Glucocorticoid Receptor Blockade Research Articles

Colonization by Enterobacteriaceae is crucial for acute inflammatory responses in murine small intestine via regulation of corticosterone production

ABSTRACT Although dysbiosis in the gut microbiota is known to be involved in several inflammatory diseases, whether any specific bacterial taxa control host response to inflammatory stimuli is still elusive. Here, we hypothesized that dysbiotic indigenous taxa could be involved in modulating host response to inflammatory triggers. To test this hypothesis, we conducted experiments in germ-free (GF) mice and in mice colonized with dysbiotic taxa identified in conventional (CV) mice subjected to chemotherapy-induced mucositis. First, we report that the absence of microbiota decreased inflammation and damage in the small intestine after administration of the chemotherapeutic agent 5-fluorouracil (5-FU). Also, 5-FU induced a shift in CV microbiota resulting in higher amounts of Enterobacteriaceae, including E. coli, in feces and small intestine and tissue damage. Prevention of Enterobacteriaceae outgrowth by treating mice with ciprofloxacin resulted in diminished 5-FU-induced tissue damage, indicating that this bacterial group is necessary for 5-FU-induced inflammatory response. In addition, monocolonization of germ-free (GF) mice with E. coli led to reversal of the protective phenotype during 5-FU chemotherapy. E. coli monocolonization decreased the basal plasma corticosterone levels and blockade of glucocorticoid receptor in GF mice restored inflammation upon 5-FU treatment. In contrast, treatment of CV mice with ciprofloxacin, that presented reduction of Enterobacteriaceae and E. coli content, induced an increase in corticosterone levels. Altogether, these findings demonstrate that Enterobacteriaceae outgrowth during dysbiosis impacts inflammation and tissue injury in the small intestine. Importantly, indigenous Enterobacteriaceae modulates host production of the anti-inflammatory steroid corticosterone and, consequently, controls inflammatory responsiveness in mice.

Changes in c-fos and p-CREB signaling following exposure to forced swim stress or exogenous corticosterone during morphine-induced place preference are dependent on glucocorticoid receptor in the basolateral amygdala.

Neural circuitry comprising the nucleus accumbens (NAc), prefrontal cortex (PFC), amygdala (AMY), and hippocampus (HIP) are the main components of the reward circuit. Our previous behavioral data showed that forced swim stress (FSS) and corticosterone administration could inhibit the acquisition of morphine-induced conditioned place preference (CPP), and this effect was blocked by intra-basolateral amygdala (BLA) administration of RU38486, glucocorticoid receptor (GR) antagonist. Therefore, we tried to evaluate the effect of intra-BLA administration of the GR antagonist during the conditioning phase on the c-fos and p-CREB/CREB ratio expression in the AMY, NAc, PFC, and HIP of rats that underwent FSS or received exogenous corticosterone (10 mg/kg; i.p.) before morphine injection (5 mg/kg; s.c.) during 3 conditioning days. Our results showed that morphine-induced CPP could increase c-fos level and p-CREB/CREB ratio in all regions (except in the HIP). In addition, c-fos expression was elevated by FSS in all regions and blockade of GR decreased this effect. In the PFC, in addition to FSS, corticosterone could raise c-fos expression, which was blocked by RU38486. In conclusion, it seems that the intra-BLA administration of RU38486 differently modulates the effect of morphine-induced CPP on the expression of c-fos and p-CREB/CREB ratio in animals that underwent FSS or corticosterone administration.

Invariant NKT cells are unusually refractory to glucocorticoid-induced apoptosis but exhibit impaired functional fitness and fail to trigger antitumor immunity following physiological stress

Abstract The nervous system serves critical roles in the regulation of immune responses. Consequently, the neuroimmune functional interface can be disrupted by physiological stress, potentially impeding our ability to combat malignancies. Invariant natural killer T (iNKT) cells are innate-like T cells that, upon activation by lipids such as αGC, participate in antitumor immune surveillance. However, whether mediators of stress impact iNKT cell functions in this context remains unexplored. Here, we subjected mice to prolonged psychological stress due to physical restraint or chronic stress due to heterotypic stressors before administering αGC. We report that stress abrogates TH1-type cytokine production by iNKT cells in vivo, an effect that is reversible by glucocorticoid receptor (GR) blockade and that is diminished in mice selectively lacking GRs in their T cell compartment. Instead, iNKT cell responses are skewed towards an abnormal anti-inflammatory and TH17-type cytokine signature. Gene expression analysis revealed that iNKT cells in stressed mice express reduced levels of critical genes supporting their typical differentiation states. Accordingly, iNKT cells in stressed mice fail to protect against pulmonary metastases of B16-F10 melanoma and are also unable to initiate killing activity against MHC class I-deficient splenocytes in vivo or YAC-1 lymphoma cells in vitro. Finally, the suppressive effects of stress on iNKT cells are not due to their apoptotic death. Unlike conventional T cells, human and mouse iNKT cells are remarkably resistant to glucocorticoid-induced apoptosis. Collectively, our findings define a novel mechanism of stress-induced immunosuppression involving innate-like but not conventional T cells.

Glucocorticoid receptor activation stimulates the sodium-chloride cotransporter and influences the diurnal rhythm of its phosphorylation.

The sodium-chloride cotransporter (NCC) in the distal convoluted tubule contributes importantly to sodium balance and blood pressure (BP) regulation. NCC phosphorylation determines transport activity and has a diurnal rhythm influenced by glucocorticoids. Disturbing this rhythm induces “nondipping” BP, an abnormality that increases cardiovascular risk. The receptor through which glucocorticoids regulate NCC is not known. In this study, we found that acute administration of corticosterone to male C57BL6 mice doubled NCC phosphorylation without affecting total NCC abundance in both adrenalectomized and adrenal-intact mice. Corticosterone also increased the whole kidney expression of canonical clock genes: period circadian protein homolog 1 (Per1), Per2, cryptochrome 1, and aryl hydrocarbon receptor nuclear translocator-like protein 1. In adrenal-intact mice, chronic blockade of glucocorticoid receptor (GR) with RU486 did not change total NCC but prevented corticosterone-induced NCC phosphorylation and activation of clock genes. Blockade of mineralocorticoid receptor (MR) with spironolactone reduced the total pool of NCC but did not affect stimulation by corticosterone. The diurnal rhythm of NCC phosphorylation, measured at 6-h intervals, was blunted by chronic GR blockade, and a similar dampening of diurnal variation was seen in GR heterozygous null mice. These effects on NCC phosphorylation did not reflect altered rhythmicity of plasma corticosterone or serum and glucocorticoid-induced kinase 1 activity. Both mineralocorticoids and glucocorticoids emerge as regulators of NCC, acting via distinct receptor pathways. MR activation provides maintenance of the NCC protein pool; GR activation dynamically regulates NCC phosphorylation and establishes the diurnal rhythm of NCC activity. This study has implications for circadian BP homeostasis, particularly in individuals with abnormal glucocorticoid signaling as is found in chronic stress and corticosteroid therapy.

Corticosterone-mediated microglia activation affects dendritic spine plasticity and motor learning functions in minimal hepatic encephalopathy

Minimal hepatic encephalopathy (MHE) is characterized as cognitive deficits including memory and learning dysfunctions after liver injuries or hepatic diseases. Our understandings of neurological mechanisms of MHE-associated cognitive syndromes, however, are far from complete. In the current study we generated a mouse MHE model by repetitive administrations of thioacetamide (TAA), which induced hyperammonemia plus elevated proinflammatory cytokines in both the general circulation and motor cortex. MHE mice presented prominent motor learning deficits, which were associated with excess dendritic spine pruning in the motor cortex under 2-photon in vivo microscopy. The pharmaceutical blockade of glucocorticoid receptor or suppression of its biosynthesis further rescued motor learning deficits and synaptic protein loss. Moreover, MHE mice presented microglial activation, which can be alleviated after glucocorticoid pathway inhibition. In sum, our data demonstrates corticosterone-induced microglial activation, synaptic over-pruning and motor learning impairments in MHE, providing new insights for MHE pathogenesis and potential targets of clinical interventions.

Abstract LB-260: Role of the glucocorticoid receptor/beta-catenin interaction in prostate cancer stem cell survival and chemoresistance

Abstract Prostate cancer (PCa) is the second leading cause of cancer deaths in U.S. men, disproportionately affecting African American men and certain Hispanic/Latino populations. Patients with advanced PCa are treated with androgen deprivation therapy (ADT) and docetaxel (DTX)-based chemotherapy with effective but transient results due to therapy resistance. While the role of glucocorticoid receptor (GR) in promoting ADT resistance has been well characterized, comparatively little is known about its contribution to PCa stemness and chemoresistance. Our group demonstrated recently that DTX-resistance is associated with upregulation of a stemness transcriptomic program and enhanced cancer stem cell (CSC) properties in PCa cells. We also demonstrated that GR signaling upregulates the DTX-resistance associated proteins Clusterin and LEDGF/p75 in a panel of PCa cells. The proto-oncogene β-catenin promotes CSC survival in many cancers, including PCa, and several studies have demonstrated a crosstalk between AR and β-catenin signaling pathways contributing to ADT resistance. Given the structural similarities between AR and GR and the importance of GR in therapy resistance, this study was designed to investigate the interaction between GR and β-catenin and its contribution to CSC survival and DTX resistance. Using a panel of PCa cell lines including PC3, DU145, 22rv1, and their corresponding DTX-resistant cell lines, we showed by quantitative immunoblotting that GR is upregulated and β-catenin is activated in PC3 and 22rv1 DTX-resistant cells compared to sensitive cells. Co-immunoprecipitation studies revealed interaction between endogenous GR and β-catenin in these cells. We next assessed the effect of the potent GR ligand dexamethasone (Dex) on CSC maintenance, and observed that Dex treatment of PC3 cells increased tumorsphere number and expression of established CSC markers measured by multicolor flow cytometry. To identify potential GR target genes that could mediate the acquisition of CSC properties and DTX resistance, we performed RNA-sequencing analysis in four different Dex-treated and -untreated PCa cell lines. This analysis revealed several candidate GR target genes differentially regulated in a cell-type dependent manner. To determine the effects of the GR/β-catenin interaction on downstream GR target genes and β-catenin target genes, we performed immunoblotting of specific candidate target genes on lysates from cells treated with Dex alone or in combination with COR-10829 (selective GR inhibitor, and MSAB (β-catenin inhibitor). Interestingly, we also observed that the dual blockade of GR and β-catenin markedly reduced tumorsphere formation in DTX-resistant cells, a phenomenon not observed with either GR or β-catenin inhibition alone. In summary, our findings revealed a novel interaction between GR and β-catenin in PCa cells, and suggest that co-targeting the GR/β-catenin signaling axis in advanced PCa patients could be a potential strategy to overcome therapy resistance. Citation Format: Shannalee R. Martinez, Carlos J. Diaz Osterman, Xin S. Chen, Charles Wang, Lubo Zhang, Carlos A. Casiano. Role of the glucocorticoid receptor/beta-catenin interaction in prostate cancer stem cell survival and chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-260.

Glucocorticoid receptor antagonism prevents microglia-mediated neuronal remodeling and behavioral despair following chronic unpredictable stress

Synaptic deficits and neuronal dystrophy in the prefrontal cortex (PFC) are linked to behavioral and cognitive symptoms in depressed individuals. Preclinical studies indicate that chronic stress causes synaptic deficits on pyramidal neurons in the PFC that contribute to behavioral and cognitive impairments. Our recent work shows that chronic stress provokes microglia-mediated neuronal remodeling via neuronal colony stimulating factor (CSF)-1 signaling, leading to synaptic deficits and depressive-like behaviors. Other reports indicate that elevated corticosterone causes pyramidal neuron atrophy and microglia activation in the medial PFC, implicating glucocorticoid signaling in microglia-mediated neuronal remodeling following chronic stress. In this study, male mice were exposed to chronic unpredictable stress (CUS) and received daily administration of glucocorticoid receptor antagonist RU486 (25 mg/kg, i.p.). As expected, CUS exposure caused adrenal hypertrophy and elevated plasma corticosterone levels. Glucocorticoid receptor blockade prevented behavioral despair and cognitive impairments following CUS. Moreover, RU486 administration diminished CUS-induced CSF1 signaling in the PFC and reduced markers of phagocytosis on purified microglia. Confocal imaging in Thy1-GFP(M) mice showed that CUS increased microglia-mediated neuronal remodeling, and RU486 administration attenuated microglial engulfment of neuronal elements and prevented dendritic spine density deficits on pyramidal neurons following CUS. These results demonstrate that chronic stress-induced glucocorticoid signaling promotes CSF1 signaling and microglia-mediated neuronal remodeling in the medial PFC, which contributes to development of behavioral despair and cognitive impairments. This study presents primary evidence that neuroendocrine responses engage neuron-microglia interactions in the PFC; further implicating microglia in stress-induced neuronal remodeling, PFC dysfunction, and associated behavioral consequences.

OR28-6 Short-Term Mifepristone Treatment Improves Hepatic and Adipose Tissue Insulin Sensitivity in Overweight and Obese Subjects with Glucose Intolerance

Background: Increased tissue cortisol availability and enhanced glucocorticoid action have been implicated in abnormal glucose and fat metabolism in patients with obesity, metabolic syndrome, and type 2 diabetes. Objective: To test the hypothesis that blockade of glucocorticoid receptor (GR) with mifepristone ameliorates insulin resistance in overweight/obese subjects with glucose intolerance. Design: We conducted a randomized, placebo-controlled, double-blind, crossover trial of mifepristone (50 mg orally, every six hr) in overweight/obese subjects (BMI 25-37 kg/m2), with abnormal glucose tolerance (n=16, women=7). Participants (age:56 ± 8 yr) had prediabetes (n=12) or mild type 2 DM (n=4, A1c < 7%) on diet control or stable dose of metformin for 3 months (n=2). Mifepristone or placebo treatment for one week was followed by a 6-8-week washout and crossover to the other arm. At baseline, and after each treatment period, insulin sensitivity was assessed by a frequently sampled intravenous glucose tolerance test (FSIVGTT) and an oral glucose tolerance test (OGTT). Whole body glucose disposal index of insulin sensitivity (SI) was derived using a Minimal model analysis. Adipose tissue-SI, which reflects insulin-induced free fatty acid (FFA) suppression, was estimated by the log-linear slope of FFA levels during the FSIVGTT. Matsuda index, oral glucose insulin sensitivity index (OGIS, ml/min/m2), and hepatic insulin resistance index (HIRI, mg/dL×µU/mL×105) were derived from OGTT. Adipo-IR, a surrogate measure of adipocyte insulin resistance, was calculated by multiplying fasting concentrations of FFA and insulin (mmol×pmol/L). Comparison of various post-treatment parameters was performed using crossover ANOVA. This analysis takes into account specific treatment arm, treatment order, and treatment effects. Thus, we explicitly tested for carryover effects in our crossover study. Results: GR blockade did not affect body weight (p=0.56) or fasting glucose (p=0.67), but significantly decreased fasting insulin levels when compared with placebo [mean (95%CI): -40.7 (-68.53 to -12.91), p=0.007]. Mifepristone administration for one week did not alter whole body glucose disposal indices of insulin sensitivity (FSIVGTT-Si: p=0.52, Matsuda Index: p=0.74; and OGIS: p=0.75). However, GR blockade reduced HIRI [-19.80 (-40.76 to 1.15), p=0.05], Adipo-IR [-15.54 (-31.47 to 0.37), p=0.04], and improved Adipose-SI [-19.75 (-31.41 to -8.08), p=0.003]. Conclusions: In patients with abnormal glucose tolerance, short-term GR blockade improved hepatic and adipose tissue insulin sensitivity without significantly altering whole-body insulin sensitivity. These findings suggest that GR may be a therapeutic target in insulin-resistant states. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Involvement of glucocorticoid and mineralocorticoid receptors in lipid accumulation and depressed G6PD activity in the livers of rats treated with postpartum oral estrogen-progestin.

Postpartum contraception is an important step for preventing closely spaced pregnancy. Combined oral contraceptive (COC) has been linked to cardiometabolic disturbances. We therefore hypothesized that postpartum oral estrogen-progestin use induces hepatic lipid accumulation that is associated with glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation via adenosine deaminase (ADA)/xanthine oxidase (XO)/uric acid (UA)-dependent pathway. Female Wistar rats weighing 130-150g were mated to achieve timed pregnancy and delivery. Twenty-four (24) dams were randomly assigned to receive vehicle (po), COC (1.0μg ethinylestradiol and 5.0μg levonorgestrel; po), COC with GR blockade (mifepristone; 80.0mg/kg; po) and COC with MR blockade (spironolactone; 0.25mg/kg; po) daily between 3rd and 11th week postpartum. Data showed that postpartum COC resulted in glucose dysregulation, increased visceral adiposity, liver weight, plasma corticosterone, aldosterone, circulating and hepatic free fatty acid (FFA), triglyceride (TG), adenosine deaminase (ADA), uric acid production, lactate production, and oxidative marker injury. On the other hand, G6PD-dependent antioxidant defenses were depressed by postpartum COC. However, these effects were attenuated by GR or MR blockade. Our data demonstrate that enhanced G6PD-dependent antioxidant defenses and suppressed ADA/XO/UA pathway in the liver by GR or MR blockade improves glucose dysregulation and hepatic TG accumulation induced by postpartum COC. This study implies a plausible involvement of GR and MR via defective G6PD-dependent antioxidant barrier and increased activity of ADA/XO/UA pathway in postpartum COC-induced hepatic lipid accumulation.

Abstract # 2066 Chronic stress-induced glucocorticoid signaling drives microglia-mediated neuronal remodeling and depressive-like behaviors

Converging evidence indicates that chronic stress elicits neurobiological responses, including pyramidal neuron remodeling and synaptic loss in the medial prefrontal cortex (PFC), that contribute to development of affective disorders. Our recent studies show that chronic stress provokes microglia-mediated neuronal remodeling via increased colony stimulating factor (CSF)-1 signaling in the medial PFC, leading to synaptic deficits and development of depressive-like behaviors. Separate studies show that elevated glucocorticoids cause neuronal remodeling and microglia activation in the medial PFC, implicating glucocorticoid receptor activation in stress-induced CSF1 signaling and microglia-mediated neuronal remodeling in the medial PFC. In the present studies C57BL/6 male mice were exposed to chronic unpredictable stress (CUS) and received daily administration of glucocorticoid receptor antagonist RU486 (25 mg/kg, i.p.). Glucocorticoid receptor blockade prevented depressive-like behaviors and cognitive impairments following CUS. Moreover, RU486 administration dampened CUS-induced CSF1 levels in the PFC and reduced CSF1 receptor along with markers of phagocytosis on sorted microglia. Confocal imaging in Thy1-GFP(M) mice revealed that CUS increased microglia-mediated neuronal remodeling and reduced spine density on apical dendrites of pyramidal neurons in the medial PFC. Notably, RU486 administration reduced microglia engulfment of neuronal elements and prevented dendritic spine density deficits on pyramidal neurons after CUS. These findings demonstrate that chronic stress-induced glucocorticoid signaling drives CSF1 signaling in the medial PFC, which provokes microglia-mediated neuronal remodeling and subsequent depressive-like behaviors and cognitive impairments.

Glucocorticoid receptor signaling during prolonged psychological stress compromises the ability of invariant NKT cells to participate in antitumor immune surveillance

Abstract The nervous system serves critical roles in the regulation of immune responses. Consequently, the neuroimmune functional interface can be disrupted by psychological stress, potentially impeding our ability to combat malignancies. Invariant natural killer T (iNKT) cells are innate-like T cells that, upon activation by glycolipid antigens and/or select environmental cues such as inflammatory cytokines, participate in antitumor immune responses. However, how mediators of stress may impact iNKT cell functions in this context remains unexplored. We subjected BALB/c and/or C57BL/6 mice to prolonged psychological stress, in a well-established model of physical restraint, before administering α-GalCer (a glycolipid agonist of iNKT cells) or a combination of IL-12 and IL-18. We report that stress abrogates IFN-γ production by activated iNKT cells in vivo, an effect that is reversible by blockade of glucocorticoid receptors. Accordingly, α-GalCer-stimulated iNKT cells in stressed mice failed to protect against pulmonary metastasis of B16-F10 melanoma. We also observed that otherwise α-GalCer-transactivated NK cells from stressed mice had a diminished capacity to lyse YAC-1 lymphoma cells. Finally, the suppressive effect of stress on iNKT cells was not due to their apoptotic death. In fact, unlike conventional T cells, iNKT cells were resistant to glucocorticoid-induced apoptosis. Taken together, our findings define a novel mechanism underlying stress-induced immunosuppression, which involves innate-like but not conventional T lymphocytes.

Endotoxin rapidly desensitizes the gonads to kisspeptin-induced luteinizing hormone release in male Siberian hamsters (Phodopus sungorus).

Activation of the immune system induces rapid reductions in hypothalamic-pituitary-gonadal (HPG) axis activity, which in turn decreases secretion of sex steroids. This response is likely adaptive for survival by temporarily inhibiting reproduction to conserve energy; however, the physiological mechanisms controlling this response remain unclear. The neuropeptide kisspeptin is a candidate to mediate the decrease in sex hormones seen during sickness through its key regulation of the HPG axis. In this study, the effects of acute immune activation on the response to kisspeptin were assessed in male Siberian hamsters (Phodopus sungorus). Specifically, an immune response was induced in animals by a single treatment of lipopolysaccharide (LPS), and reproductive hormone concentrations were determined in response to subsequent injections of exogenous kisspeptin. Saline-treated controls showed a robust increase in circulating testosterone in response to kisspeptin; however, this response was blocked in LPS-treated animals. Circulating luteinizing hormone (LH) levels were elevated in response to kisspeptin in both LPS- and saline-treated groups and, thus, were unaffected by LPS treatment, suggesting gonad-level inhibition of testosterone release despite central HPG activation. In addition, blockade of glucocorticoid receptors by mifepristone did not attenuate the LPS-induced inhibition of testosterone release, suggesting that circulating glucocorticoids do not mediate this phenomenon. Collectively, these findings reveal that acute endotoxin exposure rapidly renders the gonads less sensitive to HPG stimulation, thus effectively inhibiting sex hormone release. More broadly, these results shed light on the effects of immune activation on the HPG axis and help elucidate the mechanisms controlling energy allocation and reproduction.

Combined glucocorticoid and mineralocorticoid receptor blockade in the hypothalamic paraventricular nucleus prevents the pathogenesis of depression induced by chronic corticosterone via drinking water

Combined glucocorticoid and mineralocorticoid receptor blockade in the hypothalamic paraventricular nucleus prevents the pathogenesis of depression induced by chronic corticosterone via drinking water

Early life stress-induced alterations in rat brain structures measured with high resolution MRI.

Adverse experiences early in life impair cognitive function both in rodents and humans. In humans this increases the vulnerability to develop mental illnesses while in the rodent brain early life stress (ELS) abnormalities are associated with changes in synaptic plasticity, excitability and microstructure. Detailed information on the effects of ELS on rodent brain structural integrity at large and connectivity within the brain is currently lacking; this information is highly relevant for understanding the mechanism by which early life stress predisposes to mental illnesses.Here, we exposed rats to 24 hours of maternal deprivation (MD) at postnatal day 3, a paradigm known to increase corticosterone levels and thereby activate glucocorticoid receptors in the brain. Using structural magnetic resonance imaging we examined: i) volumetric changes and white/grey matter properties of the whole cerebrum and of specific brain areas; and ii) whether potential alterations could be normalized by blocking glucocorticoid receptors with mifepristone during the critical developmental window of early adolescence, i.e. between postnatal days 26 and 28.The results show that MD caused a volumetric reduction of the prefrontal cortex, particularly the ventromedial part, and the orbitofrontal cortex. Within the whole cerebrum, white (relative to grey) matter volume was decreased and region-specifically in prefrontal cortex and dorsomedial striatum following MD. A trend was found for the hippocampus. Grey matter fractions were not affected. Treatment with mifepristone did not normalize these changes.This study indicates that early life stress in rodents has long lasting consequences for the volume and structural integrity of the brain. However, changes were relatively modest and–unlike behavior- not mitigated by blockade of glucocorticoid receptors during a critical developmental period.

2325

OBJECTIVES/SPECIFIC AIMS: Intestinal stem cells (ISC) primarily act in the repair of ulcerated epithelium, and their proliferative capacity relies on Wnt/β-catenin signaling. However, the role of GCs on basal epithelial cell signaling has not been fully characterized. The objective of this study was to interrogate a mechanism by which steroids may limit ISC activation. GCs inhibit NFκB signaling, which has been shown to play a role in nuclear β-catenin activation in epithelial cells. We hypothesized that GCs limit Wnt/β-catenin signaling required for ISC activation and epithelial restitution by inhibiting NFκB activation in epithelial cells. METHODS/STUDY POPULATION: To examine the effects of GCs on intestinal epithelial cells, we treated a nontransformed human colonic epithelial cell line (NCM460) with dexamethasone and observed the effects on NFκB and Wnt/β-catenin signaling events. We isolated mouse epithelial cells from the distal colon for stem cell culture as 3D “organoids.” We obtained pure epithelial cell preparations from mucosal biopsies isolated from patients treated at GI clinics at the University of Kentucky Chandler Hospital and VA Medical Center, Lexington. Steroid treated patients with equivalent levels of inflammation, but no mucosal ulceration were used as controls. RESULTS/ANTICIPATED RESULTS: In steroid-treated NCM460 cells, we saw an increase in steroid-responsive genes GILZ and SGK1. We saw a significant decrease in transcripts for Wnt target genes, including Axin2 and cmyc; NFκB target genes, including IFNG and IL6; and the shared NFκB and Wnt pathway co-activator CREBBP, despite unchanged transcript levels for β-catenin (CTNNB1). This data was corroborated in 3D stem cell cultures from cells isolated from mouse colon tissue, which had significant decreases in transcripts for stem cell markers Lgr5 and Ascl2, proliferative markers KI67 and PCNA, and Wnt target Axin2. NCM460s transfected with a lentivirus carrying a TCF/LEF luciferase construct showed a 2.5-fold decrease in TNF-stimulated luciferase activity with dexamethasone treatment. Interestingly, this effect can be rescued by glucocorticoid receptor (GR) blockade with RU-486. Intestinal epithelial cells from patient biopsies showed significant decreases in colitis-induced Axin2, p-LRP6 (a positive marker of Wnt Signaling) and nuclear β-catenin, which correlated with decreased p-p65 protein levels. DISCUSSION/SIGNIFICANCE OF IMPACT: Together, these data suggest that steroid therapy inhibits Wnt/β-catenin signaling at multiple levels, and effects stem cell proliferation in pure stem cell cultures. Decreases in TCF/LEF transcriptional activation (nuclear β-catenin’s DNA binding target) can be reversed with steroid receptor blockade with RU-486, suggesting that a receptor level interaction may be occurring. Interestingly, the required co-activator CBP, shared between NFκB and Wnt pathways, has decreased transcription following steroid treatment, which may provide a mechanism for limited Wnt activation following steroid therapy. Although steroids play a significant role in regulating the amount of inflammatory damage that occurs during IBD treatment, our data suggest that they may be limiting pathways required for effective healing as well.

EARLY LIFE STRESS ACCELERATES AMYLOID PATHOLOGY AND COGNITIVE DECLINE IN APPSWE/PS1DE9 MICE, BUT CAN BE RESCUED BY BRIEFLY BLOCKING GLUCOCORTICOID RECEPTORS AT MIDDLE AGE

Exposure to stress or elevated glucocorticoid hormone levels in adult life has been associated with cognitive deficits and an increased risk for Alzheimer's disease (AD). Since exposure to stress during early life enhances stress-responsiveness and lastingly affects cognition in adult life, we here investigated; i) whether early life stress (ELS) affects AD pathology and cognition in adult and middle-aged APPswe/PS1dE9 mice, and ii) whether it is still possible to later rescue these effects by blocking glucocorticoid receptors (GR) at middle age. Transgenic APPswe/PS1dE9 mice were subjected to ELS by housing the dams with limited nesting and bedding material only from postnatal days 2–9 (1,2). At 6 and 12 months of age, different amyloid species, MR/GR, corticosterone and BACE1 levels, and learning and memory performance were determined. The same measures were determined after GRs were blocked at 12 months of age by giving mifepristone for 3 days (3). In 6 and 12 months old offspring, enhanced amyloid-b (Aβ)-40 and -42 levels, enhanced BACE1 levels and cognitive deficits were found, that correlated well with the Aβ42 levels. Surprisingly, blocking GRs for only 3 days at 12 months of age reduced corticosterone levels, reduced hippocampal Ab40/42 and BACE1 levels, and notably, rescued the cognitive deficits in 12 months old APPswe/PS1dE9 mice. This demonstrates that exposure to stress during the sensitive period of early life can modify the emergence of later amyloid pathology, and as such, may increase the vulnerability to AD. The fact that a short treatment with a GR blocker at middle age lastingly reduced Aβ levels and rescued the cognitive deficits after ELS, highlights the therapeutic potential of this drug for reducing amyloid pathology. References 1) Naninck EFG et al., Chronic early life stress alters developmental and adult neurogenesis and impairs cognitive function in mice. Hippocampus 2015. 2) Lesuis SL et al., Positive and negative early life experiences differentially modulate long term survival and amyloid protein levels in a mouse model of Alzheimer's disease. Oncotarget 2016. 3) Krugers HJ et al., Blockade of glucocorticoid receptors rapidly restores hippocampal CA1 synaptic plasticity after exposure to chronic stress. Eur J Neurosci 2006.

Blocking glucocorticoid receptors at adolescent age prevents enhanced freezing between repeated cue-exposures after conditioned fear in adult mice raised under chronic early life stress

Early life adversity can have long-lasting impact on learning and memory processes and increase the risk to develop stress-related psychopathologies later in life. In this study we investigated (i) how chronic early life stress (ELS) - elicited by limited nesting and bedding material from postnatal day 2 to 9 - affects conditioned fear in adult mice and (ii) whether these effects can be prevented by blocking glucocorticoid receptors (GRs) at adolescent age. In adult male and female mice, ELS did not affect freezing behavior to the first tone 24h after training in an auditory fear-conditioning paradigm. Exposure to repeated tones 24h after training also resulted in comparable freezing behavior in ELS and control mice, both in males and females. However, male (but not female) ELS compared to control mice showed significantly more freezing behavior between the tone-exposures, i.e. during the cue-off periods. Intraperitoneal administration of the GR antagonist RU38486 during adolescence (on postnatal days 28–30) fully prevented enhanced freezing behavior during the cue-off period in adult ELS males. Western blot analysis revealed no effects of ELS on hippocampal expression of glucocorticoid receptors, neither at postnatal day 28 nor at adult age, when mice were behaviorally tested. We conclude that ELS enhances freezing behavior in adult mice in a potentially safe context after cue-exposure, which can be normalized by brief blockade of glucocorticoid receptors during the critical developmental window of adolescence.

Attenuation of cold stress-induced exacerbation of cardiac and adipose tissue pathology and metabolic disorders in a rat model of metabolic syndrome by the glucocorticoid receptor antagonist RU486.

Objectives:Chronic stress affects the central nervous system as well as endocrine, metabolic and immune systems. However, the effects of cold stress on cardiovascular and metabolic disorders in metabolic syndrome (MetS) have remained unclear. We recently characterized DahlS.Z-Leprfa/Leprfa (DS/obese) rats, derived from a cross between Dahl salt-sensitive and Zucker rats, as a new animal model of MetS. We have now investigated the effects of chronic cold stress and glucocorticoid receptor (GR) blockade on cardiac and adipose tissue pathology as well as on metabolic parameters in this model.Methods:DS/obese rats were exposed to cold stress (immersion in ice-cold water to a depth of 1–2 cm for 2 h per day) with or without subcutaneous injection of the GR antagonist RU486 (2 mg kg−1day−1) for 4 weeks beginning at 9 weeks of age. Age-matched homozygous lean (DahlS.Z-Lepr+/Lepr+) littermates served as a control.Results:Chronic cold stress exacerbated hypertension as well as left ventricular (LV) hypertrophy, fibrosis and diastolic dysfunction in DS/obese rats in a manner sensitive to RU486 treatment. Cold stress with or without RU486 did not affect body weight or fat mass. In contrast, cold stress further increased cardiac oxidative stress as well as macrophage infiltration and proinflammatory gene expression in LV and visceral fat tissue, with all of these effects being attenuated by RU486. Cold stress also further increased GR and 11β-hydroxysteroid dehydrogenase type 1 mRNA and protein abundance in LV and visceral adipose tissue, and these effects were again inhibited by RU486. In addition, RU486 ameliorated the stress-induced aggravation of dyslipidemia, glucose intolerance and insulin resistance in DS/obese rats.Conclusions:Our results implicate GR signaling in cold stress-induced exacerbation of cardiac and adipose tissue pathology as well as of abnormal glucose and lipid metabolism in a rat model of MetS.

Dual Effect of Catecholamines and Corticosterone Crosstalk on Pineal Gland Melatonin Synthesis

Background/Aim: The nocturnal production of melatonin by the pineal gland is triggered by sympathetic activation of adrenoceptors and may be modulated by immunological signals. The effect of glucocorticoids on nocturnal melatonin synthesis is controversial; both stimulatory and inhibitory effects have been reported. During pathophysiological processes, an increased sympathetic tonus could result in different patterns of adrenoceptor activation in the pineal gland. Therefore, in this investigation, we evaluated whether the pattern of adrenergic stimulation of the pineal gland drives the direction of the glucocorticoid effect on melatonin production. Methods: The corticosterone effect on the pineal hormonal production induced by β-adrenoceptor or β+α₁-adrenoceptor activation was evaluated in cultured glands. We also investigated whether the in vivo lipopolysaccharide (LPS)-induced inhibition of melatonin is dependent on the interaction of glucocorticoids and the α₁-adrenoceptor in adrenalectomized animals and on the in vivo blockade of glucocorticoid receptors (GRs) or the α₁-adrenoceptor. Results: Corticosterone potentiated β-adrenoceptor-induced pineal melatonin synthesis, whilst corticosterone-dependent inhibition was observed when melatonin production was induced by β+α₁-adrenoceptors agonists. The inhibitory effect of corticosterone is mediated by GR, as it was abolished in the presence of a GR antagonist. Moreover, LPS-induced reduction in melatonin nocturnal plasma content was reversed by adrenalectomy and by antagonizing GR or α₁-adrenoceptors. Conclusions: The dual effect of corticosterone on pineal melatonin synthesis is determined by the activation pattern of adrenoceptors (β or β+α₁) in the gland during GR activation, suggesting that increased activation of the sympathetic system and the hypothalamic-pituitary-adrenal axis are necessary for the control of melatonin production during defense responses.

Fibroblast Growth Factor 2 Modulates Hypothalamic Pituitary Axis Activity and Anxiety Behavior Through Glucocorticoid Receptors

BackgroundDespite strong evidence linking fibroblast growth factor 2 (FGF2) with anxiety and depression in both rodents and humans, the molecular mechanisms linking FGF2 with anxiety are not understood. MethodsWe compare 1) mice that lack a functional Fgf2 gene (Fgf2 knockout [KO]), 2) wild-type mice, and 3) Fgf2 KO with adult rescue by FGF2 administration on measures of anxiety, depression, and motor behavior, and further investigate the mechanisms of this behavior by cellular, molecular, and neuroendocrine studies. ResultsWe demonstrate that Fgf2 KO mice have increased anxiety, decreased hippocampal glucocorticoid receptor (GR) expression, and increased hypothalamic-pituitary-adrenal axis activity. FGF2 administration in adulthood was sufficient to rescue the entire phenotype. Blockade of GR in adult mice treated with FGF2 precluded the therapeutic effects of FGF2 on anxiety behavior, suggesting that GR is necessary for FGF2 to regulate anxiety behavior. The level of Egr-1/NGFI-A was decreased in Fgf2 KO mice and was reestablished with FGF2 treatment. By chromatin immunoprecipitation studies, we found decreased binding of EGR-1 to the GR promoter region in Fgf2 KO mice. Finally, we examined anxiety behavior in FGF receptor (FGFR) KO mice; however, FGFR1, FGFR2, and FGFR3 KO mice did not mimic the phenotype of Fgf2 KO mice, suggesting a role for other receptor subtypes (i.e., FGFR5). ConclusionsThese data suggest that FGF2 levels are critically related to anxiety behavior and hypothalamic-pituitary-adrenal axis activity, likely through modulation of hippocampal glucocorticoid receptor expression, an effect that is likely receptor mediated, albeit not by FGFR1, FGFR2, and FGFR3.