Glucocorticoid Receptor Sites Research Articles

Chronic social stress alters dexamethasone sensitivity of glucocorticoid receptor target genes

Glucocorticoids are well-known for their role in adapting to physical and psycho-emotional stress. The prefrontal cortex (PFC) is a crucial target-tissue for glucocorticoid receptors (GR) that coordinates the stress response. Transcriptome sequencing was conducted on the prefrontal cortex of male C57Bl/6 mice subjected to 30 days of chronic social defeat stress (CSDS). Prior to tissue extraction, the mice were injected with either 2 µg/g dexamethasone or saline, resulting in four groups: CSDS+sal, CSDS+dex, control+sal, and control+dex. The study sought to identify genes regulated by GR within the differentially expressed genes (DEG) by analyzing five public GR ChIPseq experiments performed on rodent brain tissue. This endeavor aimed to elucidate the role of GR in the PFC stress response. GR binding sites that were situated –5k to +1k bp from tss were categorized as regulatory regions. The closest genes were then identified. For further analysis, 3023 genes recognized as GR-regulated by at least two studies were selected. Of these, 320 genes were demonstrated to be expressed in the PFC based on our RNAseq data. We found a significant increase in GR sites among PFC DEGs that responded to DEX treatment in both the control group (control+sal vs control+dex: OR=2.17, p 0.001) and CSDS (CSDS+sal vs CSDS+dex: OR=1.86, p 0.001). However, chronic stress alone did not result in enrichment of genes regulated by GR. Notably, genes that responded differently to DEX treatment in CSDS and control showed a higher OR value (dex*CSDS: OR=2.32, p 0.01). Common GR-target genes between DEX-con and DEX-csds exhibited the same expression change direction, except for the Sft2d2 gene, which encodes a vesicle transport protein. These genes are involved in PDZ domain binding (Fzd2, Mpp3), serine/threonine kinase activity (Rps6ka5, Akt2, Camkk1), and oxidoreductase activity (Prodh, Smox). GR-regulated genes specific to the CSDS group participate in cytokine production (e.g., Ltbp1, P2rx7, Dhx33, Hdac9, Bcl6, Lgr4, etc.) and modulate chemical synaptic transmission (e.g., Arc, Syt12, Cacng3, etc.), including components of the glutamatergic synapse (e.g., Magi2, Erc2, Dnm1, Clstn2, and Itgb1). Changes in expression of structural component genes, including those involved in membrane raft (Cavin1, Smpd2, and Slc2a1) and anchoring junction (B4galt1, Gjb6, Fzd4, and Limk1) genes, indicate the control group’s response to DEX treatment. A total of 14 genes showed differential regulation by GR in both CSDS and control groups. Among these genes are those involved in axon elongation (Link1, Rasgrf1), synaptic morphology (Clstn2), and vesicle endocytosis (Dnm1). Additionally, vital genes for axonal regeneration (Tubb3), neuroprotection (Hspb8), regulation of apoptosis (Bugalt1), and microglia activation (Cavin1) are included. In conclusion, we aimed to decipher the pathways of GR regulation triggered by social stress and DEX treatment in the PFC. Chronic stress resulted in alterations in GR regulatory networks in the PFC that impacted processes related to synapse function and the inflammatory response.

Regulation of the kiss2 promoter in yellowtail clownfish (Amphiprion clarkii) by cortisol via GRE-dependent GR pathway.

Kisspeptin plays a vital role in mediating the stress-induced reproductive regulation. Cortisol, known as a stress-related hormone, is involved in gonadal development and sexual differentiation by binding with glucocorticoid receptor (GR) to regulate the expression of kiss gene. In the present study, cortisol treatment in yellowtail clownfish (Amphiprion clarkii) showed that the expression of kiss (kiss1 and kiss2) and gr (gr1 and gr2) genes were increased significantly. We demonstrated that the yellowtail clownfish Kiss neurons co-express the glucocorticoid receptors in the telencephalon, mesencephalon, cerebellum, and hypothalamus. We further cloned the promoter of kiss2 gene in yellowtail clownfish and identified the presence of putative binding sites for glucocorticoid receptors, estrogen receptors, androgen receptors, progesterone receptors, AP1, and C/EBP. Applying transient transfection in HEK293T cells of the yellowtail clownfish kiss2 promoter, cortisol (dexamethasone) treatment was shown to enhance the promoter activities of the yellowtail clownfish kiss2 gene in the presence of GRs. Deletion analysis of kiss2 promoter indicated that cortisol-induced promoter activities were located between position −660 and −433 with GR1, and −912 and −775 with GR2, respectively. Finally, point mutation studies on the kiss2 promoter showed that cortisol-stimulated promoter activity was mediated by one GRE site located at position −573 in the presence of GR1 and by each GRE site located at position −883, −860, −851, and −843 in the presence of GR2. Results of the present study provide novel evidence that cortisol could regulate the transcription of kiss2 gene in the yellowtail clownfish via GRE-dependent GR pathway.

HNF4A modulates glucocorticoid action in the liver.

SummaryThe glucocorticoid receptor (GR) is a nuclear receptor critical to the regulation of energy metabolism and inflammation. The actions of GR are dependent on cell type and context. Here, we demonstrate the role of liver lineage-determining factor hepatocyte nuclear factor 4A (HNF4A) in defining liver specificity of GR action. In mouse liver, the HNF4A motif lies adjacent to the glucocorticoid response element (GRE) at GR binding sites within regions of open chromatin. In the absence of HNF4A, the liver GR cistrome is remodeled, with loss and gain of GR recruitment evident. Loss of chromatin accessibility at HNF4A-marked sites associates with loss of GR binding at weak GRE motifs. GR binding and chromatin accessibility are gained at sites characterized by strong GRE motifs, which show GR recruitment in non-liver tissues. The functional importance of these HNF4A-regulated GR sites is indicated by an altered transcriptional response to glucocorticoid treatment in the Hnf4a-null liver.

Glucocorticoid receptor number and affinity differ between peripheral blood mononuclear cells and granulocytes in domestic pigs

The aim of the present study was to characterize the number and affinity of glucocorticoid receptors (GR) in peripheral blood mononuclear cells (PBMC) and granulocytes of domestic pigs because glucocorticoid signaling is considered important for animal health and welfare. To investigate GR binding characteristics in intact porcine immune cells, blood samples of 6 castrated male pigs were collected via indwelling vein catheters. Porcine PBMC and granulocytes were isolated using two-layer density gradients, followed by radioligand binding assays to determine the number of GR sites per cell and the dissociation constant Kd as a measure for GR binding affinity. The present study revealed a greater number of GR sites per cell (P = 0.039) in PBMC (mean ± SEM: 1,953 ± 207 sites/cell) compared to granulocytes (1,561 ± 159 sites/cell) in domestic pigs. Furthermore, porcine PBMC had a higher GR binding affinity than porcine granulocytes (P = 0.003) as the dissociation constant Kd of PBMC (1.8 ± 0.2 nM) was lower than that of granulocytes (3.5 ± 0.4 nM). Our results point to differences in underlying mechanisms of glucocorticoid signaling in different porcine leukocyte populations.

NeuroD Factors Discriminate Mineralocorticoid From Glucocorticoid Receptor DNA Binding in the Male Rat Brain.

In the limbic brain, mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) both function as receptors for the naturally occurring glucocorticoids (corticosterone/cortisol) but mediate distinct effects on cellular physiology via transcriptional mechanisms. The transcriptional basis for specificity of these MR- vs GR-mediated effects is unknown. To address this conundrum, we have identified the extent of MR/GR DNA-binding selectivity in the rat hippocampus using chromatin immunoprecipitation followed by sequencing. We found 918 and 1450 nonoverlapping binding sites for MR and GR, respectively. Furthermore, 475 loci were co-occupied by MR and GR. De novo motif analysis resulted in a similar binding motif for both receptors at 100% of the target loci, which matched the known glucocorticoid response element (GRE). In addition, the Atoh/NeuroD consensus sequence was found in co-occurrence with all MR-specific binding sites but was absent for GR-specific or MR-GR overlapping sites. Basic helix-loop-helix family members Neurod1, Neurod2, and Neurod6 showed hippocampal expression and were hypothesized to bind the Atoh motif. Neurod2 was detected at rat hippocampal MR binding sites but not at GR-exclusive sites. All three NeuroD transcription factors acted as DNA-binding-dependent coactivators for both MR and GR in reporter assays in heterologous HEK293 cells, likely via indirect interactions with the receptors. In conclusion, a NeuroD family member binding to an additional motif near the GRE seems to drive specificity for MR over GR binding at hippocampal binding sites.

Regulation of the two kiss promoters in goldfish (Carassius auratus) by estrogen via different ERα pathways

Kisspeptin stimulates the synthesis and release of gonadotropin via controlling the secretion of gonadotropin releasing hormone in vertebrates. It also mediates the positive or negative feedback regulation of sex steroids on the hypothalamus–gonadotropic axis. In contrast to mammals, two paralogous genes of kisspeptin (kiss1 and kiss2) have been identified in several teleosts, implying the multiplicity of their physiological functions. In the present study, we cloned the promoters of kiss1 and kiss2 genes in goldfish (Carassius auratus), and identified the presence of putative binding sites for estrogen receptors, glucocorticoid receptors, Sp1, AP1, C/EBP and Oct-1. We further demonstrated that the goldfish Kiss neurons co-express the estrogen receptors, with era1 and erb1 in the habenula Kiss1 neurons and era1, era2 and erb1 in the preoptic and hypothalamic Kiss2 neurons. Using transient transfection in HEK293T cells of the two goldfish kiss gene promoters cloned upstream of a luciferase reporter, estrogen (E2, 17β-estradiol) treatment was shown to enhance the promoter activities of the two goldfish kiss genes in the presence of ERα. Deletion analysis of kiss1 promoter indicated that the E2-induced promoter activity was located between position −633 and −317 where no half ERE motifs were found. Point mutation studies on the kiss2 promoter indicated that the E2-stimulated promoter activity was mediated by a half ERE site located at position −57. Results of the present study provide evidence that E2 is capable of exerting positive feedback regulation on the expression of kiss1 and kiss2 in goldfish via ERE-independent or ERE-dependent ERα pathway, respectively.

Age, body mass index, and serum level of DHEA-S can predict glucocorticoid receptor function in women with polycystic ovary syndrome

Glucocorticoid receptor (GR) transduces the glucocorticoid (GC) signal that could lead to metabolic derangements depending on the tissue responsiveness to GC. We aimed to investigate possible causative relation of the GR functional properties in peripheral blood mononuclear cells of women with polycystic ovary syndrome (PCOS), with their clinical and biochemical characteristics. Thirty women with PCOS [mean age: 26.5 ± 5.1 years, mean body mass index (BMI) 24.5 ± 5 kg/m(2)], and thirty respective controls were analyzed for the number of GR sites per cell (B (max)), apparent equilibrium dissociation constant (K (d)), and binding potency (GR potency). A strong association between B (max) and K (d) (r = 0.70, P < 0.0001), and GR potency with age (r = 0.49, P = 0.009) was observed in PCOS women. The multiple regression analyses within the PCOS group revealed that independent predictors for K (d) were BMI, total cholesterol, and dehydroepiandrosterone-sulfate (DHEA-S) (r = 0.58, P = 0.038), while for GR potency (r = 0.687, P = 0.013) were age, BMI, DHEA-S, and basal cortisol concentration. The results suggest that PCOS pathophysiology may be related to alterations of a cross stalk between glucocorticoid signaling, age, and metabolic parameters. These findings should be further explored in studies on the role of GR in PCOS-related metabolic derangements.

Suppression of Peroxisome Proliferator-Activated Receptor γ-Coactivator-1α Normalizes the Glucolipotoxicity-Induced Decreased BETA2/NeuroD Gene Transcription and Improved Glucose Tolerance in Diabetic Rats

Peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1alpha) is significantly elevated in the islets of animal models of diabetes. However, the molecular mechanism has not been clarified. We investigated whether the suppression of PGC-1alpha expression protects against beta-cell dysfunction in vivo and determined the mechanism of action of PGC-1alpha in beta-cells. The studies were performed in glucolipotixicity-induced primary rat islets and INS-1 cells. In vitro and in vivo approaches using adenoviruses were used to evaluate the role of PGC-1alpha in glucolipotoxicity-associated beta-cell dysfunction. The expression of PGC-1alpha in cultured beta-cells increased gradually with glucolipotoxicity. The overexpression of PGC-1alpha also suppressed the expression of the insulin and beta-cell E-box transcription factor (BETA2/NeuroD) genes, which was reversed by PGC-1alpha small interfering RNA (siRNA). BETA2/NeuroD, p300-enhanced BETA2/NeuroD, and insulin transcriptional activities were significantly suppressed by Ad-PGC-1alpha but were rescued by Ad-siPGC-1alpha. PGC-1alpha binding at the glucocorticoid receptor site on the BETA2/NeuroD promoter increased in the presence of PGC-1alpha. Ad-siPGC-1alpha injection through the celiac arteries of 90% pancreatectomized diabetic rats improved their glucose tolerance and maintained their fasting insulin levels. The suppression of PGC-1alpha expression protects the glucolipotoxicity-induced beta-cell dysfunction in vivo and in vitro. A better understanding of the functions of molecules such as PGC-1alpha, which play key roles in intracellular fuel regulation, could herald a new era of the treatment of patients with type 2 diabetes mellitus by providing protection from glucolipotoxicity, which is an important cause of the development and progression of the disease.

Glucocorticoid Receptor Activation of the Ciz1-Lcn2 Locus by Long Range Interactions

The cellular response to glucocorticoid receptor (GR) activation involves a highly orchestrated series of regulatory actions influenced at multiple levels by a variety of mechanisms including the action of transcription factors and chromatin modifiers. Because the majority of GR binding sites (glucocorticoid-responsive elements (GREs)) are distant from promoters, it is likely that interactions at a distance play an important role in GR action. To determine whether long range chromosomal associations play a role in transcription regulation by GR, we utilized a chromosome conformation capture-based technique (associated chromosome trap) to identify unknown, remote sequences that interact with the GR-induced Lipocalin2 (Lcn2) gene. Our screen revealed that the Lcn2 GRE interacts with the Ciz1 gene, nearly 30 kb upstream. Ciz1 was subsequently found to be a novel GR-responsive gene. The GRE proximal to the Lcn2 promoter apparently functions to regulate both the Lcn2 gene and the distal Ciz1 gene. Using quantitative chromosome conformation capture, we find that a loop structure is organized between these two genes. This structure is hormone-independent and present only in cell types where the genes are active. The strong correlation between gene expression and loop structure in different cell lines suggests that high order interactions play a role in determining tissue-specific gene regulation.

Cloning and functional analysis of promoters of three GnRH genes in a cichlid

Mechanisms regulating gonadotropin-releasing hormone (GnRH) types, a key molecule for reproductive physiology, remain unclear. In the present study, we cloned the promoters of GnRH1, GnRH2, and GnRH3 genes in the tilapia, Oreochromis niloticus; and found putative binding sites for glucocorticoid receptors, Sp1, C/EBP, GATA, and Oct-1, but not for androgen receptors in all three GnRH promoters using computer analysis. The presence of binding sites for progesterone receptors in GnRH1, estrogen receptors in GnRH1 and GnRH2, and thyroid hormone receptors in GnRH1 and GnRH3 suggests direct action of steroid hormones on GnRH types. Our observation of SOX and LINE-like sequences exclusively in GnRH1, COUP in GnRH2, and retinoid X receptors in GnRH3 suggests their role in sexual differentiation, midbrain segmentation, and visual cue integration, respectively. Thus, the characteristic binding sites for nuclear receptors and transcription factors support the notion that each GnRH type is regulated differently and has distinct physiological roles.

Identification of the pregnancy hormone relaxin as glucocorticoid receptor agonist.

The insulin-like peptide relaxin is a central hormone of pregnancy, but it also produces anti-fibrotic, myocardial, renal, central-nervous, and vascular effects. Recently, two G protein-coupled receptors, LGR7 and LGR8, have been identified as relaxin receptors. Prompted by reports on immunoregulatory effects of relaxin, we investigated possible interactions with the human glucocorticoid receptor (GR). Relaxin blunted the endotoxin-induced production of inflammatory cytokines (IL-1, IL-6, TNF-alpha) by human macrophages--an effect that was suppressed by the GR antagonist RU-486. In three different cell lines, relaxin induced GR activation, nuclear translocation, and DNA binding as assessed in GRE-luciferase assays. Co-immunoprecipitation experiments revealed physical interaction of endogenous and exogenous relaxin with cytoplasmic and nuclear GR. Relaxin competed with GR agonists for GR binding, both in vivo in whole-cell assays, and in vitro in fluorescence polarization assays. Relaxin was shown to up-regulate GR protein expression as well as the number of functionally active GR sites. In LGR7/8-free cells, the relaxin-mediated activation of GR was preserved. In conclusion, relaxin acts as GR agonist--a pathway pivotal to its effects on cytokine secretion by human macrophages. These findings may deepen our understanding of relaxin's abundant physiological actions, as well as our insights into general principles of hormone signaling.

Characterization and functional significance of glucocorticoid receptors in patients with major depression: modulation by antidepressant treatment

Hyperactivity of the hypothalamic pituitary adrenal (HPA) axis in patients with major depression is one of the most consistent findings in biological psychiatry. Experimental data support the idea that glucocorticoid-mediated feedback via glucocorticoid receptors (GR) is impaired in major depression. The aim of the present work was to assess the putative changes in GR density of peripheral blood mononuclear cells (PBMCs) in a group of patients with major depression and to determine modulation of these GR sites by antidepressant treatment. In addition, susceptibility of PBMCs to glucocorticoid effects was also studied using a functional end-point analysis in vitro, such as cortisol inhibition of mitogen-induced lymphocyte proliferation. Cortisol levels were also measured before and after dexamethasone suppression test (DST). The results showed a decrease in GR density in depressed patients compared with healthy subjects, mainly in those patients that showed basal cortisol levels in the upper normal range and were refractory to DST. Regarding the functional significance of this variation, two representative groups emerged from our study: a) free-medication patients with GR function comparable to healthy controls, and b) patients showing diminished GR activity. These results suggest a lack of relationship between GR density and cortisol-induced inhibition of lymphocyte proliferation. Patients treated with different antidepressant drugs showed a marked increase in the number of GR sites per cell compared to non-treated. Interestingly, this increase was even higher than in normal subjects. Hence, restoration of GR density after an efficient antidepressant treatment could be an index of an effective modulatory action of drugs on GR expression and highlights the possibility that GR levels might be used as markers of a successful treatment.

Effects of the heterogeneous nuclear ribonucleoprotein U (hnRNP U/SAF-A) on glucocorticoid-dependent transcription in vivo

The glucocorticoid receptor (GR) is a ligand dependent transcription factor, which regulates the transcription of multiple hormone-dependent genes. The transcriptional regulation by GR takes place by interaction of GR with the basal transcription machinery and by recruiting glucocorticoid receptor interacting proteins (GRIPs). Previously we identified hnRNP U/SAF-A as a factor interfering with GR-dependent transcription by repressing glucocorticoid induced activation. To gain insight into the mechanisms that govern this interference, we have now investigated the transcription of GR-dependent reporter genes in Ltk − cells transiently transfected with a variety of hnRNP U constructs. We demonstrate that a hnRNP U construct lacking the GR-binding domain acts as a dominant negative factor that now enhances GR-driven transcription. In addition, hnRNP U repression of glucocorticoid induced transcription was found to be dependent on the amount of cotransfected GR, where a high amount of GR leads to ligand-inducible repression of GR-dependent reporter gene activity by hnRNP U, whereas low amounts of GR showed nearly no effect. The relative concentrations of GR, hnRNP U and DNA-binding sites for GR are important for the effect of hnRNP U on transcription, suggesting a model where hnRNP-U acts as a storage site for intranuclear GR.

Effect of Glucocorticoid Therapy on Glucocorticoid Receptors in Children with Autoimmune Diseases

Low-dose glucocorticoids (GC) achieve their action completely by classical genomic effects, mediated by the glucocorticoid receptor (GCR). In high doses of GC, nongenomic effects have also been found, but it is still unclear to what extent they contribute to a beneficial outcome. In this study, we present a determination of the number of lymphocyte GCR sites and the binding affinity in healthy children and children with autoimmune diseases. We further assess the effect of GC administration, especially of high-dose pulse therapy on the number of binding sites. The number of GCR sites per cell was analyzed with [(3)H]-dexamethasone radioligand binding assay and binding affinity (Kd given in nM) in peripheral blood mononuclear cells isolated from 48 healthy children and 35 patients. The patients were divided into three groups based on GC treatment: 0 mg/kg (group 1), 0.01-0.3 mg/kg orally (group 2), and 10-15 mg/kg i.v. pulse therapy (group 3) of prednisolone equivalent per day. Gender- and age-independent normal values of 4338 +/- 1687 sites/lymphocytes and Kd 6.7 +/- 2.2 nM were found. At 3463 +/- 1574, the number of receptor sites in patients without GC (group 1) was significantly lower than that of healthy volunteers (p < 0.05). In patients receiving GC treatment, this value was reduced to 2952 +/- 512 (group 2). Significant down-regulation to a minimum of 479 +/- 168 (group 3) was found after pulse therapy compared with untreated patients (p < 0.01). In pulse therapy, GC lead to a fast and dramatic receptor down-regulation. We suppose that the increase in therapeutic success of pulse-therapy may partly be mediated through additional nongenomic effects.

Somatostatin Increases Glucocorticoid Binding and Signaling in Macrophages by Blocking the Calpain-specific Cleavage of Hsp 90

Somatostatin has direct anti-inflammatory actions and participates in the anti-inflammatory actions of glucocorticoids, but the mechanisms underlying this regulation remain poorly understood. The objective of this study was to evaluate whether somatostatin increases glucocorticoid responsiveness by up-regulating glucocorticoid receptor (GR) expression and signaling. Somatostatin promoted a time- and dose-dependent increase in [(3)H]dexamethasone binding to RAW 264.7 macrophages. Cell exposure to 10 nM somatostatin for 18 h promoted a 2-fold increase in the number of GR sites per cell without significant modification of the affinity. Analysis of GR heterocomplex components demonstrated that somatostatin increased the level of heat shock protein (Hsp) 90, whereas the level of GR remained almost unchanged. The increase in Hsp 90 was associated with a decrease in the cleavage of its carboxyl-terminal domain. Evidence for the involvement of calpain inhibition in this process was obtained by the demonstration that 1) somatostatin induced a dose-dependent decrease in calpain activity and 2) calpain inhibitors, calpain inhibitor I and calpeptin, both abolished the cleavage of Hsp 90 and induced a dose-dependent increase in [(3)H]dexamethasone binding. Increases in glucocorticoid binding after somatostatin treatment were associated with similar increases in the ability of GR to transactivate a minimal promoter containing two glucocorticoid response elements (GRE) and to interfere with the activation of nuclear factor-kappaB (NF-kappaB). Thus, the present findings indicate that somatostatin increases glucocorticoid binding and signaling by limiting the calpain-specific cleavage of GR-associated Hsp 90. This mechanism may represent a novel target for intervention to increase glucocorticoid responsiveness.

Structure and hormone responsiveness of the gene encoding the alpha-subunit of the rat amiloride-sensitive epithelial sodium channel.

The rat amiloride-sensitive epithelial sodium channel (rENaC) is the rate-limiting step for vectorial transport of Na+ across tight epithelia. The complex is composed of three subunits, alpha, beta, and gamma. Expression of the subunits has been shown to be tissue-specific and developmentally and hormonally regulated. To study mechanisms involved in transcriptional regulation of alpharENaC, we determined the genomic organization of the alpharENaC gene. By 5' rapid amplification of cDNA ends and primer extension, two transcriptional start sites were detected 453 base pairs (bp) apart, resulting in alternative 5' untranslated region (UTR) lengths of 515 or 62 bp. The longer 5' UTR is more prevalent in fetal lung than in adult lung or kidney. The 5' untranslated and coding regions are contained within 12 exons, with the translation start site located within the first exon. Sequence analysis of approximately 1,500 bp of 5' flanking DNA identified putative binding sites for transcription factors PEA3, SP1, AP-1, nuclear factor-kappaB, and thyroid and glucocorticoid receptors. alpharENaC promoter-reporter gene constructs produced low levels of reporter gene activity in transiently transfected cells, which could be increased by dexamethasone (DEX) treatment. Tri-iodothyronine treatment alone had no effect but potentiated stimulation by DEX.

Effect of Serotonin Inhibition on Glucocorticoid and Mineralocorticoid Expression in Various Brain Structures

Many studies have shown the existence of functional interactions between central neurotransmitter systems and the hypothalamo-pituitary adrenal axis. Mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) are regulated by multiple factors including glucocorticoids themselves. Neurotransmitters such as serotonin (5-hydroxytryptamine: 5-HT) can regulate brain corticosteroid receptors in a complex way. The present study examined the short-term (48 h) effects of parachlorophenylalanine (PCPA), a drug which specifically inhibits 5-HT synthesis, on corticosteroid receptor levels and on the expression of their respective messenger ribonucleic acids (mRNA) in the rat hippocampus, hypothalamus and brain stem. The study was performed in bilaterally adrenalectomized animals, in order to avoid potential drug-induced changes in plasma corticosterone levels, which could secondarily regulate MR and GR. Short-term inhibition of 5-HT synthesis by PCPA significantly increased the number of hippocampal MR-binding sites. PCPA treatment did not alter the number of GR-binding sites in the hippocampus, hypothalamus and brain stem. We observed no change in the affinities of GR and MR sites in all the structures studied. In PCPA-treated rats, restoration of control 5-HT levels by injection of its immediate precursor, 5-hydroxytryptophan (5-HTP) brings the number of hippocampal MR-binding sites back to control levels. It can therefore be concluded that the increase in number of MR-binding sites induced by acute PCPA treatment is dependent on the decrease in 5-HT levels. The increase in hippocampal MR binding sites was correlated with an induction of their messengers, suggesting that 5-HT modulates the synthesis of MR protein. Although PCPA did not modify the number of hippocampal GR-binding sites, a decrease in hippocampal GR mRNA expression was observed. This study shows that 5-HT inhibits hippocampal mineralocorticoid receptor synthesis and that this effect is not mediated by changes in corticosterone hormone secretion, and illustrates the existence of complex mechanisms for corticosteroid receptor regulation in the hippocampus.

Continuous blockade of brain glucocorticoid receptors facilitates spatial learning and memory in rats

Previously, a corticosterone surge associated with a learning task was shown to facilitate cognitive processes through brain glucocorticoid receptors (GR) while chronic overexposure to this stress hormone impaired cognition. In the present study we tested the hypothesis that opposing effects on learning and memory might also occur after either phasic or continuous blockade of brain GR by intracerebroventricular (i.c.v.) administration of the GR antagonist RU38486 (aGR). We used a Morris water maze procedure to assess spatial learning and memory abilities in male Wistar rats. The effect of phasic brain GR blockade was studied following daily pretraining administration of 10 and 100 ng/microL aGR i.c.v. on 3 consecutive days. This repetitive aGR treatment impaired spatial learning and memory dose-dependently in comparison with vehicle controls. For continuous brain GR blockade, animals received an i.c.v., infusion of aGR (10 and 100 ng/0.5 microL per h or vehicle) over 10 days. Infusion of 100 ng aGR per hour resulted in a long-lasting facilitation of spatial performance. The 10 ng aGR infusion also caused initially a facilitating effect, which was, however, transient and performance became impaired during retest. Possible anxiolytic properties of the drugs were excluded in view of the animals' behaviour in the elevated plus maze. Both doses of aGR infusion reduced the number of mineralocorticoid receptors in the hippocampus, but only the high dose of aGR resulted in a significant reduction of available GR sites. In conclusion, continuous administration of GR antagonist improves cognitive function, while phasic blockade of brain GR function causes a cognitive deficit.

Mechanism of Interaction between the Glucocorticoid Receptor and Stat5: Role of DNA-Binding

The functional interaction between the glucocorticoid receptor (GR) and the signal transducer and activator of transcription-5 (Stat5) was investigated by studying the synergistic activation of β-casein gene transcription by prolactin and glucocorticoids. The synergism was shown to be mediated by a complex hormone response region with multiple binding sites for Stat5, the glucocorticoid receptor, and CCAAT/enhancer binding proteins (C/EBP). HC11 mammary epithelial cells, which contain physiological levels of GR and Stat5, and COS-7 cells overexpressing GR and Stat5 were employed. In both cell types intact binding sites for Stat5 and the GR were a prerequisite for the synergism, whereas C/EBP sites were only required in HC11 cells. Interestingly, the GR sites employed for the synergism were non-classical, half palindromic sites, which did not function in the absence of activated Stat5 to mediate the action of the GR on transcription. The interaction of GR and Stat5 triggered by the unusual configuration of binding sites appears to represent a novel mechanism by which these two distinct types of transcription factors cooperate. The mode of interaction provides an efficient means to restrict gene expression to conditions where both Stat5 and the GR are activated.

17 alpha (haloacetamidoalkyl) estradiols alkylate the human estrogen receptor at cysteine residues 417 and 530.

Results obtained in a previous study suggested that cysteine residues in the estrogen receptor were covalent attachment sites for four 17 alpha-(haloacetamidoalkyl) estradiols (halo, bromo or iodo; alkyl, methyl, ethyl, or propyl). To identify the putative concerned cysteines, we expressed wild-type and various cysteine --> alanine mutants of the human estrogen receptor in COS cells and determined their ability to be alkylated by the four electrophiles. The quadruple mutant, in which all the cysteines (residues 381, 417, 447, and 530) of the hormone-binding site were changed to alanines, showed very little electrophile labeling, whereas the four single mutants (C381A, C417A, C447A, and C530A) were alkylated as efficiently as the wild-type receptor. These results (i) demonstrate that cysteine residues were covalent attachment sites of electrophiles and (ii) indicate that more than one cysteine residue could be alkylated. Analysis of three double mutants (C381A/C530A, C417A/C530A, and C447A/C530A) provided strong evidence that only C417 and C530 were sites for electrophile covalent attachment. Since C530 was also alkylated by tamoxifen aziridine, a nonsteroidal affinity-labeling agent, we propose a selective mode of superimposition of tamoxifen-class antiestrogens with estradiol, which could account for the relative positioning of the two types of ligands in the receptor hormone-binding pocket. According to the structure of the hormone-binding pocket of nuclear receptors, as inferred from crystallographic studies and general sequence alignment of hormone-binding domains, C417 and C530 appear to be (1) located at the extreme border or in structural elements involved in delineation of the hormone-binding pocket, (2) spatially in close proximity to each other, and (3) in positions highly homologous to those of glucocorticoid receptor sites alkylated by affinity- and photoaffinity-labeling agents, respectively.