Inhibition of the production of mediators of inflammation by corticosteroids is a glucocorticoid receptor-mediated process

In order to find an explanation for corticosteroid resistance we assessed whether inhibition by dexamethasone (DEX) of the stimulated production of TNF- proportional, variant, IL-6, PGE(2) and LTB(4) by peripheral blood mononuclear cells (MNC) depends on binding to the glucocorticoid receptor (GR), and whether it is determined by the number or the affinity of the GR of these cells. GR number and affinity of MNC were determined by means of a whole cell DEX binding assay. MNC were incubated with DEX and LPS or A23187 in the absence or presence of RU486, a potent steroid antagonist. DEX caused a concentration dependent inhibition of TNF- proportional, variant, IL-6 and PGE(2) production but had no effect on LTB(4) production. RU486 significantly blocked the effect of DEX, but no correlations were found between the inhibition of mediator release and the K(d) or receptor number.     

Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes

Glucocorticoids are widely used in the therapy of inflammatory, autoimmune, and allergic diseases. As the end-effectors of the hypothalamic-pituitary-adrenal axis, endogenous glucocorticoids also play an important role in suppressing innate and cellular immune responses. Previous studies have indicated that glucocorticoids inhibit Th1 and enhance Th2 cytokine secretion. IL-12 promotes Th1 cell-mediated immunity, while IL-4 stimulates Th2 humoral-mediated immunity. Here, we examined the regulatory effect of glucocorticoids on key elements of IL-12 and IL-4 signaling. We first investigated the effect of dexamethasone on IL-12-inducible genes and showed that dexamethasone inhibited IL-12-induced IFN-gamma secretion and IFN regulatory factor-1 expression in both NK and T cells. This occurred even though the level of expression of IL-12 receptors and IL-12-induced Janus kinase phosphorylation remained unaltered. However, dexamethasone markedly inhibited IL-12-induced phosphorylation of Stat4 without altering its expression. This was specific, as IL-4-induced Stat6 phosphorylation was not affected, and mediated by the glucocorticoid receptor, as it was antagonized by the glucocorticoid receptor antagonist RU486. Moreover, transfection experiments showed that dexamethasone reduced responsiveness to IL-12 through the inhibition of Stat4-dependent IFN regulatory factor-1 promoter activity. We conclude that blocking IL-12-induced Stat4 phosphorylation, without altering IL-4-induced Stat6 phosphorylation, appears to be a new suppressive action of glucocorticoids on the Th1 cellular immune response and may help explain the glucocorticoid-induced shift toward the Th2 humoral immune response.     

Role of glucocorticoids in the stress-induced suppression of testicular steroidogenesis in adult male rats.

We have examined the role of glucocorticoids in the stress-induced inhibition of testicular steroidogenesis. Immobilization (3 hr) reduced plasma testosterone (T) levels to 24% of control values but did not affect plasma LH levels. This reduction was partially reversed by in vivo injections of the antiglucocorticoid, RU486, prior to the stress session at a dose of 10 mg/kg BW, but not at 1.0 or 50 mg/kg BW. Stressed rats that were treated with 10 mg/kg BW RU486 had twofold higher plasma T levels than vehicle-treated stressed animals. Injections of RU486 did not affect plasma LH levels in control or stressed rats and did not affect T levels of unstressed rats. Stressed rats had eightfold higher plasma corticosterone levels than controls, and RU486 had no effect on control or stress levels of corticosterone. The possible role of glucocorticoids in mediating the effect of stress on testicular T production was investigated also in vitro by incubating testicular interstitial cells from unstressed rats for 3 hr with corticosterone (0, 0.01, 0.1, or 1.0 microM) or dexamethasone (0, 0.001, 0.01, or 0.1 microM), followed by an additional 2 hr with hCG (0, 25, 50, or 100 microIU). Both corticosterone and dexamethasone inhibited hCG-stimulated T production in a dose-dependent manner. Cells incubated with the highest concentration of either of the glucocorticoids showed significantly reduced responses to hCG stimulation. In the absence of hCG, in vitro T production was not affected by dexamethasone or 0.01 and 0.1 microM corticosterone. However, the highest dose of corticosterone (1.0 microM) produced a 63% elevation in basal T production. Coincubation of testicular interstitial cells with corticosterone (1.0 microM) or dexamethasone (0.1 microM) and RU486 (0.01, 0.1, and 1.0 microM) reversed the glucocorticoid-induced suppressions of T production in a dose-dependent manner. Our results suggest that during stress increases in plasma levels of glucocorticoids in male rats act via glucocorticoid receptors on testicular interstitial cells to suppress the testicular response to gonadotropins, and that the decline of testosterone production during immobilization stress is in part mediated by a direct action of glucocorticoids on the testis.     

Endogenous corticosteroids modulate Clostridium difficile toxin A-induced enteritis in rats

We examined the role of glucocorticoids in acute inflammatory diarrhea mediated by Clostridium difficile toxin A. Toxin A (5 microg) or buffer was injected in rat ileal loops, and intestinal responses were measured after 30 min to 4 h. Ileal toxin A administration increased plasma glucocorticoids after 1 h, at which time the toxin-stimulated secretion was not significant. Administration of the glucocorticoid analog dexamethasone inhibited toxin A-induced intestinal secretion and inflammation and downregulated toxin A-mediated increase of macrophage inflammatory protein-2. Adrenalectomy followed by replacement with glucocorticoids at various doses suggested that intestinal responses to toxin A were related to circulating levels of glucocorticoids. Administration of the glucocorticoid receptor antagonist RU-486 enhanced toxin A-mediated intestinal secretion and inflammation. We conclude that C. difficile toxin A causes increased secretion of endogenous glucocorticoids, which diminish the intestinal secretory and inflammatory effects of toxin A.     

RU486 inhibits induction of aromatase by dexamethasone via glucocorticoid receptor in cultured human skin fibroblasts

Effects of RU486 on the induction of aromatase by dexamethasone via glucocorticoid receptor were determined using cultured human skin fibroblasts. Competition of [3H]dexamethasone binding to the cytosol receptor was 7 times stronger with RU486 than with dexamethasone. The order of the strength of competition was RU486 greater than dexamethasone greater than betamethasone greater than prednisolone greater than hydrocortisone. RU486 abolished a specific 8.6 S [3H]dexamethasone binding peak in the cytosol, determined using a sucrose density gradient analysis. Dexamethasone markedly induced aromatase and this event was strongly suppressed by RU486, in a dose-dependent manner, in the cultured skin fibroblasts. A linear correlation between the strength of competition and the induction of aromatase of various glucocorticoids was observed. RU486 non-competitively inhibited aromatase induction by dexamethasone determined from a double reciprocal plot of aromatase activity, with respect to [3H]androstenedione concentration in the presence of RU486. These results show that RU486 is a peripheral noncompetitive antiglucocorticoid on aromatase induction by glucocorticoid in human skin fibroblasts and that aromatase induction is a good marker for the biological function of glucocorticoid receptor in human skin fibroblasts.     

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.     

Inhibition of glucocorticoid-induced cataracts in chick embryos by RU486: a model for studies on the role of glucocorticoids in development

Cataract formation can be induced by glucocorticoid treatment of developing chick embryos. We show here that this response can be blocked very effectively by use of the antiglucocorticoid RU486. When dexamethasone (0.02 micromol/egg) was administered from day 13 to 16 chick embryos, their lenses (over 80%) became cataract (GC-induced cataract; stage IV-V) within 48 hrs. These GC-induced cataract formations were prevented by administration of RU486 (0.2 micromol/egg) on day 9. However, RU486 also inhibited hatching even though the embryos showed normal growth and appearance. In control embryos, more than 90% live chicks (39/42 chicks) were hatched on day 22. Chick embryos treated with RU486 on day 9 appeared to grow normally until 21, but could not hatch. When chick embryos were treated with RU486 (0.2 micromol/egg) on day 15, more than 80% live embryos (34/42 chicks) were hatched on day 23 with normal appearance, which was one day delay comparing to the control. These observations indicate that endogenous glucocorticoids are involved in the ability to hatch and that RU486 is able to block the actions of endogenous glucocorticoids. Thus, RU486 should be a very useful tool for studies on other biochemical and physiological aspects of chick embryo development that are under glucocorticoid control.     

Dual effect of dexamethasone on CYP3A4 gene expression in human hepatocytes. Sequential role of glucocorticoid receptor and pregnane X receptor.

Although CYP3A induction by dexamethasone has been extensively documented, its mechanism is still unclear because both the role of the glucocorticoid receptor and the ability of dexamethasone to activate the human pregnane X receptor have been questioned. In an attempt to resolve this problem, we investigated the response of CYP3A4 to dexamethasone (10 nm-100 microm) in primary human hepatocytes and HepG2 cells, using a variety of methods: kinetic analysis of CYP3A4 and tyrosine aminotransferase expression, effects of RU486 and cycloheximide, ligand binding assay, cotransfection of HepG2 cells with CYP3A4 reporter gene constructs and vectors expressing the glucocorticoid receptor, pregnane X receptor or constitutively activated receptor. In contrast to rifampicin (monophasic induction), dexamethasone produces a biphasic induction of CYP3A4 mRNA consisting of a low-dexamethasone component (nmol concentrations) of low amplitude (factor of 3-4) followed by a high-dexamethasone component (supramicromolar concentrations) of high amplitude (factor of 15-30). We show that the low-dexamethasone component results from the glucocorticoid receptor-mediated expression of pregnane X receptor and/or constitutively activated receptor which, in turn, are able to transactivate CYP3A4 in a xenobiotic-independent manner. At supramicromolar concentrations (>10 microm), dexamethasone binds to and activates pregnane X receptor thus producing the high-dexamethasone component of CYP3A4 induction. We conclude that, in contrast to the other xenobiotic inducers of CYP3A4, glucocorticoids play a dual role in CYP3A4 expression, first by controlling the expression of PXR and CAR under physiological conditions (submicromolar concentrations) through the classical glucocorticoid receptor pathway, and second by activating the pregnane X receptor under bolus or stress conditions (supramicromolar concentrations).     

Intracrine induction of 11beta-hydroxysteroid dehydrogenase type 1 expression by glucocorticoid potentiates prostaglandin production in the human chorionic trophoblast

Glucocorticoids are involved in the modulation of the release of parturition hormones from the fetal membranes and placenta, where their actions are determined by the prereceptor glucocorticoid metabolizing enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD). Two distinct isozymes of 11beta-HSD have been characterized. In the fetal membranes, 11beta-HSD1 is the predominate isozyme; it converts biologically inert 11-ketone glucocorticoid metabolites into active glucocorticoids. Sequence analysis of the cloned 11beta-HSD1 gene revealed a putative glucocorticoid response element in the promoter region. However, whether glucocorticoids modulate 11beta-HSD1 expression in the fetal membranes is unknown. In this study, 11beta-HSD1 and glucocorticoid receptor (GR) were coexpressed in the chorionic trophoblast. Radiometric conversion assay and Northern blot analysis revealed that both 11beta-HSD1 reductase activity and mRNA levels were increased by dexamethasone (1 microM, 0.1 microM) in the cultured chorionic trophoblast, and the effects were blocked by GR antagonist RU486 (1 microM). Prior induction of 11beta-HSD1 by dexamethasone potentiated the subsequent stimulation of prostaglandin H synthetase 2 expression and secretion of prostaglandin E(2) by cortisone in the chorionic trophoblast. There is colocalization of 11beta-HSD1 and GR in the chorionic trophoblast. By binding to GR, glucocorticoids induce the expression of 11beta-HSD1 by a possible intracrine mechanism, thereby amplifying the actions of glucocorticoids on prostaglandin production in the fetal membranes. This cascade of events initiated by glucocorticoids may play an important role in the positive feed-forward mechanisms of labor.     

Effect of glucocorticoids on C3 gene expression by the A549 human pulmonary epithelial cell line.

The third component of C, C3, is the key opsonin of the C cascade and is produced locally within the lung by pulmonary epithelial cells, macrophages, and fibroblasts. Because glucocorticoids regulate the maturation and expression of several physiologically important genes in pulmonary epithelial cells, we examined the effects of glucocorticoids on C3 mRNA expression and C3 synthesis by the human pulmonary epithelial cell line, A549. Treatment with dexamethasone enhanced C3 production in a time- and dose-dependent fashion such that concentrations of dexamethasone greater than or equal to 0.001 microM significantly increased C3 production on day 3 of culture. Natural glucocorticoids, corticosterone, cortisol, and 11-deoxycortisol also increased C3 concentrations in A549 supernatants. Both cycloheximide and the glucocorticoid receptor antagonist, RU486, individually inhibited the effect of dexamethasone on C3 production. Northern analysis demonstrated that the steady state 5.2-kb C3 message increased in A549 cells within 10 h of treatment with dexamethasone. RU486 inhibited the effect of dexamethasone on C3 mRNA expression. The integrity of the C3 thiolester bond, as measured by [3H]iodoacetic acid titration and hemolytic assay, was not disrupted by dexamethasone. We conclude that glucocorticoids such as dexamethasone enhance the expression of C3 mRNA and increase the production of functionally active C3 by A549 cells by a mechanism that is mediated by the intracellular glucocorticoid receptor.     

Glucocorticoids inhibit the apoptotic actions of UV-C but not Fas ligand in hepatoma cells: direct evidence for a critical role of Bcl-xL

Our laboratory has shown that glucocorticoids can inhibit apoptosis in rat hepatoma cells; however, the mechanisms are incompletely understood. To address this issue we sought to determine if glucocorticoid inhibition is effective when death is induced by stimuli that more selectively activate either the intrinsic (UV-C) or extrinsic (FasL) apoptotic pathways. Using flow cytometric analysis, we show that pretreatment of HTC cells with dexamethasone (Dex) inhibits UV-C- but not FasL-induced apoptosis. This inhibition requires Dex pretreatment and can be abrogated by the glucocorticoid antagonist RU486 indicating glucocorticoid receptor-mediated action. Dex increases anti-apoptotic Bcl-x(L) at both mRNA and protein levels. The Bcl-x(L) protein level remains elevated even after apoptosis induction with either UV-C or FasL although only UV-C-induced cell death is inhibited. Repression of Bcl-x(L) protein with siRNA abrogates the anti-apoptotic effect of glucocorticoids. Together these data provide direct evidence that Bcl-x(L) mediates glucocorticoid inhibition of UV-C induced apoptosis.     

Acute activation of NHE3 by dexamethasone correlates with activation of SGK1 and requires a functional glucocorticoid receptor

Glucocorticoids stimulate the intestinal absorption of Na⁺ and water partly by regulation of the Na⁺/H⁺ exchanger 3 (NHE3). Previous studies have shown both genomic and nongenomic regulation of NHE3 by glucocorticoids. Serum and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be part of this cascade, where phosphorylation of NHE3 by SGK1 initiates the translocation of NHE3 to the cell surface. In the present work, we examined a series of changes in SGK1 and NHE3 induced by glucocorticoids using human colonic Caco-2 and opossum kidney cells. We found that dexamethasone rapidly stimulated SGK1 mRNAs, but a significant change in protein abundance was not detected. Instead, there was an increase in SGK1 kinase activity as early as at 2 h. An increase in NHE3 protein abundance was not detected until 12 h of dexamethasone exposure, although the transport activity was significantly stimulated at 4 h. These data demonstrate that the changes of SGK1 precede those of NHE3. Chronic regulation (24 h) of NHE3 was blocked completely by prevention of protein synthesis with cycloheximide or actinomycin D and by the glucocorticoid receptor blocker RU486. The acute effect of dexamethasone was similarly abrogated by RU486, but was insensitive to cycloheximide and actinomycin D. Similarly, the stimulation of SGK1 activity by dexamethasone was blocked by RU486 but not by actinomycin D. Together, these data show that the acute effect of glucocorticoids on NHE3 is mediated by a glucocorticoid receptor dependent mechanism that activates SGK1 in a nongenomic manner. Na⁺/H⁺ exchanger 3; serum and glucocorticoid-inducible kinase 1     

Plasma concentrations and receptor binding of RU 486 and its metabolites in humans

Using Chromosorb chromatography and HPLC, we measured the plasma concentrations of RU 486, and its monodemethylated (RU 42633), didemethylated (RU 42848) and alcoholic nondemethylated (RU 42698) metabolites up to 72 h following oral ingestion of 100 mg of RU 486 by five female volunteers. The peak plasma level of RU 486 (4.5 mumol/l) occurred within 1 h after ingestion of the compound; at this point significant amounts of the metabolites were also present in the plasma. After the initial redistribution within 6 h the plasma concentrations of RU 486 and three of its metabolites measured remained stable for 24 h. Concentrations of the monodomethylated metabolite exceeded those of the parent steroid during the time period measured, whereas the concentrations of the didemethylated and alcoholic metabolites were lower than those of RU 486, but still notable. At 72 h the concentrations of all the four steroids were still in the micromolar range. The relative binding affinities of these metabolites to human endometrial and myometrial progesterone receptors as well as to human placental glucocorticoid receptors were determined in vitro. The affinity of RU 486 for the human uterine progesterone receptor (Kd = 1.3 X 10(-9) M for RU 486) was higher than that of progesterone but lower than that of ORG-2058, a potent synthetic progestin. The relative binding affinities of the monodemethylated, alcoholic and didemethylated metabolites to the progesterone receptor were 21, 15 and 9%, respectively, compared with the parent compound RU 486; each was lower than that of progesterone (43%). RU 486 had an approx. 4-fold higher relative binding affinity to the glucocorticoid receptor than dexamethasone. Interestingly, the relative binding affinities of the metabolites studied to the human glucocorticoid receptor exceeded those of dexamethasone or cortisol. Compared with the parent compound RU 486, they were 61, 48 and 45% for the monodemethylated, alcoholic and didemethylated metabolites, respectively; each was higher than that of dexamethasone (23%). The affinity of dexamethasone to the human glucocorticoid receptor was 1.6 X 10(-9) M. These data indicate that the pool of certain metabolites of RU 486 may contribute to a significant extent to the antiprogestagenic (23-33%) and even greater extent to the antiglucocorticoid (47-61%) effects of RU 486.     

Amniotic fluid lipoxygenase metabolites during spontaneous labor and after RU486 treatment during late pregnancy in rhesus macaques

To determine changes in amniotic fluid (AF) lipoxygenase metabolites prior to spontaneous labor and after RU486 administration, we implanted AF and vascular catheters and myometrial electromyographic (EMG) electrodes in 8 rhesus macaques at 120-130 days of pregnancy (term = 167 days). Four animals had AF samples taken serially until they delivered their infants normally at term. The other four animals received RU486 (20 mg/kg/day) for 3 days. AF samples were collected every 2-3 days and at 12 hour intervals for 72 hours before and after treatment with RU486. Uterine activity was monitored continuously. LTB4, 5-HETE and 15-HETE were measured by radioimmunoassay. In untreated animals, LTB4 and 5-HETE concentrations in AF increased significantly (P less than 0.05) 4 days before delivery with no change in 15-HETE. After RU486, mean levels of LTB4 and 5-HETE were increased although the difference was not statistically significant. No change in 15-HETE levels was observed. In conclusion, LTB4 and 5-HETE increase in AF before the onset of spontaneous labor. Progesterone receptor blockade by RU486 does not reproduce the changes in AF lipoxygenase metabolites observed during normal parturition.