
and 3.1 kilobases (kb). The 3.1-kb fragment that encodes full-length
PR-B was gel purified and cloned into the BamHI site of the
multiple cloning cassette of a mammalian cell expression plasmid under
the control of the CMV enhancer/promoter (pcDNA/I, Invitrogen, San
Diego, CA) to yield pCMV-hPRB. The correct orientation and presence of
the PR-B cDNA insert were confirmed by DNA sequencing across the
cloning junctures (Sequenase 2.0, U.S. Biochemical Corp., Cleveland,
OH).
To construct a human PR-VP16 chimeric receptor, a 2.93-kb AflII-Asp718 fragment of YEphPR-B (21) was filled in with Klenow and cloned into the unique BamHI restriction site of pGAD424 (Clontech, Palo Alto, CA) that had been blunt-ended with Klenow. The resultant plasmid, pGAD424.hPR-B, contains a fusion between the transcriptional activation domain of GAL4 and hPR-B. pGAD424.hPR-B was used as the source of hPR-B cDNA for fusion with the VP16 protein of the herpes simplex virus. A 2.9-kb EcoRI-PstI fragment from pGAD424.hPR-B was cloned into the EcoRI-PstI restriction sites of pVP16. pVP16 is a mammalian cell expression plasmid containing the acid activation domain sequences of VP16 (Clontech). This places the VP16 activation domain immediately amino-terminal to hPR cDNA. The fusion gene was sequenced (Sequenase 2.0, U.S. Biochemical) to confirm that VP16 and hPR-B were correctly oriented and fused in the correct reading frame.
Transient transfections
COS-1 cells were plated into six-well dishes (Falcon, Oxnard,
CA) at a density of 1.75 x 105/well in DMEM (Life
Technologies, Gaithersburg, MD) supplemented with 10% FBS (HyClone,
Logan, UT). Cells were grown at 37 C for approximately 24 h and
allowed to reach 60–70% confluence. Cells were then transfected by an
adenovirus-mediated method as previously described (38). The method
involves the use of a replication-defective adenovirus coupled with
poly-L-lysine to bind plasmid DNA noncovalently. This
facilitates cellular uptake of the plasmid by receptor-mediated
endocytosis. Briefly, cells were incubated for 2 h at 37 C in
serum-free DMEM with poly-L-lysine-coupled virus and
plasmid DNA. Virus was added at 250–500 particles/cell, and the PR
expression plasmid over a range of 1.0–50 ng/well and the promoter
interference reporter plasmid were added. Two nanograms per well of
pBR55 carrier DNA and empty vector CMV promoter DNA (pCMV6C) were added
so that the total amounts of DNA and molecules of CMV promoter were
equalized in all cultures. For experiments to measure PR as a
trans-activator, the E1b-DHRE-CAT reporter plasmid was added
at 500 ng/well, and the PR expression plasmid was added over a range of
1.0–50 ng/well. After incubation for 2 h, an equal volume of DMEM
medium containing 10% FBS (to bring the serum to a 5% final
concentration) was added to each well, and the cells were incubated for
another 48 h at 37 C. To treat cells with various PR ligands, DMEM
medium plus 10% FBS containing either vehicle (0.001% ethanol) or the
concentrations of ligands indicated in figure legends were added and
incubated for 48 h at 37 C.
Stable transfection of T47D cells with promoter interference
plasmids
All promoter interference constructs were stably introduced into
T47D cells by the calcium phosphate/DNA precipitation method (39). T47D
breast cancer cells were plated at a density of 1 x
106 cells in 10-cm culture dishes (Corning, Corning, NY)
and allowed to grow for 24 h. A 1-ml solution of calcium phosphate
mixed with 20 µg promoter interference plasmid DNA and 1 µg
selection plasmid pSV-2 neo (containing the neomycin resistance gene)
was added to the culture dish in 10 ml growth medium. After incubation
at 37 C for 4 h, DNA and media were removed, and cells were washed
in serum-free MEM and subjected to glycerol shock for 3 min at 37 C.
The shock medium contained 10% glycerol in HTB (127 mM
NaCl, 5 mM Na2HPO4, 6
mM dextrose, and 21 mM HEPES, pH 7.1). Cells
were then washed with serum-free MEM and returned to growth medium for
48 h. At that time medium was replaced with a 50:50 mixture of
fresh and conditioned growth medium containing 400 µg/ml of the
neomycin analog G-418 for growth selection of stably transfected cells.
Individual colonies that grew in G-418 were harvested with cloning
rings and expanded in the presence of G-418. Several clones of each
transfection were isolated and examined in the presence and absence of
various ligands. Clones that exhibited a significant R5020 reduction of
CAT expression were then pooled to obtain a polyclonal mix of cells
expressing the respective promoter interference plasmid. To study
hormone-mediated PR-DNA binding, cells were plated in six-well dishes
at a density of 2.5 x 105 cells/well. They were
allowed to grow for 24 h, were incubated in the presence or
absence of hormone for an additional 42–72 h, and then were harvested
and assayed for CAT activity.
Stable transfection of T47D cells with MMTV-luciferase and human GR
expression plasmid
The MMTV-luciferase gene (MMTV-LUC) and a GR expression vector
(pGRneo) were stably introduced into T47D cells (A1-2), and clones were
selected for functional GR protein and integration of the MMTV-LUC
reporter gene as described previously (40). A1-2 cells were plated at a
density of 1 x 106 cells in 10-cm culture dishes and
allowed to grow for 24 h. Cells were then treated with
dexamethasone (Dex), R5020, ZK112993, and ZK98299 alone or in
combination as indicated in the figure legends and allowed to grow for
7 h. Cells were then harvested and assayed for luciferase activity
or endogenous alkaline phosphatase.
Reporter gene assays
Cell monolayers in six-well dishes were washed with a CAT wash
buffer [40 mM Tris-HCl (pH 7.4), 150 mM NaCl,
and 1 mM EDTA] and lysed directly in the well by addition
of 300 µl 0.5% Triton X-100 lysis buffer (20 mM
potassium phosphate, 5 mM MgCl2, and 0.5%
Triton X-100). The lysates were removed from the well and centrifuged
at 12,000 rpm for 10 min at 4 C. The supernatants were assayed for
protein concentration by Bradford assay (41), and equal amounts of
protein (30 µg) were added to each CAT assay. CAT enzyme activity was
assayed by the radiometric/organic phase extraction method as
previously described (42), and activity was calculated as the counts
per min of [3H]acetylcoenzyme A converted/µg protein in
the cell lysate. Cell treatment groups were performed in duplicate, and
CAT assays were also conducted in duplicate for each lysate. Values for
CAT activity were calculated as averages from four assay
determinations. With transiently transfected promoter interference
plasmids, the CAT activity obtained in the absence of transfected PR
was set as 100%, and the activity obtained in the presence of
transfected PR was set as a fraction of 100%. Normalizing activity to
100% in the absence of PR allowed calculation of average values and
the SEM between multiple independent experiments. With
stably transfected promoter interference plasmids, CAT activity was set
at 100% for cells treated without hormone and a fraction of 100% for
cells treated with PR ligands. Normalization of CAT to 100% in the
absence of ligand also allowed calculation of average values and
SEMs between multiple independent experiments.
For quantification of luciferase activity, cell monolayers were rinsed twice with wash buffer [40 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM EDTA], and cells were lysed by the addition of 0.5 ml lysis buffer [20 mM K2HPO4 (pH 7.8), 5 mM MgCl2, and 0.5% Triton X-100]. Lysates were then centrifuged for 2 min to pellet particulates. Luciferase assays were performed using a Monolight 2001 luminometer (Analytical Luminescence Laboratories, San Diego, CA). Extract (50 µl) was added to 0.35 ml luciferase assay buffer [100 mM K2HPO4 (pH 7.8), 15 mM MgSO4, 5 mM ATP, and 1 mM dithiothreitol (DTT)]. Luciferase-mediated light output was assessed for 10 sec, with a built-in 2-sec delay after the injection of 100 µl 1 mM luciferin into the reaction chamber. The protein concentration of each extract was determined by the Bradford assay, as previously described (41).
For determination of alkaline phosphatase levels, A1-2 cells were plated at 6.0 x 105 cells/60-mm dish. After 24 h, cells were treated with hormones as indicated in the figure legends and allowed to grow for 24 h. Cell monolayers were then washed and harvested as described for luciferase assay. Lysates were centrifuged for 5 min at 4 C to pellet particulates. Extracts (20 µl) were prepared for assay according to Phospha-Light kit instructions (Tropix, Bedford, MA) with modifications in the Phospha-Light Reaction Buffer (0.2 M diethanolamine and 2 mM MgCl2). Samples were incubated for 20 min at room temperature, and luminescence was measured using a Monolight 2001 luminometer (Analytical Luminescence Laboratories).
Whole cell binding assay
COS-1 cells plated in six-well dishes at a density of 1.75
x 105 cells/well were grown overnight at 37 C in DMEM-10%
FBS. Cells were then transfected with 50 ng PR-B expression plasmid by
the adenovirus-mediated technique described above. After 48 h,
culture medium was removed and replaced with 1.5 ml/well DMEM-10% FBS
containing 1 nM [3H]R5020 with or without
unlabeled 100 nM R5020 for 4 h at 37 C. Medium was
then removed, and cell monolayers were washed five times with 5 ml
ice-cold PBS. To extract R5020, ethanol (1 ml/well) was added to cell
monolayers and incubated for 30 min at room temperature. Ethanol was
removed and counted for [3H]R5020 in 5 ml liquid
scintillation fluid. Parallel transfected cultures were lysed directly
in the six-well dish with 250 µl/well lysis buffer [20
mM potassium phosphate (pH 7.4), 5 mM
MgCl2, and 0.5% Triton X-100] and measured for protein
concentration by the Bradford assay. Receptor values were normalized to
protein and calculated as picomoles of steroid binding per mg total
protein.
Preparation of PR
T47D cells were plated at a density of 4 x
106/tissue culture flask under conditions described
previously and allowed to grow for 8 days at 37 C. Two hours before
trypsin-EDTA harvest, cells were incubated with medium containing
vehicle, R5020 (100 nM), RU486 (100 nM),
ZK112993 (100 nM), ZK98734 (100 nM), or ZK98299
(500 nM) at 37 C. Harvested cell pellets were washed first
in serum-free MEM and then in cold TEG [10 mM Tris-OH, 1
mM EDTA (pH 7.4), and 10% glycerol]. Pellets were
homogenized in TEDG [10 mM Tris-OH (pH 7.4), 1
mM EDTA, 1 mM DTT, and 10% glycerol]
containing a cocktail of protease inhibitors (33). Homogenates were
centrifuged at 100,000 x g for 30 min in a Beckman 50
Ti rotor (Palo Alto, CA). Pelleted nuclei were extracted with TEDG
containing 0.5 M NaCl for 1 h at 4 C and then
centrifuged for 30 min at 100,000 x g to yield soluble
nuclear supernatant. PR levels in nuclear extracts of T47D cells were
measured by a single saturating dose of [3H]R5020 (10
nM) in the presence and absence of a 100-fold excess of
unlabeled R5020 to detect nonspecific binding. Free and bound
[3H]R5020 were separated by dextran-coated charcoal, and
specific picomoles of R5020 binding to PR were calculated as previously
described (3).
EMSAs
For EMSA, we used a 28-bp oligonucleotide containing a PRE/GRE
derived from the MMTV long terminal repeat (3). T47D nuclear extracts
(nanomoles of PR are indicated in figure legends) were incubated for
1 h at 4 C with 32P-labeled DNA (0.3 ng) in a total
reaction volume of 25 µl. Also included was 1 µg
poly(dA-dT)/poly(dA-dT) as nonspecific competitor DNA. For standard
conditions, the DNA binding buffer contained 10 mM Tris
base (pH 7.4), 50 mM NaCl, 5 mM DTT, 2
mM MgCl2, 10% glycerol, and 50 ng/ml of a
carrier protein (3, 18, 26). To attempt to maintain less stable PR-DNA
complexes, EMSA conditions were altered by the addition of 2.5%
glycerol to polyacrylamide gels and reduction of NaCl from 50 to 5
mM in the binding reaction. Samples (25 µl) were
electrophoresed on 5% polyacrylamide gels prepared at a 40:1 (wt/wt)
acrylamide-bis-acrylamide ratio using 20 mM Tris-acetate
and 0.5 mM EDTA in the gels and as the electrode buffer. To
maintain constant temperature during electrophoresis, 4 C water was
recirculated through the gel apparatus. Gels were dried and subjected
to autoradiography. Quantification of PR-DNA complexes was carried out
by direct scanning of dried gels for radioactivity using a series 400
Molecular Dynamics PhosphorImager (Sunnyvale, CA).
SDS-PAGE and immunoblotting
SDS-PAGE and immunoblotting were carried out with PR-specific
monoclonal antibodies (AB-52 or 1294/H9) using
[35S]protein A and autoradiography of dried
nitrocellulose as the detection method (3, 33).
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This positions the PREs between the
TATA box and the start of transcription. In principle, binding of PR to
PREs will disrupt assembly of the general transcription complex by
steric hindrance, resulting in a reduction of expression of the
constitutively active CMV-driven CAT reporter gene. As controls to
determine the extent to which reduction of CAT expression is dependent
on PREs, we used CMV-TATA-CAT reporter constructs containing two ERE
oligonucleotides inserted into the SacI site of CMV-TATA-CAT
(either wild-type ERE or mutant EREs; Fig. 1). This type of promoter
interference assay was initially developed to detect estrogen receptor
(ER) binding to EREs in intact cells (34) and more recently was used to
study the influence of different ligands on androgen receptor (AR)
binding to androgen response elements (43). Inserting up to three PRE
oligonucleotides into CMV-TATA-CAT did not disrupt the expression of
CAT activity in the absence of PR (data not shown). Reese and
Katzenellenbogen (34) also reported that insertion of up to three ERE
oligonucleotides in the absence of ER did not reduce promoter activity.
Disruption of activity by merely inserting DNA sequence required
insertion of four EREs (34); we did not attempt more than three
PREs.
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, the number of receptor-binding
sites increased in a dose-responsive manner, reaching nearly 1 pmol
receptors/mg protein at the highest amount (50 ng) of transfected PR-B
expression plasmid. This is quantitatively comparable to the level of
native PR in breast cancer cell lines such as T47D and MCF-7 (1–2.5
pmol/mg), indicating that transfected PR in COS-1 cells is within
physiological levels and is not overexpressed. When COS-1 cells were
cotransfected with increasing amounts of the PR-B expression plasmid
and a positive PRE controlled reporter gene (pDHRE-E1b-CAT), R5020
induction of CAT activity was obtained at all amounts of transfected PR
(Fig. 2B). Additionally, the fold hormone induction increased in a
dose-dependent manner with increasing amounts of the PR plasmid (1–50
ng). Thus, under these transfection conditions, PR functions as an
activator, with no evidence of nonspecific inhibitory effects.
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taken
together with the results of similar dose-response assays showing that
25 ng/well produces a maximal effect on the activity of promoter
interference constructs (data not shown). As shown in Fig. 3A, expression of PR-B in the absence of
ligand resulted in a 45% reduction of CAT activity. By treating cells
with R5020, CAT activity was reduced another 2-fold to a level
approximately 80% below the control value (Fig. 3A). Treatment of
cells with the type II antagonist RU486 or the type I antagonist
ZK98299 also resulted in a reduction of CMV-PRE3-CAT
activity comparable to that of the hormone agonist R5020 (Fig. 3A). In
contrast, PR-B expression in COS-1 cells had a minimal effect on the
activity of the ERE (CMV-ERE2-CAT)-containing promoter
interference construct regardless of whether cells were treated with or
without PR ligands (Fig. 3B). These results taken together indicate
that the reduction of CAT activity in these assays is both PR and PRE
dependent. All antagonists used in this study (see Table 1) have a binding affinity for PR equal
to R5020, except ZK98299, which has a 10-fold lower affinity (28)
(Edwards, D. P., and M. Altmann, unpublished data). To compensate
for its lower affinity, cells were treated here and in subsequent
experiments with 500 nM ZK98299. This concentration is
based on previous studies in which 500 nM ZK98299 was
determined from dose-response curves to be required for complete
antagonism of hormone activation of PR within the cell (47). Thus, the
concentration of ZK98299 used here is that which produces complete
antagonism of PR activation. R5020 and other antagonists were used at
100 nM, which has been determined previously to be
sufficient for maximal activation of PR by R5020 and for complete
antagonism of PR by RU486 and other type II antagonists (47, Thackray,
V. G., B. A. Lieberman, and S. K. Nordeen, unpublished
data). Thus, the results with transiently transfected PR and promoter
interference constructs suggest that unliganded PR is capable of
binding to PREs within the cell and that hormone agonists and both
types of antagonists further enhance PR binding to PREs.
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). Thus, differences in total cellular levels of PR
should not be a factor when comparing results from transient and stably
transfected cells.
Because PR is expressed endogenously in T47D cells, one limitation of
stably integrated promoter interference constructs is an inability to
detect effects of unliganded PR on promoter activity. However, the
time-course experiment in Fig. 4 shows
that these stably transfected T47D cells are useful to compare the
abilities of different ligands to enhance PR binding to target DNA
sequences. In the absence of ligand, CAT activity expressed from
CMV-PRE3-CAT increased continuously between 24 h after
plating of cells up to 96 h (Fig. 4). As CAT enzyme is fairly
stable, this increased cellular level probably reflects an accumulation
of CAT protein over this period. Treatment of cells with R5020 or
RU486, which was initiated 24 h after plating, prevented the
increased accumulation of CAT at each time point examined (Fig. 4). We
next investigated the effects of different progestin antagonists by
treating cells for a total of 48 h, beginning 24 h after
plating. As with transient transfection experiments, the CMV-ERE-CAT
promoter interference constructs were used as controls to determine
whether reductions in CAT activity are PRE dependent. As shown in Fig. 5A, R5020, several different progestin
antagonists (including ZK98299, RU486, ZK98734, and ZK112993), and the
androgen dihydrotestosterone (DHT) had no effect on expression of the
control CMV-ERE2-CAT promoter interference construct. In
contrast, treatment with R5020 and all antagonists tested resulted in
significant reduction of CAT expression by cells containing stably
integrated CMV-PRE3-CAT constructs (Fig. 5B).
Interestingly, all antagonists suppressed CAT activity to a greater
extent than R5020 (Fig. 5B). As a control for steroid specificity, only
PR ligands reduced CAT activity, as DHT (Fig. 5B) and estrogen (not
shown) had no effect.
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). R5020 treatment caused a 50%
reduction below control levels with stably transfected promoter
interference constructs containing two or three PREs (Fig. 5C). In
contrast, inhibition of promoter activity by antagonists was
independent of the number of PREs. RU486 and ZK98299 both produced
approximately 70–75% reductions below the control value with a single
PRE, and the extent of reduction was comparable with constructs
containing two or three PREs (Fig. 5C).
The PR-ZK98299 complex competes with GR binding to GRE/PREs in
whole cells
As alternatives to the promoter interference assay, we used two
additional approaches to test the ability of antagonists to enhance PR
binding to PREs in the intact cell. One is based on the PR-antagonist
complex competing with the GR agonist for interaction with a GRE/PRE
positively controlled reporter gene. For these experiments we used a
human breast cancer T47D (A1-2) cell line that has been engineered to
constitutively express GR and PR and contains a stably integrated
MMTV-LUC gene. The MMTV-LUC gene in this cell line is robustly induced
by glucocorticoids. However, progestins fail to promote chromatin
remodeling of the promoter and induce luciferase expression as
glucocorticoids do (44). Coadministration of Dex and R5020 resulted in
a 4- to 5-fold reduction of the response to Dex, indicating that the PR
agonist competes with GR for occupancy of the GRE/PRE (40, 44). We used
this system to assess whether PR antagonists likewise compete with GR
for occupancy of the MMTV-GRE/PRE.
The large differential induction of MMTV-LUC by PR and GR in A1-2 cells
is shown in Fig. 6A. Treatment with R5020
along with Dex at doses at which cross-binding of the progestin to GR
is negligible, resulted in a reduction of the response to Dex similar
to that observed previously (44). Cotreatment with Dex and progestin
antagonist ZK112993 or ZK98299 also reduced the glucocorticoid-mediated
induction. This indicates that both types of PR-antagonist complexes
compete with GR for occupancy of the GRE/PRE.
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). Together, these induction
data indicate that the PR-ZK98299 complex, which itself cannot
trans-activate MMTV-LUC, can inhibit
trans-activation by the GR-agonist complex by competing for
binding to the GRE/PRE within the cell.
ZK98299 and other antagonists induce trans-activation by a PR-VP16
chimeric receptor
As a second alternative approach to detect PR binding to PREs in
the cell, we have investigated the abilities of ligands to activate
transcription of a PR-VP16 chimeric receptor. In theory, any ligand
that is capable of delivering PR-VP16 to DNA in whole cells should
stimulate the expression of a PRE-controlled gene by virtue of bringing
the constitutively active acidic activation domain of VP16 into the
proximity of the target gene promoter. The chimeric PR-VP16 construct
when transfected into COS-1 cells produced a protein detected by
Western blot with a PR-specific mAb that was the expected size compared
with that of native PR, and the expression level was similar to that of
transfected wild-type nonfusion PR as well as native PR in T47D cells
(not shown). Additionally, the expressed PR-VP16 fusion protein
exhibited specific R5020 binding in whole cells that was
indistinguishable from that of nonfusion transfected PR and native PR
in T47D cells (not shown). Thus, fusion of PR to VP16 at the
amino-terminus did not adversely affect the expression or
steroid-binding activity of PR. When COS-1 cells were cotransfected
with either wild-type human PR (pSVhPR-B) or PR-VP16 expression
plasmids and a positive PRE controlled reporter gene (pDHRE-E1b-CAT),
both wild-type PR and PR-VP16 strongly trans-activated the
reporter gene in response to R5020 treatment (Fig. 7). With wild-type PR, treatment of cells
with various antagonists, including RU486, ZK112993, and ZK98299,
caused no induction of DHRE-E1b-CAT expression over that in the vehicle
control. In contrast, both type I (ZK98299) and type II (RU486 and
ZK112993) antagonists induced PR-VP16 trans-activation when
added to cells at receptor-saturating concentrations (Fig. 7). Although
the level of PR-VP16 trans-activation caused by antagonists
was much less than that stimulated by agonist, the fold CAT induction
over the no hormone vehicle control value was significant. These
results indicate that treatment of cells with ZK98299 as well as other
antagonists stimulated the interaction of PR-VP16 with target PREs.
|
PR in T47D cells was bound to R5020 or different type I and II
antagonists in the cell before extraction from nuclei. Saturation DNA
binding analysis was performed under standard and altered EMSA
conditions using increasing amounts of PR in nuclear extracts
(nM of PR determined by steroid-binding assay) against a
constant amount of [32P]PRE oligonucleotide probe. Gels
were autoradiographed (Figs. 8A and 9A), DNA complexes were quantitated by
phosphorimage analysis, and the results were plotted as a percentage of
the upshifted PR-PRE complexes (Figs. 8B and 9B). Under standard EMSA
conditions, unliganded PR and PR complexed to ZK98299 bound poorly to
the PRE oligonucleotide. In contrast, R5020 and all the other progestin
antagonists tested induced high affinity DNA binding that saturated in
the low nanomolar range of PR (Fig. 8A, autoradiography results; Fig. 8B, quantitative phosphorimage results). Interestingly, based on the
leftward shifting of the saturation DNA binding curves, RU486 and the
other type II antagonists induced a higher affinity interaction of PR
with PREs than the agonist R5020 with the rank order of ZK98734 >
ZK112993 > RU486 > R5020. Under altered EMSA conditions,
ZK98299 was observed to stimulate a substantial increase in PR
interaction with PRE compared with unliganded PR (Fig. 9A, autoradiograph results; Fig. 9B, quantitative results). However, the
affinity of the interaction was still less than that with receptors
bound to R5020 or other antagonists. PR-DNA binding induced by ZK98299
was specific, as demonstrated by supershift with a PR-specific
monoclonal antibody (Fig. 10A) and by
competition with excess unlabeled PRE oligonucleotide, but not with an
ERE oligonucleotide (Fig. 10B).
|
|
|
. As shown by autoradiography (Fig. 11, upper panel) and quantitation of DNA complexes at
different time points (Fig. 11, lower panel), PR
dissociation from DNA was faster in the presence of ZK98299 than in the
presence of R5020.
|
, PR-PRE complexes induced by ZK98299
do not exhibit this faster mobility. Instead, they have a mobility that
is equal to or slightly slower than that induced by R5020. This
suggests that ZK98299 induces a conformational change within PR that is
unique from that induced by RU486. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial experiments to examine the effects of various antiprogestins on PR binding to DNA in whole cells involved the use of a promoter interference assay. This approach has been used previously to study the effects of various ligands on ER and AR binding to their respective target DNAs in whole cells (34, 43). In COS-1 cells transiently cotransfected with a PR expression plasmid, activity expressed from CMV-PRE3-CAT was suppressed in the absence of added ligand by 45% below that in control cells that lack PR. Exposure of PR-transfected cells to hormone agonist resulted in a further suppression to levels that were approximately 80% below those in the no ligand treatment controls. This reduction of promoter activity did not appear to be due to sequestering of limiting general transcription factors and was shown to be dependent on PR and the PRE. The interaction of unliganded PR with PREs in the whole cell did not appear to be due to the presence of endogenous progesterone in the culture medium. We obtained the same results with cells grown in whole serum and serum treated with dextran-coated charcoal to remove endogenous steroids (data not shown). Also, no endogenous progesterone was detected by RIA of FBS, indicating that progesterone levels are less than 10-12 M, a level insufficient to induce PR trans-activation in mammalian cells (data not shown). Thus, PR appears to be capable of binding to PREs in the whole cell in the absence of added ligand, whereas hormone agonist further increased this interaction.
Previous promoter interference studies of ER binding to DNA in whole cells showed that ER in the absence of added ligand suppressed promoter activity, whereas estrogen suppressed activity to a greater extent (34). Thus, similar to the present results with PR, unliganded ER appears to be capable of interacting with EREs in the whole cell, and estrogen stimulates these interactions. In contrast, no suppression of promoter activity by unliganded AR was observed from an androgen response element promoter interference construct. Suppression of promoter activity was detected only by treatment of cells with AR ligands (43). Collectively, these promoter interference studies suggest that ER and PR may be capable of interacting to some extent with their cognate target DNA sequences in whole cells, whereas the AR-DNA interaction may be more dependent upon ligand.
Transiently transfected cells treated with two different classes of progesterone antagonists, ZK98299 and RU486, also resulted in a substantial suppression of CAT activity from the CMV-PRE3-CAT interference construct. The extent of suppression by ZK98299 and RU486 was comparable to that caused by R5020, with RU486 producing a slightly greater suppression than ZK98299 and R5020. These results support the conclusion that PR-ZK98299 and PR-RU486 complexes are both capable of binding to PREs within the cell. As detected by promoter interference assays, two classes of antiestrogens have also been reported to enhance ER interaction with EREs in whole cells. The nonsteroidal partial antagonist hydroxytamoxifen and the pure steroidal antiestrogen ICI 163,384 were both observed to suppress promoter activity to the same extent as estrogen (34). Interestingly, in AR promoter interference studies, some androgen antagonists, including hydroxyflutamide, ICI 176,334, and RU23908, blocked AR binding to DNA in whole cells, whereas cyproterone acetate and RU486 stimulated AR-DNA binding activity, although to a lesser extent than androgen agonists (43). Thus, it appears that androgen antagonists can be separated into distinct mechanistic classes based on whether they impair or enhance AR binding to DNA in whole cells. Such distinctions for progesterone and estrogen antagonists in whole cells have not been detected.
To examine the ability of PR to interact with target DNA sequences that have been integrated into chromatin, T47D cells were stably transfected with promoter interference constructs. Results with these cell lines were consistent with our transient transfection data. The agonist R5020 and all antagonists tested produced significant reductions in CAT activity. Interestingly, in these experiments all antagonists tested reduced CAT activity to a greater extent than the agonist R5020. This suggests that antagonists stimulate a stronger enhancement of PR-DNA interactions in whole cells than hormone agonists. In further support of this are the results with stably integrated promoter interference constructs containing a single or multiple PREs. Antagonists mediated a suppression of CAT activity from constructs containing a single PRE insert. In contrast, suppression by agonist was not observed with a single PRE; this required at least two PREs. For reasons that are unclear, R5020 consistently produced a significant 75% increase in promoter activity with constructs containing a single PRE. One interpretation of these results is that antagonists enhance or stabilize the interaction of PR with PREs to a greater extent than agonist, so that only a single receptor dimer is required to disrupt promoter activity. With hormone agonist, two receptor dimers that can bind in a cooperative manner may be needed because of a weaker interaction with DNA. Alternatively, the reduction of promoter activity by the receptor-antagonist complex may not be due solely to steric hindrance, but to the added effect of recruiting corepressors that actively inhibit transcription (23, 24). In previous ER and AR promoter interference studies, it was observed that receptor-mediated reduction of promoter activity was dependent upon the number of inserts, using between one and three HREs (34, 43). The same dependence on the number of PREs was not detected here. This may reflect a difference between transient and stably transfected promoter interference constructs. Our studies comparing from one to three inserted PREs were performed with stably transfected constructs only. All transient transfections were performed with constructs containing three PRE inserts.
The creation of cell lines with stably integrated promoter interference reporter genes allows for the convenient screening of ligands for their effects on PR-DNA binding in whole cells. Stably transfected cell lines were also constructed in the present study to help resolve conflicting reports on the ability of antagonists to induce PR binding to PREs within the cell. By genomic footprinting assays, it was reported that neither RU486 nor ZK98299 was able to promote binding of PR to MMTV under conditions where R5020 was effective (29). In transient cotransfection assays, RU486 and ZK98299, both acting through wild-type PR, inhibited a constitutively active PR mutant lacking the ligand-binding domain. This suggested that both compounds stimulated PR binding to target DNA in the cell (28). These conflicting results were proposed to be due to an ability of the PR-antagonist complex to gain access to PREs present as multiple copies on extra chromosomal plasmids, but not to PREs integrated as a single copy into chromatin. Our results with stably integrated and transiently transfected promoter interference constructs do not agree with this conclusion. Why RU486 and ZK98299 both failed to stimulate an in vivo PR genomic footprint is not clear (29). This may be a property of the specific integration locus. However, we found similar results with antagonists using multiple different clones of T47D containing stably integrated promoter interference constructs (data not shown).
Because the mechanism by which receptor interaction disrupts the activity of promoter interference constructs is not precisely known, we used two alternative approaches to confirm the observed effects of progesterone antagonists on PR interaction with DNA in whole cells. One approach used a previously engineered cell line (T47D/A1-2) in which PR-DNA interaction is detected as an inhibition of GR-agonist trans-activation of a GRE/PRE controlled reporter gene. The other assessed the abilities of ligands to activate a PR-VP16 chimeric receptor. Results with both of these approaches were in agreement with promoter interference assays. In A1-2 cells, RU486 and ZK98299 both inhibited GR-agonist induction of MMTV-LUC under conditions where both ligands interacted minimally with GR. Because PR-ZK98299 and PR-RU486 complexes do not themselves trans-activate, inhibition of GR activity is consistent with PR competing with GR for binding to GRE/PREs in cells treated with either ZK98299 or RU486. When COS-1 cells were cotransfected with a PR-VP16 chimeric receptor, ZK98299 and RU486 both stimulated PR trans-activation of a positive PRE-controlled reporter gene, indicating that PR-VP16 was bound to DNA within cells. Thus, by all three experimental approaches, ZK98299 enhanced PR interaction with target DNA sequences in whole cells. This implies that ZK98299 does not inactivate PR by failing to promote PR binding to DNA as initially thought, but must block a step(s) downstream of DNA binding. One possible contributing factor for why these transfection experiments are capable of detecting ZK98299 stimulation of PR interaction with PREs when previous in vitro EMSAs have not is that the transfection assays are more sensitive. Transfection assays are dynamic and measure changes in CAT accumulation over many hours, whereas EMSA detects binding at a single time point.
Metzger et al. (50) also used an ER-VP16 chimeric receptor as an approach to test the effects of different classes of antiestrogens on ER binding to EREs within the cell. Both ICI 164,384 and hydroxytamoxifen induced trans-activation of a chimeric ER containing the VP16 acidic activation domain in place of the amino-terminal domain of ER. This indicates that ER-VP16 binds to DNA in the presence of both classes of antiestrogens. Because receptor sequence regions outside the DNA-binding domain can influence DNA binding, it could not be unequivocally concluded from these results whether wild-type receptor could also bind DNA in response to antiestrogen treatment of cells. Therefore, it was also shown that wild-type ER can bind to EREs within the cell in response to both classes of antiestrogens, as measured by ICI or hydroxytamoxifen inhibition of a GAL4-VP16 activator on a reporter gene that contains overlapping EREs and GAL4-binding sites (50). Full-length PR was used in all of our transient transfection experiments, and endogenous PR was used in stable transfected T47D cells. Thus, the present results are consistent with wild-type PR being capable of interacting with PREs in whole cells in response to two classes of progestin antagonists.
To attempt to explain the apparent discrepancy between previous reports that ZK98299 failed to stimulate binding of PR to PREs in vitro and the observation of the present report that ZK98299 stimulated PR binding to PREs in whole cells, EMSA conditions were altered to attempt to detect ZK98299-induced DNA complexes. We postulated that PR-DNA complexes in the presence of ZK98299 may be less stable than those induced by other PR ligands, such that they are not maintained during electrophoresis. By simply reducing the ionic strength of the DNA-binding reaction and adding glycerol to the gels, ZK98299 induction of PR-PRE complexes was detected in vitro. Under these conditions, unliganded PR continued to exhibit little or no specific DNA binding, and ZK98299-induced DNA complexes were specific, as shown by supershift with PR-specific mAbs and by competition with appropriate excess unlabeled DNA. Examination of PR off-rates from DNA provided a possible reason why this complex has escaped previous detection in vitro. Under altered EMSA conditions, the rate of PR dissociation from PRE in the presence of ZK98299 was faster than that in the presence of R5020. In previous studies, we and others showed that PR-DNA complexes formed in the presence of RU486 exhibited a slower dissociation from PREs than complexes formed in the presence of agonist (3, 17). Therefore, it appears that R5020, RU486, and ZK98299 have different effects on the stability of PR-PRE complexes in vitro, with a descending order of RU486 > R5020 > ZK98299.
Differences in off-rate and stability could also be explained by distinct effects of ligands on receptor conformation. We and others have previously shown that PR-DNA complexes in the presence of RU486 and other structurally related antagonists exhibit a faster electrophoretic gel mobility than that of agonists (3, 16, 17, 47). We now show that the PR-ZK98299 complex bound to DNA exhibits a slower mobility than the PR-RU486 complex. Ligand-induced differences in electrophoretic mobilities in nondenaturing gels have also been shown with ER-DNA complexes in the presence of estrogen, hydroxytamoxifen, and ICI 164,385. Each complex exhibits a distinct mobility, indicating that ER conformation is differentially altered by estrogen and both classes of antiestrogens (49, 50).
There is increasing evidence that steroid agonists and antagonists induce distinct conformational changes within the ligand-binding domains of ER and PR (18, 19, 31, 32, 51, 52). It also appears that ER and PR are able to assume multiple different conformations depending on the nature of the ligand. Several classes of ER ligands and three classes of PR ligands have been identified based on distinct alterations in receptor structure (51, 52). Interestingly, these different classes of ligands display pure agonist, pure antagonist, or varying degrees of mixed agonist/antagonist activity, suggesting a relationship between ligand-induced receptor structure and biological activity (51, 52). The idea that ligands with strong antagonist activity can be further separated into mechanistic classes based on inducing distinct conformations within the receptor is supported in the present study by the different electrophoretic mobilities exhibited by PR-DNA complexes in the presence of ZK98299 or RU486. The results of partial proteolytic digestion assays have also indicated that PR assumes a different conformation upon binding ZK98299 and RU486. Allan et al. (31) screened a wide variety of proteases, finding that clostripain generated different PR proteolytic peptide maps when bound to ZK98299 and RU486. Additionally, analysis of receptor mutants suggested that RU486 and ZK98299 differentially expose the carboxyl-terminal transcriptional activation domain function-2 to proteolytic attack (31). Similar studies by Clemm et al. (32) showed subtle differences in limited tryptic digestion patterns when PR was bound to ZK98299 or RU486. These studies taken together indicate that progesterone antagonists can be classified based upon their inductions of distinct conformations within PR. This ability to induce multiple different conformations may be a central mechanism to explain the biological activities of different classes of antagonists, as different conformations may modulate how PR interacts with downstream coactivators/corepressors or with general transcription factors. The aim in classifying progesterone antagonists is to correlate these compounds with pharmacological activity for endocrine therapies. Further classification of antagonists is expected to evolve as pharmacological and molecular mechanisms continue to be elucidated.
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Acknowledgments |
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Footnotes |
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Received August 14, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Substituted
analogs of the antiprogestin RU486 induce a unique conformation in the
human progesterone receptor resulting in mixed agonist activity. Proc
Natl Acad Sci USA 93:8739–8744This article has been cited by other articles:
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