Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[3H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90,000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [3H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [3H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37 degrees C. SDS-polyacrylamide gel electrophoresis of [3H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37 degrees C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei as well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[³H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90 000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [³H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [³H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37°C. SDS-polyacrylamide gel electrophoresis of [³H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37°C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei aas well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Glucocorticoid coordinate regulation of type I procollagen gene expression and procollagen DNA-binding proteins in chick skin fibroblasts

Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro alpha 1(I) and pro alpha 2(I) mRNAs but not beta-actin mRNA. Fibroblasts receiving dexamethasone and [5,6-3H]uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not beta-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, we determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the alpha 2(I) procollagen gene. Nuclear protein blots were probed with the 32P-end-labeled pBR322 vector DNA and 32P-end-labeled alpha 2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the alpha 2(I) procollagen promoter containing DNA were calculated. Three nonhistone DNA-binding proteins of Mr 90,000, 50,000, and 30,000 had altered specificities following dexamethasone treatment.     

Dexamethasone Treatment of Newborn Rats Decreases Cardiomyocyte Endowment in the Developing Heart through Epigenetic Modifications

The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1), 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2''-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner.     

Factor-assisted DNA binding as a possible general mechanism for steroid receptors. Functional heterogeneity among activated receptor-steroid complexes

We previously reported that activated glucocorticoid receptor-steroid complexes from rat HTC cell cytosol exist as at least two sub-populations, one of which requires a low molecular weight (700–3000 Da) factor(s) for binding to DNA. This factor is removed by Sephadex G-50 chromatography and is found predominantly in extracts of crude HTC cell nuclei. We have now determined that factor is not limited to HTC cells since an apparently identical factor(s) was found in nuclear extracts of rat kidney and liver as well as human HeLa and MCF-7 cells. Furthermore, the DNA binding of a sub-population of human glucocorticoid receptors depends on factor. While these results were obtained with agonist (dexamethasone) bound receptors, a sub-population of HTC cell receptors covalently labeled by the antiglucocorticoid dexamethasone 21-mesylate also displayed factor-dependent DNA binding. This receptor heterogeneity was not an artifact of cell-free activation since the cell-free nuclear binding of dexamethasone mesylate labeled complexes was, as in intact cells, less than that for dexamethasone bound complexes. Earlier results suggested that the increased DNA binding with factor involved a direct interaction of receptor with factor(s). We now find that the factor-induced DNA binding is retained by amino terminal truncated (42 kDa) glucocorticoid receptors from HTC cells. Thus the ability of receptor to interact with factor(s) is encoded by the DNA and/or steroid binding domains. Two dimensional gel electrophoresis analysis of dexamethasone-mesylate labeled 98 kDa receptors revealed multiple charged isoforms for both sub-populations but no differences in the amount of the various isoforms in each sub-population. Finally, activated progesterone and estrogen receptor complexes were also found to be heterogeneous, with a similar, if not identical, small molecular weight factor(s) being required for the DNA binding of one sub-population. The observations that functional heterogeneity of receptors is not unique to glucocorticoid receptors, whether bound by an agonist or antagonist, and that the factor(s) is neither species nor tissue specific suggests that factor-assisted DNA binding may be a general mechanism for all steroid receptors.     

Corticosteroid binding by fetal rat and rabbit lung in organ culture

To further characterize glucocorticoid action in fetal lung cells, we investigated corticosteroid metabolism and binding in explants of fetal rat and rabbit lung. Cortisone (E) was concerted to cortisol (F) and bound by receptor with a time course only somewhat slower than for F. Production of F (0.243 pmol/min/mg DNA) was the same in male and female rabbits and was not affected by prior exposure to glucocorticoid in utero or in culture. The t 1/2 for dissociation of nuclear-bound [3H]F was 84 min on changing the culture medium and 21 min on addition of excess non-labeled dexamethasone. Dissociation of [3H]dexamethasone was approx 5-fold slower by both procedures. The KD for nuclear binding of dexamethasone, F, E, and corticosterone in rabbit lung were 0.7, 7.3, 6.8 and 70.6 nM, respectively. In rat lung, the KD for dexamethasone was 6.8 nM. The concentrations of dexamethasone and F required for half-maximal stimulation of phosphatidylcholine synthesis were similar to the KD values. Dexamethasone binding capacity (sites/mg DNA) increased with age in both rat (+103% increase from day 16 to 22) and rabbit (+47% between day 23 and 30). Receptor concentration was the same in both sexes, and there were no developmental changes in non-specific binding, nuclear:cytoplasmic distribution, or KD. In 27-day rabbit fetuses, the rate of choline incorporation was higher in lungs with greater binding capacity. We conclude that (1) E is rapidly converted to F in rabbit lung to become an active glucocorticoid, whereas corticosterone probably has little physiologic activity, (2) there is a species difference in the affinity of dexamethasone binding which is reflected in responsiveness (3) there is no difference between sexes in E conversion, receptor capacity, or phosphatidylcholine synthesis, and (4) the concentration of binding sites per lung cell increases during fetal development. We suggest that developmental increases in both F production and receptor may be important factors in the expression of endogenous glucocorticoid effects.     

Identification of a glucocorticoid-induced nuclease in thymocytes. A potential "lysis gene" product

Glucocorticoids initiate a cytolytic process in lymphoid cells that is characteristic of programmed cell death. In vivo treatment of adrenalectomized rats with glucocorticoids results in the rapid degradation of the thymocyte genome at internucleosomal sites. This DNA degradation occurs prior to cell death, and considerable evidence indicates that this nucleolytic event is central to the initiation of lymphocytolysis. To further characterize this process, we have searched for the gene products in thymocytes which may be responsible for steroid-induced DNA degradation. Adrenalectomized rats were treated in vivo with dexamethasone or a vehicle control; nuclear thymocyte proteins were extracted with 0.6 M NaCl and analyzed for protein content or nuclease activity on sodium dodecyl sulfatepolyacrylamide gels containing calf thymus DNA. Glucocorticoid treatment resulted in the induction of two major protein families, a 30-32-kDa protein doublet and a series of 3-4 proteins of 12-19 kDa, both of which express prominent DNase activity. Induction of the lower molecular weight nucleases increased with time after steroid treatment and paralleled the time course of glucocorticoid-mediated DNA degradation. Nuclease induction was blocked by the glucocorticoid antagonist RU 486, indicating a steroid receptor-mediated process. When nuclei from glucocorticoid-resistant cells were incubated with nuclear extracts from glucocorticoid-treated rats, the DNA was cleaved at internucleosomal sites, whereas extracts from vehicle-treated animals were virtually inactive. Based on these findings we propose that glucocorticoids, acting via a receptor-mediated pathway, induce a nucleolytic "lysis gene" product(s) responsible for lymphocytolysis.     

Dexamethasone modulates melatonin MT2 receptor expression in splenic tissue and humoral immune response in mice

Melatonin modulates immune function through its membrane-bound MT1 and MT2 receptors in mammalian system. Adrenal glucocorticoid, an important metabolic hormone is known as a immuno-compromising agent. In the present study, we investigated the effect of dexamethasone on melatonin receptor proteins in spleen tissue and anti-klh-IgG response in Swiss albino mice. Melatonin treatment increased the MT1 and MT2 receptor proteins and anti-klh-IgG than control mice. Dexamethasone treatment increased MT2 receptor protein and anti-klh-IgG than melatonin-treated group. Dexamethasone treatment to melatonin-treated mice showed additive effects and maximally increased the anti-klh-IgG than other experimental groups. A decrease in glucocorticoid receptor (GR) protein was noted in melatonin treated as well as dexamethasone-treated mice. Dexamethasone significantly increased MT2 melatonin receptor protein in spleen and anti-klh-IgG and additively increased anti-klh-IgG when supplemented along with melatonin. Therefore, the present study may suggest that dexamethasone increased humoral immune response permissively by enhancing MT2 receptor expression in splenic tissue of mice.     

Dexamethasone suppresses estrogen action at the pituitary level without modulating estrogen receptor dynamics

The administration of glucocorticoid combined with antiestrogen such as clomiphene has been shown to be effective for the induction of ovulation in patients with anovulation. The present study was undertaken to examine the effects of glucocorticoid on estrogen-induced changes in the pituitary gland. A single intraperitoneal (i.p.) administration of 10 micrograms estradiol-17 beta (E2) in ovariectomized and adrenalectomized rats resulted in a significant stimulation of pituitaries with regard to wet tissue weight and progesterone receptor content. An i.p. administration of 1 mg dexamethasone in these animals had no effects on both the values. However, the E2-induced increases in pituitary weight and progesterone receptor content were significantly inhibited by pretreatment with 1 mg of dexamethasone. The pretreatment with dexamethasone, on the other hand, had no significant effect on the dynamics of pituitary estrogen receptor induced by the injection of E2, i.e. the degree of nuclear translocation, occupancy and cytoplasmic receptor replenishment. The inhibitory effect of dexamethasone, therefore, does not seem to be mediated through estrogen receptor system in the pituitary. These results suggest that dexamethasone acts directly on the pituitary gland to suppress the action of E2, and which may be involved in the process of induction of ovulation by glucocorticoid-clomiphene treatment.     

Effect of intravenously infused dexamethasone on collagen metabolism in skin of merino sheep.

The effects of an 8-day intravenous infusion of dexamethasone (7.6 mg kg-0.75 body weight) on collagen biosynthesis and wool growth in skin were examined in four Merino wethers. Plasma dexamethasone concentrations reached their peaks during the first 24 h infusion, which were followed by relatively stable levels (c. 1 X 10(-7) M) for the next 4--5 days. Minor increases in plasma dexamethasone levels were recorded during the final 2 days of treatment. Dexamethasone concentrations quickly fell below the level of detection once infusion ceased. Marked decreases in the wet weight, thickness and protein content of skin were observed at the end of infusion. DNA content was reduced to 42.4 +/- 4.9% s.e.m. during the first 2 days of treatment, but in the next 4 days of infusion gradually increased to 85.0 +/0 12.5% of controls. The level of collagen (expressed as hydroxyproline content of its acid hydrolysate) was elevated throughout the infusion and then gradually declined in 3 weeks to about 60% of controls. The biosynthesis of collagen measured by the rate of [14C]hydroxyproline formation and the activity of proline, 2-oxoglutarate dioxygenase (EC 1.14.11.2. formerly prolyl hydroxylase) was reduced during the first half of treatment to a greater extent than the rate of [14C]proline incorporation into proteins. Wool growth was reduced by 80.4 +/- 11.6% in the post-infusion period which allowed three sheep out of four to be defleeced. Inhibition of collagen biosynthesis in sheep skin was due initially to a decrease in the activity of proline, 2-oxoglutarate dioxygenase and later to the reduced rate of proline incorporation into proteins. It was also evident that changes in biosynthetic rate of collagen were not reflected in the total level of skin collagen. The extent of wool growth depression in individual animals paralleled the changes in DNA content and the extent of collagen biosynthesis inhibition.     

Glucocorticoids Enhance Histamine-Evoked Catecholamine Secretion from Bovine Chromaffin Cells

Abstract: The synthetic glucocorticoid dexamethasone enhanced histamine-evoked catecholamine secretion from cultured bovine chromaffin cells. Dexamethasone enhanced the effects of histamine on both adrenergic (epinephrine-rich) and noradrenergic (norepinephrine-rich) chromaffin cells but had a more dramatic effect on noradrenergic cells. Histamine-evoked secretion in noradrenergic cells appeared to become rapidly inactivated, whereas the rate of secretion in adrenergic cells was nearly constant for up to 2 h; dexamethasone treatment attenuated the inactivation seen in noradrenergic cells. The effect of dexamethasone appeared after a lag of several hours and was maximal by 24 h. The EC₅₀ for dexamethasone was ∼1 n M . The effect of dexamethasone was mimicked by the glucocorticoid agonist RU 28362 and was blocked by the antagonist RU 38486, indicating that the effects of these steroids were mediated by the glucocorticoid or type II corticosteroid receptor. Histamine-evoked catecholamine secretion in both dexamethasone-treated and untreated cells was blocked by the H₁ histamine receptor antagonist mepyramine but was not affected by the H₂ antagonist cimetidine; thus, dexamethasone appeared to enhance an H₁ receptor-mediated process. In the absence of glucocorticoids, H₁ receptor mRNA levels were higher in adrenergic than in noradrenergic cells. Dexamethasone increased H₁ receptor mRNA levels in both cell types. The increased expression of H₁ receptors presumably contributes to the enhancement of histamine-evoked catecholamine secretion by glucocorticoids. Glucocorticoids may play a physiological role in modulating the responsiveness of chromaffin cells to histamine and other stimuli.     

Growth factor- and dexamethasone-induced proteins in Swiss 3T3 cells. Relationship to DNA synthesis

Dexamethasone synergistically enhances the stimulation of DNA synthesis in quiescent Swiss 3T3 cells by cartilage-derived growth factor (CDGF) while having no consistent effect when added with platelet-derived growth factor (PDGF) or serum. We examined the hypothesis that this difference might be attributed to selective synthesis of individual proteins early in the G1 phase of the cell cycle. Swiss 3T3 cells were treated with CDGF, PDGF, and fetal bovine serum for 3 h, with or without dexamethasone, and [35S]methionine-labeled proteins were separated by two-dimensional electrophoresis on giant gels. Over 3300 proteins could be distinguished; 34 of these were consistently induced more than 3-fold by all three factors, while an additional 30 inductions were variably present. Dexamethasone by itself induced 8 other proteins, and at least 9 growth factor inductions were synergistically enhanced by addition of the hormone. To identify proteins intimately associated with growth control, we looked for inductions that reflected the dexamethasone synergy with CDGF on DNA synthesis and lack of such an effect with PDGF. The induction of only one group of proteins, the Band 1 isoforms (44-46 kDa, pI 6.1-5.9) displayed such selective synergy. The majority of the other growth factor inductions were inhibited by dexamethasone, even in the context of maximal DNA synthesis, implying that their increased synthesis is not required for growth. When 3T3 cells were treated with increasing doses of CDGF with and without dexamethasone, autoradiographic densities of induced proteins varied in a dose-responsive fashion. However, only levels of the Band 1 proteins bore a constant linear relationship to DNA synthesis, suggesting that they play an important role in early control of the cell cycle.     

Activation of hepatic and inactivation of renal xanthine dehydrogenase activity by dexamethasone during the prenatal period in chick eggs

In the experiments reported here, the effect of dexamethasone on hepatic and renal xanthine dehydrogenase toward the end of incubation was studied. Dexamethasone injected on day 17 of incubation into the chick eggs increased the hepatic and decreased the renal activity of xanthine dehydrogenase. Furthermore, dexamethasone was found to accelerate fetal development as measured by the precocious decrease of thymidine uptake into nuclear DNA and by the increased fetus weight/egg weight ratio. The data suggest that an activation of xanthine dehydrogenase is caused by a hatching-independent factor and by a factor related to the hatching process.     

Monocytes and platelets share the glycoproteins IIb and IIIa that are absent from both cells in Glanzmann''s thrombasthenia type I.

The possibility that thyroxine (T4) itself exerts the hormonal effect in vivo on the rat liver nuclear receptor was studied with the aid of iopanoic acid (IOP), an inhibitor of the conversion of T4 into tri-iodothyronine (T3). After administration of 2.4 micrograms of T4/100 g body weight to hypothyroid rats for 7 days, T4 and T3 concentrations in serum and in the liver nuclear non-histone protein (NHP) were all increased to the hyperthyroid range. Hepatic mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) activity and DNA content increased significantly. The equilibrium association constant (Ka) of the nuclear T3 receptor was unchanged and the maximal binding capacity (Cmax.) increased 1.4-fold. Simultaneous administration of IOP (5 mg/100 g body weight) to the rats given 2.4 micrograms of T4/100 g body weight completely blocked the conversion into T3. The serum T4 was even more increased, whereas the serum T3 decreased to the hypothyroid range. Although the NHP-bound T4 was at a concentration comparable with the rats given T4 alone, no NHP-bound T3 was detected. Yet the alpha-GPD activity was elevated 2.8-fold and the DNA content increased to the same extent as observed in the rats given T4 alone. The Ka and Cmax. of the nuclear receptor were significantly decreased. After administration of 48 or 480 micrograms of T4/100 g body weight for 3 days, serum T4 and T3 were markedly increased. The NHP-bound T3 was also increased, but no NHP-bound T4 was detected. The alpha-GPD activity was markedly elevated, but the DNA content was unchanged. The Cmax. per g of liver was increased, whereas the Ka remained unchanged. Simultaneous administration of IOP to these animals could not completely block the T4 conversion. The observed hormonal effects in the absence of nuclear T3 indicate that T4 possesses the intrinsic hormonal activities on the rat liver. T4 is less potent in induction of alpha-GPD activity but as potent in increment of hepatic DNA as T3. Although the binding site for T4 is not fully characterized, it appears to be acidic NHP. T4 is an active hormone, yet is also a prohormone of T3, offering the closest analogy with testosterone.     

Influence of dexamethasone on growth and development of rat fetuses: changes in nucleic acids and protein content

Pregnant rats were treated with dexamethasone in drinking water (10 micrograms/ml) from the 15th to the 22nd day of pregnancy. Dexamethasone significantly reduced the weight of rat fetuses and concentration of DNA, RNA and proteins in fetal adrenal glands, liver, placenta, brain, kidneys, heart, lung, testes and pituitary from the 17th to the 22nd day of pregnancy. These data show that dexamethasone given to pregnant rat may lead to potentially deleterious effects on fetal rat development.     

Evaluation of the role of ligand and thermal activation of specific DNA binding by in vitro synthesized human glucocorticoid receptor

We have used a DNA-binding/immunoprecipitation assay to analyze the capacity of human glucocorticoid receptor (hGR), generated in rabbit reticulocyte lysates, to bind DNA. In vitro translated hGR was indistinguishable from native hGR, as determined by migration on sodium dodecyl sulfate-polyacrylamide gels, sedimentation on sucrose density gradients, and reactivity with antipeptide antibodies generated against hGR. In addition, cell-free synthesized hGR was capable of specific binding to glucocorticoid response element (GRE)-containing DNA fragments. Using this assay system, we have evaluated the contributions of ligand binding and heat activation to DNA binding by these glucocorticoid receptors. In vitro translated hGR was capable of selective DNA binding even in the absence of glucocorticoid. Treatment with dexamethasone or the antiglucocorticoid RU486 had no additional effect on the DNA-binding capacity when receptor preparations were maintained at 0 C (no activation). In contrast, addition of either ligand or antagonist in combination with a heat activation step promoted DNA binding by approximately 3-fold over that of heat-activated unliganded receptors. Agonist (dexamethasone) was slightly more effective in supporting specific DNA binding than antagonist (RU486). DNA binding by in vitro synthesized GR was blocked by the addition of sodium molybdate to the receptor preparations before steroid addition and thermal activation. Addition of KCl resulted in less DNA binding either due to blockage of DNA-receptor complex formation or disruption of the complexes. The specificity of DNA binding by cell-free synthesized hGR was analyzed further by examining the abilities of various DNAs to compete for binding to a naturally occurring GRE found in the mouse mammary tumor virus-long terminal repeat. Oligonucleotides containing the consensus GRE were the most efficient competitors, and fragments containing regulatory sequences from glucocorticoid-repressible genes were somewhat competitive, whereas single stranded oligonucleotides were unable to compete for mouse mammary tumor virus-long terminal repeat DNA binding, except when competitor was present at extremely high concentrations. Together these studies indicate that hGR synthesized in rabbit reticulocyte lysates displays many of the same properties, including GRE-specific DNA binding, observed for glucocorticoid receptor present in cytosolic extracts of mammalian cells and tissues. Similarities between the effects of dexamethasone and RU486 suggest that the antiglucocorticoid properties of RU486 do not occur at the level of specific DNA binding.     

Acceptor sites for the oestrogen receptor in hen oviduct chromatin.

Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.