Nuclear Glucocorticoid Receptor Research Articles

Inhibition of corticosterone synthesis impairs cued water maze consolidation, but it does not affect the expression of BDNF, CK2 and SGK1 genes in dorsal striatum.

Corticosterone (CORT) release during learning experiences is associated with strong memories and activity of the glucocorticoid receptor. It has been shown that lesions of the dorsal striatum (DS) of rats trained in the cued version of the Morris water maze impair memory, and that local injection of CORT improves its performance, suggesting that DS activity is involved in procedural memory which may be modulated by CORT. We trained rats in cued Morris water maze and analyzed the effect of CORT synthesis inhibition on performance, CORT levels, expression of plasticity-involved genes, such as the brain derived neurotrophic factor (BDNF), casein kinase 2 (CK2), and the serum/glucocorticoid regulated kinase 1 (SGK1), as well as the presence of phosphorylated nuclear glucocorticoid receptor in serine 232 (pGR-S232) in the DS. The inhibition of CORT synthesis by metyrapone reduced CORT levels in plasma, prevented its increment in DS and impaired the performance of cued water maze. Additionally, there was an increase of CK2 and SGK1 mRNAs expression in trained subjects, which was unrelated to CORT levels. Finally, we did not observe changes in nuclear pGR-S232 in any condition. Our findings agree with evidence demonstrating that decreasing CORT levels hinders acquisition and consolidation of the spatial version of the Morris water maze; these novel findings broaden our knowledge about the involvement of the DS in the mechanisms underlying procedural memory.

Personalized genotype markers of the atopic disorders phenotypes in children

The goal of the study was to elucidate the impact of the single nucleotide variants rs11466749 of the thymic stromal lymphopoietin gene, rs_7216389 of the orsomucoid-1-like protein 3 gene, and rs10052957 of the human nuclear glucocorticoid receptor subfamily 3, group C, member 1 gene on the development of the mono-organic phenotype “atopic eczema” or the poly-organic “atopic eczema + allergic rhinitis/allergic rhino-conjunctivitis”. We recruited 101 patients into the main and 105 into control groups aged from 3 to 18 years old. Patients of the main group suffered from atopic eczema (58 children) and atopic eczema + allergic rhinitis/allergic rhino-conjunctivitis (43 children). Patients of the control group suffered from the digestive tract pathology. Main group patients were genotyped for the A/A, A/G, G/G of rs11466749, C/T, C/C and T/T of rs_7216389 and A/A, A/G and G/G of rs10052957; patients of the control group were genotyped for the A/A, A/G, G/G of rs11466749, C/T, C/C and T/T of rs_7216389 by polymerase chain reaction in real time with restricted fragment length polymorphism. Results: no significant differences in rs11466749 among the main and control groups, the most common variant is A/A – 55.2% (mono-organic) and 55.8% (poly-organic); T/T rs_7216389 is significantly the most common in poly-organic phenotype – 39.5%; rs10052957: A/G variant is significantly most common in mono-organic phenotype – 51.7% and G/G – in the poly-organic phenotype – 62.8%. The G/G rs11466749 variant has a trending to significance direct 0.173 association and increased odds ratio = 5.85 (0.63-54.31) for the poly-organic phenotype and protective impact onto the mono-organic phenotype -0.173 (0.17 (0.02-1.59); T/T rs7216389 variant increases the risk of poly-organic phenotype: 0.227, odds ratio = 2,79 (1.14-6.85) and decreases the risk of mono-organic” phenotype: -0.227, 0.36 (0.15-0.88); A/G rs_10052957 variant significantly increases the risk the mono-organic phenotype: 0.215, odds ratio = 2.5 (1.08-5.56)) and decreases risk of poly-organic phenotype: 0.215, odds ratio = 0.4 (0.18-0.93); G/G rs_10052957 variant significantly increases the risk of the poly-organic phenotype: 0.263, odds ratio = 2.97 (1.31-6.74)) and decreases for the mono-organic phenotype: -0.263, odds ratio = 0.34 (0.15-0.76)). Genotype variant T/T rs_7216389 of the orsomucoid-1-like protein 3 gene significantly increases the risk of developing the poly-organic atopic phenotype by 2.79 times and protects against the mono-organic atopic phenotype by 0.34 times. G/G genotype variant of rs10052957 of the human glucocorticoid receptor subfamily, group C, member 1 gene significantly increases the risk of developing the poly-organic phenotype by 2.97 times, protecting against mono-organic atopic phenotype by 0.34 times.

Генотип-асоційований прогноз розвитку моноорганних та поліорганних фенотипів атопічного маршу в дітей

Purpose - to research the influence of the single nucleotide variants (SNV) as of thymic stromal lymphopoietin genes (rs11466749 TSLP), orsomucoid-1-like protein 3 (rs7216389 ORMDL3) and human nuclear glucocorticoid receptor type 3 subfamily C member 1 gene (rs 10052957 h-GR/NR3C1) on the risk of developing AM phenotypes «AD», «AD+AR/ARC», «AD+AR/ARC+BA». Materials and methods. 127 children in the main and 105 in the control group aged from 3 to 18 were recruited into the study. All patients underwent the oral cavity mucosa swab, the material of which was subjected genotyping by the means of real-time polymerase chain reaction for SNV variants rs11466749 TSLP, rs7216389 ORMDL3 and rs 10052957 h-GR/NR3C1. For statistical processing the Pearson’s ꭕ2 criteria, Fisher’s exact test, Student’s test was used; the results were considered significant at p<0.05, trending to significance - at p=0.05-0.1. Results. The impact of the studied SNV on the risk of the poly-organ phenotype «AD+AR/ARC» development correlated to the mono-organ ««AD»: G/G rs11466749 TSLP: rs=0.173, OR=5.85 (p=0.08); C/T rs7216389 ORMDL3: rs=0.227, OR=0.36 (p<0.05), T/T rs7216389 ORMDL3: rs=0.227, OR=2.79 (p<0.05); A/G rs 10052957 h-GR/NR3C1: rs=0.215, OR=0.40 (p<0.05), G/G rs 10052957 h-GR/NR3C1: rs=0.263, OR=2.97 (p<0.01). The impact of the studied SNV on the development of the full poly-organ AM phenotype «AD+AR/ARC+BA» correlated to the mono-organ «AD»: A/A rs11466749 TSLP: rs=0.207, OR=2.71 (p=0.09), A/G rs11466749 TSLP: rs=0.310, OR=0.17 (p<0.01), G/G rs11466749 TSLP: rs=0.213, OR=7.43 (p=0.09). Conclusions. Different SNV variants of rs11466749 TSLP, rs7216389 ORMDL3, rs 10052957 h-GR/NR3C1 have both inducing and protective impact on the development of mono-organ and poly-organ AM phenotypes in children. The risk of the mono-organ phenotype «AD» developing into the poly-organ «AD+AR/ARC» is directly associated and significantly increased in carriers of the genotypes T/T rs7216389 ORMDL3 and G/G rs 10052957 h-GR/NR3C1, trending to significance - within G/G rs11466749 TSLP. The bespoke risk is significantly reduced in carriers of C/T rs7216389 ORMDL3 and A/G rs 10052957 h-GR/NR3C1 genotypes. The risk of developing a full poly-organ AM phenotype «AD+AR/ARC+BA» from mono-organ «AD» is significantly reduced in carriers of the A/G rs11466749 TSLP genotype, and is with trend to significance increased within homozygous A/A and G/G rs11466749 TSLP genotypes. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of the participating institution. The informed consent of the patients and children’s parents was obtained for conducting the studies. No conflict of interests was declared by the author.

Intact Recognition Memory and Altered Hippocampal Glucocorticoid Receptor Signaling in Fkbp5-deficient Mice Following Acute Uncontrollable Stress.

The FK506 binding protein 5 (FKBP5) is a co-chaperone that regulates the activity of the glucocorticoid receptor (GR) and has been reported to mediate stress resilience. This study aimed to determine the effects of Fkbp5 deletion on acute stress-induced recognition memory impairment and hippocampal GR signaling. Wild-type and Fkbp5-knockout mice were subjected to acute uncontrollable stress induced by restraint and electrical tail shock. First, we assessed the cognitive status of mice using a novel object recognition task. Next, we measured plasma corticosterone, GR levels, and the levels of GR phosphorylation at serine 211 in the hippocampus. Wild-type mice exhibited stress-induced memory impairments, whereas Fkbp5-knockout mice did not. Plasma corticosterone and GR levels did not differ between the non-stressed wild-type and Fkbp5-knockout mice, but the levels of phosphorylated GR were lower in Fkbp5-knockout mice than in wild-type mice. Wild-type and Fkbp5-knockout mice showed increased nuclear GR levels following stress, indicating GR translocation. However, cytosolic phosphorylated GR levels were lower in the hippocampi of Fkbp5-knockout mice following stress than in those of wild-type mice. These results suggest that FKBP5 deficiency increases resilience to acute stress by altering GR signaling.

Experimental methods and mathematical modeling quantify and predict DUSP1 RNA expression dynamics in response to dexamethasone.

Overactivation of mitogen-activated protein kinase (MAPK) signaling pathways is key to multiple inflammatory responses, and synthetic glucocorticoids (GC), such as Dexamethasone (Dex), have long been used to treat inflammatory pathologies. Upon cell entry, Dex binds to the glucocorticoid receptor (GR), which initiates translocation to the nucleus. Nuclear GR then interacts with Glucocorticoid Regulatory Elements (GRE's) to promote transcription of anti-inflammatory genes including dual-specificity phosphatase 1 (DUSP1). In turn, DUSP1 encodes for the protein mitogen-activated protein kinase phosphatase 1 (MKP-1), which regulates the cell's anti-inflammatory response through dephosphorylation of the P38 and JUN N-terminal kinase (JNK) MAPK pathways. In this presentation, we combine single-cell measurements and discrete stochastic models to elucidate the spatiotemporal mechanisms by which Dex modulates GR localization and DUSP1 transcription. Using single-molecule inexpensive fluorescent in situ hybridization, we quantify the nascent transcription, spatial localization, and cellular heterogeneity of DUSP1 mRNA for hundreds of individual HeLa cells at 20 different time points following application of continuous 100 nM Dex stimulation. We observe a rapid increase in DUSP1 transcription sites (TS's) as early as 10-20 minutes, followed by a four-fold increase in expression of mature DUSP1 mRNA that peaks at 75-90 minutes and a temporary decrease in DUSP1 expression at 120-150 minutes post stimulation. We found that expression levels stay elevated during longer time course experiments, and high cellular heterogeneity is observed throughout the post-stimuli response. We used these experimental data to infer parameters and mechanisms for a discrete stochastic model that quantitatively reproduces the time-varying probability distributions for single-cell DUSP1 expression at all time points following Dex stimulation. Finally, we use this model inferred from DUSP1 expression to predict GR translocation dynamics and compare to direct measurement of GR localization dynamic using single-cell immunocytochemistry.

Chronic Corticosterone Exposure Suppresses Copper Transport through GR-Mediated Intestinal CTR1 Pathway in Mice.

Numerous studies have discovered that chronic stress induces metabolic disorders by affecting iron and zinc metabolism, but the relationship between chronic stress and copper metabolism remains unclear. Here, we explore the influence of chronic corticosterone (CORT) exposure on copper metabolism and its regulatory mechanism in mice. Mice were treated with 100 μg/mL CORT in drinking water for a 4-week trial. We found that CORT treatment resulted in a significant decrease in plasma copper level, plasma ceruloplasmin activity, plasma and liver Cu/Zn-SOD activity, hepatic copper content, and liver metallothionein content in mice. CORT treatment led to the reduction in duodenal expression of copper transporter 1 (CTR1), duodenal cytochrome b (DCYTB), and ATPase copper-transporting alpha (ATP7A) at the mRNA and protein level in mice. CORT treatment activated nuclear glucocorticoid receptor (GR) and down-regulated CRT1 expression in Caco-2 cells, whereas these phenotypes were reversible by an antagonist of GR, RU486. Chromatin immunoprecipitation analysis revealed that GR bound to the Ctr1 promoter in Caco-2 cells. Transient transfection assays in Caco-2 cells demonstrated that the Ctr1 promoter was responsive to the CORT-activated glucocorticoid receptor, whereas mutation/deletion of the glucocorticoid receptor element (GRE) markedly impaired activation of the Ctr1 promoter. In addition, CORT-induced downregulation of Ctr1 promoter activity was markedly attenuated in Caco-2 cells when RU486 was added. These findings present a novel molecular target for CORT that down-regulates intestinal CTR1 expression via GR-mediated trans-repression in mice.

The mineralocorticoid receptor modulates timing and location of genomic binding by glucocorticoid receptor in response to synthetic glucocorticoids in keratinocytes.

Glucocorticoids (GCs) exert potent antiproliferative and anti-inflammatory properties, explaining their therapeutic efficacy for skin diseases. GCs act by binding to the GC receptor (GR) and the mineralocorticoid receptor (MR), co-expressed in classical and non-classical targets including keratinocytes. Using knockout mice, we previously demonstrated that GR and MR exert essential nonoverlapping functions in skin homeostasis. These closely related receptors may homo- or heterodimerize to regulate transcription, and theoretically bind identical GC-response elements (GRE). We assessed the contribution of MR to GR genomic binding and the transcriptional response to the synthetic GC dexamethasone (Dex) using control (CO) and MR knockout (MREKO ) keratinocytes. GR chromatin immunoprecipitation (ChIP)-seq identified peaks common and unique to both genotypes upon Dex treatment (1h). GREs, AP-1, TEAD, and p53 motifs were enriched in CO and MREKO peaks. However, GR genomic binding was 35% reduced in MREKO , with significantly decreased GRE enrichment, and reduced nuclear GR. Surface plasmon resonance determined steady state affinity constants, suggesting preferred dimer formation as MR-MR > GR-MR ~ GR-GR; however, kinetic studies demonstrated that GR-containing dimers had the longest lifetimes. Despite GR-binding differences, RNA-seq identified largely similar subsets of differentially expressed genes in both genotypes upon Dex treatment (3h). However, time-course experiments showed gene-dependent differences in the magnitude of expression, which correlated with earlier and more pronounced GR binding to GRE sites unique to CO including near Nr3c1. Our data show that endogenous MR has an impact on the kinetics and differential genomic binding of GR, affecting the time-course, specificity, and magnitude of GC transcriptional responses in keratinocytes.

Expression of glucocorticoid receptor (GR) and clinical significance in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare endocrine tumor, and most cases present with hormone excess with poor prognosis. Our research aims to determine the clinical and biological significance of glucocorticoid receptor (GR) expression using large cohorts of ACC patients. Immunohistochemistry was used to assess the expression of GR in 78 ACC cases from the West China Hospital (WCH) cohort. RNA-seq data were retrieved from The Cancer Genome Atlas database (TCGA, n=79). Clinicopathological and follow-up data were obtained from two cohorts. The correlation between the GR gene and tumor immune status was estimated using TIMER and GEPIA2. Kaplan–Meier analysis was performed to identify the prognostic value of GR in ACC. In the WCH cohort, positive nuclear GR staining was identified in 90% of the primary ACC cases. Cortisol-secreting ACCs demonstrated significantly lower GR protein expression than did nonfunctioning tumors (P<0.001). This finding was validated by the mRNA data analysis of the TCGA cohort (P = 0.030). GR expression was found to be positively correlated with the immune cell infiltration level and immune-checkpoint-related gene expression in ACC. Survival comparison and multivariate analysis showed that GR expression is an independent prognostic predictor of disease-free survival and overall survival in ACC patients in both cohorts. Our findings suggest that low GR expression is significantly correlated with excess cortisol, immune signatures and poor survival in ACC patients. We propose that GR signaling may play an important role in ACC behavior and thus may be a therapeutic target, which deserves further research.

Acute stress induces an aberrant increase of presynaptic release of glutamate and cellular activation in the hippocampus of BDNFVal/Met mice.

Stressful life events are considered major risk factors for the development of several psychiatric disorders, though people differentially cope with stress. The reasons for this are still largely unknown but could be accounted for by individual genetic variants, previous life events, or the kind of stressors. The human brain-derived neurotrophic factor (BDNF) Val66Met variant, which was found to impair intracellular trafficking and activity-dependent secretion of BDNF, has been associated with increased susceptibility to develop several neuropsychiatric disorders, although there is still some controversial evidence. On the other hand, acute stress has been consistently demonstrated to promote the release of glutamate in cortico-limbic regions and altered glutamatergic transmission has been reported in psychiatric disorders. However, it is not known if the BDNF Val66Met single-nucleotide polymorphism (SNP)affects the stress-induced presynaptic glutamate release. In this study, we exposed adult male BDNFVal/Val and BDNFVal/Met knock-in mice to 30 min of acute restraint stress. Plasma corticosterone levels, glutamate release, protein, and gene expression in the hippocampus were analyzed immediately after the end of the stress session. Acute restraint stress similarly increased plasma corticosterone levels and nuclear glucocorticoid receptor levels and phosphorylation in both BDNFVal/Val and BDNFVal/Met mice. However, acute restraint stress induced higher increases in hippocampal presynaptic release of glutamate, phosphorylation of cAMP-response element binding protein(CREB), and levels of the immediate early gene c-fos of BDNFVal/Met compared to BFNFVal/Val mice. Moreover, acute restraint stress selectively increased phosphorylation levels of synapsin I at Ser9 and at Ser603 in BDNFVal/Val and BDNFVal/Met mice, respectively. In conclusion, we reporthere that the BDNF Val66Met SNP knock-in mice display an altered response to acute restraint stress in terms of hippocampal glutamate release, CREB phosphorylation, and neuronal activation, compared to wild-type animals. Taken together, these results could partially explain the enhanced vulnerability to stressful events of Met carriers reported in both preclinical and clinical studies.

Diethyl phosphate disrupts hypothalamus-pituitary-adrenal axis endocrine hormones via nuclear receptors GR and Nur77: Integration of evidences from in vivo, in vitro and in silico approaches

Plenty of population epidemiology and cohort studies have found dialkyl phosphates (DAPs) in the urine were related to endocrine hormone disorders. However, we did not know whether these effects were caused by parent organophosphorus pesticides (OPs) or metabolite DAPs, especially the non-specific metabolite diethyl phosphate (DEP), which was the metabolic end product of most widely used diethyl OPs. In this study, animal experiments (in vivo), cell experiments (in vitro), small molecule-protein binding interaction experiments and computer molecular simulations (in silico) were used to explore the disturbing effects and molecular mechanisms of DEP on the hypothalamic-pituitary-adrenal (HPA) axis endocrine hormones. The animal experiments showed that chronic DEP exposure significantly disturbed the serum contents of HPA axis hormones in adult male rats. The target genes of glucocorticoid receptor (GR) in rat liver, including 11β-hsd1 and Pepck1 and PEPCK protein expressions, were down-regulated. Moreover, the gluconeogenic abilities of rats were impaired. However, it did not affect the expression of GR in the rat hypothalamus. These results indicated that the physiological functions of glucocorticoids and GR were damaged. Furthermore, spectroscopy experiments, cell experiments, molecular docking and molecular dynamics simulations also suggested that DEP can bind to nuclear receptors GR and Nur77, affecting their transcription factor functions, and the transcriptional expression levels of their downstream target genes were reduced. The biosynthesis and secretion of adrenocorticotropic hormone and glucocorticoids were blocked. Therefore, DEP can inhibit the production and physiological functions of HPA axis endocrine hormones by disrupting these related proteins and antagonizing nuclear receptors. These results were considered to provide a theoretical basis for strictly controlling the residue limits of OPs and their metabolites in foods, agricultural products and the environment. They also revealed new targets for evaluating the toxicities and risks of pesticide metabolites.

The DUX4 protein is a co-repressor of the progesterone and glucocorticoid nuclear receptors.

DUX4 is a transcription factor required during early embryonic development in placental mammals. In this work, we provide evidence that DUX4 is a co-repressor of nuclear receptors (NRs) of progesterone (PR) and glucocorticoids (GR). The DUX4 C-ter and N-ter regions, including the nuclear localization signals and homeodomain motifs, contribute to the co-repressor activity of DUX4 on PR and GR. Immunoprecipitation studies, using total protein extracts of cells expressing tagged versions of DUX4 and GR, support that these proteins are physically associated. Our studies suggest that DUX4 could modulate gene expression by co-regulating the activity of hormone NRs. This is the first report highlighting a potential endocrine role for DUX4.

Abstract 1165: Therapeutic targeting of glucocorticoid receptor in ETS rearranged cancer

Abstract The Nuclear receptor Glucocorticoid receptor (GR) acts as a ubiquitous hormone-dependent transcription factor, which regulates many cellular functions. Here, we discovered that some ETS (E-twenty-six) family transcription factors form physical complexes with GR. Two members of the family, FLI1 and ERG exhibited stronger binding with the ligand-activated GR. GR-FLI1 interaction enhance the transcriptional activity of DNA-bound GR. Antagonizing GR or lowering cortisol levels in Ewing sarcoma (ES) animal models, a pediatric bone malignancy driven by EWS-FLI1 fusion, robustly retarded tumor growth, as well as inhibited bone to lung metastasis. These findings prompted us to identify prognostic, GR-regulated gene signature in ES. Because the genomic binding sites of GR in ES are still unknown, using chromatin immunoprecipitation followed by high-throughput DNA sequencing in A673 cells we found previously known and new target genes of GR. By utilizing the protein-fragment complementation assay (PCA), we discovered that GR physically interacts with additional ETS factors. Disruption of this complex is enabled by clinically approved GR antagonist RU486 or newly developed selective GR modulators (i.e., SGRMs without AR or PR cross-reactivity). ETS proteins has been strongly associated with tumor progression and metastasis in several cancers. Taken together, we hypothesised that these mechanisms are also relevant for other ETS rearranged cancers. Results of co-immunoprecipitation analysis confirm the interaction of GR and ERG/ETV4 in ETS-positive prostate cancer cells. In addition, migration and proliferation were reduced when we knocked-down or antagonized GR. It is important noting that the pharmacology of GR is very well developed and some GR-targeting drugs are among the most prescribed medicines. These discoveries will open the way to clinical tests and eventual application of anti- hormone therapy in the discipline of pediatric sarcomas and ETS-rearranged cancers. Citation Format: Arunachalam Sekar, Nishanth Belugali Nataraj, Yosef Yarden. Therapeutic targeting of glucocorticoid receptor in ETS rearranged cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1165.

Crosstalk of hepatocyte nuclear factor 4a and glucocorticoid receptor in the regulation of lipid metabolism in mice fed a high-fat-high-sugar diet

BackgroundHepatocyte nuclear factor 4α (HNF4α) and glucocorticoid receptor (GR), master regulators of liver metabolism, are down-regulated in fatty liver diseases. The present study aimed to elucidate the role of down-regulation of HNF4α and GR in fatty liver and hyperlipidemia.MethodsAdult mice with liver-specific heterozygote (HET) and knockout (KO) of HNF4α or GR were fed a high-fat-high-sugar diet (HFHS) for 15 days. Alterations in hepatic and circulating lipids were determined with analytical kits, and changes in hepatic mRNA and protein expression in these mice were quantified by real-time PCR and Western blotting. Serum and hepatic levels of bile acids were quantified by LC-MS/MS. The roles of HNF4α and GR in regulating hepatic gene expression were determined using luciferase reporter assays.ResultsCompared to HFHS-fed wildtype mice, HNF4α HET mice had down-regulation of lipid catabolic genes, induction of lipogenic genes, and increased hepatic and blood levels of lipids, whereas HNF4α KO mice had fatty liver but mild hypolipidemia, down-regulation of lipid-efflux genes, and induction of genes for uptake, synthesis, and storage of lipids. Serum levels of chenodeoxycholic acid and deoxycholic acid tended to be decreased in the HNF4α HET mice but dramatically increased in the HNF4α KO mice, which was associated with marked down-regulation of cytochrome P450 7a1, the rate-limiting enzyme for bile acid synthesis. Hepatic mRNA and protein expression of sterol-regulatory-element-binding protein-1 (SREBP-1), a master lipogenic regulator, was induced in HFHS-fed HNF4α HET mice. In reporter assays, HNF4α cooperated with the corepressor small heterodimer partner to potently inhibit the transactivation of mouse and human SREBP-1C promoter by liver X receptor. Hepatic nuclear GR proteins tended to be decreased in the HNF4α KO mice. HFHS-fed mice with liver-specific KO of GR had increased hepatic lipids and induction of SREBP-1C and PPARγ, which was associated with a marked decrease in hepatic levels of HNF4α proteins in these mice. In reporter assays, GR and HNF4α synergistically/additively induced lipid catabolic genes.Conclusionsinduction of lipid catabolic genes and suppression of lipogenic genes by HNF4α and GR may mediate the early resistance to HFHS-induced fatty liver and hyperlipidemia.Graphical abstract

Immune modulatory effects of progesterone on oxLDL-induced trained immunity in monocytes.

Atherosclerotic cardiovascular diseases (CVD) are among the leading causes of death in the world. Monocyte‐derived macrophages are key players in the pathophysiology of atherosclerosis. Innate immune memory following exposure of monocytes to atherogenic compounds, such as oxidized low‐density lipoproteins (oxLDL), termed trained immunity, can contribute to atherogenesis. The current study aimed to elucidate intracellular mechanisms of oxLDL‐induced trained immunity. Using untargeted intracellular metabolomics in isolated human primary monocytes, we show that oxLDL‐induced trained immunity results in alterations in the balance of intracellular steroid hormones in monocytes. This was reflected by a decrease in extracellular progesterone concentrations following LPS stimulation. To understand the potential effects of steroid hormones on trained immunity, monocytes were costimulated with oxLDL and the steroid hormones progesterone, hydrocortisone, dexamethasone, β‐estradiol, and dihydrotestosterone. Progesterone showed a unique ability to attenuate the enhanced TNFα and IL‐6 production following oxLDL‐induced trained immunity. Single nucleotide polymorphisms in the nuclear glucocorticoid, progesterone, and mineralocorticoid receptor were shown to correlate with ex vivo oxLDL‐induced trained immunity in 243 healthy volunteers. Pharmacologic inhibition experiments revealed that progesterone exerts the suppression of TNFα in trained immunity via the nuclear glucocorticoid and mineralocorticoid receptors. Our data show that progesterone has a unique ability to suppress oxLDL‐induced trained immunity. We hypothesize that this effect might contribute to the lower incidence of CVD in premenopausal women.

Abstract P015: Biomarker results supporting selection of RP2D from a phase 1b study of ORIC-101, a glucocorticoid receptor antagonist, in combination with enzalutamide in patients with metastatic prostate cancer progressing on enzalutamide

Abstract Background: Preclinical studies have shown that activation of the glucocorticoid receptor (GR) leads to resistance to antiandrogens, which is reversed by GR inhibition. ORIC-101 is a potent, selective, orally bioavailable small molecule GR antagonist undergoing clinical development in combination with enzalutamide in patients with metastatic prostate cancer (NCT04033328). Methods: Ten patients were enrolled in the dose escalation portion of the Phase 1b study during which three dose levels of ORIC-101 given once daily were evaluated in combination with 160 mg enzalutamide. Translational markers were measured both pre-treatment and on study, including PD biomarkers, blood cortisol, GR and AR protein levels, and genomic alterations. PD biomarkers FKBP5, GILZ and PER1 were assessed by RT-qPCR in peripheral blood mononuclear cells (PBMCs), collected on multiple days and times in the first two cycles before and after dosing. PD modulation and target coverage were evaluable in 9 out of 10 patients. In tumor biopsies with &amp;gt;50 tumor cells, GR, AR and PTEN protein status were evaluated by immunohistochemistry (IHC). Liquid biopsies were collected before treatment, at the end of Cycle 2, and/or the end of treatment: nuclear GR and ARv7 protein levels were measured on circulating tumor cells (CTCs) by immunofluorescence (Epic Sciences, San Diego, CA), mutations and copy number alterations in cancer genes were captured in plasma with the 500-gene GuardantOMNI® ctDNA assay (Redwood City, CA). Results: PD suppression after one dose of ORIC-101 was observed in PBMCs of all patients. ORIC-101 reached steady-state target engagement within the first cycle in 6 out of 9 evaluable patients across dose levels, including at the RP2D. Nuclear GR protein was detected by IHC in the 6 pre-treatment biopsies, with H-scores ranging from 11 to 298 (median 95, IQR 55-148). Reduction of GR protein was observed in 3 out of 4 matched on study biopsies (pre-treatment H-scores 110-298), indicating potential elimination of GR positive tumor cells, while the fourth patient had low baseline GR levels (H-score 11). Pre-treatment AR protein levels were consistently high (IHC H-scores 230-300). AR alterations observed in ctDNA or CTCs were L702H (n=1 patient), H875Y (n=1), amplification (n=2), and ARv7 (n=2). PTEN protein aberrations were identified in two patients. The amount of tumor shedding in this cohort ranged from 0.24 to 82% (median 2.2%, IQR 0.54-12.1%), comparable to a clinical cohort of ~10,000 prostate tumors profiled with Guardant360® (Guardant Health). Prevalent genomic alterations at time of enrollment included TP53, RB1, BRCA2, ATM and SPOP, with minimal new genomic changes occurring on treatment. Conclusions: Biomarker data from patients enrolled in the Phase 1b study provide evidence of PD modulation, steady-state target coverage, and on-target tumor cell eradication at all dose levels. Tumor characterization in association with clinical outcome provides an opportunity to refine the patient population during the ongoing dose expansion portion of the study. Citation Format: Anneleen Daemen, Shravani Barkund, Ann Johnson, Aleksandr Pankov, Amber W. Wang, Haiying Zhou, Pratik S. Multani, Edna Chow Maneval, Lori S. Friedman, Rupal Patel. Biomarker results supporting selection of RP2D from a phase 1b study of ORIC-101, a glucocorticoid receptor antagonist, in combination with enzalutamide in patients with metastatic prostate cancer progressing on enzalutamide [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P015.

Overexpression of the GR Riborepressor LncRNA GAS5 Results in Poor Treatment Response and Early Relapse in Childhood B-ALL.

Simple SummaryAlthough childhood acute lymphoblastic leukemia (chALL) management is considered as one of the success stories in modern clinical oncology, the increased incidence of relapse in high-risk patients and the severe toxicity/long-term health effects due to chemotherapy intensity highlight the need for further improvements in patients’ risk stratification and personalized management. Synthetic glucocorticoids (GCs) are the core agents in chALL chemotherapy, exerting their role through the nuclear glucocorticoid receptor (GR), while GAS5 lncRNA suppresses the GCs-GR axis through binding to GR’s DNA binding domain. The objective of the study was the evaluation of GAS5 prognostic utility in chALL. GAS5 overexpression was strongly associated with a higher risk for short-term relapse and poor treatment outcome, independently of patients’ clinicopathological data. Moreover, “GAS5-including” multivariate models resulted in superior risk stratification and clinical benefit for disease prognosis, supporting precision medicine decisions in chALL.Glucocorticoids (GCs) remain the cornerstone of childhood acute lymphoblastic leukemia (chALL) therapy, exerting their cytotoxic effects through binding and activating of the glucocorticoid receptor (GR). GAS5 lncRNA acts as a potent riborepressor of GR transcriptional activity, and thus targeting GAS5 in GC-treated chALL could provide further insights into GC resistance and support personalized treatment decisions. Herein, to study the clinical utility of GAS5 in chALL prognosis and chemotherapy response, GAS5 expression was quantified by RT-qPCR in bone marrow samples of chB-ALL patients at diagnosis (n = 164) and at end-of-induction (n = 109), treated with ALL-BFM protocol. Patients’ relapse and death were used as clinical end-points for survival analysis. Bootstrap analysis was performed for internal validation, and decision curve analysis assessed the clinical net benefit for chALL prognosis. Our findings demonstrated the elevated GAS5 levels in blasts of chALL patients compared to controls and the significantly higher risk for short-term relapse and poor treatment outcome of patients overexpressing GAS5, independently of their clinicopathological data. The unfavorable prognostic value of GAS5 overexpression was strongly validated in the high-risk/stem-cell transplantation subgroup. Finally, multivariate models incorporating GAS5 levels resulted in superior risk stratification and clinical benefit for chALL prognostication, supporting personalized prognosis and precision medicine decisions in chALL.

Biomarker results supporting selection of RP2D from a phase 1b study of ORIC-101, a glucocorticoid receptor antagonist, in combination with nab-paclitaxel in patients with advanced solid tumors.

3110 Background: Preclinical studies have shown that activation of the glucocorticoid receptor (GR) leads to resistance to chemotherapeutics (eg taxanes) and antiandrogens across multiple tumor types, while GR inhibition enhances therapeutic efficacy. ORIC-101 is a potent, selective, and orally bioavailable small molecule GR antagonist undergoing clinical development in combination with nab-paclitaxel in patients with advanced solid tumors and in combination with enzalutamide in patients with metastatic prostate cancer. Methods: 21 patients were enrolled in the dose escalation portion of the phase 1b study, which evaluated both intermittent (5 days on, 2 days off for 21 days) and continuous dosing regimens of ORIC-101 across 5 cohorts (NCT03928314). Tumor tissue was obtained pre-treatment for 19 out of 21 patients, and on study or at the end of treatment for 11 patients. GR protein status was retrospectively evaluated using a proprietary IHC assay optimized for staining nuclear GR in the epithelial compartment of major tumor type tissues. Biopsies were also profiled with RNA-seq to evaluate a proprietary GR activation signature as a potentially predictive and pharmacodynamic (PD) biomarker. Blood-derived peripheral blood mononuclear cells (PBMCs) were collected for 20 patients along with the pre-treatment biopsy, in the morning of days 1, 5 and/or 8 of Cycle 1, and 2.5 or 6 hours after ORIC-101 administration. Blood cortisol levels were also simultaneously measured. PD modulation in PBMCs was assessed by RT-qPCR for biomarkers FKBP5, GILZ and PER1, selected for their consistent stimulation by GR and reversal with ORIC-101 in preclinical studies and observed PD modulation in healthy volunteers administered ORIC-101. Results: Nuclear GR protein was detected in most pre-treatment biopsies regardless of tumor type, and on treatment reduction of GR protein was observed across dose levels. At physiological systemic cortisol levels, ORIC-101 demonstrated PD suppression in PBMCs on days 1, 5 and 8 in the majority of patients. Cortisol levels increased post-dose in these patients due to negative feedback between cortisol and GR. Steady-state target engagement was not consistently demonstrated with the intermittent regimen. In healthy volunteer studies of ORIC-101, steady-state target suppression was consistently achieved after 7 consecutive daily doses of 200 or 350 mg of ORIC-101. Thus, continuous ORIC-101 administration was selected as the recommended phase 2 dose (RP2D) regimen, aimed at achieving sustained GR suppression for optimal chemotherapy re-sensitization. Conclusions: Biomarker data from patients enrolled in the phase 1b study provide evidence of on-target tumor cell eradication and PD modulation and support the RP2D and the tumor types selected for the ongoing dose expansion portion of the study. Clinical trial information: NCT03928314.

Early postnatal allergic airway inflammation induces dystrophic microglia leading to excitatory postsynaptic surplus and autism-like behavior

Microglia play key roles in synaptic pruning, which primarily occurs from the postnatal period to adolescence. Synaptic pruning is essential for normal brain development and its impairment is implicated in neuropsychiatric developmental diseases such as autism spectrum disorders (ASD). Recent epidemiological surveys reported a strong link between ASD and atopic/allergic diseases. However, few studies have experimentally investigated the relationship between allergy and ASD-like manifestations, particularly in the early postnatal period, when allergic disorders occur frequently. Therefore, we aimed to characterize how allergic inflammation in the early postnatal period influences microglia and behavior using mouse models of short- and long-term airway allergy. Male mice were immunized by an intraperitoneal injection of aluminum hydroxide and ovalbumin (OVA) or phosphate-buffered saline (control) on postnatal days (P) 3, 7, and 11, followed by intranasal challenge with OVA or phosphate-buffered saline solution twice a week until P30 or P70. In the hippocampus, Iba-1-positive areas, the size of Iba-1-positive microglial cell bodies, and the ramification index of microglia by Sholl analysis were significantly smaller in the OVA group than in the control group on P30 and P70, although Iba-1-positive microglia numbers did not differ significantly between the two groups. In Iba-1-positive cells, postsynaptic density protein 95 (PSD95)-occupied areas and CD68-occupied areas were significantly decreased on P30 and P70, respectively, in the OVA group compared with the control group. Immunoblotting using hippocampal tissues demonstrated that amounts of PSD95, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor 2, and N-methyl-D-aspartate (NMDA) receptor 2B were significantly increased in the OVA group compared with the control group on P70, and a similar increasing trend for PSD95 was observed on P30. Neurogenesis was not significantly different between the two groups on P30 or P70 by doublecortin immunohistochemistry. The social preference index was significantly lower in the three chamber test and the number of buried marbles was significantly higher in the OVA group than in the control group on P70 but not on P30, whereas locomotion and anxiety were not different between the two groups. Compared with the control group, serum basal corticosterone levels were significantly elevated and hippocampal glucocorticoid receptor (GR) amounts and nuclear GR translocation in microglia, but not in neurons or astrocytes, were significantly decreased in the OVA group on P70 but not on P30. Gene set enrichment analysis of isolated microglia revealed that genes related to immune responses including Toll-like receptor signaling and chemokine signaling pathways, senescence, and glucocorticoid signaling were significantly upregulated in the OVA group compared with the control group on P30 and P70. These findings suggest that early postnatal allergic airway inflammation induces dystrophic microglia that exhibit defective synaptic pruning upon short- and long-term allergen exposure. Furthermore, long-term allergen exposure induced excitatory postsynaptic surplus and ASD-like behavior. Hypothalamo-pituitary-adrenal axis activation and the compensatory downregulation of microglial GR during long-term allergic airway inflammation may also facilitate these changes.

Glucocorticoid signaling and lipid metabolism disturbances in the liver of rats treated with 5α-dihydrotestosterone in an animal model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex reproductive disorder often associated with obesity, insulin resistance, and dyslipidemia. Hormonal changes in PCOS may also include altered glucocorticoid signaling. Our aim was to examine whether alterations in hepatic glucocorticoid signaling are associated with disturbances of glucose and lipid metabolism in animal model of PCOS. Female rats, 3 weeks old, were subcutaneously implanted with 5α-dihydrotestosterone (DHT) or placebo pellets for 90 days to induce PCOS. Expression of 11β-hydroxysteroid dehydrogenase 1 (11βHSD1) and A-ring reductases (5α and 5β), as well as intracellular distribution of glucocorticoid receptor (GR) and expression of its regulated genes were examined in the liver. Proteins of hepatic lipid and carbohydrate metabolism and markers of inflammation were also assessed. DHT treatment induced increase in body and liver mass, as well as in triglycerides and free fatty acids levels in plasma. Elevation of 11βHSD1 and reduction of 5α-reductase expression was observed together with increased hepatic corticosterone concentration and nuclear GR activation. Induced expression of Krüppel-like factor 15 and decreased expression of genes for proinflammatory cytokines and de novo lipogenesis (DNL) were detected in the liver of DHT-treated rats, while DNL regulators and proinflammatory markers were not changed. However, increased mRNA levels of stearoyl-CoA desaturase and apolipoprotein B were observed in DHT animals. DHT treatment stimulated hepatic glucocorticoid prereceptor metabolism through increased corticosterone availability which is associated with enhanced GR activation. This does not affect gluconeogenesis and DNL, but could be linked to stimulated triglyceride synthesis and hypertriglyceridemia.

Regulation of p38 MAPK and glucocorticoid receptor activation by hydrocortisone in mono-and co-cultured pancreatic acinar and stellate cells

Background/objectivesAcute pancreatitis develops as an inflammatory response to pancreatic tissue injury. Postoperative pancreatitis has recently been associated with increased occurrence of complications. Activation of the mitogen-activated protein kinase p38 (p38 MAPK) pathway occurs early in acute pancreatitis and its inhibition has been suggested to alleviate pancreatic inflammation. Glucocorticoids are potent anti-inflammatory steroids whose use in the management of acute pancreatitis remains controversial. Our aim was to examine the effect of crosstalk between pancreatic acinar cells (PACs) and stellate cells (PSCs) on p38 MAPK and glucocorticoid receptor (GR) activation and to assess the impact of hydrocortisone on these events. MethodsThe long-term co-culture setting for mouse PACs and PSCs developed in our laboratory was used. Parallel 4d mono- and co-cultures with or without 10 nM hydrocortisone were performed followed by immunocytochemical analysis of nuclear GR and phospho-p38 MAPK (pp38 MAPK). ResultsHydrocortisone inhibited pp38 MAPK up-regulation evoked by co-culture in PACs and PSCs and increased nuclear translocation of GR in PAC monocultures and in co-cultured PACs and PSCs. In PSC monocultures and co-cultured PACs, ligand-independent expression of nuclear GR was observed. In the former no change in nuclear GR but a significant decrease in total GR as analyzed by Western blot was caused by hydrocortisone. ConclusionsCellular microenvironment plays a significant role on p38 MAPK and GR activation in PACs and PSCs. Hydrocortisone is an effective means to inhibit p38 MAPK activation in PACs and PSCs. Both ligand-dependent and -independent regulatory roles for GR are suggested in the exocrine pancreas.