Glucocorticoid Receptor Binding Research Articles

Intranasal Corticosteroids: Topical Potency, Systemic Activity and Therapeutic Index

Intranasal corticosteroid (INCS) therapy is the preferred treatment option for allergic rhinitis (AR). Although all INCSs for the treatment of AR are considered safe and effective, differences in potency, molecular structure features and physicochemical and pharmacokinetic properties could result in differences in clinical efficacy and safety. Higher glucocorticoid receptor (GR) binding affinity of INCS is associated with higher lipophilicity, nasal tissue retention and topical potency. Higher topical potency is also accompanied by low oral bioavailability and high systemic clearance conferring low systemic exposure, reduced potential for systemic adverse effects and an improved therapeutic index. It has been shown that adverse events related to systemic exposure of INCSs in children are low. Although INCSs mostly produce low systemic effects, use of an INCS with low systemic exposure in patients on multiple corticosteroid (CS) therapies could help reduce the total systemic burden of CS therapy. Despite differences in topical potency, physicochemical and pharmacokinetic properties between INCSs, clinical studies of INCSs in the treatment of AR generally show no clinically important differences between these compounds, and poor correlation between INCS topical potency and clinical response. However, the lack of head-to-head comparisons of INCSs in clinical studies conducted in more severe AR patients should be noted. This narrative review provides an assessment of the therapeutic relevance of topical potency and the physicochemical and pharmacokinetic properties of INCSs and describes for the first time the relationship between topical potency and therapeutic index using pharmacological features of INCSs. It concludes that higher GR binding affinity and topical potency can potentially improve the therapeutic index of an INCS. Therefore, both efficacy and systemic exposure profiles should be considered when comparing INCS regimens in terms of therapeutic equivalence, to aid clinical decision-making and avoid the assumption that all INCS formulations are the same when considering treatment options.

Bromodomain containing 9 (BRD9) regulates macrophage inflammatory responses by potentiating glucocorticoid receptor activity

In macrophages, homeostatic and immune signals induce distinct sets of transcriptional responses, defining cellular identity and functional states. The activity of lineage-specific and signal-induced transcription factors are regulated by chromatin accessibility and other epigenetic modulators. Glucocorticoids are potent antiinflammatory drugs; however, the mechanisms by which they selectively attenuate inflammatory genes are not yet understood. Acting through the glucocorticoid receptor (GR), glucocorticoids directly repress inflammatory responses at transcriptional and epigenetic levels in macrophages. A major unanswered question relates to the sequence of events that result in the formation of repressive regions. In this study, we identify bromodomain containing 9 (BRD9), a component of SWI/SNF chromatin remodeling complex, as a modulator of glucocorticoid responses in macrophages. Inhibition, degradation, or genetic depletion of BRD9 in bone marrow-derived macrophages significantly attenuated their responses to both liposaccharides and interferon inflammatory stimuli. Notably, BRD9-regulated genes extensively overlap with those regulated by the synthetic glucocorticoid dexamethasone. Pharmacologic inhibition of BRD9 potentiated the antiinflammatory responses of dexamethasone, while the genetic deletion of BRD9 in macrophages reduced high-fat diet-induced adipose inflammation. Mechanistically, BRD9 colocalized at a subset of GR genomic binding sites, and depletion of BRD9 enhanced GR occupancy primarily at inflammatory-related genes to potentiate GR-induced repression. Collectively, these findings establish BRD9 as a genomic antagonist of GR at inflammatory-related genes in macrophages, and reveal a potential for BRD9 inhibitors to increase the therapeutic efficacies of glucocorticoids.

Computational resources to define alleles and altered regulatory motifs at genomically edited candidate response elements.

Unequivocal functional assessment of candidate genomic regulatory regions, such as transcriptional response elements, requires genetic alteration at their native chromosomal loci. Targeted DNA cleavage by Cas9 or other programmable nucleases enables analysis at virtually any genomic region, and diverse alleles generated by editing can be defined by deep sequencing for functional analysis. Interpretation of disrupted response elements, however, presents a special challenge, as these regions typically comprise clustered DNA binding motifs for multiple transcriptional regulatory factors (TFs); DNA sequence differences, natural or engineered, that affect binding by one TF can confer loss or gain of binding sites for other TFs. To address these and other analytical complexities, we created three computational tools that together integrate, in a single experiment, allele definition and TF binding motif evaluation for up to 9216 clones isolated, sequenced and propagated from Cas9-treated cell populations. We demonstrate 1) the capacity to functionally assess edited TF binding sites to query response element function, and 2) the efficacy and utility of these tools, by analyzing cell populations targeted by Cas9 for disruption of example glucocorticoid receptor (GR) binding motifs near FKBP5, a GR-regulated gene in the human adenocarcinoma cell line A549.

Regulation of Melanophilin (Mlph) gene expression by the glucocorticoid receptor (GR)

Mlph plays a crucial role in regulating skin pigmentation through the melanosome transport process. Although Mlph is a major component involved in melanosome transport, the mechanism that regulates the expression of the Mlph gene has not been identified. In this study, we demonstrate that Mlph expression is regulated by the glucocorticoid receptor (GR). Alteration of GR activity using a specific GR agonist or antagonist only regulated the expression of Mlph among the 3 key melanosome transport proteins. Translocation of GR from the cytosol into the nucleus following Dex treatment was confirmed by separating the cytosol and nuclear fractions and by immunofluorescence staining. In ChIP assays, Dex induced GR binding to the Mlph promoter and we determined that Dex induced the GR binding motif on the Mlph promoter. Our findings contribute to understanding the regulation of Mlph expression and to the novel role of GR in Mlph gene expression.

Naturalistic Stress Hormone Levels Drive Cumulative Epigenomic Changes along the Cellular Lifespan.

Environmental stress is ubiquitous in modern societies and can exert a profound and cumulative impact on cell function and health phenotypes. This impact is thought to be in large part mediated by the action of glucocorticoid stress hormones, primarily cortisol in humans. While the underlying molecular mechanisms are unclear, epigenetics—the chemical changes that regulate genomic function without altering the genetic code—has emerged as a key link between environmental exposures and phenotypic outcomes. The present study assessed genome-wide DNA (CpG) methylation, one of the key epigenetic mechanisms, at three timepoints during prolonged (51-day) exposure of cultured human fibroblasts to naturalistic cortisol levels, which can be reached in human tissues during in vivo stress. The findings support a spatiotemporal model of profound and widespread stress hormone-driven methylomic changes that emerge at selected CpG sites, are more likely to spread to nearby located CpGs, and quantitatively accrue at open sea, glucocorticoid receptor binding, and chromatin-accessible sites. Taken together, these findings provide novel insights into how prolonged stress may impact the epigenome, with potentially important implications for stress-related phenotypes.

Corticosterone pattern-dependent glucocorticoid receptor binding and transcriptional regulation within the liver.

Ultradian glucocorticoid rhythms are highly conserved across mammalian species, however, their functional significance is not yet fully understood. Here we demonstrate that pulsatile corticosterone replacement in adrenalectomised rats induces a dynamic pattern of glucocorticoid receptor (GR) binding at ~3,000 genomic sites in liver at the pulse peak, subsequently not found during the pulse nadir. In contrast, constant corticosterone replacement induced prolonged binding at the majority of these sites. Additionally, each pattern further induced markedly different transcriptional responses. During pulsatile treatment, intragenic occupancy by active RNA polymerase II exhibited pulsatile dynamics with transient changes in enrichment, either decreased or increased depending on the gene, which mostly returned to baseline during the inter-pulse interval. In contrast, constant corticosterone exposure induced prolonged effects on RNA polymerase II occupancy at the majority of gene targets, thus acting as a sustained regulatory signal for both transactivation and repression of glucocorticoid target genes. The nett effect of these differences were consequently seen in the liver transcriptome as RNA-seq analysis indicated that despite the same overall amount of corticosterone infused, twice the number of transcripts were regulated by constant corticosterone infusion, when compared to pulsatile. Target genes that were found to be differentially regulated in a pattern-dependent manner were enriched in functional pathways including carbohydrate, cholesterol, glucose and fat metabolism as well as inflammation, suggesting a functional role for dysregulated glucocorticoid rhythms in the development of metabolic dysfunction.

Distinct regulation of hippocampal neuroplasticity and ciliary genes by corticosteroid receptors

Glucocorticoid hormones (GCs) — acting through hippocampal mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) — are critical to physiological regulation and behavioural adaptation. We conducted genome-wide MR and GR ChIP-seq and Ribo-Zero RNA-seq studies on rat hippocampus to elucidate MR- and GR-regulated genes under circadian variation or acute stress. In a subset of genes, these physiological conditions resulted in enhanced MR and/or GR binding to DNA sequences and associated transcriptional changes. Binding of MR at a substantial number of sites however remained unchanged. MR and GR binding occur at overlapping as well as distinct loci. Moreover, although the GC response element (GRE) was the predominant motif, the transcription factor recognition site composition within MR and GR binding peaks show marked differences. Pathway analysis uncovered that MR and GR regulate a substantial number of genes involved in synaptic/neuro-plasticity, cell morphology and development, behavior, and neuropsychiatric disorders. We find that MR, not GR, is the predominant receptor binding to >50 ciliary genes; and that MR function is linked to neuronal differentiation and ciliogenesis in human fetal neuronal progenitor cells. These results show that hippocampal MRs and GRs constitutively and dynamically regulate genomic activities underpinning neuronal plasticity and behavioral adaptation to changing environments.

Individual-specific functional epigenomics reveals genetic determinants of adverse metabolic effects of glucocorticoids

Glucocorticoids (GCs) are widely used as anti-inflammatory drugs, but their long-term use has severe metabolic side effects. Here, by treating multiple individual adipose stem cell-derived adipocytes and induced pluripotent stem cell-derived hepatocytes with the potent GC dexamethasone (Dex), we uncovered cell-type-specific and individual-specific GC-dependent transcriptomes and glucocorticoid receptor (GR) cistromes. Individual-specific GR binding could be traced to single-nucleotide polymorphisms (SNPs) that altered the binding motifs of GR or its cooperating factors. We also discovered another set of genetic variants that modulated Dex response through affecting chromatin accessibility or chromatin architecture. Several SNPs that altered Dex-regulated GR binding and gene expression controlled Dex-driven metabolic perturbations. Remarkably, these genetic variations were highly associated with increases in serum glucose, lipids, and body mass in subjects on GC therapy. Knowledge of the genetic variants that predispose individuals to metabolic side effects allows for a precision medicine approach to the use of clinically relevant GCs.

Hypothalamus volume and DNA methylation of stress axis genes in major depressive disorder: A CAN-BIND study report

Dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis is considered one of the mechanisms underlying the development of major depressive disorder (MDD), but the exact nature of this dysfunction is unknown. We investigated the relationship between hypothalamus volume (HV) and blood-derived DNA methylation in MDD. We obtained brain MRI, clinical and molecular data from 181 unmedicated MDD and 90 healthy control (HC) participants. MDD participants received a 16-week standardized antidepressant treatment protocol, as part of the first Canadian Biomarker Integration Network in Depression (CAN-BIND) study. We collected bilateral HV measures via manual segmentation by two independent raters. DNA methylation and RNA sequencing were performed for three key HPA axis-regulating genes coding for the corticotropin-binding protein (CRHBP), glucocorticoid receptor (NR3C1) and FK506 binding protein 5 (FKBP5). We used elastic net regression to perform variable selection and assess predictive ability of methylation variables on HV. Left HV was negatively associated with duration of current episode (ρ = −0.17, p = 0.035). We did not observe significant differences in HV between MDD and HC or any associations between HV and treatment response at weeks 8 or 16, overall depression severity, illness duration or childhood maltreatment. We also did not observe any differentially methylated CpG sites between MDD and HC groups. After assessing functionality by correlating methylation levels with RNA expression of the respective genes, we observed that the number of functionally relevant CpG sites differed between MDD and HC groups in FKBP5 (χ2 = 77.25, p < 0.0001) and NR3C1 (χ2 = 7.29, p = 0.007). Cross-referencing functionally relevant CpG sites to those that were highly ranked in predicting HV in elastic net modeling identified one site from FKBP5 (cg03591753) and one from NR3C1 (cg20728768) within the MDD group. Stronger associations between DNA methylation, gene expression and HV in MDD suggest a novel putative molecular pathway of stress-related sensitivity in depression. Future studies should consider utilizing the epigenome and ultra-high field MR data which would allow the investigation of HV sub-fields.

Mineralocorticoid receptors dampen glucocorticoid receptor sensitivity to stress via regulation of FKBP5.

SUMMARYResponding to different dynamic levels of stress is critical for mammalian survival. Disruption of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) signaling is proposed to underlie hypothalamic-pituitary-adrenal (HPA) axis dysregulation observed in stress-related psychiatric disorders. In this study, we show that FK506-binding protein 51 (FKBP5) plays a critical role in fine-tuning MR:GR balance in the hippocampus. Biotinylated-oligonucleotide immunoprecipitation in primary hippocampal neurons reveals that MR binding, rather than GR binding, to the Fkbp5 gene regulates FKBP5 expression during baseline activity of glucocorticoids. Notably, FKBP5 and MR exhibit similar hippocampal expression patterns in mice and humans, which are distinct from that of the GR. Pharmacological inhibition and region- and cell type-specific receptor deletion in mice further demonstrate that lack of MR decreases hippocampal Fkbp5 levels and dampens the stress-induced increase in glucocorticoid levels. Overall, our findings demonstrate that MR-dependent changes in baseline Fkbp5 expression modify GR sensitivity to glucocorticoids, providing insight into mechanisms of stress homeostasis.

Glucocorticoid signaling in pancreatic islets modulates gene regulatory programs and genetic risk of type 2 diabetes.

Glucocorticoids are key regulators of glucose homeostasis and pancreatic islet function, but the gene regulatory programs driving responses to glucocorticoid signaling in islets and the contribution of these programs to diabetes risk are unknown. In this study we used ATAC-seq and RNA-seq to map chromatin accessibility and gene expression from eleven primary human islet samples cultured in vitro with the glucocorticoid dexamethasone at multiple doses and durations. We identified thousands of accessible chromatin sites and genes with significant changes in activity in response to glucocorticoids. Chromatin sites up-regulated in glucocorticoid signaling were prominently enriched for glucocorticoid receptor binding sites and up-regulated genes were enriched for ion transport and lipid metabolism, whereas down-regulated chromatin sites and genes were enriched for inflammatory, stress response and proliferative processes. Genetic variants associated with glucose levels and T2D risk were enriched in glucocorticoid-responsive chromatin sites, including fine-mapped variants at 51 known signals. Among fine-mapped variants in glucocorticoid-responsive chromatin, a likely casual variant at the 2p21 locus had glucocorticoid-dependent allelic effects on beta cell enhancer activity and affected SIX2 and SIX3 expression. Our results provide a comprehensive map of islet regulatory programs in response to glucocorticoids through which we uncover a role for islet glucocorticoid signaling in mediating genetic risk of T2D.

Differential Activity and Utilization of Glucocorticoid Receptor Binding Sites Yields Transcriptional Heterogeneity

Synthetic glucocorticoids such as Dexamethasone (Dex) are widely prescribed drugs used to treat a variety of human diseases including auto-immune disorders, asthma, cancer, and COVID-19. The transcriptional response to glucocorticoids is elicited by the Glucocorticoid Receptor (GR), which enters the nucleus upon Dex treatment and interacts with thousands of enhancer elements throughout the genome. We recently demonstrated that the Dex response in human breast cancer cells is highly heterogeneous and that individual cells have unique transcriptional responses to Dex. To examine whether this heterogeneity arises from differential utilization of distinct GR-bound enhancers, we focused on the Dex response at the DNA Damage Inducible Transcript 4 (DDIT4) gene. Using a variety of genomic techniques, we identified four GR binding sites (GBSs) 18-30kb upstream of the DDIT4 TSS with differential patterns of chromatin accessibility, histone acetylation, SWI/SNF recruitment, and enhancer RNA (eRNA) transcription. To determine whether these GBSs had unique requirements for DDIT4 transcription, we used CRISPR-CAS9 to generate homozygous deletions of each site. Using ChIP-seq, 4C-seq, single molecule fluorescent in situ hybridization (smFISH), and RT-PCR, we demonstrated GR binding to these GBSs was independent and each GBS deletion had unique effects on DDIT4 and eRNA transcription, local histone acetylation, and chromatin looping. Deletion of any of the first three GBSs resulted in delayed and/or decreased induction of DDIT4 transcription whereas deletion of the fourth GBS resulted in significant upregulation of both DDIT4 and eRNA transcription. Thus, three of the GBSs acted as enhancers of DDIT4 expression while the fourth functioned as a suppressor. Strikingly, smFISH also revealed that these enhancers contributed to cellular heterogeneity, as deleting the GBSs altered the frequency and amplitude of DDIT4 transcription across cell populations. Taken together, these results demonstrate that individual GBSs uniquely contribute to cell-to-cell heterogeneity within the transcriptional response of DDIT4 to Dex. Furthermore, they underscore the possibility that targeted modification of individual GBSs could be utilized to tailor custom, patient-specific strategies for the treatment of human diseases.

NF‐κB and Glucocorticoid Receptor Synergize to Enable Pro‐inflammatory Signaling via Toll‐like Receptor 2 in Pulmonary Epithelial Cells

Rationale Inhaled glucocorticoids (GCs) are the primary treatment for mild-moderate asthma, but show limited efficacy to treat neutrophilic inflammation in severe disease or during exacerbations. GCs act via the GC receptor (GR) to reduce inflammation by repressing the expression of numerous inflammatory genes, but preserve innate immune functions. The consequences of such events are poorly understood. Toll-like receptor 2 (TLR2) is a pattern recognition receptor that activates innate immune responses to bacteria and viruses. GCs, along with inflammatory stimuli, enhance TLR2 expression in pulmonary epithelial cells. This study investigates the mechanisms and consequences of TLR2 escape from GC-repression. Methods Alveolar epithelial A549 and primary human bronchial epithelial (pHBE) cells were treated with interleukin-1β (IL1B) and GCs (dexamethasone/budesonide) to induce TLR2 expression. Chromatin immunoprecipitation (ChIP) was used to test GR and RELA binding. Cells harbouring NF-κB and IRF1-dependent luciferase reporters were examined following treatment with the TLR2 ligands, pam2csk4 and pam3csk4. Results Stimulating A549 and pHBE cells with IL1B+GCs produced delayed synergy on TLR2 expression. IL1B+GC-induced TLR2 expression was dependent on NF-κB and GR, as assessed using the dominant inhibitor of NF-κB, IκBαΔN, and siRNAs targeting GR. Upon IL1B and GC treatment, ChIP-qPCR in A549 and pHBE cells confirmed binding of RELA and GR to distal and proximal regions, R1 and R2, located 32 and 7.3 kb, respectively, upstream of the TLR2 transcription start site. To assess their ability to drive transcription, these regions were cloned separately, or together, upstream of a basal luciferase reporter and stably transfected in A549 cells. In cells harbouring the R1 construct, GC increased luciferase activity at 6, 8 and 24 h, which was unaffected by IL1B. With the R2 reporter, IL1B increased luciferase activity at 6, 8 and 24 h, which was partially repressed by GC. In case of cells harbouring R1+R2, luciferase activity was significantly induced by IL1B as well as by GC at 6, 8 and 24 h with synergistic effects due to cotreatment at each time. This suggests that interaction between the NF-κB and GR-binding regions, R1 and R2 may contribute to synergy in TLR2 expression upon IL1B+GC co-treatment in pulmonary epithelial cells. To assess the functional consequences of TLR2 synergy, A549 cells were pre-treated with IL1B+GC, followed by pam2cks4 and pam3csk4. The TLR2 ligands strongly induced NF-κB and IRF1 reporter activity, and both IL8 and CCL5 release. These effects were abolished by siRNAs targeting TLR2. Thus IL1B+GC synergy on TLR2 expression allows NF-κB and IRF1-dependent signaling and expression of inflammatory cytokines following TLR2 agonism. Conclusion IL1B+GC-induced TLR2 synergy via interaction of the proximal and distal NF-κB- and GR-binding regions upstream of TLR2 gene enables pro-inflammatory events in pulmonary epithelial cells. This represents a mechanism by which GCs act via GR to preserve innate immune responses which might impact the pathophysiology in severe asthma patients and/or during infection-triggered exacerbations.

Inferring molecular mechanisms of dexamethasone therapy in severe COVID-19 from existing transcriptomic data.

Dexamethasone, a synthetic glucocorticoid, has previously shown mortality benefit in severe coronavirus disease 2019 (COVID-19) in a randomized controlled trial. As the illness is considered to reflect a hyperinflammatory state, this therapeutic effectiveness is presumably ascribed to broad anti-inflammatory activities of glucocorticoids. Here, an unbiased analysis of available transcriptomic data on lung and blood immune cells from severe COVID-19 patients and matching cellular models of dexamethasone treatment is presented that supports this presumption. Comparison of differentially expressed genes in severe COVID-19 with that in dexamethasone treated cells reveals a small set of genes that are regulated in opposite direction between the disease and the drug, and are enriched for genes and processes related to glucocorticoid pathway and receptor binding. This expression signature differentiates as a whole various cytokines from a set of anti-cytokine/anti-inflammatory agents, with the former resembling COVID-19 and the latter dexamethasone in gene regulation. The signature apparently relates to TNF- α, IL-1α, IL-1β, IFN-α, IFN-β, and IFN-γ signaling, but not IL-6 signaling, suggesting that therapeutic effect of dexamethasone in COVID-19 does not involve IL-6 pathway. However, as all these observations are purely based on bioinformatic analysis, experimental evidence will be required to validate the inferences drawn. In conclusion, the present analysis seems to provide a proof of concept for therapeutic mechanisms of dexamethasone in COVID-19.

Mutant glucocorticoid receptor binding elements on the interleukin-6 promoter regulate dexamethasone effects

BackgroundGlucocorticoids (GCs) have been extensively used as essential modulators in clinical infectious and inflammatory diseases. The GC receptor (GR) is a transcription factor belonging to the nuclear receptor family that regulates anti-inflammatory processes and releases pro-inflammatory cytokines, such as interleukin (IL)-6.ResultsFive putative GR binding sites and other transcriptional factor binding sites were identified on theIL-6 promoter, and dexamethasone (DEX) was noted to reduce the lipopolysaccharide (LPS)-induced IL-6 production. Among mutant transcriptional factor binding sites, nuclear factor-kappa B (NF-κB), activator protein (AP)-1, and specificity protein (Sp)1–2 sites reduced basal and LPS-induced IL-6 promoter activities through various responses. The second GR binding site (GR2) was noted to play a crucial role in both basal and inducible promoter activities in LPS-induced inflammation.ConclusionsWe concluded that selective GR2 modulator might exert agonistic and antagonistic effects and could activate crucial signaling pathways during the LPS-stimulated inflammatory process.

Human DNA methylation signatures differentiate persistent from resolving MRSA bacteremia

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is life threatening and occurs in up to 30% of MRSA bacteremia cases despite appropriate antimicrobial therapy. Isolates of MRSA that cause antibiotic-persistent methicillin-resistant S. aureus bacteremia (APMB) typically have in vitro antibiotic susceptibilities equivalent to those causing antibiotic-resolving methicillin-resistant S. aureus bacteremia (ARMB). Thus, persistence reflects host-pathogen interactions occurring uniquely in context of antibiotic therapy in vivo. However, host factors and mechanisms involved in APMB remain unclear. We compared DNA methylomes in circulating immune cells from patients experiencing APMB vs. ARMB. Overall, methylation signatures diverged in the distinct patient cohorts. Differentially methylated sites intensified proximate to transcription factor binding sites, primarily in enhancer regions. In APMB patients, significant hypomethylation was observed in binding sites for CCAAT enhancer binding protein-β (C/EBPβ) and signal transducer/activator of transcription 1 (STAT1). In contrast, hypomethylation in ARMB patients localized to glucocorticoid receptor and histone acetyltransferase p300 binding sites. These distinct methylation signatures were enriched in neutrophils and achieved a mean area under the curve of 0.85 when used to predict APMB using a classification model. These findings validated by targeted bisulfite sequencing (TBS-seq) differentiate epigenotypes in patients experiencing APMB vs. ARMB and suggest a risk stratification strategy for antibiotic persistence in patients treated for MRSA bacteremia.

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.

Low-activity programming of the PDGFRβ/FAK pathway mediates H-type vessel dysplasia and high susceptibility to osteoporosis in female offspring rats after prenatal dexamethasone exposure

Dexamethasone is a common synthetic glucocorticoid drug that can promote foetal lung maturity. An increasing number of studies have shown that prenatal dexamethasone exposure (PDE) can cause a variety of short-term and long-term hazards to offspring, including bone development toxicity. H-type vessels are a newly discovered subtype of blood vessels associated with promoted bone formation and maintenance of bone mass. In this study, we aimed to explore whether H-type blood vessels are involved in PDE-induced long bone development toxicity in offspring and its mechanism. In vivo, we injected dexamethasone (0.2 mg/kg.d) subcutaneously at gestational days 9–20 and observed the H-type vessel abundance and bone mass at different time points in the offspring rats. In vitro, we investigated the effect of dexamethasone (0, 20, 100, and 500 nM) on the tube formation function of rat bone marrow-derived endothelial progenitor cells (EPCs) and explored its mechanism. Our results showed that the adult PDE female offspring rats were susceptible to osteoporosis. In addition, PDE inhibited bone mass, H-type vessel formation and the expression of bone platelet-derived growth factor receptor β (PDGFRβ)/focal adhesion kinase (FAK) pathway-related genes in antenatal and postnatal female offspring. Moreover, PDE promoted the expression of bone glucocorticoid receptor (GR), CCAAT and enhancer binding protein α (C/EBPα) and miR-34c in female foetuses. Dexamethasone suppressed the tube formation of rat bone marrow-derived EPCs and the activity of the PDGFRβ/FAK pathway, which was mediated by GR/C/EBPα/miR-34c signalling activation. In summary, PDE can cause H-type vessel dysplasia and high susceptibility to osteoporosis in female offspring, and its mechanism is related to the low-activity programming of the PDGFRβ/FAK pathway induced by GR/C/EBPα/miR-34c signalling activation. This study enhances the understanding of the molecular mechanism of dexamethasone-induced bone development toxicity and provides new insights for exploring the early intervention and therapeutic targets of foetal-derived osteoporosis.

Genomic determinants implicated in the glucocorticoid-mediated induction of KLF9 in pulmonary epithelial cells

Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However, some genes, including Krüppel-like factor 9 (KLF9), a putative transcriptional repressor, demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air–liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells, glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5ʹ-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO) sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confirmed in A549 cells, plus submersion, and ALI culture of HBE cells. Cloning each GBS into luciferase reporters revealed glucocorticoid-induced activity requiring a glucocorticoid response element (GRE) within each distal GBS. While the proximal GBSs drove modest reporter induction by glucocorticoids, this region exhibited basal eRNA production, RNA polymerase II enrichment, and looping to the TSS, plausibly underlying constitutive KLF9 expression. Post glucocorticoid treatment, interactions between distal and proximal GBSs and the TSS correlated with KLF9 induction. CBP/P300 silencing reduced proximal GBS activity, but negligibly affected KLF9 expression. Overall, a model for glucocorticoid-mediated regulation of KLF9 involving multiple GBSs is depicted. This work unequivocally demonstrates that mechanistic insights gained from cell lines can translate to physiologically relevant systems.

Microwave-assisted green synthesis of bile acid derivatives and evaluation of glucocorticoid receptor binding.

Herein, we present microwave-assisted AlCl3 catalyzed oxidation of bile acid hydroxyl groups in the presence of Oxone® in water media. Significant rate enhancements were observed for Wolff-Kishner reduction of synthesized bile acids oxo derivatives to the 5β-cholanic acid. Reaction of amidation of the simplest bile acid and aminolysis of the deoxycholic acid was accomplished in the absence of solvent and catalysts under sealed vessel microwave conditions. Because 5β-cholanic acid reportedly modulates glucocorticoid receptor signaling in cell models of Parkinson's disease, we tested the affinity of 5β-cholanic acid and deoxycholic acid derivatives for the glucocorticoid receptor in vitro using a yeast-based fluorescent screen. Treatment of GR-expressing yeast with prednisolone resulted in a dose-dependent increase in fluorescence; whereas 5β-cholanic acid binds to the glucocorticoid receptor with more moderate affinity. Similarly, molecular docking also suggests that 5β-cholanic acid can bind to the glucocorticoid receptor, with similar geometry to known GR ligands.