Molecular and Kinetic Basis for the Mixed Agonist/Antagonist Activity of Estriol
Michal Melamed,
Enrique Castaño,
Angelo C. Notides1 and
Shlomo Sasson2
Department of Pharmacology (M.M., S.S.) School of Pharmacy
Faculty of Medicine Hebrew University of Jerusalem Jerusalem
91120, Israel Departments of Environmental Medicine and
Biophysics & Biochemistry (E.C., A.C.N.) University of
Rochester School of Medicine and Dentistry Rochester, New York
14642
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ABSTRACT
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Estriol acts as a weak estrogen when administered
in a single dose into immature or ovariectomized laboratory animals,
but produces full estrogenic responses upon chronic administration.
However, when estriol is injected together with estradiol it acts as an
antiestrogen. We studied the dual agonist/antagonist properties of
estriol, using recombinant human estrogen receptor (hER) in
ligand-binding assay, cell-free transcription assay, electrophoretic
mobility shift assay with cVitII estrogen response element (ERE), and
ERE-Sepharose chromatography. We show that the weak estrogenic activity
of estriol results from impaired hER-ERE interaction. The
antiestrogenic activity of estriol was demonstrated in a cell-free
transcription assay where it reduced estradiol-dependent transcription
in a dose-dependent manner. Estriol interfered with estradiol-induced
positive cooperative binding and receptor dimerization, and binding of
hER complexes to ERE. These effects of estriol were maximal at a
10-fold molar excess over estradiol; under these conditions
estradiol-dependent transcription was decreased by 85%, although
[3H]estradiol binding was reduced by only
50%. We propose that when hER, estradiol, and estriol are
coequilibrated, several receptor species are formed: unliganded hER
monomers and dimers; estradiol-hER monomers and dimers, estriol-hER
monomers and dimers; and presumably mixed estradiol-estriol dimers.
Since estrogen-hER complexes bind cooperatively to ERE sequences, the
concentrations of transcriptionally active complexes (estriol- and
estradiol-hER dimers) are reduced to low levels that fail to bind
cooperatively with ERE and initiate transcription. We discuss our
results in relation to the massive estriol production during pregnancy
and to the "Estriol Hypothesis" on the protective role for estriol
in opposing carcinogenic effects of estradiol.
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INTRODUCTION
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Estriol has been described as a weak or short-acting estrogen.
When administered in a single dose into ovariectomized or immature
rats, it produces partial uterotropic effects compared with the full
response under a similar treatment with estradiol (1, 2). The nuclear
retention time of estriol in estrogen-sensitive tissues is
significantly shorter than that of estradiol (2, 3); nevertheless, a
full estrogenic response to estriol is observed when it is administered
continuously into similar animals (3, 4). The antagonistic activity of
estriol is evident when an excess of it is simultaneously injected with
estradiol into immature rats: under these conditions estriol diminishes
the uterotropic effects of estradiol (2, 5).
Early studies showed that the binding affinity of estriol to the
estrogen receptor (ER) was slightly lower than that of estradiol (2).
The binding interaction of estriol with calf uterine ER was found to be
partially cooperative in comparison with the full positive cooperative
interaction of estradiol (6, 7). It was concluded that estriol had an
altered capacity to activate the receptor and induce suitable
conformational changes in the receptor complex required for a full
interaction with estrogen-sensitive genes. The antagonistic properties
of estriol were correlated to its capacity to abolish the positive
cooperative binding interaction of estradiol with the receptor (8, 9).
These studies also showed that the ability of estriol to decrease the
receptors cooperativity was not proportionally related to its
capacity to compete with [3H]estradiol binding. Thus,
50% inhibition of [3H]estradiol binding by estriol was
sufficient to entirely abolish the positive cooperative interaction of
estradiol with the receptor, despite the fact that half of the binding
sites were occupied with estradiol and that the remaining sites bound
the weaker agonist, estriol (8, 9).
These early studies suggest that estriol-induced changes in the ER lead
to an altered receptor conformation that fails to interact properly
with promoters of specific genes. However, this model does not explain
the full agonist activity of continuously administered estriol, nor
does it clarify its antiestrogenic activity. The present study was
conducted to investigate the duality of estriol action at the level of
the ER and its interaction with an estrogen response element (ERE).
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RESULTS
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Partial Agonist Properties of Estriol
The human ER (hER) bound both estradiol and
estriol in a positive cooperative manner (Fig. 1
), indicative of estrogen-dependent
receptor homodimerization (6, 10). The magnitude of the positive
cooperative interaction of each estrogen with the receptor, reflected
by the Hill coefficient (nH), was dependent on receptor concentration.
The efficacy of estradiol and estriol in inducing maximal positive
cooperative interaction was similar (nH =1.38 and 1.42, respectively),
and was attained at receptor concentrations higher than 8
nM. However, the potency of the two estrogens was
different: half maximal nH values
[0.5x(nHmax-1.0)] were obtained at 2 and 4
nM hER for estradiol and estriol binding, respectively.
Figure 2
shows that the affinity of
estriol to the hER was approximately 10-fold lower than that of
estradiol, as measured in a competitive binding assay (EC50
values: 2.5 x 10-7 and 4 x 10-8
M, respectively). The relative association constant of the
interaction of estriol with hER, calculated according to Korenman (11),
was 12.5% that of estradiol.

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Figure 1. Relationship between hER Concentration and Hill
Coefficient of [3H]Estradiol and
[3H]Estriol Equilibrium Binding
Summary of equilibrium binding experiments performed as described in
Materials and Methods. Each point
represents an individual [3H]estradiol () or
[3H]estriol ( ) binding experiment.
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Figure 2. Competition between [3H]Estradiol and
Estriol for the Binding to hER
Human ER (10 nM) was incubated with
[3H]estradiol (100 nM) and the indicated
concentrations of unlabeled estradiol () or estriol ( ). The
specifically bound [3H]estradiol was measured after
3 h incubation at 25 C, as described in Materials and
Methods.
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ERE-Sepharose isocratic salt chromatography was employed to study the
kinetics and determine the association constants of the interaction
between estradiol- and estriol-hER complexes and their cognate cVitII
ERE. Figure 3
depicts the elution
profiles of [3H]estradiol- and
[3H]estriol-hER complexes, prepared under equilibrium
conditions at 25 C, and eluted from the column under identical
conditions. The association constant of estriol-hER interaction with
ERE was half that of estradiol-hER: 42.0 x 106
M-1 and 83.3 x 106
M-1, respectively.

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Figure 3. ERE-Sepharose Chromatography of hER
[3H]Estradiol- () and [3H]estriol-bound
hER ( ) complexes (15 nM), prepared for 1 h at 25 C,
were eluted from the ERE-Sepharose column as described in
Materials and Methods. The recovery of receptor eluted
from the column was 90100%.
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The capacity of estradiol- and estriol-hER complexes to induce
transcription of specific G-free transcripts from the expression vector
pEC(7) in vitro is shown in Fig. 4
. The transcriptional activation induced
by both complexes was dose-dependent. The efficacy of both complexes in
inducing transcription was similar, but the potency of estriol was
10-fold lower than that of estradiol (EC50 values: 4
x 10-8 and 4 x 10-9 M,
respectively). It is important to note that the transcripts from the
control plasmid MLP-C2AT(190), a major late promoter plasmid with
G-free cassette of 190 bp made of repetition of CCAT, were not
increased by unliganded or liganded hER. Thus, it was used as a
standard to normalize the transcription data of pEC(7). Preincubation
of hER with anti-hER antibody caused an 80% decrease in specific
pEC(7) transcripts (data not shown).The addition of a 100-fold molar
excess of ERE-oligonucleotide in the assay reduced transcription by
90% (data not shown).

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Figure 4. Cell-Free Transcriptional Activation by Estradiol-
and Estriol-hER Complexes
The hER (9 nM) was incubated (1 h at 20 C) with increasing
concentrations of estradiol or estriol, and the hER-dependent
transcription of the pEC(7) template was assayed in
vitro as described in Materials and Methods. A,
In vitro transcripts from the pEC(7) and MLP-C2AT(190)
templates in the absence of hER (lane 1); in the presence of unliganded
hER (lane 2); in the presence of hER complexes with
10-9-10-7 M estradiol (lanes
38); and in the presence of hER complexes with 5 x
10-10-10-6 M estriol (lanes
914). B, Percent transcription vs. increasing
estradiol () or estriol ( ) concentrations. Data are plotted as
the relative intensity of each band as determined by volume integration
and normalized to the intensity of the MLP-C2AT(190) transcripts. 100%
indicates transcriptional activity with 10-7 M
estradiol. The background activity with unliganded hER was subtracted
from each value.
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Antagonist Properties of Estriol
Estriol displayed a potent antagonistic activity when it was
present together with estradiol (at 1:1 to 1:10 estradiol-estriol molar
ratio) in the cell-free transcription assay (Fig. 5
). Estriol decreased the specific
production of pEC(7) transcripts in a dose-dependent manner: the
greatest inhibition was observed at 1:10 estradiol-estriol molar ratio
(85% reduction in comparison to the control). However, at a 50-fold
molar excess of estriol, no further inhibition of transcriptional
activity was detected; on the contrary, it was restored to 64% of the
control level.

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Figure 5. Effect of Estriol on Estradiol-Dependent
Transcription of pEC(7) Template
The hER (9 nM) was incubated (3 h at 20 C) with 100
nM estradiol in the absence or presence of increasing
estriol concentrations, and the transcription assay was carried as
described in Materials and Methods. The background
activity with unliganded hER was subtracted from each value. 100%
indicates transcriptional activity with 10-7 M
estradiol. Mean ± SEM (n = 2).
Inset, pEC(7) and MLP-C2AT(190) transcripts.
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Figure 6
compares the ability of estriol
to compete with [3H]estradiol binding with hER and to
inhibit estradiol-dependent transcription. While at a 10-fold molar
excess, estriol competed for only 50% of estradiol-binding sites, it
nearly abolished transcription. Similar disproportional ability of
estriol to suppress estradiol-induced transcription and compete for
binding to hER was also evident at lower estradiol-estriol molar
ratios. Nevertheless, at 50-fold molar excess of estriol this pattern
was reversed: the [3H]estradiol binding was decreased to
minimum, but the transcriptional activity was partially restored.

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Figure 6. Comparison of the Effects of Estriol on Equilibrium
Binding of [3H]Estradiol with hER and on hER-Dependent
Transcription
The data were retrieved from the experiments presented in Figs. 2 and 5 .
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ERE Promotes Estradiol-hER, but Not Estriol-hER, Positive
Cooperative Binding Interaction
To further investigate the dual agonist/antagonist activity of
estriol, we designed the following binding experiments in the presence
of cVitII ERE. Recombinant hER (3 nM) was preincubated with
double-stranded ERE-containing sequences for 30 min at 4 C. The molar
ratio of hER/ERE varied from 1:0 to 1:10. Labeled estradiol was then
added (0.130 nM), and the mixtures were further incubated
for 1 h at 25 C. The bound [3H]estradiol was
separated from the free, and the binding data were analyzed according
to Hill (Table 1
). As expected, the
binding interaction of [3H]estradiol with 3
nM hER in the absence of ERE was partially cooperative
(nH = 1.23). However, preincubation of hER with ERE at 1:0.5 molar
ratio augmented the positive cooperativity of estradiol-hER binding to
maximum (nH = 1.42). Higher excess of hER over ERE did not alter
the binding interaction of estradiol, which remained partially
cooperative, with nH values similar to the control.
Figure 7
summarizes similar experiments
over a wide range of hER concentrations without or with preincubation
of the receptor with cVitII ERE at 1:0.5 hER/ERE molar ratio. The
control curve shows a correlation between hER concentration and the
degree of cooperativity, similar to previously reported data (12, 13).
Preincubation of hER with ERE increased the [3H]estradiol
cooperative binding to its maximal level over the entire range of hER
concentrations, even at a concentration as low as
1 nM.
The nH of estradiol binding in the absence of ERE was already maximal
at 8 nM hER, and preincubation of the hER with ERE did not
increase it further. Therefore, concentrations of 4 nM hER
and 2 nM ERE were chosen to study the effect of estriol in
this experimental system.

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Figure 7. The Effect of cVitII ERE on the Relationship
between hER Concentration and Hill Coefficient of Equilibrium
[3H]Estradiol Binding
Summary of experiments, similar to those presented in Table 1 ,
performed over a wide range of hER concentrations without () or with
( ) a prior incubation (30 min at 4 C) of hER with cVitII ERE (1:0.5
hER/ERE molar ratio). Each point represents an
individual experiment.
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Both [3H]estradiol and [3H]estriol
interacted with 4 nM hER in a partially cooperative manner
(Table 2
). However, while cVitII ERE
increased the nH of [3H]estradiol-hER interaction to
maximum, it did not exert such an effect on
[3H]estriol-hER complexes. However, estriol, at a 10-fold
molar excess over estradiol, abolished both the basal and the
ERE-augmented positive cooperative binding of
[3H]estradiol, reducing the Hill coefficient value to
approximately 1. The corresponding Scatchard plots became linear rather
than convex (data not shown). Hence, estriol transformed estradiol
binding interaction to simple and noncooperative and rendered ERE
inactive in augmenting the positively cooperative
[3H]estradiol binding. We have employed a previously
described control experiment (8) and incubated hER with
[3H]estradiol without or with 1-fold molar excess of
unlabeled estradiol. Under these conditions the specific binding of
[3H]estradiol was reduced by 50%, similar to the
inhibition caused by estriol at a 10-fold molar excess. Unlike the
effect of estriol, the binding interaction of
[3H]estradiol in the presence of unlabeled estradiol
remained positively cooperative, as in the control (data not shown).
This indicates that the loss of the positive cooperative interaction
between the hER and [3H]estradiol in the presence of
unlabeled estriol was not due to isotopic dilution of
[3H]estradiol in the binding assay.
Effects of Estriol on the Mobility of hER-ERE Complex in an
Electrophoretic Mobility Shift Assay (EMSA)
The kinetics of the interactions of estradiol- and estriol-hER
complexes with ERE was investigated. Increasing hER concentrations
(120 nM) were incubated with saturating concentrations of
estradiol or estriol (10200 nM) and allowed to interact
with labeled ERE probes in an EMSA. Figure 8
shows that: 1) the mobility of
estriol-hER-ERE complexes was retarded in comparison with that
estradiol-hER-ERE complexes. 2) The relative binding interaction of
estriol-hER complexes with ERE was approximately 50% of that of
estradiol-hER complexes. 3) The binding kinetics of both types of
complexes with ERE was nonlinear and biphasic: the first phase in which
the interaction of both types of complexes was minimal (16
nM hER), and a second, linearly increasing phase, above 6
nM hER. The respective intensities of estradiol and estriol
complexes with 20 nM hER were 10- and 7-fold stronger than
those observed at 6 nM hER. The first two observations
indicate that each estrogen induces a distinctive conformation in hER
that determines its stereospecific interaction with ERE.

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Figure 8. EMSA for the hER-cVitII ERE Interaction
Increasing concentrations of hER were incubated with saturating
concentration (10200 nM) of estradiol () or estriol
( ) for 1 h at 20 C, then further incubated with
32P-labeled cVitII ERE probe and electrophoresed as
described in Materials and Methods. Data are plotted as
the relative intensity of each band as determined by volume
integration. Inset, Autoradiographed bands.
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The inhibitory effect of estriol on the interaction of hER-ERE is
presented in Fig. 9
. The mobility of
estradiol- and estriol-hER complexes differed significantly from each
other (lanes 3 and 4) and from that of the unliganded receptor (lane
2). When estriol was present together with estradiol, it reduced the
intensity of estradiol-hER-ERE interaction in a dose-dependent manner
(lanes 57): at 10-fold molar excess, estriol reduced it by nearly
70% (compare lanes 7 and 3). No additional bands, corresponding to
specific estriol-hER complexes interacting with ERE, were evident in
these lanes. However, at 50-fold molar excess of estriol, the only band
detected corresponded to that of the estriol-hER-ERE band (compare
lanes 8 and 4). Treatment of the receptor with anti-hER serum
supershifted the complex (lane 1). Similar results were obtained with
50 nM hER and saturating concentrations of estradiol,
estriol, and estradiol-estriol mixtures (data not shown).

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Figure 9. Effect of Estriol on the Electrophoretic Mobility
of hER-cVitII ERE Complexes
Human ER (9 nM) was incubated (3 h at 20 C) with anti-ER
serum (Ab), 100 nM estradiol (E2), 100
nM estriol (E3), or with 100 nM
estradiol in the presence of estriol at the indicated molar excess,
then further incubated with [32P]-labeled cVitII ERE and
electrophoresed as described in Materials and Methods.
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DISCUSSION
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The results of the present study have yielded findings that
clarify the dual agonist/antagonist activity of estriol. These results
demonstrate that the impeded estrogen activity of estriol results from
estriol-specific conformational changes in hER entailing an impaired
interaction with its cognate ERE. The antiestrogenic properties of
estriol are explained by the complex kinetics of the interaction
between hER, estradiol, estriol, and ERE.
Estriol as a Weak Estrogen Agonist
The weak estrogen activity of estriol was demonstrated in a
cell-free transcription assay (Fig. 4
) where it was 1 order of
magnitude less potent than estradiol in producing transcripts from the
expression vector pEC(7). Several findings indicate that this impeded
action of estriol results from altered conformational changes in
estriol-hER complex that affect its interaction with ERE: 1) The
relative association constant of estriol with hER was 12.5% that of
estradiol (Fig. 2
), similar to that reported by Katzenellenbogen (14)
for human endometrium ER. 2) The potency of estriol in inducing
positive cooperativity in hER, and thus promoting receptor
dimerization, was half that of estradiol, though the efficacy of both
estrogens was similar (Fig. 1
). 3) The association constant of the
interaction between the estriol-hER complex and ERE was half that of
estradiol-hER complexes (Fig. 3
), but twice that of unliganded hER
(12). 4) Estriol-hER-ERE complexes exhibited retarded mobility compared
with estradiol-containing complexes in EMSA (Figs. 8
and 9
). These
findings are compatible with the suggestion of Lewis et al.
(15) that the 16
-hydroxyl group of estriol interferes with the
17
-hydrogen bond to glutamate-419 in hER, hindering hydrogen bonding
at the D ring and engendering an altered receptor conformation. Other
experimental approaches, such as comparing the protease digestion
pattern of hER-estradiol and estriol complexes, may be used further to
examine the conformations of these complexes.
It has been shown that the ligand-free ER may exist as a dimer and that
estradiol augments its further dimerization (16, 17). Evidence for an
interaction of ligand-free ERs with ERE sequences and subsequent
ligand-induced dimerization comes from other studies (12, 18, 19, 20). Our
study also shows that ligand-free hER further dimerizes upon the
addition of estradiol or estriol (respective dimerization constants: 2
and 4 nM hER, Fig. 1
). ERE augmented the positive
cooperative binding interaction of [3H]estradiol only at
1:0.5 hER/ERE molar ratio (Table 1
). At higher ratios each ERE sequence
may bind a single hER monomer rather than two, thus negating the
condition for an efficient receptor subunit dimerization on a single
ERE. Figure 7
shows that in the presence of hER and ERE (at 1:0.5 molar
ratio) and estradiol, but not estriol, the dimerization constant of hER
significantly decreased to less than 1 nM. This results
from binding of two ligand-free hER monomers and/or a dimer to a single
palindromic ERE sequence (19, 21, 22, 23). This proximity of receptor
monomers accelerates their dimerization and subsequent conformational
changes upon the addition of estradiol, as reflected by the increased
Hill coefficient of estradiol binding at very low hER concentrations.
These observations agree with the findings of Fritsch et al.
(24) and Lefstin et al. (25), who have suggested that ERE
acts as an allosteric modulator of functional domains in ER, necessary
for receptor activation and subsequent transcriptional function. Sabbah
et al. (26) have also suggested ER-dependent bending of cVit
ERE, which augments the association of the receptor to ERE and
subsequent dimerization. In contrast to estradiol binding, the degree
of the positive cooperativity of estriol-hER interaction was not
augmented in the presence of ERE (Table 2
), suggesting a distinct
conformation of estriol-hER complexes inadequate for such ERE-dependent
allosteric modulations.
Chronic administration of estriol produces full estrogenic responses in
animal models tested (2). Indeed, estriol-hER complexes, despite their
unfavorable conformation, had a similar efficacy as estradiol-hER
complexes in inducing transcriptional activation function (Fig. 4
).
Thus, although the main factors responsible for the weak estrogenicity
of estriol upon acute treatments are the low association constant
between estriol-hER and ERE and the inability of ERE to augment
dimerization of estriol-hER complexes, it is clear that the continuous
presence and availability of estriol-hER complex for interaction with
ERE are sufficient for long-term activation of the transcriptional
machinery and full estrogenic responses (2).
Estriol as an Estrogen Antagonist
The antiestrogenic activity of estriol in the presence of
estradiol was demonstrated in the cell-free transcription assay where
it reduced the production of specific pEC(7) transcripts in a
dose-dependent manner (Fig. 5
). In parallel, estriol effectively
decreased the binding interaction of estradiol-hER with cVitII ERE, as
shown in EMSA (Fig. 9
). Nevertheless the impeded interaction of
estradiol-hER complex with ERE in the presence of estriol was not
accompanied with an appearance of specific estriol-hER-ERE complexes.
These complexes reappeared only when estriol was added at a 50-fold
molar excess over estradiol. At this molar ratio more than 90% of hER
binding sites are occupied by estriol (Fig. 2
). Under these conditions
the hER-dependent transcription was partially recovered (Fig. 5
).
These data indicate that the antagonistic effects of estriol may result
from its ability to inhibit the positive cooperativity and subsequent
dimerization of estradiol-hER complexes, both in the absence or
presence of ERE in the binding assay (Table 2
). However, a
disproportional correlation exists between the capacity of estriol to
compete with [3H]estradiol binding and inhibit the
positive cooperative interactions and its ability to decrease
hER-dependent transcription (Fig. 6
): between 1:1- and 1:10-fold molar
excess estriol over estradiol, the magnitude of estriol-induced
inhibition of transcription exceeded that of the inhibition of
[3H]estradiol-specific binding. These findings agree with
previous studies that showed a similar discrepancy in the ability of
estriol to compete with estradiol for binding and to diminish the
receptors positive cooperativity (8, 9). It is therefore clear that
the antagonistic properties of estriol cannot be explained merely by
simple competition between estradiol and estriol for hER-binding
sites.
To explain this disproportional correlation we suggest that when
estradiol and estriol are present together, a complex equilibrium is
attained among several receptor species: unliganded hER monomers and
dimers; estradiol-hER monomers and dimers; estriol-hER monomers and
dimers; and possibly estriol-estradiol-hER dimers. The relative
abundance of each complex is directly dependent on the
estradiol-estriol molar ratio. Moreover, these distinct receptor
species bind to ERE sequences with different affinities, as indicated
by ERE-Sepharose chromatography (Fig. 3
). In addition, we have recently
shown (12) a hER dose-dependent effect on the magnitude of the
association constant of estradiol-hER interaction with ERE, where
half-maximal and maximal association constant (Ka) values
were obtained at hER concentrations of 3 nM and >7
nM hER, respectively (12). Therefore, the association of
transcriptionally active estradiol- and estriol-hER complexes with ERE
is reduced when estriol competes with estradiol for binding. These
complex kinetics are apparent in the biphasic interaction of
estradiol- and estriol-hER complexes with ERE: between 16
nM of hER estradiol- or estriol-hER complexes, this
interaction was minimal, while at 620 nM it increased in
a hER concentration-dependent manner (Fig. 8
). Previous studies
(27, 28, 29, 30, 31) also show that the binding of estrogen-hER complexes to ERE is
biphasic and possibly synergistic and/or positively cooperative.
Carlsson and Häggblad (29) have suggested that such nonlinear
kinetics result from the binding of one estradiol-hER monomer to an
ERE, which increases the binding of a second monomer and allows
dimerization.
We propose that the first phase correlates to receptor concentrations
that allow only partial positive cooperativity of estradiol- and
estriol-hER interactions and weak association with ERE, while receptor
concentrations within the second phase support full positive
cooperativity and stronger interaction with ERE. Thus, the
concentrations of transcriptionally active dimeric hER complexes, at
1:10 estradiol-estriol molar ratio, are reduced to the range that fail
to (estriol-hER) or partially (estradiol-ERE) interact cooperatively
with ERE. Below and above this ratio, the respective concentrations of
estradiol- or estriol-hER complexes are high enough and fall within the
second phase of interaction with ERE.
We postulate that a conformationally distorted mixed
estradiol-estriol-hER dimer, incapable of interacting with ERE, is also
formed. In fact, we could not detect such a complex in an EMSA even
with 50 nM hER (data not shown). Bocquel et al.
(32) proposed such mixed-ligand dimers, incapable of interacting with
response elements, for the human progesterone receptor incubated with
the agonist R5020 and the antagonist ZK98,299. If such
estradiol-estriol mixed dimers are formed, the relative concentrations
of estradiol and estriol-hER complexes are further reduced to levels
that preclude efficient hER-ERE interaction and subsequent
transcription.
Endogenous corepressors and coactivators of transcription have been
proposed as regulators of steroid receptors transcriptional
activation. Jackson et al. (51) have recently suggested that
pharmacological agents can function as antagonists or partial agonist
depending on the relative abundance of such regulatory factors at the
promoter-bound receptor. Smith et al.(52) have recently
shown that the expression of coactivators and corepressors can activate
or inhibit 4-hydroxytamoxifen-induced transcription. It remains to be
investigated whether such factors interact and regulate promoter-bound
estriol-hER and further direct its agonist or antagonist
activities.
Physiological Aspects
During late human pregnancy the most abundant estrogen is estriol,
predominantly produced in the placenta from fetal androgen precursors
and secreted into the maternal circulation (33, 34). The ratio of
estriol to estradiol and estrone is nearly 1 in nonpregnant women, and
it rises to 10 and more in late pregnancy (34, 35, 36). In his article on
the early history of estriol Diczfalusy (37) writes "... an
estrogen with relatively weak binding and short elimination half-life
is the ideal compound for modulating estrogen action under special
circumstances, such as gestation." Our study suggests that estriol,
at distinct concentrations and excess over estradiol, can effectively
protect maternal estrogen-sensitive tissues from excessive
estrogen-dependent stimuli by virtue of its antiestrogenic activity,
while at a high enough excess over estradiol it may act as a weaker
estrogen agonist.
A controversy exists regarding the "Estriol Hypothesis,"
which ascribes a protective role to estriol as an antiestrogen opposing
carcinogenic effects of estradiol. The hypothesis proposed by Cole and
MacMahon (38) was supported by clinical and epidemiological studies
(39) and studies in animal models (40). However, this hypothesis has
been contested by other clinical observations (41) and work done mostly
in cultured breast cancer cells (42, 43). We suggest that some of these
results be reconsidered in light of the model presented above. Estriol
is not expected to effectively antagonize estradiol unless the molar
ratio of estradiol to estriol falls within a narrow range (
1:10).
Below this ratio, estradiol acts as a potent estrogen, being only
partially or minimally antagonized by estriol. Above this ratio,
estriol displaces estradiol from the majority of ER-binding sites,
leading to the formation of predominantly estriol-ER dimers capable of
inducing partial estrogenic responses.
 |
MATERIALS AND METHODS
|
---|
Materials
The 17ß-[6,7-3H]estradiol (47.2 Ci/mmol) and
-[32P]-UTP (800 Ci/mmol) were purchased from DuPont
New England Nuclear (Boston, MA). The [2,4-3H]estriol
(20.0 Ci/mmol) was obtained from Rotem Industries (Beer-Sheva, Israel).
Activated charcoal, bovine
-globulin, BSA, chymostatin,
CNBr-activated Sepharose, dithiothreitol (DTT), EDTA, EGTA,
glycerol, leupeptin, HEPES, ovalbumin, pepstatin,
phenylmethylsulfonyl fluoride, and salmon sperm DNA were obtained from
Sigma (St. Louis, MO). Graces insect culture medium and yeast
transfer RNA were from GIBCO BRL (Grand Island, NY). Yeastolate and
lactalbumin were obtained from Difco (Detroit, MI). FCS serum was
purchased from Biological Industries (Kibbutz Beth-Haemek, Israel).
Nucleoside triphosphates and 3'-O-methylGTP were purchased
from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Ribonuclease
(RNase) T1 was purchased from Boehringer Mannheim (Indianapolis, IN),
RNase inhibitor from Arapahoe (Boulder, CO), and HeLa cell nuclear
extract from Promega (Madison, WI). The anti-hER antibody 6 used is a
polyclonal antibody made by immunizing rabbits with a peptide
corresponding to amino acids 259278 of hER. All other chemicals were
reagent grade.
Purification of Recombinant hER from Sf9 Cells
The production of recombinant baculovirus, AcNVP-hER, carrying
the complementary DNA of the hER has been described (13). The
recombinant hER was partially purified by ammonium sulfate (40%)
precipitation of Sf9 cell lysates as described (13).
Cell-Free Transcription Assay
The pEC(7) plasmid was constructed as described (44) by removing
the fragment between the EcoRI and BglII sites of
pLovTATA and cloning in the fragment of the vitellogenin II sequence
from position -744 to -313, which includes a perfect ERE. The plasmid
MLP-C2AT(190) [provided by B. W. OMalley, (45)] contains
guanine free-sequences that are not digested by RNase T1. Incubation of
hER (9 nM) was carried out at 20 C for 1 h in the
absence or presence of 100 nM estradiol and/or estriol, and
the cell-free transcription assay was carried out as described (44).
The transcript pellet was dissolved in loading dye mixture [10
mM EDTA, 98% (vol/vol) formamide, 0.1% (wt/vol) xylen
cyanol, and 0.1% (wt/vol) bromophenol blue] and heated at 90 C for 10
min. It was then loaded on a 10% acrylamide-urea gel and run at 500 V
for 5 h. The gel was then dried and relative band intensities were
quantified by volume integration, using Image Quant software and
Phosphoimager SF (Molecular Dynamics, Sunnyvale, CA).
Equilibrium Binding Analysis of
[3H]Estrogen Binding
Ammonium sulfate precipitates of the hER were dissolved in TDEE
Buffer (40 mM Tris-HCl, pH 7.4, 1 mM DTT, 1
mM EDTA, 1 mM EGTA) containing 0.1
mM phenylmethylsulfonyl fluoride, 0.2 mM
leupeptin, 1 µg/ml chymostatin, 1 µg/ml pepstatin, 10% (vol/vol)
glycerol, 0.2 M KCl, and 10 mg/ml
-globulin. The
equilibrium binding assay was performed as previously described (12).
The effect of estriol on the positive cooperative binding of
[3H]estradiol was measured as described (46). Briefly,
estriol was added at a fixed molar ratio relative to each
[3H]estradiol concentration and equilibrated with the
receptor for 3 h at 25 C. The nonspecific binding was less than
5% of total bound [3H]estradiol. Determination of
specific [3H]estrogen binding and Scatchard and Hill
analyses were performed as previously described (6). Stability of the
receptor was assayed in each binding experiment as described previously
(6).
The effect of ERE on these binding interactions was measured in a
similar binding assay. The hER was first incubated with ERE (annealed
5'-GTCCAAAGTCAGGTCACAGTGACCTGATCAAAGTT-3' and
5'-AACTTTGATCAGGTCACTG-TGACCTGACTTTGGAC-3' nucleotide
sequences) for 30 min at 4 C, then bound to estrogens as described
above.
EMSA
End-labeled 35-bp probe encoding the cVitII ERE was prepared as
described (47). The receptor was dissolved in TDEE containing 10
mM NaF, 5 mM Na2PO4, 1
mM NaVO3, and 10% (vol/vol) glycerol, bound to
estradiol and/or estriol and mixed (1:10 ratio) with
32P-labeled probe in 20 mM HEPES, pH 7.5, 0.2
mM EDTA, 0.5 mM DTT, 100 mM KCl, 1
mM MgSO4, 0.5 mM leupeptin, 10%
(vol/vol) glycerol, 25 mg/ml BSA, 0.25 µM
32P-labeled probe and 125 µg/ml
poly(deoxyinosinic-deoxycytidylic)acid. After incubation (30 min at 4
C), samples (20 µl) were loaded on a prerun 5% polyacrylamide gel
and electrophoresed, as described (47).
Preparation of ERE-Sepharose
ERE-Sepharose was prepared as described by Kadonaga and Tjian
(48). Complementary ERE-containing nucleotide sequences
(5'-GTCCAAAGTCAGGTCACAGTGACCTGATCAAAGTT-3' and
5'-AACTTTGATCAGGTCACTGT-GACCTGACTTTGGAC-3') were annealed,
ligated, and coupled to CNbr-activated Sepharose 4B as described (13, 48). Phosphorous content in the ERE-Sepharose was 10.5 µg/ml packed
gel, as determined by inductivity-coupled plasma atomic emission
spectrometry. Thus, the calculated concentration of double-stranded ERE
sites was 6.51 nmol/ml packed gel.
ERE-Sepharose Chromatography
The partially purified hER was dissolved in TEED buffer
containing 0.2 mM leupeptin, 10% (vol/vol) glycerol, 1
µg/ml pepstatin, 1 µg/ml chymostatin, and 10 mg/ml bovine
-globulin and incubated with [3H]estradiol or
[3H]estriol as indicated. At the end of the incubation
period, 2 ml of the binding mixture were loaded on a 1 ml ERE-Sepharose
column, allowed to equilibrate, and eluted as previously described
(12). The concentration of hER in the collected fractions was
determined by the hydroxylapatite adsorption assay (49). The
Ka of the hER-ERE interaction was calculated according to
deHaseth et al. (50), using the equation Ka
= V/k ·DT, where V is the volume of
each collected fraction, DT is the concentration of
double-stranded ERE coupled to the column, and k is the
slope of the exponential elution profile.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Shlomo Sasson, Ph.D., Department of Pharmacology, Hebrew University School of Medicine, P.O. Box 12272, Jerusalem 91120, Israel.
This work was supported in part by grants from the Israel Ministry of
Health, The Israel Cancer Association (to S.S.), and NIH Grants
HD-06707 and ES-01247 (to A.C.N.)
1 The authors dedicate this paper to their beloved mentor and friend,
Angelo C. Notides, who passed away November 10, 1996. 
2 Member of The David R. Bloom Center for Pharmacy at the Hebrew
University of Jerusalem. 
Received for publication July 15, 1997.
Accepted for publication August 28, 1997.
 |
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