Interplay between estradiol and prolactin in the regulation of steroid hormone receptor levels, nature, and functionality in normal mouse mammary tissue.
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Estrogen-insensitive progesterone receptors in a human breast cancer cell line: characterization of receptors and of a ligand exchange assay.
In modified culture conditions, T47D human breast cancer cells synthesize extraordinary amounts of progesterone receptors (PgR), but, unlike other progesterone target cells, the PgR are entirely independent of estrogen controls. In the present studies we characterize some physicochemical properties of the PgR in T47D cells. We also describe an exchange assay for cytoplasmic and nuclear forms of the receptors which has enabled us to demonstrate that after progesterone treatment, translocation is stoichiometric. Despite the anomalous regulation of PgR levels, these receptors are typical of steroid receptors; they sediment at 7-8S on sucrose density gradients, they bind ligands with high affinity (Kd approximately 4 nM for R5020; Kd approximately 2 nM for progesterone), they bind only progestins specifically, and they are thermolabile (t1/2 at 37 C is approximately 15 min). Receptor levels range from 15-40 pmol/mg DNA, or more than 300,000 sites/cell. The ability of ligands to dissociate from and rebind to the receptors was measured and used in an exchange assay for nuclear PgR. The synthetic progestin R5020 dissociates readily from receptors (t1/2 approximately 3 h at 0 C and 1.5 h at 10 C), and the dissociation of progesterone is even faster (t1/2 approximately 30 min at 0 C). To quantify steroid exchange, receptor levels were measured in mixtures of hormone-filled and unfilled cytosols. These studies assess ligand dissociation and subsequent ligand rebinding. At 0 C for 4-18 H or at 10 C for 4 h, unlabeled progesterone dissociates from receptors, and R5020 rebinds all sites, resulting in 100% exchange. In contrast, despite the use of a variety of incubation times and temperatures, no more than 50% of receptors previously filled with R5020 can exchange for [3H]R5020. The progesterone to [3H]R5020 exchange assay was used to measure salt-extracted nuclear progesterone receptors. In cells treated for 5 min with 0.1 microM progesterone, all depleted cytoplasmic sites were quantitatively recovered from nuclei. These cells provide a new model system to study the molecular biology of human PgR.
Breast Neoplasms↗
Direct effects of sex steroids on prolactin release at the anterior pituitary level: interactions with dopamine, thyrotropin-releasing hormone, and isobutylmethylxanthine.
When present alone for 4 or 8 days, 5 alpha-dihydrotestosterone (DHT) or the pure progestin R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) inhibits spontaneous PRL release by 33--50% in rat anterior pituitary cells in primary culture. This inhibitory effect of DHT and R5020 can only be partially reversed by 17 beta-estradiol (E alpha). DHT and R5020 inhibit spontaneous PRL release in E2-primed cells at ED50 values of 0.5 and 3 nM, respectively. While E2 diminishes by 30--60% the maximal inhibitory effect of dopamine on PRL release and increases by 10-fold the ED50 value of dopamine action, DHT and R5020 can prevent by 30--60% the action of E2 and thus increase the potency of dopamine to inhibit PRL release. The inhibitory action of DHT and R5020 as well as the stimulatory action of E2 on spontaneous PRL release are similarly expressed on TRH- and 3-isobutyl-1-methylxanthine-induced PRL release, thus suggesting that at least part of the highly effective modulatory effects of sex steroids are exerted at a step after cAMP formation.
1-Methyl-3-isobutylxanthine↗
Progesterone receptors in mammary gland of the pregnant rat.
The characteristics of pregnant rat mammary gland progesteronee receptors have been studied by the use of two synthetic probes, [3H]R5020 (3H-labeled 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione) amd [3H]ORG 2058 (3H-labeled 16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione). [3H] ORG 2058 bound with high affinity (Kd at 4 C, approximately 0.5 nM) to an apparent single class of displaceable sites, with a hierarchy of competition as follows: ORG 2058 greater than R5020 greater than or equal to progesterone greater than dexamethasone. In contrast, [3H]R5020 showed binding in mammary gland cytosols which was nonlinear with protein concentration, of intermediate affinity (Kd at 4 C, approximately 5 nM), and poorly displaced by nonradioactive R5020; the hierarchy of competitors was R5020 greater than progesterone greater than dexamethasone = ORG 2058. Parallel studies in rat uterine cytosols showed both [3H]ORG 2058 and [3H]R5020 to bind with high affinity to an apparently identical class of sites; the hierarchy of competition for both was ORG 2058 greater than or equal to R5020 greater than progesteron greater than dexamethasone. We conclude that in pregnant rat mammary gland, as opposed to rat uterus and other tissues, R5020 is unsuitable as a progesterone receptor probe; however, the affinity, capacity, ad specificity characteristics of progesterone receptors in this tissue can be described by the use of ORG 2058.
Animals↗
Modulation of rat uterine progesterone receptor levels and peroxidase activity by tamoxifen citrate, LY117018, and estradiol.
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Animals↗
Hypothalamic progestin receptors: evidence for two classes of high affinity binding sites.
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Adrenalectomy↗
Progestin augmentation of gonadotropin-stimulated progesterone production by cultured rat granulosa cells.
Ovarian progesterone production is stimulated by FSH and LH. Concomitant treatment with a synthetic progestin, R5020, (10(-6)M) increases the FSH-stimulated production of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) in cultured rat granulosa cells. Likewise, R5020 augments the LH-stimulated progestin production in FSH-primed cells. Furthermore, the FSH stimulation of pregnenolone biosynthesis is enhanced by 10(-6) M of progesterone or R5020. These in vitro findings suggest that progestins may exert an autoregulatory positive feedback action to enhance gonadotropin-stimulated production of progesterone and 20 alpha-OH-P.
Animals↗
The subunit structure of human breast cancer progesterone receptors: characterization by chromatography and photoaffinity labeling.
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Affinity Labels↗
In situ photolinked nuclear progesterone receptors of human breast cancer cells: subunit molecular weights after transformation and translocation.
The subunit structure of mammalian cytoplasmic progesterone receptors (PR) has been difficult to study because these proteins are subject to in vitro proteolysis; the structure of nuclear PR is unknown. We have now developed an in situ photoaffinity labeling method for PR that permits study of their subunits with minimal in vitro incubations. The strategy is to use [3H]R5020, a synthetic photoactive progestin, and suitable incubation temperatures to place receptors into their precise intracellular sites in intact cells. The cells, still intact, are then irradiated with UV at 300 nm for 2 min. This irradiation efficiently (approximately 15%) yields covalently linked hormone-receptor complexes at any intracellular location. Cells are than rapidly ruptured, nuclei are separated, and receptors are extracted with salt and/or directly solubilized with detergents before the subunits are displayed on denaturing polyacrylamide gels. With this as well as with modified in vitro methods, we show here that untransformed human breast cancer PR have two dissimilar subunits (mol wt, 115,000 and 81,000) present in equimolar amounts. The same subunits, with apparently unmodified mass, can be demonstrated in nuclei after they have been translocated by progestin treatment. Therefore, PR transformation and acquisition of nuclear binding capacity does not require prior proteolytic processing of subunits or other major structural modifications that are detectable on single dimension gels.
Affinity Labels↗
Uterine steroid receptor changes associated with progesterone withdrawal during pregnancy and pseudopregnancy in rabbits.
Cytosolic and nuclear progesterone (P) and estrogen (E) receptors (PR and ER, respectively) were measured in uterine tissues of rabbits at the end of pregnancy (days 25, 27, 29, 30, and 31 and 0-10 h postpartum) and pseudopregnancy (days 13 and 18). At both times, plasma P concentrations fell dramatically after a period of prolonged elevation, effecting changes in uterine function. Plasma concentrations of P remained elevated until day 29 of pregnancy and then fell continuously until postpartum, whereas plasma estradiol levels did not change. The numbers of nuclear ER and PR were constant from days 25-29. Concomitant with the fall in plasma P on day 30, levels of nuclear ER and PR doubled and remained elevated on day 31 and postpartum. In all cases, the numbers of nuclear receptors were negatively correlated with P concentrations. The number of cystosolic ER in the myometrium increased on day 30 of pregnancy and remained elevated on day 31 and postpartum. Although levels of cytosolic ER in the endometrium appeared to rise similarly, the change was not significant. The number of cytosolic PR in endometrium and myometrium did not change significantly. Between days 13 and 18 of pseudopregnancy, plasma P declined while plasma estradiol remained constant, as during pregnancy. At this time, cytosolic PR increased 4-fold, and nuclear PR doubled. Nuclear ER also increased between days 13 and 18, but the apparent increase in cytosolic ER was not significant. These data suggest that receptors for both steroids increase as the ratio of plasma estradiol to P increases at the end of pregnancy and pseudopregnancy. Surprisingly, nuclear PR levels are highest at term, when the influence of P in suppressing myometrial activity and preventing the onset of labor is removed. Thus, in the rabbit, the level of PR does not directly reflect the physiological response to P.
Animals↗
High affinity binding of [3H]R5020 and [3H]progesterone by putative progesterone receptors in cytosol and nuclear extract of turtle oviduct.
The purpose of this investigation was to identify and characterize putative cytosolic and nuclear forms of progesterone receptor in the female reproductive tract of a turtle, Chrysemys picta. A dextran-coated charcoal adsorption assay and DNA-cellulose affinity chromatography were used as the primary methodologies with [3H]R5020 [3H-labeled 17 alpha-dimethyl-19-norpregna-4,9-diene-3,20-dione) and [3H]progesterone (P4) as the ligands. The receptor was of high affinity (Kd = 4.7 X 10(-10) M for [3H]P4; 2.2 X 10(-10) M for [3H]R5020) and limited capacity (500-6000 fmol/g tissue wet wt). Association was rapid (apparent equilibrium being reached in 30-40 min), as was dissociation (t1/2 = 45 min for [3H]P4 and 180 min for [3H] R5020). The putative receptor demonstrated strict steroid specificity, binding progestins but not estrogens, androgens, or glucocorticoids. Heterogeneity of the cytosolic receptor was demonstrated as two forms eluting off DNA-cellulose columns at 0.2- and 0.3-M salt concentrations. Binding of cytosolic receptor to DNA-cellulose was not increased by preexposure of cytosol to 25 C for 30 min. Some variations in cytosolic, but not nuclear, receptor were associated with different stages of the reproductive cycle and were positively correlated with body weight. Preliminary studies using an explant culture system suggest that the progesterone receptor in turtle oviduct may be maintained by estrogen and translocated from the cytosol to the nucleus by P4. In summary, we have partially characterized a putative P4 receptor in the oviduct of the turtle that is similar to mammalian and avian P4 receptors in specificity, affinity, and other physicochemical properties, supporting the idea that steroid receptor proteins have been highly conserved in vertebrate evolution. However, temperature sensitivity of activation and DNA affinity are different in the turtle and suggest modifications that may be related to physiological adaptation in such a poikilothermic species.
Animals↗
Regulation of estrogen and progesterone receptor levels in mouse mammary epithelial cells grown in serum-free collagen gel cultures.
The effect of collagenase dissociation of virgin mouse mammary glands on the level of mammary epithelial cytosolic estrogen receptors (ER) and progesterone receptors (PR) was assessed. After cell dissociation, ER was present in mammary epithelial cells at concentrations similar to those found in the whole gland. However, PR appeared to be affected by the collagenase treatment. The regulation of ER and PR in mouse mammary epithelial cells isolated by collagenase dissociation and grown within collagen gels was then determined. After 7 days in culture under serum-free conditions inside a collagen gel, PR and, to a lesser extent ER, as characterized by high affinity binding and specificity, were present in the epithelial cells. Although at a low level, the ER were determined to be functional, since estradiol (E2) was able to promote nuclear accumulation of ER and to induce PR. PRL was able to increase cytosolic ER and PR concentrations. The combination of progesterone (P) and PRL was more effective than PRL or P alone in increasing PR. The induction of PR by P and PRL was inhibited when epidermal growth factor was present in the culture medium. Previous studies have shown that P, PRL, and epidermal growth factor, but not E2 (either alone or in combination with these factors) are able to stimulate cell proliferation in vitro. We conclude that the effects of E2 on protein synthesis and proliferation are dissociated in vitro. The difference between the effect of E2 and PRL or P on growth may be related either to the initial concentrations of their respective receptors or estrogen may stimulate growth indirectly.
Animals↗
Cultured granulosa cells produce two plasminogen activators and an antiactivator, each regulated differently by gonadotropins.
Although treatment of cultured granulosa cells with gonadotropins increases their fibrinolytic activity, the biochemical nature of this effect is unclear. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fibrin autography techniques to characterize the fibrinolytic components secreted by granulosa cells. The fibrinolytic activity of these cells results from the production of both a tissue-type plasminogen activator (t-PA) and a urokinase-like activator (u-PA). The cells also produce an inhibitor of fibrinolysis (antiactivator). FSH and LH stimulate t-PA activity and suppress antiactivator activity, while u-PA activity is not affected by the gonadotropins. The differential regulation of these molecules by the gonadotropins may be essential for ovulation.
Animals↗
Estrogen responsiveness of normal mouse mammary cells in primary cell culture: association of mammary fibroblasts with estrogenic regulation of progesterone receptors.
Estrogens enhance proliferation of normal mouse mammary cells in vivo. However, when cultured alone, normal mouse mammary epithelial cells fail to exhibit a proliferative response to estrogen in vitro; the basis for this lack of in vitro responsiveness to estrogen is not known. The purpose of the present study is to determine if cultured normal mouse mammary cells possess estrogen receptors (ER) and/or progesterone receptors (PgR) and if the ER mechanism is functional, as measured by the ability of estrogens to regulate PgR. Recent findings that mammary fibroblasts can influence the behavior of mammary epithelial cells in vitro led us to investigate their effect on epithelial cell responsiveness to estrogen. In these studies, collagenase-dissociated mammary glands of midpregnant BALB/c mice were the source of mixed cultures (containing both epithelial cells and fibroblasts) and epithelial or fibroblast cultures. The purity of epithelial or fibroblast cultures was quantified immunocytochemically using antivimentin antibody as a fibroblast marker. Steroid hormone binding was quantified in intact cultured cells using [3H]R5020 and 17 beta-[3H]estradiol as the ligands. Specific high affinity binding sites for estrogen (Kd = 3.1 +/- 0.8 X 10(-10] and progestins (Kd = 3.3 +/- 1.2 X 10(-9) M) were detected in mixed cultures. To assess the possible role of mammary fibroblasts, we investigated cultures containing only fibroblasts which were derived by differential centrifugation. When 17 beta-estradiol was added to the culture medium, a significant (P less than 0.01) increase in PgR concentration was observed in mixed cultures. While mixed cultures maintain responsiveness to estrogen in vitro, as measured herein, the epithelial cultures, derived by differential centrifugation and Percoll gradient sedimentation, did not. However, estrogenic regulation of PgR appears to be specific to epithelial cells in mixed cultures, since fibroblast cultures neither contained PgR nor displayed estrogen-inducible PgR. The lack of responsiveness of epithelial cultures is not due to a loss or decrease in the ER concentration. Thus, the presence of mammary fibroblasts appears to be associated with epithelial cell responsiveness to estrogen in vitro.
Animals↗
The antiprogestin RU38 486: receptor-mediated progestin versus antiprogestin actions screened in estrogen-insensitive T47Dco human breast cancer cells.
Despite the theoretical promise of synthetic antiprogestational agents as anticancer agents, experimental tools, midcycle contraceptives, and implantation inhibitors, none has been available for either basic or clinical studies. However, a candidate antiprogestin, RU38 486 [17 beta-hydroxy-11 beta-(4-dimethylaminophenyl)17 alpha-(1-propynl)estra-4,9 -dien-3-one], has recently been described that has antiprogestational and antiglucocorticoid activities in early clinical trials. Its mechanisms of action are unclear. Furthermore, development of this drug underscores an old bioassay problem: that biological screening of progestins and antiprogestins is complex because of the physiological requirement that progestational effects must be superimposed upon an estrogenized system. This has made it difficult to distinguish among progestational, antiprogestational, and antiestrogenic properties of unknown agents. Here we describe the use of T47Dco human breast cancer cells to circumvent these problems. T47Dco cells are rich in progesterone receptors (PR), but are resistant to estrogens and antiestrogens. Their PR are estrogen-independent, and this permits progestins to be studied in an estrogen-free system. We have used these cells to assess the receptor-binding properties and the biological actions of RU38 486. Since RU38 486 absorbs UV at approximately 300 nm, this wavelength was used to covalently photolink the drug to PR in situ. Like the synthetic progestin R5020, low concentrations (10 nM) of [3H]RU38 486 bind two PR subunits in nuclei of T47Dco; glucocorticoid receptors are not bound. RU38 486 has a high affinity for PR in vitro (Kd approximately 2 nM at 0-4 C), and in intact cells, low concentrations (6-8 nM) transform more than 95% of PR to a high affinity nuclear binding state. In contrast to progesterone, the compound is not metabolized, so that it chronically (3-6 days) suppresses PR replenishment. These biochemical properties of RU38 486 are typical of synthetic progestins, but distinguish it from pure glucocorticoids. To bioassay RU38 486, we have measured growth and insulin receptors, since in T47Dco, physiological concentrations of progestins inhibit proliferation and increase the number of cell surface insulin-binding sites. Like progestins, RU38 486 is growth inhibitory; unlike progestins, it fails to stimulate insulin receptors and partially blocks their stimulation by R5020. Thus, RU38 486 has dual progestin agonist/antagonist actions depending on the biological response measured.
Affinity Labels↗
E-17 alpha-(2-[125I]iodovinyl)-19-nortestosterone: the synthesis of a gamma-emitting ligand for the progesterone receptor.
We have synthesized a gamma-emitting steroid, E-17 alpha-(2-[125I]iodovinyl)-19-nortestosterone (E-IVNT), which is a useful ligand for the sensitive and accurate assay of the progesterone receptor. The synthetic scheme is rapid and is performed with readily available materials. This compound, [125I]E-IVNT, is stable and binds with high affinity to the progesterone receptor.
Affinity Labels↗
Modulation of aromatase activity in human endometrial stromal cells by steroids, tamoxifen and RU 486.
The regulation of aromatase activity (AA) in human endometrial stromal cells by various steroids was studied in primary cell culture. Various progestins, but not androgens or glucocorticoids, stimulated AA. Medroxyprogesterone acetate (MPA) was the most potent progestin. Estrogen (E) alone did not change the activity but it potentiated the stimulation of AA by progestin. Biphasic regulation of AA by progestin was noted in both time- and dose-dependent manners. Endometrial AA was stimulated by MPA and reached the maximum rate between 2-5 days of incubation with subsequent decline of AA in prolonged culture. When stromal cells were treated with MPA (0.03 to 30 microM) for 3 days, AA was increased over the control at all the concentrations tested. The maximum was found at doses between 0.1-1 microM. The activities reduced steadily from the maximum stimulation to less than 50% when the concentration of MPA increased from 1-30 microM. In addition, initial treatment of stroma cells with MPA (1-3 days) resulted in further increase of activity after progestin withdrawal. The enhancement of the induction of AA by E did not alter the biphasic pattern regulated by progestin alone, i.e. E enhanced both the stimulation and the decay of AA. The time study of the effect of E showed that enhancement of AA required at least 10 h of incubation of E with MPA conditioned cells. The effect of E is dose dependent between 0.04-40 nM and shows the greatest effect in the presence of MPA between 0.01-1 microM. The optimal concentrations of E and progestin that stimulate AA in culture are similar to the plasma concentrations after pregnancy, suggesting that the physiological function of the endometrial aromatase is at the time of decidualization. The effects of antiprogestin, Ru 486, and antiestrogen, tamoxifen (TAM), on AA were studied. Ru 486 or TAM alone did not alter AA. Ru 486 inhibited the MPA stimulated AA in a dose-dependent manner suggesting that the effect of progestin may be mediated through a receptor mechanism. Enhancement, but no inhibitory effect, was observed when cells were treated with TAM + MPA and TAM + MPA + E. The effectiveness of Ru 486 to inhibit the induction of AA in endometrial cells may be of primary importance for contraception.
Adult↗
Preparation of monoclonal antibodies to the avian progesterone receptor.
In an effort to obtain additional probes for analysis of the avian progesterone receptor, this receptor was isolated and used to prepare several monoclonal antibodies. Progesterone receptor purified from oviduct cytosol by chromatography on deoxycorticosterone-Sepharose and heparin-agarose was used as the immunizing antigen. Twenty-nine hybridoma cultures which tested positive in an enzyme-linked immunosorbent assay against the receptor preparation were subcloned resulting in establishment of 12 stable cell lines. Of these, 5 produced antibodies capable of complexing receptor-bound progesterone from cytosol as measured by adsorption of receptor-antibody complexes onto antimouse immunoglobulin G-agarose. Each was used to generate ascites and the purified antibodies were designated alpha PR 6, 11, 13, 16, and 22. In addition to precipitating receptor-bound progesterone from cytosol, the antibodies were also effective in increasing the sedimentation velocity of progesterone receptor centrifuged on glycerol gradients, and in recognizing receptor proteins that were resolved by denaturing gel electrophoresis and transferred to nitrocellulose (Western blots). Immunoisolation of receptor was also demonstrated using receptor labeled covalently with the synthetic progestin, R5020. The antibodies were specific for progesterone receptor and did not cross-react with estrogen receptor from the oviduct or glucocorticoid receptor from chick liver. Two antibodies, alpha PR 6 and alpha PR 22, also recognized some mammalian forms of the progesterone receptor. Both antibodies reacted with progesterone receptor from the rabbit uterus and alpha PR 6 recognized human progesterone receptor. Four of the antibodies recognized both A and B forms of the avian receptor while alpha PR6 was specific for the B form.
Animals↗