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PubMed1979

Glucocorticoid receptors.

Glucocorticoid receptors are found in most mammalian tissues and have been studied in detail in a number of tissue culture systems. With cells that have not been exposed to steroids, the receptors are found in the cytoplasmic fraction from which they can be isolated and studied. Methods for studying glucocorticoid receptors depend on their high-affinity specific binding of radioactive steroids. The reversible interaction is intracellular. It follows Michaelian kinetics, at least in cell-free cytosol, and involves a thermodynamically homogeneous population of about 10 000 sites per cell. The receptor is an asymmetric, slightly acidic protein of about 100 000 daltons. It is very labile, especially in the unbound form. Binding activity depends on the integrity of thiol groups and perhaps on phosphorylation of amino acid residues. Although indirect, the evidence is overwhelmingly convincing that this protein is the physiologic glucocorticoid receptor. The time-kinetics of binding and dissociation are consistent with the sequence of events in glucocorticoid action. Various steroid analogs display binding characteristics predictable from their glucocorticoid activity. Loss of the binding protein from certain cultured cell lines is accompanied by unresponsiveness to glucocorticoids. The extensive tissue distribution of receptors parallels the extensive role of glucocorticoids in regulation. Finally, there is a strong correlation between nuclear binding of receptors and nuclear effects of the steroid. The glucocorticoid receptor can be distinguished from other glucocorticoid-binding proteins, based on their steroid specificity and physicochemical properties. There is no clear-cut demonstration that the receptor differs from tissue to tissue, and it is in fact very similar in various species. Unlike in other systems, receptor concentration does not seem to be regulated by its ligand or by other hormones. However, certain cases of hypo- as well as hypersensitivity to glucocorticoids appear to result from changes at the receptor level. The data indicate that the receptor can exist in inactive and active forms. The former predominate in the absence of steroid or when an angatonist is bound. Glucocorticoid agonists bind the active form, allowing it to be "activated" and subsequently bound to the nucleus. All of the receptors in isolated cytosol do not appear to be available for immediate occupancy by an agonist and this may be due to the time required for conversion of the receptors from inactive to active forms. The correlations between receptor binding and the glucocorticoid response indicate that the receptor is a rate-limiting factor in the magnitude and kinetics of the response, and this finding has important implications regarding mechanisms.

Animals↗
PubMed1975

Binding of glucocorticoid receptors to DNA.

DNA has been implicated as the nuclear acceptor for receptor-glucocorticoid complexes. The present study concerns the interaction of these complexes, isolated from cultured rat hepatoma cells, with purified DNA. This association is rapid, reaching a maximum within a few minutes at 0 degrees, whereas dissociation requires several hours. DNA binds neither free glucocorticoids nor those complexed with transcortin or cytosol proteins different from the receptor. Receptors which are not complexed by steroid have little or no affinity for DNA. "Activation," necessary for the binding of receptor-steroid complexes to isolated nuclei, also enhances DNA binding. The capacity of DNA for binding receptor-steroid complexes is large; saturation was not observed at the complex concentrations studied, using either crude or partially purified receptor preparations. The association of complexes with DNA is inhibited by divalent cations, at increasing ionic strengths, and by mercurial reagents. Complexes bind equally well to bacterial, bacteriophage, or rat DNA; however, there was either no or substantially reduced binding by bacterial 23 S rRNA. The binding of complexes to native DNA is roughly 3-fold greater than to denatured DNA. These characteristics are consistent with the possibility that DNA is the nuclear acceptor for receptor-glucocorticoid complexes; however, the actual composition of the acceptor sites remains unknown.

Animals↗
PubMed1978

[Analysis of the interaction of glucocorticoid receptors obtained from rat liver with DNA in a aqueous dextran-polyethylene glycol biphasic system].

The binding of the receptor-glucocorticoid complexes from rat liver cells to DNA has been studied in an aqueous dextran-polyethylene glycol biphasic system. It has been shown that the capacity of DNA for binding hormone-receptor complexes is very large, and that no saturation is observed at physiological concentrations of the glucocorticoid receptor. The linear binding curve is indicative of low affinity of the DNA acceptor sites for the complexes. Cell cytosol contains substances which inhibit the binding of the receptor-dexamethasone complexes to homologous DNA.

Animals↗
PubMed1976

Glucocorticoid receptors in corticosensitive and corticoresistant thymocyte subpopulations. I. characterization of glucocorticoid receptors and isolation of a corticoresistant subpopulation.

1. Separation of mouse thymocytes by centrifugation on a discontinuous bovine serum albumin gradient leads to the isolation of four subpopulations of cells. 2. The study of I13H]uridine incorporation in vitro by these subpopulation in the presence of steroid shows that one of them is corticoresistant. 3. However, the binding capacity of these subpopulations measured by incubation with [3H]dexamethasone is very similar. 4. It is therefore concluded that in mouse thymus, in contrast with lymphoma cells, corticoresistance may not be explained by a defect of cytoplasmic glucocorticoid receptors.

Animals↗
PubMed1978

Glucocorticoid receptors and glucocorticoid-sensitive secretion of neutral proteinases in a macrophage line.

A continuous line of mouse macrophages (P388D1) has been shown to secrete elastase, collagenase, and plasminogen activator at activities comparable to those of macrophages elicited by an inflammatory stimulus in vivo. At physiologic concentrations anti-inflammatory glucocorticoids selectively and reversibly inhibited secretion of the three proteinases but did not inhibit secretion of lysozyme, a constitutive enzyme produced by the P388D1 cells. The secretion of the neutral proteinases was inhibited 50% by 2 to 10 nM dexamethasone. Proliferation of the macrophages was also glucocorticoid sensitive. The P388D1 macrophages contained about 4000 saturable glucocorticoid-binding sites per cell. Concentrations of hormone saturating the high affinity receptor site (for dexamethasone the dissociation constant for steroid-receptor binding, Kd, was 4 nM) correlated well with concentrations inhibiting secretion of the proteinases. Only glucocorticoids and progesterone competed for binding to the specific receptors. Temperature-sensitive translocation of hormone-receptor complexes from "cytoplasm" to nucleus similar to that found with rat thymocytes was demonstrated. Thus, the interaction between glucocorticoids and the P388D1 cell line provides a model for the regulation of macrophage secretion of neutral proteinases under normal and stress conditions.

Cell Line↗
PubMed1978

An exchange assay for the cytoplasmic glucocorticoid receptor in the liver of the rat.

Cytosolic receptor for glucocorticoids can exist in either the free or bound form; assays now in use measure only the free form. In order to assay the total glucocorticoid receptor content of rat liver, free plus bound, we have developed an exchange assay wherein specifically bound [3H]dexamethasone is shown to be a valid measure of receptor in the presence of high concentrations of corticosterone. The exchange between [3H]dexamethasone and corticosterone is able to proceed because, unter the conditions of the assay, corticosterone is almost completely metabolized.

Adrenalectomy↗
PubMed1976

Glucocorticoid receptors: evidence for a second, non-glucocorticoid binding site.

Previous studies on cytoplasmic glucocorticoid receptors and enzyme induction led to the classification of steroids as inducers (optimal or sub-optimal), antagonists, or inactive steroids, with respect to their activity as glucocorticoids. The receptor was postulated to exist in allosteric equilibrium between two conformational states, one "active" and the other "inactive". Steroids behaved as inducers (optimal or sub-optimal), antagonists, or inactive steroids depending on their relative affinity for the active and inactive conformational state of the receptor. Another possible model would invoke multiple binding sites on a single receptor with interactions between the binding sites depending upon the particular steroid bound. To test this latter possibility, an experimental technique was developed to measure the rate of dissociation of tritiated dexamethasone ([3H]DM) or tritiated aldosterone ([3H]A) from the glucocorticoid receptor of rat liver or kidney cytosol. The dissociation of the [3H]DM-receptor at 25 C was not due to irreversible denaturation, and minimal recombination of the receptor with [3H]DM occurred. Progesterone and a number of other steroids consistently and significantly increased the dissociation rate of [3H]DM-receptor complexes in both liver and kidney cytosol. An identical effect was seen with hepatic glucocorticoid receptors labelled with [3H]A, like dexamethasone an optimal inducer. All steroids which enhanced glucocorticoid-receptor dissociation were either antagonists or sub-optimal inducers. Thus, it is postulated that glucocorticoid receptors have at least two classes of binding sites, and that occupation of the second site increases the dissociation rate of agonists from glucocorticoid receptors.

Aldosterone↗
PubMed1977

Glucocorticoid receptors in Morris hepatomas and host liver and the correlation of biological activity with receptor levels.

Glucocorticoid-binding macromolecules were examined in Morris hepatomas 7787, 5123tc, 3683F, 7800, and 3683 and the Reuber hepatoma H-35 with the use of the synthetic glucocorticoid, triamcinolone acetonide. The physical properties of the triamcinolone acetonide-binding macromolecules of the hepatomas indicate that they are specific glucocorticoid receptors. The equilibrium association constants (Ka), sedimentation coefficients, and sensitivity to sulfhydryl-blocking reagents were found to be similar when hepatoma receptors were compared with the known properties of the liver receptor. Probably the most convincing criterion that the triamcinolone acetonide-binding macromolecules from the hepatomas are specific receptors is that 50 to 90% of the receptor can be depleted from hepatoma cytosol by treating rats with cortisol. In adrenalectomized tumor-bearing rats, the receptor levels in hepatomas 7787, 7800, 5123tc, and H-35 are comparable to or greater than receptor levels of host liver. However, tryptophan oxygenase was not responsive to glucocorticoids in hepatoma 7800 although receptor levels were quite high, and there were no indications that the receptor molecules were altered. Hepatomas 3683 and 3686F have low levels of receptor which may be related to resistance of these tumors to glucocorticoid treatment.

Animals↗
PubMed1979

The ontogony of the human placental glucocorticoid receptor and inducibility of heat-stable alkaline phosphatase.

The human placenta was found to contain a cytosol receptor for glucocorticoids. The concentration of this receptor in term placenta was 27-fold higher than that found in cytosol from first trimester placenta. The levels of cytosol glucocorticoid receptor in three trophoblastic cell lines (JAr, BeWo, and JEG) were also determined and all were found to be low. The ability of prednisolone, a potent glucocorticoid, to stimulate heat-stable alkaline phosphatase activity found in these cells was tested. Although control experiments demonstrated that the conditions were adequate to stimulate HeLa cell alkaline phosphatase, none of the trophoblastic lines responded to prednisolone administration. This result may be explained by the observation that the JAr cells lacked any detectable glucocorticoid receptor and the receptor levels in cytosol prepared from JEG and BeWo cells were 12% and 2%, respectively, of those measured in HeLa cytosol. Our studies also suggest that the increase in serum levels of heat-stable alkaline phosphatase observed during pregnancy may reflect increasing placental sensitivity to glucocorticoids as a result of increased receptor levels.

Alkaline Phosphatase↗
PubMed1977

Steroid hormone receptors in normal human lymphocytes. Induction of glucocorticoid receptor activity by phytohemagglutinin stimulation.

The presence of specific steroid hormone receptors in human lymphocytes was investigated in unstimulated and phytohemagglutinin-stimulated glass wool column-purified peripheral blood lymphocytes. Specific steroid binding in intact cells was determined by a whole cell competitive binding assay. Non-phytohemagglutinin-stimulated lymphocytes had about 2700 specific glucocorticoid binding sites per cell; phytohemagglutinin stimulation induced a 2 to 3-fold increase in glucocorticoid receptor activity within 16 h of culture. No estrogen, androgen, or progestin binding sites were detected in either unstimulated or phytohemagglutinin-stimulated peripheral blood lymphocytes. Scatchard analysis of glucocorticoid binding was consistent with a single class of receptor sites with a dissociation constant (Kd) of about 5.5 x 10(-9) M (correlation coefficient r = -0.96). Significant competition for radiolabeled dexamethasone binding was not observed with steroids lacking glucocorticoid activity. There was good agreement between relative binding affinities of various steroids to glucocorticoid receptor in lymphocytes and ability of these steroids to inhibit phytohemagglutinin-stimulated thymidine incorporation.

Binding Sites↗
PubMed1976

Evidence for glucocorticoid receptor in human leukocytes.

The glucocorticoid uptake in vitro by human periferal leukocytes was studied. The uptake showed 2 main components, one saturable and one non-saturable. The saturable component was compared with the uptake by the specific glucocorticoid receptor in rabbit granulocytes. The similarities with the rabbit receptor in structural specificity, time course of uptake at 37 degrees C, sensitivity to metabolic inhibition by PCMS and the physiological concentration for half saturation indicate that the saturable component corresponds to a specific glucocorticoid receptor. Cells from chronic lymphatic leukemia and chronic myeloic leukemia were also studied. Only the former had a saturable glucocorticoid uptake.

Adult↗
PubMed1975

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.

Binding Sites↗
PubMed1978

Interaction of glucocorticoid receptors from lymphoid cell lines with their nuclear acceptor sites.

Procedures have been developed which provide simple means of determining binding constants of steroid receptors for glucocorticoids in mouse lymphoid cell lines and of characterizing the interaction of the steroid--receptor complex with the nucleus. An average of 70% of the steroid--receptor complexes is found associated with the nuclear fraction in three investigated cell lines, whereas 30% of the steroid--receptor complexes is found in the cytosol fraction. This distribution of the steroid-receptor complex within the cell is independent of whether steroid uptake of the cells is performed at low or at high steroid concentration. Part of the binding of the steroid receptor to the nuclear fraction is sensitive to high ionic strength and to high pH. A larger fraction of the steroid--receptor complex binding to the nuclear fraction is insensitive to high ionic strength and pH when the steroid uptake is performed at low steroid concentrations than when performed at high steroid concentrations. Steroid--receptor complex is released from the nuclear fraction by DNAase treatment but not by RNAase treatment. The possible correlation between the sensitivity to ionic strength and pH and the specificity of the binding is discussed.

Animals↗
PubMed1975

Glucocorticoid receptors in lung. Comparison between nonactivated and activated forms of the cytoplasmic glucocorticoid binding protein and their relationship to the nuclear binding protein of fetal rabbit lung.

In the absence of salt the cytoplasmic glucocorticoid receptor of fetal rabbit lung sediments at 7 S while the nuclear receptor sediments at 4 S. However, if nuclear extracts are mixed with receptor-depleted cytosol preparations in dilute buffer solutions without added salt, the nuclear 4 S receptor sediments as a 7 S species similar to that observed for the cytoplasmic form under the same conditions suggesting an interaction of the nuclear receptor with other cytosol proteins rather than with itself. In addition, both cytoplasmic and nuclear receptors sediment at 4 S in 0.4 M KCl and a major fraction of the nuclear receptor has an agarose elution profile identical to that of the cytoplasmic receptor. Thus a major fraction of the nuclear receptors is indistinguishable from the cytoplasmic receptors by the methods used. Since the cytoplasmic receptor sediments at 4 S in 0.15 M KCl, it is suggested that in vivo the glucocorticoid receptor may exist as a 4 S species and that the 7 S form described previously may result from an interaction of the 4 S component with other cytosol proteins in hypotonic media. About 25% of the receptor present in nuclear extracts has an agarose elution profile different from that of the cytoplasmic receptor in 0.4 M KCl. This suggests that either the nuclear receptor associates with itself or other nuclear proteins or that more than one form of nuclear receptor exists. Earlier observations suggested that in the absence of hormone the glucocorticoid receptor is localized exclusively in the cytoplasm of lung cells and that the nuclear receptor is formed by a transfer of the cytoplasmic steroid-receptor complex into the nucleus. A prerequisite for this transfer seems to be a modification of the receptor to an active form which can bind to nuclei. This receptor transfomration, referred to in this paper as activation of the receptor, can occur in the absence of nuclei and is highly dependent on temperature and ionic strength. Cytoplasmic receptors activated either by heating or by exposure to high ionic strength are indistinguishable from nonactivated receptors by sucrose density gradient analysis or by agarose gel filtration in solutions containing 0.4 M KCl. Simiarly, no significant difference in the absence of salt is observed after activation by heating. These results suggest that activation of the cytoplasmic glucocorticoid receptor involves conformational changes which favor its transfer and/or binding to nuclear sites rather than conversion of a 4 S species to a faster-sedimenting form by dimerization or by addition of another protein unit as has been proposed for the activation of the estrogen receptor of the rat uterus.

Animals↗
PubMed1979

Molybdate inhibition of glucocorticoid receptor inactivation and transformation.

The inactivation of glucocorticoid receptors that occurs when cytosol is heated at 25 degrees C is blocked reversibly by molybdate and slowed by some other phosphatase inhibitors such as fluoride and glucose 1-phosphate. Molybdate is also capable of preventing nonenzymatic inactivation of unbound receptors caused by exposure to salt or precipitation with ammonium sulfate at 0 degrees C. Inactivation of unbound receptors caused by Sephadex G-50 gel filtration is prevented by all three inhibitors. Both molybdate and tungstate block temperature-dependent transformation of glucocorticoid.receptor complexes to the DNA-binding state, where fluoride and glucose 1-phosphate have no effect. Transformation brought about at 0 degrees C by salt, ammonium sulfate precipitation, or gel filtration is also blocked by both molybdate and tungstate. Tungstate differs from molybdate in that it has little or no effect on receptor inactivation. Fluoride and glucose 1-phosphate do not inhibit transformation. These observations support the proposal that molybdate and tungstate are interacting through a reversible association with the glucocorticoid receptor itself. We propose that they may act by forming a complex with a phosphate moiety on the receptor.

Adrenalectomy↗
PubMed1978

Interaction of glucocorticoids with macrophages. Identification of glucocorticoid receptors in monocytes and macrophages.

Glucocorticoid binding was measured in resident and thioglycollate-elicited mouse peritoneal macrophages, rabbit alveolar macrophages, and human monocytes. Two assays of binding were used--an assay with intact cells in suspension or monolayers, and an assay of cytosol and nuclear forms of glucocorticoid receptors. The mononuclear phagocytes contained approximately equal to 4--10 X 10(3) high affinity receptor sites per cell, with dissociation constants of approximately equal to 2--8 nM dexamethasone. The binding to the saturable sites was specific for steroids with glucocorticoid or antiglucocorticoid activity. Cortisol, corticosterone, and progesterone competed with dexamethasone for binding, whereas estradiol, dihydrotestosterone, and 11-epicortisol competed very little. Binding of dexamethasone to cytosol and nuclear forms of the receptor complex and temperature-sensitive translocation of cytosol forms to nuclear forms were shown. At 37 degrees C the predominant form of the hormone-receptor complex was nuclear. These results demonstrate that corticosteroids interact with macrophages at physiological concentrations.

Animals↗