Glucocorticoid Receptor Response Research Articles

Glucocorticoid receptor response and glucocorticoid-induced leucine zipper expression in neutrophils of critically ill patients with traumatic and non-traumatic brain injury.

Critically ill patients, including those with brain injuries (BI), are frequently hospitalized in an intensive care unit (ICU). As with other critical states, an adequate stress response is essential for survival. Research on the hypothalamic-pituitary-adrenal gland (HPA) axis function in BI has primarily focused on assessing ACTH and cortisol levels. However, the immunological, metabolic, and hemodynamic effects of glucocorticoids (GCs) are mediated through the glucocorticoid receptor (GCR), a ubiquitously distributed intracellular receptor protein. Data on GCR-α expression and its signaling in acute BI injury are lacking. We designed a prospective observational study, carried out in one academic multi-disciplinary ICU. Forty-two critically ill patients with acute (BI)were included. These patients suffered from traumatic BI (N= 20), subarachnoid hemorrhage (N= 12), intracranial hemorrhage (N= 7), or ischemic stroke (N= 3). All patients were steroid-free. Twenty-four age and sex-matched healthy controls were used for comparison. Expression of GCR-α and the glucocorticoid-inducible leucine zipper (GILZ), serum cortisol, interleukins (IL) 6, 8, 10 and TNF- α, and the BI biomarkers glial fibrillary acidic protein (GFAP) and total Tau were measured on ICU admission (within 48 hours) and 5-7 days from admission. Compared to healthy controls, in the critically ill patients with BI, GCR-α mRNA expression was significantly downregulated on admission, and after 5-7 days in the ICU (2.3-fold, p<0.05 and 2.6-fold, p<0.01, respectively). Even though GCR-α was downregulated, its downstream gene, GILZ, was expressed at the same levels as in normal controls on admission and was significantly upregulated 5-7 days following admission (2-fold, p<0.001). TNF-α levels were undetectable at both time-points. GCR-α expression levels inversely correlated with IL-6. The levels of cortisol and the BI biomarkers did not differ between the 2 time-points. We provide novel evidence on the downregulated expression and upregulated signaling of the ligand-binding and functionally active GCR-α isoform in the polymorphonuclear neutrophils (PMNs) of critically ill patients with BI. The increased GILZ expression indicates an increased GC sensitivity in the PMNs of BI critically ill patients.

Sex differences in the transcriptional response to acute inflammatory challenge: A randomized controlled trial of endotoxin

BackgroundSex differences in immune-based disorders are well-established, with female sex associated with a markedly heightened risk of autoimmune disease. Female sex is also overrepresented in other conditions associated with elevated inflammation, including depression, chronic pain, and chronic fatigue. The mechanisms underlying these disparities are unclear. This study used an experimental model of inflammatory challenge to interrogate molecular mechanisms that may contribute to female vulnerability to disorders with an inflammatory basis. MethodIn this analysis of a secondary outcome from a randomized controlled trial, 111 participants (67 female) received either a bolus injection of endotoxin (n = 59) or placebo (n = 52). Participants provided blood samples before and 0.5 h post-injection for assessment of differential activation of key pro-inflammatory (i.e., activator protein (AP)-1; nuclear factor (NF)-κB) and immunoregulatory (i.e., glucocorticoid receptor (GR); cAMP response element binding protein (CREB)) signaling pathways via genome-wide expression profiling and promoter-based bioinformatics analyses. ResultsRelative to males, females exhibited greater endotoxin-induced increases in bioinformatic measures of CREB transcription factor activity (p's < 0.01). However, contrary to hypotheses, female vs. male sex was not associated with greater increases in activation of NF-κB, AP-1, or GR in response to endotoxin vs. placebo administration. ConclusionsThis work suggests CREB signaling as a critical upstream biological pathway that should be further interrogated as a mechanism of female vulnerability to immune-related disorders. Future work should clarify whether increased CREB signaling indicates sex differences in activity of the sympathetic nervous system or other physiological pathways that signal through CREB, such as prostaglandin release.

Channel Expansion in the Ligand-Binding Domain of the Glucocorticoid Receptor Contributes to the Activity of Highly Potent Glucocorticoid Analogues.

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR) and are commonly used as anti-inflammatory and immunosuppressant medications. Chronic GC use has been linked with unwanted complications such as steroid-induced diabetes mellitus (SIDM), although the mechanisms for these effects are not completely understood. Modification of six GC parent molecules with 2-mercaptobenzothiazole resulted in consistently less promoter activity in transcriptional activation assays using a 3xGRE reporter construct while constantly reducing inflammatory pathway activity. The most selective candidate, DX1, demonstrated a significant reduction (87%) in transactivation compared to commercially available dexamethasone. DX1 also maintained 90% of the anti-inflammatory potential of dexamethasone while simultaneously displaying a reduced toxicity profile. Additionally, two novel and highly potent compounds, DX4 and PN4, were developed and shown to elicit similar mRNA expression at attomolar concentrations that dexamethasone exhibits at nanomolar dosages. To further explain these results, Molecular Dynamic (MD) simulations were performed to examine structural changes in the ligand-binding domain of the glucocorticoid receptor in response to docking with the top ligands. Differing interactions with the transcriptional activation function 2 (AF-2) region of the GR may be responsible for lower transactivation capacity in DX1. DX4 and PN4 lose contact with Arg611 due to a key interaction changing from a stronger hydrophilic to a weaker hydrophobic one, which leads to the formation of an unoccupied channel at the location of the deacylcortivazol (DAC)-expanded binding pocket. These findings provide insights into the structure-function relationships important for regulating anti-inflammatory activity, which has implications for clinical utility.

Role of Carbohydrate response element-binding protein in mediating dexamethasone-induced glucose transporter 5 expression in Caco-2BBE cells and during the developmental phase in mice

ABSTRACT Glucose transporter 5 (GLUT5), the main fructose transporter in mammals, is primarily responsible for absorbing dietary fructose in the small intestine. The expression of this intestinal gene significantly increases in response to developmental and dietary cues that reach the glucocorticoid receptor and carbohydrate response element-binding protein (ChREBP), respectively. Our study demonstrates that ChREBP is involved in the dexamethasone (Dex)-induced expression of GLUT5 in Caco-2BBE cells and the small intestine of both wild-type and ChREBP-knockout mice. Dex, a glucocorticoid, demonstrated an increase in GLUT5 mRNA levels in a dose- and time-dependent manner. While the overexpression of ChREBP moderately increased GLUT5 expression, its synergistic increase in the presence of Dex was noteworthy, whereas the suppression of ChREBP significantly reduced Dex-induced GLUT5 expression. Dex did not increase ChREBP protein levels but facilitated its nuclear translocation, thereby increasing the activity of the GLUT5 promoter. In vivo experiments conducted on 14-day-old mice pups treated with Dex for three days revealed that only wild-type mice (not ChREBP-knockout mice) exhibited Dex-mediated Glut5 gene induction, which further supports the role of ChREBP in regulating GLUT5 expression. Collectively, our results provide insights into the molecular mechanisms involved in the regulation of GLUT5 expression in response to developmental and dietary signals mediated by glucocorticoids and ChREBP. General significance: The transcription factor ChREBP is important for Dex-mediated Glut5 gene expression in the small intestine.

Specificity protein 1 (Sp1) and glucocorticoid receptor (GR) stimulate bovine alphaherpesvirus 1 (BoHV-1) replication and cooperatively transactivate the immediate early transcription unit 1 promoter.

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1+ TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency.IMPORTANCEFollowing acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli.

Slug, a Stress-Induced Transcription Factor, Stimulates Herpes Simplex Virus 1 Replication and Transactivates a cis-Regulatory Module within the VP16 Promoter.

Stress-mediated activation of the glucocorticoid receptor (GR) and specific stress-induced transcription factors stimulate herpes simplex virus 1 (HSV-1) productive infection, explant-induced reactivation, and immediate early (IE) promoters that drive expression of infected cell protein 0 (ICP0), ICP4, and ICP27. Several published studies concluded the virion tegument protein VP16, ICP0, and/or ICP4 drives early steps of reactivation from latency. Notably, VP16 protein expression was induced in trigeminal ganglionic neurons of Swiss Webster or C57BL/6J mice during early stages of stress-induced reactivation. If VP16 mediates reactivation, we hypothesized stress-induced cellular transcription factors would stimulate its expression. To address this hypothesis, we tested whether stress-induced transcription factors transactivate a VP16 cis-regulatory module (CRM) located upstream of the VP16 TATA box (-249 to -30). Initial studies revealed the VP16 CRM cis-activated a minimal promoter more efficiently in mouse neuroblastoma cells (Neuro-2A) than mouse fibroblasts (NIH-3T3). GR and Slug, a stress-induced transcription factor that binds enhancer boxes (E-boxes), were the only stress-induced transcription factors examined that transactivated the VP16 CRM construct. GR- and Slug-mediated transactivation was reduced to basal levels when the E-box, two 1/2 GR response elements (GREs), or NF-κB binding site was mutated. Previous studies revealed GR and Slug cooperatively transactivated the ICP4 CRM, but not ICP0 or ICP27. Silencing of Slug expression in Neuro-2A cells significantly reduced viral replication, indicating Slug-mediated transactivation of ICP4 and VP16 CRM activity correlates with enhanced viral replication and reactivation from latency. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latency in several types of neurons. Periodically cellular stressors trigger reactivation from latency. Viral regulatory proteins are not abundantly expressed during latency, indicating cellular transcription factors mediate early stages of reactivation. Notably, the glucocorticoid receptor (GR) and certain stress-induced transcription factors transactivate cis-regulatory modules (CRMs) essential for expression of infected cell protein 0 (ICP0) and ICP4, key viral transcriptional regulatory proteins linked to triggering reactivation from latency. Virion protein 16 (VP16) specifically transactivates IE promoter and was also reported to mediate early stages of reactivation from latency. GR and Slug, a stress-induced enhancer box (E-box) binding protein, transactivate a minimal promoter downstream of VP16 CRM, and these transcription factors occupy VP16 CRM sequences in transfected cells. Notably, Slug stimulates viral replication in mouse neuroblastoma cells suggesting Slug, by virtue of transactivating VP16 and ICP4 CRM sequences, can trigger reactivation in certain neurons.

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.

Glucocorticoid receptor, a potential mediator of differential regulation on amygdala neurons by chronic stress

The amygdala, a key target for chronic stress, forms the hub of the brain’s emotional circuits with the medial prefrontal cortex (mPFC) and ventral hippocampus (vHPC). The structural and functional remodeling of the amygdala, especially the basolateral amygdala (BLA), caused by chronic stress is closely related to attendant changes in mood disorders such as anxiety disorders. Our previous study found that chronic stress differentially regulated BLA projection neurons (PNs) in different circuits, as evidenced by the fact that spine density and glutamatergic signaling only in BLA PNs targeting vHPC (BLA→vHPC PNs) were remodeled by chronic restraint stress (CRS), whereas BLA PNs targeting mPFC (BLA→mPFC PNs) and nucleus accumbens (BLA→NAc PNs) were not markedly altered. However, the underlying mechanisms are unclear. These BLA PNs integrated in different neural circuits are in an almost-the-same microenvironment, but triggered different reactions to stress, suggesting that they may have different signal receiving or transmitting mechanisms. Considering the critical role of glucocorticoids in the modulation of brain structure and function by stress, we hypothesize that glucocorticoid receptor (GR) may be a potential mediator of the differential regulation of BLA neurons by chronic stress. In this study, we sought to clarify whether the expression levels of GR on BLA PNs in different circuits and their response patterns to CRS are different. And the results showed that CRS significantly enhanced the GR expression of BLA→vHPC PNs, but had no obvious effect on the GR expression of BLA→mPFC PNs and BLA→NAc PNs. Moreover, the increase of corticosterone concentration in mice caused by prolonged stress only selectively caused the elevated response of GR in BLA→vHPC PNs. Taken together, our findings hint that the GR may be a potential and essential mediator of chronic stress-caused differential regulation on BLA PN.

OR07-5 PARP1 Contributes to the Glucocorticoid Receptor Transcriptional Response And Influences the Skeletal Muscle Phenotype

Abstract Background NAD+-dependent PARP1 (Poly-(ADP-ribosyl)polymerase 1) generates the post-translational modification ADP-Ribosylation (ADPR). Given PARP1-ADPR determines cellular NAD+ availability, which in turn impacts a range of hormonally governed actions, we sought to identify the molecular roles of PARP1 in relation to glucocorticoids in the endocrine sensitive tissue skeletal muscle. Importance: Activity of the glucocorticoid receptor (GR) is dictated by molecular partners whose actions skew the frequency with which GR produces meaningful transcriptional outcomes. As a result, our molecular understanding of this steroid hormone receptor remains incomplete. Hypothesis PARP1 through ADPR actions impacts skeletal muscle phenotype and co-regulates glucocorticoid-mediated transcriptional responses. Experimental Design: Murine muscle myoblasts (C2C12) or primary murine muscle cultures were treated with PARP inhibitors (PJ34(10uM) or BYK204165(1uM)) or transfected with silencing RNA targeting PARP1. Cells were treated with ± dexamethasone (1uM). Major Results Analysis of differentiating muscle revealed PARP1 and ADPR are dynamic at days 0-5 (measured by Western immunoblot. p&amp;lt;0.001; n=6). Chemical inhibition of PARP1 significantly decreased ADPR (Western immunoblot. p&amp;lt;0.001; n=9) but did not prevent myotube formation. To identify if initial ADPR dynamics held consequence for fully differentiated myotubes, cells were treated with a single dose of the PARP1 inhibitor BYK204165 at induction of differentiation. Myotube lysates were collected 5 days later and were subjected to unlabelled SWATH-MS. This measured the abundance of 180 proteins as significantly different in PARP inhibitor-treated cells (n=7). Analyses of these proteins revealed over-representation of pathways governing muscle development (FDR 8.99-08), contraction (FDR 9.47-08, myofiber assembly (FDR 8.06-05), and energy metabolism (FDR 2.93-06). Indicating PARP1-ADPR regulates myogenesis and plays a role in the striated muscle contractile response. ChIP assays for PARP1 binding in cells showed a (30-fold enrichment ±3.2SEM) at GR response elements and detectable ADPR was reduced when exposed to dexamethasone (Western immunoblot n=5). These data support ADPR as responsive to glucocorticoid exposure and PARP1 influences GR recruitment to the genome. RNAseq of siPARP1 (n=4) versus scrambled myoblasts (n=5) identified differential expression of 165 genes. Gene ontology revealed over-representation in siPARP1 cells of those pathways regulating muscle differentiation, including actin-binding, cytoskeletal structure, and NAD+ binding genes. SiRNA of PARP1 had no influence over the ability of glucocorticoids to activate archetype target genes (DUSP1, MURF1, ATROGIN1 &amp; GILZ), but differentially impacted the expression of 434 other genes. Conclusions PARP1 mediated ADPR is critical to the 'early phase' events of muscle cell differentiation. PARP1 influences short-term transcriptional behaviors of the glucocorticoid receptor; in turn, glucocorticoids themselves impact PARP1-genome interaction events. These findings are important for the understanding of general GR mechanisms and have relevance for the widespread functional decline observed during tissue glucocorticoid excess. Presentation: Saturday, June 11, 2022 12:30 p.m. - 12:45 p.m.

Independent Cis-Regulatory Modules within the Herpes Simplex Virus 1 Infected Cell Protein 0 (ICP0) Promoter Are Transactivated by Krüppel-like Factor 15 and Glucocorticoid Receptor.

A corticosteroid antagonist impairs Herpes Simplex Virus 1 (HSV-1) productive infection and explant-induced reactivation from latency, suggesting corticosteroids and the glucocorticoid receptor (GR) mediate certain aspects of these complex virus–host interactions. GR-hormone complexes regulate transcription positively and negatively, in part, by binding GR response elements (GREs). Recent studies revealed infected cell protein 0 (ICP0), ICP4, and ICP27 promoter/cis-regulatory modules (CRMs) are cooperatively transactivated by GR and Krüppel-like factor 15 (KLF15), which forms a feed-forward transcription loop. We hypothesized the ICP0 promoter contains independent CRMs that are transactivated by GR, KLF15, and the synthetic corticosteroid dexamethasone (DEX). This hypothesis is based on the finding that the ICP0 promoter contains multiple transcription factor binding sites, and GR and KLF15 cooperatively transactivate the full-length ICP0 promoter. ICP0 promoter sequences spanning −800 to −635 (fragment A) were efficiently transactivated by GR, KLF15, and DEX in monkey kidney cells (Vero), whereas GR and DEX significantly enhanced promoter activity in mouse neuroblastoma cells (Neuro-2A). Furthermore, ICP0 fragment B (−458 to −635) was efficiently transactivated by GR, KLF15, and DEX in Vero cells, but not Neuro-2A cells. Finally, fragment D (−232 to −24) was transactivated significantly in Vero cells by GR, KLF15, and DEX, whereas KLF15 and DEX were sufficient for transactivation in Neuro-2A cells. Collectively, these studies revealed efficient transactivation of three independent CRMs within the ICP0 promoter by GR, KLF15, and/or DEX. Finally, GC-rich sequences containing specificity protein 1 (Sp1) binding sites were essential for transactivation.

α-Actinin 4 Links Vasopressin Short-Term and Long-Term Regulation of Aquaporin-2 in Kidney Collecting Duct Cells.

Water permeability of the kidney collecting ducts is regulated by the peptide hormone vasopressin. Between minutes and hours (short-term), vasopressin induces trafficking of the water channel protein aquaporin-2 to the apical plasma membrane of the collecting duct principal cells to increase water permeability. Between hours and days (long-term), vasopressin induces aquaporin-2 gene expression. Here, we investigated the mechanisms that bridge the short-term and long-term vasopressin-mediated aquaporin-2 regulation by α-actinin 4, an F-actin crosslinking protein and a transcription co-activator of the glucocorticoid receptor. Vasopressin induced F-actin depolymerization and α-actinin 4 nuclear translocation in the mpkCCD collecting duct cell model. Co-immunoprecipitation followed by immunoblotting showed increased interaction between α-actinin 4 and glucocorticoid receptor in response to vasopressin. ChIP-PCR showed results consistent with α-actinin 4 and glucocorticoid receptor binding to the aquaporin-2 promoter. α-actinin 4 knockdown reduced vasopressin-induced increases in aquaporin-2 mRNA and protein expression. α-actinin 4 knockdown did not affect vasopressin-induced glucocorticoid receptor nuclear translocation, suggesting independent mechanisms of vasopressin-induced nuclear translocation of α-actinin 4 and glucocorticoid receptor. Glucocorticoid receptor knockdown profoundly reduced vasopressin-induced increases in aquaporin-2 mRNA and protein expression. In the absence of glucocorticoid analog dexamethasone, vasopressin-induced increases in glucocorticoid receptor nuclear translocation and aquaporin-2 mRNA were greatly reduced. α-actinin 4 knockdown further reduced vasopressin-induced increase in aquaporin-2 mRNA in the absence of dexamethasone. We conclude that glucocorticoid receptor plays a major role in vasopressin-induced aquaporin-2 gene expression that can be enhanced by α-actinin 4. In the absence of vasopressin, α-actinin 4 crosslinks F-actin underneath the apical plasma membrane, impeding aquaporin-2 membrane insertion. Vasopressin-induced F-actin depolymerization in one hand facilitates aquaporin-2 apical membrane insertion and in the other hand frees α-actinin 4 to enter the nucleus where it binds glucocorticoid receptor to enhance aquaporin-2 gene expression.

Reprogramming of glucocorticoid receptor function by hypoxia.

Here, we investigate the impact of hypoxia on the hepatic response of glucocorticoid receptor (GR) to dexamethasone (DEX) in mice via RNA-sequencing. Hypoxia causes three types of reprogramming of GR: (i) much weaker induction of classical GR-responsive genes by DEX in hypoxia, (ii) a number of genes is induced by DEX specifically in hypoxia, and (iii) hypoxia induces a group of genes via activation of the hypothalamic-pituitary-adrenal (HPA) axis. Transcriptional profiles are reflected by changed GR DNA-binding as measured by ChIP sequencing. The HPA axis is induced by hypothalamic HIF1α and HIF2α activation and leads to GR-dependent lipolysis and ketogenesis. Acute inflammation, induced by lipopolysaccharide, is prevented by DEX in normoxia but not during hypoxia, and this is attributed to HPA axis activation by hypoxia. We unfold new physiological pathways that have consequences for patients suffering from GC resistance.

TAZ inhibits glucocorticoid receptor and coordinates hepatic glucose homeostasis in normal physiological states.

The elucidation of the mechanisms whereby the liver maintains glucose homeostasis is crucial for the understanding of physiological and pathological states. Here, we show a novel role of hepatic transcriptional co-activator with PDZ-binding motif (TAZ) in the inhibition of glucocorticoid receptor (GR). TAZ is abundantly expressed in pericentral hepatocytes and its expression is markedly reduced by fasting. TAZ interacts via its WW domain with the ligand-binding domain of GR to limit the binding of GR to the GR response element in gluconeogenic gene promoters. Therefore, liver-specific TAZ knockout mice show increases in glucose production and blood glucose concentration. Conversely, the overexpression of TAZ in mouse liver reduces the binding of GR to gluconeogenic gene promoters and glucose production. Thus, our findings demonstrate that hepatic TAZ inhibits GR transactivation of gluconeogenic genes and coordinates gluconeogenesis in response to physiological fasting and feeding.

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.

Regulation of neurotropic herpesvirus productive infection and latency-reactivation cycle by glucocorticoid receptor and stress-induced transcription factors.

Neurotropic α-herpesvirinae subfamily members, herpes simplex virus type 1 (HSV-1) and bovine herpesvirus 1 (BoHV-1), are important viral pathogens in their respective hosts. Following acute infection on mucosal surfaces, these viruses establish life-long latency in neurons within trigeminal ganglia (TG) and central nervous system. Chronic or acute stress (physiological or psychological) increases the frequency of reactivation from latency, which leads to virus shedding, virus transmission, and recurrent disease. While stress impairs immune responses and inflammatory signaling cascades, we predict stressful stimuli directly stimulate viral gene expression and productive infection during early stages of reactivation from latency. For example, BoHV-1 and HSV-1 productive infection is impaired by glucocorticoid receptor (GR) antagonists but is stimulated by the synthetic corticosteroid dexamethasone. Promoters that drive expression of key viral transcriptional regulatory proteins are cooperatively stimulated by GR and specific Krüppel like transcription factors (KLF) induced during stress induced reactivation from latency. The BoHV-1 immediate early transcription unit 1 promoter and contains two GR response elements (GRE) that are essential for cooperative transactivation by GR and KLF15. Conversely, the HSV-1 infected cell protein 0 (ICP0) and ICP4 promoter as well as the BoHV-1 ICP0 early promoter lack consensus GREs: however, these promoters are cooperatively transactivated by GR and KLF4 or KLF15. Hence, growing evidence suggests GR and stress-induced transcription factors directly stimulate viral gene expression and productive infection during early stages of reactivation from latency. We predict the immune inhibitory effects of stress enhance virus spread at late stages during reactivation from latency.

Moderate Aerobic Exercise Training Prevents the Augmented Hepatic Glucocorticoid Response Induced by High-Fat Diet in Mice.

Glucocorticoids (GCs) are critical regulators of energy balance. Their deregulation is associated with the development of obesity and metabolic syndrome. However, it is not understood if obesity alters the tissue glucocorticoid receptor (GR) response, and moreover whether a moderate aerobic exercise prevents the alteration in GR response induced by obesity. Methods: To evaluate the GR response in obese mice, we fed C57BL6J mice with a high-fat diet (HFD) for 12 weeks. Before mice were sacrificed, we injected them with dexamethasone. To assess the exercise role in GR response, we fed mice an HFD and subjected them to moderate aerobic exercise three times a week. Results: We found that mice fed a high-fat diet for 12 weeks developed hepatic GC hypersensitivity without changes in the gastrocnemius or epididymal fat GR response. Therefore, moderate aerobic exercise improved glucose tolerance, increased the corticosterone plasma levels, and prevented hepatic GR hypersensitivity with an increase in epididymal fat GR response. Conclusion: Collectively, our results suggest that mice with HFD-induced obesity develop hepatic GR sensitivity, which could enhance the metabolic effects of HFD in the liver. Moreover, exercise was found to be a feasible non-pharmacological strategy to prevent the deregulation of GR response in obesity.

ACUTE ANDROGEN AND GLUCOCORTICOID RECEPTOR RESPONSE FOLLOWING A MODERATE INTENSITY RESISTANCE EXERCISE BOUT

PURPOSE: The purpose of this study was to examine the acute androgen receptor (AR) and glucocorticoid receptor (GR) response to a moderate intensity resistance exercise bout in resistance trained (RT) and untrained men (UT). METHODS: RT men (n = 10; X ± SD, age = 21.3 ± 1.7 yrs, height = 175.8 ± 6.8 cm, body mass = 84.5 ± 13.5 kg, back squat 1RM = 154.3 ± 19.3 kg, training history = 5.4 ± 2.0 yrs) and UT men (n = 9; X ± SD, age = 20.8 ± 3.1 yrs, height = 178.7 ± 8.9 cm, body mass = 81.0 ± 14.0 kg, squat 1RM = 108.1 ± 13.7 kg, training history = 0.7 ± 1.7 yrs) volunteered for this study. Prior to the RE bout, subjects were strength tested for back squat (BS) and leg extension (LE). Subjects returned 4-7 days later between 10am-2pm, and completed a RE bout consisting of 6 sets of 10 repetitions of BS at 75% 1RM, immediately followed by 4 sets of 10 repetitions of LE at 75% 1RM with 1.5 min rest between all sets. Muscle samples were collected from the vastus lateralis prior to exercise (PRE) and 10 min (10P), 30 min (30P), 60 min (60P), and 180 min (180P) post exercise. Total AR and GR expression was determined via western blotting. Receptor data were not normally distributed, thus all receptor data were analyzed using Mann-Whitney U test, Friedman test, and Wilcoxon signed-rank test. RESULTS: For total AR expression, there were no differences between time points within the RT group (p > .05); however, there were differences between time points within the UT group (p = .016). In the UT group, total AR expression significantly decreased at 30P (-19%∆, z = -2.192, p = .027) and 60P (-11%∆, z = -2.192, p = .027) post exercise, but returned to baseline values by 180P (z = -.178, p > .05). For Total GR content, there were no differences between time points within the RT or UT groups (p > .05). Total GR content was significantly greater in the RT group compared to the UT group at 10P (Mann-Whitney U = 19, z = -2.123, p = .035). CONCLUSIONS: While no changes were observed for AR expression in the RT group, the UT subjects experienced a significant decrease in AR expression at 30P and 60P suggesting acute AR responses vary depending on training status. No differences were seen across time for the GR in either group; however, RT and UT subjects were different from each other at 10P. It is unclear if these responses are related to the acute hormonal response; therefore, future research will address this.

OR12-02 When the Glucocorticoid Receptor Meets the Mineralocorticoid Receptor in the Nucleus of Human Cells

Adrenal corticosteroids, such as glucocorticoids and mineralocorticoids, are indispensable for mediating response to stress, development, limiting inflammation, and maintaining energy and fluid homeostasis. These hormones exert their actions via binding to two closely related nuclear receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). The GR has low affinity for corticosteroids, but is expressed in nearly every cell. In contrast, the MR shows a higher affinity for corticosteroids and its expression is largely confined to those tissues where electrolyte exchange and fluid balance are required. GR and MR act as ligand-activated transcription factors which, following interaction with co-regulators and DNA responsive elements, either promote or repress gene transcription. The affinity for the same ligands, structural homology, and binding to the same DNA regions suggest GR and MR can compensate for each other’s actions. Yet, there are specific glucocorticoid and mineralocorticoid-mediated responses indicating GR-MR functional diversity. To investigate this interplay, we developed U-2 OS (human osteosarcoma) cell lines stably expressing GR, MR, and both GR and MR (GRMR). Immunofluorescence analysis showed that treatment of these cell lines with 1 nM of the synthetic glucocorticoid dexamethasone (Dex) induced nuclear traslocation of GR and MR. Conversely, treatment with 1 nM of aldosterone (Aldo) promoted nuclear translocation of the MR only. Moreover, Proximity Ligation Assay revealed that, in the absence of ligand, GR associated with MR in the cytoplasm and, upon 1 nM Dex exposure, GR-MR dimers were detected in the nucleus of GRMR cells. Surprisingly, nuclear GR-MR dimers were also detected in the presence of Aldo, suggesting that it is necessary to activate at least one receptor to induce nuclear traslocation of the heterocomplex. To decipher the functional contribution of GR-MR dimers in the transcriptional response of GR to Dex and MR to Aldo, we performed RNA-seq in GR, MR, and GRMR cells treated with 1 nM of Dex or Aldo. Transcriptome analysis revealed that Dex-activated GR regulated the transcription of 6180 genes. Co-expression of MR resulted in a blunted Dex-mediated gene response which affected only 1608 genes, suggesting a functional antagonism of MR. Aldo-activated MR regulated the transcription of 1660 genes. However, co-expression of GR expanded the Aldo-mediated gene response to 3150 genes. Strikingly, 74% of these genes were also regulated by Dex via GR, suggesting that GR-MR dimers in the presence of aldosterone are able to mimic the glucocorticod transcriptional response. Our data suggest that the role of distinct GR and MR homo- and hetero-dimers is relevant for regulating gene expression. Dissecting the mechanism and investigating the cross-talk between GR and MR may be useful to understanding these two receptors in heath and disease.

Bidirectional crosstalk between Hypoxia-Inducible Factor and glucocorticoid signalling in zebrafish larvae.

In the last decades in vitro studies highlighted the potential for crosstalk between Hypoxia-Inducible Factor-(HIF) and glucocorticoid-(GC) signalling pathways. However, how this interplay precisely occurs in vivo is still debated. Here, we use zebrafish larvae (Danio rerio) to elucidate how and to what degree hypoxic signalling affects the endogenous glucocorticoid pathway and vice versa, in vivo. Firstly, our results demonstrate that in the presence of upregulated HIF signalling, both glucocorticoid receptor (Gr) responsiveness and endogenous cortisol levels are repressed in 5 days post fertilisation larvae. In addition, despite HIF activity being low at normoxia, our data show that it already impedes both glucocorticoid activity and levels. Secondly, we further analysed the in vivo contribution of glucocorticoids to HIF activity. Interestingly, our results show that both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) play a key role in enhancing it. Finally, we found indications that glucocorticoids promote HIF signalling via multiple routes. Cumulatively, our findings allowed us to suggest a model for how this crosstalk occurs in vivo.

The role of glucocorticoid receptors in the induction and prevention of hippocampal abnormalities in an animal model of posttraumatic stress disorder.

Since the precise mechanisms of posttraumatic stress disorder (PTSD) remain unknown, effective treatment interventions have not yet been established. Numerous clinical studies have led to the hypothesis that elevated glucocorticoid levels in response to extreme stress might trigger a pathophysiological cascade which consequently leads to functional and morphological changes in the hippocampus. To elucidate the pathophysiology of PTSD, we examined the alteration of hippocampal gene expression through the glucocorticoid receptor (GR) in the single prolonged stress (SPS) paradigm, a rat model of PTSD. We measured nuclear GRs by western blot, and the binding of GR to the promoter of Bcl-2 and Bax genes by chromatin immunoprecipitation-qPCR as well as the expression of these 2 genes by RT-PCR in the hippocampus of SPS rats. In addition, we examined the preventive effects of a GR antagonist on SPS-induced molecular, morphological, and behavioral alterations (hippocampal gene expression of Bcl-2 and Bax, hippocampal apoptosis using TUNEL staining, impaired fear memory extinction (FME) using the contextual fear conditioning paradigm). Exposure to SPS increased nuclear GR expression and GR binding to Bcl-2 gene, and decreased Bcl-2 mRNA expression. Administration of GR antagonist immediately after SPS prevented activation of the glucocorticoid cascade, hippocampal apoptosis, and impairment FME in SPS rats. The activation of GRs in response to severe stress may trigger the pathophysiological cascade leading to impaired FME and hippocampal apoptosis. In contrast, administration of GR antagonist could be useful for preventing the development of PTSD.