In vitro activation and DNA binding affinity of human lymphoid (CEM-C7) cytoplasmic receptors labeled with the antiglucocorticoid RU 38486

The data reported here demonstrate that the synthetic steroid RU 38486 functions as an optimal antagonist in the glucocorticoid-sensitive human leukemic cell line CEM-C7. This steroid blocks the ability of the potent agonist triamcinolone acetonide (TA) to induce glutamine synthetase activity and to ultimately cause cell lysis, but when given alone does not exhibit partial agonist activity. Both [3H]RU 38486 and [3H]TA bind with high affinity and specificity to cytosolic glucocorticoid receptors in this cell line. However, under a variety of in vitro conditions (elevated temperature and presence of exogenous ATP), [3H]TA promotes receptor activation more effectively than [3H]RU 38486. This difference in the extent of activation was verified by two independent techniques: DEAE-cellulose chromatography and DNA-cellulose binding. [3H]RU 38486 and [3H]TA dissociate at the same rate from the unactivated receptors but at 25 degrees C (not 0 degree C) [3H]RU 38486 dissociates slightly more rapidly from the activated receptors. The defective receptors in the glucocorticoid-resistant subclone 3R7 appear to be "activation labile" (rapid dissociation of ligand from activated form) using either tritiated steroid. Once activated in vivo, the CEM-C7 [3H]TA- and [3H]RU 38486-receptor complexes undergo similar nuclear translocation and those activated complexes generated in vitro appear to bind to nonspecific DNA-cellulose with the same relative affinities. Thus the precise mechanism(s) by which RU 38486 exerts its potent antiglucocorticoid effect in this human cell line cannot be easily explained in terms of a defect in one of the crucial steps (specific high affinity binding, activation, translocation, DNA binding) required to elicit a physiological response. However, the data presented here do suggest that when comparing an antagonist and agonist which both bind to receptors with the same relative high affinity, the agonist may be more effective in facilitating the conformational change associated with in vitro activation.     

Binding studies of the antiglucocorticoid RU38486 in Daudi and Raji lymphoma cells

The activity of RU38486 has been studied in Burkitt's lymphoma cells which are Epstein-Barr virus (EBV) positive. The early antigens (EA) of the virus are induced by dexamethasone (DXM) in Daudi but not in Raji cells, whereas a growth factor (transforming growth factor-beta, TGF-beta) induces the EA in both cell lines. RU38486 blocks the EA induction obtained by DXM or by TGF-beta in either cell line. In order to understand the interaction of RU38486, we considered its binding to specific receptors. We first investigated the binding of the antagonist in whole cells at 22 degrees C. A number of specific binding sites higher for RU38486 than for DXM was found, suggesting that RU38486 may bind to the glucocorticoid receptor and also to other cellular structures which we called the antiglucocorticoid binding sites ("AGBS"). To support this hypothesis, competition experiments have been conducted between RU38486 and other steroid hormones (progesterone and testosterone) since it is known that RU38486 is also able to interact with their cognate receptors. Binding studies of RU38486 in vitro at 4 degrees C in the presence of cytosolic extracts from Daudi and Raji cells led to conclusions similar to those drawn from the whole cell experiments: more complexes were formed with RU38486 than with DXM. Finally, the steroid-receptor complexes were incubated with DNA-cellulose. Since the binding measured for RU38486 was higher than for DXM, we suspect that sites different from the classical glucocorticoid receptor sites are also able to interact with DNA. The blockage exerted by RU38486 on the EA induced by glucocorticoids or by non-steroidal molecules and the lack of responsiveness to glucocorticoids in Raji cells are discussed in the light of the present findings.     

Physicochemical characteristics of the interaction of the glucocorticoid antagonist RU38486 with glucocorticoid receptors in vitro, and the role of molybdate

The physicochemical properties of complexes formed between the glucocorticoid antagonist, RU38486, and the glucocorticoid receptor in rat thymus cytosol were investigated and compared with those of complexes formed with the potent agonist, triamcinolone acetonide. The equilibrium dissociation constant for the interaction of [3H]RU38486 with the molybdate-stabilized glucocorticoid receptor was lower than that for [1,2,4-3H]triamcinolone acetonide at 0 degree C but higher at 25 degrees C, suggesting that hydrophobic interactions play a major role in the binding of RU38486. Differences in equilibrium constants were reflected in corresponding differences in dissociation rate constants; association rate constants for the two steroids were similar. The rate of dissociation of [3H]RU38486 from the glucocorticoid receptor was higher in the absence of molybdate than in its presence both at 0 degree C and at 25 degrees C, suggesting that molybdate modifies the physical state of the antagonist-receptor complex, but other physical properties were similar both in the presence and in the absence of molybdate. The rate of inactivation of the unoccupied glucocorticoid receptor at 25 degrees C in the absence of molybdate was lower in phosphate buffer than in Tris-HCl buffer but the rate of dissociation of [3H]RU38486 was the same in both buffers. The binding of RU38486 afforded little, if any, protection against inactivation in either buffer; [3H]RU38486 dissociated irreversibly from the inactivated receptor at the same rate as from the non-inactivated complex but molybdate had no effect on the dissociation kinetics of the inactivated complex. It is concluded that RU38486 interacts with the ground state of the glucocorticoid receptor in a manner which neither promotes receptor transformation nor prevents receptor inactivation.     

Effect of the antiglucocorticoid agent RU 38486 on the dexamethasone inhibition of Friend cell differentiation

Glucocorticoid hormones are known to inhibit the erythroid differentiation of Friend cells. The mechanism of action of these hormones has been questioned, and results suggesting an action not involving the nuclear binding of the receptors have been published. We have used the antiglucocorticoid RU 38486 to block the inhibitory effect of dexamethasone on the induced differentiation of Friend cells. Our results strongly suggest a glucocorticoid action involving the binding of classical receptors to the cell nucleus.     

The glucocorticoid antagonist 17 alpha-methyltestosterone binds to the 10 S glucocorticoid receptor and blocks agonist-mediated dissociation of the 10 S oligomer to the 4 S deoxyribonucleic acid-binding subunit

The glucocorticoid antagonist 17 alpha-methyltestosterone inhibits binding of the agonist [3H]triamcinolone acetonide ot the glocucorticoid receptor in cytosol prepared from rat pituitary tumor GH1 cells. Competitive binding studies indicate that the dissociation constant for 17 alpha-methyltestosterone is about 1 microM. After incubation of intact GH1 cells with 10 nM [3H]triamcinolone acetonide at 37 C and subsequent cell fractionation at 4 C, three glucocorticoid receptor forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent incubation with 17 alpha-methyltestosterone reduces the amount of [3H]triamcinolone acetonide bound to each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with cytosolic 10 S receptor. Immunochemical analysis using monoclonal antibody BuGR2 indicates that 17 alpha-methyltestosterone does not cause substantial accumulation of glucocorticoid receptors in GH1 cell nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17 alpha-methyltestosterone does not reduce the total amount of cellular glucocorticoid receptor and does not reduce receptor half-life. These results are consistent with a model for glucocorticoid receptor transformation in which binding of agonist promotes the dissociation of an oligomeric 10 S cytosolic receptor protein to its DNA-binding 4 S subunit. The antagonist 17 alpha-methyltestosterone competes with agonist for binding to the 10 S cytosolic receptor but does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor.     

Antagonism of glucocorticoid induction of Epstein-Barr virus early antigens by different steroids in Daudi lymphoma cells

Four antiglucocorticoids, RU38486, RU5020, RU25055 and progesterone were found to antagonize the induction of latent Epstein-Barr virus (EBV) information by dexamethasone. The dose response studies show that the antagonization was more prominent with the synthetic steroids than with the natural hormone. Specific binding characteristics of dexamethasone measured in whole cells indicate the presence of glucocorticoid receptors. Total cellular receptor contents deduced from binding data give values similar to those reported for B-lymphoblasts. Competition experiments between dexamethasone and RU38436 strongly suggest that RU38486 binds to two distinct sites in the whole cell; one is the glucocorticoid receptor but the nature of the other site is unknown. Inhibition by antiglucocorticoids differs from antagonism by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) since the latter does not compete for any sites interacting with RU38486.     

Molybdate-sensitive and molybdate-resistant activation-labile glucocorticoid-receptor mutants of the human lymphoid cell line CEM-C7

The basis for the glucocorticoid resistance of three (3R7, 3R43 and 4R4) genetically independent mutants derived from the glucocorticoid-sensitive human lymphoid cell line CEM-C7 was examined. Each mutant contained significant, albeit reduced, amounts of steroid binding activity measured in both whole and broken cell assays. These activities were of similar high affinity and specificity to that seen in the sensitive parent, suggesting that the steroid binding portion of the receptors in these mutants was normal. However, nuclear translocation of steroid-receptor (SR) complexes was defective in all three clones. Analysis of SR complex activation by DEAE-cellulose chromatography established that receptors from the mutant clones were extremely labile under conditions which would activate normal SR complexes. Such lability was not exhibited by the unoccupied or occupied but unactivated forms of these receptors, indicating that all three were "activation labile" (act1). SR complexes of clones 3R7 and 4R4 were completely protected by the inclusion of molybdate during attempted activation and were classified as act1:molybdate-sensitive. However, SR complexes of clone 3R43 were unstable during attempted activation, even in the presence of 50 mM molybdate, and were thus classified as act1:molybdate-resistant. Our results suggest that while the act1 phenotype may predominate among spontaneously derived glucocorticoid-resistant mutants derived from CEM-C7, this phenotype may be the consequence of at least two different mutations.     

RU 38486: a potent antiglucocorticoid in vitro and in vivo

The antiglucocorticoid activity of RU 38486, was studied both in vitro and in vivo. In vitro studies, RU 38486 was characterized by a high affinity (3 times higher than that of dexamethasone) for the cytosolic glucocorticoid receptor in rat hepatoma tissue culture (HTC) cells. This high affinity was due to a very low dissociation rate of the complexes formed with the receptor. In whole cells it was a potent full antagonist of dexamethasone-induced tyrosine aminotransferase (TAT) activity: the IC50 was 6-7 times lower than the concentration of the dexamethasone used. It was devoid of any glucocorticoid activity up to a concentration of 10 microM. In in vivo studies using adrenalectomized rats, RU 38486 totally inhibited dexamethasone-induced hepatic tryptophan oxygenase (TO) activity. It is also the first pure antagonist of dexamethasone-induced hepatic TAT. However, doses as high as 5 mg/kg of body weight were required for a 50% inhibition of the effect of dexamethasone at 0.01 mg/kg. RU 38486 did not display any glucocorticoid effect on these two responses up to 50 mg/kg.     

Interactions of corticosterone, 5alpha-dihydrocorticosterone and dexamethasone with proteins in rat-liver cytosol.

The intracellular binding of [3H]corticosterone and [3H]dexamethasone and their metabolites to macromolecules in rat liver cytosol was studied in vivo and in vitro. The macromolecules binding corticosterone and its metabolites were characterized as (a) a steroid conjugate-binding (Stokes radius 2.5 nm and sedimentation coefficient 4.1 S in high ionic strength; pI 8.7, (b) transcortin and (c) a glucocorticoid "receptor". Competition experiments indicate that corticosterone and dexamethasone bind to the same site of the glucocorticoid receptor molecule. Different Stokes radii between the corticosterone-receptor and the dexamethasone-receptor complexes (6.9 and 6.3 nm, respectively, in high ionic strength) indicate that the two ligands induce different conformations of the receptor protein. This may be of importance when explaining the qualitative differences between the cellular effects of natural and synthetic glucocorticoids. 5alpha-Dihydrocorticosterone, on the other hand, competed to a very limited extent with dexamethasone for binding sites on the receptor. An assay of the inductive effect on liver tyrosine aminotransferase and tryptophan oxygenase indicated that 5alpha-dihydrocorticosterone was practically devoid of glucocorticoid activity. It is concluded that 5alpha-dihydrocorticosterone probably does not act as the mediator of corticosterone action in rat liver.     

Modifications of rat liver glucocorticoid receptor by insulin-induced hypoglycemia

Stability-, equilibrium- and kinetic binding parameters, transformation rate and sedimentation properties of liver cytosol glucocorticoid receptor from insulin-treated rats were studied. 40% elevation of cytosolic glucocorticoid binding and a lower affinity of the receptor for ligand were observed in hypoglycemic rats as compared to the controls. A small but significant decrease of [3H]triamcinolone acetonide-receptor complexes association rate and an increase of dissociation rate were also found. The rate and the extent of activation of the complexes from insulin-treated rats were somewhat higher compared to the controls, and the complexes from both groups showed higher affinity for the nuclei isolated from insulin-treated animals. Mixing experiments suggested that insulin treatment lead to alterations at the level of both the receptor protein and the nuclear binding sites. Sedimentation properties of transformed and untransformed receptor remained unchanged upon insulin treatment. The physiological relevance of the data was confirmed by hypoglycemia-related stimulation of tyrosine aminotransferase induction by dexamethasone.     

Antagonism of glucocorticoid action in cultured hepatoma cells

We report here our recent data on the effects of antiglucocorticoids in two established cell lines (HTC, FAZA). These steroid hormone target tissues were designed to consider the problem of differential antagonism and lack of correlation between antiglucocorticoid activity and competition for the glucocorticoid receptor. In the systems chosen, several responses were considered which may be differentially antagonized: induction of tyrosine aminotransferase (TAT), alanine aminotransferase (AAT) and tryptophan oxygenase (TPO). By testing the anti-inducing capacities of a number of steroids, we found a few synthetic compounds like promegestone and R 25055 which exert a stronger antagonism against TAT and AAT induction than natural steroids like progesterone. The availability of highly radioactive antagonists (promegestone, progesterone) has greatly facilitated our "whole cell" study and allowed us to detect the antagonists in isolated nuclei whose purity and morphological integrity were controlled by specific criteria; our results suggest that the binding of the antagonists to the nucleus proceeds via the glucocorticoid receptor. The appearance of promegestone and progesterone in the nucleus suggests that the nucleus may be involved in the mechanism of action of anti-glucocorticoids.     

Effect of antiglucocorticoids on dexamethasone-induced inhibition of uridine incorporation and cell lysis in isolated mouse thymocytes

We have compared in isolated mouse thymocytes the action of progesterone, cortexolone, DXH (a 17-beta carboxamide derivative of dexamethasone) and RU 38486 (a new antiglucocorticoid molecule), on dexamethasone-induced inhibition of uridine incorporation and cell lysis, with the affinities of these drugs for glucocorticoid receptors. Our results show that progesterone, cortexolone and DXH which possess similar affinities for glucocorticoid receptors may exhibit variable, weak agonist and antagonist activities according to the parameter studied. RU 38486 was a potent competitor of dexamethasone and was able, when present in a 10-fold excess, to counteract almost completely the inhibitory action as well as the lytic action of 5 X 10(-8) M dexamethasone. This compound which exerts almost no agonist activity may therefore represent a useful tool to investigate the mode of action of antiglucocorticoids.     

Activation of rat liver glucocorticoid receptor bound to the antiglucocorticoid RU38486

The kinetics of steroid binding to rat liver glucocorticoid receptor (GR) and receptor denaturation were dependent upon the nature of the molecule occupying GR. Both the agonist [triamcinolone acetonide (TA)] and the antagonist (Ru38486) however competed for the same saturable binding site. Despite opposing physiological action, both steroid analogues permitted receptor activation as evident by binding to DNA-cellulose and 9S to 4S shift on sucrose gradient sedimentation. It therefore seems necessary to reevaluate a current notion that antagonist action of RU38486 in rat liver is a result of impaired receptor activation.     

In vitro activation of rat cardiac glucocorticoid antagonist- versus agonist-receptor complexes

The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [3H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25 degrees C for 30 min or at 15 degrees C for 30 min in the presence of 5 mM pyridoxal 5'-phosphate. Once thermally activated, the cardiac [3H]triamcinolone acetonide and [3H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215-250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.     

H-7 reduces the nuclear binding of [3H]dexamethasone in rat liver slices but does not affect the phosphorylation of glucocorticoid receptor.

The effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, on the nuclear binding of [3H]dexamethasone and on the phosphorylation of glucocorticoid receptor was studied in rat liver slices to ascertain the role of protein kinase C in the expression of glucocorticoid action. H-7 reduces the nuclear binding of [3H]dexamethasone in rat liver slices. It does not affect the extent of phosphorylation of glucocorticoid receptor both in the absence or in the presence of glucocorticoid. These findings indicate that protein kinase C may be involved in the nuclear binding of glucocorticoid receptor but does not directly influence the receptor phosphorylation.     

Progestins stimulate the differentiation of 3T3-L1 preadipocytes

The effect of progesterone on the differentiation of the 3T3-L1 preadipocytes was investigated and compared with other sex steroids (estradiol and testosterone), with cortisol, with the synthetic progestin R5020 and with the progestin/glucocorticoid antagonist RU38486. At 10⁻⁸ M, progesterone stimulated the activity of glycerol-3-phosphate dehydrogenase and triglyceride deposition. Progesterone, R5020, cortisol, and RU38486 increased triglycerides about 2-fold at 10⁻⁷ M. Only minimal effects were observed with testosterone and estradiol even at 10⁻⁶ M. When the cells were cultured in presence of 10⁻⁵ M metyrapone the effect of progesterone was unchanged, suggesting that the progesterone was not metabolized to a glucocorticoid. Progesterone, R5020 and RU38486 competed efficiently with [³H]dexamethasone for the glucocorticoid receptor in 3T3-L1 cytosol. These results indicate a significant, reproducible dose-dependent effect of progestins on differentiation of the preadipocytes, which appears to be mediated via the glucocorticoid receptor.     

In vitro activation of rat cardiac glucocorticoid antagonist-versus agonist-receptor complexes

The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [³H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25°C for 30 min or at 15°C for 30 min in the presence of 5 mM pyridoxal 5′-phosphate. Once thermally activated, the cardiac [³H]triamcinolone acetonide and [³H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215–250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.     

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.     

Human recombinant IL-1 alters glucocorticoid receptor function in Reuber hepatoma cells

Exposure of Reuber hepatoma cells (RHC) to 30 and 300 fM human rIL-1 (hurIL-1) for 4 h significantly decreased cytosolic glucocorticoid binding. Scatchard analysis indicated that the 30 and 300 fM doses of hurIL-1 significantly decreased the Bmax (maximum number of available binding sites), but did not alter the Kd (affinity of the glucocorticoid receptor for ligand). The decrease in cytosolic glucocorticoid binding, expressed relative to cytosol protein, did not result from increased intracellular protein in hurIL-1-treated RHC. In addition, the receptor binding reaction in RHC treated with 300 fM hurIL-1 could be resolved only by computer application of a three-parameter model. Sucrose density gradient ultracentrifugation analysis confirmed significantly less untransformed (8 to 10S) receptor-ligand complexes in hurIL-1-treated RHC, which is biologically significant because hurIL-1 (300 fM) also inhibited the glucocorticoid induction of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK). Altered transformation of the receptor-ligand complex, a possible mechanism of action for hurIL-1-mediated inhibition of PEPCK induction, was examined. However, receptor transformation, verified by in vitro activation by high salt (0.3 M KCl) of glucocorticoid receptor-ligand complexes and subsequent sucrose density gradient ultracentrifugation analysis, was not affected by hurIL-1. Furthermore, cytoplasmic glucocorticoid binding, determined in intact cell dexamethasone uptake experiments, was decreased in hurIL-1-treated RHC. The decrease in cytoplasmic glucocorticoid binding was reflected subsequently in decreased nuclear binding. The results support our hypothesis that, during acute infection and inflammation, mediators alter metabolic pathways in the liver by interfering with glucocorticoid action.