Latent agonist activity of the steroid antagonist, RU486, is unmasked in cells treated with activators of protein kinase A

RU486 is a glucocorticoid and progesterone antagonist. In glucocorticoid-responsive fibroblasts, it mediates little or no induction of a truncated, hormone-responsive mouse mammary tumor virus promoter; moreover, it abrogates the induction mediated by the glucocorticoid agonist, dexamethasone. However, when the fibroblasts are treated with activators of protein kinase A, 8-Br-cAMP or forskolin, along with RU486, the steroid now acts as a partial agonist, capable of mediating an induction of hormone-responsive reporter genes. In addition, the ability of RU486 to block the action of the glucocorticoid agonist, dexamethasone, is compromised by concomitant treatment with 8-Br-cAMP. Activators of protein kinase C fail to elicit these phenomena. Induction of gene expression in the presence of 8-Br-cAMP is dependent on the dose of RU486 over a range consistent with a glucocorticoid receptor-mediated mechanism. An antagonist, ZK98 299, which unlike RU486 is not thought to permit receptor binding to DNA, is not activated by 8-Br-cAMP. The elicitation of RU486 agonist activity cannot be attributed solely to idiosyncrasies of the cell line or the promoter. Similar phenomena are observed in another glucocorticoid-responsive fibroblast line. Furthermore, RU486 can induce a minimal promoter bearing two copies of a synthetic receptor target site. However, we have identified at least one promoter toward which RU486 still behaves as an antagonist despite 8-Br-cAMP treatment. These observations suggest that the unmasking of latent agonist activity in a type II antagonist is not an isolated phenomenon and may, therefore, be seen with other receptors and antagonists. The finding that modulation of cellular signal transduction pathways can unmask agonist activity in an otherwise effective steroid antagonist has significant implications for the use of steroid antagonists in the clinical setting and could represent a heretofore unrecognized mechanism for the development of steroid resistance.     

Inhibition of gene expression by steroid hormone receptors via a negative glucocorticoid response element: evidence for the involvement of DNA-binding and agonistic effects of the antiglucocorticoid/antiprogestin RU486

We have used a negative glucocorticoid response element (nGRE) from the bovine prolactin promoter linked to the gene for chloramphenicol acetyltransferase (PRL3CAT) to study the inhibition of gene expression by steroid hormone receptors. This nGRE increased basal expression from a heterologous promoter in COS-7 cells. In the presence of cotransfected glucocorticoid (GR), androgen, or progesterone receptor (PR) expression vectors and their cognate ligands, the expression of PRL3CAT could be repressed, indicating that these steroid receptor subfamily members could function through the same negative response element. No repression was observed with the estrogen receptor, showing that the repressive effect was specific for members of the GR-subfamily. Mutation of three amino acids within the GR-DNA binding domain that determine the specificity of GR-GRE interaction abolished the ability of the GR to inhibit the expression of PRL3CAT, demonstrating the requirement for DNA binding of the GR in the mechanism of repression. The antiglucocorticoid/antiprogestin RU486 when bound to PR or GR also repressed the expression of the PRL3CAT, but higher concentrations of RU486 were required to obtain an effect with the GR when compared to the PR. RU486 was unable to antagonize the effect of progestins on PRL3CAT and only partially antagonized the glucocorticoid repression. Thus, regarding the repression of PRL3CAT, RU486 acted as an agonist when bound to the PR and as a partial agonist when bound to the GR.     

Effects of neonatal RU486 on adult sexual, parental, and fearful behaviors in rats.

Exposure to gonadal hormones during perinatal life influences later behavior. The finding that sex differences exist in progestin receptor expression in the perinatal rat brain suggests differential sensitivity of male and female brains to progesterone (C. K. Wagner, A. N. Nakayama, & G. J. De Vries, 1998). Because these sex differences are in neural sites that influence sexually differentiated sexual, parental, and fearful behaviors in adults, this study examined the effects of administering the progestin receptor antagonist RU486 for the first 10 days after birth on these behaviors in adulthood. Neonatal RU486 significantly reduced sexual behavior in males but did not impair reproduction in females. Neonatal RU486 did not affect parental responses of virgin rats exposed to pups (sensitization) but reduced fear in the elevated plus-maze in both sexes. Treatment of pups with RU486 affected neither mother–litter interactions nor plasma testosterone levels in males during or after treatment. These results suggest that neonatal exposure to progesterone, in addition to androgens and estrogens, influences behavioral development in rats. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effects of adrenalectomy and glucocorticoid receptor antagonist, RU38486, on pituitary growth hormone-releasing hormone receptor gene expression in rats

We examined the effects of adrenalectomy and a glucocorticoid receptor antagonist, RU38486, on pituitary GH-releasing hormone (GRH) receptor gene expression in rats. GRH receptor mRNA levels were significantly decreased in adrenalectomized rats and replacement of dexamethasone reversed the decrease to normal. GH secretion was inhibited by adrenalectomy, whereas dexamethasone replacement failed to restore the impaired GH secretion. A high dose of RU38486 had an agonistic effect on GRH receptor mRNA levels. These results suggest that endogenous glucocorticoid is necessary for normal expression of pituitary GRH receptor mRNA in rats.     

Biological mechanisms underlying the clinical effects of RU 486: modulation of cultured endometrial stromal cell stromelysin-1 and prolactin expression

During in vitro decidualization of human endometrial stromal cells (HESCs), medroxyprogesterone acetate (MPA) inhibits expression of the potent extracellular matrix (ECM)-degrading protease stromelysin-1 (MMP-3), but enhances PRL expression. Consistent with its priming role in vivo, estradiol (E2) augments these effects. In the current study, immunoblot analysis revealed that coincubation with 10(-6) M RU 486 blocked the inhibition in HESC-secreted MMP-3 levels (50,000 mol wt) evoked by 10(-8) M E2 + 10(-7) M MPA. Although MPA can act as a glucocorticoid, the HESCs were refractory to 10(-7) M dexamethasone added alone or with E2. Because E2 elevates progesterone but not glucocorticoid receptor levels, MPA and RU 486 control MMP-3 expression as a progestin and antiprogestin, respectively. To study RU 486 involvement in steroid withdrawal leading to menstruation, HESCs were decidualized during 10 days incubation with E2 + MPA, and parallel cultures were kept in E2 + MPA or withdrawn to either control or RU 486-containing medium. Compared with E2 + MPA-suppressed HESCs, increases in levels of secreted MMP-3 (2.0-fold), and its 2.1-kilobase messenger RNA (10-fold) were observed in HESCs after 4 days of withdrawal to control medium, with much greater increases seen in RU 486-containing medium (10-fold protein, 100-fold messenger RNA). Previously, we showed that RU 486 up-regulated E2 + MPA-inhibited plasminogen activator expression in the cultured HESCs. Extrapolation of these in vitro observations to endometrial events following RU 486 administration suggests that coordinate enhancement of MMP-3 and plasminogen activator expression promotes proteolysis of the stromal/decidual ECM, which leads to endometrial sloughing. Moreover, destabilization of endometrial microvessels resulting from degradation of their surrounding ECM is consistent with the heavy menstrual bleeding stemming from RU 486 administration. However, in contrast to the marked RU 486-initiated reversal of MMP-3 expression, RU 486 did not significantly reverse E2 + MPA-enhanced PRL secretion by the cultured HESCs. Interestingly, decidual PRL, unlike decidual MMP-3, does not appear to play a role in menstruation. Interleukin-1 beta counteracted E2 + MPA-mediated inhibition of secreted MMP-3 levels, implying that leukocyte/trophoblast-derived cytokines can modulate steroid-regulated MMP-3 expression by stromal/decidual cells during menstruation and pregnancy.     

Evidence for mineralocorticoid receptor facilitation of glucocorticoid receptor-dependent regulation of hypothalamic-pituitary-adrenal axis activity

These studies further evaluated the relative role of mineralocorticoid (type I) and glucocorticoid (type II) receptors in mediating corticosteroid feedback regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Acute treatment of rats with the selective mineralocorticoid receptor antagonist, RU28318 (50 mg/kg sc), produced elevated basal corticosterone levels in the morning, but had no effect on basal corticosterone levels in the evening or on restraint stress corticosterone levels at either time of day. Acute treatment with the selective glucocorticoid receptor antagonist, RU40555 (30 mg/kg sc) had no effect on basal or restraint stress corticosterone levels at either time of day. However, combined treatment with RU28318 and RU40555 produced an elevation of evening basal corticosterone levels (and morning basal on one occasion) and produced an increase in corticosterone levels during and after stress at both times of day. In a separate experiment conducted in the morning, the combined RU28318 and RU40555 treatment also produced elevated ACTH responses during restraint stress. Based on available corticosteroid receptor measures, the RU28318 treatment was estimated to selectively occupy approximately 85% of mineralocorticoid receptors in rat brain, whereas the RU40555 treatment was estimated to selectively occupy approximately 50% of glucocorticoid receptors in rat brain. We conclude that mineralocorticoid receptor activation is necessary and sufficient to maintain low basal corticosterone levels during the circadian trough, whereas glucocorticoid receptor activation is necessary to constrain corticosterone secretion during the circadian peak or during acute stress. However, even during the circadian peak or acute stress, mineralocorticoid receptor activation plays an important role in facilitating the glucocorticoid receptor dependent regulation of HPA axis activity by corticosterone.     

Effectors of cyclic adenosine 5'-monophosphate up-regulating-oxytocin receptors in rabbit amnion cells: isoproterenol, parathyroid hormone-related protein, and potentiation by cortisol

Forskolin (FSK; an activator of adenylyl cyclase) and cortisol synergistically increase the concentration of oxytocin receptors (OTRs) in rabbit amnion cells. The aims of this study were to characterize potential physiological regulators of OTR concentrations acting through adenylyl cyclase and to clarify the mechanisms of potentiation by cAMP and cortisol. Both isoproterenol (ISO) and parathyroid hormone-related protein (PTHrP) elevated amnion cell cAMP levels and OTR concentrations. The effects of ISO and PTHrP on OTR were potentiated by cortisol. Cortisol had no effect on the ability of ISO or PTHrP to stimulate adenylyl cyclase activity, and cAMP did not affect the number or affinity of glucocorticoid receptors in whole cells or in cytosol. Adenylyl cyclase activation, however, caused conversion of mifepristone (RU486) from a glucocorticoid antagonist to agonist. Thus, mifepristone elevated OTR receptor concentrations in the presence of FSK. In contrast, a structurally related glucocorticoid antagonist, onapristone (ZK98 299), was unaffected by cAMP. Because glucocorticoid receptors bound to mifepristone are capable of interacting with DNA, whereas onapristone-occupied receptors are not, we conclude that cAMP affects glucocoticoid receptor-DNA interactions, accounting for the synergistic effects of cAMP and cortisol on OTRs.     

Persistence of premenstrual syndrome during low-dose administration of the progesterone antagonist RU 486

OBJECTIVE: To test whether progesterone or progesterone receptors are important mediators of premenstrual syndrome (PMS) and whether progesterone antagonist RU 486 would alleviate symptoms. METHODS: Following extensive screening including physical and psychological assessment, seven women with severe PMS participated in a 6-month, randomized, double-blind, placebo-controlled, crossover study. The treatment included 3 months of low-dose RU 486 (5 mg alternate days for four doses, beginning 3 days after the urinary LH surge) or placebo, administered in a similar fashion. Symptoms were evaluated using the Calendar of Premenstrual Experiences, Beck Depression Inventory, State-Trait Anxiety Inventory, and the Profile of Mood States. RESULTS: Symptoms of PMS were similar during RU 486 and placebo treatments. CONCLUSION: Luteal-phase administration of low-dose RU 486 does not significantly reduce the physical or behavioral manifestations of PMS.     

Evidence that RU 24969-induced locomotor activity in C57/B1/6 mice is specifically mediated by the 5-HT1B receptor

1. The behavioural effects of the 5-HT1B receptor agonists, RU 24969 and CGS 12066B, have been investigated in C57/B1/6 mice. 2. RU 24969 (1-30 mg kg-1) produced intense and prolonged hyperlocomotion and other behavioural changes. 3. CGS 12066B caused similar effects, but they were much less pronounced, inconsistent and transient irrespective of whether this drug was given i.p. (1-15 mg kg-1) or i.c.v. (0.2-40 micrograms). However, CGS 12066B (7.5 and 15 mg kg-1) caused a dose-related inhibition of RU 24969 (7.5 mg kg-1)-induced hyperlocomotion indicating that the former is a 5-HT1B partial agonist. 4. RU 24969 (7.5 mg kg-1 i.p.)-induced hyperlocomotion was inhibited by the (-)-, but not (+)-isomers of pindolol (4 mg kg-1) and propranolol (20 mg kg-1) but not by metoprolol (10 mg kg-1) or ICI 118,551 (5 mg kg-1), consistent with an involvement of 5-HT1A or 5-HT1B receptors. 5. The response was not altered by the selective 5-HT1A receptor antagonist, WAY 100135 (5 mg kg-1, s.c.), the 5-HT2A/5-HT2C receptor antagonist, ritanserin (0.1 mg kg-1), the selective 5-HT3 receptor antagonist, ondansetron (1 mg kg-1) or the non-selective 5-HT receptor antagonists methysergide (3 mg kg-1) and metergoline (3 mg kg-1). 6. Although spiroxatrine (0.1 mg kg-1) and ketanserin (1 mg kg-1) inhibited RU 24969-induced hyperlocomotion, these effects were probably due to antagonism of dopamine D2 receptors and alpha 1-adrenoceptors respectively. 7. Taken together, these results indicate that RU 24969-induced hyperlocomotion results specifically from activation of central 5-HTIB receptors.8. Lesioning of 5-HT neurones with 5,7-dihydroxytryptamine (75 microg, i.c.v.) or depletion with pchlorophenylalanine(200 mg kg-1, i.p. for 14 days) had no effect on RU 24969-induced hyperlocomotiondemonstrating that the 5-HTIB receptors involved are postsynaptic and that they do not show super sensitivity.9. The involvement of other monoamine neurotransmitter systems in RU 24969-induced hyperlocomotionwas also examined. The response was inhibited by the al-adrenoceptor antagonist, prazosin(1 mg kg-1), the dopamine DI receptor antagonist, SCH 23390 (0.05 mg kg-1) and the dopamine D2 receptor antagonist, BRL 34778 (0.03 mg kg-1), but not by the M2-adrenoceptor antagonist, idazoxan(1 mg kg-1). Lesioning noradrenergic neurones with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine(100 mg kg-1) markedly attenuated this behaviour. These results show that the hyperlocomotion is expressed via noradrenergic and dopaminergic neurones acting on alpha 1-adrenoceptors, DI and D2 receptors.10. RU 24969 decreased brain concentrations of 5-hydroxyindoleacetic acid whilst simultaneously increasing 5-HT, consistent with the reduction of 5-HT neuronal activity by activation of 5-HTlA and 5-HTIB autoreceptors. RU 24969 increased brain 3-methoxy-4-hydroxyphenylglycol, but not noradrenaline, concentrations which supports the involvement of noradrenergic neurones in the expression of hyperlocomotion. RU 24969 did not alter dopamine, dihydroxyphenylacetic acid or homovanillic acid concentrations in the nucleus accumbens suggesting that the dopaminergic neurones terminating there are not directly involved.     

Neuroendocrine regulation of cytokine production during experimental influenza viral infection: effects of restraint stress-induced elevation in endogenous corticosterone

A murine model of influenza viral infection was used to examine the neuroendocrine regulation of cytokine production. Restraint stress (RST) was used to activate the hypothalamic-pituitary-adrenal axis and elevate corticosterone (CORT) levels in influenza A/Puerto Rico/8/34 (PR8) virus-infected C57BL/6 mice. The type II glucocorticoid receptor antagonist RU486 was used to specifically examine the modulation of PR8 virus-specific cytokine responses by CORT. RST suppressed the PR8 virus- specific production of Th1-type (IL-2 and IFN-gamma) and Th2-type IL-10) cytokines by cells from the regional lymph nodes and spleens. In addition, IL-6 production by splenocytes was inhibited by RST; however, IL-6 production by cells from the regional lymph nodes was enhanced. Treatment of mice with RU486 prevented the effects of RST, suggesting that the RST-induced alterations in cytokine responses were mediated by CORT. Furthermore, CORT was shown to inhibit the PR8 virus-specific production of both Thl-type and Th2-type cytokines in vitro at doses corresponding to the physiologic range of free plasma CORT following hypothalamic-pituitary-adrenal axis activation.     

RU486 is a potent inhibitor of aromatase induction in human breast adipose tissue stromal cells

Aromatization of circulating androgens in adipose tissue is a major source of estrogens in postmenopausal women. As part of our efforts to elucidate the mechanism of aromatase induction in human breast adipose tissue, we tested the effects of the antiglucocorticoid and antiprogestin, RU486, on aromatase induction in primary cultures of adipose tissue stromal cells in a serum-free system devoid of phenol-red. Under these conditions 1 microM cortisol alone induces low levels of aromatase activity within one day, whereas platelet-derived growth factor BB (PDGF) potentiates this effect two- to three-fold. The well-known strong inductive effect of dibutyryl-cAMP (db-cAMP) is also augmented by cortisol, but is inhibited by PDGF in the absence of cortisol. RU486 completely prevented aromatase induction by cortisol and PDGF. Induction by db-cAMP in the presence and absence of cortisol was significantly inhibited by RU486. Even lower activities were measured when RU486 was added to cells stimulated with PDGF and db-cAMP in the absence or presence of cortisol. Similar results were obtained after prolonged incubation. The inhibitory effects of RU486 are dose dependent, less than 1 microM completely blocking the effects of cortisol, whereas 10 microM are needed to block db-cAMP induction. RU486 does not affect cell number, cellular protein, viability or house-keeping enzymes such as lactate dehydrogenase (LDH), and therefore seems to act specifically. The time course of RU486 action on the db-cAMP induction of aromatase indicates that it acts via a newly synthetized mediator or target in stromal cells. These results suggest that all known inducers of aromatase in adipose tissue depend upon the action of signalling molecules (probably members of the nuclear receptor superfamily) which can be blocked by RU486. The inhibitory action of PDGF seems to be independent of steroid hormone action, as seems some basal activity induced by db-cAMP. In conclusion, this in vitro study suggests that RU486 might be a useful tool for the therapy of estrogen-dependent tumours through its inhibition of aromatase induction.     

Altered vulnerability to acute opiate withdrawal following stress: Roles of N-methyl-{d}-aspartate and glucocorticoid receptors.

Five experiments studied the modulation of acute opiate withdrawal by restraint stress. Rats were subjected to a 2-hr restraint stress, and 1, 3, or 7 days later they received a single injection of morphine followed by injection of naloxone. Naloxone precipitated a withdrawal syndrome. This syndrome was enhanced when it occurred 1 day after stress but was reduced when it occurred 7 days after stress. The enhancement of withdrawal by restraint stress was prevented by treatment with the N-methyl-{d}-aspartate (NMDA) receptor antagonist MK801 or the glucocorticoid receptor antagonist RU486 prior to stress. Together these experiments show that restraint stress alters vulnerability to opiate withdrawal and identify activation of NMDA and glucocorticoid receptors as causal to this vulnerability. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

RU486 suppresses prolactin production in explant cultures of leiomyoma and myometrium

OBJECTIVE: To assess the action of RU486 (mifepristone), in the presence and absence of P, on PRL production by explant cultures of leiomyoma and myometrium. DESIGN: Explant cultures using tissue from nine premenopausal women undergoing hysterectomy in the proliferative phase of the menstrual cycle; immunohistochemical staining of tissue sections from five patients for P receptor (PR) subtype. MAIN OUTCOME MEASURES: Prolactin secretion (measured by RIA), lactate dehydrogenase secretion (measured by quantitative colorimetric assay), and immunohistochemistry for PR subtype. RESULTS: Prolactin secretion was decreased in leiomyomas by RU486 at concentrations of 10(-7) M and 10(-5)M when normal serum-containing medium was used. In experiments with all detectable P removed from serum, PRL secretion was suppressed in both leiomyomas and myometrium at an RU486 concentration of 10(-7)M. Immunohistochemistry results suggest that the A form of the PR is the dominant form in both leiomyomas and myometrium. CONCLUSIONS: Prolactin production is suppressed in both leiomyomas and myometrium after treatment with RU486 in vitro, and this suppression may serve as a marker for the clinical effectiveness of agents used in the treatment of leiomyomas.     

Protection against hypoxic-ischemic damage with corticosterone and dexamethasone: inhibition of effect by a glucocorticoid antagonist RU38486

We investigated whether the neuroprotection provided by dexamethasone against neonatal hypoxic-ischemic damage can be inhibited by a glucocorticoid antagonist and whether corticosterone, the endogenous glucocorticoid in the rat, also provides protection. Rats (6 days old) were treated with either vehicle (0.1 ml/10 g), corticosterone (3.5-80 mg/kg, s.c.) or dexamethasone alone or in combination with RU38486 (20-80 mg/kg, s.c.) 15 min prior to dexamethasone (0.1 mg/kg, i.p.). At 7 days of age, cerebral hypoxia-ischemia was produced by right carotid artery ligation under anesthesia and subsequent exposure to 2 h of hypoxia. Damage was quantified from brains perfusion-fixed and processed 2 days later. The reduction in somatic growth, thymus weight and the relatively elevated blood glucose levels at the end of hypoxia-ischemia were inhibited by RU38486. The protective effect of dexamethasone was also prevented by RU38486 (P < 0.001). Similar to pre-treatment with dexamethasone, administration of corticosterone (40-80 mg/kg) markedly reduced the extent of infarction compared to vehicle-treated controls (P < 0.0001). Thus, the endogenous glucocorticoid in the rat also provides protection against hypoxic-ischemic damage. RU38486 inhibits the beneficial effects of dexamethasone demonstrating that the neuroprotection observed with dexamethasone is a glucocorticoid receptor-mediated effect.     

Interaction and dissociation by ligands of estrogen receptor and Hsp90: the antiestrogen RU 58668 induces a protein synthesis-dependent clustering of the receptor in the cytoplasm

The in vivo interaction of estrogen receptor (ER) and Hsp90, demonstrated in the absence of hormone by a nuclear cotranslocation assay of the cytoplasmic Hsp90 with the karyophilic receptor, was disrupted by agonist and antagonist ligands, which, after dissociating the Hsp90, allowed the chaperone protein to be relocalized in the cytoplasm. The pure antiestrogen RU 58668 (RU), which was unable to stimulate an estrogen-dependent reporter gene and completely inhibited its estradiol-induced activity, also profoundly modified the subcellular distribution of ER in a specific time- and dose-dependent manner; ER appeared as speckled fluorescent clusters mainly located in the perinuclear region of the cytoplasm. The kinetics of appearance and reversal of the RU-dependent ER mislocalization in the presence or absence of cycloheximide demonstrated 1) that this effect was reversed by RU withdrawal or estradiol (E2) treatment, and 2) that cycloheximide with RU inhibited and reversed the ER cytoplasmic mislocalization induced by RU alone. These results point to a protein synthesis-dependent step in the mechanism of action of this antiestrogen. After RU treatment, a large portion of ER was found in the particulate fraction of the cytoplasm. However, confocal and electron microscopic analysis showed that ER clusters were not associated with specific cytoplasmic organelles or compartments. Using ER mutants, it was found that the ligand binding domain was sufficient for RU to produce receptor mislocalization, while the constitutive nuclear localization signals were dispensable. We propose that the antiestrogenic properties of RU are primarily due to the induction of an aggregation-prone receptor conformation that cannot undertake the constitutive and the ligand-induced nuclear localization function of the receptor because it is sequestered in the cytoplasm by fast turning over protein(s). We predict that antiestrogens able to block ER nuclear localization will behave as pure antihormones and will inhibit all the nuclear action of ER elicited by agonistic ligands or by ligand-independent mechanisms such as growth factor stimulation.