Glucocorticoid Receptor Protein Research Articles

Discovery of novel ocotillol derivatives modulating glucocorticoid receptor/NF-κB signaling for the treatment of sepsis

Glucocorticoids (GCs) have been used in the treatment of sepsis because of their potent anti-inflammatory effects. However, their clinical efficacy against sepsis remains controversial because of glucocorticoid receptor (GR) downregulation and side effects. Herein, we designed and synthesized 30 ocotillol derivatives and evaluated their anti-inflammatory activities. Ocotillol 24(R/S) differential isomers were stereoselective in their pharmacological action. Specifically, 24(S) derivatives had better anti-inflammatory activity than their corresponding 24(R) derivatives. Compound 20 most effectively inhibited NO release (85.97% reduction), and it exerted dose-dependent inhibitory effects on interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels. Mechanistic studies revealed that compound 20 reduces the degradation of GR mRNA and GR protein. Meanwhile, compound 20 inhibited the activation of nuclear factor-κB (NF-κB) signaling, thereby inhibiting the nuclear translocation of p65 and attenuating the inflammatory response. In vivo studies revealed that compound 20 attenuated hepatic, pulmonary, and renal pathology damage in mice with sepsis and suppressed the production of inflammatory mediators. These results indicated that compound 20 is a promising lead compound for designing and developing anti-sepsis drugs.

Abstract 6603: A TBX2-driven signaling switch from androgen receptor to glucocorticoid receptor confers enzalutamide resistance in prostate cancer

Abstract Background: Recent studies suggest that glucocorticoid receptor (GR) activation can cause enzalutamide resistance in advanced prostate cancer (PCa) via functional bypass of androgen receptor (AR) signaling. However, the specific molecular mechanism(s) driving this process remain unknown. In an effort to identify drivers of prostate cancer progression, in a previous study, we determined that TBX2, a developmental T-box transcription factor (TF) master regulator, is over-expressed in CRPC and drives bone metastatic progression. A recent report confirmed that TBX2 and GR are two of the four TFs that drive enzalutamide resistance in advanced PCa. Our current study demonstrates that TBX2 with known repressor and activator functions, is the molecular switch that represses AR levels while activating GR expression thereby resulting in the replacement of AR signaling to control tumor growth. Methods: We genetically modulated TBX2 using multiple approaches: a) dominant negative, DN, to block TBX2 (TBX2DN), and b) overexpression, OE, to increase TBX2 expression (TBX2OE), c) shRNA mediated knockdown (shTBX2). RNA-seq was performed, and qRT-PCR, Western blot and immunohistochemical (IHC) analyses were used for validation. Further, we used chromatin immunoprecipitation (ChIP) and site directed mutagenesis (SDM) to confirm TBX2 binding on the AR promoter. We also used co-immunoprecipitation (Co-IP) to determine protein partners of TBX2. Results: Mechanistically, our studies revealed that TBX2 bound to the promoters of both AR and GATA2, an AR coregulator, thereby resulting in a bimodal repression of AR expression. Conversely, TBX2 upregulated GR via direct GR promoter binding and TBX2-GR protein-protein interaction. Together, concurrent repression of the AR and activation of GR resulted in enzalutamide resistance. Importantly, we found that SP2509, an allosteric inhibitor of the demethylase-independent function of LSD1, a TBX2-interacting protein in the COREST complex, can disrupt both TBX2-LSD1 and TBX2-GR protein-protein interactions thereby uncovering a unique mode of SP2509 action in CRPC. Together, our study supports a molecular model of CRPC wherein: 1) TBX2, LSD1 and GR proteins interface with each other, and 2) pharmacological inhibition of LSD1 blocks the TBX2-driven AR-to-GR switch through disruption of TBX2-GR interaction. Conclusions: In summary, our study identifies TBX2 as the molecular switch that drives the AR to GR signaling bypass thereby conferring enzalutamide resistance. Further, our study provides key insights into a potential therapeutic modality for targeting the AR to GR signaling switch via disruption of the TBX2-LSD1 and TBX2-GR protein-protein interactions Citation Format: Sayanika Dutta, Girijesh Kumar Patel, Hamed Khedmatgozar, Daniel Latour, Jonathan Welsh, Manisha Tripathi, Srinivas Nandana. A TBX2-driven signaling switch from androgen receptor to glucocorticoid receptor confers enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6603.

Abstract 1953: Posttranslational modification in glucocorticoid receptor in treatment resistant prostate cancers

Abstract Prostate Cancer (PCa) remains the most common cancer in men in the United States and the second leading cause of cancer-related death in men. An evolving concept that contributes to our understanding of resistance to androgen deprivation therapy (ADT) and AR antagonists in advanced PCa is the ability of cells to turn on compensatory hormone receptor signaling for survival. Preclinical and clinical studies demonstrate that induction of glucocorticoid receptor (GR) expression confers resistance to AR-targeted therapy. Previous studies from our lab demonstrated that the inhibition of AKT signaling blocks pro-oncogenic GR activity and overcomes resistance to AR-targeted therapy. AKT1 is predicted to phosphorylate GR at s134, however, the implications of pGR(s134) in advanced PCa remains unclear. Although ligands (e.g., Dex) activates GR, we have found GR activation modulated by post-translational modification (PTM) and persistent in treatment resistant PCa. Therefore, we hypothesize pro-oncogenic GR activation through AKT1-induced PTM. The rationale is that by inhibiting AKT signaling, we block residual GR signaling that can potentially drive lethal prostate cancers and consequently be re-sensitized to the standard of care. We validated our findings by Western blot analysis, we used an AKT1-specific inhibitor (AKT1i) and observed decreased AKT1 activity and decreased pGR(s134) without affecting total GR protein expression. Functional studies using shRNA against AKT1 showed reduced pGR(s134) expression without affecting the total GR expression Our preliminary data suggests that pro-oncogenic GR activity is modulated through the phosphorylation of GR on s134 by AKT1. Citation Format: Surendra Gulla, Ephraim Gardner, Shreya Shyam Sundar, Praveen Jaiswal, Ning Gao, Lynda Bennet, Suzanne Conzen, Remi Adelaiye-Ogala. Posttranslational modification in glucocorticoid receptor in treatment resistant prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1953.

Dysregulation of Glucocorticoid Receptor Homeostasis and Glucocorticoid-Associated Genes in Umbilical Cord Endothelial Cells of Diet-Induced Obese Pregnant Sheep.

Maternal obesity (MO) is associated with offspring cardiometabolic diseases that are hypothesized to be partly mediated by glucocorticoids. Therefore, we aimed to study fetal endothelial glucocorticoid sensitivity in an ovine model of MO. Rambouillet/Columbia ewes were fed either 100% (control) or 150% (MO) National Research Council recommendations from 60 d before mating until near-term (135 days gestation). Sheep umbilical vein and artery endothelial cells (ShUVECs and ShUAECs) were used to study glucocorticoid receptor (GR) expression and function in vitro. Dexamethasone dose-response studies of gene expression, activation of a glucocorticoid response element (GRE)-dependent luciferase reporter vector, and cytosolic/nuclear GR translocation were used to assess GR homeostasis. MO significantly increased basal GR protein levels in both ShUVECs and ShUAECs. Increased GR protein levels did not result in increased dexamethasone sensitivity in the regulation of key endothelial gene expression such as endothelial nitric oxide synthase, plasminogen activator inhibitor 1, vascular endothelial growth factor, or intercellular adhesion molecule 1. In ShUVECs, MO increased GRE-dependent transactivation and FKBP prolyl isomerase 5 (FKBP5) expression. ShUAECs showed generalized glucocorticoid resistance in both dietary groups. Finally, we found that ShUVECs were less sensitive to dexamethasone-induced activation of GR than human umbilical vein endothelial cells (HUVECs). These findings suggest that MO-mediated effects in the offspring endothelium could be further mediated by dysregulation of GR homeostasis in humans as compared with sheep.

Role of Carbohydrate response element-binding protein in mediating dexamethasone-induced glucose transporter 5 expression in Caco-2BBE cells and during the developmental phase in mice

ABSTRACT Glucose transporter 5 (GLUT5), the main fructose transporter in mammals, is primarily responsible for absorbing dietary fructose in the small intestine. The expression of this intestinal gene significantly increases in response to developmental and dietary cues that reach the glucocorticoid receptor and carbohydrate response element-binding protein (ChREBP), respectively. Our study demonstrates that ChREBP is involved in the dexamethasone (Dex)-induced expression of GLUT5 in Caco-2BBE cells and the small intestine of both wild-type and ChREBP-knockout mice. Dex, a glucocorticoid, demonstrated an increase in GLUT5 mRNA levels in a dose- and time-dependent manner. While the overexpression of ChREBP moderately increased GLUT5 expression, its synergistic increase in the presence of Dex was noteworthy, whereas the suppression of ChREBP significantly reduced Dex-induced GLUT5 expression. Dex did not increase ChREBP protein levels but facilitated its nuclear translocation, thereby increasing the activity of the GLUT5 promoter. In vivo experiments conducted on 14-day-old mice pups treated with Dex for three days revealed that only wild-type mice (not ChREBP-knockout mice) exhibited Dex-mediated Glut5 gene induction, which further supports the role of ChREBP in regulating GLUT5 expression. Collectively, our results provide insights into the molecular mechanisms involved in the regulation of GLUT5 expression in response to developmental and dietary signals mediated by glucocorticoids and ChREBP. General significance: The transcription factor ChREBP is important for Dex-mediated Glut5 gene expression in the small intestine.

Early social isolation differentially affects the glucocorticoid receptor system and alcohol-seeking behavior in male and female Marchigian Sardinian alcohol-preferring rats

Adverse early life experiences during postnatal development can evoke long-lasting neurobiological changes in stress systems, thereby affecting subsequent behaviors including propensity to develop alcohol use disorder. Here, we exposed genetically selected male and female Marchigian Sardinian alcohol-preferring (msP) and Wistar rats to mild, repeated social deprivation from postnatal day 14 (PND14) to PND21 and investigated the effect of the early social isolation (ESI) on the glucocorticoid receptor (GR) system and on the propensity to drink and seek alcohol in adulthood. We found that ESI resulted in higher levels of GR gene and protein expression in the prefrontal cortex (PFC) in male but not female msP rats. In female Wistars, ESI resulted in significant downregulation of Nr3c1 mRNA levels and lower GR protein levels. In male and female msP rats, plasma corticosterone levels on PND35 were similar and unaffected by ESI. Wistar females exhibited higher levels of corticosterone compared with males, independently from ESI. In alcohol self-administration experiments we found that the pharmacological stressor yohimbine (0.0, 0.312, 0.625, and 1.25 mg/kg) increased alcohol self-administration in both rat lines, regardless of ESI. After extinction, 0.625 mg/kg yohimbine significantly reinstated alcohol seeking in female rats only. ESI enhanced reinstatement in female msP rats. Overall, the present results indicate that repeated social deprivation during the third week of postnatal life affects GR expression in a strain- and sex-dependent manner: such effect may contribute, at least partially, to the heightened sensitivity of female msP rats to the effects of yohimbine-induced alcohol seeking.

The effect of cold exposure on the levels of glucocorticoids, 11-hydroxysteroid dehydrogenase 2, and placental vascularization in a rat model.

Cold exposure (CE) before birth is one of the initial stressors that may impact mammalian pregnancy, changing placental and fetal development and affecting the health of the offspring. While glucocorticoids (GCs) participate in the body's response to the stress of CE, the specific mechanisms of their action are unclear. This study aims to determine the effect of CE stress on the placenta and to test whether stress, caused by cold exposure in pregnancy impairs fetal development by changing placental angiogenesis via excessive GC expression. CE rat model was created by exposing 30 SD rats to cold preconception, or during the first, second, and third weeks of pregnancy. Serum cortisol and soluble fms-like tyrosine kinase-1 (sFlt-1) expression levels, physiological index changes (food intake, body weight change and blood pressure), and pregnancy outcomes (fetal rat weight, number of live fetal rats, and placental weight) were collected at baseline and at different time points after the conception. Protein expression levels of 11 β-hydroxysteroid dehydrogenase 2 (11β-HSD2), glucocorticoid receptor, vascular endothelial growth factor A (VEGF-A), placental growth factor (PIGF), and sFlt-1 in placental tissues were measured by western blotting. Cytokeratin (CK) and laminin (LN) in trophoblasts, and α-actin in vascular smooth muscle of the spiral arteries of pregnant rats after the systemic cold treatment were assessed by immunofluorescence and visualized by fluorescent microscopy. To test the effect of 11β-HSD2 levels on the placental recasting, human first-trimester extravillous trophoblast cells (HTR8/SVneo) underwent knockdown using specific 11β-HSD2 siRNA constructs. Expression levels of 11β-HSD2 were analyzed by quantitative real-time PCR (qPCR) and into HTR8 cells, and the expression levels of the 11β-HSD2 gene in each group were measured using qPCR. Cell migration and invasion was assessed by Transwell migration assay, and sFlt-1 levels in HTR8 cells were measured by ELISA. CE pre-conception led to consistently increasing serum corticosterone and sFlt-1 levels throughout pregnancy, and persistently increased diastolic blood pressure (DBP) in rat CE model compared to control animals. CE during the second week of gestation (Gp.3) was associated with significantly lower placental weight (p=0.0003). Cold exposure in the third week (Gp.4) was associated with significantly (p=0.001) lower fetal weight. CE pre-conception was associated with significantly decreased placental levels of 11β-HSD2, glucocorticoid receptor, VEGF-A, PIGF, and sFlt-1 proteins and α-actin compared to the control group. Silencing 11β-HSD2 by siRNA led to reduced cell migrations and invasion, and markedly increased expression levels of sFlt-1 in HTR8/SVneo cells (p<0.05). Pre-conception cold exposure and during early pregnancy leads to increased GCs levels and impaired placental 11β-HSD2 activity. We suggest that the subsequent 11β-HSD2-induced increase in the sFlt-1expression during early pregnancy may affect placental vascular remodeling and change placental morphological structure and function.

Glucocorticoid treatment influences prostate cancer cell growth and the tumor microenvironment via altered glucocorticoid receptor signaling in prostate fibroblasts

Despite significant therapeutic advances in recent years, treatment of metastatic prostate cancer (PCa) remains palliative, owing to the inevitable occurrence of drug resistance. There is increasing evidence that epithelial glucocorticoid receptor (GR) signaling and changes in the tumor-microenvironment (TME) play important roles in this process. Since glucocorticoids (GCs) are used as concomitant medications in the course of PCa treatment, it is essential to investigate the impact of GCs on stromal GR signaling in the TME. Therefore, general GR mRNA and protein expression was assessed in radical prostatectomy specimens and metastatic lesions. Elevated stromal GR signaling after GC treatment resulted in altered GR-target gene, soluble protein expression, and in a morphology change of immortalized and primary isolated cancer-associated fibroblasts (CAFs). Subsequently, these changes affected proliferation, colony formation, and 3D-spheroid growth of multiple epithelial PCa cell models. Altered expression of extra-cellular matrix (ECM) and adhesion-related proteins led to an ECM remodeling. Notably, androgen receptor pathway inhibitor treatments did not affect CAF viability. Our findings demonstrate that GC-mediated elevated GR signaling has a major impact on the CAF secretome and the ECM architecture. GC-treated fibroblasts significantly influence epithelial tumor cell growth and must be considered in future therapeutic strategies.

Integrating Proteomics and Functional Genomics to Identify Targets for Improving Glucocorticoid-Based Treatment of B-Lymphoblastic Leukemia

Glucocorticoids are a critical component of successful combination chemotherapy regimens for B-lymphoblastic leukemia (B-ALL). Despite advances in immunotherapies, glucocorticoid-based chemotherapy continues to be the essential frontline treatment of patients with B-ALL. This approach has led to high cure rates but at the risk of both acute, potentially life-threatening complications and chronic toxicities. Additionally, glucocorticoids remain ineffective for some patients with B-ALL, which predicts poor outcomes. To achieve better outcomes and reduce the toxicity of treatment, we take a functional genomics approach to understand how glucocorticoids induce cell death and identify strategies to make them more effective. Glucocorticoids work through the glucocorticoid receptor (GR), a ligand-activated transcription factor, to induce cell death in B-ALL. By integrating gene expression and RNA interference screening, we have identified both effector genes (genes whose regulation by glucocorticoids contributes to B-ALL cell death) and sources of resistance that can be targeted to improve treatment (Kruth et al Blood 2017). This identified the network of BCL-2 family proteins (e.g., BCL2, BCL2L11, BMF), whose regulation contributes to apoptosis in B-ALL. Surprisingly, it also showed that suppression of B-cell development genes (e.g., EBF1, PAX5, IL7R) contributes to B-ALL cell death. However, only a fraction of the genes that affect the glucocorticoid sensitivity of B-ALL are effector genes. We therefore sought to identify functional interaction partners of GR, defined as proteins that physically interact with GR and influence glucocorticoid sensitivity. To identify proteins that physically interact with GR, we used proximity labeling. We fused an ascorbate peroxidase domain (APEX2) to the N-terminus of GR and expressed it REH cells, a B-ALL cell line that does not express GR protein. After verifying that APEX2-GR restored glucocorticoid sensitivity in the REH cells, we performed proximity labeling in the presence and absence of dexamethasone. After optimizing these reactions, cells were lysed and labeled proteins were isolated using magnetic streptavidin beads and identified by mass spectrometry. We were able to detect more than 4,000 proteins that interact with GR, including hundreds that are enriched upon addition of dexamethasone. To identify functional interaction, we intersected the proteins enriched upon addition of dexamethasone with proteins shown by us to modulate glucocorticoid sensitivity in B-ALL (Kruth et al Blood 2017). Supporting our working model, we found functional interaction partners that are involved in B-cell development and apoptosis. We found that GR interacts with EBF1 and MEF2C, B-cell development transcription factors that restrain glucocorticoid-induced cell death. This indicates that GR activity and glucocorticoid sensitivity is directly blunted by B-cell development, and further supports the model that glucocorticoids restrain B-cell development. The mechanism of how these interactions influence glucocorticoid sensitivity will guide rational targeting to improve treatment of B-ALL both in general and in different B-ALL subtypes. Additionally, the mitochondrial voltage dependent anion channel 2 (VDAC2) also interacts with GR in a dexamethasone-dependent manner and is integral to glucocorticoid-induced cell death. VDAC2 is important for nucleating and perhaps triggering pore formation through BAK and BAX. How GR influences this activity is not known but suggests a non-genomic role of GR in glucocorticoid-induced cell death. In summary, integration of functional genomics and proteomics provides rich data sets that will help understand the mechanism of glucocorticoid-induced B-ALL cell death. Targeting of non-canonical (B-cell development) and non-genomic (VDAC2) functions of GR will allow for optimization of glucocorticoid-based chemotherapy regimens.

Comparative Studies on the Anti-Inflammatory and Apoptotic Activities of Four Greek Essential Oils: Involvement in the Regulation of NF-κΒ and Steroid Receptor Signaling.

Essential oils (EOs) are well-known for their anti-fungal, anti-microbial, anti-inflammatory and relaxing activities. Steroid hormones, especially glucocorticoids, are also well-known for their anti-inflammatory activities and control of the hypothalamus-pituitary-adrenal (HPA) axis and glucose homeostasis. The biological activities of glucocorticoids render them the most widely prescribed anti-inflammatory drugs, despite their adverse side effects. In this study, comparative studies of the anti-inflammatory activities and interference with glucocorticoids receptor (GR) and estrogen receptor (ER) signaling of EOs from Greek Oregano, Melissa officinalis, Lavender and from the Chios Mastic, produced from the Greek endemic mastic tree, were performed in Human Embryonic Kidney 293 (HEK-293) cells. Chios Mastic (Mastiha) and oregano EOs exhibited the highest anti-inflammatory activities. The former showed a reduction in both NF-κB activity and protein levels. Mastic essential oil also caused a reduction in GR protein levels that may compensate for its boosting effect on dexamethasone (DEX)-induced GR transcriptional activation, ending up in no induction of the gluconeogenic phoshoenolpyruvate carboxykinase (PEPCK) protein levels that constitute the GR target. Oregano, Melissa officinalis and lavender EOs caused the suppression of the transcriptional activation of GR. Furthermore, the most active EO, that taken from Melissa officinalis, showed a reduction in both GR and PEPCK protein levels. Thus, the anti-inflammatory and anti-gluconeogenic activities of the EOs were uncovered, possibly via the regulation of GR signaling. Moreover, cytotoxic actions of Melissa officinalis and lavender EOs via the induction of mitochondrial-dependent apoptosis were revealed. Our results highlight these essentials oils' anti-inflammatory and apoptotic actions in relation to their implication on the regulation of steroid hormones' actions, uncovering their potential use in steroid therapy, with many applications in pharmaceutical and health industries as anti-cancer, anti-hyperglycemic and anti-inflammatory supplements.

The potential role of GSK-3β signaling pathway for amelioration actions of ketamine on the PTSD rodent model

RationalePost-traumatic stress disorder (PTSD) is a complex, chronic psychiatric disorder typically triggered by life-threatening events and, as yet, lacks a specialized pharmacological treatment. The potential therapeutic role of ketamine, an N-methyl-D-aspartate receptor antagonist, in mitigating PTSD has been the subject of investigation. ObjectiveThe aim of this study was to elucidate alterations in the glycogen synthase kinase-3β (GSK-3β) signaling pathway in response to ketamine intervention, using the single prolonged stress (SPS) model of PTSD at a molecular level. MethodsPTSD-like symptoms were simulated using the SPS model. Ketamine (10 mg/kg) and GSK-3β antagonist SB216763 (5 mg/kg) were then administered intraperitoneally. Stress-related behavior was evaluated through the open field test (OFT) and the elevated plus maze test (EMPT). Additionally, brain activity was analyzed using quantitative electroencephalography (qEEG). Changes in protein and mRNA expressions of glucocorticoid receptor (GR), brain-derived neurotrophic factor (BDNF), GSK-3β, phosphorylated ser-9 GSK-3β (p-GSK-3β), FK506 binding protein 5 (FKBP5), and corticotropin-releasing hormone (CRH) were assessed in the hypothalamus via western blot and qPCR. ResultsSPS-exposed rats exhibited reduced distance and time spent in the center of the open arms, a pattern divergent from control rats. qEEG readings revealed SPS-induced increases in alpha power, low gamma and high gamma power. Furthermore, SPS triggered an upregulation in the protein and gene expression of GSK-3β, GR, BDNF, p-GSK-3β, and FKBP5, and downregulated CRH expression in the hypothalamus. Ketamine administration following the SPS procedure counteracted these changes by increasing the time spent in the center of the OFT, the distance traversed in the open arms of the EMPT, and mitigating SPS-induced alterations in cerebral cortex oscillations. Moreover, ketamine reduced the protein levels of GSK-3β, GR, p-GSK-3β, and altered the ratio of p-GSK-3β to GSK-3β. Gene expression of GSK-3β, GR, BDNF, and FKBP5 decreased in the SPS-Ket group compared to the SPS-Sal group. ConclusionsKetamine appeared to remediate the abnormal GSK-3β signaling pathway induced by SPS. These findings collectively suggest that ketamine could be a promising therapeutic agent for PTSD symptoms, working through the modulation of the GSK-3β signaling pathway.

Prognostic Value of SGK1 and Bcl-2 in Invasive Breast Cancer.

It is crucial to understand molecular alterations in breast cancer and how they relate to clinicopathologic factors. We have previously shown that the glucocorticoid receptor (GCR) protein expression was reduced in invasive breast carcinoma compared to normal breast tissue. Glucocorticoids, signaling through the GCR, regulate several cellular processes via downstream targets such as serum/glucocorticoid-regulated kinase 1 (SGK1) and B-cell lymphoma 2 (Bcl-2). We measured the expression of SGK1 and Bcl-2, in respective breast cancer tissue arrays, from a multiracial cohort of breast cancer patients. Higher cytoplasmic SGK1 staining was stronger in breast cancer tissue compared to normal tissue, especially in hormone receptor-negative cases. Conversely, the expression of cytoplasmic Bcl-2 was reduced in breast cancer compared to normal tissue, especially in hormone receptor-negative cases. Bcl-2 staining was associated with the self-reported racial/ethnic category, an earlier clinical stage, a lower histological grade, and a higher survival rate. Bcl-2 expression was associated with longer survival in models adjusted for age and race (HR = 0.32, 95% CI: 0.15, 0.65), and Bcl-2 expression remained strongly positively associated with protection from breast cancer death, with additional adjustments for ER/PR status (HR = 0.41, 95% CI: 0.2, 0.85). SGK1 and Bcl-2 may play biological roles in breast cancer development and/or progression.

Mineralocorticoid receptor knockout in Schwann cells alters myelin sheath thickness.

Myelination allows fast and synchronized nerve influxes and is provided by Schwann cells (SCs) in the peripheral nervous system. Glucocorticoid hormones are major regulators of stress, metabolism and immunity affecting all tissues. They act by binding to two receptors, the low-affinity glucocorticoid receptor (GR) and the high-affinity mineralocorticoid receptor (MR). Little is known about the effect of glucocorticoid hormones on the PNS, and this study focuses on deciphering the role of MR in peripheral myelination. In this work, the presence of a functional MR in SCs is demonstrated and the expression of MR protein in mouse sciatic nerve SC is evidenced. Besides, knockout of MR in SC (SCMRKO using Cre-lox system with DesertHedgeHog (Dhh) Cre promoter) was undertaken in mice. SCMRKO was not associated with alterations of performance in motor behavioral tests on 2- to 6-month-old male mice compared to their controls. No obvious modifications of myelin gene expression or MR signaling gene expression were observed in the SCMRKO sciatic nerves. Nevertheless, Gr transcript and GR protein amounts were significantly increased in SCMRKO nerves compared to controls, suggesting a possible compensatory effect. Besides, an increase in myelin sheath thickness was noted for axons with perimeters larger than 15 µm in SCMRKO illustrated by a significant 4.5% reduction in g-ratio (axon perimeter/myelin sheath perimeter). Thus, we defined MR as a new player in peripheral system myelination and in SC homeostasis.

Modelling acute glucocorticoid transcriptome response in human embryonic stem cell derived neural cultures

Our goal is to demonstrate and characterize acute glucocorticoid transcriptome response in human embryonic stem cell (hESC) derived neural cultures. Toward this, we confirmed the differentiation of hESC lines H9 and H1 into post-mitotic neurons and astrocytes, in addition to the expressions of glucocorticoid receptor (GR) protein, and the GR co-chaperone FK506 binding protein 51 (FKBP5). In a series of experiments in hESC-derived neural cultures treated with dexamethasone (Dex) for 6 h, glucocorticoid hormone (GH) response was detected through the transcriptional upregulation of GH-responsive genes, FKBP5 and PER1. Both genes responded to Dex treatment in a dose-dependent fashion, and FKBP5 protein was significantly upregulated after a 12-hour Dex exposure. We additionally examined the transcriptome-wide effects of acute GH exposure in hESC-derived cultures and identified FKBP5 as the most highly up-regulated gene. We identified 30 additional differentially expressed (DE) genes common to cultures derived from both H9 and H1 hESCs whose expression levels changed in both lines with similar magnitudes and direction.

Short-term acute bright light exposure induces a prolonged anxiogenic effect in mice via a retinal ipRGC-CeA circuit

Light modulates mood through various retina-brain pathways. We showed that mice treated with short-term acute bright light exposure displayed anxiety-related phenotypes in a prolonged manner even after the termination of the exposure. Such a postexposure anxiogenic effect depended upon melanopsin-based intrinsically photosensitive retinal ganglion cell (ipRGC) activities rather than rod/cone photoreceptor inputs. Chemogenetic manipulation of specific central nuclei demonstrated that the ipRGC-central amygdala (CeA) visual circuit played a key role in this effect. The corticosterone system was likely to be involved in this effect, as evidenced by enhanced expression of the glucocorticoid receptor (GR) protein in the CeA and the bed nucleus of the stria terminalis and by the absence of this effect in animals treated with the GR antagonist. Together, our findings reveal a non-image forming visual circuit specifically designed for "the delayed" extinction of anxiety against potential threats, thus conferring a survival advantage.

Opposing effects of KDM6A and JDP2 on glucocorticoid sensitivity in T-ALL.

Glucocorticoids (GCs) are the cornerstone of acute lymphoblastic leukemia (ALL) therapy. Although mutations in NR3C1, which encodes the GC receptor (GR), and other genes involved in GC signaling occur at relapse, additional mechanisms of adaptive GC resistance are uncertain. We transplanted and treated 10 primary mouse T-lineage acute lymphoblastic leukemias (T-ALLs) initiated by retroviral insertional mutagenesis with GC dexamethasone (DEX). Multiple distinct relapsed clones from 1 such leukemia (T-ALL 8633) exhibited discrete retroviral integrations that upregulated Jdp2 expression. This leukemia harbored a Kdm6a mutation. In the human T-ALL cell line CCRF-CEM, enforced JDP2 overexpression conferred GC resistance, whereas KDM6A inactivation unexpectedly enhanced GC sensitivity. In the context of KDM6A knockout, JDP2 overexpression induced profound GC resistance, counteracting the sensitization conferred by KDM6A loss. These resistant "double mutant" cells with combined KDM6A loss and JDP2 overexpression exhibited decreased NR3C1 mRNA and GR protein upregulation upon DEX exposure. Analysis of paired samples from 2 patients with KDM6A-mutant T-ALL in a relapsed pediatric ALL cohort revealed a somatic NR3C1 mutation at relapse in 1 patient and a markedly elevated JDP2 expression in the other. Together, these data implicate JDP2 overexpression as a mechanism of adaptive GC resistance in T-ALL, which functionally interacts with KDM6A inactivation.

Epigenetic programming of TBX2/CX43 mediates lower sperm quality in male offspring induced by prenatal dexamethasone exposure.

Decreased sperm quality is the main cause of male infertility. Studies have found that prenatal dexamethasone exposure (PDE) decreases sperm quality in male offspring after birth, but the mechanism is unclear. Wistar pregnant rats were subcutaneously injected with 0.1, 0.2 and 0.4 mg/kg.d dexamethasone at gestational day 9-20. The testes and sperm of first-generation (F1) offspring were collected, and F1 offspring were mated with wild-type female rats to obtain F2. Compared with the control group, F1 offspring in PDE group had lower sperm count and motility after birth, and the deformity rate increased. F2 fetal rats' body length and weight decreased, and the intrauterine growth retardation rate increased. Meanwhile, PDE decreased the expression of connexin 43 (CX43) in offspring testes, while T-box transcription factor 2 (TBX2) promoter region histone 3 lysine 9 acetylation (H3K9ac) level and its expression were increased. Traced back to F1 fetus testes, PDE increased the expression of glucocorticoid receptor (GR) and P300, activated GR protein into the nucleus, and made GR act on the TBX2 promoter region. Further, a series of Sertoli cell interventions confirmed that dexamethasone promoted GR to recruit P300, increased the H3K9ac level of TBX2 promoter region and its expression, and inhibited the expression of CX43. This study confirmed that PDE decreased sperm quality of male offspring, which is related to the epigenetic programming of TBX2/CX43 in the Sertoli cells, provided a theoretical and experimental basis for guiding the rational use of drugs during pregnancy.

Dexamethasone activates c-Jun NH2-terminal kinase (JNK) which interacts with GR and protects it from ubiquitin-mediated degradation in NSCLC cells

Dexamethasone-mediated pharmacological activation of the glucocorticoid receptor (GR) is widely used in the treatment regimen of hematological malignancies and solid cancers. However, DEX sensitivity towards patients primarily depends on the endogenous protein levels of GR. We observed that DEX treatment leads to an increase in GR protein levels despite inhibition of neo-protein synthesis in non-small cell lung cancer (NSCLC) cells. Mechanistically, DEX-stimulation concomitantly increased the JNK phosphorylation and GR protein levels, however the JNK stimulation preceds GR upregulation. Moreover, we also observed that DEX-mediated phosphorylation is partially mediated by upregulation in MEKK1 phosphorylation. Further, GR protein levels were significantly decreased in JNK inhibitor (JNKi, SP600125) treated cells whereas MG132 treatment restored GR levels indicating that DEX induced JNK activity regulated the GR protein levels through proteasomal-degradation pathway. Next, we showed that DEX led to JNK activation which physically interacts with GR and protects it from ubiquitination-mediated degradation. Furthermore, at basal level GR interacts with JNK in cytoplasm whereas upon DEX stimulation GR and pJNK both localized to nucleus and interact with each other. Next, we show that JNK-mediated GR stabilization affects its nuclear transcriptional functional activity in NSCLC cells. In line with these in vitro data, patient dataset analysis also shows that increased levels of both JNK and GR contributes towards better prognosis of NSCLC patients. Taken together, our data shows that DEX treatment may lead to positive feedback regulation of GR by activating JNK and thus highlights importance of GR-JNK crosstalk in NSCLC.

PFO5DoDA disrupts hepatic homeostasis primarily through glucocorticoid signaling inhibition

Legacy per- and polyfluoroalkyl substances (PFASs) are a worldwide health concern due to their potential bioaccumulation and toxicity in humans. A variety of perfluoroether carboxylic acids (PFECAs) have been developed as next-generation replacements of legacy PFASs. However, information regarding their possible environmental and human health risks is limited. In the present study, we explored the effects of PFECAs on mice based on long-term exposure to environmentally relevant doses of perfluoro-3,5,7,9,11-pentaoxadodecanoic acid (PFO5DoDA). Results showed that PFECAs exposure suppressed many cellular stress signals and resulted in hepatomegaly. PFO5DoDA acted as an agonist of the peroxisome proliferator-activated receptor (PPAR) in vitro and modulated PPAR-dependent gene expression in the liver. Importantly, PFECAs had an inhibitory effect on the glucocorticoid receptor (GR), which may contribute to the extensive suppression of stress signals. Of note, the GR suppression induced by PFECAs was not reported by legacy perfluorooctanoic acid (PFOA). PFO5DoDA-induced changes in both GR and PPAR signals remodeled hepatic metabolic profiles, including decreased fatty acids and amino acids and increased β-oxidation. Mechanistically, PFO5DoDA inhibited GR transactivation by degradation of GR proteins. Our results emphasize the potential risk of PFECAs to human health, which were introduced to ease concerns regarding legacy PFASs.

Prolactin and glucocorticoid receptors in the prefrontal cortex are associated with anxiety‐like behavior in prenatally stressed adolescent offspring rats

Prenatal stress (PS) causes anxiety in mothers and their offspring and chewing is a commonly observed behavior during maternal stress. Prolactin (PRL) is an anti-anxiety factor that suppresses the hypothalamic-pituitary-adrenal axis. Here, we studied the roles of PRL, corticosterone (CORT), and their receptors in PS-induced anxiety-like behavior in dams and their offspring. We further investigated whether chewing during maternal stress could prevent PS-induced harmful consequences. Pregnant rats were randomly divided into PS, PS + chewing, and control groups. Anxiety-like behaviors of dams and their adolescent offspring were assessed using the open field test and elevated plus maze. Serum levels of PRL and CORT were measured by ELISA. Expression of mRNA and protein of PRLR and glucocorticoid receptor (GR) in the prefrontal cortex (PFC) were evaluated by qRT-PCR and western blotting, respectively. Compared to the control rats, dams and their female offspring, but not male offspring, in the PS group showed increased anxiety-like behaviors. The PS-affected rats had a lower serum PRL level and increased PRLR expression in the PFC. In contrast, these rats had a higher serum CORT level and decreased GR expression in the PFC. Chewing ameliorated anxiety-like behaviors and counteracted stress-induced changes in serum PRL and CORT, as well as the expression of their receptors in the PFC. Conclusion: PS-induced anxiety-like behavior is associated with changes in the serum levels of PRL and CORT and expression of their receptors in the PFC. Moreover, chewing blunts the hormonal and receptor changes and may serve as an effective stress-coping method for preventing PS-induced anxiety-like behavior.