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 It is only after the addition of hormone agonist to RU486 withdrawn v-mos-transformed cells that GRs are depleted from nuclei and subsequently redistributed to the cytoplasm.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.     

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.     

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.     

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.     

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.     

Glucocorticoid receptors: ATP-dependent cycling and hormone-dependent hyperphosphorylation

The dependence of hormone binding to glucocorticoid receptors (GRs) on cellular ATP levels led us to propose that GRs normally traverse an ATP-dependent cycle, possibly involving receptor phosphorylation, and that without ATP they accumulate in a form that cannot bind hormone. We identified such a form, the null receptor, in ATP-depleted cells. GRs are basally phosphorylated, and become hyperphosphorylated after treatment with hormone (but not RU486). In mouse receptors we have identified 7 phosphorylated sites, all in the N-terminal domain. Most are on serines and lie within a transactivation region. The time-course of hormone-induced hyperphosphorylation indicates that the primary substrates for hyperphosphorylation are the activated receptors; unliganded and hormone-liganded nonactivated receptors become hyperphosphorylated more slowly. After dissociation of hormone, most receptors appear to be recycled and reutilized in hyperphosphorylated form. From these and related observations, we have concluded that the postulated ATP-dependent cycle can be accounted for by hormone-induced or spontaneous dissociation of receptor-Hsp90 complexes, followed by reassociation of unliganded receptors with Hsp90 via an ATP-dependent reaction like that demonstrated in cell-free systems. Other steroid hormone receptors might traverse a similar cycle. Four of the 7 phosphorylated sites in the N-terminal domain are in consensus sequences for p34^cdc2 kinases important in cell cycle regulation. This observation, along with the known cell cycle-dependence of sensitivity to glucocorticoids and other evidence, point to a role for receptor phosphorylation in controlling responses to glucocorticoids through the cell cycle.     

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.     

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.     

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.     

Evidence for a divergence in function between two glucocorticoid receptors from a basal teleost

ABSTRACT: BACKGROUND: Duplicated glucocorticoid receptors (GR) are present in most teleost fish. The evolutionary advantage of retaining two GRs is unclear, as no subtype specific functional traits or physiological roles have been defined. To identify factors driving the retention of duplicate GRs in teleosts, the current study examined GRs in representatives of two basal ray-finned fish taxa that emerged either side of the teleost lineage whole genome duplication event (WGD) event, the acipenseriform, Acipenser ruthenus, (pre-WGD) and the osteoglossimorph, Pantodon buchholzi, (post-WGD). RESULTS: The study identified a single GR in A. ruthenus (ArGR) and two GRs in P. buchholzi (PbGR1 and PbGR2). Phylogenetic analyses showed that ArGR formed a distinct branch separate from the teleosts GRs. The teleost GR lineage was subdivded into two sublineages, each of which contained one of the two P. buchholzi GRs. ArGR, PbGR1 and PbGR2 all possess the unique 9 amino acid insert between the zinc-fingers of the DNA-binding domain that is present in one of the teleost GR lineages (GR1), but not the other (GR2). A splice variant of PbGR2 produces an isoform that lacked these 9 amino acids (PbGR2b). Cortisol stimulated transactivation activity of ArGR, PbGR2b and PbGR1 in vitro; with PbGR2b and PbGR1, the glucocorticoid 11-deoxycortisol was a more potent agonist than cortisol. The hormone sensitivity of PbGR2b and PbGR1 differed in the transactivation assay, with PbGR2b having lower EC50 values and greater fold induction. CONCLUSIONS: The difference in transactivation activity sensitivity between duplicated GRs of P. buchholzi suggests potential functional differences between the paralogs emerged early in the teleost lineage. Given the pleiotropic nature of GR function in vertebrates, this finding is in accordance with the hypothesis that duplicated GRs were potentially retained through subfunctionalisation followed by gene sharing. A 9 amino acid insert in the DNA-binding domain emerged in basal ray-finned fish GRs. However, the presence of a PbGR2 splice variant that lacks this insert, as well as the loss of the exon encoding these amino acids in the genes encoding for other teleost GR2 suggests the selection of two receptors with different DNA-binding domain structures in teleosts.     

Role of activation function domain-1, DNA binding, and coactivator GRIP1 in the expression of partial agonist activity of glucocorticoid receptor-antagonist complexes

The determinants of the partial agonist activity of most antisteroids complexed with steroid receptors are not well understood. We now examine the role of the N-terminal half of the glucocorticoid receptor (GR) including the activation domain (AF-1), the DNA binding site sequence, receptor contact with DNA, and coactivator binding on the expression of partial agonist activity in two cell lines for GRs bound by five antiglucocorticoids: dexamethasone mesylate (Dex-Mes), dexamethasone oxetanone (Dex-Ox), progesterone (Prog), deoxycorticosterone (DOC), and RU486. Using truncated GRs, we find that the N-terminal half of GR and the AF-1 domain are dispensable for the partial agonist activity of antiglucocorticoids. This contrasts with the AF-1 domain being required for the partial agonist activity of antisteroids with most steroid receptors. DNA sequence (MMTV vs a simple GRE enhancer) and cell-specific factors (CV-1 vs Cos-7) exert minor effects on the level of partial agonist activity. Small activity differences for some complexes of GAL4/GR chimeras with GR- vs GAL-responsive reporters suggest a contribution of DNA-induced conformational changes. A role for steroid-regulated coactivator binding to GRs is compatible with the progressively smaller increase in partial agonist activity of Dex-Mes > Prog > RU486 with added GRIP1 in CV-1 cells. This hypothesis is consistent with titration experiments, where low concentrations of GRIP1 more effectively increase the partial agonist activity of Dex-Mes than Prog complexes. Furthermore, ligand-dependent GRIP1 binding to DNA-bound GR complexes decreases in the order of Dex > Dex-Mes > Prog > RU486. Thus, the partial agonist activity of a given GR-steroid complex in CV-1 cells correlates with its cell-free binding of GRIP1. The ability to modify the levels of partial agonist activity through changes in steroid structure, DNA sequence, specific DNA-induced conformational changes, and coactivator binding suggests that useful variations in endocrine therapies may be possible by the judicious selection of these parameters to afford gene and tissue selective results.     

Glucocorticoid receptor and sequential P53 activation by dexamethasone mediates apoptosis and cell cycle arrest of osteoblastic MC3T3-E1 cells

Glucocorticoids play a pivotal role in the proliferation of osteoblasts, but the underlying mechanism has not been successfully elucidated. In this report, we have investigated the molecular mechanism which elucidates the inhibitory effects of dexamethasone on murine osteoblastic MC3T3-E1 cells. It was found that the inhibitory effects were largely attributed to apoptosis and G1 phase arrest. Both the cell cycle arrest and apoptosis were dependent on glucocorticoid receptor (GR), as they were abolished by GR blocker RU486 pre-treatment and GR interference. G1 phase arrest and apoptosis were accompanied with a p53-dependent up-regulation of p21 and pro-apoptotic genes NOXA and PUMA. We also proved that dexamethasone can't induce apoptosis and cell cycle arrest when p53 was inhibited by p53 RNA interference. These data demonstrate that proliferation of MC3T3-E1 cell was significantly and directly inhibited by dexamethasone treatment via aberrant GR activation and subsequently P53 activation.     

Evidence from pulse-chase labeling studies that the antiglucocorticoid hormone RU486 stabilizes the nonactivated form of the glucocorticoid receptor in mouse lymphoma cells

A pulse-chase labeling technique was used to determine the properties of glucocorticoid receptors occupied by the antiglucocorticoid hormone RU486 in S49.1 mouse lymphoma cells. Cells were pulse-labeled with [35S]methionine and then at the beginning of the chase, either no hormone (control), dexamethasone, or RU486 was added to cells. At 4 h into the chase, cytosol was prepared and receptors were immunoadsorbed to protein A-Sepharose using the BuGR2 antireceptor antibody. Immunoadsorbed proteins were resolved by gel electrophoresis and analyzed by autoradiography. The 90 kDa heat shock protein (hsp90) coimmunoadsorbed with receptors from control cells when protein A-Sepharose pellets were washed with 250 mM NaCl but not when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that hsp90-receptor complexes are disrupted by a high concentration of salt in the absence of molybdate. hsp90 coimmunoadsorbed with receptors from RU486-treated cells even when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that RU486 stabilizes the association of hsp90 with the glucocorticoid receptor. In contrast, hsp90 did not coimmunoadsorb with receptors from dexamethasone-treated cells, consistent with earlier evidence that hsp90 dissociates from the receptor when the receptor binds glucocorticoid hormone. Dexamethasone induced a rapid quantum decrease in the amount of normal receptor recovered from cytosol but did not induce a decrease in the amount of nuclear transfer deficient receptor recovered from cytosol, consistent with tight nuclear binding of normal receptors occupied by dexamethasone. In contrast, RU486 did not induce a quantum decrease in the recovery of normal receptors from cytosol, indicating that receptors occupied by RU486 are not tightly bound in the nuclear fraction. We conclude that the antiglucocorticoid hormone RU486, in contrast to the glucocorticoid hormone dexamethasone, stabilizes the association between the glucocorticoid receptor and hsp90. The decreased affinity of receptors occupied by RU486 for the nuclear fraction may be due to their association with hsp90 and may account for the failure of RU486 to exert agonist activity.