Glucocorticoid Receptor Inhibition Research Articles

White light emitting diode induces autophagy in hippocampal neuron cells through GSK-3-mediated GR and RORα pathways.

Autophagy plays an important role in cell survival under diverse stress conditions. Here, we show that white LED light exposure for 24 h significantly activated autophagy-related genes and increased autophagosome formation in hippocampal neural cells (HT-22). Concurrently, the rhythmic pattern of clock-related gene expression was disrupted, which was associated with augmented expression of SIRT1, AMPK and retinoid-related orphan receptor alpha (RORα). SR1001, a specific inhibitor of RORα, protected the cells from light-induced activation of autophagy. Moreover, light exposure increased glucocorticoid receptor (GR) phosphorylation and nuclear translocation. GR inhibitor RU486 prevented light-induced up-regulation of RORα and the activation of autophagy. These changes were associated with enhanced glycogen synthase kinase-3 (GSK-3) activity and its specific inhibitor CHIR-99021 significantly rescued light-induced autophagy and augmented GR, RORα and autophagy-related proteins. Furthermore, GSK-3 was identified as an upstream regulator of GR/RORα signaling as it was not affected by GR or RORα inhibitors. Taken together, our data demonstrate that GSK-3-mediated GR/RORα signaling pathway is involved in white LED light-induced autophagy in hippocampal neuron cells.

Cytoplasmic Interactions between the Glucocorticoid Receptor and HDAC2 Regulate Osteocalcin Expression in VPA-Treated MSCs.

Epigenetic regulation has been considered an important mechanism for influencing stem cell differentiation. In particular, histone deacetylases (HDACs) have been shown to play a role in the osteoblast differentiation of mesenchymal stem cells (MSCs). In this study, the effect of the HDAC inhibitor, valproic acid (VPA), on bone formation in vivo by MSCs was determined. Surprisingly, VPA treatment, unlike other HDAC inhibitors, produced a well-organized lamellar bone tissue when MSCs–collagen sponge constructs were implanted subcutaneously into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, although a decrease of osteocalcin (OC) expression was observed. Consequently, we decided to investigate the molecular mechanisms by which VPA exerts such effects on MSCs. We identified the glucocorticoid receptor (GR) as being responsible for that downregulation, and suggested a correlation between GR and HDAC2 inhibition after VPA treatment, as evidenced by HDAC2 knockdown. Furthermore, using co-immunoprecipitation analysis, we showed for the first time in the cytoplasm, binding between GR and HDAC2. Additionally, chromatin immunoprecipitation (ChIP) assays confirmed the role of GR in OC downregulation, showing recruitment of GR to the nGRE element in the OC promoter. In conclusion, our results highlight the existence of a cross-talk between GR and HDAC2, providing a mechanistic explanation for the influence of the HDAC inhibitor (namely VPA) on osteogenic differentiation in MSCs. Our findings open new directions in targeted therapies, and offer new insights into the regulation of MSC fate determination.

Leonurine promotes neurite outgrowth and neurotrophic activity by modulating the GR/SGK1 signaling pathway in cultured PC12 cells.

Depression is a common psychiatric disorder that affects almost 10% of children and adolescents worldwide. Numerous synthetic chemical antidepressants used to treat depression have adverse side effects. Therefore, new therapeutic approaches for depression treatment are urgently needed. Leonurus cardiaca has recently been shown to be effective for the treatment of nervous system diseases such as depression, but its mechanism is not clear. In this study, we aimed to reveal the mechanism underlying leonurine’s antidepressant activity. Leonurine was used to treat corticosterone-induced PC12 cells to examine its effect on neurite outgrowth and neurotrophic factors after treatment with the inhibitor of glucocorticoid receptor (GR) and serum-inducible and glucocorticoid-inducible kinase 1 (SGK1). Methyl thiazolyl tetrazolium assays were used to evaluate the viability of cells. High content analysis was used to detect cell area, total neurite length, maximum neurite length, and expression of GR, SGK1, brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), and B-cell lymphoma-2 (BCL-2). The results showed that leonurine increased cell viability in a concentration-dependent manner, with the maximal prosurvival effect at 60 μM. Leonurine increased cell area, total neurite length, and maximum neurite length of corticosterone-induced PC12 cells, increased the expression of GR, BDNF, NT-3, and BCL-2, and decreased the expression of SGK1. After treatment with GR inhibitor RU486, the expressions of GR, BDNF, NT-3, and BCL-2 were significantly decreased and SGK1 was increased. In contrast, treatment with GSK650394 had the opposite effect of RU486. Our data indicate that leonurine promotes neurite outgrowth and neurotrophic activity in cultured PC12 cells, and its potential mechanism may involve the GR/SGK1 signaling pathway.

Abstract A218: Modulating glucocorticoid receptor-mediated signaling for enhancement of cancer immunotherapy

Abstract Glucocorticoids are a class of steroid hormones essential to the modulation of a number of physiologic processes, including cellular metabolism, neural development and immune system function. Functions of glucocorticoids are mediated through the glucocorticoid receptor (GR). Synthetic glucocorticoids are front-line therapies in the clinic for leukemia and lymphoma due to their potent induction of lymphocyte death. Glucocorticoids are also prescribed as adjuvant therapies to chemotherapies to alleviate adverse effects including nausea, emesis and allergic reactions. In the current regimen of immune checkpoint blockade therapies against solid tumors, glucocorticoids are incorporated to manage symptoms related to acute immune hyperactivation. However, given that glucocorticoids preferentially cause lymphocyte death, their inclusion in immune checkpoint blockade regimens which capitalize on cytotoxic T-cell-mediated killing of tumor cells is controversial. The precise effects of systematic glucocorticoids use on tumor development, and particularly, on the tumor-immune microenvironment comprised tumor-infiltrating leukocytes (TILs) remains unknown. Conversely, methods to antagonize glucocorticoid signaling could potentially boost lymphocyte number and function and potentiate cytotoxic T-cell-mediated killing of tumor cells. Presently, the treatment of “immune cold” solid tumor types remains a challenge for immunotherapy due low level of TILs and presence of immunosuppressive cells (including regulatory T-cells and myeloid-derived suppressor cells) in the tumor microenvironment. Pharmacologic modulation of GR presents a unique opportunity to regulate immune cell homeostasis and function with the goal of promoting a favorable tumor immune microenvironment to augment the effectiveness of cancer immunotherapies in solid tumors. Our research seeks to understand the impact of glucocorticoid modulation in the context of immune checkpoint blockade therapies in preclinical models, and to provide a rationale for selective pharmacologic modulation of GR to promote a favorable tumor immune microenvironment and increased efficacy of immunotherapies in solid tumors. We investigated GR activation and inhibition on the immune tumor microenvironment in murine syngeneic melanoma (B16), breast cancer (4T1) and colorectal cancer (CT26) models. Mice bearing allograft tumors were randomized into groups receiving synthetic glucocorticoids or GR inhibitors in combination with anti-PD-1 and anti-CTLA4 therapies. Following treatment, tumors were assessed for composition of intratumoral leukocyte infiltration by flow cytometry phenotyping. In the mouse breast cancer model (4T1), we found evidence supporting substantial lymphodepletion by dexamethasone (a synthetic glucocorticoid compound). Treatment with PD-1 and CTLA-4 blocking monoclonal antibodies increased lymphocyte infiltration into the tumor. Interestingly, immune checkpoint blockade therapies significantly raised the number of more CD4+ T helper cells than CD8+ cytotoxic T-cells in the tumor. Our future plan includes investigation of the effects of GR inhibition on TILs in murine solid tumor models, and utilization of RNA-sequencing on TILs for identification of genes and molecular pathways that provide opportunities to boost TIL function within the tumor. In summary, our work can provide insight into the fundamental molecular mechanisms by which the glucocorticoid receptor modulates the tumor immune microenvironment and provide novel avenues for enhancing cancer immunotherapies in solid tumors. Citation Format: Qin Tang, Myles A. Brown. Modulating glucocorticoid receptor-mediated signaling for enhancement of cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A218.

Effect of glucocorticoid receptor function on the behavior of rats with attention deficit hyperactivity disorder

To investigate the ideal animal models for attention deficit hyperactivity disorder (ADHD) subtypes and the effect of glucocorticoid receptor (GR) function on the behavior of ADHD rats by comparing behavioral differences between spontaneously hypertensive rats (SHRs), Wistar Kyoto (WKY) rats, and Sprague-Dawley (SD) rats. A total of 24 male SHRs aged 21 days were randomly divided into GR agonist group, GR inhibitor group, and SHR group, with 8 rats in each group. Eight male WKY rats and 8 male SD rats, also aged 21 days, were enrolled as WKY group and SD group respectively. The GR agonist group was treated with intraperitoneal injection of dexamethasone (0.5 mg/kg daily); the GR inhibitor group was treated with intraperitoneal injection of mifepristone (RU486) (54 mg/kg daily); the SHR, WKY, and SD groups were treated with intraperitoneal injection of normal saline (0.5 mL/kg daily). The course of treatment was 14 days for all groups. The open field test and Lat maze test were used to evaluate spontaneous activity and non-selective attention. The open field test showed that before drug intervention the SHR group had significantly higher numbers of line crossings and rearings than the WKY and SD groups (P<0.05); the WKY group had a significantly higher number of line crossings than the SD group (P<0.05); the SD group had a significantly higher number of groomings than the WKY group (P<0.05). After drug intervention, the GR agonist group had significantly lower numbers of line crossings and groomings than the SHR group (P<0.05). The Lat maze test indicated that before drug intervention the SHR group had significantly higher numbers of corner crossings and rearings than the WKY and SD groups (P<0.05); the WKY group had significantly higher numbers of rearings and leanings than the SD group (P<0.05). After drug intervention, the GR agonist group had significantly lower numbers of corner crossings and rearings than the SHR group (P<0.05); the GR inhibitor group had a significantly higher number of rearings than the SHR group (P<0.05); the WKY group had significantly higher numbers of rearings and leanings than the SD group (P<0.05). SHR is an ideal animal model for mixed subtype ADHD, and further studies are needed to determine whether WKY rats can be used as an animal model for attention-deficit subtype ADHD. GR agonist can effectively improve spontaneous activity and non-selective attention in SHRs.

Modulation of glucocorticoid receptor in human epileptic endothelial cells impacts drug biotransformation in an in vitro blood–brain barrier model

SummaryObjectiveNuclear receptors and cytochrome P450 (CYP) regulate hepatic metabolism of several drugs. Nuclear receptors are expressed at the neurovascular unit of patients with drug‐resistant epilepsy. We studied whether glucocorticoid receptor (GR) silencing or inhibition in human epileptic brain endothelial cells (EPI‐ECs) functionally impacts drug bioavailability across an in vitro model of the blood–brain barrier (BBB) by CYP‐multidrug transporter (multidrug resistance protein 1, MDR1) mechanisms.MethodsSurgically resected brain specimens from patients with drug‐resistant epilepsy, primary EPI‐ECs, and control human brain microvascular endothelial cells (HBMECs) were used. Expression of GR, pregnane X receptor, CYP3A4, and MDR1 was analyzed pre‐ and post‐GR silencing in EPI‐ECs. Endothelial cells were co‐cultured with astrocytes and seeded in an in vitro flow‐based BBB model (DIV‐BBB). Alternatively, the GR inhibitor mifepristone was added to the EPI‐EC DIV‐BBB. Integrity of the BBB was monitored by measuring transendothelial electrical resistance. Cell viability was assessed by glucose‐lactate levels. Permeability of [3H]sucrose and [14C]phenytoin was quantified. CYP function was determined by measuring resorufin formation and oxcarbazepine (OXC) metabolism.ResultsSilencing and inhibition of GR in EPI‐ECs resulted in decreased pregnane X receptor, CYP3A4, and MDR1 expression. GR silencing or inhibition did not affect BBB properties in vitro, as transendothelial electrical resistance and Psucrose were unaltered, and glucose metabolism was maintained. GR EPI‐EC silencing or inhibition led to (1) increased Pphenytoin BBB permeability as compared to control; (2) decreased CYP function, indirectly evaluated by resorufin formation; (3) improved OXC bioavailability with increased abluminal (brain‐side) OXC levels as compared to control.SignificanceOur results suggest that modulating GR expression in EPI‐ECs at the BBB modifies drug metabolism and penetration by a mechanism encompassing P450 and efflux transporters. The latter could be exploited for future drug design and to overcome pharmacoresistance.

Abstract 1968: A novel glucocorticoid receptor (GR) antagonist overcomes GR-mediated chemoresistance in triple-negative breast cancer

Abstract The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily, which is activated by its endogenous steroid hormone ligand cortisol, and by synthetic glucocorticoids such as dexamethasone. Several preclinical studies have shown that GR mediates resistance to both targeted therapies and conventional chemotherapies in a variety of epithelial cancers including prostate, lung, bladder, renal, ovarian and triple-negative breast cancers (TNBC) (Gassler et al., 2005; Li et al., 2017; Zhang et al., 2007). In TNBC, both GR activation and a disrupted cortisol secretion cycle are associated with chemotherapy resistance, increased disease recurrence and poor prognosis (Pan et al., 2011; Skor et al., 2013). Therefore a molecule that inhibits GR activation could attenuate the development of therapy resistance and improve patient outcomes. We have developed novel GR inhibitors that effectively block GR transcriptional activity in cells by competing for ligand binding and by blocking GR-coactivator interactions. In vitro, treatment of TNBC cells with the GR antagonist OP-3713 blocks GR transcriptional activity and enhances the efficacy of chemotherapeutic agents. In rodents the predominant glucocorticoid is corticosterone (Siswanto et al., 2008), which is a weak agonist of human GR. Therefore, to fully activate GR in human xenograft cancer models it is necessary to provide exogenous cortisol. Using xenograft models of TNBC, we find that tumors grown in mice with physiologically relevant circulating cortisol levels to activate GR are significantly less sensitive to chemotherapy than those grown in the absence of cortisol. Furthermore, inhibition of GR by OP-3713 prevents tumor relapse following chemotherapy treatment. We have begun to elucidate the mechanisms by which GR mediates chemoresistance in TNBC, and the basis for the reversal by OP-3713. Our findings underscore the important role of GR as a mediator of resistance in TNBC and highlight the therapeutic potential of GR inhibitors in combination with clinically relevant chemotherapeutic agents. Citation Format: Nadine Jahchan, Haiying Zhou, Wayne Kong, Dan Mc Weeney, Ted Tracy, James Stice, Dena Sutimantanapi, Chelsea Chen, Tom Huang, Yosup Rew, Xiauhui Du, Tatiana Zavorotinskaya, Daqing Sun, Qiuping Ye, Erica Jackson, Valeria Fantin. A novel glucocorticoid receptor (GR) antagonist overcomes GR-mediated chemoresistance in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1968.

Glucocorticoid Upregulates Thioredoxin-interacting Protein in Cultured Neuronal Cells

Previous studies have shown that chronic stress and chronic stress hormone treatment induce oxidative damage in rodents. Thioredoxin (Trx) is a small redox protein that plays an important role in regulation of oxidative protein cysteine modification. A Trx reduced state is maintained by thioredoxin reductase (TrxR), and the thioredoxin-interacting protein (Txnip) is an endogenous inhibitor of Trx. The purpose of this study was to investigate the effects of chronic treatment with stress hormone corticosterone on Trx, TrxR and Txnip in cultured neuronal cells. Using immunoblotting analysis we found that although chronic corticosterone treatment had no effect on Trx and TrxR protein levels, this treatment significantly increased Txnip protein levels. Using immunocytochemistry we also found that chronic corticosterone treatment increased Txnip in both nucleus and cytosol, while glucocorticoid receptor inhibitor RU486 can block corticosterone-increased Txnip protein levels. Using biotin switch, dimedone conjugation and CRISPR/Cas9 methods we found that chronic corticosterone treatment increased protein nitrosylation and sulfenylation, while knocking out Txnip blocked corticosterone-induced protein nitrosylation and sulfenylation. Since Trx can reduce cysteine oxidative protein modification such as nitrosylation and sulfenylation, our findings suggest that chronic corticosterone treatment may upregulate Txnip by targeting glucocorticoid receptor, subsequently inhibiting Trx activity and enhancing oxidative protein cysteine modification, which contributes to corticosterone-caused oxidative damage.

Glucocorticoid Signaling Enhances Expression of Glucose-Sensing Molecules in Immature Pancreatic Beta-Like Cells Derived from Murine Embryonic Stem Cells In Vitro

Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. In this study, we aim to identify small molecules that affect immature beta cells. A cell-based assay, using pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative reverse transcription-polymerase chain reaction analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GRflox/flox mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM d-glucose and stimulated by 17 mM d-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells.

Programming for increased expression of hippocampal GAD67 mediated the hypersensitivity of the hypothalamic\u2013pituitary\u2013adrenal axis in male offspring rats with prenatal ethanol exposure

An imbalance of excitatory and inhibitory signals in the brain has been proposed to be one of the main pathological features of various diseases related to hypothalamic–pituitary–adrenal axis (HPAA) dysfunction. Excessive glutamate release induces neuronal excitotoxicity, while glutamic acid decarboxylase (GAD) 67 promotes the transformation of excessive glutamate to γ-aminobutyric acid (GABA). Our previous studies demonstrated that prenatal ethanol exposure (PEE) causes foetal over-exposure to maternal corticosterone and hypersensitivity of the HPAA after birth, but its intrauterine programming mechanism is unknown. In this study, PEE was shown to lead to an enhanced potential excitatory ability of the hypothalamus and hypersensitivity of the HPAA, as well as mild abnormal hippocampal morphology, demethylation of the -1019 to -691-bp region in the hippocampal GAD67 promoter and upregulation of GAD67 expression accompanied by a reduction in glutamatergic neurons and increase in GABAergic neurons in PEE male offspring. Similar changes were also found in PEE male foetal rats. Furthermore, corticosterone increased the expression of the glucocorticoid receptor (GR) and GAD67 in foetal hippocampal H19-7 cells in a concentration-dependent manner, accompanied by demethylation of the GAD67 promoter, a decrease in glutamatergic neurons and increase in GABAergic neurons. The GR inhibitor, mifepristone, reversed the effects of corticosterone on H19-7 cells. These results suggested that PEE-induced excessive corticosterone can lead to upregulation of GAD67 through epigenetic modification mediated by the GR in the male foetal hippocampus, thereby weakening the negative regulation of the HPAA by the hippocampus and increasing the potential excitatory ability of the hypothalamus. These changes persisted until after birth, resulting in hypersensitivity of the HPAA. However, gender differences were observed in the hippocampal development, morphology and GAD67 expression associated with PEE. Programming for the increased expression of hippocampal GAD67 is a potential mechanism responsible for the hypersensitivity of the HPAA in PEE male rats.

Recombinant relaxin protects liver transplants from ischemia damage by hepatocyte glucocorticoid receptor: From bench-to-bedside.

This translational study underscores the role of rhRLX-GR signaling as a regulator of hepatocellular protection against IR stress in OLT. In the context of a recent phase III clinical trial demonstrating positive outcomes of rhRLX in patients with acute heart failure, studies on rhRLX for the management of IRI in OLT recipients are warranted. (Hepatology 2018;68:258-273).

The Influence of Reactive Oxygen Species and Glucocorticoids on Dry Skin in a Mouse Model of Arthritis

Background: Dry skin induced by rheumatoid arthritis (RA) causes itching, which negatively influences a patient’s quality of life. We previously reported that mast cells are related to dry skin in arthritic mice. However, the mechanism of mast cell activation is unclear. In this study, we examined the mechanism underlying the formation of dry skin induced by mast cells in arthritis that involves thymic stromal lymphopoietin (TSLP), neutrophils, reactive oxygen species (ROS), and glucocorticoids. Methods: Mice with DBA/1JJmsSlc collagen-induced arthritis were treated with inhibitors or neutralizing antibodies. We measured transepidermal water loss (TEWL) to examine the modulating signal of mast cells. Results: TEWL, the number of mast cells, and the plasma levels of TSLP, ROS, and corticosterone in the arthritic mice were increased when compared with the control mice. However, the mice treated with TSLP- and neutrophil-neutralizing antibodies and ROS and glucocorticoid receptor inhibitors (N-acetyl-L-cysteine [NAC] and RU-486, respectively) experienced an improvement. The ameliorating effect was most remarkable following treatment with NAC + RU-486. Conclusion: This study suggested that inhibiting ROS and glucocorticoids is important to ameliorate dry skin in arthritis, which may provide a novel treatment option for dry skin in RA patients.

P.2.015 - The differential effects of short-term exposure to high fat diet on hippocampal function during juvenility and adulthood

Environmental and metabolic challenges during early life can have long-term devastating effects on cognitive and emotional functions. We recently showed in two lines of experiments that the juvenile brain is different from that of the adult brain; we showed that stress has opposite effects on prefrontal cortex plasticity and extinction [1]. Specifically, while stress results in impaired long-term potentiation (LTP) and extinction in the adult animal, it resulted in enhanced extinction and LTP in the juveniles. Similarly, exposure to long-term high fat diet (HFD) from juvenility until adulthood was associated with enhanced activation and plasticity in the amygdala and impaired activation and plasticity in the hippocampus, while similar exposure starting at adulthood was not associated with these effects [2]. Both stress and HFD are associated with activation of the hypothalamic-pituitary-adrenal (HPA) axis. Based on these findings we focused on the effects of short- term exposure to HFD on synaptic plasticity in Schaffer Collateral- CA1 pathway and hippocampal-dependent spatial memory in adults [post-natal day (PND 60)] and juveniles (PND 27). Rats were exposed to HFD for 7-10 days and CA1 LTP was examined or spatial recognition. In the spatial paradigm, two different objects were located in opposite corners; each rat was allowed to explore the objects for 5 min, 24h after the sample phase the rat was presented with the same two objects one of the objects was placed at a new location. A discrimination index calculated for each animal was expressed as TN/(TN+TF) (TN = time spent exploring the object in the novel location; TF = time spent exploring the object in the familiar location). In the last part of this work we examined the ability of the glucocorticoid receptor (GR) antagonist (RU486) to reverse the effects of the HFD on LTP in the CA1 pathway and on spatial memory. The main finding of this experiment was that short-term HFD deferentially modulates plasticity in the adult animals as compared to the juveniles. Specifically, in the Schaffer Collateral- CA1 pathway, in contrast to the adult animals where exposure to short-term HFD enhanced the induction of LTP; in the juvenile animals’ exposure to the same duration of HFD inhibited LTP in the CA1. Furthermore, corresponding to these results, short-term HFD enhanced spatial memory in adult animals while it abolished spatial memory in juvenile rats. In addition, after systemic application of GRs antagonist the effects of HFD were no longer observed; the antagonist abolished the induction of LTP in the HFD-fed adults while enhancing induction in HFD-fed juveniles and reversed the HFD effects on spatial memory.The results were presented as mean ± S.E.M. For statistical analysis, independent t-test and ANOVA for repeated measures tests were used as indicated. These results suggest developmental differences in the way short-term HFD affects CA1 plasticity and the spatial memory. These effects are differently modulated by GRs; inhibition of GRs normalized the potentiation in CA1. These effects of HFD may be relevant to the increase prevalence of emotional and behavioral disorders in children exposed to western diet.

Expression and regulation of M-type K+ channel in PC12 cells and rat adrenal medullary cells.

M-type K+ channels contribute to the resting membrane potential in the sympathetic ganglion neurons of various animals, whereas their expression in adrenal medullary (AM) cells has been controversial. The present experiment aims to explore the expression of M channels comprising the KCNQ2 subunit in the rat AM cell and its immortalized cell line PC12 cells at the protein level and how its expression in PC12 cells is regulated. The KCNQ2 isoform was recognized in homogenates of PC12 cells but not the rat adrenal medullae by immunoblotting and KCNQ2-like immunoreactivity (IR) was detected in PC12 cells but not in rat AM cells. When the PC12 cells were maintained in a dexamethasone-containing medium, KCNQ2-like IR in the cells was suppressed, whereas the removal of fetal bovine serum from the culture medium for 1day resulted in an increase in KCNQ2-like IR. A similar enhancement occurred when PC12 cells were cultured under conditions where glucocorticoid receptor (GR) and/or mineralocorticoid receptor (MR) activities were suppressed. These morphological findings were confirmed in functional analysis. The cells cultured in the presence of an inhibitor of either GR or MR exhibited larger amplitudes of Ca2+ signal in response to an M channel inhibitor than did the cells in its absence, whereas the resting Ca2+ level in the former was lower than that in the latter. These results indicate that the M channel is not expressed in rat AM cells and this absence of expression may be ascribed to the suppression by glucocorticoid activity.

The Glucocorticoid Receptor Is a Key Player for Prostate Cancer Cell Survival and a Target for Improved Antiandrogen Therapy.

Purpose: The major obstacle in the management of advanced prostate cancer is the occurrence of resistance to endocrine therapy. Although the androgen receptor (AR) has been linked to therapy failure, the underlying escape mechanisms have not been fully clarified. Being closely related to the AR, the glucocorticoid receptor (GR) has been suggested to play a role in enzalutamide and docetaxel resistance. Given that glucocorticoids are frequently applied to prostate cancer patients, it is essential to unravel the exact role of the GR in prostate cancer progression.Experimental Design: Assessment of GR expression and functional significance in tissues from 177 prostate cancer patients, including 14 lymph node metastases, as well as in several human prostate cancer models, including androgen-dependent, androgen-independent, and long-term antiandrogen-treated cell lines.Results: Although GR expression is reduced in primary prostate cancer tissue, it is restored in metastatic lesions. Relapse patients with high GR experience shortened progression-free survival. GR is significantly increased upon long-term abiraterone or enzalutamide treatment in the majority of preclinical models, thus identifying GR upregulation as an underlying mechanism for cells to bypass AR blockade. Importantly, GR inhibition by RNAi or chemical blockade results in impaired proliferation and 3D-spheroid formation in all tested cell lines.Conclusions: GR upregulation seems to be a common mechanism during antiandrogen treatment and supports the notion that targeting the GR pathway combined with antiandrogen medication may further improve prostate cancer therapy. Clin Cancer Res; 24(4); 927-38. ©2017 AACR.

Abstract A133: Development of murine models to evaluate the impact of glucocorticoid receptor (GR) inhibition on chemotherapy response

Abstract The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. GR is activated by its endogenous steroid hormone ligand, cortisol, and by synthetic glucocorticoid drugs such as dexamethasone. Several preclinical studies have established a role for GR in mediating resistance to both targeted therapies and conventional chemotherapies in epithelial cancers. Glucocorticoids have been reported to confer resistance to antimetabolites, taxanes, and platinum compounds in lung, prostate, bladder, renal, ovarian, and triple-negative breast cancers (Gassler et al., 2005; Li et al., 2017; Zhang et al., 2007). GR has also been reported to confer resistance to antiandrogen therapies in prostate cancer (Arora et al., 2013). Therefore, a molecule that inhibits GR activation could attenuate the development of resistance to cancer therapeutics. We are developing novel GR inhibitors that effectively block GR transcriptional activity in cells by competing for ligand binding and blocking GR-Coactivator interactions. In vitro, GR inhibition enhances the efficacy of chemotherapeutic agents under conditions supporting GR activation. In humans, the predominant glucocorticoid is cortisol, while in mice the predominant glucocorticoid is corticosterone (Siswanto et al., 2008). Corticosterone is a potent agonist of murine GR, but a weak agonist of human GR. When human tumor cells are grown in mice, GR is not active in the tumor cells due to the lack of circulating cortisol. Therefore it is necessary to provide exogenous cortisol to fully activate GR. Using xenograft models of ovarian and triple-negative breast cancer, we find that tumors grown in mice with physiologically relevant circulating cortisol levels are significantly less sensitive to standard-of-care chemotherapy than those grown in the absence of cortisol. Inhibition of GR is effective at restoring the response to chemotherapy in these models. These results demonstrate the therapeutic potential of GR inhibitors in combination with clinically utilized chemotherapeutics. Furthermore, the in vivo models developed provide a milieu for testing and developing GR inhibitors. Citation Format: Nadine Jahchan, Haiying Zhou, James Stice, Wayne Kong, Dan McWeeney, Daqing Sun, Yosup Rew, Xiaohui Du, Liusheng Zhu, Qiuping Ye, Erica L. Jackson, Valeria Fantin. Development of murine models to evaluate the impact of glucocorticoid receptor (GR) inhibition on chemotherapy response [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A133.

Adrenergic and glucocorticoid receptor antagonists reduce ozone-induced lung injury and inflammation

Recent studies showed that the circulating stress hormones, epinephrine and corticosterone/cortisol, are involved in mediating ozone-induced pulmonary effects through the activation of the sympathetic-adrenal-medullary (SAM) and hypothalamus-pituitary-adrenal (HPA) axes. Hence, we examined the role of adrenergic and glucocorticoid receptor inhibition in ozone-induced pulmonary injury and inflammation. Male 12-week old Wistar-Kyoto rats were pretreated daily for 7days with propranolol (PROP; a non-selective β adrenergic receptor [AR] antagonist, 10mg/kg, i.p.), mifepristone (MIFE; a glucocorticoid receptor [GR] antagonist, 30mg/kg, s.c.), both drugs (PROP+MIFE), or respective vehicles, and then exposed to air or ozone (0.8ppm), 4h/d for 1 or 2 consecutive days while continuing drug treatment. Ozone exposure alone led to increased peak expiratory flow rates and enhanced pause (Penh); with greater increases by day 2. Receptors blockade minimally affected ventilation in either air- or ozone-exposed rats. Ozone exposure alone was also associated with marked increases in pulmonary vascular leakage, macrophage activation, neutrophilic inflammation and lymphopenia. Notably, PROP, MIFE and PROP+MIFE pretreatments significantly reduced ozone-induced pulmonary vascular leakage; whereas PROP or PROP+MIFE reduced neutrophilic inflammation. PROP also reduced ozone-induced increases in bronchoalveolar lavage fluid (BALF) IL-6 and TNF-α proteins and/or lung Il6 and Tnfα mRNA. MIFE and PROP+MIFE pretreatments reduced ozone-induced increases in BALF N-acetyl glucosaminidase activity, and lymphopenia. We conclude that stress hormones released after ozone exposure modulate pulmonary injury and inflammatory effects through AR and GR in a receptor-specific manner. Individuals with pulmonary diseases receiving AR and GR-related therapy might experience changed sensitivity to air pollution.

Glucocorticoid receptor inhibition prevents lasting endocrine but not behavioral effects of adolescent stress

Glucocorticoid receptor inhibition prevents lasting endocrine but not behavioral effects of adolescent stress

Triamcinolone Acetonide Suppresses Inflammation and Facilitates Vascular Barrier Function in Human Retinal Microvascular Endothelial Cells.

Diabetic macular edema (DME) is resulted from the retinal microvascular leakage that accompanies the breakdown of blood-retinal barrier. Triamcinolone acetonide (TA) is a therapeutic agent for DME, but since the detailed mechanism of action of TA is not known, part of its action was examined. In vitro model to enhance the permeability of human retinal microvascular endothelial cells (HRMECs) was constructed by using DME-related cytokines such as vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). The efficacy of TA and anti-VEGF antibody against retinal permeability was evaluated. Furthermore, the localization of ZO-1 in HRMECs was examined using immunofluorescence staining and the expression level of tight junction proteins (ZO-1, Occludin, and Claudin-5) was examined using immunoblot. TA and anti-VEGF antibody showed inhibitory effects against VEGF-induced permeability enhancement, and TA also inhibited the increase in permeability induced by TNF-α and IL-1β. In addition to the inhibitory effects against cytokine-induced hyperpermeability, TA enhanced the barrier function of HRMECs and reduced the vascular leakage. TA altered the localization of tight junction proteins, but did not increase the upregulation of tight junction proteins. Moreover, the enhancement of barrier function by TA was inhibited by 17-AAG (glucocorticoid receptor inhibitor). It was suggested that TA reinforced the barrier function through the glucocorticoid receptor. In this study, we found that TA suppressed the inflammation caused by VEGF, TNF-α and IL-1β, and decreased the retinal vascular hyperpermeability.

Glucocorticoids/glucocorticoid receptors effect on dopaminergic neurotransmitters in ADHD rats

Dexamethaone (DEX, glucocorticoid receptor agonist) and RU486 (glucocorticoid receptor inhibitor) may affect the behavior of attention deficit hyperactivity disorder (ADHD) rats, by changing the level of dopamine and noradrenaline and dopamine transporter in different regions of their brain. In this study, we have investigated the effect and the underlying mechanism of Glucocorticoids/Glucocorticoid receptors on dopaminergic neurotransmitters in ADHD Rats. Thirty male Wistar Kyoto rats (WKY) and 30 male spontaneously hypertensive rats (SHR) were respectively divided into 3 groups randomly as follows: a GR agonist group (DEX), a GR inhibitor group (RU486) and a control group (CON). Open field test and Y maze were performed respectively to assess the behavior of the rats. The levels of dopamine, norepinephrine, and dopamine transporter (DAT) in the prefrontal cortex and striatum were also tested. Our results showed that, the behavior of rats were improved after DEX treatment. We also found that the level of DA and NE increased in DEX group, but decreased significantly in RU486 group. Immunohistochemical assay showed that DAT expression level in DEX group was significantly less than that in RU486 and CON group. In conclusion, by regulation of glucocorticoid receptor, GR agonist can decrease DAT expression, resulting in the increase of DA and NE levels in brain that ameliorate hyperactivity and attention deficit in ADHD rats. Our results suggest that the effects of glucocorticoid receptor on dopaminergic neurotransmitter in the central nervous system may be involved in the pathogenesis of ADHD.