Dexamethasone Receptor Research Articles

5′-Deoxypyridoxal inhibition of glucocorticoid receptor binding in HeLa S 3 cells and rat thymocytes

Recent evidence suggests a possible role for pyridoxal 5′-phosphate, the active physiological form of vitamin B 6, in steroid hormone action (1–3). We now report that 5′-deoxypyridoxal, a synthetic vitamin B 6 antagonist, causes a rapid and complete loss of dexamethasone receptor binding in cytosol preparations in whole HeLa S 3 cells or in rat thymocytes. This effect is concentration and time dependent, and is specific for 5′-deoxypyridoxal. In whole cell incubations of either HeLa S 3 cells or rat thymocytes at 37°C, a 50% reduction in [ 3H]dexamethasone binding was observed in the presence of 0.25 mM 5′-deoxypyridoxal. Cytotoxicity was not evident in rat thymocytes or HeLa S 3 cells incubated with 3.0mM or 1.0mM 5′-deoxypyridoxal respectively. Pyridoxal 5′-phosphate, 5′-deoxypyridoxine and 5′-deoxypyridoxamine were ineffective in causing a loss of steroid receptor binding. These studies suggest that 5′-deoxypyridoxal may be an effective non-steroidal compound which can effect the binding of glucocorticoids to specific receptor proteins in whole cells.

Aggregation-induced stability of dexamethasone receptors in rat kidney

The stability of the dexamethasone-receptor complex of rat kidney cytosol was studied using homogenizing media of varying pH and composition. It was found that the complex is less stable with buffers usually employed for the study of steroid receptors such as Tris, EDTA etc., whereas the stability increases considerably with a solution composed of monothioglycerol 12 mmol/l and glycerol 5% in distilled water. Sepharose 4-B column chromatography revealed that the increased stability was associated with a larger form (probably an aggregate) of the receptor-steroid complex.

Detection of glucocorticoid receptors on Friend erythroleukemia cells.

The inhibition of dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells by steroids led us to examine these cells for the presence of glucocorticoid receptors. Direct assessement of dexamethasone binding revealed high-affinity dexamethasone receptors on the untreated cells. The specific binding of [3H]dexamethasone was dose dependent. At a concentration of 10(-8) M, almost all binding sites were occupied. The mean number of binding sites per cell in two separate experiments was 8045 and 7191, respectively, and the Kd varied between 3.38 and 3.49 x 10(-9) M. Dimethyl sulfoxide treatment led to a decrease in the number of dexamethasone binding sites on the cells induced to differentiate. After 5 days of treatment, the mean number of sites per cell was reduced to 1216 and 896 in two experiments, with a Kd of 5 x 10(-9) M. Dexamethasone treatment resulted in a moderate decrease in the efficiency of colony formation within 72 hr after the cells were plated in methylcellulose. The mechanism of this inhibitory effect is unknown. However, it was also dose dependent and could be abrogated by appropriate concentrations of progesterone or 11-deoxycortisone. These results suggest that the steroid effects on growth and differentiation of the erythroleukemia cells may be mediated via glucocorticoid receptors.

Sex Hormone and Glucocorticoid Receptors in the Bursa of Fabricius of Immature Chicks

Abstract Estrogen, androgen, progestin, and glucocorticoid binding was measured in the immature chick bursa of Fabricius. Studies with whole bursal cytosol indicated the presence of distinct and noninteracting estradiol, dihydrotestosterone, progesterone, and dexamethasone receptors with mean dissociation constants of 0.1, 0.3, 7.8, and 12 nM and average binding site concentrations of 1.9, 13.6, 42, and 520 fmoles/mg protein, respectively. Binding to saturable sites was specific for steroids with estrogen, androgen, progestin, or glucocorticoid activity. For example, estrone and diethylstilbestrol competed with estradiol, whereas progesterone, dihydrotestosterone, and dexamethasone did not. Similarly, testosterone competed for dihydrotestosterone binding, whereas estradiol, progesterone, and dexamethasone competed very little. In the presence of 5 µM cortisol, progestosterone binding was competed for by the anti-progestin R-5020, but not by dexamethasone, estradiol, and dihydrotestosterone. Competition for dexamethasone binding was observed with cortisol and corticosterone but not with estradiol, dihydrotestosterone, and progesterone. When cytosol was prepared from bursal cell pellets that were predominantly lymphoid in nature, glucocorticoid but not estrogen, androgen, or progestin binding was identified. Since only glucocorticoid binding was found, it suggests that estrogen, androgen, and progestin receptors are present in epithelial cells of the bursa. Binding of dexamethasone in intact cells to cytosol and nuclear forms of the receptor complex and the temperature-dependent translocation of receptor complex from the cytosol to the nucleus were also shown. The results presented indicate that estrogens, androgens, progestins, and glucocorticoids interact with cells in the bursa of Fabricius at physiologic concentrations.

Heterogeneity and properties of hepatic dexamethasone-binding proteins.

Evidence from experiments in vivo and in vitro is presented for the presence of three species of dexamethasone-binding proteins in rat liver, which are identified by chromatography on Sepharose 6B or by isoelectric focusing. Although two of these species (DI and DII) possess properties characteristic of a true receptor, the third binding protein (i.e. DIII), which migrates most slowly on Sepharose 6B, but has stability properties similar to protein DII, exhibits a 3-fold lower affinity for dexamethasone and the activated complex neither binds to DNA-cellulose nor translocates to the nucleus. Only the predominant liver receptor (DI), which is eluted first from Sepharose 6B, is present in Novikoff-hepatoma cytosol, suggesting that the major and minor species are not interconverted through simple dissociation during their isolation. The binding activities of all three species in the liver cytosol increase approx. 2-fold in vivo after adrenalectomy and show a transient 2-fold fall in vivo after the administration of cortisol. These changes in vivo in protein DIII shows a marked lag compared with those in proteins DI and DII, which change in parallel. It is therefore proposed that rat liver cytosol contains two dexamethasone receptors and a dexamethasone-binding protein that may be derived from these receptors.

Heterogeneity of nuclear glucocorticoid receptor interactions.

When thymocytes are incubated with glucocorticoids at 37 degrees, 60--70% of the receptor bound steroid is associated with the nucleus. Under conditions where the rate of steroid-receptor formation is not limiting the transfer of steroid-receptors from the cytoplasm to the nucleus occurs rapidly with a T 1/2 of 30 seconds. These observations have led us to investigate whether or not all glucocorticoid receptor complexes are associated with the nucleus in the same manner. To this end, nuclear glucocorticoid-receptor complexes have been extracted by differential salt extraction and DNase I and DNase II digeston. Of the nuclear dexamethasone receptor complex initially bound, 70--75% is resistant to 0.2 M KCl extraction (designated N2) and 25--30% is resistant to 0.4 extraction (designated N4). N2 can be further extracted with 0.4 M KCl whereas N4 is resistant to reextraction with either 0.2 M KCl, suggesting that N2-N4 (N2-4) and N4 represent distinct physical forms of nuclear dexamethasone receptor. In intact cells, N2 and N4 differ under the following physiological condition. (1) N4 binding occurs prior to N2-4; (2) a cold chase of unlabeled dexamethasone decreases N2-4 by 70% but N4 binding by only 10%; (3) N4 binding decreases more rapidly than N2-4 following a decrease in hormone concentration by dilution; (4) a cold chase of either cortexolone or progesterone preferentially decreases N2-4 and has little effect on N4. In addition, the nuclear N2-4 and N4 distribution differ for cortisol, dexamethasone and triamcinolone acetonide, three steroids having different in vitro biological potencies. DNase I treatment of nuclei solubilizes approximately 60% of nuclear DNA yet releases only 20--30% of nuclear receptor, whereas DNase II solubilizes only 10% of nuclear DNA and releases 76--80% of nuclear receptor. As seen with salt extraction, the resistance of nuclear glucocorticoid-receptor complexes to a DNase I and II is dependent on the steroid molecule which is associated with the receptor. Of the steroids we have tested, nuclear triamcinolone acetonide and dexamethasone receptor complexes are most resistant to nuclease attack. Nuclear cortisol receptor complexes are readily solubilized by either DNase I or II under conditions where little dissociation of steroid from receptor occurs. These data represent evidence for physiologically distinct forms of nuclear glucocorticoid receptor interaction. In addition, they demonstrate the importance of the steroid portion of the steroid receptor in directing the nature and/or location of steroid receptors within or on the nucleus.

Interaction between dexamethasone receptor complexes and isolated nuclei from zajdela hepatoma and rat liver cells

Temperature-activation of the hormone-receptor complex (HRC) was shown to be necessary to ensure its translocation from cytoplasm to nucleus both in the rat liver and hepatoma. Hepatoma nuclei bind 20 times less HRC derived from homologous hepatoma cytosol (0.15 pmol/mg DNA), but twice as much (5.6 pmol/mg DNA) of HRC from heterologous liver cytosol, as compared with the binding of HRC from normal liver cytosol by liver nuclei (3 pmol/mg DNA), Ka of HRC with the acceptor sites in hepatoma and liver nuclei were found to be practically of the same order of magnitude. The above findings suggest an inhibition of cytosol-nucleus translocation of HRC from the cytosol of hepatoma cells as a possible cause of the nonresponsiveness of the latter to the hormone.

Extraction of nuclear glucocorticoid-receptor complexes with pyridoxal phosphate

Rat thymic lymphocytes have saturable, specific receptors for glucocorticoids, which are localized predominantly in the nucleus following exposure of thymocytes to dexamethasone at 37°C. The present results demonstrate the dose-dependent extraction by pyridoxal phosphate of dexamethasone-receptor complexes from isolated thymocyte nuclei. On an equal molar basis, pyridoxal phosphate is considerably more effective than pyridoxal; pyridoxine, pyridoxamine phosphate and 5-deoxypyridoxal are ineffective. The release of the nuclear dexamethasone receptor complex is dependent on the integrity of the C4′ carboxaldehyde group of pyridoxal phosphate as evidenced by the inhibition of extraction of dexamethasone-receptor complexes by either hydroxylamine or semicarbazide. The dexamethasone which pyridoxal phosphate liberates from thymus nuclei is bound to a macromolecule which is of smaller size than unactivated cytoplasmic dexamethasone receptor.

172 DECREASED DNA-CELLULOSE AFFINITY OF LUNG GLUCOCORTICOID RECEPTORS IN THE FETUS OF HYPERCLYCEMIC RATS

Respiratory distress syndrome (RDS) is due to a deficiency of dipalmitoyl lecithin (DPL), the developmental appearance of which is controlled in the fetal lung by glucocorticoid (GC) induction of choline phos-photransferase (CPT). GC produce their effects by binding to cytosolic receptors which in turn bind to nuclei and induce DNA-transcription and consequently enzyme synthesis. Since infants of diabetic mothers (IDM) have an increased incidence of RDS, we investigated this relationship by producing hyperglycemic rats with streptozotocin (STZ), prior to implantation. Mothers were consistently hyperglycemic (> 250mg%). One day prior to term, fetuses were obtained. The concentration of fetal lung cytosol dexamethasone receptor at saturating concentration (2×10-8M) was unchanged (500.4 vs. 515.8 fmole/mg prot.). However, in vitro DNA-cellulose affinity and CPT activity were decreased in the fetuses of treated animals (128.6 vs. 236.0 f.mole/mg prot. and 1.28 vs. 4.24 p.mole/min./mg, protein respectively; p < 0.05 for both). It is concluded that the fetus of the STZ-treated hyperglycemic rat is a useful model for RDS in the IDM, and suggests that steroid receptor DNA-affinity may affect DPL production and the incidence of RDS.

Physical measurements of the liver glucocorticoid receptor.

Physical measurements were made on the cytosolic form of the liver [3H]dexamethasone receptor. These include a Stokes radius of 3.5 nm, determined by gel filtration, and sedimentation coefficients of 5.1 and 7-8S, by sucrose-density-gradient centrifugation. From these measurements, the following physical properties were calculated: apparent mol. wt. 78000 (the 5.1 S form); D app. 6.1 X 10(-7) cm2-S-1; f/fo 1.25; axial ratio 4.7; these indicate a globular protein. Measurements of sedimentation coefficient of cytosol steroid-receptor complexes previously subjected to various activating conditions gave different values and lead to the conclusion that the mechanism of activation in vitro enabling the steroid-receptor complex to bind to DNA is more complex than simple disaggregation to a uniform size.

Cytoplasmic dexamethasone receptors in embryonic chick cartilage: Nature and ontogeny

We have studied the binding of [ 3H]-dexamethasone to a cytoplasmic component in embryonic chick cartilage as a function of age and nature. Binding was observed by 9 days of embryonic development and increased to a maximum between 13 and 16 days. Injection of the embryo with 12.5 μg (30nmol) dexamethasone markedly reduced the binding of [ 3H]-dexamethasone to 10–20% of the control value. The eytosol binding component exhibited saturation at 2.5 × 10 −8 M dexamethasone and showed high affinity for [ 3H]-dexamethasone with K D = 7.4 × 10 −9 M. A single class of binding sites was suggested by Scatchard analysis. The binding molecule is a protein (binding ability was destroyed by pronase but not DNase and RNase) and binding was dependent on the integrity of —SH groups. Maximum binding occurred at pH 7.5 and markedly inhibited at pH less than 6.0 or at temperatures greater than 37°C.

The ontogeny of corticosterone and dexamethasone receptors in rat brain

The ontogeny of [ 3Hcorticosterone and [ 3Hdexamethasone receptor macromolecules in cytosol derived from brains of adrenalectomized rats 2–30 days of age was studied. The binding of [ 3Hcorticosterone in male rats perfused at sacrifice with ice-cold isotonic sucrose in buffer, 2–8 days of age, ranged from 100 to 133 fmoles/mg protein. Binding increased from 133 to 356 fmoles/mg protein between 8 and 19 days of age, then decreased slightly between 19 and 30 days of age. [ 3HDexamethasone binding under similar conditions was comparable to that of corticosterone from 3 to 8 days of age, but in older animals the amount of receptor-bound [ 3Hdexamethasone was two-thirds that of corticosterone. Binding of these steroids in female rats was similar to that in male rats. The apparent dissociation constants for [ 3Hcorticosterone and [ 3Hdexamethasone were equal, and they did not change with age. In all age groups studied, unlabeled corticosterone competed more strongly for [ 3Hcorticosterone binding sites than did unlabeled dexamethasone, but both steroids competed equally well for [ 3Hdexamethasone binding sites. Although qualitatively similar at all ages, competition by unlabeled corticosterone and dexamethasone binding sites showed quantitative differences as the animal matured. For example, there was less competition for binding sites in 5-day-old animals than in older animals. These results indicate that binding sites for [ 3Hcorticosterone and [ 3Hdexamethasone in neonatal brain may be less specific than in the more mature brain. Also, the increase in the number of binding sites with age can be correlated with known physiological changes in the developing pituitary-adrenal axis.

Formation and characterisitcs of hepatic dexamethasone-receptor complexes of different molecular weight

The dexamethasone-binding receptor protein in rat liver cytosol has a Stokes radius of 61 Å and a sedimentation coefficient of 4.0 S. In contrast, cell nuclei labelled with [ 3H]dexamethasone in vivo or in vitro (reconstitution experiments with [ 3H]dexamethasone-labelled cytosol and isolated unlabelled nuclei) contain a high-salt-extractable dexamethasone-receptor complex with a Stokes radius of 30–36 Å and a sedimentation coefficient of 3.2 S. Exposure of liver homogenate or 1000 × g homogenate supernatant to low ionic strenght during preparation of cytosol resulted in conversion of the 61 Å to a 36 Å complex very similar to the intranuclear form of dexamethasone receptor. 61 → 36 Å complex-verting activity was present in both the 100 × g −10 000 × g sediment of liver homogenate, from which it could be extracted by hypotonic media, and in the liver cell nuclei, from which it could be extracted by hypertonic media. Mild digestion of the 61 Å dexamethasone-receptor complex with trypsin also gave rise to a complex with a Stokes radius of 36 Å. Reconstitution experiments with isolated liver cell nuclei indicated that both the 61 Å and 36 Å dexamethasone-receptor complexes were taken up by the nuclei; reextraction of the nuclei incubated with the 61 Å complex revealed that this form had been converted to the 30–36 Å complex. Further digestion of teh 61 and 36 Å [ 3H]dexamethasone-receptor complexes with hypotonic extract of the 1000 × g −10 000 × g sediment of liver homogenate or with trypsin resulted in formation of a third complex with a Stokes radius of 19 Å and a sedimentation coefficient of 2.5 S. The approximate molecular weights of the 61, 36 and 19 Å dexamethasone-receptor complexes were calculated as 102 000, 46 00 and 19 000, respectively, and the frictional ratios of the molecules as 1. 84, 1. 38 amd 1.00, respectively. It is concluded that the nuclear 30–36 Å dexamethasone-receptor complex is formed from the cytosol 61 Å complex by proteolytic digestion and that this latter protein contains at least two sites with a relatively high sensitivity to protelytic cleavage.

Studies on steroid hormone receptors (5alpha-dihidrotesterone, estradiol, and dexamethasone) in cultured human fibroblasts and amniotic fluid cells.

Cultured human fibroblasts and amniotic fluid cells (AF cells) were examined for the presence of steroid hormone receptors. In both cell types, the androgen (DHT) or glucocorticoid (dexamethasone) receptor was detected, but not the estrogen receptor. Binding parameters in fibroblasts were: for androgen: KD = 3.7 X 10(-9) M, Bmax = 13 fmol/mg; for dexamethasone: KD = 4.5 X 10(-9) M, Bmax = 120 fmol/mg. Binding parameters in AF cells were: for androgen: KD = 4 X 10(-9) M, Bmax = 8 fmol/mg; for dexamethasone: KD = 1.9 X 10(-8) M, Bmax = 375 fmol/mg. Cultured cells derived from the gonads (of a patient with 17beta-ketosteroid reductase deficiency) seem to have more receptors than cells from extragenital body parts (Bmax = 21 fmol). With the aid of gel chromatography, the molecular weight of the androgen receptor was estimated to be 30-40 000D.

Dramatic protective effect of ligand against thermal degradation on mineralo- and glucocorticoid receptors of rat kidney

Rat kidney cytosol contains both mineralo- and glucocorticoid receptors. The stability of these receptors was studied by [ 3H-aldosterone and [ 3H]-dexamethasone respectively. Stability experiments at 0°C and 20°C showed more rapid decay of aldosterone binding sites than of dexamethasone receptors. Moreover, a pronounced protective effect of ligand on the stability of binding sites was indicated by a more rapid disappearance rate of aldosterone ∗ and dexamethasone ∗ receptors in the absence of their ligand. The protective effect of ligand on [ 3H]-aldosterone binding sites was further documented by Scatchard analysis and competition experiments. When aldosterone receptors were protected during cytosol preparation by adding [ 3H]-aldosterone after homogenization: (1) Scatchard plot of [ 3H[-aldosterone showed two slopes, the steeper slope ( K DI = 3.9 × 10 −9M) corresponding to the mineralocorti-coid receptor; (2) competition experiments showed that aldosterone was the best competitor for [ 3H]-aldosterone binding sites. When [ 3H]-aldosterone was added after cytosol preparation: (1) only the lower slope ( k dh = 9 × 10 −8M) corresponding to glucocorticoid receptor was detected; (2) dexamethasone was the best competitor for [ 3H]-aldosterone binding sites. Various attempts were made to stabilize aldosterone receptors: glycerol and EDTA-containing buffers appeared to be the most effective buffers. No further stability was obtained by the addition of several protease inhibitors.

Concanavalin A-induced glucocorticoid resistance in rat thymus cells: Decreased cytoplasmic and nuclear receptor binding of dexamethasone

Concanavalin A (Con A) rapidly stimulates glucose uptake in isolated rat thymocytes, whereas cortisol has been shown to reduce the uptake of glucose. If Con A is added to thymocytes before or together with cortisol, no decrease in glucose uptake effect is observed. But if cortisol is added as little as 10 min before Con A (in the absence of glucose) the typical glucocorticoid response is seen superimposed on the Con A effect. The Con A-induced glucocorticoid antagonism may be accounted for in part by alteration in thymocyte glucocorticoid receptor metabolism. Con A exposure within 30 min lowers saturable dexamethasone binding by 40%. At concentrations as low as 20 μg/ml Con A significantly reduces dexamethasone binding to thymocytes in both cytoplasmic and nuclear components, but does not alter the temperature-sensitive cytoplasmic-to-nuclear translocation of dexamethasone receptor complexes. When Con A exposure follows dexamethasone, only a reduction in the concentration of cytoplasmic binding sites occurs. At all concentrations tested, Con A did not affect binding of dexamethasone to isolated cytoplasmic glucocorticoid receptors. Con A reduces the number of thymocyte glucocortocoid receptors without altering the affinity of the r

Stabilization and characterization of the dexamethasone-binding proteins in rat liver cytosol

Conditions were worked out for maximal stabilization of dexamethasone binding activity of rat liver cytosol in the absence of the protective steroid ligand. Important stabilization factors are ionic strength, thiol-protecting agents, glycerol and pH. Maximal stability of the cytosol is observed in a buffer consisting of 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 25 mM β-mercaptoethanol and 20% glycerol. Chromatography of cytosol on DEAE-cellulose revealed the existence of three dexamethasone receptors, binder DE-1, present in the flow-through fraction and binders DE-2 and DE-3, eluting from the column with salt concentrations of 100 and 190 mM, respectively. Binders DE-2 and DE-3 are not adsorbed on phosphocellulose at pH 7.5, whereas binder DE-1 is. All three receptors are retained to varying degrees on DNA-cellulose columns: binder DE-1 is eluted with salt concentrations of 270 mM, whereas binders DE-2 and DE-3 are eluted between 180 and 200 mM NaCl. The dexamethasone receptors also bind natural glucocorticoids, but to varying degrees, the highest binding being observed to binder DE-2. The receptors obtained after chromatography on DEAE-cellulose, but not on phosphocellulose, cannot be to an appreciable extent charged with dexamethasone.

Effect of endogenous corticosterone on the determination of dexamethasone receptor levels in rat liver cytosol

The effect of endogenous corticosterone on the quantitative measurement of dexamethasone receptors in liver cytosols from developing rats has been studied. Liver cytosols from adrenalectomized rats were preincubated with increasing concentrations of nonlabeled corticosterone and the levels of detectable dexamethasone receptors were subsequently determined either directly or after removal of unbound corticosterone. Corticosterone concentrations of 50 nM or lower had no significant effect on the specific binding of labeled dexamethasone. Higher concentrations of corticosterone resulted in under-estimation of dexamethasone receptor levels. The mean levels of endogenous corticosterone in liver cytosols from 19.5- to 21.5- day fetuses, 22-day fetuses, 6-day-old immature rats and adult rats were 27.40, 11.91, 0.81 and 4.05 nM, respectively. It is concluded that variations in the levels of circulating corticosterone in the rat under normal physiological conditions have no significant effect on the quantitative measurement of total (occupied and unoccupied) receptor sites for dexamethasone in liver cytosol. This is supported by the finding that prior treatment of liver cytosols, from rats at different stages of development, with charcoal to remove unbound steroids has no effect on the amount of detectable dexamethasone receptors.

The dexamethasone receptor in the Novikoff Hepatoma. Characterization and changes in concentration and nuclear uptake during glucocorticoid therapy

Novikoff hepatoma ascites cells, grown intraperitoneally in rats, are shown to possess a high-affinity, low-capacity dexamethasone-binding protein. The receptor protein has an intracellular localization and concentration, association constant (Ka), glucocorticoid specificity and nuclear uptake in the presence of dexamethasone comparable with those of the G-protein of rat liver. During therapy of the tumour-bearing animal with cortisol, marked cyclic variations were observed in the concentration and nuclear uptake of the putative G-protein in the tumor cells; more transient variations were also observed in the Ka value of the receptor protein.