Autoradiographic localization of specific [3H]dexamethasone binding in fetal lung

The cellular and subcellular localization of specific [3H]dexamethasone binding was examined in fetal mouse lung at various stages of development and in human fetal lung at 8 weeks of gestation using a rapid in vitro steroid incubation technique followed by thaw-mount autoradiography. Competition studies with unlabeled steroids demonstrate the specificity of [3H]dexamethasone labeling, and indicate that fetal lung mesenchyme is a primary glucocorticoid target during lung development. Quantitative binding studies, involving incubation of intact tissue with competing ligand and subsequent subcellular fractionation, show this to be specific, nuclear binding characteristic of glucocorticoid receptors. Autoradiographs of [3H]dexamethasone binding in lung tissue at early stages of development demonstrate that the mesenchyme directly adjacent to the more proximal portions of the bronchiolar network is heavily labeled. In contrast, the epithelium which will later differentiate into bronchi and bronchioles, is relatively unlabeled. Distal portions of the growing epithelium, destined to become alveolar ducts and alveoli, do show nuclear localization of [3H]dexamethasone. Because of the known importance of the mesenchyme in controlling lung development and the ability of glucocorticoids to stimulate lung development, these results suggest that many of the growth-promoting effects of glucocorticoids may be mediated through the mesenchyme. In addition, by utilizing a technique which allows the simultaneous examination of extracellular matrix components and [3H]dexamethasone binding, a relationship is observed between extensive mesenchymal [3H]dexamethasone binding and extensive extracellular matrix accumulation. Since glucocorticoids stimulate the synthesis of many extracellular matrix components, these results suggest a role for these hormones in affecting mesenchymal-epithelial interactions during lung morphogenesis.     

Effect of pyridoxine on the inhibition by dexamethasone of murine erythroleukemia cell differentiation

Glucocorticoids are known to inhibit the induced differentiation of murine erythroleukemia cells. I show here that the binding of glucocorticoid receptors to the intact nuclei of these cells is reduced by exogenous extracellular pyridoxine (1–4 mM). This reduction of glucocorticoid-receptor binding did not abrogate the inhibition by dexamethasone of the induced differentiation.     

Suppression of rat mammary tumor cell growth in vitro by glucocorticoids requires serum proteins. Characterization of wild type and glucocorticoid-resistant epithelial tumor cells

CON8 is a single-cell derived subclone of the 13762NF transplantable, hormone-responsive rat mammary tumor that proliferates rapidly in serum-free medium. Addition of either glucocorticoids or calf serum alone caused a slight stimulation of CON8 proliferation. However, glucocorticoids required the presence of specific serum proteins to strongly suppress CON8 cell growth. Furthermore, the anchorage-independent growth of CON8 cells was significantly reduced in the presence of glucocorticoids and serum. We have designated this serum activity GMGSF, for glucocorticoid modulating growth suppression factor. Inhibition of cell growth was limited to steroids with strong glucocorticoid biological activity, while exposure to the glucocorticoid antagonist RU38486 prevented this response. Half-maximal growth inhibition and half-maximal expression of a glucocorticoid-inducible gene product (2 nM) occurred slightly below the half-maximal receptor binding of [3H]dexamethasone (10nM). We have also selected a variant mammary epithelial tumor cell line, derived from CON8, denoted 8RUV7, whose proliferation and soft agar colony formation failed to be suppressed by glucocorticoids in the presence of serum. These glucocorticoid-resistant variant cells possess functional glucocorticoid receptors, competently produce the glucocorticoid-responsive gene product plasminogen activator inhibitor, and along with CON8 cells express milk fat globule protein antigens on their cell surface, indicative of their mammary epithelial cell character. We are using this variant line to genetically dissect the molecular mechanism of the glucocorticoid/GMGSF growth suppression pathway in mammary epithelial tumor cells.     

Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. We have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with [3H]dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified approximately 100-kDa steroid-binding subunit was eluted from gel slices and subjected to enzymatic digestion. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single [3H]dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the [3H]dexamethasone 21-mesylate was located at position 5 from the amino terminus. Dual-isotope labeling studies with [3H]dexamethasone 21-mesylate and [35S]methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of [3H]dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, our data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of glucocorticoid receptor in HeLa-S3 cells

Glucocorticoid receptor of the human cell line HeLa-S3 has been characterized and has been compared to rat and to mouse glucocorticoid receptors. If HeLa cells were lysed in the absence of glucocorticoid, glucocorticoid receptor was isolated in a nonactivated form, which did not bind to DNA-cellulose. If HeLa cells were preincubated with glucocorticoid, glucocorticoid receptor was isolated in an activated, DNA-binding form. HeLa cell glucocorticoid receptor bound [3H]triamcinolone acetonide with a dissociation constant (KD = 1.3 nM at 0 degrees C) that was similar to those of mouse and rat glucocorticoid receptors. Similarly, the relative binding affinities for steroid hormones decreased in the order of triamcinolone acetonide greater than dexamethasone greater than promegestone greater than methyltrienolone greater than aldosterone greater than or equal to moxestrol. Nonactivated and activated receptors were characterized by high-resolution anion-exchange chromatography (FPLC), DNA-cellulose chromatography, and sucrose gradient centrifugation. Human, mouse, and rat nonactivated glucocorticoid receptors had very similar ionic and sedimentation properties. Activated glucocorticoid receptors were eluted at similar salt concentrations from DNA-cellulose columns but at different salt concentrations from the FPLC column. A monoclonal mouse anti-rat liver glucocorticoid receptor antibody [Westphal, H.M., Mugele, K., Beato, M., & Gehring, U. (1984) EMBO J. 3, 1493-1498] did not cross-react with HeLa cell glucocorticoid receptor. Glucocorticoid receptors of HeLa, HTC, and S49.1 cells were affinity labeled with [3H]dexamethasone and with [3H]dexamethasone 21-mesylate. The molecular weights of [3H]dexamethasone 21-mesylate labeled glucocorticoid receptors (MT 96 000 +/- 1000) were undistinguishable by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid derivatives for electrophilic affinity labelling of glucocorticoid binding sites: interaction with the glucocorticoid receptor and biological activity

To investigate the possible use of electrophilic affinity labelling for the characterization of glucocorticoid receptors, different chemically reactive derivatives of deoxycorticosterone (deoxycorticosterone 21-mesylate and deoxycorticosterone 21-(1-imidazole) carboxylate), dexamethasone (dexamethasone 21-mesylate, dexamethasone 21-iodoacetate and dexamethasone 21-bromoacetate) and progesterone (21-chloro progesterone) were tested for their ability to bind irreversibly to the glucocorticoid receptor from goat lactating mammary gland. Using partially purified receptor, only one of the steroids tested, dexamethasone 21-mesylate (DXM-M) was found more effective than dexamethasone (DXM) in preventing exchange of radioactive dexamethasone in the receptor binding site. The affinity of DXM-M for the glucocorticoid receptor, measured by competitive binding assay, was 1/15 that of DXM. Polyacrylamide gel electrophoresis in sodium dodecyl sulphate of the [3H]-DXM-M labeled glucocorticoid receptor revealed a specific covalently radiolabeled fraction corresponding to an apparent molecular weight of 75,000 to 80,000. The biological activity of DXM-M was studied in RPMI 3460-clone 6 Syrian hamster melanoma cells, a cell line which is sensitive to growth inhibition by glucocorticoids. Like DXM, DXM-M inhibits the growth of RPMI 3460-clone 6 cells and it acts as a slowly reversible glucocorticoid agonist at concentrations which correlate with the affinity of DXM-M for the glucocorticoid receptor in vitro.     

Glucocorticoid-receptor interaction and induction of murine mammary tumor virus.

The relationship between the cellular uptake of glucocorticoid hormones, the binding of these hormones to specific in vitro receptors, and the induction of mouse mammary tumor viruses in an established mouse mammary tumor cell line was highly correlated. These results suggest that the induction of mouse mammary tumor virus by glucocorticoid hormones is a physiological process acting through a mechanism of high affinity, saturable steroid-receptors. A temperature-sensitive or salt-dependent step following glucocorticoid-receptor interaction was required for nuclear uptake of the steroid. Induction studies with different adrenocorticoids indicate that the synthetic glucocorticoid, dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), is the most potent inducer of mouse mammary tumor viruses and all steroids which caused significant induction were glucocorticoids. Other glucocorticoids appear to stimulate murine mammary tumor virus production by a mechanism similar to that of dexamethasone; for example, corticosterone competes with dexamethasone for binding to the glucocorticoid receptor and blocks the uptake of dexamethasone into cells. Progesterone also blocks the cellular uptake of dexamethasone and can bind to the glucocorticoid receptor at low concentrations (10-7 to 10-8 M) but progesterone does not consistently induce virus at hormone concentrations even as high as 10-4 M. Thus, in this system, binding to a cytoplasmic receptor is necessary but not sufficient for induction by glucocorticoids. Estrogens and androgens interfere with receptor binding and cellular uptake of dexamethasone but only at much higher concentration (10-4 M) than progesterone, and do not induce mammary tumor virus production. Although there was a positive correlation between steroid structure, binding, and biologic induction, other factors clearly affect the physiological manifestations of steroid actions. Mouse cells with comparable cytoplasmic receptor levels and comparable nuclear uptake differed absolutely in their degree of murine mammary tumor virus induction following hormone treatment. Although all mouse cells examined contain comparable levels of murine mammary tumor virus DNA, only cells producing constitutive levels of murine mammary tumor virus RNA could be induced to higher levels by a variety of glucocorticoids.     

Differential binding of dexamethasone to ammonium sulfate precipitates of human adipose tissue cytosols

Saturation analysis of the binding of [3H]dexamethasone [( 3H]DEX) to ammonium sulfate precipitates (ASPs) confirmed the presence of a limited-capacity, high-affinity binder in human adipose tissue cytosols. Various non-radioactive steroids competed with [3H]DEX for binding to the ASPs in the following sequence: dexamethasone (DEX) approximately equal to triamcinolone acetonide (TA) greater than progesterone (P) much greater than estradiol (E2). The steroid specificity of the binder precipitated by AS was consistent with the specificities reported for glucocorticoid receptors in a number of systems. In order to investigate possible regional differences, glucocorticoid binding to ASPs derived from adipose tissues removed from two different sites in the same subject was quantitated. ASPs of human omental adipose tissue bound significantly more [3H]DEX than did similar preparations of subcutaneous adipose tissue from the abdominal wall (116 +/- 32 vs. 50 +/- 22 fmol/mg protein; mean +/- SD; p less than 0.02). The findings are consistent with reports from other laboratories suggesting that intra-abdominal fat is more responsive to glucocorticoids than is subcutaneous adipose tissue.     

Antiprogesterone and antiglucocorticoid actions of RU 486 on rabbit mammary gland explant cultures. Evidence for a persistent inhibitory action of residual progesterone upon the mammary tissue

The antiprogesterone and antiglucocorticoid compound RU 486 added to pregnant rabbit mammary gland explant cultures had no effect alone but significantly stimulated casein production in the presence of ovine prolactin (PRL) in a dose dependent manner. This stimulation was inhibited by progesterone (Pg) and the Pg agonist R5020. When the explants were cultured for 5 days with two changes of medium, to eliminate all steroids, and hormones added afterwards, the effect of PRL was potentiated, Pg was no longer inhibitory and RU 486 had no effect, RU 486 also could inhibit the stimulatory action of glucocorticoids added to the cultures along with PRL. The compound was able to displace [3H]dexamethasone and [3H]R 5020 from mammary gland glucocorticoid and Pg receptors respectively and proved to have a high relative binding affinity (RBA) for both receptors when compared with typical ligands for each receptor. The RBAs of RU 486 and the steroids used in this study to mammary gland glucocorticoid and Pg receptors correlated well with the ability of RU 486 to block their biological activities. These results demonstrate that RU 486 has both antiglucocorticoid and antiprogesterone activities in pregnant rabbit mammary glands as well as the existence of a strong inhibitory residual action of Pg in the gland that persists during the first 48 h of culture and that can be eliminated by RU 486 or after several days of culture with no hormones.     

Cortisol 17 beta acid, transcortin, and the heterogeneity of rat brain glucocorticoid receptors

Binding of tracer or competing steroids to transcortin can compromise specificity studies on receptors for adrenal steroids. Recently Alexis et al. have used cortisol 17 beta acid at high concentrations to prevent steroid binding to any transcortin possibly contaminating rat brain cytosol preparations. On the basis of limited specificity studies of [3H]dexamethasone and [3H]corticosterone binding under such conditions, it was claimed that binding sites for the two steroids are indistinguishable, and it is thus unnecessary to invoke distinct binding sites for each glucocorticoid. We have extended these competition studies in the presence of cortisol 17 beta acid, and shown that in rat hippocampus Type I, corticosterone-preferring glucocorticoid receptors can be clearly distinguished both from transcortin and from Type II, dexamethasone-binding glucocorticoid receptors.     

Binding of glucocorticoid receptors to mammary chromatin acceptor sites

We have recently characterized the interaction of mouse mammary estrogen receptors (ER) with mammary chromatin acceptor sites and demonstrated that ER from estrogen resistant lactating mammary glands do not bind to chromatin. In this study we have characterized the chromatin binding of the glucocorticoid receptor from mouse mammary glands isolated from nulliparous and lactating mice in order to better understand the relationship between receptor binding to chromatin and steroidogenic sensitivity of the tissue. Mammary chromatin was linked covalently to cellulose and deproteinized sequentially by 0-8 M Gdn-HCl. Binding to intact chromatin as well as to chromatin deproteinized by Gdn-HCl was determined using partially purified [3H]dexamethasone labelled glucocorticoid-receptor complexes (GR) obtained by fractionation on DEAE-cellulose columns. The binding of [3H]GR from mammary glands of nulliparous mice to chromatin fractions from the same tissue revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 5-6 M Gdn-HCl. Binding of [3H]GR was of high affinity (Kd = 0.2 nM) and saturable. A simultaneous comparison of the chromatin binding patterns for [3H]ER and [3H]GR isolated from mammary glands of nulliparous mice revealed that the chromatin subfractions obtained with 4-6 M Gdn-HCl extraction contained acceptor sites for both [3H]ER and [3H]GR; however, while the [3H]ER bound to a 4.5 M and a 5.5 M site, the [3]GR bound a 5 M and a 6 M site. Competition experiments supported the steroid receptor specificity of the chromatin acceptor sites. Thus, the 4-6 M chromatin fractions contain distinct acceptor sites for the glucocorticoid receptor and for the estrogen receptor. In addition our studies reveal that the binding patterns of [3H]GR isolated from mammary glands of nulliparous and lactating mice to their homologous chromatin is essentially similar. Thus, in contrast to estrogen receptors, glucocorticoid receptors from lactating mammary glands are able to effectively bind to chromatin acceptor sites which supports our previous suggestion that the estrogenic insensitivity of lactating mouse mammary glands may at least be in part due to the impeded interaction of ER with chromatin acceptor sites.     

Stimulation and inhibition of cellular functions by glucocorticoids. Correlations with rapid influences on chromatin structure.

The effect of media conditions on the glucocorticoid response has been examined in three types of cultured cells. In rat pituitary tumor cells (GC cells) growth hormone production was stimulated by glucocorticoids provided fresh culture media was present (enriched media conditions). In contrast, dexamethasone either failed to induce or deinduce growth hormone synthesis if added to cultures which had not received fresh media for 3 days (depleted media condition). With human skin fibroblasts, cortisol stimulated [3H]thymidine incorporation in the enriched condition but inhibited this response in the depleted condition. In mouse lymphoma (S49) cells the enriched media conditions significantly delayed the killing response to glucocorticoids (20% killing after 24 h versus 90% killing after 24 h for the depleted condition). Thus, the magnitude and in some cases, the direction of the glucocorticoid response are sensitive to the conditions to which the cells are exposed. In all three cell types the steroid also rapidly (detectable by 15 min, maximal by 2 h) altered chromatin structure as detected by a change in the number of initiation sites for Escherichia coli RNA polymerase assayed under cell-free conditions. This early nuclear response could be in a positive or negative direction and was also affected by the culture conditions; enriched media favored a positive or less negative effect on the initiation sites by the steroid, while depleted media favored a steroid-induced inhibition of this chromatin function. In S49 and GC cells the kinetics and magnitude of the change in chromatin closely followed receptor . glucocorticoid complex binding to nuclei while removal of dexamethasone from the culture media resulted in a rapid (t 1/2 = approximately 20 min) disappearance of the effect which paralleled loss of bound hormone from the nucleus. The glucocorticoid effect on chromatin was not observed in two lines of glucocorticoid-resistant mutant S49 cells. One line (R-) lacks detectable glucocorticoid receptors; the other line (Nti) has receptors that bind the hormone normally, but the receptor . glucocorticoid complexes bind more avidly to the nucleus. These results suggest that the receptor is involved in both the stimulatory and the inhibitory effects on chromatin. The findings in the Nti cells and of a slight lag between nuclear binding of receptors and initiation site alteration implies that some receptor property, in addition to nuclear binding per se, is responsible for the influence on chromatin. These results are discussed in terms of a model in which steroid hormones initiate their actions by influencing a reaction that modifies chromatin structure. The direction and magnitude of the reaction, and its effect on the expression of specific genes, are dictated by the metabolic state and differentiation of the cell.     

Glucocorticoid receptors in mouse mammary tumors: Specific binding to nuclear components.

The specific interaction of glucocorticoids with nuclei of mouse mammary tumor was studied in vitro by incubation of the tissue with [3H]dexamethasone at 25 degrees. It was demonstrated that the mammary tumors contain a limited number of specific nuclear binding sites which were saturated with low hormone concentrations (10-8 M)9 The concentrations of specific binding sites in the nuclei were related to the concentration of cytoplasmic binding sites of unincubated tissues and varied between individual tumors. The binding component in the nuclei appeared to be a protein and was easily solubilized with 0.4 M KCl containing buffers. The ability of various corticoids to block the nuclear localization of the steroid correlated well with their glucocorticoid potency. Estradiol and progesterone at concentrations of 10-6 M were also effective in competing for the glucocorticoid receptor binding sites. However, while the glucocorticoids such as hydrocortisone and corticosterone translocated to nuclear sites also specific for dexamethasone, estradiol and progesterone competed for the cytoplasmic binding sites and did not translocate to the nucleus. The possible significance of the interaction of various steroids with the glucocorticoid receptors in mammary tumors is discussed.     

Depletion of [3H]methyltrienolone cytosol binding in glucocorticoid-induced muscle atrophy

The present study was undertaken to determine cytosol binding properties of [3H]methyltrienolone, a synthetic androgen, in comparison with [3H]dexamethasone, a synthetic glucocorticoid, under conditions of glucocorticoid excess in skeletal muscle. Male hypophysectomized rats received either seven daily subcutaneous injections of cortisone acetate (CA) (100 mg X kg-1 body wt) or the vehicle, 1% carboxymethyl cellulose. Following treatment, both [3H]dexamethasone and [3H]methyltrienolone-receptor concentrations were decreased from those in vehicle-treated rats by more than 90 and 80%, respectively, in CA-treated animals. Scatchard analysis of [3H]methyltrienolone binding in muscles of vehicle-treated animals became nonlinear at high concentrations of labeled ligand and were reanalyzed by a two-component binding model. The lower affinity, higher capacity component, which was attributed to binding of methyltrienolone to a "dexamethasone" component, disappeared in muscles of CA-treated rats and Scatchard plots were linear. Receptor concentrations of the higher affinity lower capacity "methyltrienolone" component were similar in muscles of vehicle-treated and CA-treated groups. From competition studies, the high relative specificities of glucocorticoids for [3H]methyltrienolone binding in muscles of vehicle-treated animals were markedly reduced by CA treatment. In addition, the binding specificity data also showed strong competition by progesterone and methyltrienolone for [3H]dexamethasone binding and estradiol-17 beta for [3H]methyltrienolone binding. These results demonstrate that most of the [3H]methyltrienolone binding is eliminated under in vivo conditions of glucocorticoid excess. Furthermore, the competitiveness of various steroids for receptor binding suggests that rat muscle may not contain classic (ligand-specific) glucocorticoid and androgen receptors.     

IL-2 protects T lymphocytes from glucocorticoid-induced DNA fragmentation and cell death

In the IL-2-dependent T cell clone CTLL-2, dexamethasone, a synthetic glucocorticoid, induces a suicide program characterized by the early degradation of chromatin in oligonucleosome-length fragments which precedes the loss of cell viability by 2 to 4 h. These effects are most likely mediated through the interaction with a specific glucocorticoid receptor as suggested by the structure-activity relationship of the various steroids tested. Incubation of nuclei of glucocorticoid-untreated cells in the presence of calcium and magnesium ions induces the cleavage of DNA in the linker region between nucleosomes, suggesting that fragmentation of chromatin in intact cells by glucocorticoids may involve the activation of a preexisting endonuclease. Interestingly, the presence of a saturating dose of IL-2 during the treatment of CTLL-2 cells with glucocorticoids completely blocks the cell death program.     

Inhibition of T cell-mediated cytotoxicity by anti-inflammatory steroids

We have tested the capacity of glucocorticoids to modulate the effector function of splenic cytotoxic T lymphocytes (CTL) obtained after i.p. immunization with allogeneic cells. Although acute exposure to glucocorticoids did not inhibit the activity of freshly obtained splenic CTL, preincubation of these CTL for several hours with subnanomolar concentrations of several different glucocorticoids caused marked inhibition. The relative inhibitory potency of the steroids tested correlated with their reported activity both in glucocorticoid receptor binding assays and in assays of anti-inflammatory potency in man. The inhibitory effects of low concentrations (10(-10) M to 10(-9) M) of dexamethasone were reversed by human or mouse interleukin 2 (IL 2)-containing supernatants, but were not reversed by IL 1-containing supernatants. The inhibitory effects of higher concentrations (10(-8) M to 10(-7) M) of dexamethasone could not be reversed even by very high doses of mouse IL 2. In contrast to previous reports of minimal direct glucocorticoid effects on CTL activity, the present results suggest that after preincubation, splenic CTL from in vivo-immune mice are sensitive to inhibition by glucocorticoids, and that the glucocorticoids may act both indirectly (on IL 2 production) and directly on the CTL.     

Steroid inhibition of calcitriol-induced prolactin production in GH4C1 cells. Specificity and sensitivity.

The induction of prolactin (PRL)-gene expression by calcitriol (1,25-dihydroxyvitamin D3, 1,25-dihydroxycholecalciferol) in clonal rat pituitary tumour (GH4C1) cells was selectively inhibited by cortisol [IC50 (concentration causing 50% inhibition) = 3.2-4.1 nM]. The steroid specificity of this effect was investigated and various steroids were found to inhibit calcitriol-stimulated PRL production with the following relative potencies: cortisol, 1; dexamethasone, 8; 11-deoxycortisol, 0.5; corticosterone, 0.4; aldosterone, 0.07; testosterone and oestradiol, less than 0.003. The steroid antagonist RU 38486 did not affect basal or calcitriol-stimulated PRL production, but antagonized the effect of 10 nM-cortisol in a concentration-dependent manner. Neither progesterone nor 11-deoxycortisol antagonized the effect of 10 nM-cortisol. Calcitriol-induced PRL production was 14 times more sensitive to dexamethasone inhibition than was non-stimulated PRL production. Growth-hormone production was stimulated by dexamethasone, in the presence or absence of calcitriol, with a concentration-dependence similar to that of dexamethasone inhibition of basal PRL production. These data indicate that steroid inhibition of calcitriol-stimulated PRL production is a specific glucocorticoid effect. The sensitivity of calcitriol-stimulated PRL production to dexamethasone was 14-26-fold greater than that of other measured responses in the same cells. Two of the possible explanations for this selectively increased sensitivity to glucocorticoids are: amplification of the glucocorticoid effect via an induced mediator; and the presence of very-high-affinity glucocorticoid-receptor-binding sites on DNA.     

Kinetic pulse-chase labeling study of the glucocorticoid receptor in mouse lymphoma cells. Effect of glucocorticoid and antiglucocorticoid hormones on intracellular receptor half-life

A kinetic pulse-chase labeling technique was used to measure the intracellular half-life of the glucocorticoid receptor in S49 mouse lymphoma cells. Cells were pulse-labeled with [35S]methionine for 30 min and then cultured in the presence of unlabeled methionine (chase). Labeled receptors were quantitated at periodic time points during the chase by immunoadsorption to protein A-Sepharose using the BuGR2 monoclonal antireceptor antibody. The decay of labeled receptors during the chase was linear on a semilog plot, consistent with first order kinetics. Receptor half-life was 9 h when cells were cultured in either phenol red-containing medium supplemented with fetal calf serum or in phenol red free-medium supplemented with charcoal extracted serum, indicating that endogenous steroids do not affect receptor half-life. Receptor half-life was also unchanged when cells were cultured in the presence of 0.1 microM dexamethasone, a glucocorticoid hormone, or 0.1 microM RU486 (11 beta-(4-dimethylamino-phenyl)-17 beta-hydroxy-17 alpha-(propynylestra-4,9- diene-3-one), an antiglucocorticoid hormone. We conclude that the intracellular half-life of the glucocorticoid receptor in S49 mouse lymphoma cells is not regulated by either glucocorticoid or antiglucocorticoid hormones.     

Characterization of cortisol binding sites in chicken liver plasma membrane

1. The presence of sites specifically binding [3H]cortisol in plasma membrane isolated from chicken liver has been determined. The kinetic parameters of this binding are: Kd = 4.5 nM and Bmax = 2225 fmol/mg protein in presence of 10(-6) M progesterone. 2. The affinities of several natural and synthetic steroids for the membrane binding site respect to the binding of 4 nM [3H]cortisol without competitor increased in the following order: Testosterone less than pregnenone less than dexamethasone less than progesterone less than prednisolone less than corticosterone less than deoxycorticosterone. 3. Other steroids such as estradiol, ouabain and triamcinolone acetonide does not bind to the plasma membrane. 4. Metal ions such as Ca2+ and Mg2+ did not modify the binding of [3H]cortisol. 5. Neither propranolol nor phentolamine, beta- and alpha-adrenergic antagonists affected [3H]cortisol binding to the plasma membranes. 6. The result suggest that the binding site detected is more specific for glucocorticoids and it is different of nuclear glucocorticoid receptor and progesterone receptor.     

Glucocorticoid--receptor interactions. Studies of the negative co-operativity induced by steroid interactions with a secondary, hydrophobic, binding site.

The effects of steroids on the binding of [1,2-3H]dexamethasone and [1,2-3H]progesterone to the glucocorticoid receptor of rat thymus cytosol were studied. Although both glucocorticoid agonists and antagonists competed with [1,2-3H]dexamethasone for binding to the receptor under equilibrium conditions, only glucocorticoid antagonists of partial agonists, at micromolar concentrations, were capable of accelerating the rate of dissociation of previously bound [1,2-3H]dexamethasone from the receptor. Antagonists or partial agonists also enhanced the rate of dissociation of [1,2-3H]progesterone from the glucocorticoid receptor, with identical specificity and concentration--response characteristics. These effects are attributed to the presence on the receptor of a secondary, low-affinity, binding site for glucocorticoid antagonists, the occupancy of which produces negatively co-operative interactions with the primary glucocorticoid-binding site. In contrast with the interactions with the primary site, the interactions of steroids with the negatively co-operative site appear to be primarily hydrophobic in nature, and the site resembles the steroid-binding site of progestin-binding proteins in its specificity, though not its affinity. The results also suggest that the initial interactions of both glucocorticoid agonists and antagonists with the receptor under equilibrium conditions are with one primary site on a receptor existing in one conformation only.