Estrogen receptors and growth response in cultured human periodontal ligament cells

The periodontal ligament (PDL) is a connective tissue involved in the remodeling process associated with tooth development and positioning. PDL cells grown in culture were analyzed for the capacity to specifically bind steroid hormones and for growth response to estradiol-17 beta. Using [3H]estradiol-17 beta as the ligand, PDL cells in first passage cultures exhibited a specific estrogen binding capacity of 881 fmol/mg cell protein. With [3H]dexamethasone as a ligand, the binding capacity of the glucocorticoid receptor was 143 fmol/mg protein. With [3H]R5020 as a ligand, the progestin receptor exhibited a binding capacity of 5 pmol/mg protein. Scatchard analysis of estradiol binding at 37 degrees revealed a dissociation constant of 2.7 X 10(-9) M, representative of the estrogen receptor. The addition of estradiol-17 beta at concentrations of 10(-9) and 10(-8) M to culture media induced a dose-dependent decrease in growth (DNA content) to 62% and 38% control values, respectively. The addition of the antiestrogens tamoxifen and 4-hydroxytamoxifen at concentrations of 10(-7) and 10(-6) M similarly depressed cell growth. These results show that PDL cells contain high affinity receptors for several steroid hormones and further that these cells are targets for the action of estrogens.     

Hepatic estrogen receptor in the turtle, Chrysemys picta: partial characterization, seasonal changes and pituitary dependence

A hepatic estrogen receptor is described from female turtles, Chrysemys picta. The receptor adheres to DNA after incubation with [3H]estradiol and can be eluted with a linear salt gradient as a single component with an elution maximum of 0.21 M. It is steroid-specific, binding estrogens, but not androgens or progestins. Specific binding saturates between 3 and 7 nM [3H]estradiol-17 beta and Scatchard analysis gave a Kd of 2 X 10(-9) M and a maximal binding capacity of 3.02 fmol/mg protein. Hypophysectomy reduces hepatic estradiol receptor from 70 fmol/g tissue in control animals to non-detectable levels. Growth hormone replacement partially restored the receptor to 36% of control. Significant changes in receptor occur during the ovarian cycle.     

Identification of estrogen receptors in granulosa cells of immature rats

Nuclear and cytoplasmic binding sites for estradiol (E2-17 beta) in granulosa cells of immature rats were characterized. These binding sites for estrogen were high affinity, low capacity with an affinity constant (Kd) of 1.9 X 10(-10)M (binding capacity, Ro = 80 pM) for nuclear sites and a Kd = 3.5 X 10(-10) M (Ro = 45 pM) for cytosol sites. Binding was specific for biologically active estrogens. The estrogen receptor in granulosa cells is a protein and heat-labile as treatment with protease or pre-incubation at 37 degrees C for 1 h significantly diminished binding. RNase and DNase had no effect on estrogen binding. Sedimentation coefficients for nuclear and cytosol binding components were 5S and 8S respectively, similar to values obtained with uteri. Finally, translocation was demonstrated after a s.c. injection of E2-17 beta. Forty-five minutes post-injection, cytosol binding sites for estradiol were depleted concomitant with accumulation of nuclear binding sites. We concluded that granulosa cells of immature rats have binding sites specific for estradiol which have characteristics similar to the classical estrogen receptor in uteri.     

Quantification of cytosolic steroid receptors in secretory and non-secretory epithelial cells of the canine prostate

Radiolabelled methyltrienolone, dihydrotestosterone and estradiol were used as ligands to identify and quantify androgen and estrogen receptors in freshly dispersed cells from the canine prostate. Soluble extracts (cytosols) were obtained from secretory and non-secretory epithelial cells separated on the basis of their density in Percoll gradients. For both cell types, as well as for the whole prostate, Scatchard plot analyses were linear and showed a single class of high affinity binding sites: Kd values of 3.6 +/- 2.2 X 10(-9) M and 3.0 +/- 1.2 X 10(-10) M were measured for the androgen and estrogen receptors, respectively. The number of binding sites for the cytosolic androgen receptor, expressed per mg of protein or per mg of DNA, was 2.4- to 6.7-fold higher in the non-secretory cells compared to the secretory cells. However, these two cell types contained a similar number of specific sites for the estrogens. The specificities of the androgen and estrogen receptors were shown to be identical for the two cell types: the binding of [3H]R1881 was strongly inhibited by unlabelled R1881, 5 alpha-androstane-3 alpha, 17 beta-diol and dihydrotestosterone, while 5 alpha-androstane-3 beta, 17 beta-diol, estradiol and estrone did not displace bound R1881. The addition of triamcinolone acetonide did not alter the binding of R1881 in extracts of either cell type or in the whole prostate. The binding of [3H]estradiol to the estrogen receptor was highly specific since a strong displacement was only observed with estradiol (83%).     

In vitro mycotoxin binding to bovine uterine steroid hormone receptors

The mycotoxins, aflatoxin B(1), aflatoxin M(1), aflatoxicol and zearalenone were tested for binding to bovine endometrial estrogen and progestin receptors. Radioinert estradiol-17beta, estrone, testosterone, and cholesterol were evaluated for binding to the estrogen receptor. Zearalenone and aflatoxicol but not aflatoxins B(1) and M(1) competed with estradiol-17beta for the estrogen receptor. The order of binding affinities for the estrogen receptor were zearalenone > estradiol-17beta > estrone > aflatoxicol. The affinity of zearalenone for the estrogen receptor was 2-3 times that of estradiol-17beta. Progesterone, cortisol, radioinert R 5020, and cholesterol were evaluated for binding to the progestin receptor. None of the tested compounds except R 5020 and progesterone competed for the progestin receptor. The significance of aflatoxicol binding to the estrogen receptor is unclear. It is proposed that aflatoxicol binding to the receptor may alter gene expression in target tissues or act at the level of the hypothalamus to inhibit gonadotropin secretion and ovulation. These effects could explain reports of reduced fertility in domestic animals following ingestion of aflatoxin contaminated feedstuffs. It is also suggested that the mechanism of adverse effects on fertility of chronic aflatoxin ingestion in cattle and other livestock should be more thoroughly investigated.     

Estrogen receptor in the thymus of the castrated mice.

Sucrose density gradient ultracentrifugation and dextran-coated charcoal adsorption permitted us to characterize the estrogen-binding proteins in cytosols obtained from the thymus, spleen and mesenteric lymph node of the castrated male and female mice of C57BL strain. The thymic cytosol from both sexes incubated with 3H-estradiol-17 beta in the presence of excess unlabeled steroids showed a specific estrogenbinding 4 S protein with its binding capacity of 10(-14) moles/mg protein for males and 4 x 10(-15) moles/mg protein for females, respectively. The dissociation constant was of 4 x 10(-10) M for males and 3 x 10(-10) M for females, respectively. No specific binding was, however, found in the cytosols of the spleen and mesenteric lymph node. Steroid analysis by thin-layer chromatography of the thymic cytosols after incubation of them with 3H-estradiol-17 beta showed that a fair amount (around 60%) of radioactivity was from the undegradated radioactive steroid still bound to 4 S binder in both sexes. Enzyme study and heat experiment revealed that the estrogen specific 4 S binding component in the thymic cytosols bears at least protein in nature and is of heat-labile nature. These results strongly suggest that the thymus of the castrated mice contain a specific estrogen receptor, the nature of which is in part protein and heat-labile.     

Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells

The effects of estrogens on ovarian aromatase activity were investigated in vitro using granulosa cells from immature hypophysectomized estrogen-primed rats. The cells were cultured for 3 days in an androgen-free medium in the presence of follicle-stimulating hormone (FSH), with or without the specified estrogen. After washing, the cells were reincubated for 5 h with 10(-7) M androstenedione, and the formation of estrogens was measured. Estrogen production by control and diethylstilbestrol-treated cells was negligible, while FSH stimulated aromatase activity. Furthermore, concomitant treatment with diethylstilbestrol led to dose-dependent increases in the FSH-induced aromatase activity with an ED50 value of 4 X 10(-9) M and an apparent Vmax value 12- to 16-fold higher than those induced by FSH alone. The direct stimulatory effect of estrogens was time-dependent and was not accounted for by increases in cell protein. Various native and synthetic estrogens also augmented the FSH induction of aromatases (native estrogens: estradiol-17 beta = estrone greater than estradiol-17 alpha greater than estriol; synthetic estrogens: hexestrol greater than moxestrol greater than ethinyl estradiol much greater than chlorotrianisene and mestranol). The effect of estradiol-17 beta was dose-dependent with an ED50 value of 9 X 10(-9) M, which is within the physiological levels of follicular estradiol-17 beta. Although treatment with androgens also enhanced the FSH-induced aromatases, treatment with a progestin (R5020) or a mineralocorticoid (aldosterone) was without effect. Thus, estrogens directly augment the stimulation of granulosa cell aromatase activity by FSH. Follicular estrogens may activate intraovarian autoregulatory positive feedback mechanisms to enhance their own production, resulting in selective follicle maturation and the preovulatory estrogen surge.     

Biochemical and immunological characterization of estrogen binding components in human neoplastic adrenocortical tissues

The estrogen binding components in human adrenocortical tissues were examined. Two adrenocortical cancer cytosols were found to contain the binder with a relative low affinity (Kd 5 X 10(-9) M) for estradiol. The association of [3H]estradiol to these cytosols was inhibited by a large dose of unlabeled estrone, estradiol or estriol, but neither by diethylstilbestrol nor by dihydrotestosterone. Incubation of cultured cells derived from these cancers with [3H]estradiol also showed the presence of this low-affinity estradiol binder. The addition of bovine serum albumin into these cytosols surprisingly resulted in a marked increase in estradiol binding capacity in a concentration-dependent manner. This component sedimented at 5 S in the low salt sucrose density gradient. This binding ability was found to be heat-labile in the absence of estradiol, but preformation of complexes with estradiol markedly stabilized its binding ability against thermal inactivation. In addition, experiments using monoclonal antibodies to human estrogen receptor revealed that the estrogen binder from one adrenocortical cancer cytosol shared antigenic determinants with human estrogen receptor. These results suggest that the unique estrogen binder in some adrenocortical cancer has the characteristics similar to estrogen receptors in terms of thermal stability and immunological cross-reactivity to antibodies.     

Estrogen binding protein of rat liver.

An estrogen binding protein for estradiol-17beta is present in the liver cytosol of female intact and one day oophorectomized rats. The dissociation constant reveals high affinity binding (Kd: 0.69 +/- 0.14 times 10(-10) M). Quantitation of EBP using a dextran-coated charcoal method shows that this specific macromolecular binding is much less than in the rat uterus, but similar to that in DMBA-induced mammary tumors. Sucrose density gradient analysis shows sedimentation at 8-9 S and 4-5 S when compared to bovine serum albumin.     

Estrogen receptor in rat liver and its dependence on prolactin.

Estrogen receptor is shown to be present in the livers of adult rats. The receptor binds estradiol-17beta with a Kd of 1 x 10(-10) M and sediments at 8 S in sucrose gradients. Other estrogens and anti-estrogens compete for estradiol binding, while nonestrogenic steroids do not. Receptor levels fall dramatically after hypophysectomy, but can be partially restored within 18 hours by a single injection of prolactin. It is known that prolactin critically regulates the level of its own receptor in the liver, and we now suggest that it also exerts a primary control over the availability of liver estrogen receptor.     

Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to estrogen receptors from human breast cancers

The binding of catechol estrogens, epoxyenones and methoxyestrogens was evaluated using estrogen receptors in cytosol prepared from human breast cancers. The relative affinity of 2-hydroxyestradiol, a metabolite formed in vitro from estradiol-17 beta by breast cancer cells, was indistinguishable from that of estradiol-17 beta. 4-Hydroxyestradiol, which is also a metabolite of estradiol-17 beta, associated with the estrogen receptor with a relative affinity approximately 1.5-fold greater than that of estradiol-17 beta. Epoxyenones and methoxyestrogens were weak competitors compared to the binding of estradiol-17 beta, exhibiting relative affinities 3% or less than the affinity of estradiol-17 beta. Sucrose density gradient centrifugation revealed that both 2- and 4-hydroxyestradiol inhibited the binding of estradiol-17 beta to both the 4S and 8S isoforms of the estrogen receptor in a competitive manner, with a Ki = 0.94 nM for 2-hydroxyestradiol and a Ki = 0.48 nM for 4-hydroxyestradiol. It can be concluded that these data demonstrate a specific receptor-mediated estrogenic action for both of these catechol estrogens.     

The characteristic binding of catechol estrogens to estrogen receptors in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

The binding of catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestradiol) to estrogen receptors in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor cytosols was investigated. Cytosol estrogen receptors exhibited high affinities (Ka = 1.12-1.88 X 10(8) M-1) for all catechol estrogens as well as estradiol. The receptor level of catechol estrogens (46.1-97.5 fmol/mg protein) was 1.6-3.0 times higher than that of estradiol; especially the binding of 4-hydroxyestrone to estrogen receptors was the highest of all catechol estrogens and estradiol. In judging the receptor level of more than 20 fmol/mg protein to be positive, the binding of catechol estrogens to estrogen receptors was approximately correlated with that of estradiol. The positive receptor level of catechol estrogens was found in a half of tumor cytosols which showed the negative receptor level of estradiol. These results suggested that characteristic estrogen receptors indicating high affinities for catechol estrogens might be present in rat mammary tumor cytosols.     

Binding of [3H]methyltrienolone to androgen receptor in rat liver

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [3H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the Kd value was 3.3 X 10(-8) M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the Kd value was 2.5 X 10(-8) M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to 3/4 of that in the untreated cytosol. The profile of glycerol gradient centrifugation indicated that [3H]methyltrienolone-bound receptor migrated in the 8-9 S region in both untreated and triamcinolone-blocked cytosols, but the 8-9 S peak in triamcinolone-blocked cytosol was reduced to about 3/4 of that of untreated cytosol.     

Active immunization of female rats against 17 beta-estradiol. Preliminary studies on 17 beta-estradiol binding in uterine and pituitary cytosols

Female rats were immunized with 17 beta-estradiol-6-carboxymethyloxime-bovine serum albumin. They developed antibodies to estradiol and, to a very low extent, antibodies to BSA. Anti-estradiol antibodies possessed tight specificity to estradiol-17 beta, without cross-reactivities with other estrogens. It was demonstrated that the specific estradiol binding in uterine and pituitary cytosols gradually decreased when antiserum titres increased. In uterine cytosols, the presence of progesterone receptor was studied using promegestone (R50 20) as ligand. No significant variations in promegestone binding were observed. Competition experiments however, questioned the permanence in immunized rats of the actual progesterone receptor or of a promegestone binding protein.     

Synthesis and study of the estrogenic activity of 2,4-bis(bromomethyl)estradiol-17 beta 3-methyl ether.

Synthesis of 2,4-bis(bromomethyl)estradiol-17 beta 3-methyl ether (BBE2M) was accomplished by reducing a methanolic solution of 2,4-bis(bromomethyl)estrone methyl ether with sodium borohydride. In 0.5 M phosphate buffer, pH 7.0, 25 degrees, BBE2M readily reacts with Ellman's anion and alkylates cysteine to form a steroid-amino acid conjugate. Stoichiometry of the reaction indicates that the bromosteroid is divalent with cysteine. Tryptophan and histidine react more slowly with the bromosteroid. Estrogenic activity of BBE2M was evaluated in ovariectomized rats by uterine intraluminal administration and quantitation of glucose-6-phosphate dehydrogenase (D-glucose-6-P:NADP+ oxidoreductase, EC 1.1.1.49) activity in the uterus. BBE2M induced glucose-6-phosphate dehydrogenase activity as did estradiol-17 beta or estradiol-17 beta 3-methyl ether (E2M). BBE2M was more persistent in activity than E2M. Histological examination of uterus following BBE2M treatment shows classic estrogenic morphology. BBE2M covalently binds to the cytoplasmic estrogen receptor of calf uterus. Such binding is prevented by pretreatment of the receptor protein with estradiol-17 beta. The covalently bound steroid-receptor complex appears to stimulate RNA synthesis in isolated nuclei from calf endometrium.     

Secretion of soluble functional insulin receptors by transfected NIH3T3 cells

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.     

Effect of active immunization against 17 beta-estradiol on cytosolic estrogen receptors in the rat uterus

Following active immunization of female rats against estradiol-17 beta, the amount of specific binding sites for estrogen decreased in uterine cytosol as a function of antiserum titres. They were undetected when antibodies titres were higher than 1/2000. Moreover, a binding protein specific for estradiol-17 beta appeared. Estradiol binding was not displaced with an excess of unlabeled DES nor precipitated with protamine sulfate. The sedimentation coefficient of the hormone-protein complex (7-8 S) was not modified in medium of high ionic strength (0.4 M KCl). That protein represented antibodies to Estradiol-17 beta which could be precipitated with antiserum to rat IgG.     

Nongenomic inhibition of oxytocin binding by progesterone in the ovine uterus

Progesterone (P4) has been reported to inhibit oxytocin (OT) binding to its receptor in isolated murine endometrial membranes. The purpose of the present research was to 1). examine the in vivo and in vitro effect of P4 on the binding of OT to its receptor in the ovine endometrium and 2). determine whether the endometrial plasma membranes have high-affinity binding sites for P4. Ovariectomized ewes were pretreated with a sequence of estradiol-17beta (2 days) and P4 (5 days) before being treated with estradiol-17beta plus either vehicle (corn oil), P4, or P4 + mifepristone (RU 486) for 3 consecutive days. Treatment of ewes with 10 mg P4/day for 3 days suppressed binding of OT (P < 0.01) compared with that of controls, whereas concomitant treatment with the progestin antagonist RU 486 (10 mg/day) blocked the effect of P4. Similarly, incubation of endometrial plasma membranes with P4 (5 ng/ml) inhibited binding of OT (P < 0.05), whereas this effect of P4 was blocked by the presence of RU 486 (10 ng/ml). By radioreceptor assay, the endometrial plasma membranes were found to contain a high-affinity binding site for P4 and the progestin agonist promegestone (Kd 1.2 x 10-9 and 1.74 x 10-10M, respectively). Incubation of endometrial plasma membranes with P4 (5 ng/ml) significantly increased the concentration of progestin binding sites. Binding of labeled promegestone (R 5020) was competitively inhibited by excess unlabeled R 5020, P4, RU 486, and OT but not by estradiol-17beta, cortisol, testosterone, and arginine vasopressin. These data suggest a direct suppressive action of P4 on the binding of OT to OT receptors in the ovine endometrial plasma membrane.     

Presence of two types of estrogen binding sites in mouse testis

The characteristics of cytosol estrogen binding sites in BALB/c mouse testis were investigated. The cytosol prepared from the whole testis contained two classes of the specific estrogen binding sites by Scatchard and Rosenthal plot analyses. The first binding site (first binder) had high affinity for 17 beta-estradiol (E2; Kd = 4.9 X 10(-9) M) and binding specificity as observed in the typical estrogen receptor. The second binding site (second binder) had lower affinity for E2 (Kd = 4.8 X 10(-8) M) and the binding was inhibited less vividly by diethylstilbestrol (DES) and antiestrogens in comparison with that for the first binder. Postlabeled sucrose density gradient analysis in a low salt medium revealed that the major radioactive peak of the first binder appeared at 7S region, while that of the second binder sedimented at 4S region. The 7S component showed an appreciable binding to the nuclei, while the 4S component did not show a significant binding ability to the nuclei. Much higher concentrations of the first and the second binders were found in Leydig cells preparations. These results demonstrate the presence of two types of the specific estrogen binding sites in the mouse testis especially in Leydig cells.