Effect of adrenalectomy and steroid treatment on rat skin cytosol glucocorticoid receptor

Using an exchange assay to measure occupied and unoccupied binding sites, the glucocorticoid receptor in rat skin cytosol has been measured after adrenalectomy and parenteral steroid administration. Adrenalectomy increased the number of receptor sites with maximal effect after 5 days, after which numbers decreased to those of intact animals. Injection of adrenalectomized animals with the unlabelled agonist corticosterone resulted in a rapid dose-related decrease in the number of cytosolic receptor sites at 30 min whereas the antagonist progesterone had no effect. It is concluded that changes in glucocorticoid concentration lead to rapid inverse changes in cytosolic receptor.     

Interleukin-6 upregulates glucocorticoid receptor numbers in human osteoblast-like cells

Zusammenfassung Interaktionen des Endokriniums, vor allem der Hypothalamus-Hypophyse-Nebennierenrinden-Achse und dem Immunsystem sind gut bekannt. Nur wenige Publikationen befassen sich allerdings mit den komplexen Einflüssen der Zytokine auf die Wirksamkeit der Glukokortikoide (GK) im Gewebe. Osteoblastenkulturen sind ein etabliertes Modell, um Interaktionen von GK und Zytokinen im Knochen zu studieren. Wir untersuchten deshalb zwei Zelllinien des humanen Osteosarkoms (Saos-2 und MC-63) mit unterschiedlichen Differenzierungsgrad und unterschiedlicher konstitutiver IL-6-Produktion (3,4ǂ,3 (SEM) und 3309눊 pg/10⁶ Zellen). Wir bestimmten die Dichte des Glukokortikoidrezeptors (GR) und seine Affinität in Abhängigkeit unterschiedlicher IL-6-Mengen. Inkubationen von IL-6 über 20 h in zunehmenden Konzentrationen bis maximal 2000 pg/ml ergaben eine signifikante Zunahme der Bindungsstellen für den GR in beiden Zelllinien. Weiter konnte IL-6 den Hemmeffekt von Dexamethason (1 7mol/l) auf den GR aufheben. In MG-63-Zellinien, die eine höhere Konzentration des GR verursachen, führte eine Abnahme von IL-6 mittels eines spezifischen IL-6-Antikörpers (100 ng/ml) zu einer signifikanten Abnahme des GR. In den Saos-2-Zellen dagegen, welche eine niedrigere Konzentration des GR verursachen, erhöhte eine 40-stündige Inkubation mit humanem IL-1# sowohl die IL-6- als auch die GR-Konzentration. Die gleichzeitige Behandlung mit dem IL-6-Antikörper hob diesen Effekt wieder völlig auf. Nach unseren Ergebnissen besitzt IL-6 deshalb eine positive autokrin-modulierende Wirkung auf die Dichte des GR in humanen Osteoblasten. Summary Interactions between the endocrine and immune systems are well known with special regard to the hypothalamic-pituitary-adrenal axis. Little of the literature focuses on the complex effects of cytokines on tissue responsiveness to glucocorticoids (GC). In bone tissue, osteoblasts represent a valuable model for studying GC-cytokine interactions. Hence, we have studied two human osteosarcoma cell lines (Saos-2 and MG-63) with different degrees of differentiation and different constitutive IL-6 production (3.4ǂ.3 (mean -SE) and 3309눊 pg/10⁶ cells). We measured glucocorticoid receptor (GR) number and affinity as a function of the exposure of cells to different amounts of IL-6. Incubation for 20 h with IL-6 at increasing concentrations up to 2000 pg/ml yielded a significant increase of GR binding sites in both cell lines. IL-6 was also able to reverse the inhibitory effect of dexamethasone (1 7mol/l) on GR in both cell lines. In MG-63 cells, expressing higher concentrations of GR, IL-6 deprivation via a specific anti-IL-6 antibody (100 ng/ml) significantly decreased GR. In Saos-2 cells, expressing lower concentrations of GR, a 40 h treatment with human IL-1# (10 ng/ml) significantly increased both IL-6 production and GR. This latter effect was completely abolished by co-treating the cells with the anti-IL-6 antibody. Our results provide evidence that IL-6 is an autocrine positive modulator of GR number in human osteoblasts.     

A single common electrophoretic abnormality of glucocorticoid receptors in human leukemia cells

We determined the mol wt of glucocorticoid receptors in human leukemia cells in order to detect glucocorticoid receptor defects that might cause glucocorticoid resistance. Glucocorticoid receptors in intact cells were affinity labeled with [3H]dexamethasone-21-mesylate and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Receptors in normal human peripheral blood mononuclear cells and six human leukemia cell lines had mol wt of 97,000. Malignant cells from ten of 25 patients with leukemia contained electrophoretically abnormal glucocorticoid receptors having mol wt of 55,000 in addition to normal size receptors (Mr = 97,000). The receptor abnormality was not restricted to a particular type of leukemia and was seen in cells from both newly diagnosed patients and patients who had received prior chemotherapy, including prednisone. The abnormal receptor was not generated when cells having only normal size receptors were assayed under conditions that favor proteolysis or when cytosol from cells containing the abnormal receptor form was mixed with cytosol from cells containing only normal size receptors. The mol wt of the abnormal receptors in human leukemia cells was the same as the mol wt of receptors in mutant mouse lymphoma cell lines, S49 143R and S49 55R, which have the nuclear transfer-increased phenotype of glucocorticoid resistance. This work describes for the first time a single common electrophoretic abnormality of glucocorticoid receptors in human leukemia cells. Further investigation of glucocorticoid receptor defects in human leukemia cells could lead to an improved understanding of the mechanisms of glucocorticoid resistance in leukemia as well as a method of predicting which patients are likely to be resistant to glucocorticoid therapy.     

Expression of the glucocorticoid receptor and its isoforms in relation to glucocorticoid resistance in childhood acute lymphocytic leukemia

In vitro prednisolone resistance is a poor prognostic factor in the treatment of childhood acute lymphoblastic leukemia (ALL). In a cohort of 54 children with ALL, a lower expression of the glucocorticoid receptor (GR), but not the relative expression levels of the GR-alpha, GR-beta and GR-P isoforms, was associated with in vitro prednisolone resistance.     

Acquired deficit of forebrain glucocorticoid receptor produces depression-like changes in adrenal axis regulation and behavior

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a hallmark of major depressive disorder. A number of studies have shown that this dysregulation is correlated with impaired forebrain glucocorticoid receptor (GR) function. To determine whether a primary, acquired deficit in forebrain GR signaling is an etiologic factor in the pathogenesis of depression, we generated a line of mice with time-dependent, forebrain-specific disruption of GR (FBGRKO). These mice develop a number of both physiological and behavioral abnormalities that mimic major depressive disorder in humans, including hyperactivity of the HPA axis, impaired negative feedback regulation of the HPA axis and, increased depression-like behavior. Importantly, a number of these abnormalities are normalized by chronic treatment with the tricyclic antidepressant, imipramine. Our findings suggest that imipramine's proposed activities on forebrain GR function are not essential for its antidepressant effects, and that alteration in GR expression may play a causative role in disease onset of major depressive disorder.     

Expression of the glucocorticoid receptor alpha and beta isoforms in human nasal mucosa and polyp epithelial cells

The lower sensitivity of the inflamed nasal mucosa to glucocorticoids might be related to an increased expression of the glucocorticoid receptor (GR) beta isoform. We investigated GRalpha and GRbeta mRNA expression in epithelial cells from nasal mucosa and nasal polyps. GRalpha mRNA was at least 1000 times more expressed than GRbeta mRNA in both tissues. GRbeta expression (mean+/-SEM of 10(3) cDNA copies/microg of total RNA) was higher in nasal polyps (1.15+/-0.19; n=27; P<0.01) than in nasal mucosa (0.62+/-0.10; n=32). Nasal polyps with > 3% of inflammatory cells had higher GRbeta levels (1.40+/-0.29; n=16) than both nasal mucosa (P<0.01) and polyps with < or = 3% of inflammatory cells (0.80+/-0.18; n=11; P<0.05). No difference in GRbeta expression was found between nasal mucosa and polyps with < or = 3% of inflammatory cells. GRbeta expression correlated with the inflammatory cell number, especially with mast cells (r=0.50, P<0.0001). There was no difference in GRalpha mRNA expression between nasal mucosa and nasal polyps. In summary, GRalpha is far more expressed than GRbeta in both tissues. The increased expression of GRbeta may be related to the presence of inflammatory cells.     

Immunocytochemical localization of glucocorticoid receptor in target cells

Antiserum prepared against highly purified glucocorticoid receptor was used for immunocytochemical studies. Rat liver and pituitary were chemically fixed, and histological sections were examined for immunoreactivity by an indirect immunoperoxidase procedure. In liver, immunoperoxidase staining was observed in the nuclei and cytoplasm of most hepatocytes. Kupffer, endothelial, and bile duct cells were not significantly stained. Control sections treated with preimmune serum remained unstained; preadsorption of antisera with purified liver glucocorticoid receptor resulted in a significant decrease in immunocytochemical staining. In adrenalectomized rats, nuclear staining was markedly reduced, whereas in adrenalectomized rats treated with cortisol acetate, the density of nuclear staining was comparable to and often slightly higher than that in intact animals. In the anterior pituitary, numerous cells were immunoreactive; their nuclei, cytoplasm, or both were stained. Alternate histological sections to those stained with antireceptor antibodies were processed for the localization of ACTH. It was found that the number of anterior pituitary cells that stained with the antireceptor antibodies exceeded the number of corticotrophs. Cells of the intermediate lobe pituitary were devoid of staining, whereas cells in the posterior lobe were stained. The presence of immunoreactive glucocorticoid receptors in pituitary cytosolic fractions was biochemically confirmed by immunoadsorption studies with [3H]triamcinolone acetonide-receptor complexes. This morphological localization of glucocorticoid receptor in liver and pituitary tissues demonstrated that immunocytochemistry can be successfully used to study localization of the glucocorticoid receptor. The known lack of response of intermediate pituitary cells to glucocorticoid may be secondary to a very small number or even an absence of glucocorticoid receptors.     

Clinical implications of glucocorticoid receptor studies in childhood acute lymphoblastic leukemia

We have performed in parallel, in 19 children with acute lymphoblastic leukemia, a quantitative determination of glucocorticoid levels, in vitro steroid induced inhibition of nucleic acid precursors, and a short-term clinical trial of corticosteroids alone, before the treatment was given, which included corticosteroids and other drugs. From our results it appears that high glucocorticoid receptor levels in acute lymphoblastic leukemia of children do not guarantee a clinical response to corticosteroids. On the other hand, glucocorticoid receptors may turn out to be of value in predicting a poor response to corticosteroids only if their levels are considerably low.     

Neutrophil elastase mutations and risk of leukaemia in severe congenital neutropenia

Severe congenital neutropenia (SCN) is a heterogeneous bone marrow failure syndrome predisposing to myelodysplastic syndrome and acute myeloid leukaemia (MDS/AML). We studied 82 North American and Australian SCN patients enrolled in the Severe Chronic Neutropenia International Registry who were on long-term treatment with granulocyte colony-stimulating factor and for whom the neutrophil elastase ( ELA2 ) gene was sequenced. There was no significant difference in the risk of MDS/AML in patients with mutant versus wild-type ELA2 : the respective cumulative incidences at 15 years were 36% and 25% ( P  =   0·96). Patients with either mutant or wild-type ELA2 should be followed closely for leukaemic transformation.     

Studies on the glucocorticoid receptor in human peripheral lymphocytes. II. Regulation by glucocorticoid

The effect of glucocorticoid (GC) on the GC receptor in human peripheral lymphocytes was studies in vivo and in vitro. Excluding the effect of receptor occupancy, the GC receptor was measured by whole cell binding assay using [3H] dexamethasone as a ligand. The numbers of GC receptor in lymphocytes (whole cell, 8.42 +/- 1.88; nucleus, 3.56 +/- 1.38 fmoles/10(6) cells (M +/- SD) of patients treated with prednisolone (n = 26, mean: 33.7 mg/day) were significantly reduced when compared with those of normal subjects (n = 21; 10.48 +/- 2.40, 5.68 +/- 1.53) and those of patients without GC therapy (n = 9: 10.56 +/- 2.10, 5.26 +/- 1.07), respectively. The dissociation constants among these subjects were almost similar (5-7 nM). There were negative correlations between the receptor numbers (both whole cell and nucleus) of patients and daily dosage of prednisolone. The GC receptor numbers in both the whole cell and the nucleus were significantly decreased after 48 h in vitro preincubation with active GC. When new protein synthesis was blocked by cycloheximide (1 microgram/ml), GC did not reduce its own receptor number in vitro. Thus, the new protein synthesis may be required for this reduction in receptor number by GC. These results strongly suggest that GC down-regulates its own receptor in human peripheral lymphocytes.