Identification of the functional domain of glucocorticoid receptor involved in RU486 antagonism

Mifepristone, also known as RU486, is a potent glucocorticoid receptor (GR) antagonist that inhibits GR-mediated transactivation. As an alternative to existing antidepressants, RU486 has been shown to rapidly reverse psychotic depression, most likely by blocking GR. Although a number of studies have demonstrated RU486-induced GR antagonism, the precise mechanism of action still remains unclear. To identify the GR domain involved in RU486-induced suppression, GR transactivation and nuclear translocation were examined using cells transfected with human GR (hGR), Guyanese squirrel monkey GR (gsmGR), and GR chimeras into COS-1 cells. RU486 showed a much more potent suppressive effect in gsmGR-expressing cells versus hGR-expressing cells, without significant cortisol- or RU486-induced changes in nuclear translocation. A GR chimera containing the gsmGR AF1 domain (amino acids 132–428) showed a marked decrease in luciferase activity, suggesting that this domain plays an important role in RU486-induced GR antagonism. Furthermore, fluorescence recovery after photobleaching (FRAP) analysis indicated that, in the presence of RU486, gsmGR AF1 domain contributes to GR mobility in living COS-1 cells. Taken together, these results demonstrate, for the first time, that the antagonistic effects of RU486 on GR transactivation involve a specific GR domain.     

Contribution of glucocorticoid-mineralocorticoid receptor pathway on the obesity-related adipocyte dysfunction

AimsMineralocorticoid receptor (MR) blockade ameliorated insulin resistance with improvements in adipocytokine dysregulation, inflammation, and excess of reactive oxygen species (ROS) in obese adipose tissue and adipocytes, but its mechanism has not been clarified. The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), producing active glucocorticoids, is highly expressed in adipocytes and glucocorticoids bind to MR with higher affinity than to glucocorticoid receptor (GR). We investigated whether glucocorticoids effect on adipocytokines and ROS through MR in adipocytes. In addition, fat distributions of MR and GR were investigated in human subjects.Methods and ResultsCorticoid receptors and their target genes were examined in adipose tissue of obese db/db mice. 3T3-L1 adipocytes were treated with glucocorticoids, H₂O₂, MR antagonist eplerenone (EP), GR antagonist RU486 (RU), MR-siRNA, and/or N-acetylcysteine. Human adipose tissues were obtained from seven patients who underwent abdominal surgery. The mRNA levels of MR and its target gene were higher in db/db mice than in control db/m + mice. In 3T3-L1 adipocytes, glucocorticoids, similar to H₂O₂, caused the dysregulation of mRNA levels of various genes related to adipocytokines and the increase of intracellular ROS. Such changes were rectified by MR blockade, not by GR antagonist. In human fat, MR mRNA level was increased in parallel with the increase of body mass index (BMI) and its increase was more significant in visceral fat, while there were no apparent correlations of GR mRNA level to BMI or fat distribution.ConclusionGlucocorticoid-MR pathway may contribute to the obesity-related adipocytokine dysregulation and adipose ROS.     

Recycling and desensitization of glucocorticoid receptors in v-mos transformed cells depend on the ability of nuclear receptors to modulate gene expression

In v-mos transformed cells, glucocorticoid receptor (GR) proteins that bind hormone agonist are not efficiently retained within nuclei and redistribute to the cytoplasmic compartment. These cytoplasmic desensitized receptors cannot be reutilized and may represent trapped intermediates derived from GR recycling. We have used the glucocorticoid antagonist RU486 to examine whether v-mos effects can be exerted on any ligand-bound GR. In the rat 6m2 cell line that expresses a temperature-sensitive p85gag-mos oncoprotein, RU486 is a complete antagonist and suppresses dexamethasone induction of metallothionein-1 mRNA at equimolar concentrations. Using indirect immunofluorescence, we observe efficient nuclear translocation of GR in response to RU486 treatment in either the presence or absence of v-mos oncoproteins. However, in contrast to the redistribution of agonist-bound nuclear receptors to the cytoplasm of v-mos-transformed cells, RU486-bound GRs are efficiently retained within nuclei. Interestingly, withdrawal of RU486 does not lead to efficient depletion of nuclear GR in either nontransformed or v-mos transformed cells. It is only after the addition of hormone agonist to RU486 withdrawn v-mos-transformed cells that GRs are depleted from nuclei and subsequently redistributed to the cytoplasm. Thus, only nuclear GRs that are agonist-bound and capable of modulating gene activity can be subsequently processed and recycled into the cytoplasm.     

Glucocorticoid ligands specify different interactions with NF-kappaB by allosteric effects on the glucocorticoid receptor DNA binding domain

Glucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing NF-kappaB function. Ligand binding to the C-terminal of GR promotes the nuclear translocation of the receptor and binding to NF-kappaB through the GR DNA binding domain. We sought how ligand recognition influences the interaction between NF-kappaB and GR. Both dexamethasone (agonist) and RU486 (antagonist) promote efficient nuclear translocation, and we show occupancy of the same intranuclear compartment as NF-kappaB with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-kappaB transactivation. This failure may stem from altered co-factor recruitment or altered interaction with NF-kappaB. Using both glutathione S-transferase pull-down and bioluminescence resonance energy transfer approaches, we identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-kappaB, with RU486 inhibiting recruitment compared with dexamethasone. Using the bioluminescence resonance energy transfer assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore, RU486 promotes differential protein recruitment to both the C-terminal and DNA binding domain of the receptor. Importantly, using chromatin immunoprecipitation, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-kappaB-responsive region of the interleukin-8 promoter, again in contrast to dexamethasone that significantly increased GR binding. We demonstrate that ligand-induced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication.     

Role of the low-affinity glucocorticoid receptor in the regulation of behavior and energy metabolism in the migratory red knot Calidris canutus islandica

Plasma corticosterone increases in association with migratory flight in the red knot Calidris canutus islandica, suggesting that corticosterone may promote migratory activity and/or energy mobilization in this species. This hypothesis is supported by general effects of glucocorticoids, which include stimulation of locomotion and the mobilization of energy depots. We experimentally examined the role of elevated corticosterone levels in the migratory red knot by comparing foraging behavior, flight frequency, and plasma metabolites between vehicle-injected controls and birds treated with RU486, an antagonist to the genomic low-affinity glucocorticoid receptor (GR). We predicted that RU486 treatment would interfere with energy mobilization. However, we expected no effects on flight activity because recent studies suggest that glucocorticoids affect locomotion through a nongenomic receptor. Finally, because glucocorticoids exert permissive effects on food intake, we postulated that RU486 treatment in the red knot would interfere with feeding. Results were consistent with the latter prediction, suggesting that the GR participates in the promotion of hyperphagia, the intense feeding state that is characteristic of the migratory condition. RU486 treatment did not affect flight frequency, suggesting that corticosterone may support migratory activity through a receptor other than the GR. Energy metabolism (as determined through plasma metabolites) was also unaffected by RU486, possibly because energetic demands experienced by captive birds were low.     

RU486对早孕猕猴海马GR的影响

目的探讨早孕猕猴给予大剂量RU486 3天后海马糖皮质激素受体(GR)的表达变化.方法 15只早孕猕猴随机分为空白对照组、赋形剂组和RU486组.空白对照组不予任何处理,赋形剂组和RU486组分别鼻饲赋形剂和RU486 3天.应用单克隆抗体链菌素亲生物蛋白过氧化酶(SP)免疫组织化学方法观察海马GR的表达情况,并用电子计算机图象分析技术进行处理.结果 RU486组猕猴妊娠终止,该组的海马GR表达显著下降,与对照组的差异有显著性.空白对照组和赋形剂组猕猴妊娠没有终止,其海马GR表达无差异.结论 RU486可使早孕猕猴海马的GR表达下降,推测这可能是该药终止妊娠的中枢作用机理之一.     

New analogues of mifepristone with more dissociated antiprogesterone activities

Mifepristone (RU 486 or RU 38486) possesses strong antiprogesterone and antiglucocorticoid along with moderate antiandrogen properties, which would limit its use in some therapeutic applications. In a search for more dissociated derivatives, the hydroxy substituent and the propynyl group in position 17 of the RU 486 series was replaced by a spiroether group, which is known to induce specific affinity for the progestin receptor in steroid series. The substituents in the para position of the 11 beta-phenyl group, leading to the most potent derivatives in the RU 486 series, were retained. The new derivatives have been studied in vitro for their relative binding affinities (RBAs) for the steroid receptor and in vivo for their hormonal and antihormonal activities. The selected compounds, RU 46556 and RU 49295 display the following properties: in vitro, like RU 486, they show a strong RBA for the rabbit progestin receptor, but a much lower one for the rat thymus glucocorticoid receptor; in vivo they are about three times more active than RU 486 for inducing abortion in rats, but unlike the latter they are devoid of any antiglucocorticoid activity on the thymus weight in rats. These antiprogesterone effects have been confirmed on the deciduoma formation in rats and on the endometrial proliferation in rabbits. However, in contrast to RU 486 in the latter test, some progestomimetic activity has been observed. RU 46556 and RU 49295 are now under extensive pharmacological study.     

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.     

Dissociation of steroid receptor coactivator 1 and nuclear receptor corepressor recruitment to the human glucocorticoid receptor by modification of the ligand-receptor interface: the role of tyrosine 735

Within the human glucocorticoid receptor (GR) steroid binding pocket, tyrosine 735 makes hydrophobic contact with the steroid D ring. Substitution of tyrosine735 selectively impairs glucocorticoid transactivation but not transrepression. We now show, using both mammalian two-hybrid and glutathione-S-transferase pull downs, that such substitutions reduce interaction with steroid receptor coactivator 1, both basally and in response to agonist binding. Using a yeast two-hybrid screen we identified one of the three nuclear receptor interacting domains (NCoR-N1) of nuclear receptor corepressor (NCoR) as interacting with the GR C terminus in an RU486-specific manner. This was confirmed in mammalian two-hybrid experiments, and so we used the NCoR-N1 peptide to probe the GR C-terminal conformation. Substitution of Tyr735phe, Tyr735val, and Tyr735 ser, which impaired steroid receptor coactivator 1 (SRC1) interaction, enhanced NCoR-N1 recruitment, basally and after RU486. RU486 did not direct SRC1 recruitment to any of the GR constructs, and dexamethasone did not allow NCoR-N1 recruitment. Using a glutathione-S-transferase pull-down approach, the NCoR-N1 peptide was found to bind the full-length GR constitutively, and no further induction was seen with RU486, but it was reduced by dexamethasone. As both SRC1 and NCoR are predicted to recognize a common hydrophobic cleft in the GR, it seems that changes favorable to one interaction are detrimental to the other, thus identifying a molecular switch.     

Glucocorticoid receptors take part in the apoptotic process of human lens epithelial cells, but the glucocorticoid receptor antagonist RU486 does not rescue the cells fully

To identify an agent with specific activity against human lens epithelial cells (HLECs), we confirmed the presence of glucocorticoid receptors (GRs) and GR-α genes and evaluated whether GRs have a relationship with the apoptotic process in cultured HLECs. We also determined whether the inhibitor RU486 could rescue the cells from apoptosis when the HLECs were exposed to dexamethasone (Dex), a steroid, in 4 concentrations for 4 periods, or were co-treated with the antagonist RU486. We found that Dex, which has been used as a medical agent for a long time, resulted in increased expression of GRE-luciferase, the GR-α gene and GR-protein and, in contrast, decreased the viability of HLECs. The expression of Bax protein was increased in an earlier stage in contrast to the expression of Bcl-2 protein, which was increased in a later stage. Caspase-3 activity was significantly increased under lower concentrations of Dex in the last stage. The nuclear morphology of HLECs showed an obvious apoptotic phenomenon under greater concentrations of Dex in the last stage. However, RU486, a GR antagonist, could partially inhibit GR and Bax expressions and the expression of caspase-3 was increased so that there was not a decrease in the ratio of apoptotic cells and an increase in the viability of HLECs. Our data showed that GRs had a partial relationship to the apoptotic process of HLECs when exposed to Dex and RU486 did not rescue the cells fully. Because of its toxicity, RU486 did not provide a therapeutic benefit in a glucocorticoid induced cataract (GIC) for the in vitro model, however, its activity and pathway targeting should still be studied further with appropriate drug combinations.     

Regulation of basal ACTH secretion by corticosterone is mediated by both type I (MR) and type II (GR) receptors in rat brain.

The mechanisms involved in the physiology of the secretion of ACTH are reviewed. The secretion is regulated by the biological consequences of the occupancy of high affinity mineralocorticoid (MR) and lower affinity glucocorticoid receptors (GR) for corticosterone at specific sites of the rat brain. The regulation by this mechanism of basal secretion during the circadian rhythm, the effect of adrenalectomy and of corticosterone replacement is discussed. Experiments with RU486, a specific glucocorticoid antagonist, suggest that occupancy of both MR and GR is required for normal control of ACTH at the time of peak activity. The occupancy of the GR for a few hours per day apparently suffices to maintain steady levels of the products of GR-responsive genes throughout the body.     

In vitro effects of RU486 on proliferation and differentiation capabilities of human bone marrow mesenchymal stromal cells

Although exogenous glucocorticoids (GC) play a role in the regulation of bone marrow mesenchymal stem/stromal cells (MSCs) proliferation and differentiation, the function of endogenous GC is not well understood. The purpose of this study was to investigate the effect of the blockage of endogenous GC using RU486, an antagonist of the glucocorticoid receptor, on the in vitro proliferation and differentiation capabilities of human MSCs. We quantitatively measured cell proliferation of human MSCs after treatment with increasing concentrations of RU486. We also evaluated multiple MSC differentiation capabilities, as well as the expression of stemness and senescence genes after proliferation of these human cells in vitro in the presence of RU486 at 10(-8)M. It was observed that RU486 treatment significantly increases the proliferation of human MSCs, although the optimal dose of RU486 for this increase in proliferation differs depending on the gender of the MSC donor. This improvement in MSC proliferation with RU486 treatment was higher in MSCs from male donors than that from females. No effect of RU486 on MSC proliferation was observed in a steroid-free medium. RU486 pretreatment significantly increased the expression of mRNA for alkaline phosphatase in human MSCs and the mRNA expression of osteocalcin of these cells up-regulated earlier after their exposure to osteogenic differentiation medium. Although no statistical significance in terms of chondrogenic differentiation markers was detected, mRNA expression for aggrecan and collagen type 2 were higher in a majority of the RU486-pretreated donor MSCs than their untreated controls. No significant difference in terms of MSC adipogenic differentiation capabilities were observed after RU486 treatment. RU486 treatment up-regulated the expressions of FGF-2 and Sox-11 in human MSCs. These results indicate that blockage of endogenous GCs may be developed as a novel approach to effectively improve the proliferation and osteochondral differentiation capabilities of human MSCs for potential clinical applications. Additional studies will be required to determine the potential long-term effects of RU486 treatment on these bone marrow cells.     

Expression and induction of CYP3As in human fetal hepatocytes

CYP3A4 and CYP3A7 mRNA expression levels were markedly up-regulated by dexamethasone (DEX), but not by rifampicin (RIF). CYP3A5 mRNA level was not increased significantly by DEX, RIF, or phenobarbital. Testosterone 6beta-hydroxylase activity was induced to about 2-fold of control by DEX. However, concomitant treatment with RIF did not alter DEX-mediated induction of CYP3A mRNA expression and testosterone 6beta-hydroxylase activity. DEX-mediated induction of CYP3A mRNA was suppressed in a dose-dependent manner by RU486, a glucocorticoid receptor (GR) antagonist. At 5microM RU486, DEX-mediated induction of CYP3A4, CYP3A5, and CYP3A7 mRNA expression was inhibited almost completely. These results suggest that, in human fetal hepatocytes, PXR is not involved in DEX-mediated induction of CYP3A4 and CYP3A7, and that the induction is mediated directly by GR.     

Hepatic glucocorticoid receptor behaves differently when its hormone binding site is occupied by agonist (triamcinolone acetonide) or antagonist (RU486) steroid ligands

We have examined the influence of sulfhydryl (SH)-group modifying agents on the interaction of the rat liver glucocorticoid receptor (GR) with its known agonist triamcinolone acetonide (TA) and the newly synthesized antagonist mifepristone (RU486). In the freshly prepared cytosol, [3H]TA or [3H]RU486 bound to macromolecule(s) which sediment as 8-9 moieties: the binding of either ligand can be competed with radioinert TA or RU486. The presence of 2-10 mM dithiothreitol (DTT), beta-mercaptoethanol (beta-MER), and monothioglycerol (MTG) caused a 2-3 fold increase in the [3H]TA and [3H]RU486 binding to GR. Iodoacetamide (IA) and N-ethylmaleimide (NEM) decreased the agonist binding significantly. In contrast, the [3H]RU486 binding to GR increased by 50 percent in the presence of IA. IA and NEM inhibited the binding of the heat-transformed [3H]TA-receptor complex to DNA-cellulose by 70-90 percent whereas DNA binding of [3H]RU486-bound GR was inhibited only slightly. These results indicate that either a) the interaction of GR with the agonist or antagonist steroid ligands causes differential structural alterations, which are more readily detectable in the presence of SH-modifying agents or b) the agonist and the antagonist interact with distinct steroid binding sites.     

Glucocorticoid receptor blockade inhibits brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus

When animals are under stress, glucocorticoids commonly inhibit adult neurogenesis by acting through glucocorticoid receptors (GRs). However, in some cases, conditions that elevate glucocorticoids promote adult neurogenesis, and the role of glucocorticoid receptors in these circumstances is not well understood. We examined the involvement of GRs in social enhancement of brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus. In this species, long-term social interaction simultaneously elevates plasma cortisol, enhances brain cell addition and increases production of aggressive electrocommunication signals (“chirps”). We implanted isolated and paired fish with capsules containing nothing (controls) or the GR antagonist, RU486, recorded chirp production and locomotion for 7 d, and measured the density of newborn cells in the periventricular zone. Compared to isolated controls, paired controls showed elevated chirping in two phases: much higher chirp rates in the first 5 h and moderately higher nocturnal rates thereafter. Treating paired fish with RU486 reduced chirp rates in both phases to those of isolated fish, demonstrating that GR activation is crucial for socially induced chirping. Neither RU486 nor social interaction affected locomotion. RU486 treatment to paired fish had a partial effect on cell addition: paired RU486 fish had less cell addition than paired control fish but more than isolated fish. This suggests that cortisol activation of GRs contributes to social enhancement of cell addition but works in parallel with another GR-independent mechanism. RU486 also reduced cell addition in isolated fish, indicating that GRs participate in the regulation of cell addition even when cortisol levels are low.     

In vivo effects of the steroid analogue RU486 on some aspects of intermediary and thyroid metabolism of brook charr, Salvelinus fontinalis.

The glucocorticoid analogue RU486 was administered by intraperitoneal injection to brook charr (Salvelinus fontinalis) to further explore the role of cortisol on aspects of intermediary and thyroid hormone metabolism of the species. RU486 significantly elevated the hepatosomatic index, hepatic G3PDH activity, and hepatic glycogen content, but was without effect on hepatic protein content, hepatic FPBase activity, or plasma glucose concentration. However, the stressor-related increase in plasma glucose concentration that was evident in brook charr 24 h following handling and injection was suppressed in RU486-treated groups. The distribution volume, turnover rates, and metabolic clearance rates of [3H]cortisol were similar in RU486- and vehicle-treated groups. Plasma T3 and T4 concentrations were similar in RU486- and vehicle-treated groups, but hepatic T3 production and hepatic T3 content were lower in RU486-treated fish; TSH had no effect on hepatic T3 content of vehicle-treated brook charr but significantly increased T3 content in the RU486-treated group. These observations support the concept of a role of cortisol in the control of peripheral monodeiodination of T4 in salmonid fish and suggest that RU486 may be a useful drug for evaluating the role of cortisol in fish in vivo.     

Heat shock preconditioning and pretreatment with glucocorticoid antagonist RU 486 protect rat myogenic cells H9c2 against glutamate-induced cell death

We have observed that the treatment of rat-heart derived H9c2 myoblasts for 20 h with the excitatory amino acid glutamate resulted in cell death in a dose dependent manner as determined by LDH release. The optimum cardiotoxicity was seen at 25 mM glutamate. Preconditioning with either sublethal heat shock (42°C for 30 min) or pretreatment with 500 nM of the glucocorticoid antagonist RU 486 for 24 h almost completely protected H9c2 cells against subsequent 20 h treatment with 25 mM lethal glutamate. In addition, we have observed that glutamate treatment resulted in intense nuclear localization of glucocorticoid receptors (GR) in H9c2 cells as judged by the confocal immunofluorescence microscopy. Furthermore, pretreatment with either heat shock or RU 486 followed by glutamate treatment resulted in dramatic decrease in GR nuclear localization which was almost comparable to that observed with control untreated cells. In conclusion, we have shown for the first time using H9c2 cells that (i) protection from glutamate cardiotoxicity occurs with prior treatment with sub lethal heat shock or RU 486 and (ii) these measures down regulate the intense nuclear localization of GR induced by glutamate. The block to GR nuclear localization is likely to be involved in cardioprotective effects offered against glutamate toxicity by pretreatment with heat shock or RU 486.