Glucocorticoid Receptor Signaling Research Articles

Multi-omics in nasal epithelium reveals three axes of dysregulation for asthma risk in the African Diaspora populations

Asthma has striking disparities across ancestral groups, but the molecular underpinning of these differences is poorly understood and minimally studied. A goal of the Consortium on Asthma among African-ancestry Populations in the Americas (CAAPA) is to understand multi-omic signatures of asthma focusing on populations of African ancestry. RNASeq and DNA methylation data are generated from nasal epithelium including cases (current asthma, N = 253) and controls (never-asthma, N = 283) from 7 different geographic sites to identify differentially expressed genes (DEGs) and gene networks. We identify 389 DEGs; the top DEG, FN1, was downregulated in cases (q = 3.26 × 10−9) and encodes fibronectin which plays a role in wound healing. The top three gene expression modules implicate networks related to immune response (CEACAM5; p = 9.62 × 10−16 and CPA3; p = 2.39 × 10−14) and wound healing (FN1; p = 7.63 × 10−9). Multi-omic analysis identifies FKBP5, a co-chaperone of glucocorticoid receptor signaling known to be involved in drug response in asthma, where the association between nasal epithelium gene expression is likely regulated by methylation and is associated with increased use of inhaled corticosteroids. This work reveals molecular dysregulation on three axes – increased Th2 inflammation, decreased capacity for wound healing, and impaired drug response – that may play a critical role in asthma within the African Diaspora.

Dexamethasone-Integrated Mesenchymal Stem Cells for Systemic Lupus Erythematosus Treatment via Multiple Immunomodulatory Mechanisms.

The therapeutic application of mesenchymal stem cells (MSCs) has good potential as a treatment strategy for systemic lupus erythematosus (SLE), but traditional MSC therapy still has limitations in effectively modulating immune cells. Herein, we present a promising strategy based on dexamethasone liposome-integrated MSCs (Dexlip-MSCs) for treating SLE via multiple immunomodulatory pathways. This therapeutic strategy prolonged the circulation time of dexamethasone liposomes in vivo, restrained CD4+T-cell proliferation, and inhibited the release of proinflammatory mediators (IFN-γ and TNF-α) by CD4+T cells. In addition, Dexlip-MSCs initiated cellular reprogramming by activating the glucocorticoid receptor (GR) signaling pathway to upregulate the expression of anti-inflammatory factors such as cysteine-rich secretory protein LCCL-containing domain 2 (CRISPLD2) and downregulate the expression of proinflammatory factors. In addition, Dexlip-MSCs synergistically increased the anti-inflammatory inhibitory effect of CD4+T cells through the release of dexamethasone liposomes or Dex-integrated MSC-derived exosomes (Dex-MSC-EXOs). Based on these synergistic biological effects, we demonstrated that Dexlip-MSCs alleviated disease progression in MRL/lpr mice more effectively than Dexlip or MSCs alone. These features indicate that our stem cell delivery strategy is a promising therapeutic approach for clinical SLE treatment.

Glucocorticoid receptor-mediated oncogenic activity is dependent on breast cancer subtype

Breast cancer incidence has been steadily rising and is the leading cause of cancer death in women due to its high metastatic potential. Individual breast cancer subtypes are classified by both cell type of origin and receptor expression, namely estrogen, progesterone and human epidermal growth factor receptors (ER, PR and HER2). Recently, the importance and context-dependent role of glucocorticoid receptor (GR) expression in the natural history and prognosis of breast cancer subtypes has been uncovered. In ER-positive breast cancer, GR expression is associated with a better prognosis as a result of ER-GR crosstalk. GR appears to modulate ER-mediated gene expression resulting in decreased tumor cell proliferation and a more indolent cancer phenotype. In ER-negative breast cancer, including GR-positive triple-negative breast cancer (TNBC), GR expression enhances migration, chemotherapy resistance and cell survival. In invasive lobular carcinoma, GR function is relatively understudied, and more work is required to determine whether lobular subtypes behave similarly to their invasive ductal carcinoma counterparts. Importantly, understanding GR signaling in individual breast cancer subtypes has potential clinical implications because of the recent development of highly selective GR non-steroidal ligands, which represent a therapeutic approach for modulating GR activity systemically.

EP300/CREBBP acetyltransferase inhibition limits steroid receptor and FOXA1 signaling in prostate cancer cells

The androgen receptor (AR) is a primary target for treating prostate cancer (PCa), forming the bedrock of its clinical management. Despite their efficacy, resistance often hampers AR-targeted therapies, necessitating new strategies against therapy-resistant PCa. These resistances involve various mechanisms, including AR splice variant overexpression and altered activities of transcription factors like the glucocorticoid receptor (GR) and FOXA1. These factors rely on common coregulators, such as EP300/CREBBP, suggesting a rationale for coregulator-targeted therapies. Our study explores EP300/CREBBP acetyltransferase inhibition’s impact on steroid receptor and FOXA1 signaling in PCa cells using genome-wide techniques. Results reveal that EP300/CREBBP inhibition significantly disrupts the AR-regulated transcriptome and receptor chromatin binding by reducing the AR-gene expression. Similarly, GR’s regulated transcriptome and receptor binding were hindered, not linked to reduced GR expression but to diminished FOXA1 chromatin binding, restricting GR signaling. Overall, our findings highlight how EP300/CREBBP inhibition distinctively curtails oncogenic transcription factors’ signaling, suggesting the potential of coregulatory-targeted therapies in PCa.

Abstract 7307: Silibinin regulates glucocorticoid signaling via mast cells: Potential treatment of androgen-resistant prostate cancer

Abstract Prostate cancer (PCa) is the second most common cancer in American men. The American Cancer Society estimates that in 2023 there have been ~288,300 new cases of PCa and ~34,700 deaths from PCa in the United States. We previously showed that silibinin (SB; a natural flavonolignan from milk thistle seeds) is an effective therapeutic against PCa that inhibits cell growth and mitogenic cell survival via EGFR, IGFR-1, and NFκB signaling. However, the effect of SB on the tumor immunological microenvironment has not been elucidated. Mast cells (MCs) are archetypal solider of the immune system. The dynamics of PCa and MCs are complex, where they may contribute to tumor transformation/progression/regression; furthermore, their role in PCa is poorly understood. To address this gap and understand the role of SB in MC regulation, MCs were derived from the bone marrow (BMMCs) of C57BL/6 mice. BMMCs were generated in the presence of IL-3 and SCF, and were confirmed to be BMMCs using flow cytometry (dual staining: cKit/Fc€RI). Thereafter, the BMMCs were exposed to different doses of SB (25-100 µM) and two concentrations (25 µM; SB 25 and 100 µM; SB 100) were selected for further experiments. BMMCs treated with SB 25, SB 100, and untreated (control) were subjected to proteomic analysis using LCMS on Fusion Lumos mass spectrometer. A total of 3575 proteins were identified using the annotated mouse proteome. Statistical analysis was performed using ANOVA followed by Fisher’s posthoc analysis (p<0.05, FDR<0.01) and 166 proteins were found to be have statistically significant differential expression amongst the three groups (n=4/group). These 166 proteins were selected for further pathway analysis using Ingenuity Pathway Analysis (IPA; Qiagen). Only pathways specifically demonstrated experimentally in prostatic tissues/cells were selected for further analysis. Important nodes found for SB regulation of MCs in PCa were androgen receptor (AR), transmembrane serine protease (TMPRSS), and NK3 Homeobox 1 (NKX3-1). AR, TMPRSS, and NkX3-1 are well established in PCA. Most important pathway found was glucocorticoid receptor (GR) signaling; nuclear receptor subfamily 3 group C member 1 (NR3C1) and HS90 were the most important proteins identified in GR signaling. GR signaling may cause androgen deprivation therapy (ADT)-resistance by directly activating AR-target genes (including TMPRSS2) and/or activating an independent transcriptome. NR3C1 has been shown to share target specificity with AR and is highly upregulated in the resistant tumors. Thus, SB could play an important therapeutic role, specifically in ADT-resistant tumors via regulation of MCs. Further studies are warranted to delineate these mechanisms. Understanding these mechanistic could also potentially aid to decipher a general mechanism of resistance to antiandrogens and potential of SB in treating ADT-resistant tumors. Citation Format: Neha Mishra, Sandeep Paudel, Komal Raina, Paul Maroni, Chapla Agarwal, Rajesh Agarwal. Silibinin regulates glucocorticoid signaling via mast cells: Potential treatment of androgen-resistant 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 7307.

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.

Sex differences in single neuron function and proteomics profiles examined by patch-clamp and mass spectrometry in the locus coeruleus of the adult mouse.

This study aimed to characterize the properties of locus coeruleus (LC) noradrenergic neurons in male and female mice. We also sought to investigate sex-specific differences in membrane properties, action potential generation, and protein expression profiles to understand the mechanisms underlying neuronal excitability variations. Utilizing a genetic mouse model by crossing Dbhcre knock-in mice with tdTomato Ai14 transgenic mice, LC neurons were identified using fluorescence microscopy. Neuronal functional properties were assessed using patch-clamp recordings. Proteomic analyses of individual LC neuron soma was conducted using mass spectrometry to discern protein expression profiles. Data are available via ProteomeXchange with identifier PXD045844. Female LC noradrenergic neurons displayed greater membrane capacitance than those in male mice. Male LC neurons demonstrated greater spontaneous and evoked action potential generation compared to females. Male LC neurons exhibited a lower rheobase and achieved higher peak frequencies with similar current injections. Proteomic analysis revealed differences in protein expression profiles between sexes, with male mice displaying a notably larger unique protein set compared to females. Notably, pathways pertinent to protein synthesis, degradation, and recycling, such as EIF2 and glucocorticoid receptor signaling, showed reduced expression in females. Male LC noradrenergic neurons exhibit higher intrinsic excitability compared to those from females. The discernible sex-based differences in excitability could be ascribed to varying protein expression profiles, especially within pathways that regulate protein synthesis and degradation. This study lays the groundwork for future studies focusing on the interplay between proteomics and neuronal function examined in individual cells.

Endocrine disrupting potential of total and bioaccessible extracts of dust from seven different types of indoor environment

Information on the indoor environment as a source of exposure with potential adverse health effects is mostly limited to a few pollutant groups and indoor types. This study provides a comprehensive toxicological profile of chemical mixtures associated with dust from various types of indoor environments, namely cars, houses, prefabricated apartments, kindergartens, offices, public spaces, and schools. Organic extracts of two different polarities and bioaccessible extracts mimicking the gastrointestinal conditions were prepared from two different particle size fractions of dust. These extracts were tested on a battery of human cell-based bioassays to assess endocrine disrupting potentials. Furthermore, 155 chemicals from different pollutant groups were measured and their relevance for the bioactivity was determined using concentration addition modelling. The exhaustive and bioaccessible extracts of dust from the different microenvironments interfered with aryl hydrocarbon receptor, estrogen, androgen, glucocorticoid, and thyroid hormone (TH) receptor signalling, and with TH transport. Noteably, bioaccessible extracts from offices and public spaces showed higher estrogenic effects than the organic solvent extracts. 114 of the 155 targeted chemicals were detectable, but the observed bioactivity could be only marginally explained by the detected chemicals. Diverse toxicity patterns across different microenvironments that people inhabit throughout their lifetime indicate potential health and developmental risks, especially for children. Limited data on the endocrine disrupting potency of relevant chemical classes, especially those deployed as replacements for legacy contaminants, requires further study.

Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling

While physiologic stress has long been known to impair mammalian reproductive capacity through hormonal dysregulation, mounting evidence now suggests that stress experienced prior to or during gestation may also negatively impact the health of future offspring. Rodent models of gestational physiologic stress can induce neurologic and behavioral changes that persist for up to three generations, suggesting that stress signals can induce lasting epigenetic changes in the germline. Treatment with glucocorticoid stress hormones is sufficient to recapitulate the transgenerational changes seen in physiologic stress models. These hormones are known to bind and activate the glucocorticoid receptor (GR), a ligand-inducible transcription factor, thus implicating GR-mediated signaling as a potential contributor to the transgenerational inheritance of stress-induced phenotypes. Here, we demonstrate dynamic spatiotemporal regulation of GR expression in the mouse germline, showing expression in the fetal oocyte as well as the perinatal and adult spermatogonia. Functionally, we find that fetal oocytes are intrinsically buffered against changes in GR signaling, as neither genetic deletion of GR nor GR agonism with dexamethasone altered the transcriptional landscape or the progression of fetal oocytes through meiosis. In contrast, our studies revealed that the male germline is susceptible to glucocorticoid-mediated signaling, specifically by regulating RNA splicing within the spermatogonia, although this does not abrogate fertility. Together, our work suggests a sexually dimorphic function for GR in the germline, and represents an important step towards understanding the mechanisms by which stress can modulate the transmission of genetic information through the germline.

Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling.

While physiologic stress has long been known to impair mammalian reproductive capacity through hormonal dysregulation, mounting evidence now suggests that stress experienced prior to or during gestation may also negatively impact the health of future offspring. Rodent models of gestational physiologic stress can induce neurologic and behavioral changes that persist for up to three generations, suggesting that stress signals can induce lasting epigenetic changes in the germline. Treatment with glucocorticoid stress hormones is sufficient to recapitulate the transgenerational changes seen in physiologic stress models. These hormones are known to bind and activate the glucocorticoid receptor (GR), a ligand-inducible transcription factor, thus implicating GR-mediated signaling as a potential contributor to the transgenerational inheritance of stress-induced phenotypes. Here, we demonstrate dynamic spatiotemporal regulation of GR expression in the mouse germline, showing expression in the fetal oocyte as well as the perinatal and adult spermatogonia. Functionally, we find that fetal oocytes are intrinsically buffered against changes in GR signaling, as neither genetic deletion of GR nor GR agonism with dexamethasone altered the transcriptional landscape or the progression of fetal oocytes through meiosis. In contrast, our studies revealed that the male germline is susceptible to glucocorticoid-mediated signaling, specifically by regulating RNA splicing within the spermatogonia, although this does not abrogate fertility. Together, our work suggests a sexually dimorphic function for GR in the germline, and represents an important step towards understanding the mechanisms by which stress can modulate the transmission of genetic information through the germline.

Glucocorticoid and Adrenergic Receptor Distribution Across Human Organs and Tissues: A Map for Stress Transduction.

Psychosocial stress is transduced into disease risk through energy-dependent release of hormones from the hypothalamic-pituitary-adrenal and sympathetic-adrenal-medullary axes. The levels of glucocorticoid and adrenergic hormones, together with the sensitivity of tissues to their signaling, define stress responses. To understand existing pathways responsible for the psychobiological transduction of stressful experiences, we provide a quantitative whole-body map of glucocorticoid and adrenergic receptor (AR) expression. We systematically examined gene expression levels for the glucocorticoid receptor (GR), α- and β-ARs (AR-α1B, AR-α2B AR-β2, and AR-β3), across 55 different organs using the Human Protein Atlas and Human Proteome Map datasets. Given that mitochondria produce the energy required to respond to stress, we leveraged the Human Protein Atlas and MitoCarta3.0 data to examine the link between stress hormone receptor density and mitochondrial gene expression. Finally, we tested the functional interplay between GR activation and AR expression in human fibroblast cells. The GR was expressed ubiquitously across all investigated organ systems, whereas AR subtypes showed lower and more localized expression patterns. Receptor co-regulation, meaning the correlated gene expression of multiple stress hormone receptors, was found between GR and AR-α1B, as well as between AR-α1B and AR-α2B. In cultured human fibroblasts, activating the GR selectively increased AR-β2 and AR-α1B expression. Consistent with the known energetic cost of stress responses, GR and AR expressions were positively associated with the expression of specific mitochondrial pathways. Our results provide a cartography of GR and AR expression across the human body. Because stress-induced GR and AR signaling triggers energetically expensive cellular pathways involving energy-transforming mitochondria, the tissue-specific expression and co-expression patterns of hormone receptor subtypes may in part determine the resilience or vulnerability of different organ systems.

Protective effects of Jing-Si-herbal-tea in inflammatory cytokines-induced cell injury on normal human lung fibroblast via multiomic platform analysis.

The protective effects and related mechanisms of Jing-Si herbal tea (JSHT) were investigated in cellular damage mediated by pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α, on normal human lung fibroblast by multiomic platform analysis. The in silico high-throughput target was analyzed using pharmacophore models by BIOVIA Discovery Studio 2022 with ingenuity pathway analysis software. To assess cell viability, the study utilized the MTT assay technique. In addition, the IncuCyte S3 ZOOM System was implemented for the continuous monitoring of cell confluence of JSHT-treated cytokine-injured HEL 299 cells. Cytokine concentrations were determined using a Quantibody Human Inflammation Array. Gene expression and signaling pathways were determined using next-generation sequencing. In silico high-throughput target analysis of JSHT revealed ingenuity in canonical pathways and their networks. Glucocorticoid receptor signaling is a potential signaling of JSHT. The results revealed protective effects against the inflammatory cytokines on JSHT-treated HEL 299 cells. Transcriptome and network analyses revealed that induction of helper T lymphocytes, TNFSF12, NFKB1-mediated relaxin signaling, and G-protein coupled receptor signaling play important roles in immune regulatory on JSHT-treated cytokine-injured HEL 299 cells. The findings from our research indicate that JSHT holds promise as a therapeutic agent, potentially offering advantageous outcomes in treating virus infections through various mechanisms. Furthermore, the primary bioactive components in JSHT justify extended research in antiviral drug development, especially in the context of addressing coronavirus.

Identification of FDA-approved drugs that increase mevalonate kinase in hyper IgD syndrome.

Mevalonate kinase deficiency (MKD) is an autoinflammatory metabolic disorder caused by bi-allelic loss-of-function variants in the MVK gene, resulting in decreased activity of the encoded mevalonate kinase (MK). Clinical presentation ranges from the severe early-lethal mevalonic aciduria to the milder hyper-IgD syndrome (MKD-HIDS), and is in the majority of patients associated with recurrent inflammatory episodes with often unclear cause. Previous studies with MKD-HIDS patient cells indicated that increased temperature, as caused by fever during an inflammatory episode, lowers the residual MK activity, which causes a temporary shortage of non-sterol isoprenoids that promotes the further development of inflammation. Because an increase of the residual MK activity is expected to make MKD-HIDS patients less sensitive to developing inflammatory episodes, we established a cell-based screen that can be used to identify compounds and/or therapeutic targets that promote this increase. Using a reporter HeLa cell line that stably expresses the most common MKD-HIDS variant, MK-V377I, C-terminally tagged with bioluminescent NanoLuc luciferase (nLuc), we screened the Prestwick Chemical Library®, which includes 1280 FDA-approved compounds. Multiple compounds increased MK-V377I-nLuc bioluminescence, including steroids (i.e., glucocorticoids, estrogens, and progestogens), statins and antineoplastic drugs. The glucocorticoids increased MK-V377I-nLuc bioluminescence through glucocorticoid receptor signaling. Subsequent studies in MKD-HIDS patient cells showed that the potent glucocorticoid clobetasol propionate increases gene transcription of MVK and other genes regulated by the transcription factor sterol regulatory element-binding protein 2 (SREBP-2). Our results suggest that increasing the flux through the isoprenoid biosynthesis pathway by targeting the glucocorticoid receptor or SREBP-2 could be a potential therapeutic strategy in MKD-HIDS.

Activation of glucocorticoid receptor signaling inhibits KSHV-induced inflammation and tumorigenesis.

Kaposi's sarcoma (KS) is the most common cancer in HIV-infected patients caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Hyperinflammation is the hallmark of KS. In this study, we have shown that KSHV mediates hyperinflammation by inducing IL-1α and suppressing IL-1Ra. Mechanistically, KSHV miRNAs and vFLIP induce hyperinflammation by activating the NF-κB pathway. A common anti-inflammatory agent dexamethasone blocks KSHV-induced hyperinflammation and tumorigenesis by activating glucocorticoid receptor signaling to suppress IL-1α and induce IL-1Ra. This work has identified IL-1-mediated inflammation as a potential therapeutic target and dexamethasone as a potential therapeutic agent for KSHV-induced malignancies.

Glucocorticoid Receptor Antagonism Improves Glucose Metabolism in a Mouse Model of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a complex metabolic disorder associated with obesity, insulin resistance, and dyslipidemia. Hyperandrogenism is a major characteristic of PCOS. Increased androgen exposure is believed to deregulate metabolic processes in various tissues as part of the PCOS pathogenesis, predominantly through the androgen receptor (AR). Notably, various metabolic features in PCOS are similar to those observed after excess glucocorticoid exposure. We hypothesized that glucocorticoid receptor (GR) signaling is involved in the metabolic symptoms of PCOS. In a PCOS model of chronic dihydrotestosterone (DHT) exposure in female mice, we investigated whether GR signaling machinery was (de)regulated, and if treatment with a selective GR antagonist alleviated the metabolic symptoms. We observed an upregulation of GR messenger RNA expression in the liver after DHT exposure. In white adipose tissues and liver we found that DHT upregulated Hsd11b1, which encodes for the enzyme that converts inactive into active glucocorticoids. We found that preventive but not therapeutic administration of a GR antagonist alleviated DHT-induced hyperglycemia and restored glucose tolerance. We did not observe strong effects of GR antagonism in DHT-exposed mice on other features like total fat mass and lipid accumulation in various tissues. We conclude that GR activation may play a role in glucose metabolism in DHT-exposed mice.

Protective Effects of AR1001 in Alzheimer’s Disease Models: Polypharmacological Mechanisms

AbstractBackgroundNitric oxide/cGMP/CREB signaling is critical for learning and memory processes and is disturbed by Abeta amyoid and in Alzheimer’s disease. AR1001 is an inhibitor of phosphodiesterase (PDE)‐5, which degrades specifically cGMP, thus having a potential to reverse AD pathology.MethodFor the in vivo study, AR1001 was intraperitoneally administered (4 mg/kg/day) to APP‐C105 mice for 4 weeks. Then after behavioral tests (Morris‐water maze and passive avoidance tests) were done, hippocampal levels of Abeta1‐42 and p‐Tau (S199/202) were determined. In the in vitro study using human neuroblastoma SH‐SY5Y cells and mouse hippocampal HT‐22 cells, the effects of AR1001 on Abeta1‐42‐induced cytotoxicity and intracellular signaling were examined.ResultAR1001 markedly improved cognitive functions in both Morris‐water maze and passive avoidance tests and reduced hippocampal Abeta1‐42 and p‐Tau levels in the APP‐C105 mice. In the in vitro experiments, AR1001 inhibited Abeta1‐42‐induced cell death, mitochondrial membrane potential derangement, and apoptotic markers (cleaved caspase‐3 and cleaved PPAR) concentration‐dependently. AR1001 reversed Abeta1‐42‐induced decrease in phosphorylated CREB (p‐CREB), and NGF/BDNF expression. Further, AR1001 blocked Abeta1‐42‐induced pSer9‐GSK3beta decrease and p25/p35 increase. Interestingly, compared with sildenafil, vardenafil and tadalafil, AR1001 was more effective in inhibiting glucocorticoid receptor (GR) dimerization and Wnt antagonist Dickkopf‐1 (Dkk‐1) expression, and in enhancing Wnt3a expression.ConclusionAR1001 had protective effects both in vivo and in vitro models of AD. Furthermore, AR1001 showed more pronounced activity in the modulation of GR and Wnt signaling among the various PDE‐5 inhibitors. It is suggested that the multimodal activity of AR1001 contributes to the therapeutic effects of AR1001 observed in the phase 2 clinical trial in AD patients (NCT03625622).

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.

Intratumoral cortisol associated with aromatase in the endometrial cancer microenvironment

Glucocorticoids bind to glucocorticoid receptors (GR). In the peripheral tissues, active cortisol is produced from inactive cortisone by 11β-hydroxysteroid dehydrogenase (HSD)1. 11β-HSD2 is responsible for this reverse catalysis. Although GR and 11β-HSDs have been reported to be involved in the malignant behavior of various cancer types, the concentration of glucocorticoids in cancer tissues has not been investigated. In this study, we measured glucocorticoids in serum and cancer tissues using liquid chromatography-tandem mass spectrometry and clarified, for the first time, the intratumoral “intracrine” production of cortisol by 11β-HSD1/2 in endometrial cancer. Intratumoral cortisol levels were high in the high-malignancy type and the cancer proliferation marker Ki-67-high group, suggesting that cortisol greatly contributes to the malignant behavior of endometrial cancer. A low expression level of the metabolizing enzyme 11β-HSD2 is more important than a high expression level of the synthase 11β-HSD1 for intratumoral cortisol action. Intratumoral cortisol was positively related to the expression/activity of estrogen synthase aromatase, which involved GR expressed in fibroblastic stromal cells but not in cancer cells. Blockade of GR signaling by hormone therapy is expected to benefit patients with endometrial cancer.

SAT016 Small Compounds Modulate Glucocorticoid Receptor Function By Binding To A Novel Allosteric Pocket

Abstract Disclosure: A. Jimenez-Panizo: None. The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. This receptor exerts its functions by binding to corticosteroids through its ligand-binding domain (LBD), which harbors a hydrophobic pocket where ligands are encapsulated. Synthetic glucocorticoids, as Dexamethasone, are widely used in the clinic for the treatment of chronic inflammatory diseases. Unfortunately, their prolonged use is associated with critical side effects and most of the mechanisms underlying these collateral issues are still unknown. In this work, we present a deep biochemical and biophysical analysis showing how small molecules are able to bind a new allosteric pocket in solution and how these compounds can modulate GR transcriptional activity in cells. These findings can be essential to further understand GR signaling and how to better design synthetic corticosteroids to avoid side effects. Presentation: Saturday, June 17, 2023