Glucocorticoid Receptor Signaling Pathway Research Articles

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.

Discovery of a glucocorticoid receptor (GR) activity signature correlates with immune cell infiltration in adrenocortical carcinoma

BackgroundAdrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine malignancy, of which >40% present with glucocorticoid excess. Glucocorticoids and glucocorticoid receptor (GR) signaling have long been thought to suppress...

Effects of hydrostatic compression on milk production-related signaling pathways in mouse mammary epithelial cells

Mammary epithelial cells (MECs) secrete milk into the mammary alveolar lumen during lactation. The secreted milk accumulates in the alveolar lumen until milk ejection occurs, and excess milk accumulation downregulates milk production in alveolar MECs. Intramammary hydrostatic pressure also increases in the alveolar lumen in a manner dependent on milk accumulation. In this study, we investigated whether high hydrostatic compression directly affects lactating MECs, using a commercial compression device and a lactation culture model of MECs, which have milk production ability and less permeable tight junctions. High hydrostatic compression at 100 kPa for 8 h decreased β-casein and increased claudin-4 levels concurrently with inactivation of STAT5 and glucocorticoid receptor signaling pathways. In addition, high hydrostatic compression for 1 h inactivated STAT5 and activated p38 MAPK signaling. Furthermore, repeated rises and falls of the hourly hydrostatic compression induced activation of positive (Akt, mTOR) and negative (STAT3, NF-κB) signaling pathways for milk production concurrently with stimulation of casein and lactoferrin production in MECs. These results indicate that milk production-related signaling pathways in MECs change in response to hydrostatic compression. Hydrostatic compression of the alveolar lumen may directly regulate milk production in the alveolar MECs of lactating mammary glands.

Accumulation and glucocorticoid signaling suppression by four emerging perfluoroethercarboxylic acids based on animal exposure and cell testing

Various perfluoroethercarboxylic acids (PFECA) have emerged as next-generation replacements of legacy per- and polyfluoroalkyl substances (PFAS). However, there is a paucity of information regarding their bioaccumulation ability and hazard characterization. Here, we explored the accumulation and hepatotoxicity of four PFECA compounds (HFPO-DA, HFPO-TA, PFO4DA, and PFO5DoDA) in comparison to perfluorooctanoic acid (PFOA) after chronic low-dose exposure in mice. Except for HFPO-DA, the levels of all tested PFAS in the liver exceeded that in serum. High molecular weight PFECA compounds (PFO5DoDA and HFPO-TA) showed stronger accumulation capacity and longer half-lives (t1/2) than low molecular weight PFECA compounds (HFPO-DA and PFO4DA) and even legacy PFOA. Although hepatomegaly is a common apical end point of PFAS exposure, the differentially expressed gene (DEG) profiles in the liver suggested significant differences between PFOA and the four PFECA compounds. Gene enrichment analysis supported a considerable inhibitory effect of PFECA, but not PFOA, on the glucocorticoid receptor (GR) signaling pathway. Both HFPO-TA and PFO5DoDA demonstrated a more pronounced ability to perturb RNA expression profiles in vivo and to suppress GR signaling in vitro compared to HFPO-DA and PFO4DA. Calculated reference doses (RfDs) emphasized the potential hazard of PFECA to human health. Overall, our findings indicate that PFECA alternatives do not ease the concerns raised from legacy PFAS pollution.

Folate and Cobalamin Deficiencies during Pregnancy Disrupt the Glucocorticoid Response in Hypothalamus through N-Homocysteinilation of the Glucocorticoid Receptor.

Vitamin B9 (folate)/B12 (cobalamin) deficiency is known to induce brain structural and/or functional retardations. In many countries, folate supplementation, targeting the most severe outcomes such as neural tube defects, is discontinued after the first trimester. However, adverse effects may occur after birth because of some mild misregulations. Various hormonal receptors were shown to be deregulated in brain tissue under these conditions. The glucocorticoid receptor (GR) is particularly sensitive to epigenetic regulation and post-translational modifications. In a mother-offspring rat model of vitamin B9/B12 deficiency, we investigated whether a prolonged folate supplementation could restore the GR signaling in the hypothalamus. Our data showed that a deficiency of folate and vitamin B12 during the in-utero and early postnatal periods was associated with reduced GR expression in the hypothalamus. We also described for the first time a novel post-translational modification of GR that impaired ligand binding and GR activation, leading to decrease expression of one of the GR targets in the hypothalamus, AgRP. Moreover, this brain-impaired GR signaling pathway was associated with behavioral perturbations during offspring growth. Importantly, perinatal and postnatal supplementation with folic acid helped restore GR mRNA levels and activity in hypothalamus cells and improved behavioral deficits.

Induction of SGK1 via glucocorticoid-influenced clinical outcome of triple-negative breast cancer patients.

Triple-negative breast cancer (TNBC) is a highly heterogeneous and aggressive breast malignancy. Glucocorticoid (GC)-glucocorticoid receptor (GR) pathway plays a pivotal role in the cellular responses to various stresses including chemotherapy. Serum- and glucocorticoid-induced kinase-1 (SGK1) is known as an important downstream effector molecule in the GR signaling pathway, we attempted to explore its clinicopathological and functional significance in TNBC in which GR is expressed. We first immunolocalized GR and SGK1 and correlated the results with clinicopathological variables and clinical outcome in 131 TNBC patients. We also evaluated the effects of SGK1 on the cell proliferation and migration in TNBC cell lines with administration of dexamethasone (DEX) to further clarify the significance of SGK1. The status of SGK1 in carcinoma cells was significantly associated with adverse clinical outcome in TNBC patients examined and was significantly associated with lymph node metastasis, pathological stage, and lymphatic invasion of the patients. In particular, SGK1 immunoreactivity was significantly associated with an increased risk of recurrence in GR-positive TNBC patients. Subsequent in vitro studies also demonstrated that DEX promoted TNBC cell migration and the silencing of gene expression did inhibit the cell proliferation and migration of TNBC cells under DEX treatment. To the best of our knowledge, this is the first study to explore an association between SGK1 and clinicopathological variables and clinical outcome of TNBC patients. SGK1 status was significantly positively correlated with adverse clinical outcome of TNBC patients and promoted carcinoma cell proliferation and migration of carcinoma cells.

Abstract 1933: Transcriptomic profiling of Hispanic colorectal cancer disparities

Abstract Colorectal cancer (CRC) was reported as the third most diagnosed cancer worldwide with approximately 1.93 million new cases reported in 2020. Within the U.S., the incidence and death rates differ by ethnicity. Among Hispanics, CRC accounted for 12% and 8% of all estimated new cases of cancer and 11% and 9% of all cancer deaths in men and women, respectively, in 2018. Though CRC is preventable, and the overall survival rate is approximately 90% when detected at early stages, only about 39% of CRC patients are diagnosed in the early stages, mostly because screening rates in the US are low, especially among Hispanics. Access to health care for Hispanics is limited when compared to non-Hispanic Whites (NHWs); as a result, they tend to present with later-stage CRC, which likely accounts for their inferior survival after CRC. Essential to improving treatment, prognosis, and detection strategies is the identification and validation of new ethnicity-specific transcriptomic profiles. Here, we have conducted RNA sequencing from Hispanic and NHW CRC (n=10 each) and normal adjacent tissues (NATs; n=3 each), to identify the differentially expressed genes (DEGs) in these two populations. The Illumina sequencing reads were mapped against the reference human genome, and the read counts were normalized using the median of ratios method using DESeq2 software. The transcriptomic analysis revealed that as compared to the respective NATs, 7222 and 7873 genes were observed to be differentially expressed in NHW and Hispanic CRC tissues. Among these, 213 and 363 genes were unique to the NHW and Hispanic CRC cohorts, respectively. Within the Hispanic cohort, gender-based analyses revealed that 504 and 266 genes were differentially expressed in the female and male donors (n=5 each), respectively, as compared to the NHW counterparts. Further, 271 and 475 genes were found to be differentially expressed in early (stage I/II; n=5) and late (stage III/IV; n=3) stages of Hispanic CRC tissues, respectively, as compared to the NHW counterparts. Pathway enrichment analysis of the unique DEGs from the Hispanic and NHW cohorts was conducted using Ingenuity Pathway Analysis software. The genes involved in cysteine biosynthesis, ferroptosis, and GABA receptor signaling pathways were seen to be enriched in the NHW cohort, whereas the genes involved in glucocorticoid receptor (GR) signaling, bile, and androgen biosynthesis pathways were enriched in the Hispanic cohort. Previous studies have implicated, GR signaling to promote tumor heterogeneity and invasion in CRC via CDK1, or bile acids to regulate numerous genes including p53, CDK1, cyclooxygenase 2, IL8, and various miRNAs leading to CRC metastasis. As each pathway likely regulates specific cellular behaviors, the DEGs identified from this study will constitute potential ethnicity-specific biomarkers or attractive targets for the development of novel therapeutic interventions. Citation Format: Soumya Nair, Aditi Kulkarni, Sourav Roy. Transcriptomic profiling of Hispanic colorectal cancer disparities [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 1933.

Protective effects and mechanisms of the Erzhi formula on glucocorticoid induced primary cortical neuron injury.

High concentrations of glucocorticoids (GC) can cross the blood-brain barrier into the brain parenchyma, triggering a stress state that can lead to a range of physiological changes. This study investigated whether Erzhi formula has neuroprotective effects against glucocorticoid damage by establishing a dexamethasone-induced primary cortical neuron injury model in vitro. The results showed that Erzhi formula could reduce dexamethasone-induced apoptosis in primary cultured cortical neurons and improve synaptic damage. Further, network pharmacological analysis revealed that Erzhi formula may exert antidepressant effects by multi-component, multi-target, and multi-pathway characteristics, in which Salidroside, Biochanin-A and other ingredients are key components, HSD11B1, NR3C1, and other proteins are key targets, and steroid metabolism may be a key process in its action. Moreover, our study found that the neuroprotective effect of Erzhi formula might be related to the 11β-HSD1-GC/glucocorticoid receptor (GR) signaling pathway. The Erzhi formula could significantly inhibit the activity of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in vitro using homogeneous time-resolved fluorescence. In addition to providing evidence for the pharmacological effects of the Erzhi formula, the present study lays down the foundation for subsequent experiments.

RF01 | PMON168 Hedgehog-Induced Somatostatin Receptor Expression is Inhibited by the Activation of the Glucocorticoid Receptor Signaling Pathway in Human Pituitary Adenoma Organoids

Abstract Background Cushing's disease (CD) is an endocrine disorder caused by an ACTH-secreting pituitary adenoma that is associated with increased morbidity and mortality. Because somatostatin inhibits pituitary ACTH secretion, the somatostatin receptor subtypes (SSTR) 2 and 5 have been targets of medical therapies for CD. The SSTR5 is most consistently and strongly expressed in corticotroph pituitary tumors but the SSTR dynamics in CD during treatment and responses to cortisol level changes are far from being understood. Studies in adult stomach demonstrated that the Hedgehog (Hh) signaling pathway is critical for the regulation of somatostatin and SSTR signaling. While Hh signaling is known to be essential during the embryonic development of the pituitary and in the adult gland, the mechanism by which Hh signaling regulates SSTR expression in CD is unknown. Hypothesis Activation of glucocorticoid receptor (NR3C1, GR) results in the inhibition of Hh transcription factor GLI1 leading to reduced SSTR expression. Methods We developed a human pituitary adenoma organoid model (hPITOiPSC) from induced pluripotent stem cells treated with the glucocorticoid receptor (GR) antagonist mifepristone, and co-treated them with or without SSTR agonists pasireotide or octreotide. In a separate series of experiments, the role of Hh transcription factor GLI1 was identified using hPITOiPSC treated with mifepristone with or without GANT61 (GLI inhibitor) or ketoconazole (Smoothened, SMO inhibitor). CRISPR-Cas9 gene editing of hPITOiPSC pituitary organoids were used to model the development of pituitary corticotroph adenomas in the presence of BRAF, USP48 and USP8 mutations. Human pituitary adenoma tissue harvested fresh during pituitary surgery was used to generate pituitary corticotroph subtype adenoma organoids (hPITOs). Dose responses using standard of care drugs were performed using the hPITOs. Results 1) At baseline SSTR2 was abundantly expressed within hPITOiPSC. Mifepristone induced significant increases in ACTH secretion and POMC expression that correlated with induced SSTR2 and 5. Mifepristone-induced SSTR2 and 5 expression was significantly inhibited in the presence of GANT61, whereas SMO inhibitor ketoconazole had a minimal effect on mifepristone-induced SSTR2/5, POMC expression and ACTH secretion. Dexamethasone alone significantly inhibited both GLI1 and SSTR2 and 5. 2) While pituitary organoids that were differentiated from control iPSCs (iPSCCtrl) expressed all major hormone-producing cell lineages, there was a significant increase in ACTH expression with loss of PIT1, GH, FSH, LH and PRL in iPCSs expressing mutated BRAF, USP48 and USP8. Organoids expressing a mutation in BRAF had significantly higher SSTR2 expression levels compared to controls. 3) HPITOs generated from pituitary adenomas of CD patients showed differential organoid responses to pasireotide, mifepristone, and ketoconazole. Conclusion Within pituitary adenomas, the SSTR is a transcriptional target of Gli1, and this response is blocked by the activation of the GR. These data form the basis of combination therapy for CD with mifepristone and pasireotide. Presentation: Saturday, June 11, 2022 1:37 p.m. - 1:42 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Biological pathways and comparison with biopsy signals and cellular origin of peripheral blood transcriptomic profiles during kidney allograft pathology

Kidney transplant injury processes are associated with molecular changes in kidney tissue, primarily related to immune cell activation and infiltration. How these processes are reflected in the circulating immune cells, whose activation is targeted by strong immunosuppressants, is poorly understood. To study this, we analyzed the molecular alterations in 384 peripheral blood samples from four European transplant centers, taken at the time of a kidney allograft biopsy, selected for their phenotype, using RNA-sequencing. In peripheral blood, differentially expressed genes in 136 rejection and 248 no rejection samples demonstrated upregulation of glucocorticoid receptor and nucleotide oligomerization domain-like receptor signaling pathways. Pathways enriched in antibody-mediated rejection (ABMR) were strongly immune-specific, whereas pathways enriched in T cell-mediated rejection were less immune related. In polyomavirus infection, upregulation of mitochondrial dysfunction and interferon signaling pathways was seen. Next, we integrated the blood results with transcriptomics of 224 kidney allograft biopsies which showed consistently upregulated genes per phenotype in both blood and biopsy. In single-cell RNASeq (scRNASeq) analysis of seven kidney allograft biopsies, the consistently overexpressed genes in ABMR were mostly expressed by infiltrating leukocytes in the allograft. Similarly, in peripheral blood scRNASeq analysis, these genes were overexpressed in ABMR in immune cell subtypes. Furthermore, overexpression of these genes in ABMR was confirmed in independent cohorts in blood and biopsy. Thus, our results highlight the immune activation pathways in peripheral blood leukocytes at the time of kidney allograft pathology, despite the use of current strong immunosuppressants, and provide a framework for future therapeutic interventions.

Circadian Clock Genes Are Correlated with Prognosis and Immune Cell Infiltration in Colon Adenocarcinoma.

Background Colon adenocarcinoma (COAD) is a malignancy with a high incidence and is associated with poor quality of life. Dysfunction of circadian clock genes and disruption of normal rhythms are associated with the occurrence and progression of many cancer types. However, studies that systematically describe the prognostic value and immune-related functions of circadian clock genes in COAD are lacking. Methods Genomic data obtained from The Cancer Genome Atlas (TCGA) database was analyzed for expression level, mutation status, potential biological functions, and prognostic performance of core circadian clock genes in COAD. Their correlations with immune infiltration and TMB/MSI score were analyzed by Spearman's correlation analysis. Pearson's correlation analysis was performed to analyze their associations with drug sensitivity. Lasso Cox regression analysis was performed to construct a prognosis signature. Moreover, an mRNA-miRNA-lncRNA regulatory axis was also detected by ceRNA network. Results In COAD tissues, the mRNA levels of CLOCK, CRY1, and NR1D1 were increased, while the mRNA levels of ARNTL, CRY2, PER1, PER3, and RORA were decreased. We also summarized the relative genetic mutation variation landscape. GO and KEGG pathway analyses demonstrated that these circadian clock genes were primarily correlated with the regulation of circadian rhythms and glucocorticoid receptor signaling pathways. COAD patients with high CRY2, NR1D1, and PER2 expression had worse prognosis. A prognostic model constructed based on the 9 core circadian clock genes predicted the COAD patients' overall survival with medium to high accuracy. A significant association between prognostic circadian clock genes and immune cell infiltration was found. Moreover, the lncRNA KCNQ1OT1/hsa-miRNA-32-5p/PER2/CRY2 regulatory axis in COAD was also detected through a mRNA-miRNA-lncRNA network. Conclusion Our results identified CRY2, NR1D1, and PER2 as potential prognostic biomarkers for COAD patients and correlated their expression with immune cell infiltration. The lncRNA KCNQ1OT1/hsa-miRNA-32-5p/PER2/CRY2 regulatory axis was detected in COAD and might play a vital role in the occurrence and progression of COAD.

Extracellular Vesicle Proteins and MicroRNAs Are Linked to Chronic Post-Traumatic Stress Disorder Symptoms in Service Members and Veterans With Mild Traumatic Brain Injury.

Symptoms of post-traumatic stress disorder (PTSD) are common in military populations, and frequently associated with a history of combat-related mild traumatic brain injury (mTBI). In this study, we examined relationships between severity of PTSD symptoms and levels of extracellular vesicle (EV) proteins and miRNAs measured in the peripheral blood in a cohort of military service members and Veterans (SMs/Vs) with chronic mTBI(s). Participants (n = 144) were divided into groups according to mTBI history and severity of PTSD symptoms on the PTSD Checklist for DSM-5 (PCL-5). We analyzed EV levels of 798 miRNAs (miRNAs) as well as EV and plasma levels of neurofilament light chain (NfL), Tau, Amyloid beta (Aβ) 42, Aβ40, interleukin (IL)-10, IL-6, tumor necrosis factor-alpha (TNFα), and vascular endothelial growth factor (VEGF). We observed that EV levels of neurofilament light chain (NfL) were elevated in participants with more severe PTSD symptoms (PCL-5 ≥ 38) and positive mTBI history, when compared to TBI negative controls (p = 0.024) and mTBI participants with less severe PTSD symptoms (p = 0.006). Levels of EV NfL, plasma NfL, and hsa-miR-139–5p were linked to PCL-5 scores in regression models. Our results suggest that levels of NfL, a marker of axonal damage, are associated with PTSD symptom severity in participants with remote mTBI. Specific miRNAs previously linked to neurodegenerative and inflammatory processes, and glucocorticoid receptor signaling pathways, among others, were also associated with the severity of PTSD symptoms. Our findings provide insights into possible signaling pathways linked to the development of persistent PTSD symptoms after TBI and biological mechanisms underlying susceptibility to PTSD.

Longitudinal CSF proteome profiling in mice to uncover the acute and sustained mechanisms of action of rapid acting antidepressant (2R,6R)-hydroxynorketamine (HNK)

Delayed onset of antidepressant action is a shortcoming in depression treatment. Ketamine and its metabolite (2R,6R)-hydroxynorketamine (HNK) have emerged as promising rapid-acting antidepressants. However, their mechanism of action remains unknown. In this study, we first described the anxious and depression-prone inbred mouse strain, DBA/2J, as an animal model to assess the antidepressant-like effects of ketamine and HNK in vivo. To decode the molecular mechanisms mediating HNK's rapid antidepressant effects, a longitudinal cerebrospinal fluid (CSF) proteome profiling of its acute and sustained effects was conducted using an unbiased, hypothesis-free mass spectrometry-based proteomics approach. A total of 387 proteins were identified, with a major implication of significantly differentially expressed proteins in the glucocorticoid receptor (GR) signaling pathway, providing evidence for a link between HNK and regulation of the stress hormone system. Mechanistically, we identified HNK to repress GR-mediated transcription and reduce hormonal sensitivity of GR in vitro. In addition, mammalian target of rapamycin (mTOR) and brain-derived neurotrophic factor (BDNF) were predicted to be important upstream regulators of HNK treatment. Our results contribute to precise understanding of the temporal dynamics and molecular targets underlying HNK's rapid antidepressant-like effects, which can be used as a benchmark for improved treatment strategies for depression in future.

Combined glucocorticoid resistance and hyperlactatemia contributes to lethal shock in sepsis

Sepsis is a potentially lethal syndrome resulting from a maladaptive response to infection. Upon infection, glucocorticoids are produced as a part of the compensatory response to tolerate sepsis. This tolerance is, however, mitigated in sepsis due to a quickly induced glucocorticoid resistance at the level of the glucocorticoid receptor. Here, we show that defects in the glucocorticoid receptor signaling pathway aggravate sepsis pathophysiology by lowering lactate clearance and sensitizing mice to lactate-induced toxicity. The latter is exerted via an uncontrolled production of vascular endothelial growth factor, resulting in vascular leakage and collapse with severe hypotension, organ damage, and death, all being typical features of a lethal form of sepsis. In conclusion, sepsis leads to glucocorticoid receptor failure and hyperlactatemia, which collectively leads to a lethal vascular collapse.

Forecasting early onset diminished ovarian reserve for young reproductive age women.

To investigate the biological networks associated with DOR in young women and the subsequent molecular impact on preimplantation embryos. Whole peripheral blood was collected from patients: young women presenting with diminished ovarian reserve (DOR) and age-matched young women with normal ovarian reserve. Maternal exome sequencing was performed on the NovaSEQ 6000 and sequencing validation was completed using Taqman® SNP Genotyping Assays. Blastocyst global methylome and transcriptome sequencing were also analyzed. Exome sequencing revealed 730 significant DNA variants observed exclusively in the young DOR patients. Bioinformatic analysis revealed a significant impact to the Glucocorticoid receptor (GR) signaling pathway and each young DOR female had an average of 6.2 deleterious DNA variants within this pathway. Additional stratification based on patient age resulted in a cut-off at 31 years for young DOR discrimination. Embryonic global methylome sequencing resulted in only a very small number of total CpG sites with methylation alterations (1,775; 0.015% of total) in the DOR group. Additionally, there was no co-localization between these limited number of altered CpG sites and significant variants, genes, or pathways. RNA sequencing also resulted in no biologically significant transcription changes between DOR blastocysts and controls. GR signaling DNA variants were observed in women with early-onset DOR potentially compromising oocyte production and quality. However, no significant downstream effects on biological processes appear to impact the resulting blastocyst. The ability to forecast premature DOR for young women may allow for earlier identification and clinical intervention for this patient population.

BCOR modulates transcriptional activity of a subset of glucocorticoid receptor target genes involved in cell growth and mobility

Glucocorticoid (GC) receptor (GR) is a key transcription factor (TF) that regulates vital metabolic and anti-inflammatory processes. We have identified BCL6 corepressor (BCOR) as a dexamethasone-stimulated interaction partner of GR. BCOR is a component of non-canonical polycomb repressor complex 1.1 (ncPCR1.1) and linked to different developmental disorders and cancers, but the role of BCOR in GC signaling is poorly characterized. Here, using ChIP-seq we show that, GC induces genome-wide redistribution of BCOR chromatin binding towards GR-occupied enhancers in HEK293 cells. As assessed by RNA-seq, depletion of BCOR altered the expression of hundreds of GC-regulated genes, especially the ones linked to TNF signaling, GR signaling and cell migration pathways. Biotinylation-based proximity mapping revealed that GR and BCOR share several interacting partners, including nuclear receptor corepressor NCOR1. ChIP-seq showed that the NCOR1 co-occurs with both BCOR and GR on a subset of enhancers upon GC treatment. Simultaneous depletion of BCOR and NCOR1 influenced GR target gene expression in a combinatorial and gene-specific manner. Finally, we show using live cell imaging that the depletion of BCOR together with NCOR1 markedly enhances cell migration. Collectively, our data suggest BCOR as an important gene and pathway selective coregulator of GR transcriptional activity.

Abstract PS13-22: Shedding light on the dark side of chemo: Post-GWAS functional studies of rs28714259 in anthracycline-induced cardiotoxicity

Abstract Anthracyclines are commonly used chemotherapies for breast cancer treatment, but can cause dose-related cardiotoxicities and lead to congestive heart failure (CHF) in 2% of patients. Several mechanisms of anthracycline’s cardiotoxic effect have been proposed, but the molecular pathogenesis is not fully understood. In addition, there are no clinically available biomarkers to predict cardiotoxicity. Previously we have identified and validated the association of a single nucleotide polymorphism (SNP), rs28714259 (G/A), with risk of anthracycline-induced CHF through genome-wide association study (GWAS) across three large phase III adjuvant breast cancer trials. rs28714259 (G/A) locates in a putative glucocorticoid receptor (GR) response element and the risk allele (A) is predicted to disrupt GR binding. The activation of GR signaling pathway by dexamethasone is known to protect cardiomyocytes from doxorubicin-induced apoptosis in rats. To investigate the role of rs28714259 in CHF post-GWAS, we set out to determine whether rs28714259 modulates GR signaling pathway through allele-specific GR enhancer activity. We cloned a 1kb DNA sequence on both sides of rs28714259, containing either the wildtype (G) or risk allele (A), into a luciferase reporter plasmid. Luciferase assay in iPSC-derived cardiomyocytes (iPSC-CMs) with GR activation by 100nM dexamethasone showed that cardiomyocytes transfected with wildtype construct had 60% increased activity compared to control vector with no enhancer. iPSC-CMs transfected with risk allele constructs did not show increased luciferase activity, suggesting that the A-allele disrupts GR-mediated transcriptional activation. Using electrophoretic mobility shift assay (EMSA) with nuclear extract from iPSC-CMs treated with dexamethasone, we observed a prominent band shift with either G- or A-allele probes. Furthermore, a supershift band was observed with GR antibody, confirming that GR indeed binds to the rs28714259 region. Notably, the band intensity of risk allele probes decreased by 50% compared to wildtype, suggesting weaker GR binding affinity to risk allele probes, consistent with reduced transcriptional activation. Finally, to identify genes differentially regulated by rs28714259 with anthracycline exposure, we performed RNA-Seq analysis on iPSC-CMs of each genotype. RNA-Seq data revealed that the top differentially regulated network was the death receptor pathway including: FADD, FAS, and Caspase-8; these effectors are known to induce apoptosis in response to doxorubicin. Moreover, 11 genes in the GR signaling pathway were also differentially regulated by rs28714259. Taken together, these findings suggest that the rs28714259 variant may possess allele-specific GR enhancer activity and differentially regulates genes involved in doxorubicin-induced apoptosis. Citation Format: Xi Wu, Gloria Xue, Fei Shen, Guanglong Jiang, Santosh Philips, Geneva Cunningham, Erica Cantor, Bryan P Schneider. Shedding light on the dark side of chemo: Post-GWAS functional studies of rs28714259 in anthracycline-induced cardiotoxicity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS13-22.

Macrophage migration inhibitory factor deficiency aggravates effects of fructose-enriched diet on lipid metabolism in the mouse liver.

Dietary fructose can disturb hepatic lipid metabolism in a way that leads to lipid accumulation and steatosis, which is often accompanied with low-grade inflammation. The macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with important role not only in the regulation of inflammation, but also in the modulation of energy metabolism in the liver. Thus, the aim of this study was to investigate the role of Mif deficiency in fructose-induced disturbances of hepatic lipid metabolism and ectopic lipid accumulation. Wild type (WT) and Mif deficient (MIF-/- ) C57Bl/6J mice were used to analyze the effects of 9-week 20% fructose-enriched diet on hepatic lipid metabolism (both lipogenesis and β-oxidation) and histology, inflammatory status and glucocorticoid receptor (GR) signaling. The results showed fructose-induced elevation of lipogenic genes (fatty acid synthase (Fas) and stearoyl-CoA desaturase-1 (Scd1) and transcriptional lipogenic regulators (liver X receptor (LXR), sterol regulatory element binding protein 1c (SREBP1c), and carbohydrate response element-binding protein (ChREBP)). However, microvesicular fatty changes, accompanied with enhanced inflammation, were observable only in fructose-fed Mif deficient animals, and were most likely result of GR activation and facilitated uptake and decreased β-oxidation of FFA, as evidenced by elevated protein level of fatty acid translocase (FAT/CD36) and decreased carnitine palmitoyl transferase 1 (CPT1) level. In conclusion, the results show that Mif deficiency aggravates the effects of energy-rich fructose diet on hepatic lipid accumulation, most likely through enhanced inflammation and activation of GR signaling pathway.

Regulation of Gut Microbiota Disrupts the Glucocorticoid Receptor Pathway and Inflammation-related Pathways in the Mouse Hippocampus.

An increasing number of studies have recently indicated the important effects of gut microbes on various functions of the central nervous system. However, the underlying mechanisms by which gut microbiota regulate brain functions and behavioral phenotypes remain largely unknown. We therefore used isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis to obtain proteomic profiles of the hippocampus in germ-free (GF), colonized GF, and specific pathogen-free (SPF) mice. We then integrated the resulting proteomic data with previously reported mRNA microarray data, to further explore the effects of gut microbes on host brain functions. We identified that 61 proteins were upregulated and 242 proteins were downregulated in GF mice compared with SPF mice. Of these, 124 proteins were significantly restored following gut microbiota colonization. Bioinformatic analysis of these significant proteins indicated that the glucocorticoid receptor signaling pathway and inflammation-related pathways were the most enriched disrupted pathways. This study provides new insights into the pathological mechanisms of gut microbiota-regulated diseases.

Activation of HDAC4 and GR signaling contributes to stress-induced hyperalgesia in the medial prefrontal cortex of rats

“Stress-induced hyperalgesia (SIH)” is a phenomenon that stress can lead to an increase in pain sensitivity. Epigenetic mechanisms have been known to play fundamental roles in stress and pain. Histone acetylation is an epigenetic feature that is changed in numerous stress-related disease situations. However, epigenetic mechanism for SIH is not well known. We investigated the effect of histone acetylation on pain hypersensitivity using SPS (single-prolonged stress) + CFA (complete Freund’s adjuvant) model. We showed that the glucocorticoid receptor (GR)-pERK-pCREB-Fos signaling pathway was upregulated on stress-induced hyperalgesia and the paw withdrawal threshold in the SPS + CFA group dropped significantly compared with the SPS or CFA group. Histone deacetylases 4 (HDAC4)-expressing neurons in the medial prefrontal cortex (mPFC) were increased in the SPS + CFA-exposed group compared with CFA-exposed or SPS-exposed group. And we showed that the effects of stress-induced hyperalgesia were critically regulated via reversible acetylation (HDAC4) of the GR. Inhibiting HDAC4 by microinjection of sodium butyrate into the mPFC could disrupt glucocorticoid receptor (GR) signaling pathway, which lowered SPS + CFA-caused mechanical allodynia and alleviated anxiety-like behavior. Together, our studies suggest that HDAC inhibitors might involve in the process of stress-induced hyperalgesia.