Cytoplasmic Glucocorticoid Receptor Research Articles

α1-Antitrypsin Binds to the Glucocorticoid Receptor with Anti-Inflammatory and Antimycobacterial Significance in Macrophages.

α1-Antitrypsin (AAT), a serine protease inhibitor, is the third most abundant protein in plasma. Although the best-known function of AAT is irreversible inhibition of elastase, AAT is an acute-phase reactant and is increasingly recognized to have a panoply of other functions, including as an anti-inflammatory mediator and a host-protective molecule against various pathogens. Although a canonical receptor for AAT has not been identified, AAT can be internalized into the cytoplasm and is known to affect gene regulation. Because AAT has anti-inflammatory properties, we examined whether AAT binds the cytoplasmic glucocorticoid receptor (GR) in human macrophages. We report the finding that AAT binds to GR using several approaches, including coimmunoprecipitation, mass spectrometry, and microscale thermophoresis. We also performed in silico molecular modeling and found that binding between AAT and GR has a plausible stereochemical basis. The significance of this interaction in macrophages is evinced by AAT inhibition of LPS-induced NF-κB activation and IL-8 production as well as AAT induction of angiopoietin-like 4 protein, which are, in part, dependent on GR. Furthermore, this AAT-GR interaction contributes to a host-protective role against mycobacteria in macrophages. In summary, this study identifies a new mechanism for the gene regulation, anti-inflammatory, and host-defense properties of AAT.

Topical therapy of various dermatoses with ImpoyzTM (clobetasol propionate) cream 0.025%

The most widely prescribed drugs for the treatment of a variety of dermatoses are Topical corticosteroids (TC). These medications are approved for the treatment of inflammatory and pruritic manifestations of dermatologic disorders due to their powerful symptom-relieving impact. Clobetasol propionate (CP) is the most popular (TC) used to relieve itching, redness, and oedema caused by a variety of skin disorders. Anti-inflammatory, anti-pruritic, and vasoconstrictive characteristics are all present in it. CP works by binding to cytoplasmic glucocorticoid receptors and activating glucocorticoid receptor-mediated gene expression, resulting in the production of anti-inflammatory proteins while suppressing the production of inflammatory mediators. The formulation is free from known contact allergens, such as propylene glycol, short-chain alcohols, and sorbitol-based emulsifiers, and has demonstrated hypoallergenic effects. The efficacy, safety, and clinical experience of utilizing CP 0.025% cream for the treatment of various dermatologic disorders are discussed in this case series.

Glucocorticoid gene signatures in systemic lupus erythematosus and the effects of type I interferon: a cross-sectional and in-vitro study.

Glucocorticoids, used as a therapy in systemic lupus erythematosus (SLE), interact with the cytoplasmic glucocorticoid receptor to modulate gene transcription. Minimising the use of glucocorticoids is a goal in SLE; however, pharmacological measures to support clinical guidelines are scarce. We evaluated glucocorticoid-regulated genes for their potential use as biomarkers of glucocorticoid exposure in SLE. We examined interactions between changes in gene expression that are induced by glucocorticoids and type I interferon. Genes regulated by glucocorticoids and type I interferon were analysed in relation to glucocorticoid exposure in adult patients meeting the American College of Rheumatology criteria for SLE from three cross-sectional cohorts: a local cohort from a tertiary hospital in Melbourne, VIC, Australia, and two public datasets (GSE49454, Hospital de la Conception, Marseille, France, and GSE88884, patients enrolled in a large, multicentre clinical trial). RNA sequencing was done using RNA from healthy donor leucocytes treated with the glucocorticoid dexamethasone, or type I interferon, or both. Glucocorticoid-regulated genes were analysed in a local SLE cohort (n=18) and public dataset GSE49454 (n=62). Five genes correlated with glucocorticoid dose in both cohorts and were combined to make a glucocorticoid gene signature. Validity of the glucocorticoid gene signature was tested in the public dataset GSE88884 (n=1756). A dose-dependent association was observed with glucocorticoid dose (p<0·0001), and the glucocorticoid gene signature had moderate ability to identify patients taking high-dose glucocorticoid (area under the curve [AUC]=0·77) although was less discriminatory when including all doses (AUC=0·69). We saw no effect of glucocorticoid dose on type I interferon -regulated gene expression. Patients with a high type I interferon gene signature had reduced glucocorticoid gene signature expression compared with patients with a low type I interferon gene signature matched for glucocorticoid dose, suggesting type I interferon inhibits glucocorticoid-stimulated gene expression. In RNA sequencing experiments, type I interferon impaired the expression of glucocorticoid-induced genes, whereas dexamethasone had minimal effect on the expression of type I interferon-stimulated genes. We identified genes regulated by dexamethasone but not affected by type I interferon; combined signatures using these genes also showed moderate ability to distinguish patients taking glucocorticoids. A gene signature for glucocorticoid exposure was identified, but the substantial effect of type I interferon on glucocorticoid-induced genes might limit its application in SLE. These data confirm the insensitivity of type I interferon-regulated genes to glucocorticoids, and together support the concept that type I interferon has a role in glucocorticoid resistance in SLE. Lupus Research Alliance and Australian National Health and Medical Research Council.

Tumor suppressor REIC/Dkk-3 and its interacting protein SGTA inhibit glucocorticoid receptor to nuclear transport.

REIC/Dkk-3 is a tumor suppressor, and its expression is significantly downregulated in a variety of human cancer types. A previous study performed yeast two-hybrid screening and identified the small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), known as a negative modulator of cytoplasmic androgen receptor (AR) signaling, which is a novel interacting partner of REIC/Dkk-3. The previous study also indicated that the REIC/Dkk-3 protein interferes with the dimerization of SGTA and then upregulates the AR transport and signaling in human prostate cancer PC3 cells. Since the transport of some steroid receptors to nucleus is conducted similarly by dynein motor-dependent way, the current study aimed to investigate the role of SGTA and REIC/Dkk-3 in the transport of other glucocorticoid receptors (GR). In vitro reporter assays for the cytoplasmic GR transport were performed in human prostate cancer PC3 cells and 293T cells. As for the SGTA protein, a suppressive effect on the GR transport to the nucleus was observed in the cells. As for the REIC/Dkk-3 protein, an inhibitory effect was observed for the GR transport in PC3 cells. Under the depleted condition of SGTA by short-hairpin (sh)RNA, the downregulation of GR transport by REIC/Dkk-3 was significantly enhanced compared with the non-depleted condition in PC3 cells, suggesting a compensatory role of REIC/Dkk-3 in the SGTA mediated inhibition of GR transport. The current study therefore demonstrated that SGTA inhibited the cytoplasmic transport of GR in 293T and PC3 cells, and REIC/Dkk-3 also inhibited the cytoplasmic transport of GR in PC3 cells. These results may be used to gain novel insight into the GR transport and signaling in normal and cancer cells.

Functional characterization of canine wild type glucocorticoid receptor and an insertional mutation in a dog

BackgroundGlucocorticoids, among the most widely utilized drugs in veterinary medicine, are employed to treat a wide variety of diseases; however, their use often induces adverse events in dogs. The efficacy of glucocorticoids usually depends on dosage, although differences in sensitivity to glucocorticoids in individual animals have been reported. Glucocorticoids bind to the cytoplasmic glucocorticoid receptor (GR), which is expressed in almost all cells. These receptors are key factors in determining individual sensitivity to glucocorticoids. This study examined individual differences in glucocorticoid sensitivity in dogs, focusing on reactivity of the GR to prednisolone.ResultsWe first molecularly cloned the GR gene from a healthy dog. We discovered a mutant GR in a dog suspected to have iatrogenic Cushing syndrome. The mutant GR had extra nucleotides between exons 6 and 7, resulting in a truncated form of GR that was 98 amino acids shorter than the wild-type dog GR. The truncated GR exhibited very low reactivity to prednisolone, irrespective of concentration.ConclusionsWe have identified the truncated form of canine GR in a dog with iatrogenic Cushing syndrome. This truncated form showed the very less sensitivity to glucocorticoid in vitro, unfortunately, we could not elucidate its clinical significance. However, our data is a first report about the function of canine GR, and will facilitate the analysis of canine glucocorticoid sensitivity.

Cochlear Glucocorticoid Receptor and Serum Corticosterone Expression in a Rodent Model of Noise-induced Hearing Loss: Comparison of Timing of Dexamethasone Administration

Glucocorticoid (GC) is a steroid hormone secreted from the adrenal cortex in response to stress, which acts by binding to cytoplasmic glucocorticoid receptors (GRs). Dexamethasone (DEX) is a synthetic GC exhibiting immunosuppressive effects in both human and rodent models of hearing loss. While clinical evidence has shown the effectiveness of DEX for treatment of various inner ear diseases, its mechanisms of action and the optimal timing of treatment are not well understood. In the present study, intergroup comparisons were conducted based on the time point of treatment with DEX: (1) pretreatment; (2) posttreatment; and (3) pre&post-noise. The pre&post DEX treatment group showed a significant improvement in threshold shift at 1 day post-noise exposure as compared to the TTS (transient threshold shift)-only group at 8 and 16 kHz. Both TTS and PTS (permanent threshold shift) significantly reduced cochlear GR mRNA expression and increased serum corticosterone and cochlear inflammatory cytokines. The pre&post DEX treatment group showed a significant decrease in serum corticosterone level as compared to other DEX treatment groups and TTS-treated group at 3 days after acoustic trauma. Our results suggest that the timing of DEX administration differentially modulates systemic steroid levels, GR expression and cochlear cytokine expression.

Respiratory Syncytial Virus Nonstructural Protein 1 Blocks Glucocorticoid Receptor Nuclear Translocation by Targeting IPO13 and May Account for Glucocorticoid Insensitivity.

Despite their powerful antiinflammatory effect, glucocorticoids have shown no significant clinical benefit in respiratory syncytial virus (RSV)-induced bronchiolitis, the reason for which remains unclear. Upon glucocorticoid binding, the cytoplasmic glucocorticoid receptor (GR) translocates to the nucleus with the help of importin 13 (IPO13). Here, we report that RSV infection reduced GR nuclear translocation in nasopharyngeal aspirates from RSV-infected infants, lungs of infected mice, and A549 cells, which coincided with decreased IPO13 expression. This led to repression of GR-induced antiinflammatory genes, such that dexamethasone failed to suppress airway inflammation and airway hyperresponsiveness in the infected mice. The anti-GR effect of RSV was mediated by viral nonstructural protein 1 , which likely functioned by competing with IPO13 for GR binding. Our findings provide a mechanism for the ineffectiveness of glucocorticoids in RSV-related disease and highlight the potential to target the IPO13-GR axis as a treatment for multiple glucocorticoid-related diseases.

Cross-talk between the glucocorticoid receptor and MyoD family inhibitor domain-containing protein provides a new mechanism for generating tissue-specific responses to glucocorticoids

Glucocorticoids are primary stress hormones that regulate many physiological processes, and synthetic derivatives of these molecules are widely used in the clinic. The molecular factors that govern tissue specificity of glucocorticoids, however, are poorly understood. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR). To discover new proteins that interact with GR and modulate its function, we performed a yeast two-hybrid assay. The MyoD family inhibitor domain-containing protein (MDFIC) was identified as a binding partner for GR. MDFIC associated with GR in the cytoplasm of cells, and treatment with glucocorticoids resulted in the dissociation of the GR-MDFIC complex. To investigate the function of the GR-MDFIC interaction, we performed a genome-wide microarray in intact and MDFIC-deficient A549 cells that were treated with glucocorticoids. A large cohort of genes was differentially regulated by GR depending on the presence or absence of MDFIC. These gene changes were strongly associated with inflammation, and glucocorticoid regulation of the inflammatory response was altered in MDFIC-deficient cells. At a molecular level, the interaction of MDFIC with GR altered the phosphorylation status of the receptor. We demonstrate in COS-1 cells that changes in receptor phosphorylation underlie the ability of MDFIC to regulate the transcriptional activity of GR. Finally, we show that GR directly represses the MDFIC gene, revealing a negative feedback loop by which glucocorticoids limit MDFIC activity. These findings identify a new binding partner for cytoplasmic GR that modulates the receptor transcriptome and contributes to the tissue-specific actions of glucocorticoids.

The Carboxy-Terminal Domain of Calreticulin (CALR) Exports the Glucocorticoid Receptor (GR) from the Nucleus to the Cytoplasm of Human Erythroid Cells Resetting Their Stress Response

Steady state erythropoiesis is regulated by interaction of EPO with its receptor, EPO-R which activates Ca2+signaling and terminal maturation [Miller, Blood 1989;73:1188]. Under stress, GR switches EPO-R signaling to a proliferation mode allowing erythroblast (Ery) amplification [Zhang, Nature 2013;499:92]. However, for recovery from anemia to occur, a mechanism, still to be identified, must counteract GR and switch EPO-R signaling back to a maturation mode.CALR chaperones other proteins to their active sites[Michalak, Biochem J 1999;344: 281]. By importing GR from the nucleus, CALR resets the stress response of murine fibroblasts. This function is regulated by the exposure of its C-terminal domain (C-CALR) sustained by Ca2+ [Holaska, Mol Cell Biol 2002;22:6286]. Whether CALR regulates nuclear import of GR in human Ery and how this regulation is modulated by Ca2+and EPO is unknown. Studies were performed to fill this gap.By WB, human Ery expanded in vitro from adult blood expressed robust levels of CALR, detectable with N-CALR- and C-CALR antibodies, and of GR. By flow cytometry, N-CALR and C-CALR were detectable on the surface of cells from the proerythroblast (proEry) to polychromatic stage. Expression of N-CALR, the epitope most exposed on the surface, was up-regulated by EPO. By confocal microscopy, both N-CALR and C-CALR were detected in non-permeabilized Ery but only C-CALR was detectable in cells permeabilized to reveal their cytoplasmic content. Intra-cytoplasmic C-CALR staining was robust in proEry, barely detectable in orthochromatic cells and enhanced by adding Ca2+ to the permeabilization buffer. Double staining of C-CALR with GR or LaminB1 (an inner nuclear membrane protein) indicated that C-CALR/GR were associated in the perinuclear area of the cytoplasm but that C-CALR remained distinct from Lamin B1, suggesting that C-CALR/GR co-localize at the cytoplasm/nuclear boundary. These results suggest that C-CALR mediates nuclear export of GR and that this function is regulated by Ca2+.This hypothesis was tested first by determining content and localization of GRα and C-CALR in nuclear and cytoplasmic fractions of Ery treated with Dex, EPO and SCF by WB. Although none of these stimulations altered the total content of these proteins, some of them affected their compartmentalization: Dex rapidly (15’) induced GR S211 phosphorylation and increased the levels of GRα in nuclear fractions. These effects were antagonized by addition of the GR inhibitor RU486. The nuclear levels of GRα were modestly increased also by SCF but strongly decreased by EPO. By contrast, regardless of the stimuli, C-CALR was detected mostly in cytoplasmic fractions.The effects of these stimulations were also assessed by confocal microscopy. Exposure to Dex and EPO increased by 15' but decreased by 4h the nuclear localization of GR and significantly increased the levels of C-CALR and of C-CALR/GR co-localization detected in the cytoplasm by 4h, suggesting that C-CALR drives nuclear export of GR. In agreement with this hypothesis, multi-regression analyses comparing single C-CALR and total and cytoplasmic GR signals as independent parameters against merged signals as dependent parameter in 410 individual proEry indicated that the levels of C-CALR/GR co-localization directly correlated with the cytoplasmic levels of both C-CALR and GR but that the greatest p-values were observed for C-CALR (p=0.0000 vs 0.045). Moreover, treatment with the nuclear export inhibitor Leptomycin greatly decreased C-CALR/GR co-localization, increased nuclear localization of GR in proEry, both in untreated and EPO-treated cells, and reduced Ery proliferation in response to Dex down to levels sustained by Dex with RU486.To assess the role of Ca2+ in the nuclear export activity of C-CALR confocal microscopy and proliferation studies on Ery exposed or not to the Ca2+ chelator BAPTA in combination with Ionomycin (Io, a raiser of intracellular Ca2+) or Thapsigargin (TG, a reducer of Ca2+in endoplasmic reticulum) were performed. These treatments greatly decreased C-CALR and C-CALR/GR co-localization in the cytoplasm, increased GR signals in nuclei and reduced the proliferation of Ery in response to Dex.These results suggest that conformation changes of C-CALR induced by Ca2+signaling downstream to EPO-R sustain nuclear export of GR, resetting the stress-response of normal proEry allowing them to undergo terminal maturation. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cortisol Induces Reactive Oxygen Species Through a Membrane Glucocorticoid Receptor in Rainbow Trout Myotubes.

Cortisol is an essential regulator of neuroendocrine stress responses in teleosts. Cortisol predominantly affects target tissues through the genomic pathway, which involves interacting with cytoplasmic glucocorticoid receptors, and thereby, modulating stress-response gene expressions. Cortisol also produces rapid effects via non-genomic pathways, which do not involve gene transcription. Although cortisol-mediated genomic pathways are well documented in teleosts, non-genomic pathways are not fully understood. Moreover, no studies have focused on the contribution of non-genomic cortisol pathways in compensatory stress responses in fish. In this study, rainbow trout (Oncorhynchus mykiss) skeletal myotubes were stimulated with physiological concentrations of cortisol and cortisol-BSA, a membrane-impermeable agent, resulting in an early induction of reactive oxygen species (ROS). This production was not suppressed by transcription or translation inhibitors, suggesting non-genomic pathway involvement. Moreover, myotube preincubation with RU486 and NAC completely suppressed cortisol- and cortisol-BSA-induced ROS production. Subcellular fractionation analysis revealed the presence of cell membrane glucocorticoid receptors. Finally, cortisol-BSA induced a significant increase in ERK1/2 and CREB phosphorylation, as well as in CREB-dependent transcriptional activation of the pgc1a gene expression. The obtained results strongly suggest that cortisol acts through a non-genomic glucocorticoid receptor-mediated pathway to induce ROS production and contribute to ERK/CREB/PGC1-α signaling pathway activation as stress compensation mechanisms. J. Cell. Biochem. 118: 718-725, 2017. © 2016 Wiley Periodicals, Inc.

Glucocorticoid-independent modulation of GR activity: Implications for immunotherapy.

Pharmacological doses of glucocorticoids (GCs), acting via the glucocorticoid receptor (GR) to repress inflammation and immune function, remain the most effective therapy in the treatment of inflammatory and immune diseases. Since many patients on GC therapy exhibit GC resistance and severe side-effects, much research is focused on developing more selective GCs and combination therapies, with greater anti-inflammatory potency. GCs mediate their classical genomic transcriptional effects by binding to the cytoplasmic GR, followed by nuclear translocation and modulation of transcription of target genes by direct DNA binding of the GR or its tethering to other transcription factors. Recent evidence suggests, however, that the responses mediated by the GR are much more complex and involve multiple parallel mechanisms integrating simultaneous signals from other receptors, both in the absence and presence of GCs, to shift the sensitivity of a target cell to GCs. The level of cellular stress, immune activation status, or the cell cycle phase may be crucial for determining GC sensitivity and GC responsiveness as well as subcellular localization of the GR and GR levels. Central to the development of new drugs that target GR signaling alone or as add-on therapies, is an in-depth understanding of the molecular mechanisms of GC-independent GR desensitization, priming and activation of the unliganded GR, as well as synergy and cross-talk with other signaling pathways. This review will discuss the information currently available on these topics and their relevance to immunotherapy, as well as identify unanswered questions and future areas of research.

11. The Role of Glucocorticoid Receptor Signaling in Adeno-Associated Virus 2 Infection

We previously observed that purified recombinant glucocorticoid receptor (GR) protein specifically binds to the D-sequence within the inverted terminal repeat of the AAV2 genome because the D-sequence shares partial homology to the consensus half-site of the glucocorticoid receptor-binding element (GRE). The half-site is an essential core element, which has been reported to be sufficient to mediate GR signaling. Substitution of the D-sequence with the authentic GRE further increased AAV2 vector-mediated transgene expression. Based on these observations, we systemically examined whether AAV2 infection involves the GR signaling pathway. Our results showed that following infection by wild-type (WT) AAV2, or transduction with recombinant AAV2 vectors in vitro, GR was activated. The serine phosphorylated form of GR in whole cell lysates was significantly increased in a dose-dependent manner 18 hours post viral infection/vector transduction. Translocation and accumulation of GR in the nucleus was also increased correspondingly. To further corroborate our findings in an in vivo model, C57BL/6 mice were injected via tail-vein with either WT-AAV2 (1×1012 vgs/mouse) or AAV2-EGFP vectors (5×1011 vgs/mouse) and sacrificed 16 hours post virus/vector administration. A significant increase in the serine phosphorylated form of GR was also observed in liver tissues. We also observed that in cell cultures, dexamethasone, a well-known activator of GR, significantly enhanced rAAV2 vector-mediated transgene expression in various GR-positive cell lines, but not in a GR-deficient human osteosarcoma cell line, U2OS. Furthermore, stable transfection of U2OS cells with a GR expression plasmid not only facilitated AAV2 vector transduction, but also restored the ability of dexamethasone to further increase AAV2 vector-mediated transgene expression. Together with our previous data, we propose a model for elucidating the role of glucocorticoid receptor signaling pathway in AAV2 infection/AAV2 vector transduction (Fig. 1Fig. 1). Upon viral transduction or stimulation with glucocorticoids, the cytoplasmic GR is phosphorylated and translocated into the nucleus. Following AAV2 entry into the nucleus, the viral genome is released. Upon viral second-strand DNA synthesis or annealing of the complementary DNA strands, the activated GR binds to the double-stranded D-sequence in the viral inverted terminal repeats and consequently, enhances transgene expression. The activated GR also regulates its downstream pathways, such as NF-kB, which we have previously shown to have a substantial effect on AAV2 vector-mediated transgene expression (Proc Natl Acad Sci USA, 108:3743-3738, 2011). A better understanding of the complex interaction between GR and NF-kB signaling pathways notwithstanding, the availability of the fully functional GRE site-substituted novel AAV vectors to achieve high-efficiency transgene expression has implications in the potential use of these vectors in human gene therapy.View Large Image | Download PowerPoint Slide

Chronic Psychosocial Stress and Negative Feedback Inhibition: Enhanced Hippocampal Glucocorticoid Signaling despite Lower Cytoplasmic GR Expression.

Chronic subordinate colony housing (CSC), a pre-clinically validated mouse model for chronic psychosocial stress, results in increased basal and acute stress-induced plasma adrenocorticotropic hormone (ACTH) levels. We assessed CSC effects on hippocampal glucocorticoid (GC) receptor (GR), mineralocorticoid receptor (MR), and FK506 binding protein (FKBP51) expression, acute heterotypic stressor-induced GR translocation, as well as GC effects on gene expression and cell viability in isolated hippocampal cells. CSC mice showed decreased GR mRNA and cytoplasmic protein levels compared with single-housed control (SHC) mice. Basal and acute stress-induced nuclear GR protein expression were comparable between CSC and SHC mice, as were MR and FKBP51 mRNA and/or cytoplasmic protein levels. In vitro the effect of corticosterone (CORT) on hippocampal cell viability and gene transcription was more pronounced in CSC versus SHC mice. In summary, CSC mice show an, if at all, increased hippocampal GC signaling capacity despite lower cytoplasmic GR protein expression, making negative feedback deficits in the hippocampus unlikely to contribute to the increased ACTH drive following CSC.

The contribution of hypothalamic neuroendocrine, neuroplastic and neuroinflammatory processes to lipopolysaccharide-induced depressive-like behaviour in female and male rats: Involvement of glucocorticoid receptor and C/EBP-β

Peripheral inflammation induced by lipopolysaccharide (LPS) causes behavioural changes indicative for depression. The possible mechanisms involve the interference with neuroinflammatory, neuroendocrine, and neurotrophic processes. Apart from heterogeneity in the molecular background, sexual context may be another factor relevant to the manifestation of mood disturbances upon an immune challenge. We investigated sex-dependent effects of a 7-day LPS treatment of adult Wistar rats on depressive-like behaviour and their relation with hypothalamic neuroendocrine factor, corticotrophin-releasing hormone (CRH), proplastic brain-derived neurotropic factor (BDNF), pro-inflammatory cyclooxygenase-2 (COX-2) and nuclear factor kappa beta (NFkB). Also, their regulators, the glucocorticoid receptor (GR) and CCAAT enhancer-binding protein (C/EBP) β were followed.LPS induced depressive-like behaviour in females was associated with the increased hypothalamic CRH and decreased BDNF, but not with COX-2. These changes were paralleled by an increase in nuclear GR, NFkB and 20kDa C/EBPβ. LPS also altered behaviour in males and increased CRH expression, but in contrast to females, this was accompanied with the elevated COX-2, accumulation of cytosolic GR and elevated nuclear 38kDa C/EBPβ and NFkB. In conclusion, depressive-like phenotype induced by LPS in both sexes emerges from similar HPA axis activation and sex-specific alterations of hypothalamic molecular signalling: in males it is related to compromised control of neuroinflamation connected with cytoplasmic GR retention, while in females it is related to diminished proplastic capacity of BDNF. Sex-dependent mechanisms by which inflammation alters hypothalamic processes and cause pathological behaviour in animals, could be operative in the treatment of depression-related brain inflammation.

The JAK2V617F mutation disrupts the regulated association between calreticulin and the glucocorticoid receptor observed in normal erythroid cells

Calreticulin (CALR) is a multifunctional protein normally found within the lumen of the endoplasmic reticulum that mediates the cellular response to Ca2+ by chaperoning other proteins to their acting sites. Somatic loss-of-function mutations in the CALR gene were recently discovered in 70% of patients with the Philadelphia-negative myeloproliferative neoplasm (MPN) primary myelofibrosis (PMF) who did not harbor gain-of-function mutations of JAK21,2. Nevertheless, the JAK2 pathway is constitutively activated also in patients carrying CALR mutation and treatments with JAK2 inhibitors are effective not only in MPN patients (PMF and polycythemia vera, PV) harboring JAK2 mutations but also in PMF patients harboring mutations in CALR3. We have previously reported that erythroid cells from PV and PMF patients express abnormal activity of the glucocorticoid receptor (GR), a nuclear receptor whose transcriptional activity plays an important role in the regulation of stress erythropoiesis4,5. Since GR is one of the numerous proteins regulated by CALR6, we hypothesized that in human erythroid cells CALR regulates GR functions and that this regulation is disrupted both by CALR and JAK2 mutations in MPN. In this study we tested this hypothesis by determining whether GR and CALR are associated in normal erythroid cells and whether this association is impaired in those from MPN patients. First, biochemical studies determined that human erythroblasts (Erys) expanded ex-vivo from normal stem cell sources [cord blood (CB) and adult blood (AB)] and from MPN patients contain similar levels of CALR and GR. Analyses of cell fractions indicated that in normal Erys, CALR was constitutively localized in the cytoplasm while GR was detected either in the cytoplasm or in the nucleus, depending on the growth factor (the glucocorticoid receptor agonist dexamethasone, erythropoietin or stem cell factor) to which they had been exposed. Second, robust levels of CALR and GR expression were also detected by confocal microscopy. In addition, this analyses revealed that in Erys expanded from normal sources CALR and GR are co-localized in the cytoplasm and that the cytoplasmic association between the two proteins is increased by growth factor deprivation and further enhanced by stimulation with growth factors that activate the JAK2/STAT5 signaling (dexamethasone and/or erythropoietin) while it is inhibited by stimulation with factors that do not use this pathway (stem cell factor). By contrast, in Erys expanded from MPN carrying either CALR or JAK2 mutations, CALR and GR are not associated and remain not associated when the cells are exposed to dexamethasone or erythropoietin. However, in Erys from JAK2V17F-positive MPN patients, association between CALR and GR in the cytoplasm is restored by exposing the cells to the JAK2 inhibitor ruxolitinib. These results suggest that CALR/GR association is a downstream event induced by the JAK2/STAT5 pathway and identify for the first time that CALR functions are impaired in erythroid cells from MPN patients carrying JAK2 mutations.

Changes in the glucocorticoid receptor and Ca²⁺/calreticulin-dependent signalling pathway in the medial prefrontal cortex of rats with post-traumatic stress disorder.

The glucocorticoid receptor (GR), calreticulin (CRT) and protein kinase C (PKC) have all been implicated in the Ca(2+)-dependent signalling pathway, which plays an important role in the plasticity of the central nervous system, learning and memory. The medial prefrontal cortex (mPFC) is known to be involved in mechanisms of learning and memory. In the present study, single prolonged stress (SPS) was used as an animal model of post-traumatic stress disorder (PTSD). The Morris water maze test was used to detect rats' ability for spatial memory and learning. A fluorescence spectrophotometer was used to measure the concentration of intracellular Ca(2+) in mPFC. Immunohistochemistry, immunofluorescence, western blot and reverse transcription polymerase chain reaction were used to explore changes in GR, CRT and PKC in mPFC of SPS rats. The concentration of Ca(2+) in mPFC was increased in the SPS rats. We found increased intensity of GR and CRT immunoreactivity and increased messenger RNA (mRNA) levels of GR, CRT and PKC in mPFC of the SPS groups, although the degree and time of increase was different among them. The protein levels of cytoplasmic GR, cytoplasmic CRT and cytoplasmic pPKC increased in mPFC of the SPS groups, whereas the protein level of nuclear GR decreased in comparison with the control group. As a conclusion, changed CRT and GR/PKC were involved in the mechanism of SPS-induced dysfunctional mPFC.

Accumulation of cytoplasmic glucocorticoid receptor is related to elevation of FKBP5 in lymphocytes of depressed patients.

We have previously shown that patients with the major depressive disorder (MDD) exhibited elevated phosphorylation of the lymphocyte glucocorticoid receptor (GR) at serine 226 (S226). Here, we further analyse potential alterations of GR signalization in lymphocytes of MDD patients, i.e. the cytoplasmic/nuclear distribution of GR, levels of FK506-binding protein 5 (FKBP5) and glucocorticoid-induced leucine zipper (GILZ). The FKBP5 acts as an important regulator of GR activation, by decreasing ligand binding and impeding translocation of the receptor to the nucleus, while GILZ mediates glucocorticoid anti-inflammatory effects. Our result showed that the depressed patients had significantly higher GR levels in the cytoplasm compared to controls, which was accompanied by higher FKBP5 levels. Linear regression model demonstrated significantly higher correlation between FKBP5 and cytoplasmic GR than the presence of MDD itself or phosphorylation of nuclear GR at S226. There were no differences in the levels of GILZ isoforms. Therefore, the results suggest that accumulation of the GR in cytoplasm is related to the elevation of FKBP5, adding one more step in understanding altered GR signalling in lymphocytes, and potentially brain tissue, of MDD patients.

Regulation of corticosterone function during early weaning and effects on gastric cell proliferation

ObjectivesThe development of the gastrointestinal tract depends on many elements, including glucocorticoids. In the current study, we evaluated the effects of early weaning on corticosterone function and the growth of rat gastric mucosa. MethodsBy using Wistar rats submitted to early weaning at 15 d, we analyzed plasma corticosterone, corticosteroid-binding globulin (CBG), and glucocorticoid receptor (GR) distribution in the gastric epithelium. ResultsWith the use of radioimmunoassay, we found that early weaning increased corticosterone concentration at day 16 and 17 in test subjects as compared with controls, whereas it was equivalent between groups at day 18. CBG binding capacity decreased during treatment, and it was significantly lower at day 18. At this age, GR levels and distribution in the gastric mucosa were also reduced as compared with suckling counterparts. To reduce corticosterone activity during early weaning and to explore cell proliferation responses, we administered RU486 to 15-d-old pups. We found that cytoplasmic GR reached a peak after 48 h, whereas nuclear levels remained constant, thereby confirming the inhibition of receptor function. Next, by checking gastric proliferative responses, we observed that RU486 induced higher DNA synthesis and mitotic indices in test subjects as compared with control groups. ConclusionsWe demonstrated that early weaning changed corticosterone activity by increasing hormone levels, reducing CBG binding capacity, and decreasing GR distribution in the gastric epithelium. These modifications seem to be important to the reorganization of gastric growth after the abrupt interruption of suckling.

糖皮质激素诱导移植耐受的免疫细胞调控效应及机制

糖皮质激素(Glucocorticoid; GC)是强有效的甾体类抗炎药和免疫抑制剂，在重症感染、休克、自身免疫病和器官移植等疾病中广泛应用。在器官移植领域GC一直发挥着基础性作用，它是器官移植围手术期免疫诱导必不可少的药物，是经典免疫抑制维持三联方案的重要组成部分，也是治疗急性排斥的首选用药。其作用广泛，能调节代谢、细胞发育和分化；作用机制复杂，不仅有经典的细胞核糖皮质激素受体(glucocorticoid receptor; GCR)信号通路调节DNA的转录，还有其他多种快速作用机制调节细胞功能。本文对GC诱导移植免疫耐受的免疫细胞学调控效应及其机制做以简要综述。 Glucocorticoids (GCs) are potent steroidal anti-inflammatory drugs and immunosuppressants, which are widely used in severe infection, shock, autoimmune diseases, organ transplantation and other diseases. GCs have played a fundamental role in the field of organ transplantation, including induction of immune tolerance, maintenance of immunosuppression and therapy for graft rejection. They act on various cells, and regulate metabolism, cell growth and differentiation. The modulatory mechanisms of GCs are very complex. Through the classic cytoplasmic glucocorticoid receptor (GCR) signaling pathway, it can regulate the transcription of DNA. And, it also has rapid effects on immunity via non-genomic mechanisms. This review summarized the current progresses of immunomodulation and mechanisms of glucocorticoids on the transplantation immune tolerance.

Corticosterone targets distinct steps of synaptic transmission via concentration specific activation of mineralocorticoid and glucocorticoid receptors

Hippocampal neurons are affected by chronic stress and have a high density of cytoplasmic mineralocorticoid and glucocorticoid receptors (MR and GR). Detailed studies on the genomic effects of the stress hormone corticosterone at physiologically relevant concentrations on different steps in synaptic transmission are limited. In this study, we tried to delineate how activation of MR and GR by different concentrations of corticosterone affects synaptic transmission at various levels. The effect of 3-h corticosterone (25, 50, and 100 nM) treatment on depolarization-mediated calcium influx, vesicular release and properties of miniature excitatory post-synaptic currents (mEPSCs) were studied in cultured hippocampal neurons. Activation of MR with 25 nM corticosterone treatment resulted in enhanced depolarization-mediated calcium influx via a transcription-dependent process and increased frequency of mEPSCs with larger amplitude. On the other hand, activation of GR upon 100 nM corticosterone treatment resulted in increase in the rate of vesicular release via the genomic actions of GR. Furthermore, GR activation led to significant increase in the frequency of mEPSCs with larger amplitude and faster decay. Our studies indicate that differential activation of the dual receptor system of MR and GR by corticosterone targets the steps in synaptic transmission differently.