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.     

Vitamin B6 influences glucocorticoid receptor-dependent gene expression

We have examined the influence of intracellular vitamin B6 concentration on glucocorticoid receptor function in HeLa S3 cells transfected with a glucocorticoid-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid. CAT activity is induced from this plasmid specifically by glucocorticoid hormones in a glucocorticoid receptor-dependent manner. The intracellular concentration of pyridoxal phosphate, the physiologically active form of the vitamin, was elevated by supplementation of the culture medium with the synthesis precursor pyridoxine and lowered by exposure to the pyridoxal phosphate synthesis inhibitor 4-deoxypyridoxine. Analysis of glucocorticoid responsiveness revealed that elevated concentrations of intracellular pyridoxal phosphate suppressed the amount of glucocorticoid-induced CAT activity whereas moderate deficiency enhanced the level of glucocorticoid receptor-mediated gene expression. In contrast, modulation of the intracellular pyridoxal phosphate concentration had no effect on either basal CAT activity derived from cells not stimulated with dexamethasone or on CAT activity derived from two glucocorticoid-insensitive reporter plasmids. The modulatory effects of pyridoxal phosphate concentration occur without changes in glucocorticoid receptor mRNA levels, glucocorticoid receptor protein concentration, or the steroid binding capacity of the receptor. These observations demonstrate that vitamin B6 selectively influences glucocorticoid receptor-dependent gene expression through a novel mechanism that does not involve alterations in glucocorticoid receptor concentration or ligand binding capacity.     

Regulation of the glucocorticoid receptor in fetal rat lung during development

The effect of the synthetic glucocorticoid betamethasone on the regulation of the glucocorticoid receptor mRNA and on receptor protein was studied in fetal rat lung during development. Using a glucocorticoid receptor cRNA probe, glucocorticoid receptor mRNA was examined by Northern blot hybridization and by solution hybridization. A monoclonal antibody against the glucocorticoid receptor was used to study regulation of the receptor protein by the Western immunoblotting technique. In fetal rat lungs, of 16-21 days of gestation, as well as in adult lungs, betamethasone treatment resulted in a significant decrease of glucocorticoid receptor mRNA to 50-65% of the control level. In contrast, betamethasone treatment did not down-regulate the receptor protein in rat lungs of 16-19 days of gestation, whereas a decrease of glucocorticoid receptor protein to 40-60% of control was seen in lungs of 21 days of gestation, in postnatal and adult lung. These results provide data for a change in regulation in vivo of the glucocorticoid receptor by its homologous ligand in fetal rat lung during development.     

Tissue-specific regulation of glucocorticoid receptor mRNA by dexamethasone

The effect of glucocorticoids on tissue-specific regulation of glucocorticoid receptor mRNA was studied in intact and adrenalectomized rats. Glucocorticoid receptor mRNA was examined by Northern blot hybridization and quantitated by slot blot hybridization using a glucocorticoid cRNA probe. Glucocorticoid receptor mRNA was greatest in the lung with the relative levels in other tissues as follows: spleen, 70%; brain, 55%; liver, 50%; kidney, 43%; heart, 35%; adrenal, 13%; and testis only 8%. A tissue-specific difference in glucocorticoid receptor mRNA accumulation was found after adrenalectomy. There was little change in glucocorticoid receptor mRNA levels in liver and lung, but the brain and kidney demonstrated a 40 and 80% increase in mRNA, respectively. In contrast, dexamethasone treatment resulted in a consistent decrease of 40-60% in the accumulation of glucocorticoid receptor mRNA in all tissues studied. These results provide in vivo evidence for the autoregulation of the glucocorticoid receptor by its homologous ligand and demonstrate the existence of tissue-specific regulation of the glucocorticoid receptor mRNA levels in states of glucocorticoid excess and depletion.     

Glucocorticoid regulation of hepatic cytosolic glucocorticoid receptors in vivo and its relationship to induction of tyrosine aminotransferase

Regulation of rat hepatic cytosolic glucocorticoid receptors was studied using our newly developed exchange assay. Injecting 1 mg of dexamethasone or corticosterone into 150-250 g adrenalectomized rats caused a rapid decline in glucocorticoid receptor binding. Glucocorticoid receptor levels were depressed 80-90% in less than 15 min after hormone treatment, and remained low for about 24-48 h after glucocorticoid administration. 80-90% of glucocorticoid receptor binding was regenerated by 48 h, and complete binding was recovered by 72 h. Regenerated glucocorticoid receptor binding (48-72 h after first hormone injection) could be re-depressed by a second injection of the hormone. Similar results were obtained using normal (intact) rats. Optimum induction of tyrosine aminotransferase activity was obtained within 2 h following the first hormonal injection. Induction of tyrosine aminotransferase activity (measured 2 h after a second injection of the glucocorticoid) correlated with glucocorticoid receptor levels. Thus, 1 mg of dexamethasone or corticosterone greatly enhanced the liver tyrosine aminotransferase activity in the adrenalectomized rats (not previously hormone treated) and in adrenalectomized rats previously injected (48-72 h) with 1 mg of the glucocorticoid hormone. Enhancement of tyrosine aminotransferase activity was lowest 16-24 h after the first hormone injection (when receptor levels were extremely low). These results indicate that the induction of liver tyrosine aminotransferase activity by glucocorticoid hormones is correlated with cytosolic glucocorticoid receptor levels.     

Characterization of the partially purified, ligand-free glucocorticoid receptor

A new method was developed to synthesize a cortexolone-substituted affinity matrix, based on the fast, mild and quantitative reaction between alpha-ketomesylates and thiols. The resulting cortexolone-Sepharose absorbed easily the cytosolic chick thymus glucocorticoid receptor. Owing to the relatively fast dissociation of the glucocorticoid receptor-cortexolone complex, glucocorticoid receptor could be eluted with cortexolone as well as with triamcinolone acetonide from the affinity gel with similarly good yields. We obtained 75-150-fold purification factors (yield: 20-30%) using this column procedure. The partially purified glucocorticoid receptor was obtained in non-activated form. It had a Stokes radius of 6.2 +/- 0.1 nm. It could be activated to DNA-cellulose binding form by heat or 0.3 M KCl. KCl treatment activated 30-50% of the partially purified glucocorticoid receptor. Heat activation, however, was rather poor. Cortexolone-complexed, partially purified glucocorticoid receptor dissociated easily, and partially purified free glucocorticoid receptor, capable of steroid binding, could be obtained. Binding properties of the partially purified glucocorticoid receptor were then analyzed using different steroids. Dissociation rate constants were similar to those of the cytosolic glucocorticoid complexes. Association rate constants were consistently smaller than in the case of cytosolic glucocorticoid receptor, but the relative order of rates for different steroids was basically the same for glucocorticoid receptor in the two studied systems.     

Interaction of the glucocorticoid receptor DNA-binding domain with DNA as a dimer is mediated by a short segment of five amino acids

We have previously shown that protein-protein interactions mediate cooperative binding of the glucocorticoid receptor DNA-binding domain to a glucocorticoid response element (Dahlman-Wright, K., Siltala-Roos, H., Carlstedt-Duke, J., and Gustafsson, J.-A. (1990) J. Biol. Chem. 265, 14030-14035). The cooperativity of DNA binding is lost when the distance between the two half-sites constituting a glucocorticoid responsive element is altered or when their relative orientation is changed. We show here that mutations in the responsive element which interfere with cooperative DNA binding by the glucocorticoid receptor DNA-binding domain in vitro also abolish transactivation by the full length glucocorticoid receptor in vivo. We also identify a short segment in the proximity of one of the bound zinc ions that is required for cooperative binding of the glucocorticoid receptor DNA-binding domain to a glucocorticoid response element. We suggest that this segment is involved in dimer formation of the native glucocorticoid receptor and that it is important for correct positioning of the dimeric molecule on the double helix of DNA.     

Selective regulation of bone cell apoptosis by translational isoforms of the glucocorticoid receptor

Glucocorticoids are widely used in the treatment of inflammatory and other diseases. However, high-dose or chronic administration often triggers troublesome side effects such as metabolic syndrome and osteoporosis. We recently described that one glucocorticoid receptor gene produces eight translational glucocorticoid receptor isoforms that have distinct gene-regulatory abilities. We show here that specific, but not all, glucocorticoid receptor isoforms induced apoptosis in human osteosarcoma U-2 OS bone cells. Whole human genome microarray analysis revealed that the majority of the glucocorticoid target genes were selectively regulated by specific glucocorticoid receptor isoforms. Real-time PCR experiments confirmed that proapoptotic enzymes necessary for cell death, granzyme A and caspase-6, were induced by specific glucocorticoid receptor isoforms. Chromatin immunoprecipitation assays further suggested that glucocorticoid receptor isoform-dependent induction of proapoptotic genes was likely due to selective coregulator recruitment and chromatin modification. Interestingly, the capabilities to transrepress proinflammatory genes were similar among glucocorticoid receptor isoforms. Together, these findings provide new evidence that translational glucocorticoid receptor isoforms can elicit distinct glucocorticoid responses and may be useful for the development of safe glucocorticoids with reduced side effects.     

Effects of progesterone, promegestone and RU 486 on glucocorticoid receptor levels in primary cultures of mouse mammary epithelial cells

Mammary epithelial cells isolated from midpregnant mice and cultured on collagen gels contain glucocorticoid receptors whose levels are modulated by a variety of steroids. In the absence of any added steroid to the cell culture medium, the levels of glucocorticoid receptors in the cells decline during culture, which is counteracted by the addition of a variety of glucocorticoid agonists. The effectiveness of the glucocorticoid in preventing the loss of glucocorticoid receptors is in turn counteracted by the addition of the synthetic progestin promegestone and the synthetic antiglucocorticoid RU 486. Of the two, RU 486 is the most potent in antagonizing the effect of cortisol on the GR levels. Promegestone antagonizes the effect of cortisol, too, although higher concentrations are necessary. Progesterone was without a clear effect either as a glucocorticoid agonist or an antagonist. Progesterone, however, was extensively metabolized by mammary epithelial cells in culture. Based on these observations we conclude that in mammary epithelial cells glucocorticoids positively regulate the metabolism of their own receptors and that antiglucocorticoids, such as RU 486 and progestins, can antagonize that effect.     

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)     

Glucocorticoid receptors.

Glucocorticoid hormones are secreted uniquely from the zona fasciculata of the adrenal cortex, with marked circadian variation in basal levels and acute elevation in response to stress. Glucocorticoid receptors are almost ubiquitously distributed, and mediate a wide range of tissue-specific responses; in addition to classical, [3H]dexamethasone-binding GR (Type II receptors) there is excellent evidence that Type I sites (MR) act as mineralocorticoid receptors in some tissues but high affinity glucocorticoid receptors in others. Particular issues to be addressed in the presentation include: (i) the extent to which glucocorticoid receptor occupancy is modulated by extracellular (plasma-binding enzymes) or intracellular (proto-oncogenes) factors; (ii) whether or not there are specific response elements for Type I and II receptors; (iii) putative physiological roles for Type I, high affinity glucocorticoid receptors; (iv) evidence for glucocorticoid receptors other than classical GR and "MR". In summary, glucocorticoid receptors appear to be a final common pathway mediating and/or modulating circadian rhythms and stress responses. Cell-and tissue-specificity of response to a whole-body signal is determined by local pre-receptor, receptor and genomic differences. On the basis of previous studies on glucocorticoid secretion, and recent information on glucocorticoid action, it would at last appear possible to begin to construct a coherent physiology for glucocorticoid hormones.     

Antibodies to steroid receptor deoxyribonucleic acid binding domains and their reactivity with the human glucocorticoid receptor

Functional properties of the DNA-binding domain of the human glucocorticoid receptor were investigated using high titer polyclonal antibodies produced against single synthetic peptides or a mixture of peptides whose sequences were derived from the DNA-binding domain of steroid receptor proteins. Three of seven antisera recognized both native and denatured forms of the glucocorticoid receptor, although considerably lower antisera dilutions were required for antibody binding to native receptor. Activation of the glucocorticoid receptor to its DNA-binding form was required for antibody recognition of the native receptor. Antisera to the second finger region of the DNA-binding domain caused a portion of the activated 4S glucocorticoid receptor to sediment as 7 or 9S in sucrose gradients containing 0.4 M KCl, but did not alter the sedimentation of the nontransformed 8S receptor. Specificity of the glucocorticoid receptor-antibody interaction was demonstrated by loss of reactivity after preabsorption with peptide antigens. Antisera that interacted specifically with the glucocorticoid receptor inhibited DNA binding of the activated receptor by as much as 80%. Thus, antibody probes directed against DNA-binding domain sequences provide immunological evidence that glucocorticoid receptor activation exposes the DNA-binding region of the receptor.