Conformational transitions of the estrogen receptor monomer. Effects of estrogens, antiestrogen, and temperature

The technique of aqueous two-phase partitioning has been used to study changes in estrogen receptor (ER) structure that occur upon ligand binding and/or heating in vitro. Studies with steroidal and nonsteroidal ligands indicate that the difference in partitioning properties between unoccupied and nontransformed ER is due to a ligand-induced change in this conformation of the protein. Furthermore, this conformational change is only partially induced by binding of 4-OH-tamoxifen. Although nontransformed 4-OH-tamoxifen complexes can be transformed by heat, there are significant differences in the transformation process for receptors bound to 4-OH-tamoxifen versus estrogenic ligands. A kinetic analysis of estrogen receptor transformation indicates that the process follows apparent first order kinetics, but is 2.5-fold slower for the 4-OH-tamoxifen-receptor complex. Direct heating of the unoccupied ER causes a significant change in receptor structure. Ligand binding to the heat-altered unoccupied receptor results in a further alteration of receptor structure. Experiments using polyethylene glycol palmitate indicate that the ligand-binding transition is associated with a reduction of the hydrophobic characteristics of the receptor. These results demonstrate that there are a number of independent conformational changes that occur within the monomeric ER steroid-binding subunit upon ligand binding and exposure to elevated temperature in vitro.     

Estrogen and antiestrogen interaction with estrogen receptor of MCF-7 cells--relationship between processing and estrogenicity

Overnight preincubation of MCF-7 cells with 2 x 10(-10) M estradiol (E2) produces a dramatic reduction of their specific [3H]E2 binding capacity. Scatchard plot analysis revealed that this loss of estrogen receptor (ER) concentration, usually termed "processing", occurs without any significant modification of binding properties of the unprocessed receptors. Direct measurement of ER (ER-EIA from Abbott) gave residual receptor concentrations close to those established by binding assay indicating that processing involves the loss of at least one epitope other than the steroid binding site. Incubation with increasing amounts of E2 (0.1 to 5 x 10(-10) M) resulted in an increasing reduction of binding capacity indicating that the extent of processing is associated with the hormone concentration. Steroidal estrogens other than E2 as well as antiestrogens of the triphenylethylene category behaved similarly in this regard although the latter compounds usually acted only when at higher concentrations. The processing capacity of a large series of ligands was compared with the corresponding binding affinity for ER as assessed by classical competitive inhibition of [3H]E2 binding in both cytosol and whole cells. For steroidal estrogens, a large spectrum of concordant values was found which correlated with the known uterotrophic activity of the compounds. On the contrary, weak estrogen and antiestrogens of the triphenylethylene category displayed low processing capacities which were in the order of magnitude of the binding affinities established in whole cells; these values were considerably lower than the corresponding values measured in the cytosol. These observations are consistent with the concept that the capacity of a ligand to process ER is related to its agonistic activity. They also support our hypothesis (J. steroid Biochem. 25 (1986) 677-682) that assessment of the ability of a ligand to inhibit the binding of [3H]E2 in whole cells provides an estimate of its agonistic activity, an estimate which can not be established in the corresponding cytosol assay.     

Evidence of an estrogen receptor form devoid of estrogen binding ability in MCF-7 cells

In MCF-7 breast cancer cells, hydroxytamoxifen (OH-Tam) up-regulates the estrogen receptor (ER) in a form unable to bind [(3)H]estradiol (E(2)). We show here that this property is not restricted to this antiestrogen. [(3)H]E(2) binding assays (whole cell assays, DCC assays on cell extracts) and enzyme immunoassays (Abbott) performed in parallel, establish the permanent presence of such unusual ERs in the absence of any exposure of the cells to a ligand. E(2) and the pure antiestrogen RU 58 668, which down-regulate ER, also decrease [(3)H]E(2) binding. In control cells, these ERs represent about the half of the whole receptor population; they also display a tendency to stabilize within the cell nucleus. Loss of E(2) binding ability appears irreversible, since we failed to label receptor accumulated under OH-Tam with [(3)H]E(2) or [(3)H]tamoxifen aziridine (TAZ). Cycloheximide (CHX), which blocks E(2)-induced down regulation of ER, failed to stabilize [(3)H]E(2) binding (whole cell assay) after an [(3)H]E(2) pulse (1 h), confirming that regulation of E(2) binding and peptide level are related to different regulatory mechanisms. Loss of binding ability could not be ascribed to any ER cleavage as demonstrated by Western blotting with a panel of ER antibodies raised against its various domains (67 kDa ER solely detected). We propose that loss of E(2) binding ability is related to the aging process of the receptor, i.e. it is progressively converted to a form devoted to degradation after it has accomplished its physiological role. Ligands may favor (E(2), RU 58 668) or impede (OH-Tam) this elimination process.     

The antiestrogen ICI 182,780 decreases the expression of estrogen receptor-alpha but has no effect on estrogen receptor-beta and androgen receptor in rat efferent ductules

Background  

The antiestrogen ICI 182,780 has been used successfully as an alternative experimental model for the study of estrogen action in the rodent adult male reproductive tract. Although ICI 182,780 causes severe alterations in testicular and efferent ductule morphology and function, the effects on the expression of estrogen and androgen receptors in the male have not been shown.     

Electrophoretic analysis of the estrogen receptor. Relationship of multiple receptor forms to the molybdate-stabilized form

The origin of and relationships among multiple forms of the estrogen receptor from rat uteri were investigated using electrophoretic and conventional hydrodynamic methods of analysis. Evidence is presented that the molybdate-stabilized, multimeric receptor (Stokes radius approximately 70A; S20,w approximately 9.5 S; Mr approximately 290,000) corresponds to an acidic form of the receptor that has relatively high electrophoretic mobility. This discrete form, which appears to represent the untransformed state that does not bind to DNA, was converted to a number of derived forms by exposure to conditions that result in receptor transformation and/or subunit dissociation. In crude cytosol, transformation always generated receptor forms that were excluded from polyacrylamide gels, and it was shown that these are large heterogeneous aggregates. This explains previous failed attempts to analyze the receptor by polyacrylamide gel electrophoresis. Transformation of partially purified, molybdate-stabilized receptor never led to aggregate formation, but resulted instead in the generation of two relatively basic estrogen-binding species of low electrophoretic mobility. These components may represent the free or dissociated estrogen-binding subunits. Together, the results suggest a model for the molybdate-stabilized receptor wherein at least one of its components is an acidic, nonestrogen-binding subunit.     

An evaluation of the involvement of polyamines in modulating MCF-7 human breast cancer cell proliferation and progesterone receptor levels by estrogen and antiestrogen

The present studies were undertaken to determine the importance of the polyamine biosynthetic pathway in cellular proliferation and hormone-regulated progesterone receptor synthesis in estrogen receptor-containing breast cancer cells. Treatment of MCF-7 cells with difluoromethylornithine (DFMO), the irreversible inhibitor of the enzyme ornithine decarboxylase (ODC), prevented estradiol-induced cell proliferation in a dose-dependent fashion. DFMO inhibition of estradiol-induced cell proliferation was completely recoverable by the addition of exogenous putrescine while putrescine alone did not stimulate proliferation of control cells. ODC activity was 4-fold greater in estrogen-treated cells and DFMO (5 mM) fully inhibited ODC activity. DFMO was able to suppress only slightly further the proliferation of antiestrogen (tamoxifen) treated cells and putrescine was able to recover this DFMO inhibition. In contrast to the suppressive effect of DFMO on cell proliferation, DFMO had no effect on the ability of estrogen to stimulate increased (4-fold elevated) levels of progesterone receptor. Hence, while ODC activity appears important for estrogen-induced cell proliferation, inhibition of the activity of this enzyme has no effect on the ability of estradiol to increase cellular progesterone receptor content.     

Comparative analysis of estrogen receptors covalently labeled with an estrogen and an antiestrogen in several estrogen target cells as studied by limited proteolysis

Estrogen receptors covalently labeled with the estrogen affinity label [3H]ketononestrol aziridine (KNA) or with the antiestrogen affinity label [3H]tamoxifen aziridine (TAZ) were subjected to limited proteolysis with trypsin, alpha-chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on 10-20% sodium dodecyl sulfate-polyacrylamide gradient gels followed by fluorography. The similar molecular weights of intact receptors (Mr 66,000 daltons) and the proteolytic digest patterns indicate extensive homology among estrogen receptors from MCF-7 human breast cancer cells, GH4 rat pituitary cells and rat uterus when liganded with estrogen or antiestrogen. Each protease generated a distinctive ladder of estrogen receptor fragments, and the fragmentation patterns were virtually identical for estrogen receptors labeled with estrogen (KNA) or antiestrogen (TAZ). Each protease yielded a relatively "resistant" receptor fragment of about 28,000-35,000 daltons. Trypsin and chymotrypsin at higher concentrations generated a much smaller 6,000-8,000 dalton digest product that still contained the [3H]KNA- or [3H]TAZ-labeled receptor binding site. Moreover, the receptor digest patterns were similar for estrogen receptors from the three different target cells. Our studies suggest considerable structural relatedness among these three estrogen receptors and also indicate that these two affinity labels bind to a similar, perhaps identical, region of the receptor molecule.     

Progesterone interaction with estrogen and antiestrogen in the rat uterus--receptor effects.

Daily injections of estradiol or the antiestrogen tamoxifen initially stimulate uterine weight increase and progesterone receptor synthesis, though continued tamoxifen fails to maintain the increased weight. The stimulatory actions of both estradiol and tamoxifen are inhibited or reversed by a single injection of progesterone. It has been hypothesized that progesterone antagonizes estrogen action by reducing estrogen receptor levels, but in the present experiments neither cytoplasmic nor nuclear estrogen receptor was affected. We conclude that progesterone acts at a point beyond estrogen receptor availability or translocation to antagonize estrogen action.     

Identification of cysteine 530 as the covalent attachment site of an affinity-labeling estrogen (ketononestrol aziridine) and antiestrogen (tamoxifen aziridine) in the human estrogen receptor

Radiosequence analysis of peptide fragments of the estrogen receptor (ER) from MCF-7 human breast cancer cells has been used to identify cysteine 530 as the site of covalent attachment of an estrogenic affinity label, ketononestrol aziridine (KNA), and an antiestrogenic affinity label, tamoxifen aziridine (TAZ). ER from MCF-7 cells was covalently labeled with [3H]TAZ or [3H]KNA and purified to greater than 95% homogeneity by immunoadsorbent chromatography. Limit digest peptide fragments, generated by prolonged exposure of the labeled receptor to trypsin, cyanogen bromide, or Staphylococcus aureus V8 protease, were purified to homogeneity by high performance liquid chromatography (HPLC), and the position of the labeled residue was determined by sequential Edman degradation. With both aziridines, the labeled residue was at position 1 in the tryptic peptide, position 2 in the cyanogen bromide peptide, and position 7 in the V8 protease peptide. This localizes the site of labeling to a single cysteine at position 530 in the receptor sequence. The identity of cysteine as the site of labeling was confirmed by HPLC comparison of the TAZ-labeled amino acid (as the phenylthiohydantoin and phenylthiocarbamyl derivatives) and the KNA-labeled amino acid (as the phenylthiocarbamyl derivative) with authentic standards prepared by total synthesis. Cysteine 530 is located in the hormone binding domain of the receptor, near its carboxyl terminus. This location is consistent with earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the size of the proteolytic fragments containing the covalent labeling sites for TAZ and KNA and the antigen recognition sites for monoclonal antibodies. The fact that both the estrogenic and antiestrogenic affinity labeling agents react covalently with the same cysteine indicates that differences in receptor-agonist and receptor-antagonist complexes do not result in differential covalent labeling of amino acid residues in the hormone binding domain.     

Interaction of the antiestrogen [3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.     

The effects of the antiestrogen CI-628 on sexual behavior activated by androgen or estrogen in quail

This series of experiments sought to determine whether conversion of androgen to estrogen is important in the activation of male sexual behavior in quail by seeing if an antiestrogen will block androgen stimulated copulation in this species. Experiment I compared the ability of two antiestrogens, MER-25 (5 mg/day) and CI-628 (2 mg/day), to block estrogen stimulated characteristics in female quail. Both treatments greatly reduced oviduct growth in “photically castrated” females given estradiol benzoate (EB, 50 μg/day), but only CI-628 reduced receptivity in these birds. In Experiment II surgically castrated males given 50 μg/day EB together with 2 mg/day CI-628 were much less receptive than castrated males given EB alone, and in addition copulated in fewer tests. In Experiments III, IV, and V, castrated males given testosterone propionate (TP) together with CI-628 were compared with males given TP alone. The ability of CI-628 to suppress TP-stimulated copulation increased with increasing CI/TP dosage ratio, and at the highest ratio (4:1), CI-628 effectively blocked copulation in five out of seven birds. Those birds that did copulate did so in fewer tests and performed fewer cloacal contact movements. CI-628 had no antiandrogenic effects in these experiments. These results suggest that estrogens may be important active metabolites of testosterone with respect to quail copulation.     

Antagonism of estrogen action with a new benzothiophene derived antiestrogen

A new benzothiophene derived antiestrogen, LY139481, inhibited the uterotropic action of estradiol in a dose related fashion, and at 1 mg per day suppressed more than 90 percent of estradiol's activity in immature rats. LY139481 induced minimal uterotropic activity, and that activity declined in relation to dose. The relative binding affinity of LY139481 for rat uterine cytosol estrogen receptors was greater than that of estradiol in competitive assays and increased in relation to temperature (2.9 +/- 0.5 x estradiol at 30 degrees C). LY139481 caused estradiol-induced uterine hypertrophy to regress in a manner similar to that which resulted from withdrawal of estradiol treatment. Three successive daily injections of LY139481 slightly increased uterine weight, and blocked additional uterotropic action in response to estradiol and LY139481 administration on subsequent days. Furthermore, ten daily injections of estradiol alone did not increase uterine weight in animals pretreated with LY139481 for three days. In contrast, LY139481 did not prevent the partial uterotropic action of tamoxifen administration.     

Response of estrogen receptor-positive breast cancer tumorspheres to antiestrogen treatments

Estrogen signaling plays a critical role in the pathogenesis of breast cancer. Because the majority of breast carcinomas express the estrogen receptor ERα, endocrine therapy that impedes estrogen-ER signaling reduces breast cancer mortality and has become a mainstay of breast cancer treatment. However, patients remain at continued risk of relapse for many years after endocrine treatment. It has been proposed that cancer recurrence may be attributed to cancer stem cells (CSCs)/tumor-initiating cells (TICs). Previous studies in breast cancer have shown that such cells can be enriched and propagated in vitro by culturing the cells in suspension as mammospheres/tumorspheres. Here we established tumorspheres from ERα-positive human breast cancer cell line MCF7 and investigated their response to antiestrogens Tamoxifen and Fulvestrant. The tumorsphere cells express lower levels of ERα and are more tumorigenic in xenograft assays than the parental cells. Both 4-hydroxytamoxifen (4-OHT) and Fulvestrant attenuate tumorsphere cell proliferation, but only 4-OHT at high concentrations interferes with sphere formation. However, treated tumorsphere cells retain the self-renewal capacity. Upon withdrawal of antiestrogens, the treated cells resume tumorsphere formation and their tumorigenic potential remains undamaged. Depletion of ERα shows that ERα is dispensable for tumorsphere formation and xenograft tumor growth in mice. Surprisingly, ERα-depleted tumorspheres display heightened sensitivity to 4-OHT and their sphere-forming capacity is diminished after the drug is removed. These results imply that 4-OHT may inhibit cellular targets besides ERα that are essential for tumorsphere growth, and provide a potential strategy to sensitize tumorspheres to endocrine treatment.     

Regulation of epidermal growth factor-receptor by estrogen and antiestrogen in the human breast cancer cell line MCF-7

Regulation of breast tumor proliferation depends in a large part on a variety of hormones and growth factors. In this report we show that estrogen and antiestrogen modulate epidermal growth factor-receptor (EGF-R) level in the human breast cancer MCF-7 cells with opposite mechanisms. Although a short-term treatment (24h to 48h) with estradiol leads to a decrease in EGF-R number, the addition of hormone in cell culture for 5 days increases EGF-R level with a maximal effect observed at 10(-10) M estradiol. In contrast, when cells are treated with the antiestrogen hydroxytamoxifen, a dose-dependent decrease in EGF-R level occurs. We also report that EGF is able to induce estrogen receptors and, to a lesser extent, progesterone receptors when added to MCF-7 cell cultures. These results demonstrate an interaction between both estrogen receptor and EGF receptor growth promoting systems in target cells. The implications of such an interaction in the understanding of human breast cancer hormone responsiveness and, in the development of therapies, are discussed.     

Postnatal uterine development in the rat: estrogen and antiestrogen effects on luminal epithelium

We examined the effects of the synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE), and the triphenylethylene antiestrogen, clomiphene citrate (CC), on uterine growth and development in the rat. These compounds, unlike estradiol, do not bind significantly to rat serum alphafetoprotein (AFP). Administration of DES or EE during the period of normal uterine gland genesis (postnatal days 10-14) induced luminal epithelium hypertrophy and increased uterine wet weight. The durations of these responses were dose-related. By day 26, luminal epithelium cell numbers were significantly depressed, compared to controls. Uterine gland development was delayed 6 to 9 days, depending upon estrogen dose, and the numbers of uterine glands ultimately achieved were generally less than in untreated control animals. While a daily dose of 0.1 micrograms CC/rat did not alter uterine development, 10 micrograms CC/rat caused prolonged luminal epithelium hypertrophy and inhibited uterine gland genesis without inducing the large increases in uterine weight or the decreases in luminal epithelium cell number seen after estrogen exposure. The number of stromal cells was significantly increased on day 26 after CC exposure. Together with previous studies, these data demonstrate the greater potency and developmental stage specificity of non-AFP-bound estrogens with respect to altering uterine gland development. In addition, these data suggest that the disruptive influence of antiestrogens on gland genesis may be mediated through an indirect influence on the uterine stroma.