Glucocorticoid Receptor Knockdown Research Articles

Abstract C096: The GR-LEDGF/p75 transcriptional network as a promising target to reduce therapy cross-resistance in prostate cancer

Abstract Prostate cancer (PCa) is the second-leading cause of cancer mortality in U.S. men, with African American (AA) men showing higher incidence and mortality compared to European American men. In addition, AA men are more likely to be diagnosed with more aggressive PCa resulting in reduction of treatment options, especially as tumors develop therapy resistance. Thus, understanding the mechanisms underlying PCa therapy resistance is crucial for developing new therapeutic options. The upregulation of the Lens Epithelium Derived Growth Factor p75 (LEDGF/p75) by glucocorticoid receptor (GR) is a novel mechanism of PCa therapy resistance recently identified by our group. GR upregulation in PCa bypasses the blockade of androgen receptor by inhibitors such as enzalutamide and abiraterone. LEDGF/p75 is an oncogenic transcription co-activator that promotes DNA damage repair and cancer cell survival against environmental stressors including chemotherapeutic drugs such as docetaxel (DTX). An emerging concern in PCa treatment is therapy cross-resistance, in which the molecular mechanisms of resistance to a particular drug confer resistance to another drug. Our recent studies showed that GR upregulates and interacts with LEDGF/p75 forming a transcriptional complex that includes other oncoproteins such as Menin, JPO2, c-Myc and HRP2, and promotes DTX resistance (DTX-R) in PCa cells. In addition, we reported that both GR and LEDGF/p75 are upregulated in enzalutamide resistant (ENZ-R) PCa cells. We hypothesized that the GR- LEDGF/p75 transcriptional network plays a role in PCa therapy cross-resistance to both ENZ and DTX and is a promising therapeutic target. Cell viability assays revealed that ENZ-R in PCa cells confers cross-resistance to DTX. Western blot analysis revealed upregulation of members of the GR-LEDGF/p75 network such as GR, LEDGF/p75, Menin and c-Myc in ENZ-R PCa cells, consistent with similar observations in DTX-R PCa cells. Upon knockdown of GR and LEDGF/p75 in DTX-R PCa cells, we identified by RNA-seq analysis overlapping DEGs associated with both ENZ-R and DTX-R, suggesting that the GR-LEDGF/p75 transcriptional network may contribute to therapy cross-resistance. Consistent with this, mining of publicly available RNA-seq databases identified 46 overlapping differentially expressed genes (DEGs) between ENZ-R and DTX-R PCa cells. Pathway analysis of DEGs regulated by both GR and LEDGF/p75 revealed significant enrichment of Apoptosis, p53, and DNA repair pathways, which are associated with therapy resistance. Ongoing studies are evaluating the anti-cancer properties and synergistic/additive potential of various inhibitors of the GR-LEDGF/p75 transcriptional network in cellular models of ENZ-R and DTX-R. These studies provide novel mechanistic insights into PCa therapy cross-resistance and may lead to the development of novel therapeutic strategies to reduce PCa mortality disparities. Citation Format: Pedro T. Ochoa, Evelyn S. Sanchez-Hernandez, Kai Wen Cheng, Isaac Kremsky, Charles Wang, Carlos A. Casiano. The GR-LEDGF/p75 transcriptional network as a promising target to reduce therapy cross-resistance in prostate cancer [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C096.

Mechanistic Understanding of Dexamethasone-Mediated Protection against Remdesivir-Induced Hepatotoxicity.

Remdesivir (RDV), a broad-spectrum antiviral agent, is often used together with dexamethasone (DEX) for hospitalized COVID-19 patients requiring respiratory support. Potential hepatic adverse drug reaction is a safety concern associated with the use of RDV. We previously reported that DEX cotreatment effectively mitigates RDV-induced hepatotoxicity and reduces elevated serum alanine aminotransferase and aspartate aminotransferase levels in cultured human primary hepatocytes (HPH) and hospitalized COVID-19 patients, respectively. Yet, the precise mechanism behind this protective drug-drug interaction remains largely unknown. Here, we show that through the activation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling, RDV induces apoptosis (cleavage of caspases 8, 9, and 3), autophagy (increased autophagosome and LC3-II), and mitochondrial damages (decreased membrane potential, respiration, ATP levels, and increased expression of Bax and the released cytosolic cytochrome C) in HPH. Importantly, cotreatment with DEX partially reversed RDV-induced apoptosis, autophagy, and cell death. Mechanistically, DEX deactivates/dephosphorylates p38, JNK, and ERK1/2 signaling by enhancing the expression of dual specificity protein phosphatase 1 (DUSP1), a mitogen-activated protein kinase (MAPK) phosphatase, in a glucocorticoid receptor (GR)-dependent manner. Knockdown of GR in HPH attenuates DEX-mediated DUSP1 induction, MAPK dephosphorylation, as well as protection against RDV-induced hepatotoxicity. Collectively, our findings suggest a molecular mechanism by which DEX modulates the GR-DUSP1-MAPK regulatory axis to alleviate the adverse actions of RDV in the liver. SIGNIFICANCE STATEMENT: The research uncovers the molecular mechanisms by which dexamethasone safeguards against remdesivir-associated liver damage in the context of COVID-19 treatment.

Glucocorticoid receptors involved in ginsenoside compound K ameliorate adjuvant arthritis by inhibiting the glycolysis of fibroblast-like synoviocytes via the NF-κB/HIF-1α pathway

Context Ginsenoside metabolite compound K (CK) is an active metabolite produced by ginsenosides in vivo that has an anti-arthritic effect related to the glucocorticoid receptor (GR). However, the potential mechanisms of CK remain unclear. Objective This study explores the role and potential mechanisms of CK in vivo and in vitro. Materials and methods Adjuvant arthritis (AA) model was induced in Sprague-Dawley (SD) rats; the rats were randomly divided into four groups (n = 10): normal, AA, CK (80 mg/kg), and dexamethasone (Dex) group (1 mg/kg). From day 15, rats were treated with CK (once a day, i.g.) and Dex (once every 3 days, i.p.) for 18 days. To further verify the mechanism of CK, fibroblast-like synoviocytes (FLS) were stimulated by tumour necrosis factor α (TNF-α) to establish an inflammatory model in vitro. Results CK (80 mg/kg) reduced paw swelling (52%) and arthritis global assessment (31%) compared to that in AA rats. In addition, CK (80 mg/kg) suppressed GLUT1 (38%), HK2 (50%), and PKM2 (56%) levels compared with those in AA FLS. However, the effects of CK (30 μM) on these events were weakened or enhanced after GR knockdown or overexpression in FLS stimulated by TNF-α (30 ng/mL). CK (80 mg/kg) also downregulated the expression of P65 (61%), p-IκB (92%), and HIF-1α (59%). Discussion and conclusions The inhibition of CK on glycolysis and the NF-κB/HIF-1α pathway is potentially mediated through activating GR. These findings provide experimental evidence for elucidating the molecular mechanism of CK in treating rheumatoid arthritis (RA).

Abstract A011: TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer

Abstract Background: A major obstacle in the treatment of metastatic castrate resistant prostate cancer (mCRPC) is acquired resistance to androgen deprivation therapy (ADT). It is now recognized that ADT, particularly the 2nd generation androgen receptor (AR) antagonists, such as enzalutamide, orchestrate plasticity changes/molecular alterations leading to therapy resistance. It is hypothesized in the field that the emergence of castrate resistance to 2nd generation ADT is driven by a switch/bypass from AR signaling to the glucocorticoid receptor (GR) signaling. Therefore, identifying the molecular mechanisms that concurrently drive the loss of AR and gain of GR signaling may be crucial in devising novel and effective therapeutic modalities against mCRPC. We have previously reported that TBX2, a T-box transcription factor (TF) with both repressor and activator functions, is over-expressed in CRPC and that TBX2 drives PCa bone metastatic progression. In agreement with our findings, a recent report showed that TBX2 is a key TF that drives plasticity associated with CRPC. In this study, we investigated the molecular mechanisms by which TBX2 drives plasticity associated with enzalutamide resistance. Methods: We genetically modulated TBX2 using the dominant negative (DN), sh-RNA, and overexpression (OE) approaches. RNA-seq was performed, and qRT-PCR, Western blot and immunohistochemical (IHC) analyses were used for validation. Further, we performed chromatin immunoprecipitation (ChIP) and site directed mutagenesis (SDM). Results: Using publicly available databases, we observed a negative correlation between TBX2 and AR; and a positive correlation between TBX2 and GR. Strikingly, blocking TBX2 expression in wildtype PC3 human PCa cells that do not express AR mRNA or protein led to a marked elevation of AR signature as assessed by RNA-seq analysis. These results were confirmed/validated at the protein level using additional human PCa cell lines. Further, these results were validated using all three approaches to genetically modulate TBX2, i.e. DN, shRNA, and OE. Further, ChIP and SDM analyses confirmed that TBX2 directly binds and transcriptionally represses AR. Conversely, genetic modulation of TBX2 was positively associated with GR expression, and TBX2 was found to directly bind to the GR promoter. Further, we found that knock-down of GR in LNCaP cells overexpressing TBX2 (LNCaPTBX2OE) reversed enzalutamide resistance that is associated with elevated TBX2 expression. Conclusions: Our studies suggest that TBX2 acts as the molecular switch that on one hand represses AR and on the other hand activates GR. Further our study paves the way for potential therapeutic strategies against the AR/GR switch/bypass in CRPC wherein AR and GR signaling could be co-inhibited through targeting TBX2. Citation Format: Sayanika Dutta, Girijesh Patel, Hamed Khedmatgozar, Daniel Latour, Manisha Tripathi, Srinivas Nandana. TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A011.

Abstract 1445: TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer

Abstract Background: A major obstacle in the treatment of metastatic castrate resistant prostate cancer (mCRPC) is acquired resistance to androgen deprivation therapy (ADT). It is now recognized that ADT, particularly the 2nd generation androgen receptor (AR) antagonists, such as enzalutamide, orchestrate plasticity changes/molecular alterations leading to therapy resistance. It is hypothesized in the field that the emergence of castrate resistance to 2nd generation ADT is driven by a switch/bypass from AR signaling to the glucocorticoid receptor (GR) signaling. Therefore, identifying the molecular mechanisms that concurrently drive the loss of AR and gain of GR signaling may be crucial in devising novel and effective therapeutic modalities against mCRPC. We have previously reported that TBX2, a T-box transcription factor (TF) with both repressor and activator functions, is over-expressed in CRPC and that TBX2 drives PCa bone metastatic progression. In agreement with our findings, a recent report showed that TBX2 is a key TF that drives plasticity associated with CRPC. In this study, we investigated the molecular mechanisms by which TBX2 drives plasticity associated with enzalutamide resistance. Methods: We genetically modulated TBX2 using the dominant negative (DN), sh-RNA, and overexpression (OE) approaches. RNA-seq was performed, and qRT-PCR, Western blot and immunohistochemical (IHC) analyses were used for validation. Further, we performed chromatin immunoprecipitation (ChIP) and site directed mutagenesis (SDM). Results: Using publicly available databases, we observed a negative correlation between TBX2 and AR; and a positive correlation between TBX2 and GR. Strikingly, blocking TBX2 expression in wildtype PC3 human PCa cells that do not express AR mRNA or protein led to a marked elevation of AR signature as assessed by RNA-seq analysis. These results were confirmed/validated at the protein level using additional human PCa cell lines. Further, these results were validated using all three approaches to genetically modulate TBX2, i.e. DN, shRNA, and OE. Further, ChIP and SDM analyses confirmed that TBX2 directly binds and transcriptionally represses AR. Conversely, genetic modulation of TBX2 was positively associated with GR expression, and TBX2 was found to directly bind to the GR promoter. Further, we found that knock-down of GR in LNCaP cells overexpressing TBX2 (LNCaPTBX2OE) reversed enzalutamide resistance that is associated with elevated TBX2 expression. Conclusions: Our studies suggest that TBX2 acts as the molecular switch that on one hand represses AR and on the other hand activates GR. Further our study paves the way for potential therapeutic strategies against the AR/GR switch/bypass in CRPC wherein AR and GR signaling could be co-inhibited through targeting TBX2. Citation Format: Sayanika Dutta, Girijesh Patel, Hamed Khedmatgozar, Daniel Latour, Manisha Tripathi, Srinivas Nandana. TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer [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 1445.

Abstract P2-20-01: Patient-derived triple-negative breast cancer organoids as a platform for glucocorticoid receptor- mediated mechanisms of immunotherapy response and resistance

Abstract Background: In early-stage triple-negative breast cancer (TNBC), high glucocorticoid receptor (GR) expression correlates with worse relapse-free survival across TNBC subtypes, induces oncogenic gene expression and tumor cell survival, and potentiates anti-inflammatory or an anti-immunogenic phenotype. Immune checkpoint therapy has increased pathologic complete response rates in the neoadjuvant setting, however response to immunotherapy remains modest overall and the explanatory mechanisms for this phenomenon are incompletely understood. We sought a three-dimensional model of the TNBC immune microenvironment using patient-derived organoids (PDOs) that capture tumor heterogeneity, partially recapitulates tumor microenvironment, and can be established for longer-term in vitro/in-vivo translational study after exposure to immunotherapy. We hypothesize that during chemoimmunotherapy treatment of early-stage triple-negative breast cancer, high glucocorticoid receptor activity mediates an immunosuppressive phenotype and glucocorticoid receptor modulation can restore anti-tumor immunity. Methods and Results: We are successfully growing 5 PDOs out of 8 surgically resectedion samples tumors from patients with early-stage triple-negative breast cancer treated with chemotherapy vs. chemoimmunotherapy as per KEYNOTE-522. Interrogation of complementary TNBC cell line data revealed that GR activation downregulates the expression of immune checkpoint genes (PD-L1, B7-H3, B7-H4), while GR knockdown or treatment with a selective GR modulator restores expression of these immune checkpoint genes. TNBC-intrinsic GR activation increases the proportion of regulatory T cells vs. CD8+ T cells. In a coculture system of TNBC spheroids and T cells, GR activation in TNBC results in decreased activated CD8+ CD137+ T cells and use of the selective GR modulator increases the proportion of activated CD8+ CD137+ T cells. Conclusions and Future Directions: In early-stage triple-negative breast cancer, GR activation downregulates expression of immune checkpoint genes and reduction of GR activity restores expression. In a coculture system that recapitulates T cell repertoire found in triple-negative breast cancer, tumor-intrinsic GR activation results in increased regulatory T cell population and reduction of GR activity increases activated CD8+ CD137+ T cells. Ongoing analyses of TNBC organoid-immune coculture systems modeling high GR states will be used to (i) define cooperativity amongst immune checkpoint proteins in costimulation vs. coinhibition of anti-tumor responses and (ii) define novel pairs of receptor-ligands amongst TNBC:CD8+ vs. TNBC:Treg vs. CD8+:Treg as discovery of potential new therapeutic targets. Citation Format: Christine Shiang, Candace Frerich, Ishrat Durdana, Cheryl Lewis, Lynda B. Bennett, Carlos Arteaga, Suzanne D. Conzen. Patient-derived triple-negative breast cancer organoids as a platform for glucocorticoid receptor- mediated mechanisms of immunotherapy response and resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-01.

Hippocampal glucocorticoid receptors modulate status epilepticus severity

Status epilepticus (SE) is a life-threatening medical emergency with significant morbidity and mortality. SE is associated with a robust and sustained increase in serum glucocorticoids, reaching concentrations sufficient to activate the dense population of glucocorticoid receptors (GRs) expressed among hippocampal excitatory neurons. Glucocorticoid exposure can increase hippocampal neuron excitability; however, whether activation of hippocampal GRs during SE exacerbates seizure severity remains unknown. To test this, a viral strategy was used to delete GRs from a subset of hippocampal excitatory neurons in adult male and female mice, producing hippocampal GR knockdown mice. Two weeks after GR knockdown, mice were challenged with the convulsant drug pilocarpine to induce SE. GR knockdown had opposing effects on early vs late seizure behaviors, with sex influencing responses. For both male and female mice, the onset of mild behavioral seizures was accelerated by GR knockdown. In contrast, GR knockdown delayed the onset of more severe convulsive seizures and death in male mice. Concordantly, GR knockdown also blunted the SE-induced rise in serum corticosterone in male mice. GR knockdown did not alter survival times or serum corticosterone in females. To assess whether loss of GR affected susceptibility to SE-induced cell death, within-animal analyses were conducted comparing local GR knockdown rates to local cell loss. GR knockdown did not affect the degree of localized neuronal loss, suggesting cell-intrinsic GR signaling neither protects nor sensitizes neurons to acute SE-induced death. Overall, the findings reveal that hippocampal GRs exert an anti-convulsant role in both males and females in the early stages of SE, followed by a switch to a pro-convulsive role for males only. Findings reveal an unexpected complexity in the interaction between hippocampal GR activation and the progression of SE.

Profound Effects of Dexamethasone on the Immunological State, Synthesis and Secretion Capacity of Human Testicular Peritubular Cells

The functions of human testicular peritubular cells (HTPCs), forming a small compartment located between the seminiferous epithelium and the interstitial areas of the testis, are not fully known but go beyond intratesticular sperm transport and include immunological roles. The expression of the glucocorticoid receptor (GR) indicates that they may be regulated by glucocorticoids (GCs). Herein, we studied the consequences of the GC dexamethasone (Dex) in cultured HTPCs, which serves as a unique window into the human testis. We examined changes in cytokines, mainly by qPCR and ELISA. A holistic mass-spectrometry-based proteome analysis of cellular and secreted proteins was also performed. Dex, used in a therapeutic concentration, decreased the transcript level of proinflammatory cytokines, e.g., IL6, IL8 and MCP1. An siRNA-mediated knockdown of GR reduced the actions on IL6. Changes in IL6 were confirmed by ELISA measurements. Of note, Dex also lowered GR levels. The proteomic results revealed strong responses after 24 h (31 significantly altered cellular proteins) and more pronounced ones after 72 h of Dex exposure (30 less abundant and 42 more abundant cellular proteins). Dex also altered the composition of the secretome (33 proteins decreased, 13 increased) after 72 h. Among the regulated proteins were extracellular matrix (ECM) and basement membrane components (e.g., FBLN2, COL1A2 and COL3A1), as well as PTX3 and StAR. These results pinpoint novel, profound effects of Dex in HTPCs. If transferrable to the human testis, changes specifically in ECM and the immunological state of the testis may occur in men upon treatment with Dex for medical reasons.

Upregulation of hepatic CD36 via glucocorticoid receptor activation contributes to dexamethasone-induced liver lipid metabolism disorder in mice

Glucocorticoids such as dexamethasone (DEX) are widely prescribed to treat numerous conditions and diseases. However, glucocorticoid-induced liver lipid metabolism disorder, even nonalcoholic fatty liver disease, has caused extensive attention. Since fatty acid transporters such as CD36 and FATP play crucial roles in hepatic fatty acid uptake, this work examined their potential involvement in DEX-induced liver lipid accumulation. Chronic DEX administration (1–5 mg/kg/day over 28 days) induced hepatic lipid accumulation in mice. Fatty acid uptake in HepG2 cells and mouse primary hepatocytes was also stimulated after incubation with 0.5–2 μM DEX. Meanwhile, qPCR and western blotting demonstrated dose-dependent upregulation of CD36 expression by DEX in the mouse liver and in cultured hepatocytes. Glucocorticoid receptor (GR) inhibition with mifepristone (RU486) and siRNA-mediated GR knockdown attenuated lipid accumulation in hepatocytes by inhibiting DEX-induced CD36 upregulation, and direct binding of GR to the CD36 promoter was demonstrated by luciferase reporter and chromatin immunoprecipitation assays. These results indicate that DEX promotes free fatty acid uptake leading to hepatic steatosis by upregulating CD36 expression via activation of GR. Thus, strategies aimed at inhibiting GR/CD36 expression or activity might help prevent or reduce the onset and progression of hepatic lipid metabolism disorders induced by glucocorticoid drugs.

Glucocorticoid receptors participate in epilepsy in FCDII patients and MP model rats: A potential therapeutic target for epilepsy in patients with focal cortical dysplasia II (FCDII)

ABSTRACT Background Glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs) are involved in neuronal excitability, neurogenesis, and neuroinflammation. However, the roles of GRs and MRs in epilepsy in focal cortical dysplasia II (FCDII) have not been reported. Research Design and Methods We evaluated GRs and MRs expression and distribution in FCDII patients and methylazoxymethanol-pilocarpine-induced epilepsy model rats (MP rats), and the effects of a GR agonist on neurons in human FCDII and investigated the electrophysiological properties of rats’ neurons after lentivirus-mediated GR knockdown or overexpression and GR agonist or antagonist administration. Results GR expression (not MR) was decreased in specimens from FCDII patients and model rats. GR agonist dexamethasone reduced neuronal excitatory transmission and increased neuronal inhibitory transmission in FCDII. GR knockdown increased the excitability of cultured neurons, and GR overexpression rescued the hyperexcitability of MP-treated neurons. Moreover, dexamethasone decreased neuronal excitability and excitatory transmission in MP rats, while GR antagonist exerted the opposite effects. Dexamethasone reduced the seizure number and duration by approximately 85% and 60% in MP rats within one to two hours. Conclusions These results suggested that GRs play an important role in epilepsy in FCDII and GR activation may have protective and antiepileptic effects in FCDII.

Irilone, a Red Clover Isoflavone, Combined with Progesterone Enhances PR Signaling through the Estrogen and Glucocorticoid Receptors.

Trifolium pratense L. (red clover) is a popular botanical supplement used for women's health. Irilone isolated from red clover previously demonstrated progestogenic potentiation activity. In this study, irilone enhanced progesterone signaling was determined to not occur due to post-translational phosphorylation or by reducing progesterone receptor (PR) protein levels but instead increased PR protein levels in T47D breast cancer cells, which could be blocked by estrogen receptor (ER) antagonists, suggesting an ER dependent effect. Further, irilone increased luciferase activity from a hormone responsive element in a cell line that lacked ER and PR but expressed the glucocorticoid receptor (GR). A siRNA knockdown of GR in Ishikawa PR-B endometrial cancer cells reduced irilone's ability to enhance progesterone signaling. In an ovariectomized CD-1 mouse model, irilone did not induce uterine epithelial cell proliferation. The mechanism of action of irilone gives insight into PR crosstalk with other steroid hormone receptors, which can be important for understanding botanicals that are used for women's health.

A Novel Methodology Using Dexamethasone to Induce Neuronal Differentiation in the CNS-Derived Catecholaminergic CAD Cells.

The Cath.a-differentiated (CAD) cell line is a central nervous system-derived catecholaminergic cell line originating from tyrosine hydroxylase (TH)-producing neurons located around the locus coeruleus area of the mouse brain. CAD cells have been used as an in vitro model for cellular and molecular studies due to their ability to differentiate under serum-free media conditions. However, the lack of serum-derived survival factors, limits the longevity for differentiated CAD cells to be maintained in healthy conditions; thereby, limiting their use in long-term culture studies. Here, we present a novel differentiation method that utilizes dexamethasone (Dex), a synthetic glucocorticoid receptor agonist. Specifically, we discovered that the addition of 100µM of Dex into the 1% fetal bovine serum (FBS)-supplemented media effectively induced neuronal differentiation of CAD cells, as characterized by neurite formation and elongation. Dex-differentiated CAD cells exited the cell cycle, stopped proliferating, extended the neurites, and expressed neuronal markers. These effects were dependent on the glucocorticoid receptors (GR) as they were abolished by GR knockdown. Importantly, Dex-differentiated CAD cells showed longer survival duration than serum-free differentiated CAD cells. In addition, RNA-sequencing and qPCR data demonstrate that several genes involved in proliferation, neuronal differentiation, and survival pathways were differentially expressed in the Dex-differentiated cells. This is the first study to reveal Dex as a novel differentiation methodology used to generate postmitotic neuronal CAD cells, which may be utilized as an in vitro neuronal model for cellular and molecular neurobiology research.

Knockdown of the astrocytic glucocorticoid receptor in the central nucleus of the amygdala diminishes conditioned fear expression and anxiety

The amygdala is a key structure involved in both physiological and behavioural effects of fearful and stressful stimuli. The central stress response is controlled by the activity of the hypothalamic-pituitary-adrenal (HPA) axis via glucocorticoid hormones, acting mainly through glucocorticoid receptors (GR), widely expressed among different brain regions, including the central nucleus of the amygdala (CeA). Although to date, neuronal GR was postulated to be involved in the mediating stress effects, increasing evidence points to the vital role of glial GR. Here, we aimed to evaluate the role of astrocytic GR in CeA in various aspects of the stress response. We used a lentiviral vector to disrupt an astrocytic GR in the CeA of Aldh1l1-Cre transgenic mice. Astrocytic GR knockdown mice (GR KD) exhibited an attenuated expression of fear-related memory in the fear conditioning paradigm. Interestingly, the consolidation of non-stressful memory in the novel object recognition test remained unchanged. Moreover, GR KD group presented reduced anxiety, measured in the open field test. However, knockdown of astrocytic GR in the CeA did not affect an acute response to stress in the tail suspension test. Taken together, obtained results suggest that astrocytic GR in the CeA promotes aversive memory consolidation and some aspects of anxiety behaviour.

Glucocorticoid Receptor Maintains Vasopressin‐Regulated Water Reabsorption Pathway in the Kidney Collecting Duct Cells

Aquaporin‐2 (AQP2) is a transmembrane protein expressed in the principal cells of the kidney collecting ducts, which mediate osmotic water reabsorption. AQP2 is regulated by the antidiuretic peptide hormone arginine vasopressin (AVP). When AVP binds to the vasopressin type 2 receptor (V2R) in the principal cells, it initiates a signaling network that results in 1) AQP2 trafficking to the apical plasma membrane for water transport and 2) AQP2 gene expression. In a previous study, we found that dexamethasone, a glucocorticoid receptor (GR) agonist, enhanced AVP‐induced AQP2 gene expression in the collecting duct cell model mpkCCD. Here, we showed that GR knockdown drastically decreased AVP‐induced AQP2 mRNA and protein levels, indicative of a role of GR in AVP‐induced AQP2 gene expression. We further showed that the 1,000bp region upstream to the AQP2 transcription start site was sufficient to mediate GR‐enhanced AVP‐induced promoter activity and that GR bound to the above AQP2 promoter region. RNA‐seq was used to profile the transcriptome of the GR knockdown vs. control cells. A total of 93,308 transcripts belonging to 22,025 genes were quantified. GR knockdown decreased expression of 5,266 genes and increased expression of 3,709 genes. These two gene groups respectively represent positive and negative regulators for the AVP responses. Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis showed 62 pathways associated with the positive regulators and 48 pathways associated with the negative regulators. The AVP‐regulated water reabsorption pathway was among the 62 positive pathways whose gene expression was reduced upon GR knockdown. Quantitative RT‐PCR analysis showed about 60% reduction in the V2R mRNA level upon GR knockdown. Taken together, our results implicated that GR in one hand potentiates AVP‐induced AQP2 gene expression and in the other hand maintains expression of genes involved in the AVP‐regulated water reabsorption pathway in the kidney collecting duct cells.Support or Funding InformationThis work was supported by the Ministry of Science and Technology, Taiwan (107‐2320‐B‐002‐057‐MY3).

ETS Proteins Bind with Glucocorticoid Receptors: Relevance for Treatment of Ewing Sarcoma

SummaryThe glucocorticoid receptor (GR) acts as a ubiquitous cortisol-dependent transcription factor (TF). To identify co-factors, we used protein-fragment complementation assays and found that GR recognizes FLI1 and additional ETS family proteins, TFs relaying proliferation and/or migration signals. Following steroid-dependent translocation of FLI1 and GR to the nucleus, the FLI1-specific domain (FLS) binds with GR and strongly enhances GR’s transcriptional activity. This interaction has functional consequences in Ewing sarcoma (ES), childhood and adolescence bone malignancies driven by fusions between EWSR1 and FLI1. In vitro, GR knockdown inhibited the migration and proliferation of ES cells, and in animal models, antagonizing GR (or lowering cortisol) retarded both tumor growth and metastasis from bone to lung. Taken together, our findings offer mechanistic rationale for repurposing GR-targeting drugs for the treatment of patients with ES.

Role of glucocorticoid signaling in urothelial tumorigenesis: Inhibition by prednisone presumably through inducing glucocorticoid receptor transrepression.

Glucocorticoids, including dexamethasone (DEX) and prednisone (PRED), have been prescribed in patients with neoplastic disease as cytotoxic agents or comedications. Nonetheless, it remains uncertain whether they have an impact on the development of bladder cancer. We, therefore, assessed the functional role of theglucocorticoid-mediated glucocorticoid receptor (GR) signaling in urothelial tumorigenesis. Tumor formation was significantly delayed in xenograft-bearing mice with implantation of control bladder cancer UMUC3 cells or nonneoplastic urothelial SVHUC cells undergoing malignant transformation induced by a chemical carcinogen 3-methylcholanthrene (MCA), compared with respective GR knockdown xenografts. Using the in vitro system with MCA-SVHUC cells, we screened 11 GR ligands, including DEX, and found significant inhibitory effects of PRED on their neoplastic transformation. The effects of PRED were restored by a GR antagonist RU486 in GR-positive MCA-SVHUC cells, while PRED failed to inhibit theneoplastic transformation of GR knockdown cells. Significant decreases in the expression levels of oncogenes (c-Fos/c-Jun) and significant increases in those of a tumor suppressor UGT1A were seen in MCA-SVHUC-control cells (vs GR-short hairpin RNA) or PRED-treated MCA-SVHUC-control cells (vs mock). In addition, N-butyl-N-(4-hydroxybutyl) nitrosamine induced bladder cancer in all of eight mock-treated mice vs seven (87.5%) of DEX-treated (P = .302) or four (50%) of PRED-treated (P = .021) animals. Finally, DEX was found to considerably induce both transactivation (activation of glucocorticoid-response element mediated transcription and expression of its targets) and transrepression (suppression of nuclear factor-kappa B transactivation and expression of its regulated genes) of GR in SVHUC cells, while PRED more selectively induced GR transrepression. These findings suggest that PRED could prevent urothelial tumorigenesis presumably via inducing GR transrepression.

Dihydrotestosterone promotes kidney cancer cell proliferation by activating the STAT5 pathway via androgen and glucocorticoid receptors.

Androgen receptors (ARs) are expressed on a variety of cell types, and AR signaling plays an important role in tumor development and progression in several cancers. This in vitro study evaluated the effect of dihydrotestosterone (DHT) on the proliferation of renal cell carcinoma (RCC) cells in relation to AR status. Steroid hormone receptor expression was evaluated using RT-PCR and Western blotting. The effect of DHT on cell proliferation and STAT5 phosphorylation was evaluated in RCC cell lines (Caki-2, A498, and SN12C) and primary RCC cells using cell viability assays and Western blotting. ARs and glucocorticoid receptors (GRs) were knocked down with small interfering RNAs before assessing changes in cell proliferation and STAT5 activation. DHT treatment promoted cell proliferation and increased STAT5 phosphorylation regardless of AR status. The AR antagonist bicalutamide reduced kidney cancer cell proliferation, regardless of AR status. AR and GR knockdown blocked STAT5 activation and reduced cell proliferation in all RCC cell lines. In patient-derived primary cells, DHT enhanced cell proliferation and this effect was diminished by treatment with the AR antagonists bicalutamide and enzalutamide and the GR antagonist mifepristone. DHT promotes cell proliferation through STAT5 activation in RCC cells, regardless of AR status. DHT appears to utilize the AR and GR pathways to activate STAT5, and the inhibition of AR and GR showed antitumor activity in RCC cells. These data suggest that targeting AR and GR may be a promising new approach to the treatment of RCC.

Both insufficient and excessive glucocorticoid receptor-mediated signaling impair neuronal migration

Glucocorticoids (GCs) are a class of steroid hormones that regulate numerous physiological events in the human body. Clinically, glucocorticoids are used for anti-inflammatory and immunosuppressive actions via binding with glucocorticoid receptors (GRs). Emerging evidence has also indicated that inappropriate GC and GR levels are detrimental for brain development and eventually lead to severe neurological diseases. However, the roles of GC/GR signaling in brain development are not fully understood. Here, we showed that stable GR expression levels were critical for brain development, because both GR knockdown and overexpression severely impaired neuronal migration. Further studies showed that the multipolar-bipolar transition and leading process development were interrupted in GR-knockdown and GR-overexpressing neurons. To elucidate the underlying mechanism, we screened the protein levels of downstream molecules and identified RhoA as a factor negatively regulated by the GR. Restoration of the RhoA protein level partially rescued the neuronal migration defects in the GR-knockdown and GR-overexpressing neurons, indicating that RhoA played a major role in GR-mediated neuronal migration. These data suggest that an appropriate level of GC/GR signaling is essential for precise control of neuronal migration.

Conditional deletion of glucocorticoid receptors in rat brain results in sex-specific deficits in fear and coping behaviors.

Glucocorticoid receptors (GR) have diverse functions relevant to maintenance of homeostasis and adaptation to environmental challenges. Understanding the importance of tissue-specific GR function in physiology and behavior has been hampered by near-ubiquitous localization in brain and body. Here we use CRISPR/Cas9 gene editing to create a conditional GR knockdown in Sprague Dawley rats. To test the impact of cell- and region-specific GR knockdown on physiology and behavior, we targeted GR knockdown to output neurons of the prelimbic cortex. Prelimbic knockdown of GR in females caused deficits in acquisition and extinction of fear memory during auditory fear conditioning, whereas males exhibited enhanced active-coping behavior during forced swim. Our data support the utility of this conditional knockdown rat to afford high-precision knockdown of GR across a variety of contexts, ranging from neuronal depletion to circuit-wide manipulations, leveraging the behavioral tractability and enhanced brain size of the rat as a model organism.

Repression of the Glucocorticoid Receptor Increases Hypoxic-Ischemic Brain Injury in the Male Neonatal Rat

Hypoxic-ischemic encephalopathy (HIE) resulting from asphyxia is the most common cause of neonatal brain damage and results in significant neurological sequelae, including cerebral palsy. The current therapeutic interventions are extremely limited in improving neonatal outcomes. The present study tests the hypothesis that the suppression of endogenous glucocorticoid receptors (GRs) in the brain increases hypoxic-ischemic (HI) induced neonatal brain injury and worsens neurobehavioral outcomes through the promotion of increased inflammation. A mild HI treatment of P9 rat pups with ligation of the right common carotid artery followed by the treatment of 8% O2 for 60 min produced more significant brain injury with larger infarct size in female than male pups. Intracerebroventricular injection of GR siRNAs significantly reduced GR protein and mRNA abundance in the neonatal brain. Knockdown of endogenous brain GRs significantly increased brain infarct size after HI injury in male, but not female, rat pups. Moreover, GR repression resulted in a significant increase in inflammatory cytokines TNF-α and IL-10 at 6 h after HI injury in male pups. Male pups treated with GR siRNAs showed a significantly worsened reflex response and exhibited significant gait disturbances. The present study demonstrates that endogenous brain GRs play an important role in protecting the neonatal brain from HI induced injury in male pups, and suggests a potential role of glucocorticoids in sex differential treatment of HIE in the neonate.