Glucocorticoid Receptor Stimulation Research Articles

Genetic glucocorticoid receptor variants differ between ethnic groups but do not explain variation in age of diabetes onset, metabolic and inflammation parameters in patients with type 2 diabetes.

The effect of excess glucocorticoid receptor (GR) stimulation through glucocorticoid medication or cortisol on glucose metabolism is well established. There are genetic GR variants that result in increased or decreased GR stimulation. We aimed to determine the prevalence of genetic GR variants in different ethnic groups in a cohort of patients with type 2 diabetes, and we aimed to determine their association with age of diabetes onset and metabolic and inflammation parameters. A cross-sectional analysis was performed in a multiethnic cohort (n = 602) of patients with established type 2 diabetes. Polymorphisms in the GR gene that have previously been associated with altered glucocorticoid sensitivity (TthIIII, ER22/23EK N363S, BclI and 9β) were determined and combined into 6 haplotypes. Associations with age of diabetes onset, HbA1c, hs-CRP and lipid values were evaluated in multivariate regression models. The prevalence of the SNPs of N363S and BclI was higher in Dutch than in non-Dutch patients. We observed a lower prevalence of the SNP 9β in Dutch, South(East) Asian and Black African patients versus Turkish and Moroccan patients. We did not detect an association between SNPs and diabetes age of onset or metabolic parameters. We only found a trend for lower age of onset and higher HbA1c in patients with 1 or 2 copies of haplotype 3 (TthIIII + 9β). The prevalence of genetic GR variants differs between patients of different ethnic origins. We did not find a clear association between genetic GR variants and age of diabetes onset or metabolic and inflammation parameters. This indicates that the clinical relevance of GR variants in patients with established type 2 diabetes is limited.

Abstract 3080: Characterization of steroid receptor mediated activation of GLI as driving force of breast cancer growth

Abstract Background: Hedgehog (Hh) signaling pathway plays a fundamental role in the early stages of development by regulating morphogenesis. GLI transcription factors are drivers of this signaling pathway by regulating the expression of growth related genes. Hyperactivation of GLI proteins have been associated with several cancers including medulloblastoma, glioblastoma, ovarian, prostate and breast cancers. Our study in breast cancer (BCa) suggests transcriptional activation of GLI3 in estrogen receptor alpha (ERα) positive cells upon estradiol stimulation. Loss of ERα greatly decreases GLI3 protein stability and stimulation with estradiol significantly increases GLI3 stability in ER-positive BCa cells. We have discovered that GLI3 forms nuclear complexes with ERα upon estradiol stimulation and that the loss of GLI3 reduces BCa cells growth. Therefore, we hypothesize that ERα-GLI3 complex orchestrates BCa transcriptome required for cell growth and development. Our study focuses on characterizing ERα mediated activation of GLI3 in BCa cells and the possible compensation by other steroid receptors such as the androgen receptor (AR) and the glucocorticoid receptor (GR) in ER-negative BCa cells. Method: We examined ERα- GLI transcriptional activity by RNA sequencing upon stimulation with estradiol and inhibition of GLI activity by GANT61 in ER-positive MCF7 cells. We also identified ERα domains that are essential for GLI3 binding by immunoprecipitation and proximity ligation assay (PLA). To identify ERα-GLI3 interactome, we have optimized Rapid Immunoprecipitation Mass Spectrometry of Endogenous protein (RIME). We also examined the role of GLI3 in ER-negative BCa cell growth and characterized the role of AR and GR in activating GLI3 by luciferase reporter assay.Results: Inhibition of GLI DNA binding by GANT61 reduced the expression of GLI regulated (CDC20, CDK1, UBE2C) and modified expression of ERα regulated (FOXM1, SPC24, KIF20A, BIRC5) genes, suggesting cooperative role of ERα-GLI3 in mediating growth and metastasis. The optimized RIME assay suggests immunoprecipitation of ERα with GLI3 in BCa cells upon estradiol induction. Immunoprecipitation and PLA studies suggest that ERα-N terminal, DNA binding and C-terminal domains interact with GLI3. We also showed that knockdown of GLI3 reduces growth in ER negative BCa cells and that AR or GR stimulation by dihydrotestosterone and dexamethasone respectively, are important for GLI3 transcriptional activity. Conclusion: Collectively, our results suggest a new role of steroid receptors in regulating GLI oncogenic transcriptional activity in BCa, leading to new therapeutic possibilities for patients. Citation Format: Shabnam Massah, Maria Guo, Jane Foo, Ralph Buttyan, Artem Cherkasov, Nada Lallous. Characterization of steroid receptor mediated activation of GLI as driving force of breast cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3080.

Sex-Dimorphic Glucocorticoid Receptor Regulation of Hypothalamic Primary Astrocyte Glycogen Metabolism: Interaction with Norepinephrine.

Astrocyte glycogen is a critical metabolic variable that impacts hypothalamic control of glucostasis. Glucocorticoid hormones regulate peripheral glycogen, but their effects on hypothalamic glycogen are not known. A hypothalamic astrocyte primary culture model was used to investigate the premise that glucocorticoids impose sex-dimorphic independent and interactive control of glycogen metabolic enzyme protein expression and glycogen accumulation. The glucocorticoid receptor (GR) agonist dexamethasone (DEX) down-regulated glycogen synthase (GS), glycogen phosphorylase (GP)-brain type (GPbb), and GP-muscle type (GPmm) proteins in glucose-supplied male astrocytes, but enhanced these profiles in female. The catecholamine neurotransmitter norepinephrine (NE) did not alter these proteins, but amplified DEX inhibition of GS and GPbb in male or abolished GR stimulation of GPmm in female. In both sexes, DEX and NE individually increased glycogen content, but DEX attenuated the magnitude of noradrenergic stimulation. Glucoprivation suppressed GS, GPbb, and GPmm in male, but not female astrocytes, and elevated or diminished glycogen in these sexes, respectively. Glucose-deprived astrocytes exhibit GR-dependent induced glycogen accumulation in both sexes, and corresponding loss (male) or attenuation (female) of noradrenergic-dependent glycogen build-up. Current evidence for GR augmentation of hypothalamic astrocyte glycogen content in each sex, yet divergent effects on glycogen enzyme proteins infers that glucocorticoids may elicit opposite adjustments in glycogen turnover in each sex. Results document GR modulation of NE stimulation of glycogen accumulation in the presence (male and female) or absence (female) of glucose. Outcomes provide novel proof that astrocyte energy status influences the magnitude of GR and NE signal effects on glycogen mass.

GLUCO- AND MINERALOCORTICOIDS SYNERGISTICALLY DOWNREGULATE BKCA CHANNEL TRANSCRIPTION IN VASCULAR SMOOTH MUSCLE CELLS BUT DO NOT AFFECT THE EPOXYEICOSATRIENOIC ACID PATHWAY

Objective: A major driver of cardiovascular morbidity and mortality in primary aldosteronism (PA) is endothelial dysfunction. Endothelial dysfunction in PA is well established, but the precise mechanism underlying this phenomenon is incompletely investigated. Specifically, the cytochrome P450 monoxygenase (CYP)-dependent epoxyeicosatrienoic (EET) acid pathway has not been studied comprehensively. Promiscuity of mineralocorticoid receptors due to activation by glucocorticoids further complicates understanding of mechanisms. Design and method: Primary human coronary artery smooth muscle and endothelial cells were challenged with aldosterone concentrations typically found in primary aldosteronism (1 nM) in a background of physiological cortisol levels (140 nM). In both cell types, we analysed expression levels of transcripts which are vital for the generation and inactivation of EETs as well as their proposed mode of action. Smooth muscle cell responses to EETs were assessed by calcium imaging with Fura2. Release of EETs by aldosterone and cortisol-treated endothelial cells were measured by high-pressure liquid chromatography followed by tandem mass spectrometry. Results: Glucocorticoids and mineralocorticoids synergistically reduced BKCa potassium channel transcription in smooth muscle cells. Antagonist studies demonstrated that this effect was predominantly related to glucocorticoid receptor stimulation. The aldosterone-mediated decrease in BKCa mRNA levels was not reflected at the protein level. In line with preserved BKCa protein expression, aldosterone treatment did not affect the calcium response of smooth muscle cells to 14,15 EET stimulation. Endothelial cells challenged with aldosterone showed unchanged mRNA levels of the CYP monoxygenases and epoxide hydrolases, which are relevant for the synthesis and degradation of EETs, respectively. Mass spectrometry of supernatants from acetylcholine-stimulated endothelial cells did not reveal significant differences in concentrations of any of the 4 EET regioisomeres or their respective degradation products (dihydroxyeicosatrienoic acids). Conclusions: In endothelial dysfunction related to mineralocorticoid excess, targeting the EET pathway may be a promising therapeutic approach because all constituents of EET signalling appear to be expressed and functionally intact. Soluble epoxide hydrolase inhibition may thus represent a feasible specific therapy of endothelial dysfunction in PA. The effects of glucocorticoids on BKCa channel expression warrant further investigation under conditions such as glucocorticoid co-secretion in PA and Cushing syndrome.

Sexual dimorphic role of the glucocorticoid receptor in chronic muscle pain produced by early-life stress.

Fibromyalgia and other chronic musculoskeletal pain syndromes are associated with stressful early life events, which can produce a persistent dysregulation in the hypothalamic-pituitary adrenal (HPA) stress axis function, associated with elevated plasm levels of corticosterone in adults. To determine the contribution of the HPA axis to persistent muscle hyperalgesia in adult rats that had experienced neonatal limited bedding (NLB), a form of early-life stress, we evaluated the role of glucocorticoid receptors on muscle nociceptors in adult NLB rats. In adult male and female NLB rats, mechanical nociceptive threshold in skeletal muscle was significantly lower than in adult control (neonatal standard bedding) rats. Furthermore, adult males and females that received exogenous corticosterone (via dams’ milk) during postnatal days 2–9, displayed a similar lowered mechanical nociceptive threshold. To test the hypothesis that persistent glucocorticoid receptor signaling in the adult contributes to muscle hyperalgesia in NLB rats, nociceptor expression of glucocorticoid receptor (GR) was attenuated by spinal intrathecal administration of an oligodeoxynucleotide (ODN) antisense to GR mRNA. In adult NLB rats, GR antisense markedly attenuated muscle hyperalgesia in males, but not in females. These findings indicate that increased corticosterone levels during a critical developmental period (postnatal days 2–9) produced by NLB stress induces chronic mechanical hyperalgesia in male and female rats that persists in adulthood, and that this chronic muscle hyperalgesia is mediated, at least in part, by persistent stimulation of glucocorticoid receptors on sensory neurons, in the adult male, but not female rat.

Effects of Chronic Glucocorticoid Receptor Stimulation on Circadian Locomotor Activity and Serotonergic Neurotransmission in the Basolateral Amygdala of Rats.

Clinical studies, especially those in animal models, have provided evidence that chronic stress may play a role in the etiology of psychiatric diseases, such as depression. Because chronic stress activates the hypothalamic-pituitary-adrenal (HPA) axis, resulting in the excessive secretion of glucocorticoids, the chronic stimulation of glucocorticoid receptors (GRs) may be involved in the pathogenesis of depression. To further investigate the relationship between GR activation and depression, we used the synthetic glucocorticoid dexamethasone (DEX) and the GR antagonist mifepristone to examine the effects of chronic GR stimulation on the circadian rhythms of locomotor activity and serotonergic neurotransmission in the basolateral amygdala (BLA) of rats. Chronic treatment with DEX reduced locomotor activity during the dark phase, without changing overall activity patterns. Measuring the basal release of serotonin in the BLA, using in vivo microdialysis, confirmed that chronic treatment with DEX induced serotonergic hypofunction in the BLA. The co-administration of DEX with mifepristone effectively suppressed the depressive-like symptoms caused by chronic treatment with DEX. Our results provided further evidence for a relationship between GR and depression and suggest that the pharmacological blockade of GR may increase the effectiveness of conventional pharmacotherapies used to treat depression.

Extent of cortisol suppression at baseline predicts improvement in HPA axis function during antidepressant treatment

BackgroundA dysregulation in the hypothalamic-pituitary-adrenal (HPA)-axis function has been repeatedly observed in major depressive disorders (MDD). Normalization of this dysregulation, i.e. of cortisol suppression after glucocorticoid receptor (GR)-stimulation, may be mandatory for clinical remission in some patient subgroups. However, there are no biological measures applied in the clinical setting to identify patient subgroups with HPA axis alterations. ObjectiveWe aimed to define a suppression index of cortisol concentrations before and after GR stimulation with dexamethasone to predict the variability in improvement of HPA axis activity during antidepressant treatment. MethodsA modified dexamethasone suppression test (mDST) was performed with blood withdrawal for cortisol and ACTH measurement before and 3 h after 1.5 mg dexamethasone intake at 18:00 in two cohorts of depressed patients treated in a naturalistic setting. The discovery sample consisted of 106 patients, the replication sample of 117 patients. The suppression index was defined as cCORTpreDEXcCORTpostDEX. ResultsThe baseline suppression index explained 27.4 % of the variance in changes of HPA axis activity before and after treatment with antidepressants. Age, cCORTpreDEXcACTHpreDEX at baseline and sex explained further variance up to 56.2 % (stepwise linear regression, p = 7.8e−8). A threshold of the suppression index at baseline was determined by ROC analysis and revealed, that only patients with a maximum index of 2.32 achieved a normalization of the HPA axis activity after antidepressant treatment. In the replication sample, the threshold was 2.86. However, the estimated suppression index was not associated with treatment response. ConclusionFor the first time, by establishing a short-term suppression index of cortisol before and after GR-stimulation a threshold could be identified to predict improvement of HPA axis activity during antidepressant therapy. After replication in further studies this index may help to identify patients who benefit from a specific treatment that targets components of the HPA axis in the future.

Glucocorticoid Receptor Stimulation Resulting from Early Life Stress Affects Expression of DNA Methyltransferases in Rat Prefrontal Cortex.

Early life stress initiates long-term neurobiological changes that affect stress resilience and increased susceptibility to psychopathology. Maternal separation (MS) is used to cause early life stress and it induces profound neurochemical and behavioral changes that last until adulthood. The molecular pathways of how MS affects the regulation of DNA methyltransferases (Dnmt) in brain have not been entirely characterized. We evaluated MS effects on Dnmt1, Dnmt3a and Dnmt3b expression, DNMT enzyme activity and glucocorticoid receptor (GR) recruitment to different Dnmt loci in the prefrontal cortex (PFC) of Wistar rats. We found increased plasma corticosterone levels after MS that were associated with induced Dnmt expression and enzyme activity in rat PFC at post-natal day 15 (PND15). Chromatin immunoprecipitation showed increased binding of GR at the Dnmt3b promoter after MS, suggesting that genomic signaling of GR is an important regulatory mechanism for the induced Dnmt3b expression and DNMT activity. Although GR also binds to Dnmt3a promoter and a putative regulatory region in intron 3 in rat PFC, its expression after maternal separation may be influenced by other mechanisms. Therefore, GR could be a link between early life stress experience and long-term gene expression changes induced by aberrant DNA methylation.

Glucocorticoid receptor signaling in astrocytes is required for aversive memory formation

Stress elicits the release of glucocorticoids (GCs) that regulate energy metabolism and play a role in emotional memory. Astrocytes express glucocorticoid receptors (GR), but their contribution to cognitive effects of GC’s action in the brain is unknown. To address this question, we studied how astrocyte-specific elimination of GR affects animal behavior known to be regulated by stress. Mice with astrocyte-specific ablation of GR presented impaired aversive memory expression in two different paradigms of Pavlovian learning: contextual fear conditioning and conditioned place aversion. These mice also displayed compromised regulation of genes encoding key elements of the glucose metabolism pathway upon GR stimulation. In particular, we identified that the glial, but not the neuronal isoform of a crucial stress-response molecule, Sgk1, undergoes GR-dependent regulation in vivo and demonstrated the involvement of SGK1 in regulation of glucose uptake in astrocytes. Together, our results reveal astrocytes as a central element in GC-dependent formation of aversive memory and suggest their relevance for stress-induced alteration of brain glucose metabolism. Consequently, astrocytes should be considered as a cellular target of therapies of stress-induced brain diseases.

Glucocorticoid receptor activation selectively influence performance of Wistar rats in Y-maze

Glucocorticoid receptors (GR) are ubiquitously expressed in metazoans. Different and contrasting phenotypes have been reported upon their activation. This study investigated the behavioral phenotypes characteristic of GR stimulation in male Wistar rats. Rats in each of the four groups of rats received one of the following treatments: distilled water (control) or one of three doses of dexamethasone (treatment) injected intraperitoneally for 7 days. The Rats were afterwards subjected to the Y maze, the elevated plus maze (EPM), the Morris water maze (MWM), and the novel object recognition (NOR) test. At the end of the study, the animals were anesthetized and neural activity from the prefrontal cortex recorded. Blood was collected via cardiac puncture to evaluate the levels of plasma insulin and glucose, and the prefrontal cortexes excised to determine the levels of insulin, markers of oxidative stress, and calcium in the homogenate.This study showed that treatment with dexamethasone significantly reduced the total and percentage alternation in the Y maze, but had no significant effect on object recognition in the NOR test, long-term and short-term spatial memory in the MWM, or anxiety-like behavior in the EPM. Plasma and brain insulin and calcium levels were elevated moderately following treatment with the lowest dose of dexamethasone. All doses of dexamethasone decreased brain superoxide dismutase and increased lactate dehydrogenase levels. No significant change in neural activity was observed.This study shows that activation of glucocorticoid receptors differentially affects different behavioral paradigms and provides evidence for a role for glucocorticoids in mediating insulin function in the brain.

Cortisol inhibits mTOR signaling in avascular necrosis of the femoral head

BackgroundANFH is a major health problem, to which long lasting and definitive treatments are lacking. The aim of this study is to study RNA alterations attributed to cortisol-induced ANFH.MethodsRat models were stratified into three groups: in vitro group (n = 20) for molecular biological assays, control group (n = 3), and ANFH group induced using lipopolysaccharide and dexamethasone (n = 3). Bone marrow-derived endothelial progenitor cells (BM-EPCs) were extracted from the rats. An RNA expression array was performed on BM-EPCs, and enriched genes were subject to pathway analysis. In vitro studies following findings of array results were also performed using the isolated BM-EPCs.ResultsSignificant alterations in mammalian target of rapamycin (mTOR) and HIF signaling pathways were identified in BM-EPCs of ANFH. By applying cortisol and dexamethasone to BM-EPCs, significant changes in mTOR and HIF elements were identified. The alteration of HIF pathways appeared to be downstream of mTOR signaling. Glucocorticoid receptor (GR) expression was related to glucocorticoid-dependent mRNA expression of mTOR/HIF genes. mTOR-dependent angiogenesis but not anabolism was the target of GR in ANFH. Inhibition of mTOR signaling also induced apoptosis of BM-EPCs via CHOP-dependent DR5 induction in response to GR stimulation.ConclusionDecreased mTOR signaling in response to GR stimulation leading to downregulated HIF pathway as well as increased apoptosis could be the pathophysiology.

Abstract 5213: Aberrant tumor metabolism enables GR takeover in enzalutamide-resistant prostate cancer

Abstract It is well known that castration resistant prostate cancer (CRPC) is still driven by the androgen axis. Therefore, the advent of new AR-targeting drugs, such as Enzalutamide (ENZ), has been beneficial for many patients with CRPC. ENZ is a potent FDA-approved AR antagonist which prolongs survival for patients with CRPC, even after docetaxel chemotherapy. Unfortunately, although ENZ has proven effective clinically, treatment resistance eventually arises in nearly all patients with CRPC. Emerging data suggest that the glucocorticoid receptor (GR) is upregulated in this context, stimulating expression of AR-target genes that permit continued growth despite AR blockade. Hence, we hypothesize that similar to metabolic mechanisms that elicit dihydrotestosterone (DHT) synthesis, which in turn stimulate AR in CRPC, a role for GR in ENZ resistance would be accompanied by a tumor metabolic switch that provides sustained tissue cortisol concentrations which enable GR activation. Here, we found that long-term ENZ treatment sustains tumor cortisol concentrations in models of prostate cancer and patient tissues by using [3H]-cortisol and high performance liquid chromatography. Further investigations with immunoblot, co-immunoprecipitation as well as in vitro ubiquitination assays in ENZ resistant prostate cancer models demonstrated that autocrine motility factor receptor (AMFR), an ubiquitin E3-ligase, and one of its adaptors, Erlin-2, mediate loss of 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2), which sustains tumor cortisol concentrations, resulting in the continuous stimulation of GR and ENZ resistance. Remarkably, reinstatement of 11β-HSD2 expression, or AMFR loss, reverses ENZ resistance in mouse xenograft tumors. Together, these findings reveal a surprising metabolic mechanism of ENZ resistance that may be targeted with a strategy that circumvents a requirement for systemic GR ablation. Citation Format: Jianneng Li. Aberrant tumor metabolism enables GR takeover in enzalutamide-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5213. doi:10.1158/1538-7445.AM2017-5213

Role of glucocorticoid receptor-mediated mechanisms in cocaine memory enhancement

The basolateral amygdala (BLA) is a critical site for the reconsolidation of labile contextual cocaine memories following retrieval-induced reactivation/destabilization. Here, we examined whether glucocorticoid receptors (GR), which are abundant in the BLA, mediate this phenomenon. Rats were trained to lever press for cocaine reinforcement in a distinct environmental context, followed by extinction training in a different context. Rats were then briefly exposed to the cocaine-paired context (to elicit memory reactivation and reconsolidation) or their home cages (no reactivation control). Exposure to the cocaine-paired context elicited greater serum corticosterone concentrations than home cage stay. Interestingly, the GR antagonist, mifepristone (3–10 ng/hemisphere), administered into the BLA after memory reactivation produced a further, dose-dependent increase in serum corticosterone concentrations during the putative time of cocaine-memory reconsolidation but produced an inverted U-shaped dose-effect curve on subsequent cocaine-seeking behavior 72 h later. This effect was anatomically selective, dependent on memory reactivation (i.e., not observed after home cage exposure), and did not reflect protracted hyperactivity. However, the effect was also observed when mifepristone was administered after novelty stress that mimics drug context-induced hypothalamic-pituitary-adrenal (HPA) axis activation without explicit memory reactivation. Together, these findings suggest that, similar to explicit memory retrieval, a stressful event is sufficient to destabilize cocaine memories and permit their manipulation. Furthermore, BLA GR stimulation exerts inhibitory feedback upon HPA axis activation and thus suppresses cocaine-memory reconsolidation.

Aberrant tumor metabolism to enable glucocorticoid receptor takeover in enzalutamide-resistant prostate cancer.

157 Background: Prostate cancer is driven by androgen stimulation of the androgen receptor (AR). The next-generation AR antagonist, enzalutamide, prolongs progression-free and overall survival, but resistance and lethal disease eventually prevail. Emerging data suggest that the glucocorticoid receptor (GR) is upregulated in this context, stimulating expression of approximately 50% of genes normally stimulated by AR, thereby permitting continued growth despite AR blockade. However, countering this mechanism by administration of GR antagonists is problematic because GR is essential for life. Methods: We assessed the effects of enzalutamide on metabolism of cortisol to cortisone in the LAPC4 and VCaP models of prostate cancer using [3H]-cortisol and high performance liquid chromatography. Expression of 11β-hydroxysteroid dehydrogenase-2, encoding the enzyme 11βHSD2, which converts cortisol to cortisone, was assessed by immunoblot, in models of prostate cancer, tissues from patients treated with enzalutamide and prostate tissues treated exogenously with enzalutamide. The effect of shRNA knockdown of the AMFR ubiquitin E3-ligase on 11βHSD2 protein expression and enzyme activity was assessed. Finally, the potential therapeutic effects of 11βHSD2 re-expression on enzalutamide resistance was assessed in xenograft models. Results: Enzalutamide impedes inactivation of cortisol to cortisone, thereby sustaining tumor cortisol concentrations, permitting GR stimulation and enzalutamide resistance. Impeded cortisol inactivation by enzalutamide occurs by way of 11β-HSD2 expression loss in models of prostate cancer, prostate tissues from enzalutamide-treated patients and fresh prostatic tissues treated exogenously with enzalutamide. AMFR mediates loss of 11β-HSD2, which otherwise inactivates cortisol. Remarkably, reinstatement of 11β-HSD2 expression, or AMFR loss, reverses enzalutamide resistance in mouse xenograft tumors. Conclusions: Together, these findings reveal a surprising metabolic mechanism of enzalutamide resistance that may be targeted with a strategy that circumvents a requirement for systemic GR ablation.

Glucocorticoid receptor signalling activates YAP in breast cancer

The Hippo pathway is an oncosuppressor signalling cascade that plays a major role in the control of cell growth, tissue homoeostasis and organ size. Dysregulation of the Hippo pathway leads to aberrant activation of the transcription co-activator YAP (Yes-associated protein) that contributes to tumorigenesis in several tissues. Here we identify glucocorticoids (GCs) as hormonal activators of YAP. Stimulation of glucocorticoid receptor (GR) leads to increase of YAP protein levels, nuclear accumulation and transcriptional activity in vitro and in vivo. Mechanistically, we find that GCs increase expression and deposition of fibronectin leading to the focal adhesion-Src pathway stimulation, cytoskeleton-dependent YAP activation and expansion of chemoresistant cancer stem cells. GR activation correlates with YAP activity in human breast cancer and predicts bad prognosis in the basal-like subtype. Our results unveil a novel mechanism of YAP activation in cancer and open the possibility to target GR to prevent cancer stem cells self-renewal and chemoresistance.

Requisite Role of Basolateral Amygdala Glucocorticoid Receptor Stimulation in Drug Context-Induced Cocaine-Seeking Behavior.

Background:Exposure to cocaine-associated stimuli triggers a robust rise in circulating glucocorticoid levels. Glucocorticoid receptors are richly expressed in the basolateral amygdala, a brain region that controls the reinstatement of cocaine-seeking behavior upon exposure to a previously cocaine-paired environmental context. In the present study, we investigated whether glucocorticoid receptor stimulation in the basolateral amygdala is integral to drug context-induced motivation to seek cocaine in a rat model of drug relapse.Methods:Rats were trained to lever press for cocaine reinforcement in a distinct environmental context and were then given daily extinction training sessions in a different context. At test, the rats received bilateral glucocorticoid receptor antagonist (mifepristone; 3 or 10ng/hemisphere) or vehicle microinfusions into either the basolateral amygdala or the overlying posterior caudate-putamen (anatomical control region). Immediately thereafter, drug-seeking behavior (i.e., nonreinforced lever presses) was assessed in the previously cocaine-paired context and locomotor activity was assessed in a novel context.Results:Intra-basolateral amygdala, but not intra-posterior caudate-putamen, mifepristone dose-dependently attenuated drug context-induced cocaine-seeking behavior relative to vehicle, such that responding was similar to that observed in the extinction context. In contrast, mifepristone treatment did not alter locomotor activity.Conclusions:These findings suggest that basolateral amygdala glucocorticoid receptor stimulation is necessary for drug context-induced motivation to seek cocaine.

A novel actor in skin biology: the mineralocorticoid receptor.

A novel actor in skin biology: the mineralocorticoid receptor.

Abstract 183: Improved Ca-cycling by Dexamethasone Preserves Contractility After Ischemia/Reperfusion in Diabetic Rat Cardiomyocytes

Glucocorticoid receptor (GR) stimulation is essential for normal heart function and dexamethasone (DEX) treatment has been shown to reduce ischemia reperfusion injury (IRI). Prevalence of diabetes is increasing and myocardial infarction in such patients is more severe and risk of complications is augmented. We tested the hypothesis, that GR stimulation with DEX protects diabetic myocardium from IRI by improving Ca cycling. Cardiomyocytes from type 2 diabetic Zucker diabetic fatty rats were isolated and cultured in DEX (10 μg/mL) vs. vehicle for 24h. [Ca] i and fractional shortening (FS) was measured in electrical field-stimulated cardiomyocytes loaded with Fura-2 AM (10 μM). Compared to control, DEX-treated cells display increased Ca transient amplitude (0.33±0.03 vs. 0.46±0.04, P = 0.01, n=6 animals) and FS (fig.) DEX also significantly increased sarcoplasmic reticulum (SR) Ca content (Caffeine, 10 mM) and reduced SR Ca leak measured as Ca spark frequency (SpF), fluo-4 loaded myocytes, confocal microscopy (fig.). To test, whether DEX reduced IRI, rats were treated with DEX (2 mg/kg i.p.) 24h before measurement (vehicle as control). Left ventricular developed pressure (LVDP) was monitored in Langendorff-perfused-hearts during global ischemia for 30 min and reperfusion. During reperfusion, DEX-treated hearts showed significantly greater LVDP (P < 0.05, 2way ANOVA, fig.). In diabetic rats, glucocorticoid receptor stimulation with DEX leads to a more efficient Ca cycling with reduced SR Ca leakage, which translates to an increased contractility on the cellular level and protection against ischemia reperfusion injury in the whole organ.

Corticosterone induces DNA methyltransferases expression in rat cortical neurons

Corticosterone (CORT) is the main glucocorticoid hormone involved in stress responses in rodents. It is established that CORT exerts its effects via glucocorticoid receptor (GR) and mineralocorticoid receptor that regulate downstream gene expression during development and adulthood. In our previous study, we have shown that maternal separation on postnatal day 15 increases DNA methyltransferase (DNMT) 1, 3A and 3B expression levels in rat nucleus accumbens lasting into adulthood (Anier et al. 2014). However, the exact mechanism how maternal separation alters DNMT expression is unclear. We hypothesize that stress-induced GR stimulation may increase the expression levels of DNMTs and alter long-term DNA methylation-demethylation balance in infant rat brain. Our aim is to evaluate the effect of CORT and maternal separation on the expression levels of DNMTs in rat cortex. In rat primary cortical neurons, CORT treatment increased mRNA levels of DNMT3A and DNMT3B. GR antagonist mifepristone significantly decreased CORT-induced DNMTs mRNA levels indicating GR stimulation-dependent upregulation of DNMTs expression. Higher mRNA levels of DNMT1, DNMT3A and DNMT3B in rat cortex at postnatal day 15 and increased plasma CORT levels suggest that elevated CORT upregulates DNMTs expression. Our results indicated that DNMTs are downstream targets of GR-dependent CORT stimulation and early life stress may induce aberrant DNA methylation pattern that could facilitate long-term changes in gene expression.

Stress Switches Cannabinoid Type-1 (CB1) Receptor-Dependent Plasticity from LTD to LTP in the Bed Nucleus of the Stria Terminalis

The bed nucleus of the stria terminalis (BNST) exerts a coordinated modulation of the psychoneuroendocrine responses to stress. However, how acute stress impacts on BNST in vivo plasticity is a crucial question that still remains unanswered. Here, neurons from the anterior portion of the BNST (aBNST) were recorded in vivo during and after stimulation of their medial prefrontal cortical (mPFC) afferents. In C57BL/6N mice, a 1 h restraint stress induced a switch from long-term depression (LTD) to long-term potentiation (LTP) in the aBNST after a 10 Hz mPFC stimulation. This switch was independent from glucocorticoid receptor stimulation. Because the endocannabinoid system regulates aBNST activity, we next examined the role of cannabinoid type-1 receptors (CB1-Rs) in these changes. Mutant mice lacking CB1-Rs (CB1(-/-) mice) displayed a marked deficit in the ability to develop plasticity under control and stress conditions, compared with their wild-type littermates (CB1(+/+) mice). This difference was not accounted for by genetic differences in stress sensitivity, as revealed by Fos immunohistochemistry analyses. Local blockade of CB1-Rs in the aBNST and the use of mutant mice bearing a selective deletion of CB1-Rs in cortical glutamatergic neurons indicated that stress-elicited LTP involved CB1-Rs located on aBNST excitatory terminals. These results show that acute stress reverts LTD into LTP in the aBNST and that the endocannabinoid system plays a key role therein.