The effect of antiestrogens on the nuclear binding of the estrogen receptor

Experiments were designed to determine whether or not various antiestrogens in direct competition with estradiol-17β (E₂) would inhibit the translocation of the estrogen receptor complex from the cytoplasm to nuclei in rat uterine tissue. Incubation of the antiestrogens CI-628, $\text{cis}$-clomiphene, U-11,100A and MER-25 with rat uteri caused the nuclear uptake of the antiestrogen receptor complex which was greatest for most antiestrogens at concentrations of 1 × 10^?6 to 1 × 10^?5M. At higher concentrations of CI-628, $\text{cis}$-clomiphene, and U-11,100A the nuclear binding of the antiestrogen receptor complex was greatly decreased. Incubation of the antiestrogens with E₂ resulted in a dramatic inhibition of the nuclear uptake of the estrogen receptor. $\text{Trans}$-clomiphene, a weak estrogen, did not inhibit the movement of the uterine cytoplasmic receptor into the nuclear fraction.     

Differential interactions of estrogens and antiestrogens at the 17 beta-hydroxy or counterpart function with the estrogen receptor

The action of diethylpyrocarbonate on lamb uterine estrogen receptor produced an homogeneous population of the receptor (approximately 55%) which still bound triarylethylene antiestrogens such as 4-hydroxytamoxifen with a high affinity but bound classical potent estrogens such as estradiol or diethylstilbestrol with a very low affinity. To specify the structural features of the ligands involved in the decrease of ligand affinity upon modification of the estrogen receptor, we determined the relative affinity constants of 17 steroidal estrogens or antiestrogens (deriving from estradiol by a 7 alpha- or 11 beta-substitution) and 14 nonsteroidal estrogens or antiestrogens (all including the 1,2-trans-diphenylethylene structure of diethylstilbestrol) for native and diethylpyrocarbonate-modified estrogen receptors. Then the ratio of the relative affinity constant for the native receptor to that for the modified receptor (rho) was calculated for each ligand, to compare the variation in the affinity of the ligand upon modification of the receptor to that of 4-hydroxytamoxifen (rho = 1). The results showed that the strong decrease of ligand affinity upon modification of the receptor displayed by classical estrogens (rho greater than or equal to 200) is strictly dependent on the presence of the 17 beta-hydroxyl group in steroidal compounds or its alpha-4- and beta-4-counterparts in diethylstilbestrol-related compounds. However, for the 7 alpha- or 11 beta-derivatives of estradiol displaying potent antiestrogenic properties, the relative decrease in affinity was much more limited (rho less than or equal to 19). For 11 beta-derivatives displaying a relative estrogenic activity weaker than that of estradiol itself, an average decrease in affinity was observed (23 less than or equal to rho less than or equal to 62). With the diethylstilbestrol-related compounds, bearing or not the alpha-4-hydroxyl and/or the beta-4-hydroxy functions and showing either weak relative estrogenic or antiestrogenic properties, the relative variation in affinity was weak (0.6 less than or equal to rho less than or equal to 24). These results indicate that the interaction of 7 alpha- or 11 beta-substituted steroidal antiestrogens and of 1,2-trans-diphenylethylene or triphenylethylene derivatives, displaying either weak relative estrogenic or antiestrogenic properties with the receptor, differs at the 17 beta-hydroxy or at the alpha-4-/beta-4-hydroxy functions from that of potent estrogens. They suggest that the strong decrease in the relative affinity of ligands upon receptor modification may reflect the high efficiency of the ligands to activate the receptor properly.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antagonistic effect of triphenylethylenic antiestrogens on the association of estrogen receptor to calmodulin.

Binding of (3H)-estradiol labeled estrogen receptor from uterine cytosol to calmodulin was demonstrated by both affinity chromatography and sucrose gradient sedimentation. Triphenylethylene antiestrogens (tamoxifen family) with strong antagonistic activity against the calmodulin-dependent c-AMP phosphodiesterase largely reduced the binding of the receptor. Relevance of this observation with regard to the major antiproliferative activity (cytotoxicity) of these drugs is discussed.     

Differential interactions of estrogens and antiestrogens at the 17 beta-hydroxyl or counterpart hydroxyl with histidine 524 of the human estrogen receptor alpha

We investigated the role of H524 of the human estrogen receptor alpha (ERalpha) for the binding of various estrogens [estradiol (E(2)), 3-deoxyestradiol (3-dE(2)), and 17beta-deoxyestradiol (17beta-dE(2))] and antiestrogens [4-hydroxytamoxifen (OHT), RU 39 411 (RU), and raloxifene (Ral)], which possess the 17beta-hydroxyl or counterpart hydroxyl (designated: 17beta/c-OH), with the exception of 17beta-dE(2) and OHT. The work involved a comparison of the binding affinities of these ligands for wild-type and H524 mutant ERs, modified or not with diethyl pyrocarbonate (DEPC), a selective histidine reagent. Alanine substitution of H524 did not significantly change the association affinity constant (relative to OHT) of 17beta-dE(2), whereas those of RU, Ral, E(2), and 3-dE(2) were decreased 3-fold, 14-fold, 24-fold, and 49-fold, respectively. Values of the two ligands available in radiolabeled form (E(2) and OHT) were correlated with the dissociation rate constants, which were increased 250-fold and 2-fold, respectively. The action of DEPC on wild-type ER led to a homogeneous ER population which still bound antiestrogens and 17beta-dE(2) with practically unchanged affinities (less than 4-fold decreases in relative affinity constants), while E(2) and 3-dE(2) displayed markedly decreased affinities (56-fold decrease for E(2)). Conversely, DEPC treatment of H524A mutant ER did not induce marked decreases in the relative affinities of any of the checked compounds (decreases wild-type ER) and very weakly protected H524A ER. Molecular modeling was tentatively used to interpret the biochemical results.     

Interaction of non-steroidal antiestrogens with dopamine receptor binding

The ability of various estrogen antagonists and agonists to compete with [3H]spiroperidol, [3H]domperidone, [3H]dihydroalprenolol, [3H]dihydroergocryptine, [3H]dopamine or [3H]5-hydroxytryptamine for binding to membrane preparations from rat brain tissue was tested. The non-steroidal triphenylethylene-type antiestrogens with an amine side chain--enclomiphene, nitromifene, tamoxifen and zuclomiphene--were found to be competitive inhibitors of [3H]spiroperidol (Kd = 0.12 nM; Bmax = 101 fmol/mg protein) and [3H]domperidone (Kd = 0.62 nM; Bmax = 86 fmol/mg protein) binding to striatal membranes. The Ki values ranged from 4-12 microM. Estradiol-17 beta (Ki = 480 microM) or diethylstilbestrol (Ki = 63 microM) were much less effective inhibitors exhibiting noncompetitive interaction with the in vitro binding of [3H]spiroperidol. The pharmacological relevance of the antiestrogen interactions with dopamine receptor binding is discussed with respect to adverse effects of the in vivo administered compounds such as nausea and vomiting.     

2,5-Diphenylfuran-based pure antiestrogens with selectivity for the estrogen receptor alpha

The estrogen receptor alpha (ERalpha) is understood to play an important role in the progression of breast cancer. Therefore, pure antiestrogens with a preference for this receptor form are of interest as new agents for the treatment of this malignancy. Several chemical structures with selective binding affinity for ERalpha have been identified and might be useful for the synthesis of ERalpha-selective pure antiestrogens. In this study we applied the 2,5-diphenylfuran system which is closely related to the triphenylfurans described by others. Various side chains with amino and/or sulfur functions were linked to C3 to convert the furans to estrogen antagonists without residual estrogenic activity. The degree of alpha-selectivity which ranges from 2.5- to 236-fold is strongly influenced by the alkyl group at C4. Antiestrogenic potency was determined in MCF-7/2a breast cancer cells stably transfected with a luciferase gene under the control of an ERE. The 2,5-bis(4-hydroxyphenyl)furan with an ethyl substituent and a 6-[N-methyl-N-(3-pentylthiopropyl)amino]hexyl side chain exerted the strongest antiestrogenic effect in this series with an IC(50) value of 50 nM in cells stimulated with 1 nM estradiol. The RBA values of this derivative were 18% (ERalpha) and 3.4% (ERbeta) of estradiol, respectively. It inhibited the growth of wild-type MCF-7 cells with an IC(50) value of 22 nM. The data show that the 2,5-diphenylfuran system is appropriate for the development of pure antiestrogens with preference for ERalpha.     

The binding of estrogen and estrogen antagonists to the estrogen receptor.

The model of the estrogen receptor as a dimer of identical, interacting subunits and data obtained by Sasson and Notides (1988, Mol. Endocrinol. 2, 307-312) were used to find the standard free energy changes that describe the binding of estradiol and 4-hydroxytamoxifen to the estrogen receptor. For the binding of estradiol or 4-hydroxytamoxifen to the estrogen receptor the data do not deviate systematically from the best fit to the model. The standard free energy change for binding of one molecule of estradiol at one site and one molecule of 4-hydroxytamoxifen at the second site of estrogen receptor indicates that 4-hydroxytamoxifen antagonizes the binding of estradiol to the estrogen receptor.     

Differential activation of wild-type estrogen receptor alpha and C-terminal deletion mutants by estrogens, antiestrogens and xenoestrogens in breast cancer cells

17beta-Estradiol (E2), diethylstilbestrol (DES) and several synthetic (or xenoestrogenic) compounds induced transactivation in MCF-7 or MDA-MB-231 cells transfected with wild-type estrogen receptor alpha (ERalpha) and a construct (pERE(3)) containing three tandem estrogen responsive elements (EREs) linked to a luciferase gene. In contrast, the antiestrogens ICI 182,780 and 4-hydroxytamoxifen (4-OHT) were inactive in this assay. We have investigated the effects of these compounds and several structurally-diverse estrogenic compounds on transactivation in cells transfected with pERE(3) and wild-type ERalpha, mutant ERalpha (1-553), and ERalpha (1-537) containing deletions of amino acids 595-554 and 595-538, respectively. These constructs were used to develop an in vitro assay to distinguish between different structural classes of estrogenic compounds. The results obtained using these constructs were highly cell context- and structure-dependent. Neither E2- nor diethylstilbestrol-induced transactivation in MCF-7 (or MDA-MB-231) cells transfected with pERE(3)/ERalpha (1-537) due to partial deletion of helix 12; however, octylphenol and nonlylphenol, resveratrol (a phytoestrogen), kepone and 2',3',4',5'-tetrachloro-4-biphenylol were "estrogenic" in MCF-7 cells transfected with pERE(3)/ERalpha (1-537). Moreover, the structure-dependent estrogenic activities of several synthetic estrogens (xenoestrogens) in MDA-MB-231 cells were different than those observed in MCF-7 cells. These results demonstrate that the estrogenic activity of many synthetic compounds do not require activation function 2 (AF-2) of ERalpha and are mechanistically different from E2. These data suggest that xenoestrogens are selective ER modulators (SERMs).     

Specific binding of estrogen receptor to the estrogen response element.

Gene transfer studies have shown that estrogen regulation of specific genes is mediated by estrogen response elements (ERE). We report that binding of the estrogen receptor to the ERE can be detected by a gel retardation (band shift) assay. This binding interaction was highly sequence and receptor specific. Methylation interference analysis showed that the ERE contact sites of estrogen receptor displayed a perfect twofold rotational symmetry. This is compatible with estrogen receptor binding to the ERE as a head-to-head dimer.     

Differential DNA-binding abilities of estrogen receptor occupied with two classes of antiestrogens: studies using human estrogen receptor overexpressed in mammalian cells.

We have developed a transient transfection system using the Cytomegalovirus (CMV) promoter to express the human estrogen receptor (ER) at very high levels in COS-1 cells and have used it to study the interaction of agonist and antagonist receptor complexes with estrogen response element (ERE) DNA. ER can be expressed to levels of 20-40 pmol/mg or 0.2-0.3% of total soluble protein and all of the soluble receptor is capable of binding hormone. The ER binds estradiol with high affinity (Kd 0.2 nM), and is indistinguishable from native ER in that the receptor is capable of recognizing its cognate DNA response element with high affinity, and of transactivating a transgene in an estradiol-dependent manner. Gel mobility shift assays reveal interesting ligand-dependent differences in the binding of receptor complexes to ERE DNA. Receptors occupied by estradiol or the type I antiestrogen transhydroxytamoxifen bind to DNA response elements when exposed to the ligand in vitro or in vivo. Likewise, receptors exposed to the type II antiestrogen ICI 164,384 in vitro bind to ERE DNA. However, when receptor exposure to ICI 164,384 is carried out in vivo, the ER-ICI 164,384 complexes do not bind to ERE DNA, or do so only weakly. This effect is not reversed by subsequent incubation with estradiol in vitro, but is rapidly reversible by in vivo estradiol exposure of intact COS-1 cells. This suggests there may be some cellular process involved in the mechanism of antagonism by the pure antiestrogen ICI 164,384, which is not observed in cell-free extracts.     

Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha

The activation function 2/ligand-dependent interaction between nuclear receptors and their coregulators is mediated by a short consensus motif, the so-called nuclear receptor (NR) box. Nuclear receptors exhibit distinct preferences for such motifs depending both on the bound ligand and on the NR box sequence. To better understand the structural basis of motif recognition, we characterized the interaction between estrogen receptor alpha and the NR box regions of the p160 coactivator TIF2. We have determined the crystal structures of complexes between the ligand-binding domain of estrogen receptor alpha and 12-mer peptides from the Box B2 and Box B3 regions of TIF2. Surprisingly, the Box B3 module displays an unexpected binding mode that is distinct from the canonical LXXLL interaction observed in other ligand-binding domain/NR box crystal structures. The peptide is shifted along the coactivator binding site in such a way that the interaction motif becomes LXXYL rather than the classical LXXLL. However, analysis of the binding properties of wild type NR box peptides, as well as mutant peptides designed to probe the Box B3 orientation, suggests that the Box B3 peptide primarily adopts the "classical" LXXLL orientation in solution. These results highlight the potential difficulties in interpretation of protein-protein interactions based on co-crystal structures using short peptide motifs.     

Life-long effect of a single neonatal treatment with estradiol or progesterone on rat uterine estrogen receptor binding capacity.

Rats treated with a single dose of 17 beta-estradiol or progesterone within 24 h of birth were subjected to ovariectomy at 8 weeks of age and were nine days later examined for the binding capacity of the uterine estradiol receptors by saturation and competition tests (with diethylstilbestrol used as competitor). The Bmax value of the neonatally estradiol-treated rats (6.78 x 10(-10) M) was significantly decreased relative to the control (1.99 x 10(-9) M). The competition analysis affirmed these results. Neonatal progesterone treatment also accounted for a significant decrease (1.25 x 10(-9) M) in receptor concentration relative to the control (1.66 x 10(-9) M). Considering the competition analysis the decrease was less than in the case of estradiol and not even significant by saturation analysis. The uterine mass did not differ between the experimental and control rats, but part of those treated with estradiol developed ovarian cysts. It follows that not only synthetic steroids (DES, allylestrenol), but also an excessive presence of the physiological steroid hormone during the critical period of receptor maturation can account for a decrease in uterine receptor concentration in adulthood.     

Role of estrogen receptors and antiestrogen binding sites in an early effect of antiestrogens, the inhibition of cholesterol biosynthesis

The effects of estradiol (E2), 4-hydroxy-tamoxifen (OH-Tam), and LY117018 on cholesterogenesis were investigated in two human breast cancer cell lines (MCF-7 and BT20), and in rat hepatoma (HTC) and fibroblastic (NRK-49F) cell lines. It was found that 10(-10) M E2 stimulated and 10(-8) M OH-Tam inhibited cholesterol synthesis in the estrogen-sensitive MCF-7 cell line. The OH-Tam effect occurred in less than 15 min whereas E2 only stimulated after 8 h. The inhibition of cholesterol synthesis was not reversed by E2. E2 was without effect in the HTC and estrogen-resistant BT20 cell lines whereas OH-Tam was as effective as in the MCF-7 cells. LY117018 had nearly as much effect on cholesterol synthesis as OH-Tam, in both MCF-7 and BT20 cells. Neither E2 nor OH-Tam had any effect on the NRK-49F cell line, even at micromolar concentrations. The three lines (MCF-7, BT20, HTC), whose cholesterol synthesis has been shown to be OH-Tam sensitive, appeared to contain high-affinity antiestrogen binding sites (AEBS); since the OH-Tam-resistant line (NRK) only contained low-affinity AEBS, there appears to be some relationship between OH-Tam sensitivity and high-affinity AEBS content. This suggests that the cholesterogenesis inhibition induced by antiestrogens is ER-independent and may involve AEBS. The cholesterogenesis stimulation induced by E2 occurred via a different pathway that appears to be related to the presence of ER in the cells.     

The effect of TGF-beta receptor binding peptides on smooth muscle cells

TGF-beta1 is a potent regulator of vascular smooth muscle cell (VSMC) proliferation, migration, and extracellular matrix (ECM) synthesis. In this study, we selected two peptides, IM-1 and IM-2, that bind to the TGF-beta type II receptor (TGF-beta RII) using phage display. IM-1 and IM-2 bind to the TGF-beta RII, with a K(d) of 1 microM. Like TGF-beta, IM-1 induced VSMC chemotaxis and PAI-1 mRNA expression, as determined using Boyden chambers and real time quantitative PCR. In contrast, IM-2 had no effect on VSMC chemotaxis or PAI-1 induction. Induction of ECM synthesis, involving proteins such as osteopontin and alpha-smooth muscle actin, was determined by ELISA. Osteopontin expression was inhibited by both peptides, but TGF-beta-induced alpha-smooth muscle actin expression could only be inhibited by IM-1. In conclusion, IM-1 activity on VSMC is agonistic with TGF-beta, except for ECM synthesis, whereas the IM-2 peptide is antagonistic for some examined TGF-beta functions.