Structural characterization of the 9-10S estradiol receptor from calf uterus

An affinity chromatography-based method has been developed for estrogen receptor isolation which requires the inclusion of sodium molybdate in purification buffers for maintaining the large 9-10S form of the receptor. The protein products obtained from affinity chromatography of calf uterine receptor extracts or from extracts presaturated with estradiol have been analyzed by gel electrophoresis under denaturing conditions. Major estrogen sensitive proteins were peptides with Mr approximately 90,000, 65,000 and 50,000. Two additional proteins (60,000 and 53,000) of lower abundance and with demonstrated estrogen sensitivity were also observed. Affinity labeling with [3H]tamoxifen aziridine identified the Mr 65,000 protein as the estrogen receptor and suggested that the Mr 60,000, 53,000 and 50,000 peptide components were derived proteolytically from this parent unit. The 90,000 mol. wt component was readily dissociated from heparin-sepharose immobilized estrogen receptor by elution with low salt buffers without molybdate. Peptide mapping experiments indicated that the 90,000 mol. wt component was not related to the Mr 65,000 and 50,000 estrogen receptors, but confirmed the smaller binding unit to be a proteolytic fragment of the 65,000 mol. wt receptor. The results suggest that the 90K protein associates non-covalently with the Mr 65,000 estrogen binding unit as a nonhormone binding component of the 9-10S receptor.     

Sex steroid-binding protein interacts with a specific receptor on human premenopausal endometrium membrane: modulating effect of estradiol.

Sex steroid-binding protein receptor was detected on membranes prepared from human premenopausal endometrium. The binding of sex steroid-binding protein to membranes was specific, saturable, and high affinity. Scatchard analysis showed the presence of two binding sites at different affinities. The addition of estradiol (10(-8) M) did not produce any inhibition of binding; indeed, it resulted in a modification of binding characteristics. The demonstration of sex steroid-binding protein receptor on membranes of human premenopausal endometrium indicates that the expression of receptor on membranes is not an effect of estrogen over stimulation on target tissues. Estradiol could act as a modulating factor of the binding, probably reflecting the sensitivity of tissues to different steroids.     

Rapid recovery of nuclear estrogen receptor and oxytocin receptor in the ovine uterus following progesterone withdrawal

We previously showed that progesterone rapidly down regulates nuclear estrogen receptor (Re) in the estrogen-primed rodent uterus. We have now extended these studies to test the response of the Re system in sheep uterus to progesterone withdrawal. Since the estrogen-Re complex is believed to regulate hormone-dependent gene expression, it was of interest to determine whether withdrawal of progesterone under constant estrogen stimulation would lead to the recovery of nuclear Re levels and estrogen action, i.e. oxytocin receptor (ROT) synthesis. Ovariectomized ewes were primed with estradiol-17 beta and serum steroid levels were maintained by constant infusion of estradiol (0.5 microgram/h) and progesterone (500 micrograms/h) for 5 days. The animals were anesthetized with fluothane/O2, and uterine samples were excised 1 h before and 3, 6 and 12 h after progesterone withdrawal. Estradiol infusion was continued during the experiment in order to maintain estrogen levels at a steady state (14 pg/ml plasma). Re, ROT and progesterone receptor (Rp) were measured in endometrium and myometrium using standard 3H-hormone binding assays. Following progesterone withdrawal, the nuclear Re concentration increased in both uterine compartments, and the nuclear Re level was correlated significantly with the ROT concentration in the membrane fraction of both uterine tissues (endometrium, r = 0.79; myometrium, r = 0.86). Although cytosol Re rose between 6 and 12 h in the endometrium, cytosol Re levels remained unchanged in myometrium. Cytosol Rp appeared to increase in endometrium but not in myometrium. Uterine tissue sampled from a control animal before stopping the progesterone infusion revealed that the observed changes in receptor concentration following progesterone withdrawal were not due to regional differences in receptor levels. These results demonstrate that the recovery of nuclear Re in the ovine endometrium and myometrium following progesterone withdrawal represents a selective effect on Re retention in the nucleus rather than on cytosol Re availability or Re activation which was controlled by constant estrogen infusion. Thus, these results are consistent with the hypothesis that progesterone induces an Re regulatory factor which acts to down regulate nuclear Re, and that the activity of this factor diminishes rapidly after progesterone withdrawal.     

Binding characteristics of progesterone and antiprogestin ZK 98.299 in human endometrial and myometrial cytosol

The binding of ZK 98.299, a synthetic progesterone antagonist, with human endometrium and myometrium cytosol was studied and compared with that of progesterone. Progesterone showed specific saturable binding to its receptors in both endometrium and myometrium. ZK 98.299 and progesterone were mutually competitive for binding to progesterone receptors; however, the relative binding affinity of ZK 98.299 was 16% that of progesterone. ZK 98.299 exchanged the progesterone-labelled receptor sites. [3H]ZK 98.299 showed specific binding which was linearly related to the cytosol protein concentration. The binding was not saturable at 15 nM of ligand. The binding capacity and binding affinity of ZK 98.299 receptor was less than that of progesterone. Progesterone also partially displaced the binding of [3H]ZK 98.299. This study suggest that ZK 98.299 and progesterone both bind to the same protein. However, whether ZK 98.299 binds to progesterone receptors alone or even to other functionally related sites is not known. It appears that ZK 98.299 when present in higher concentration than progesterone would be an effective receptor ligand.     

Binding of methyltrienolone to various androgen-dependent and androgen-responsive tissues in four animal species.

Comparative sucrose gradient studies of the in vitro binding of dihydrotestosterone (DHT) and of a synthetic androgen, methyltrienolone (R 1881), have been done with the cytosols of various tissues of the rat, mouse, cock and man. With rat prostate cytosol, the amount of R 1881 and DHT binding in the 8-9S region of the gradient was found to be comparable. Specific 8-9S peaks of R 1881 were also found in rat levator ani/bulbocavernosus and skeletal muscles and in the mouse kidney. Only 4-5S peaks could be demonstrated in the cock's comb while DHT under the same conditions showed both 8-9S and 4-5S binding. Binding of R 1881 to the cytosol of the hyperplastic prostate was polydispersed, and showed evidence of the presence of aggregates. Evidence was also found that R 1881 could bind to the progesterone receptor in rat uterus. Our study supports the theory that in a given species the androgen receptors are similar if not identical in all the tissues. The synthetic androgen R 1881 appears to be a useful tool for androgen receptor studies in various animal species provided that the tissue under study contains no progesterone receptor.     

Characterization of a distinct glucocorticoid-binding protein in the lactating mammary gland of the rat

Specific substances binding [³H]triamcinolane acetonide were detected in the cytosol fraction of the lactating mammary gland of the rat using sucrose gradient centrifugation. These receptors, which were protein in nature, exhibited sedimentation coefficients of 7–8 S and dissociated into lower molecular weight components sedimenting at 4–5 S when separated on sucrose gradients containing 0.4 M KCl. The cytoplasmic form of the binding protein was relatively specific for glucocorticoids although progesterone inhibited binding significantly. The dissociation constant (K_d) of the receptor-ligand complex was in the range of 10⁻⁸ M. p-Chloromercuribenzoate diminished the ligand-binding capacity of the receptor suggesting a role for sulfhydryl groups in the binding reaction. Cytosols from mammary tissue obtained from virgin and pregnant rats revealed a paucity of binding sites as compared to those in the lactating gland. Examination of ligand-binding specificity indicates that these glucocorticoid-binding sites are distinct and easily discriminated from those of either the estrogen receptor of the mammary gland or the triamcinolone-binding component in plasma.     

Sodium molybdate increases the amount of progesterone and estrogen receptor detected in certain human breast cancer cytosols

When sodium molybdate is added at a final concentration of 20 mM, additional 85 and 4S progesterone (³ H-R5020) receptor can be detected in the cytosols from a number of human breast cancers. Additional estrogen receptor also could be measured in some cytosols, and a quantitative temperaturedependent conversion of 8S to 4S binding molecules achieved. Sodium molybdate also prevented the loss of binding activity that occurred when cytosols were incubated at 30° in the absence of added estradiol. In addition to increasing the amount of progesterone receptor, and to a lesser extent estrogen receptor that may be detected, elucidation of the mechanism by which this salt stabilized receptors should contribute to further understanding of how cytosol steroid receptor content and function is regulated.     

MCF-7; a human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors.

We have identified receptors for glucocorticoids, progestins, and androgens in a human breast tumor cell line (MCF-7) known to have estrogen receptor. Sucrose density gradients show that MCF-7 cytosol contains approximately 100 fm/mg protein estradiol (E2-3H) receptor, more than 300 fm/mg protein progesterone receptor (measured with R5020-3H), about 40 fm/mg protein 5alpha-dihydrotestosterone (5alpha-DHT-3H) receptor, and 800 fm/mg glucocorticoid receptor (measured with dexamethasone-3H). Dissociation constants obtained by Scatchard analyses were approximately 0.6 x 10(-10)M (E2), 1 x 10(-9)M (R5020), 2.8 x 10(-10)M (5alpha-DHT) and 8 x 10(-9)M (dexamethasone). No cross competition was found for estrogen receptor, but progestins competed for androgen and glucocorticoid binding. The androgen, but not the glucocorticoid, partially competed for R5020 binding to progesterone receptor. This first demonstration of 4 classes of steroid receptors in human breast cancer means that MCF-7 may be an excellent in vitro model for studying the mechanism of tumor response to endocrine therapy as well as the complex relationships between binding and biological actions of these hormones.     

Oligomeric structures of cytosoluble estrogen-receptor complexes as studied by anti-estrogen receptor antibodies and chemical crosslinking of intact cells

The structure of estrogen-receptor complexes recovered in cytosolic extracts of MCF-7 cells treated with hormone at 2°C was probed by chemical crosslinking of intact cells and sample analysis with four monoclonal anti-estrogen receptor antibodies. When MCF-7 cells were treated with either glutaraldehyde or dithiobis(succinimidyl propionate), cytosoluble estrogen-receptor complexes consisted of two major forms sedimenting as 4 S monomers and 8–9 S salt-resistant oligomers. By high salt sucrose density gradient centrifugation, we could observe that the four monoclonal anti-estrogen receptor antibodies bound different forms of receptor complexes from crosslinked cells. While H222 and H226 antibodies could interact with any form we detected, the D75 and D547 monoclonals could only recognize those showing sedimentation coefficients lower than 7 S. When cytosolic extracts from [³⁵S]-methionine-labeled cells were subjected to immunoprecipitation with H222 and D75 anti-estrogen receptor antibodies, electrophoretic analysis of material extracted from immunoprecipitates revealed the presence of 65 kDa estrogen receptors. If extracts were prepared from crosslinked cells, instead, two more components with estimated molecular masses of 220 and 100 kDa were specifically immunoprecipitated by the H222 antibody, whereas only the 100 kDa component and the estrogen receptor were found in immunoprecipitates obtained with the D75 monoclonal. When estrogen-receptor complexes were immunopurified from extracts prepared after cells had been crosslinked with dithiobis(succinimidyl propionate), and the oligomers were dissociated by treatment with β-mercaptoethanol, electrophoretic analysis of our samples showed that only the 65 kDa estrogen receptor and a 50 kDa protein were selectively immunoprecipitated by anti-estrogen receptor antibodies. We concluded that the structures of cytosoluble estrogen-receptor complexes in MCF-7 cells treated with hormone at 2°C, include oligomeric forms which contain a 50 kDa non-steroid binding protein.     

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.     

An insulin effect on cytoplasmic estrogen receptor in the human breast cancer cell line MCF-7

It has recently been reported (Horwitz, K. B., Zava, D. T., Thilagar, A. K., Jensen, E. M., and McGuire, W. L. (1978) Cancer Res. 38, 2434-2437) than the human breast cancer-derived cell line MCF-7 from EG&G Mason Research Institute contains no 8 S and very little 4 S cytoplasmic estrogen receptor. Even so, we have found significant levels of cytoplasmic estrogen receptor in MCF-7 cells from this source. The receptor was found at a maximum level of 132 fmol/mg of cytoplasmic protein, and had an apparent dissociation constant at 30 degrees C of 7.3 X 10(-10) M and at 4 degrees C of 1.2 X 10(-10) M. In sucrose gradients without KCl, the receptor migrated at 6-7 S, and with 0.4 M KCl, at 3-4 S. The receptor was specific for estrogen, in that a 100-fold excess of diethylstilbestrol eliminated binding of radiolabeled estrogen, whereas hydrocortisone, aldosterone, progesterone, and testosterone had no effect. It was further demonstrated that at least part of the reason for the discrepancy between our data and those of Horwitz et al. is that the high insulin level (10 microgram/ml) used by Horwitz et al. dramatically lowers the assayable level of receptor. These results may have important implications for steroid receptor assays in other cell lines in tissue culture and in human breast cancer patients as well.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.     

Regulation of nuclear binding of the avian oviduct progesterone receptor. Changes during estrogen-induced oviduct development, withdrawal, and secondary stimulation

The progesterone receptors from various stages of estrogen induced oviduct development, estrogen withdrawal, and secondary stimulation with estrogen were examined. The progesterone receptors were characterized for their biological function (i.e. capacity for nuclear translocation, nuclear binding, and effects on RNA polymerase II activity) as well as certain physical properties. The progesterone receptors from the undeveloped or partially developed oviducts (0 to 8 days of estrogen treatment) displayed little or no nuclear translocation and binding in vivo or in vitro. Similarly, progesterone showed little or no effect in vivo on RNA polymerase II activity at the early stages of development. As development progressed from 8 to 12 days of estrogen treatment, the above parameters rapidly increased to maximal levels and plateaued through day 23 of estrogen treatment. A marked decrease in these parameters occurred within 1 day of estrogen withdrawal. The reverse series of events occurred during secondary estrogen stimulation of 10-day-old withdrawn chicks. While the receptor concentrations increased rapidly to maximum values by 2 days of restimulation, receptor function did not return until day 4. Similarly, the effects of progesterone on RNA polymerase II activity reached maximal values by day 4. The progesterone receptor isolated from oviducts during development, estrogen withdrawal, and restimulation, displayed similar patterns of cell-free binding to chromatin and nucleoacidic protein as that observed in vivo supporting the nativeness of the in vitro binding assay. In contrast, the cell-free binding of these same progesterone receptor to pure DNA were not similar to the in vivo binding, i.e. no patterns (differences) in progesterone receptor binding were observed. These data support that protein DNA complexes and not pure DNA represent the native acceptor sites for oviduct progesterone receptor. Comparison of the progesterone receptor between the functional and nonfunctional states revealed no differences in the steroid affinity for the receptor, in the apparent pI of the species, or in the sedimentation of the receptor under high salt conditions. However, the nonfunctional receptors consistently displayed a deficiency in one of the two monomer molecular species (the B species) as determined by isoelectric focusing. These results suggest that both monomer species of progesterone receptor are required for biological activity. Interestingly, the 7S "aggregate" species of the progesterone receptor was constantly detected even when only one of the monomer species was present.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of Combination of Estradiol with Selective Progesterone Receptor Modulators (SPRMs) on Human Breast Cancer Cells In Vitro and In Vivo

Use of estrogen or estrogen / progestin combination was an approved regimen for menopausal hormonal therapy (MHT). However, more recent patient-centered studies revealed an increase in the incidence of breast cancer in women receiving menopausal hormone therapy with estrogen plus progestin rather than estrogen alone. Tissue selective estrogen complex (TSEC) has been proposed to eliminate the progesterone component of MHT with supporting evidences. Based on our previous studies it is evident that SPRMs have a safer profile on endometrium in preventing unopposed estrogenicity. We hypothesized that a combination of estradiol (E2) with selective progesterone receptor modulator (SPRM) to exert a safer profile on endometrium will also reduce mammary gland proliferation and could be used to prevent breast cancer when used in MHT. In order to test our hypothesis, we compared the estradiol alone or in combination with our novel SPRMs, EC312 and EC313. The compounds were effectively controlled E2 mediated cell proliferation and induced apoptosis in T47D breast cancer cells. The observed effects were found comparable that of BZD in vitro. The effects of SPRMs were confirmed by receptor binding studies as well as gene and protein expression studies. Proliferation markers were found downregulated with EC312/313 treatment in vitro and reduced E2 induced mammary gland proliferation, evidenced as reduced ductal branching and terminal end bud growth in vivo. These data supporting our hypothesis that E2+EC312/EC313 blocked the estrogen action may provide basic rationale to further test the clinical efficacy of SPRMs to prevent breast cancer incidence in postmenopausal women undergoing MHT.     

Endocrine steroid sulfotransferases: porcine endometrial estrogen sulfotransferase

Porcine endometrial estrogen sulfotransferase has been isolated and its properties examined. This enzyme only appeared in uteri from ovariectomized gilts which had been primed with estrogen and treated with progesterone. The most stable form of the enzyme was obtained via chromatofocusing of the 100,000 g supernatant from secretory endometrium. A molecular weight of 31 KDa was determined for this sulfotransferase by molecular sieve (Sephadex G-200 Superfine) and disk-gel electrophoresis. The active protein displayed a pI of 6.1, pH optimum of 7.6-7.8 and a requirement of 10 mM Mg2+ for maximum transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to estrone (E1). Km of the reaction was 24 +/- 4.7 microM for PAPS and 24 +/- 9.8 nM for E1 as substrate. Porcine endometrial sulfotransferase thus displayed a much greater affinity for E1 than a similar enzyme previously isolated from bovine adrenals. As has been observed of sulfotransferases from other tissues, an endogenous substrate (presumed to be E1) accompanies the enzyme throughout its purification.     

Effects of temperature, storage and sodium molybdate on the analysis of estrogen and progesterone receptors in rabbit uterine tissue and gynecologic tumor

The cytoplasmic estrogen receptor (ERc) and progesterone receptor (PRc) in mammary tumors have been recognized as useful biochemical markers for predicting the objective response of patients with advanced breast cancers to endocrine therapy. These proteins are also useful in the prognosis of gynecologic carcinoma. This report presents data showing the effect of sodium molybdate in the stabilization of estrogen and progesterone receptors. In rabbit uterine tissue, molybdate (20 mM) increased the binding of progesterone and estrogen to the receptors in several ways: (a) the apparent loss of detectable receptors during lengthy sucrose gradient analysis and at elevated temperature (30 degrees C) was reduced; (b) the instability of receptors due to storage at -70 degrees C was lessened, and (c) the conversion of the 7S PRc to the 3.5S form was minimized. Similarly, molybdate caused a qualitative and/or a statistically significant quantitative difference in receptor values for some human gynecologic tumors presented herein; the molybdate-associated changes vary with tumor specimen. Of the 8 tumors for which receptor values in the presence of molybdate (M+) and its absence (M-) can be compared, detectable ERc of 6 and PRc of 7 tumors increased with molybdate, and ERc of 2 and PRc of 1 tumor showed no change. In addition to the increase in receptor values, a concomitant shift of the 3-4S molecules to the 7-8S moieties was noted for some tumors (1 of 6 for ERc and 3 of 7 for PRc). In 2 receptor-poor tumor samples, ERc was only detected in M+ cytosols. These results show that molybdate is effective in reducing receptor degradation and stabilizes the 7-8S molecules from converting to 4S moieties. The addition of molybdate may be helpful for better quantitation of steroid receptors in clinical specimens.     

Metabolism of pregnenolone by human breast cancer. Evidence for 17 alpha-hydroxylase and 17,20-lyase.

The metabolism of 7-(3)H-pregnenolone was studied in vitro using 16 human breast carcinomas. All mammary tumors transformed pregnenolone to progesterone. All estrogen receptor poor tumors and 4 out of 8 estrogen receptor rich tumors converted pregnenolone to 17-hydroxypregnenolone. Five estrogen receptor poor tumors showed the presence of 17,20-lyase as evidenced by formation of dehydroepiandrosterone and androstenedione. In two estrogen receptor poor tumors, conversions of pregnenolone to progesterone, 17-hydroxy pregnenolone, dehydroepiandrosterone, androstenedione and finally to estradiol was documented, providing a hypothetical pathway for steroid metabolism in human breast cancer. The conversion of pregnenolone to 17-hydroxypregnenolone was significantly less in receptor rich tumors and was totally absent in 4 receptor rich tumors with estrogen receptors of over 45 fmol/mg protein.