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.     

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.     

Repeated immobilization stress increases total cytosolic glucocorticoid receptor in rat liver

Glucocorticoids are well-known mediators of stress-related endocrine, autonomic, and behavioral responses in mammals and human beings. However, our understanding of the mechanisms of glucocorticoid action in response to stress remains elusive. Therefore, in the present study, an effort has been made to systematically examine glucocorticoid action during acute (2 h) and repeated (2 h daily for 7, 15, and 30 days) immobilization stress in male Sprague-Dawley rats. Prolonged 30-day stress resulted in reduced total body weight gain. There was a dramatic 3- to 4-fold increase in plasma corticosterone levels after single acute stress paradigm, which remained augmented 2- to 3-fold higher than basal control levels during the repeated 30-day immobilization conditions. There was good relationship between increased plasma corticosterone levels and elevation of tyrosine aminotransferase activity in the liver during 30 days of stress. Because repeated immobilization stress animals showed increased levels of both plasma corticosterone and tyrosine aminotransferase activity, the regulation of cytosolic glucocorticoid receptor (GR) in rat liver, a major target tissue for glucocorticoid, was carried out during repeated stress by using GR binding assay, exchange assay, and Western blotting techniques. Exposure of animals to acute and repeated stress resulted in decreased free cytosolic GR. Interestingly, the bound cytosolic GR increased remarkably in liver during prolonged stress of 7-30 days. Overall, results obtained by using both binding assays and Western blotting for the first time showed that repeated stress animals had higher levels of total hepatic cytosolic GR as compared to control animals. These novel results suggest that repeated stress influences the hypothalamic-pituitary-adrenal axis in rats by elevating both the level of plasma corticosterone and total hepatic cytosolic GR.     

Dexamethasone and corticosterone binding by glucocorticoid-resistant and glucocorticoid-sensitive thymocytes in vitro and in vivo

No correlation between the sensitivity of thymocytes to glucocorticoids and the number of glucocorticoid receptors has been revealed in studies carried out in vivo and in vitro. The micromedium of the thymus has been found capable of accumulating glucocorticoid hormones.     

Artificial induction of lactation in ewes: the involvement of progesterone and prolactin in lactogenesis.

An attempt has been made to evaluate the importance of prolactin and 'progesterone withdrawal' for lactogenesis. The experimental model system used was the ovariectomized, non-pregnant ewe induced to lactate artifically by treatment with trigger hormone (either oestrogen, glucocorticoid or oxytocin) alone or in combination with progesterone. It appears from the results that prolactin is important in the lactogenic responses elicited by oestrogen and oxytocin but not as important in the response elicited by glucocorticoid. Moreover, the results suggest that, in the ewe, an appropriate positive hormonal stimulus will overcome the inhibitory influence of progesterone on lactogenesis.     

The glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension

Glucocorticoids are used as a treatment for a variety of conditions and hypertension is a well-recognized side effect of their use. The mechanism of glucocorticoid-induced hypertension is incompletely understood and has traditionally been attributed to promiscuous activation of the mineralocorticoid receptor by cortisol. Multiple lines of evidence, however, point to the glucocorticoid receptor as an important mediator as well. We have developed a mouse model of glucocorticoid-induced hypertension, which is dependent on the glucocorticoid receptor. To determine the site(s) of glucocorticoid receptor action relevant to the development of hypertension, we studied glucocorticoid-induced hypertension in a mouse with a tissue-specific knockout of the glucocorticoid receptor in the distal nephron. Although knockout mice had similar body weight, nephron number and renal histology compared to littermate controls, their baseline blood pressure was mildly elevated. Nevertheless, distal nephron glucocorticoid receptor knockout mice and controls had a similar hypertensive response to dexamethasone. Urinary excretion of electrolytes, both before and after administration of glucocorticoid was also indistinguishable between the two groups. We conclude that the glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension in our model.     

Loss of TBL1XR1 Disrupts Glucocorticoid Receptor Recruitment to Chromatin and Results in Glucocorticoid Resistance in a B-Lymphoblastic Leukemia Model

Although great advances have been made in the treatment of pediatric acute lymphoblastic leukemia, up to one of five patients will relapse, and their prognosis thereafter is dismal. We have previously identified recurrent deletions in TBL1XR1, which encodes for an F-box like protein responsible for regulating the nuclear hormone repressor complex stability. Here we model TBL1XR1 deletions in B-precursor ALL cell lines and show that TBL1XR1 knockdown results in reduced glucocorticoid receptor recruitment to glucocorticoid responsive genes and ultimately decreased glucocorticoid signaling caused by increased levels of nuclear hormone repressor 1 and HDAC3. Reduction in glucocorticoid signaling in TBL1XR1-depleted lines resulted in resistance to glucocorticoid agonists, but not to other chemotherapeutic agents. Importantly, we show that treatment with the HDAC inhibitor SAHA restores sensitivity to prednisolone in TBL1XR1-depleted cells. Altogether, our data indicate that loss of TBL1XR1 is a novel driver of glucocorticoid resistance in ALL and that epigenetic therapy may have future application in restoring drug sensitivity at relapse.     

High faecal glucocorticoid levels predict mortality in ring-tailed lemurs (Lemur catta)

Glucocorticoid levels are commonly used as measures of stress in wild animal populations, but their relevance to individual fitness in a wild population has not been demonstrated. In this study I followed 93 ring-tailed lemurs (Lemur catta) at Berenty Reserve in Madagascar, collecting 1089 faecal samples from individually recognized animals, and recording their survival over a 2 year period. I evaluated faecal glucocorticoid levels as predictors of individual survival to the end of the study. Animals with high glucocorticoid levels had a significantly higher mortality rate. This result suggests that glucocorticoid measures can be useful predictors of individual survival probabilities in wild populations. The 'stress landscape' indicated by glucocorticoid patterns may approximate the fitness landscape to which animals adapt.     

Stimulation of IgG production by glucocorticoids in human myeloma lymphoblasts

LICR-LON-HMy2 cells (HMy2 cells), an established line of human myeloma lymphoblasts, produce and secrete IgG, and have been used for production of human-human hybridomas. We have previously shown that HMy2 cells are growth-inhibited by glucocorticoids and contain high affinity, saturable, steroid-specific glucocorticoid receptors. Here we report that treatment for 0-4 days with the synthetic glucocorticoid dexamethasone (1,4-pregnadien-9-fluoro-16 alpha-methyl-11 beta,17 alpha,21-triol-3,20-dione) leads to time-dependent increases in IgG secretion rates as measured by goat anti-human IgG antibodies in an enzyme-linked immunosorbent assay. Stimulation of IgG secretion is dependent on the concentration of dexamethasone employed, with half-maximal stimulation occurring between 1.10(-9) and 1.10(-8) M, and maximal stimulation occurring at 1.10(-7) M. Stimulation of IgG secretion is specific for active glucocorticoids such as cortisol and dexamethasone; treatment of cells with 17 beta-estradiol, progesterone, dihydrotestosterone, and aldosterone has little, if any, effect on IgG secretion. Finally, dexamethasone markedly stimulates both secreted and newly synthesized IgG, as determined by continuous and pulse labeling of extracellular and intracellular proteins, respectively, followed by binding to protein A-Sepharose, gel electrophoresis, and autoradiography. Thus, although dexamethasone effects on post-translational or secretory processes have not been ruled out, our data indicate that increased biosynthesis of IgG accounts for most, if not all, of the observed increase in IgG secretion rates. In summary our results demonstrate that despite the known immunosuppressive effects of glucocorticoids, these hormones can stimulate IgG biosynthesis and secretion in human myeloma lymphoblasts in vitro.     

T-cell glucocorticoid receptor is required to suppress COX-2-mediated lethal immune activation

Glucocorticoids, acting through the glucocorticoid receptor, potently modulate immune function and are a mainstay of therapy for treatment of inflammatory conditions, autoimmune diseases, leukemias and lymphomas. Moreover, removal of systemic glucocorticoids, by adrenalectomy in animal models or adrenal insufficiency in humans, has shown that endogenous glucocorticoid production is required for regulation of physiologic immune responses. These effects have been attributed to suppression of cytokines, although the crucial cellular and molecular targets remain unknown. In addition, considerable controversy remains as to whether glucocorticoids are required for thymocyte development. To assess the role of the glucocorticoid receptor in immune system development and function, we generated T-cell-specific glucocorticoid receptor knockout mice. Here we show that the T-cell is a critical cellular target of glucocorticoid receptor signaling, as immune activation in these mice resulted in significant mortality. This lethal activation is rescued by cyclooxygenase-2 (COX-2) inhibition but not steroid administration or cytokine neutralization. These studies indicate that glucocorticoid receptor suppression of COX-2 is crucial for curtailing lethal immune activation, and suggest new therapeutic approaches for regulation of T-cell-mediated inflammatory diseases.     

The non-activated glucocorticoid receptor: structure and activation

Glucocorticoid hormone receptors are present in the soluble fraction of target cell homogenates as large entities (Mr approximately 300,000) that are unable to interact with DNA. These large complexes contain an Mr approximately 94,000 steroid- and DNA-binding polypeptide, in association with an Mr approximately 90,000 non-ligand-binding entity, which has been identified as a heat shock protein, hsp90. This protein has been purified to near homogeneity as a component of the non-activated receptor complex. Characterization of the purified protein revealed its presence as a dimer in the large receptor form. Dissociation of the receptor-hsp90 complex can be induced by heat treatment only when ligand is bound to the receptor, as demonstrated by specific DNA-binding assay and sucrose gradient ultracentrifugation, hsp90 represents ca 1% of total proteins in rat liver cytosol, and milligram amounts were purified using a combination of high performance ion exchange and gel permeation chromatography. Monospecific antibodies were raised in rabbits. They were found to precipitate the intact non-activated glucocorticoid receptor, as well as the Mr approximately 27,000 steroid-binding fragment of the receptor generated by trypsin treatment, indicating that hsp90 interacts with the steroid-binding domain of the glucocorticoid receptor. Finally, translation of glucocorticoid receptor mRNA in reticulocyte lysate yields a protein which also interacts with hsp90 and binds to DNA only after ligand-binding and heat treatment. Thus, the glucocorticoid receptor is synthesized in a non-activated form also in vitro.