Interstrain differences in cellular glucocorticoid reception in mice and the degree of suppression of normal killer activity during immobilization stress

The correlation between the level of stress-induced natural killer (NK) depression and glucocorticoid binding to specific spleen cell receptors and hormonal profile in inbred mouse strains (CBA, BALB/c, C57BL/c, A/Sn) has been investigated. Stable interstrain differences in stress-induced natural killer activity and glucocorticoid receptor binding (Bm and Kd) have been revealed. It was demonstrated that the mechanism of NK activity depression during stress consists in genetically determined potential sensitivity of lymphoid cells to physiological fluctuations of glucocorticoid levels. This made it possible to identify stress-resistant and stress-sensitive mouse strains.     

Age-related changes in glucocorticoid and mineralocorticoid receptors in lymphocytes of healthy persons and patients with hypertension]

It has been found that the number of glucocorticoid receptors in lymphocytes of the peripheral blood of healthy elderly subjects increases, while the number of mineralocorticoid receptors decreases. The mechanisms of hormone-receptor interactions in hypertension are activated: the number of glucocorticoid and mineralocorticoid binding sites grows in hypertensive patients. Still a more essential rise in the number of receptors is observed in mid-age hypertensive patients than in elderly ones.     

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.     

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)     

Structure and specific DNA binding of the rat liver glucocorticoid receptor

During recent years major advances have been made in our understanding of glucocorticoid mechanism of action. This progress has been made possible by access to purified glucocorticoid receptor in significant amounts as well as by application of hybrid DNA technology within the field of glucocorticoid control of gene expression. Especially the mammary tumour virus genome has turned out to be a convenient experimental system suitable for such investigations. This paper summarizes some of the work carried out in our own laboratory, partially in collaboration with Dr Keith Yamamoto and his associates at the Department of Biochemistry and Biophysics, University of California, San Francisco, U.S.A.     

Lack of association between five polymorphisms in the human glucocorticoid receptor gene and glucocorticoid resistance

Glucocorticoid resistance due to mutations in the gene for the glucocorticoid receptor has been suggested to be more common than is thought at present, owing to the relative mildness of its symptoms and the difficulty of its diagnosis. To investigate the prevalence of mutations in the glucocorticoid receptor gene responsible for relative insensitivity to glucocorticoids, we carried out polymerase chain reaction/single-strand conformation analysis of the glucocorticoid receptor gene in a group of 20, otherwise healthy, persons with a reduced response in a dexamethasone suppression test and in 20 controls. We did not find mutations or polymorphisms associated with a reduced sensitivity to glucocorticoids. However, we identified five novel polymorphisms in the gene for the human glucocorticoid receptor, which may be useful in analyzing whether loss of (part of) the glucocorticoid receptor gene plays a role in glucocorticoid-resistant malignancies. Although relative resistance to glucocorticoids seems to be rather frequent in otherwise healthy persons, it is not usually associated with mutations or polymorphisms in the glucocorticoid receptor gene. Received: 17 July 1996 / Revised: 26 November 1996     

Glucocorticoid prevents brain-derived neurotrophic factor-mediated maturation of synaptic function in developing hippocampal neurons through reduction in the activity of mitogen-activated protein kinase

An increased level of glucocorticoid may be related to the pathophysiology of depressive disorder. The involvement of brain-derived neurotrophic factor (BDNF) in the antidepressive effect has also been suggested; however, the possible influence of glucocorticoid on the action of BDNF in the developing central nervous system has not been elucidated. In this study, we investigated the effect of glucocorticoid (dexamethasone, DEX) on synaptic maturation and function enhanced by BDNF in early developing hippocampal neurons. In the immature stage, BDNF increased the outgrowth of dendrites and the expression of synaptic proteins including glutamate receptors and presynaptic proteins. Pretreatment with DEX significantly inhibited the BDNF-dependent up-regulation of both dendritic outgrowth and synaptic proteins. In the more mature stage, the BDNF-reinforced postsynaptic Ca(2+) influx was decreased by DEX. BDNF-enhanced presynaptic glutamate release was also suppressed. RU486, a glucocorticoid receptor antagonist, canceled the DEX-dependent blocking effect on the action of BDNF. After down-regulation of glucocorticoid receptor by small interfering RNA application, no inhibitory effect of DEX on the BDNF-increased synaptic proteins was observed. Interestingly, the BDNF-activated MAPK/ERK pathway, which is an essential intracellular signaling pathway for the BDNF-increased synaptic proteins, was reduced by DEX. These results suggest that BDNF-mediated synaptic maturation is disturbed after neurons are exposed to high-level glucocorticoid in their development stage.     

Interactions between glucocorticoids and anti-inflammatory peptides

Both pro- and anti-inflammatory mediators regulate the anti-inflammatory actions of glucocorticoids, in part by modifying the binding of glucocorticoids to specific receptors. For instance, somatostatin has been shown to increase glucocorticoid binding and signaling in macrophages. The mechanism of this regulation does not require an increased expression of glucocorticoid receptors but, rather, a stabilization of glucocorticoid receptor-associated heat shock protein 90. This is related to a decrease in calpain activity. Thus calpain inhibition may offer a new and exciting possibility for enhancing the anti-inflammatory efficiency of glucocorticoids.     

Binding of glucocorticoid antagonists to androgen and glucocorticoid hormone receptors in rat skeletal muscle

The binding of ten steroids possessing antiglucocorticoid activity has been studied in rat skeletal muscle cytosol. The affinity of these steroids for both the androgen and the glucocorticoid receptors was determined by competition with radioactive R1881 (methyltrienolone, metribolone) and dexamethasone, respectively. The antiglucocorticoid activity of these compounds was assessed in rat hepatoma (HTC) cells by measuring their inhibitory effect on the glucocorticoid-induced tyrosine aminotransferase activity. This led to identification of five novel in vitro glucocorticoid antagonists. All the steroids tested bound to both the glucocorticoid and the androgen receptors in muscle. Four steroids had an affinity for the glucocorticoid receptor higher than for the androgen receptor. The assumption is made that the steroids tested also behave as antagonists when binding to the glucocorticoid receptor in muscle and behave as agonists when binding to the androgen receptor. On this basis, the data allow one to compute a potential anticatabolic (PAG) and a potential anabolic (PAA) index for each compound. These indices might be of predictive value to determine whether these steroids exert their anabolic action in muscle through the glucocorticoid receptor or through the androgen receptor. The data also make it unlikely that satellite cells are a preferential target for anabolic steroids in muscle.     

Characterization of the rat liver glucocorticoid receptor purified by DNA-cellulose and ligand affinity chromatography

Two rapid and high yield purification methods for the rat liver glucocorticoid receptor based on differential DNA affinity (method A) and ligand affinity (method B) chromatography are described. In method A, the amount of receptor in rat liver cytosol that can be activated and subsequently eluted from a DNA-cellulose column has been increased to 80% by introducing a second heat activation step. Using this method, 1.5 nmol of 25% pure glucocorticoid receptor can be routinely obtained per day from 15-20 rat livers. Method B yields about 2.2 nmol of 60% pure receptor with an overall yield of congruent to 60%. The quality of these purifications has been controlled by affinity labeling. In each case, more than 95% of purified binding activity represented the intact 92,000 +/- 400-Da glucocorticoid receptor polypeptide as shown by sodium dodecyl sulfate-gel electrophoresis and fluorography. No difference in the labeling pattern was observed using either [3H]triamcinolone acetonide (photoaffinity labeling) or [3H]dexamethasone 21-mesylate (electrophilic labeling). The electrophilic labeling step was performed in the cytosol prior to purification by method A to compare the labeled components thus purified with those obtained when the photoaffinity labeling was performed after the purification. Using this approach, distinct breakdown products of the glucocorticoid receptor were revealed, co-purifying during DNA affinity chromatography. Cross-linked receptor obtained by method A has been further purified to homogeneity by preparative sodium dodecyl sulfate-gel electrophoresis and successfully used as immunogen to raise glucocorticoid receptor antibodies in rabbits. These antibodies raised against glucocorticoid receptor, as well as those previously obtained using affinity chromatography-purified receptor, react with the receptor molecules irrespective of their method of purification. Glucocorticoid receptors purified by methods A and B have been analyzed for specific DNA-binding properties by the nitrocellulose filter binding assay.     

Evidence for thyroid hormone-dependent and independent glucocorticoid actions in cultured cells. Studies on the induction of growth hormone and glutamine synthetase in GH1 cells and tyrosine aminotransferase in Reuber H-35 cells.

The induction of growth hormone synthesis and mRNA by thyroid hormone in cultured GH1 cells is mediated by the thyroid hormone nuclear receptor. In addition, the regulation of the growth hormone response by glucocorticoid is highly dependent on the action of thyroid hormone. To clarify whether thyroid hormone has a general influence on glucocorticoid action in GH1 cells, the glucocorticoid induction of growth hormone and glutamine synthetase was simultaneously examined. In contrast to the growth hormone response, the induction of glutamine synthetase by glucocorticoid was not influenced by thyroid hormone. Both responses appear to be modulated by the glucocorticoid receptor, and thyroid hormone had no influence on nuclear-associated glucocorticoid receptor levels. These results suggest that the thyroid hormone control of glucocorticoid induction of growth hormone may be a selective process, and the nuclear associated receptors for both thyroid and glucocorticoid hormones interrelate to control the growth hormone response.