Evidence from pulse-chase labeling studies that the antiglucocorticoid hormone RU486 stabilizes the nonactivated form of the glucocorticoid receptor in mouse lymphoma cells

A pulse-chase labeling technique was used to determine the properties of glucocorticoid receptors occupied by the antiglucocorticoid hormone RU486 in S49.1 mouse lymphoma cells. Cells were pulse-labeled with [35S]methionine and then at the beginning of the chase, either no hormone (control), dexamethasone, or RU486 was added to cells. At 4 h into the chase, cytosol was prepared and receptors were immunoadsorbed to protein A-Sepharose using the BuGR2 antireceptor antibody. Immunoadsorbed proteins were resolved by gel electrophoresis and analyzed by autoradiography. The 90 kDa heat shock protein (hsp90) coimmunoadsorbed with receptors from control cells when protein A-Sepharose pellets were washed with 250 mM NaCl but not when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that hsp90-receptor complexes are disrupted by a high concentration of salt in the absence of molybdate. hsp90 coimmunoadsorbed with receptors from RU486-treated cells even when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that RU486 stabilizes the association of hsp90 with the glucocorticoid receptor. In contrast, hsp90 did not coimmunoadsorb with receptors from dexamethasone-treated cells, consistent with earlier evidence that hsp90 dissociates from the receptor when the receptor binds glucocorticoid hormone. Dexamethasone induced a rapid quantum decrease in the amount of normal receptor recovered from cytosol but did not induce a decrease in the amount of nuclear transfer deficient receptor recovered from cytosol, consistent with tight nuclear binding of normal receptors occupied by dexamethasone. In contrast, RU486 did not induce a quantum decrease in the recovery of normal receptors from cytosol, indicating that receptors occupied by RU486 are not tightly bound in the nuclear fraction. We conclude that the antiglucocorticoid hormone RU486, in contrast to the glucocorticoid hormone dexamethasone, stabilizes the association between the glucocorticoid receptor and hsp90. The decreased affinity of receptors occupied by RU486 for the nuclear fraction may be due to their association with hsp90 and may account for the failure of RU486 to exert agonist activity.     

Glucocorticoid negative feedback and glucocorticoid receptors after hippocampectomy in rats

In rats with dorsal hippocampectomy, glucocorticoid receptors in the hypothalamus and anterior pituitary, as well as the pituitary transcortin-like compound, are preserved, in spite of a 60% depletion of glucocorticoid receptors in the hippocampus. In the hippocampectomized group, basal levels of serum corticosterone (CORT) were increased, although there was a normal response to ether stress. Inhibition of the response to ether with dexamethasone (DEX) was dose-dependent: whereas 100 micrograms/kg completely suppressed serum CORT, 10 micrograms/kg were ineffective. However, we observed a reduced sensitivity to DEX inhibition with 25 micrograms/kg in hippocampectomized animals. These results indicate that the hippocampus is involved in negative feedback mechanisms, although different doses of DEX are needed for this demonstration. The inhibition of serum CORT due to 100 micrograms/kg DEX suggests that negative feedback at sites other than the hippocampus was still operative, in agreement with normal levels of glucocorticoid receptors in the anterior pituitary and hypothalamus of hippocampectomized rats.     

Identification and characterization of glucocorticoid receptors in liver of nude mice

Glucocorticoids regulate the expression of many liver-specific genes via glucocorticoid receptors. The presence of glucocorticoid receptors in liver has been reported in many mammalian species but not in nude mice. In the present study, we demonstrate the presence of specific glucocorticoid receptors in nude mouse liver. The binding of ligands to these receptors could be completely inhibited by RU486, and partially blocked by hydrocortisone and progesterone, whereas estrogen and testosterone had no effect. Hydrocortisone down-regulated the level of glucocorticoid receptors in livers of nude mice and correspondingly enhanced the activities of tyrosine aminotransferase and -glutamyltransferase. Our results indicate that glucocorticoid receptors in nude mouse liver are specific, fully functional, and present at levels 28.5-fold higher than in the liver of normal inbred mice. We suggest that the nude mouse is a valuable model for studies of hepatic glucocorticoid action and may provide a clue to a putative hepatic-thymic interaction.     

Effect of alcohol on the interaction of cortisol with plasma proteins, glucocorticoid receptors and erythrocytes

Alcohol ingestion stimulates glucocorticoid secretion in animals and normal men. It is generally believed that this effect is mediated through the pituitary-adrenal axis. To investigate its mechanism, we focussed on the effects of ethanol on cortisol binding to plasma proteins and to glucocorticoid receptors, and on cortisol uptake by erythrocytes. Addition of ethanol (up to 800 mg/dl) decreased cortisol binding to albumin and corticosteroid-binding globulin (CBG), causing an increase in the plasma unbound component. Ethanol also decreased cortisol binding to glucocorticoid receptors in normal human peripheral lymphocytes. The uptake of cortisol by erythrocytes was not affected at ethanol concentrations as high as 2000 mg/dl. These results provide new insight to ethanol effects in vivo. The stimulatory effect of ethanol on the pituitary-adrenal axis appears to be attributable in part to a relative ineffectiveness of cortisol in cortisol-responsive cells consequent to ethanol's ability to diminish cortisol binding to glucocorticoid receptors. A compensatory increase in ACTH secretion in response to the relative hypoglucocorticoid state perceived by corticotrophs would result in maintenance of elevated plasma unbound cortisol and cytosol cortisol levels. We conclude that altered interactions of cortisol with its receptors and transport proteins could be pathophysiological components of the changes in adrenocortical function induced by ethanol ingestion.     

A potential mechanism in medroxyprogesterone acetate teratogenesis.

MPA (medroxyprogeste)rone acetate) has been shown to be te)ratogenic in rabbits but not in rats or mice (Andrew and Staples 1977). Since normal steroid action appears to be mediated, in large part, through interaction with specific steroid receptors, it was postulated that the species difference in teratogenicity might be due to a difference in the interaction of MPA with target cells. A primary event in steroid-cell interaction is the binding of a steroid to intracellular receptors. Studies were initiated to measure the specific nature of MPA binding to glucocorticoid and progestin receptors in appropriate rat and rabbit target tissues. The competition of MPA with 3H-dexamethasone binding in liver cytosol (glucocorticoid receptor) and with 3H-progesterone binding in uterine cytosol (progesterone receptor) was determined. In rabbit liver cytosol, MPA was as effective at competing for specific dexamethasone binding as the natural glucocorticoids and considerably more effective than the nonspecific steroids. In rat liver cytosol MPA was only 10% as effective as the natural glucocorticoids and the competition could not be distinguished from that of nonspecific steroids. A similar species difference was not seen in uterine cytosol; MPA competed with progesterone in a similar fashion in both rat and rabbit. These data demonstrate a distinct species difference in the competitive nature of MPA for the glucocorticoid receptor but not for the progestin receptor. The results suggest that MPA, or possibly a metabolite, may be teratogenic in rabbits by binding with specific glucocorticoid receptors to inhibit or alter normal steroidal function in embryo-fetal development.     

Circadian and seasonal variations of glucocorticoid receptors in normal human lymphocytes

Circulating human lymphocytes are known to contain specific glucocorticoid receptors. Using a competitive binding whole cell assay, we have examined the binding of [3H] dexamethasone to peripheral lymphocytes of normal male subjects. Lymphocytes were found to contain 2000-10000 glucocorticoid receptor sites/cell and the Kd value was in the range of 0.5-9 X 10(-9) M. The number and affinity of glucocorticoid receptors did not change throughout a 1-year observation time. In contrast, there was a significant diurnal variation in receptor content (38% higher at 11 p.m. than at 8 a.m.), while receptor affinity did not change.     

Cross-reactivity of a monoclonal antiglucocorticoid receptor antibody BuGR1 with glucocorticoid and mineralocorticoid receptors of various species

The reactivity of a monoclonal antibody BuGR1, raised against glucocorticoid receptors of rat liver, with glucocorticoid and mineralocorticoid receptors of mammalian (rabbit) and amphibian (A6 cells) origin was examined. The glucocorticoid receptors of rabbit kidney and liver and of A6 cells were labeled with tritiated dexamethasone. The mineralocorticoid receptors were labeled with tritiated aldosterone in the presence or absence of RU26988, depending on whether aldosterone was bound to glucocorticoid receptors (A6 cells) or not (rabbit kidney), in addition to its binding to mineralocorticoid receptors. BuGR1 did not recognize mineralocorticoid receptors of A6 cells and rabbit kidney. BuGR1 cross-reacted with glucocorticoid receptors of rabbit liver and kidney but not of A6 cells, suggesting that the domain of glucocorticoid receptors recognized by BuRG1 could be present only in the mammalian species. The findings indicate that BuGR1 shows species differences as well as receptor class specificity.     

Characterization of an antiserum against the glucocorticoid receptor

An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor from rat liver cytosol contained specific antibodies to glucocorticoid receptor. This was shown following incubation of the [³H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [³H]TA-GR-antibody complex to protein A linked to Sepharose, (II) an increased sedimentation rate of the [³H]TA-GR-antibody complex compared to that of the [³H]TA-GR complex, and (III) an increased molecular size of the [³H]TA-GR-antibody complex when compared to that of the [³H]TA-GR complex as judged from gel filtration. The antibody fraction was characterized with regard to titer, cross-reactivity and specificity. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus), as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. In the rat liver, the antibody bound to both the nuclear and the cytosolic glucocorticoid receptor (Stokes radius 6.1 nm). It did not cross-react with the proteolytic fragments of the glucocorticoid receptor, the 3.6 nm complex or the 1.9 nm complex. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat to rat serum transcortin. With an indirect competitive ELISA (enzyme-linked immunosorbent assay) combined with various separation techniques, based on different physiocochemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. These findings also indicate an immunochemical similarity between glucocorticoid receptors in different tissues as well as in different species, but not between glucocorticoid receptors and other steroid hormone receptor proteins. The cytosolic and nuclear glucocorticoid receptors in rat liver were shown to be immunochemically similar.     

A new affinity matrix for mineralocorticoid receptors

The behavior of mineralocorticoid and glucocorticoid receptors of rabbit kidney cytosol was investigated on two affinity gels: a new affinity matrix prepared with a 3-O-derivative of carboxymethyloxime deoxycorticosterone (deoxycorticosterone gel) and a gel linked to a 17 beta-dexamethasone derivative (dexamethasone gel). Deoxycorticosterone gel was highly specific, since it retained mineralocorticoid but not glucocorticoid receptors, and dexamethasone gel exhibited high selectivity for glucocorticoid receptors since it did not bind mineralocorticoid receptors. The use of these two matrices allowed separation of mineralocorticoid and glucocorticoid receptors and further characterization of each type of cytosolic receptors after its isolation. Cytosolic mineralocorticoid and glucocorticoid receptors stabilized by tungstate were found to have a Stokes radius of approximately 6 nm, as determined by high performance size exclusion chromatography and a sedimentation coefficient of approximately 9 S, determined on a glycerol density gradient containing tungstate, under either high or low salt conditions. The hydrodynamic parameters, binding characteristics, and specificity of mineralocorticoid receptors were the same in the untreated and dexamethasone gel-treated cytosol. Similarly glucocorticoid receptor characteristics remained unchanged after deoxycorticosterone gel treatment, indicating biochemical independence of cytosolic mineralocorticoid and glucocorticoid receptors. The [3H]aldosterone receptor complex eluted from deoxycorticosterone gel was recovered with a 30-40% yield and a purification factor of about 1000. Purified mineralocorticoid receptors had the same sedimentation coefficient as cytosolic mineralocorticoid receptors (9 S) but a different Stokes radius (4 versus 6 nm). The decrease in the Stokes radius of the purified mineralocorticoid receptors was probably due to the gel filtration method. These results indicate that the newly synthesized matrix specific for mineralocorticoid receptors constitutes a powerful tool for their extensive purification.     

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)     

Hormone-independent repression of AP-1-inducible collagenase promoter activity by glucocorticoid receptors.

The role of the ligand in glucocorticoid receptor-mediated transactivation and transrepression of gene expression was investigated. Half-maximal transactivation of a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene in transfected cells expressing the human glucocorticoid receptor mutant GRL753F, from which the rate of ligand dissociation is four to five times higher than the rate of dissociation from normal receptors, required a 200- to 300-fold-higher concentration of dexamethasone than was required in cells expressing the normal receptor. Immunocytochemical analysis demonstrated that this difference was not the result of a failure of the mutant receptor to accumulate in the nucleus after steroid treatment. In contrast, in cells cotransfected with a reporter gene containing the AP-1-inducible collagenase gene promoter, the concentration of dexamethasone required for 50% transrepression was the same for mutant and normal receptors. Efficient receptor-mediated transrepression was also observed with the double mutant GRL753F/C421Y, in which the first cysteine residue of the proximal zinc finger has been replaced by tyrosine, indicating that neither retention of the ligand nor direct binding of the receptor to DNA is required. RU38486 behaved as a full agonist with respect to transrepression. In addition, receptor-dependent transrepression, but not transactivation, was observed in transfected cells after heat shock in the absence of the ligand. Taken together, these results suggest that unlike transactivation, transrepression of AP-1 activity by the nuclear glucocorticoid receptor is ligand independent.     

Effects of chronic and acute training on glucocorticoid receptors concentrations in rats

The effects of chronic endurance training and acute exercise on glucocorticoid receptors were investigated in rats. For chronic endurance training, rats were exposed to progressive running training on a motor-driven treadmill for 3, 5 and 7 weeks, twice a day and 6 days a week. The samples were taken, 34-36 hours after the last exercise bout. Some of the 7-week training rats were killed by decapitation 7 days following the last exercise bout. The glucocorticoid receptors in hepatic cytosol in 5-week and 7-week rats decreased as compared to the sedentary control. There was no significant difference between the glucocorticoid receptors in hepatic cytosol in some of the 7-week rats those who had stopped training for 7 days and those in the controls. The chronic endurance training did not lead to change of the apparent dissociation constant (Kd). The changes of glucocorticoid receptors after acute exercise have also been investigated and it showed profound decreases of glucocorticoid receptors in renal and myocardial cytosol in low intensity (swimming without an extra weight for 60 minutes) and high intensity (swimming with a weight equal to 6% of body mass for 60 minutes) training groups. The decreases in glucocorticoid receptors in renal and myocardial cytosol were less prominent after low intensity training. These results demonstrated that both acute exercise training and chronic endurance training could lead to a decrease in glucocorticoid receptors, which was in a training intensity- and training load volume-dependent manner, and the changes in glucocorticoid receptors during exercise training were reversible.     

Melatonin effects on glucocorticoid receptors in rat brain and pituitary: significance in adrenocortical regulation

1. The effects of chronic melatonin treatment on glucocorticoid binding sites in hippocampus, hypothalamus and pituitary were investigated in rats, subjected to long-term manipulation of circulating corticosterone concentrations. 2. Melatonin treatment decreased the affinity of glucocorticoid receptors. 3. The effect of melatonin was apparent in the presence of normal or enhanced systemic corticosterone levels, but not in long-term adrenalectomized animals.     

Glucocorticoids "receptors" in human gingiva.

Specific binding of [3H]-dexamethasone by the cytoplasmic fraction of normal human gingiva was carried out. Different concentrations of [3H]-dexamethasone were used with or without cold dexamethasone to determine the binding. Binding data by Scatchard plot yielded a straight line indicating a single class of specific receptors with equilibrium dissociation constant of 1.39 x 10(-9) M and B max of 80 fmol/mg protein. The proteins satisfied the high affinity and low capacity requirements of receptor.     

A test for hormonal responsiveness in a mammary epithelial cell line, NMuMg

The NMuMG cell line derived from normal mouse mammary epithelial cells was tested for responsiveness to hormones. The hormones studied included insulin, glucocorticoids (cortisol and dexamethasone), and prolactin. In addition to membrane bound insulin receptors and prolactin receptors, the cells had 2 X 10(4) cytoplasmic glucocorticoid receptors per cell. Morphological changes were observed in response to hormones. Clusters of cells appeared with greatly increased diameter, and the number of cells per plate was reduced. The rate of DNA synthesis, corrected by cell number, indicates that cell division, and hence cell turnover, was increased by the combination of all three hormones. Insulin greatly enhanced protein synthesis, but glucocorticoid and prolactin did not further increase the rate. The combination of three hormones produced a change in the synthesis of histones, consistent with the increase in cell turnover. There were substantial responses of enzyme activities to hormonal treatment of the cells. Insulin by itself induced a doubling of the activity of glyceraldehyde phosphate dehydrogenase and perhaps a modest increase in NADH-cytochrome c reductase. Lactose synthetase activity showed a three- to fourfold induction of both A and B subunits of the enzyme when the cells were treated with insulin, glucocorticoid, and prolactin, and the effect of the latter two hormones was shown to be additional to that of insulin.     

Glucocorticoid receptors.

Glucocorticoid hormones are secreted uniquely from the zona fasciculata of the adrenal cortex, with marked circadian variation in basal levels and acute elevation in response to stress. Glucocorticoid receptors are almost ubiquitously distributed, and mediate a wide range of tissue-specific responses; in addition to classical, [3H]dexamethasone-binding GR (Type II receptors) there is excellent evidence that Type I sites (MR) act as mineralocorticoid receptors in some tissues but high affinity glucocorticoid receptors in others. Particular issues to be addressed in the presentation include: (i) the extent to which glucocorticoid receptor occupancy is modulated by extracellular (plasma-binding enzymes) or intracellular (proto-oncogenes) factors; (ii) whether or not there are specific response elements for Type I and II receptors; (iii) putative physiological roles for Type I, high affinity glucocorticoid receptors; (iv) evidence for glucocorticoid receptors other than classical GR and "MR". In summary, glucocorticoid receptors appear to be a final common pathway mediating and/or modulating circadian rhythms and stress responses. Cell-and tissue-specificity of response to a whole-body signal is determined by local pre-receptor, receptor and genomic differences. On the basis of previous studies on glucocorticoid secretion, and recent information on glucocorticoid action, it would at last appear possible to begin to construct a coherent physiology for glucocorticoid hormones.     

Regulation of intracellular cholesterol synthesis in hypercholesterolemia by glucocorticoids]

The rate of endogenous cholesterol synthesis in blood lymphocytes and skin fibroblasts from patients with type IIa hyperlipidemia was found to be increased in comparison with healthy donors. The cells of hyperlipidemic patients had lowered levels of glucocorticoid receptors concomitantly with a partial loss of their sensitivity to glucocorticoids. In fibroblasts from patients with hereditary hypercholesteremia of homozygous type the number of glucocorticoid receptors did not exceed 10% of their content in normal cells. The decrease of the number of glucocorticoid receptors in patients with type IIa hyperlipidemia seems to be a compensatory response of cells culminating in activation of endogenous cholesterol synthesis.     

Binding of steroids in nuclear extracts and cytosol of rat pituitary and estrogen-induced pituitary tumors

We have determined binding sites for estrogen, progestin, androgen and glucocorticoid in anterior pituitaries from Sprague-Dawley rats, a strain with low estrogen sensitivity, and in diethylstilbestrol-induced pituitary tumors in Fischer 344 rats, a strain with high estrogen sensitivity. Binding sites differ in their quantity and subcellular distribution. Cytosolic sites for [3H]estradiol in normal pituitaries from untreated rats were high prevailing over sites for other hormones, but they were depleted in the tumors due to their retention in nuclei under the influence of estrogen. Unoccupied nuclear sites for estrogen in normal glands also prevailed over sites for other steroids, and were similar to those in tumors. Second, the progestin site labeled with [3H]R 5020 was concentrated 5.7-fold in cytosol and 8.5-fold in nuclei of the tumors over the values found in glands from normal males estrogenized for 3 days. Third, glucocorticoid receptors labeled with [3H]dexamethasone were predominantly cytosolic in normal glands, but very low in cytosol and more evident in nuclear extracts from the tumors, the reverse of the profile found in normal pituitaries. Last, limited and comparable amounts of androgen receptors were measured in the subcellular fractions of both tissues. It is suggested that the subcellular distribution of some steroid receptors may be controlled in part by the cell population of the tissue and its degree of genetic activity.     

Stability and sequence-specific DNA binding of activation-labile mutants of the human glucocorticoid receptor

The stability and DNA-binding properties of activation-labile (act1) human glucocorticoid receptors (hGRs) from the glucocorticoid-resistant mutant 3R7.6TG.4 were investigated. These receptors are able to bind reversibly associating ligands with normal affinity and specificity, but become unstable during attempted activation to the DNA binding form [Harmon et al. (1984) J. Steroid Biochem. 21, 227-236]. Affinity labeling and immunochemical analysis demonstrated that act1 receptors are not preferentially proteolyzed during attempted activation. In addition, analysis of binding to calf thymus DNA showed that after loss of ligand, act1 receptors retain the ability to bind to DNA nonspecifically. A 370 bp MMTV promoter fragment containing multiple GREs and an upstream 342 bp fragment lacking GRE sequences were used to assess the binding of act1 hGR to specific DNA sequences. Immunoadsorption of hGR-DNA complexes after incubation with 32P-end-labeled fragments showed that both normal and act1 hGR bound selectively to the GRE-containing fragment in an activation-dependent manner. Binding of both normal and act1 hGRs could be blocked with a synthetic oligonucleotide containing a perfect palindromic GRE, but not with an oligonucleotide in which the GRE was replaced by an ERE. Analogous results were obtained for normal and act1 hGR activated in the absence of ligand, or after incubation with the glucocorticoid antagonist RU 38486. These results suggest that sequence-specific binding of the hGR does not require the presence of bound ligand and suggest a role for the ligand in trans-activation of hormonally responsive genes.