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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 receptor’s 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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Partial Agonist Properties of Estriol
The human ER (hER) bound both estradiol and estriol in a positive cooperative manner (Fig. 1Go), 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 2Go 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.



View larger version (16K):
[in this window]
[in a new window]
 
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 ({circ}) binding experiment.

 


View larger version (17K):
[in this window]
[in a new window]
 
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 ({circ}). The specifically bound [3H]estradiol was measured after 3 h incubation at 25 C, as described in Materials and Methods.

 
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 3Go 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.



View larger version (16K):
[in this window]
[in a new window]
 
Figure 3. ERE-Sepharose Chromatography of hER

[3H]Estradiol- (•) and [3H]estriol-bound hER ({square}) 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 90–100%.

 
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. 4Go. 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).



View larger version (41K):
[in this window]
[in a new window]
 
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 3–8); and in the presence of hER complexes with 5 x 10-10-10-6 M estriol (lanes 9–14). B, Percent transcription vs. increasing estradiol (•) or estriol ({circ}) 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.

 
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. 5Go). 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.



View larger version (74K):
[in this window]
[in a new window]
 
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.

 
Figure 6Go 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.



View larger version (19K):
[in this window]
[in a new window]
 
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. 2Go and 5Go.

 
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 (0.1–30 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 1Go). 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.


View this table:
[in this window]
[in a new window]
 
Table 1. Effect of cVitll ERE on the Equilibrium Binding of [3H]Estradiol and hER

 
Figure 7Go 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.



View larger version (15K):
[in this window]
[in a new window]
 
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 1Go, performed over a wide range of hER concentrations without (•) or with ({circ}) 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.

 
Both [3H]estradiol and [3H]estriol interacted with 4 nM hER in a partially cooperative manner (Table 2Go). 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.


View this table:
[in this window]
[in a new window]
 
Table 2. Effect of Estriol on the Hill Coefficient of the Equilibrium Binding of [3H]Estradiol to the hER

 
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 (1–20 nM) were incubated with saturating concentrations of estradiol or estriol (10–200 nM) and allowed to interact with labeled ERE probes in an EMSA. Figure 8Go 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 (1–6 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.



View larger version (35K):
[in this window]
[in a new window]
 
Figure 8. EMSA for the hER-cVitII ERE Interaction

Increasing concentrations of hER were incubated with saturating concentration (10–200 nM) of estradiol (•) or estriol ({circ}) 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.

 
The inhibitory effect of estriol on the interaction of hER-ERE is presented in Fig. 9Go. 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 5–7): 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).



View larger version (50K):
[in this window]
[in a new window]
 
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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 4Go) 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. 2Go), 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. 1Go). 3) The association constant of the interaction between the estriol-hER complex and ERE was half that of estradiol-hER complexes (Fig. 3Go), but twice that of unliganded hER (12). 4) Estriol-hER-ERE complexes exhibited retarded mobility compared with estradiol-containing complexes in EMSA (Figs. 8Go and 9Go). These findings are compatible with the suggestion of Lewis et al. (15) that the 16{alpha}-hydroxyl group of estriol interferes with the 17{alpha}-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. 1Go). ERE augmented the positive cooperative binding interaction of [3H]estradiol only at 1:0.5 hER/ERE molar ratio (Table 1Go). 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 7Go 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 2Go), 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. 4Go). 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. 5Go). In parallel, estriol effectively decreased the binding interaction of estradiol-hER with cVitII ERE, as shown in EMSA (Fig. 9Go). 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. 2Go). Under these conditions the hER-dependent transcription was partially recovered (Fig. 5Go).

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 2Go). 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. 6Go): 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 receptor’s 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. 3Go). 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 1–6 nM of hER estradiol- or estriol-hER complexes, this interaction was minimal, while at 6–20 nM it increased in a hER concentration-dependent manner (Fig. 8Go). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
The 17ß-[6,7-3H]estradiol (47.2 Ci/mmol) and {alpha}-[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 {gamma}-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). Grace’s 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 259–278 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. O’Malley, (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 {gamma}-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 {gamma}-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. Back

2 Member of The David R. Bloom Center for Pharmacy at the Hebrew University of Jerusalem. Back

Received for publication July 15, 1997. Accepted for publication August 28, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Hisaw FLJ 1959 Comparative effectiveness of oestrogens on fluid imbibation and growth of the rat’s uterus. Endocrinology 64:276–289
  2. Clark JH, Markaverich BM 1984 The agonist and antagonist actions of estriol. J Steroid Biochem 20:1005–1013[CrossRef][Medline]
  3. Clark JH, Paszko Z, Peck EJ 1977 Nuclear binding and retention of the receptor estrogen complex: relation to the agonistic and antagonistic properties of estriol. Endocrinology 100:91–96[Abstract]
  4. Anderson JN, Peck EJ, Clark JH 1975 Estrogen-induced uterine responses and growth: relationship to receptor estrogen binding by uterine nuclei. Endocrinology 96:160–167[Abstract]
  5. Huggins C, Jensen EV 1955 The depression of oestrone-induced uterine growth by phenolic oestrogens with oxygenated functions at position 6 or 16: the impeded oestrogens. J Exp Med 102:335–346[Medline]
  6. Sasson S, Notides AC 1983 Estriol and estrone interaction with the estrogen receptor. I. Temperature-induced modulation of the cooperative binding of [3H]estriol and [3H]estrone to the estrogen receptor. J Biol Chem 258:8113–8117[Abstract/Free Full Text]
  7. Sasson S, Notides AC 1984 Inability of [3H]estriol to induce maximal cooperativity of the estrogen receptor. J Steroid Biochem 20:1027–1032[CrossRef][Medline]
  8. Sasson S, Notides AC 1983 Estriol and estrone interaction with the estrogen receptor. II. Estriol and estrone-induced inhibition of the cooperative binding of [3H]estradiol to the estrogen receptor. J Biol Chem 258:8118–8122[Abstract/Free Full Text]
  9. Sasson S, Notides AC 1984 The estriol-induced inhibition of the estrogen receptor’s positive cooperativity. J Steroid Biochem 20:1021–1026[CrossRef][Medline]
  10. Notides AC, Lerner N, Hamilton DE 1981 Positive cooperativity of the estrogen receptor. Proc Natl Acad Sci USA 78:4926–4930[Abstract]
  11. Korenman SG 1970 Relation between estrogen inhibitory activity and binding to cytosol of rabbit and human uterus. Endocrinology 87:1119–1123
  12. Melamed M, Arnold SF, Notides AC, Sasson S 1996 Kinetic analysis of the interaction of human estrogen receptor with an estrogen response element. J Steroid Biochem Mol Biol 57:153–159[CrossRef][Medline]
  13. Obourn JD, Koszewski NJ, Notides AC 1993 Hormone and DNA binding mechanisms of the recombinant human estrogen receptor. Biochemistry 32:6229–6236[Medline]
  14. Katzenellenbogen BS 1984 Biology and receptor interactions of estriol and estriol derivatives in vitro and in vivo. J Steroid Biochem 20:1033–1037[CrossRef][Medline]
  15. Lewis FV, Parker MG, King RJB 1995 Molecular modeling of the human estrogen receptor and ligand interactions based on site-directed mutagenesis and amino acid sequence homology. J Steroid Biochem Mol Biol 52:55–65[CrossRef][Medline]
  16. Sabbah M, Redeuilh G, Baulieu EE 1989 Subunit composition of the estrogen receptor. J Biol Chem 274:2397–2400
  17. Reese JC, Katzenellenbogen BS 1992 Examination of DNA-binding ability of estrogen receptor in whole cells: implications for hormone independent transactivation and action of antiestrogens. Mol Cell Biol 12:4531–4538[Abstract]
  18. Klein-Hitpass L, Tsai Y, Greene GL, Clark JH, Tsai M-J, O’Malley BW 1989 Specific binding of estrogen receptor to the estrogen response element. Mol Cell Biol 9:43–49[Medline]
  19. Kumar V, Chambon P 1988 The oestrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell 55:145–156[Medline]
  20. Metzger D, Berry M, Ali S, Chambon P 1995 Effect of antagonists on DNA binding properties of the human estrogen receptor in vitro and in vivo. Mol Endocrinol 9:579–591[Abstract]
  21. Wong C, Zhou Z, Sar M, Wilson EM 1993 Steroid requirement for androgen receptor dimerization and DNA binding. J Biol Chem 268:19004–19012[Abstract/Free Full Text]
  22. Tsai S, Carlstedt-Duke J, Weigel NL, Dahlman K, Gustafsson J-A, Tsai M-J, O’Malley BW 1988 Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation. Cell 55:361–369[Medline]
  23. Berg JM 1989 DNA binding specificity of steroid receptors. Cell 57:1065–1068[Medline]
  24. Fritsch M, Welch RD, Murdoch FE, Anderson I, Gorski J 1992 DNA allosterically modulates the steroid binding domain of the estrogen receptor. J Biol Chem 267:1823–1828[Abstract/Free Full Text]
  25. Lefstin J, Thomas JR, Yamamoto KR 1994 Influence of steroid receptor DNA-binding domain on transcriptional regulatory functions. Genes Dev 8:2842–2856[Abstract]
  26. Sabbah M, Le Ricousee S, Redeuilh G, Baulieu EE 1992 Estrogen receptor-induced bending of the Xenopus vitellogenin A2 gene hormone response element. Biochem Biophys Res Commun 185:944–952[Medline]
  27. Predki PF, Sarkar B 1995 Cooperative interaction of oestrogen receptor ’zinc finger’ domain polypeptides on DNA binding. Biochem J 305:805–810[Medline]
  28. Klinge CM, Peale FVJ, Hilf R 1992 Cooperative estrogen receptor interaction with consensus or variant estrogen responsive elements in vitro. Cancer Res 52:1073–1081[Abstract]
  29. Carlsson B, Häggblad J 1995 Quantitative determination of DNA-binding parameters for the human estrogen receptor in a solid-phase nonseparation assay. Anal Biochem 232:172–179[CrossRef][Medline]
  30. Martinez E, Wahli W 1989 Cooperative binding of estrogen receptor to imperfect estrogen-responsive DNA elements correlates with their synergistic hormone-dependent enhancer activity. EMBO J 8:3781–3791[Abstract]
  31. Chang TC, Nardulli AM, Lew D, Shapiro DJ 1992 The role of estrogen-response elements in expression of the Xenopus laevis vitellogenin-B1 gene. Mol Endocrinol 6:346–354[Abstract]
  32. Bocquel M-T, Ji J, Ylikomi T, Benhamou B, Vergezac A, Chambon P, Gronemeyer H 1993 Type II antagonists impair the DNA binding of steroid hormone receptors without affecting dimerization. J Steroid Biochem Mol Biol 45:205–215[CrossRef][Medline]
  33. Levitz M, Kander S, Young BK 1984 Intermediary metabolism of estriol in pregnancy. J Steroid Biochem 20:971–974[CrossRef][Medline]
  34. Peter M, Dorr HG, Stippell WG 1994 Changes in the concentrations of dehydroepiandrosterone sulfate and estriol in maternal plasma during pregnancy: a longitudinal study in healthy women throughout gestation and term. Horm Res 42:278–281[Medline]
  35. De Hertogh R, Bietlot TY, Vanderheyden I, Ferin J 1975 Plasma levels of unconjugated estrone, estradiol, and estriol and of HCS throughout pregnancy in normal women. J Clin Endocrinol Metab 40:93–101[Medline]
  36. Simpson ER, MacDonald PC 1981 Endocrine physiology of the placenta. Annu Rev Physiol 43:163–188[CrossRef][Medline]
  37. Diczfalusy E 1984 The early history of estriol. J Steroid Biochem 20:945–953[CrossRef]
  38. Cole P, MacMahon B 1969 Oestrogen fractions during early reproductive life in the aetiology of breast cancer. Lancet 1:604–609[CrossRef][Medline]
  39. Gross J, Modan B, Bertini B, Spira O, de Waard F, Thijssen JH, Vestergaard P 1977 Relationship between steroid patterns and breast cancer incidence in Israeli women of various origins. J Natl Cancer Inst 59:7–11[Medline]
  40. Lemon HM 1975 Estriol prevention of mammary carcinoma induced by 7,12-dimethylbenz(a)anthracene. Cancer Res 35:1341–1353[Abstract]
  41. Zumoff B 1994 Hormonal profiles in women with breast cancer. Obstet Gynecol Clin North Am 21:751–773[Medline]
  42. Jozan S, Kreitmann B, Bayard F 1981 Different effects of oestradiol, oestriol, oestrol and of oestrone on human breast cancer cells (MCF-7) in long term tissue culture. Acta Endocrinol (Copenh) 98:73–80[Medline]
  43. Lippman M, Monaco ME, Bolan G 1977 Effects of estrone, estradiol. and estriol on hormone-responsive human breast cancer in long-term tissue culture. Cancer Res 37:1901–1907[Abstract]
  44. Castaño E, Vorojeikina DP, Notides AC 1997 Phosphorylation of serine 167 on the human estrogen receptor is important for ERE-binding and transcriptional activation. Biochem J 326:149–157[Medline]
  45. Bagchi MK, Tsai SY, Wigel NL, Tsai M-J, O’Malley BW 1990 Regulation of in vitro transcription by progesterone receptor. J Biol Chem 265:5129–5134[Abstract/Free Full Text]
  46. Sasson S, Notides AC 1982 The inhibition of estrogen receptor’s positive cooperative [3H]estradiol bind-ing by the antagonist, clomiphene. J Biol Chem 257:11540–11545[Abstract/Free Full Text]
  47. Koszewski NJ, Notides AC 1991 Phosphate-sensitive binding of the estrogen receptor to its response element. Mol Endocrinol 5:1129–1136[Abstract]
  48. Kadonaga JT, Tjian R 1986 Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci USA 83:5889–5893[Abstract]
  49. Pavlik EJ, Coulson PB 1976 Hydroxylapatite "batch" assay for estrogen receptors: increased sensitivity over present receptor assays. J Steroid Biochem 7:357–368[CrossRef][Medline]
  50. deHaseth PL, Gross CA, Burgess RR, Record MT 1977 Measurement of binding constant for protein-DNA interaction by DNA-cellulose chromatography. Biochemistry 16:4777–4782[Medline]
  51. Jackson TA, Richer, JK, Bain DL, Takimoto GS, Tung L, Horwitz KB 1997 The partial agonist activity of antagonist-occupied steroid receptors is controlled by a novel hinge domain-binding coactivator L7/SPA and the corepressor N-CoR or SMRT. Mol Endocrinol 11:693–705[Abstract/Free Full Text]
  52. Smith CL, Nawaz Z, O’Malley BW 1997 Coactivator and corepressor regulation of agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. Mol Endocrinol 11:657–666[Abstract/Free Full Text]