Glucocorticoid Receptor Levels Research Articles

The Effect of Histone Deacetylase Inhibitor on the Expression Level of Glucococrticoid Receptor in Rat Forebrain under Hypoxia

The activity of the gene transcription depends to a large extent on the histone acetylation status and can be affected by different stimuli such as hypoxia. Histone deacetylase inhibitors promote gene transcription by facilitating histone acetylation and are thus considered as candidates for target therapy of posthypoxic states. In the present study, using immunohistochemistry, the effect of the histone deacetylase inhibitor trihostatin A (TSA) on the expression level of glucocorticoid receptor (GR) have been analyzed in the neocortex and hippocampus of rats in an original model of severe hypobaric hypoxia (SHH). It has been found that injections of TSA in rats facilitated GR expression after SHH in the neocortex and CA1 field but not in the dentate girus of hippocampus. GR overexpression enhances the toxicity of circulating glucocorticoid hormones on the hippocampal neurons. Stimulation of GR expression by TSA injections in the CA1 field of the hippocampus may lead to maladaptation, making possible damaging effects of unfavorable factors, such as hypoxia. The data obtained indicate that therapy using histone deacetylase inhibitors may have adverse side effects for vulnerable brain neurons.

Prenatal caffeine exposure increases the susceptibility to non-alcoholic fatty liver disease in female offspring rats via activation of GR-C/EBPα-SIRT1 pathway

This study aimed to evaluate female adult offspring induced by prenatal caffeine exposure (PCE) are susceptible to non-alcoholic fatty liver disease (NAFLD) and to explore the underlying programming mechanisms. Pregnant rats were intragastrically administered caffeine (30, 60, and 120 mg/kg.d) on gestational day (GD) 9-20. The female adult offspring were randomly divided into three groups: offspring without or with chronic stress during postnatal week (PW) 10–12 and PW28 offspring. Results showed that PW28 PCE female offspring had a higher hepatic triglyceride content and Kleiner scores, accompanied by elevated serum corticosterone levels. Moreover, the expression levels of hepatic glucocorticoid receptor (GR), CCAAT enhancer binding protein α (C/EBPα), fatty acid synthetase (FASN) and the transcription factor-sterol regulatory element binding protein 1c (SREBP1c) were increased, but SIRT1 expression was decreased. The fetal rats and PW12 offspring with chronic stress exhibited similar changes as PW28 offspring, accompanied by increased levels of H3K14ac and H3K27ac in the SREBP1c and FASN gene promoters. These effects were also observed by treating L02 cells with cortisol and were partially reversed by GR or C/EBPα siRNA or treatment with the SIRT1 agonist resveratrol. Taken together, PCE-induced high glucocorticoids levels enhanced histone modifications and expression of SREBP1c and FASN via activation of the GR-C/EBPα-SIRT1 pathway in utero. This enhanced female fetal hepatic triglyceride synthesis and continued throughout postnatal and adult life, increasing the susceptibility to adult NAFLD.

Tactile Stimulation on Adulthood Modifies the HPA Axis, Neurotrophic Factors, and GFAP Signaling Reverting Depression-Like Behavior in Female Rats.

Depression is a common psychiatric disease which pharmacological treatment relieves symptoms, but still far from ideal. Tactile stimulation (TS) has shown beneficial influences in neuropsychiatric disorders, but the mechanism of action is not clear. Here, we evaluated the TS influence when applied on adult female rats previously exposed to a reserpine-induced depression-like animal model. Immediately after reserpine model (1mg/kg/mL, 1×/day, for 3days), female Wistar rats were submitted to TS (15min, 3×/day, for 8days) or not (unhandled). Imipramine (10mg/kg/mL) was used as positive control. After behavioral assessments, animals were euthanized to collect plasma and prefrontal cortex (PFC). Behavioral observations in the forced swimming test, splash test, and sucrose preference confirmed the reserpine-induced depression-like behavior, which was reversed by TS. Our findings showed that reserpine increased plasma levels of adrenocorticotropic hormone and corticosterone, decreased brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B, and increased proBDNF immunoreactivity in the PFC, which were also reversed by TS. Moreover, TS reestablished glial fibrillary acidic protein and glucocorticoid receptor levels, decreased by reserpine in PFC, while glial cell line-derived neurotrophic factor was increased by TS per se. Our outcomes are showing that TS applied in adulthood exerts a beneficial influence in depression-like behaviors, modulating the HPA axis and regulating neurotrophic factors more effectively than imipramine. Based on this, our proposal is that TS, in the long term, could be considered a new therapeutic strategy for neuropsychiatric disorders improvement in adult life, which may represent an interesting contribution to conventional pharmacological treatment.

A miR-29a-driven negative feedback loop regulates peripheral glucocorticoid receptor signaling.

The glucocorticoid receptor (GR) represents the crucial molecular mediator of key endocrine, glucocorticoid hormone-dependent regulatory circuits, including control of glucose, protein, and lipid homeostasis. Consequently, aberrant glucocorticoid signaling is linked to severe metabolic disorders, including insulin resistance, obesity, and hyperglycemia, all of which also appear upon chronic glucocorticoid therapy for the treatment of inflammatory conditions. Of note, long-term glucocorticoid exposure under these therapeutic conditions typically induces glucocorticoid resistance, requiring higher doses and consequently triggering more severe metabolic phenotypes. However, the molecular basis of acquired glucocorticoid resistance remains unknown. In a screen of differential microRNA expression during glucocorticoid-dependent adipogenic differentiation of human multipotent adipose stem cells, we identified microRNA 29a (miR-29a) as one of the most down-regulated transcripts. Overexpression of miR-29a impaired adipogenesis. We found that miR-29a represses GR in human adipogenesis by directly targeting its mRNA, and downstream analyses revealed that GR mediates most of miR-29a's anti-adipogenic effects. Conversely, miR-29a expression depends on GR activation, creating a novel miR-29-driven feedback loop. miR-29a and GR expression were inversely correlated both in murine adipose tissue and in adipose tissue samples obtained from human patients. In the latter, miR-29a levels were additionally strongly negatively correlated with body mass index and adipocyte size. Importantly, inhibition of miR-29 in mice partially rescued the down-regulation of GR during dexamethasone treatment. We discovered that, in addition to modulating GR function under physiologic conditions, pharmacologic glucocorticoid application in inflammatory disease also induced miR-29a expression, correlating with reduced GR levels. This effect was abolished in mice with impaired GR function. In summary, we uncovered a novel GR-miR-29a negative feedback loop conserved between mice and humans, in health and disease. For the first time, we elucidate a microRNA-related mechanism that might contribute to GR dysregulation and resistance in peripheral tissues.-Glantschnig, C., Koenen, M., Gil-Lozano, M., Karbiener, M., Pickrahn, I., Williams-Dautovich, J., Patel, R., Cummins, C. L., Giroud, M., Hartleben, G., Vogl, E., Blüher, M., Tuckermann, J., Uhlenhaut, H., Herzig, S., Scheideler, M. A miR-29a-driven negative feedback loop regulates peripheral glucocorticoid receptor signaling.

Glucocorticoid receptor dysfunction orchestrates inflammasome effects on chronic obstructive pulmonary disease-induced depression: A potential mechanism underlying the cross talk between lung and brain

Depression is highly prevalent among patients with chronic obstructive pulmonary disease (COPD). However, depression with COPD comorbidity is often underdiagnosed and undertreated, and pathogenic research is also insufficient. In the present study, we characterised pulmonary and hippocampal dysfunction by researching the interaction between inflammasome-regulated cytokines and glucocorticoid receptor (GR) signalling by investigating the role of fluoxetine (FLU), one of the most widely used antidepressants in clinical practice. Mice were exposed to cigarette smoke (CS) to induce the model of COPD with comorbid depression, and pathological alterations in serum, hippocampus, lung, and bronchoalveolar lavage fluid were determined. Our results showed that the CS procedure induced the accumulation of inflammatory cells (macrophages, neutrophils, and lymphocytes), the production of cytokines, the activation of inflammasome components (NLRP3, ASC, caspase-1), depression-related behaviours, and the stimulation of GR signalling. Intriguingly, glucocorticoid resistance occurred in CS-exposed mice, with elevated serum corticosterone and suppressed hippocampal GR levels, which suggested a novel potential regulatory mechanism underlying COPD-induced depression comorbidity. Furthermore, chronic CS exposure decreased the pGR-S211/pGR-S226 ratio, increased the active nuclear GR, and impaired cytosolic GR binding capacity and GR transcriptional activity, which might be responsible for the activation of the inflammasome-induced inflammatory cascade. These alterations were reversed by chronic FLU treatment, indicating that FLU-mediated GR signalling was involved in the COPD induced inflammasome activation. Our research explored the underlying molecular mechanism of comorbid COPD/depression and provided in vivo evidence that glucocorticoid resistance occurred during CS-induced central nervous system inflammation, a potential mechanism underlying the cross talk between the lung and brain.

FKBP51 is associated with early postoperative cognitive dysfunction in elderly patients undergoing hip fracture surgery.

Enhanced inflammation response was increasingly reported in association with postoperative cognitive dysfunction (POCD). Glucocorticoid receptor (GR) signal plays a key role in suppression of inflammation. This prospective cohort study aimed to evaluate GR signaling in elderly patients undergoing selective operation.One hundred twenty-six elderly patients were scheduled for hip fracture surgery with general anesthesia. Plasma cortisol levels and the expression levels of GR and FK506 binding protein 51 (FKBP51) in leukocytes were determined at 1 day preoperatively and 7 days. Postoperatively postoperative pain was assessed following surgery using visual analog pain scale (VAS). Neuropsychological tests were performed before surgery and 1 week postoperation. A decline of 1 or more standard deviations in 2 or more tests was considered to reflect POCD.POCD incidence in participants was 28.3% at 1 week after surgery. POCD patients presented significantly higher cortisol and FKBP51 levels compared with non-POCD patients (P < .05). Compared with non-POCD patients, VAS scores at 12 hours after surgery were higher in POCD patients (P < .05). No significant difference in expression levels of GR was found between groups POCD and non-POCD patients.High expression of FKBP51 in leukocytes and glucocorticoid resistance were associated with POCD in aged patients following hip fracture surgery.

Antenatal Glucocorticoid Exposure Results in Sex-Specific and Transgenerational Changes in Prefrontal Cortex Gene Transcription that Relate to Behavioural Outcomes

Synthetic glucocorticoids (sGC) are administered to women at risk for pre-term delivery to reduce respiratory distress syndrome in the newborn. The prefrontal cortex (PFC) is important in regulating stress responses and related behaviours and expresses high levels of glucocorticoid receptors (GR). Further, antenatal exposure to sGC results in a hyperactive phenotype in first generation (F1) juvenile male and female offspring, as well as F2 and F3 juvenile females from the paternal lineage. We hypothesized that multiple courses of antenatal sGC modify gene expression in the PFC, that these effects are sex-specific and maintained across multiple generations, and that the gene sets affected relate to modified locomotor activity. We performed RNA sequencing on PFC of F1 juvenile males and females, as well as F2 and F3 juvenile females from the paternal lineage and used regression modelling to relate gene expression and behavior. Antenatal sGC resulted in sex-specific and generation-specific changes in gene expression. Further, the expression of 4 genes (C9orf116, Calb1, Glra3, and Gpr52) explained 20–29% of the observed variability in locomotor activity. Antenatal exposure to sGC profoundly influences the developing PFC; effects are evident across multiple generations and may drive altered behavioural phenotypes.

Antidepressant-like activities of live and heat-killed Lactobacillus paracasei PS23 in chronic corticosterone-treated mice and possible mechanisms

Emerging evidence indicates that ingestion of specific probiotics, known as “psychobiotics”, confer beneficial effects on mental health. This study investigated antidepressant-like effects and possible underlying mechanisms of Lactobacillus paracasei PS23 (PS23), live or heat-killed, in a mouse model of corticosterone-induced depression using fluoxetine as standard drug. PS23 were orally gavaged to mice from day 1 to 41 or fluoxetine from day 17 to 41 and injected with corticosterone from day 17 to 37. After the last corticosterone treatment, anxiety- and depression-like behaviors were tested within 4 days. On day 42, serum and brain tissue were collected 24 min after forced swim stress. Abnormal behavioral changes induced by corticosterone were ameliorated by treatment with live PS23 in open field and sucrose preference tests, with heat-killed PS23 in open field, forced swim and sucrose preference tests, and with fluoxetine in open field and forced swim tests. Furthermore, both live and heat-killed PS23 and fluoxetine reversed corticosterone-reduced protein levels of brain-derived neurotropic factor, mineralocorticoid, and glucocorticoid receptors in the hippocampus. In addition, live PS23 also reverses corticosterone-reduced serotonin levels in hippocampus, prefrontal cortex and striatum; whereas heat-killed PS23 reverses corticosterone-reduced dopamine levels in hippocampus and prefrontal cortex. And fluoxetine normalized reduced corticosterone level in serum. These studies showed that both live and heat-killed PS23 can reverse chronic corticosterone-induced anxiety- and depression-like behaviors and that may provide insights into the mechanism and a potential psychobiotic for depression management.

The selenium-containing compound 3-((4-chlorophenyl)selanyl)-1-methyl-1H-indole reverses depressive-like behavior induced by acute restraint stress in mice: modulation of oxido-nitrosative stress and inflammatory pathway.

Stress-induced alterations in oxidative and inflammatory parameters have been implicated in the pathophysiology of mood disorders. Based on the antioxidant and anti-inflammatory properties of the selenium-containing compound 3-((4-chlorophenyl)selanyl)-1-methyl-1H-indole (CMI), we assessed its ability to reverse depression-like behavioral alterations, neuroinflammation, and oxidative imbalance induced by acute restraint stress. Mice submitted to restraint for 240min received CMI (1 or 10mg/kg, orally) 10min after the end of the stress induction. Behavioral and biochemical tests were carried out after further 30min. Restraint-induced depression-like behavior in the tail suspension test (TST), splash test, and new object exploration test was reversed by CMI. None of the treatments evoked locomotor alteration. In addition, CMI abrogated restraint-induced increases in plasma levels of corticosterone and in markers of oxidative stress and impaired superoxide dismutase and catalase activity in the prefrontal cortex (PFC) and hippocampus (HC). CMI also blocked stress-induced downregulation of mRNA levels of glucocorticoid receptor and brain-derived neurotrophic factor and upregulation of nuclear factor kappa B, inducible nitric oxide synthase, tumor necrosis alpha, indoelamine-2,3-dioxygenase, and glycogen synthase kinase 3 beta in PFC and HC. These preclinical results indicate that administration of selenium-containing compounds might help to treat depression associated with inflammation and oxidative stress. Graphical abstract ᅟ.

Neonatal isolation modulates glucocorticoid-receptor function and synaptic plasticity of hippocampal and amygdala neurons in a rat model of single prolonged stress.

Early life and stressful experiences affect hippocampal and amygdala structure and function. They also increase the incidence of mental and nervous system disorders in adults. However, prospective studies have yet to show if early-life experiences affect the risk/severity of post-traumatic stress disorder (PTSD). We applied neonatal isolation (NI) alone, single prolonged stress (SPS) alone and NI + SPS to rats. We evaluated anxiety-like behavior and spatial memory of behavior using open field, elevated plus maze, and Morris water maze tests. Then, we measured expression of glucocorticoid receptors (GRs) and synaptic-related proteins by immunofluorescence, immunohistochemistry and western blotting in the hippocampus and amygdala. NI + SPS exacerbated the increased anxiety levels and impaired spatial memory induced by NI alone or SPS alone. NI alone or SPS alone induced varying degrees of change in expression of GRs and synaptic proteins (synapsin I and postsynaptic density protein-95) in the hippocampus and amygdala. There were opposite changes in GR expression in the hippocampal dentate gyrus and basolateral amygdala. The degree of such change was exacerbated considerably by NI + SPS. In addition, neuroligin (NLG)-1 and NLG-2 were distributed in postsynaptic sites of excitatory and inhibitory synapses, respectively. NI, SPS, and NI + SPS altered the patterns of NLG-1 and NLG-2 colocalization as well as their intensity. NI + SPS strengthened the increased ratio of NLG-1/NLG-2 in the hippocampus, but decreased this ratio in the amygdala. NI and SPS together induced greater degrees of change in anxiety and spatial memory, as well as GR and synaptic protein levels, in the hippocampus and amygdala than the changes induced by NI alone or SPS alone.

Characterizing changes in glucocorticoid receptor internalization in the fear circuit in an animal model of post traumatic stress disorder.

Glucocorticoid receptors (GRs) shuttle from the cytoplasm (cy) to the nucleus (nu) when bound with glucocorticoids (i.e. GR internalization) and alter transcriptional activity. GR activation within the fear circuit has been implicated in fear memory and post traumatic stress disorder (PTSD). However, no study to date has characterized GR internalization within the fear circuit during fear memory formation or examined how traumatic stress impacts this process. To address this, we assayed cy and nu GR levels at baseline and after auditory fear conditioning (FC) in the single prolonged stress (SPS) model of PTSD. Cy and nu GRs within the medial prefrontal cortex (mPFC), dorsal hippocampus (dHipp), ventral hippocampus (vHipp), and amygdala (AMY) were assayed using western blot. The distribution of GR in the cy and nu (at baseline and after FC) was varied across individual nodes of the fear circuit. At baseline, SPS enhanced cyGRs in the dHipp, but decreased cyGRs in the AMY. FC only enhanced GR internalization in the AMY and this effect was attenuated by SPS exposure. SPS also decreased cyGRs in the dHipp after FC. The results of this study suggests that GR internalization is varied across the fear circuit, which in turn suggests GR activation is selectively regulated within individual nodes of the fear circuit. The findings also suggest that changes in GR dynamics in the dHipp and AMY modulate the enhancing effect SPS has on fear memory persistence.

Effects of Glucocorticoid-Induced Transcript 1 Gene Deficiency on Glucocorticoid Activation in Asthmatic Mice.

Background:Glucocorticoid (GC) is the first-line therapy for asthma, but some asthmatics are insensitive to it. Glucocorticoid-induced transcript 1 gene (GLCCI1) is reported to be associated with GCs efficiency in asthmatics, while its exact mechanism remains unknown.Methods:A total of 30 asthmatic patients received fluticasone propionate for 12 weeks. Forced expiratory volume in 1 s (FEV1) and GLCCI1 expression were detected. Asthma model was constructed in wild-type and GLCCI1 knockout (GLCCI1-/-) mice. Glucocorticoid receptor (GR) and mitogen-activated protein kinase phosphatase 1 (MKP-1) expression were detected by polymerase chain reaction and Western blotting (WB). The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was also detected by WB.Results:In asthmatic patients, the change of FEV1 was well positively correlated with change of GLCCI1 expression (r = 0.430, P = 0.022). In animal experiment, GR and MKP-1 mRNA levels were significantly decreased in asthmatic mice than in control mice (wild-type: GR: 0.769 vs. 1.000, P = 0.022; MKP-1: 0.493 vs. 1.000, P < 0.001. GLCCI1-/-: GR: 0.629 vs. 1.645, P < 0.001; MKP-1: 0.377 vs. 2.146, P < 0.001). Hydroprednisone treatment significantly increased GR and MKP-1 mRNA expression levels than in asthmatic groups; however, GLCCI1-/- asthmatic mice had less improvement (wild-type: GR: 1.517 vs. 0.769, P = 0.023; MKP-1: 1.036 vs. 0.493, P = 0.003. GLCCI1-/-: GR: 0.846 vs. 0.629, P = 0.116; MKP-1: 0.475 vs. 0.377, P = 0.388). GLCCI1-/- asthmatic mice had more obvious phosphorylation of p38 MAPK than wild-type asthmatic mice (9.060 vs. 3.484, P < 0.001). It was still higher even though after hydroprednisone treatment (6.440 vs. 2.630, P < 0.001).Conclusions:GLCCI1 deficiency in asthmatic mice inhibits the activation of GR and MKP-1 and leads to more obvious phosphorylation of p38 MAPK, leading to a decremental sensitivity to GCs.Trial Registration:ChiCTR.org.cn, ChiCTR-RCC-13003634; http://www.chictr.org.cn/showproj.aspx?proj=5926.

A Gain of Function Mutation in the NSD2 Histone Methyltransferase Drives Glucocorticoid Resistance of Acute Lymphoblastic Leukemia

▪Acute lymphoblastic leukemia (ALL) is the most common diagnosed pediatric cancer. Despite improvements in chemotherapy that have increased the 5-year survival rate to close to 90%, 15-20% of these patients may relapse with the majority of such children succumbing to this disease. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biological processes mediated by mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in two B-ALL cell lines (RCH-ACV and SEM) and one T-ALL cell line (RPMI-8402) and inserted the E1099K mutation into three ALL cell lines (697, CEM, MOLT4). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099K cells compared to cells in which the mutation is removed demonstrate enhanced cell growth, colony formation and migration. NSD2 mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system (CNS) in xenografts.The NSD2 mutation is found prominently in children who relapse early from therapy for ALL, and NSD2E1099K cells are particularly resistant to glucocorticoids (GC). Reversion of NSD2E1099K mutation to wild type NSD2 conferred glucocorticoid sensitivity to both B and T cell lines. GC response upon disruption of mutant NSD2 was accompanied by cell cycle arrest and apoptosis. Mice xenografted with NSD2E1099K cells were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2 wild-type cells led to significant tumor reduction and survival extension. RNA-Seq analysis showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2 wild-type cells. Furthermore, in NSD2 mutant cells, there was lower basal expression level of glucocorticoid receptor (GR) and GR levels were not significantly induced by GC. Accordingly, after treatment with GC, there was significantly less DNA-binding activity of the GR in NSD2E1099K cells than that of NSD2 wild-type cells. The key pro-apoptotic regulators Bim and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. In conclusion, these studies demonstrate that the NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by causing GC resistance. Future studies will determine how NSD2 which generally activates genes paradoxically blocks the ability of GC and the GR to induce critical pro-death genes. DisclosuresLicht:Celgene: Research Funding.

Chemogenomic Approach Unveils the Increased Susceptibility of RUNX1-Mutated AML to Glucocorticoids

Abstract RUNX1 is an essential transcription factor for definite hematopoiesis and plays important roles in immune function. Mutations in RUNX1 occur in 5-13% of Acute Myeloid Leukemia (AML) patients (RUNX1mut ) and are associated with adverse outcome, highlighting the need for better genetic characterization of this AML subgroup and for the design of efficient therapeutic strategies for patients with RUNX1mut AML. Toward this goal, we performed RNA-sequencing of a cohort of RUNX1mut primary AML specimens and used a chemogenomic approach combining mutational and gene expression analysis of these specimens with assessment of their drug sensitivity profile through chemical screening. Sequencing data analysis demonstrated that samples with no remaining RUNX1 wild-type allele are clinically and genetically distinct and display a more homogeneous gene expression profile. Interestingly, these studies also unveiled the increased susceptibility of RUNX1-mutated specimens to Glucocorticoids (GCs) and revealed that RUNX1 allele dosage dictates sensitivity to these compounds in AML patient cells, unravelling a new role for RUNX1 in the Glucocorticoid Receptor (GR) pathway. GR is a nuclear receptor that modulates the expression of thousands of genes involved in several biological processes such as metabolism, immune function, skeletal growth, etc. GCs are commonly used to treat cancers of the lymphoid system, however their potential benefits for AML treatment have never been assessed formally and the mechanism of action of these drugs is not fully understood. Transcriptome analyses identified NR3C1 (encoding the GR) as one of the genes whose expression is determined by RUNX1 allele dosage, with increased expression in RUNX1mut specimens, indicating that RUNX1 inactivation could lead to GR upregulation, which might explain the increased sensitivity to GCs. We previously showed that RUNX1 silencing sensitizes human AML cell lines to GCs and that this acquired sensitivity is accompanied by the upregulation of the GR both at the transcript and protein levels. However, basal levels of GR could not explain GC sensitivity in all cases, indicating that other mechanisms are involved in the GC response. By performing co-immunoprecipitations (co-IP), we demonstrated that RUNX1 and GR physically interact in AML cells. Overexpression of FLAG-tagged RUNX1 mutants in HEK293 cells followed by co-IP identified the C-terminal inhibitory domain of RUNX1 as essential for the interaction with GR. Our results suggest that RUNX1 could be part of the GR transcriptional complex and could modulate the transcription of genes involved in the response to GCs. To identify regulators of the GC response, we are currently performing genome-wide CRISPR-Cas9-based genetic screens in AML cell lines. We generated RUNX1-deficient (GCs sensitive) AML cell lines that uniformly express Cas9 under a doxycycline-inducible promoter. These cell lines, along with parental cell lines (GC resistant) were used to conduct screens in GCs-supplemented media for the identification of genes that confer sensitivity or resistance to GCs. Mechanistic insights gained from these experiments will allow the design of additional therapeutic strategies to potentiate the effect of GCs on poor outcome AML. Disclosures No relevant conflicts of interest to declare.

Glucocorticoids-Resistant Leukemic B-Cells Undergo a Phenotypic Change That Increases Sensitivity to SRC/ABL Inhibition

▪Glucocorticoids (GCs) have a crucial role in the treatment of B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is considered a strong prognostic marker of relapse, which occurs in about 20% of pediatric B-ALL patients. GCs exert therapeutic effects by inducing cell cycle arrest and eventually, cell death. However, their precise mechanisms of action and resistance are not fully understood. We have previously demonstrated the importance of developmental state in treatment failure in B-ALL (Good Z Nat Med 2018 and Bendall SC Cell 2014), specifically at the developmental transition between pro-B to pre-B cells. Using this developmental framework, we examined the effect of GCs on healthy and malignant early B cells progenitors to better understand mediators of GC resistance.To understand the transcriptional programs mediating early B-cell differentiation, we sorted three B-cell developmental populations (pre-pro-B, pro-B/pre-B I and pre-B II) and performed RNA-seq analysis. Ingenuity pathway analysis revealed a coordinated upregulation of B cell developmental genes and Glucocorticoid Receptor (GCR) pathway genes in healthy pre-B I cells, suggesting a role of GCs in healthy B-cell development. We confirmed expression of the GCR in healthy pre-B I cells by mass cytometry (CyTOF). Further, in vitro treatment of healthy B-cells with GCs (dexamethasone, dex 1 uM) demonstrated pre-B I cycling cells to be most sensitive to GC-induced cell death.Given the importance of GCs in B-ALL treatment, we investigated the GCR pathway and its relationship with B-cell development in 18 B-ALL primary samples (14 diagnosis, 4 relapse; including Ph+=1; MLL rearranged=2; TEL-AML translocated=3). Samples were profiled by CyTOF with a 40-antibody panel including surface markers, signaling molecules, transcription factors, apoptosis and cell cycle molecules. To better comprehend the relation of GCR pathway with BCR signaling, primary cells were analyzed after 48 hours of treatment with dex (1uM), SRC/ABL inhibitor dasatinib (das, 100 nM) or their combination.Similar to results obtained in healthy B-cells, we found the highest expression of GCR in the pre B-I cells in all patients (p<0.001). Interestingly, samples from relapse or diagnostic high-risk groups demonstrated the highest expression of GCR in this cell population. As expected, dex induced cell cycle arrest and cell death in almost all the samples (88%), while two samples were completely unresponsive. Moreover, combined treatment resulted in increased cell death in 29% of patients, compared to dex or das alone.Interestingly, we also observed consistent patterns of phenotypic modulation in dex-resistant cells and to understand how these were related to B-cell development we performed developmental classification (Good Z Nat Med 2018) of leukemic cells in dex-treated versus untreated conditions. This analysis revealed a large decrease of early B-cell progenitors and corresponding increase of more mature subsets of either B and non-B cells (false discovery rate, FDR<0.001), suggesting a differentiating effect of GCs. This phenomenon was even more prominent when we performed, for some primary samples (n=4), longer culture up to six days. Moreover, dex-resistant cells showed aberrant activation of prpS6 and pCREB that was blunted by the combination with das resulting in a reversion of the phenotype and killing of the majority of the cells.To determine if this effect was directly related to GCR expression, we compared results between a GCR+ cell line (NALM6) and GCR- cell line (REH). Consistent with the primary samples, the combination of dex with das more effectively killed NALM6 cells yet the REH were unresponsive to either treatment. However, overexpressing GCR in REH cells reversed dex-resistance, also becoming more sensitive to the combined treatment with das. Similar to primary cells, NALM6 and REH GCR+ cells showed a phenotypic change when treated with dex, that will be further investigated to clarify the relationship between the dex-induced phenotype and its apoptotic effect.In summary, these data suggest a dual role of glucocorticoids on early B-cells; mediating both apoptosis and differentiation in relationship to cell cycle status and GCR level. These findings may carry therapeutic implications and suggest cell types vulnerable to BCR signaling inhibition as a therapeutic intervention to overcome GCs resistance. DisclosuresNolan:Akoya Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Sex-specific deficits in biochemical but not behavioral responses to delay fear conditioning in prenatal alcohol exposure mice.

Studies in clinical populations and preclinical models have shown that prenatal alcohol exposure (PAE) is associated with impairments in the acquisition, consolidation and recall of information, with deficits in hippocampal formation-dependent learning and memory being a common finding. The glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and extracellular signal-regulated kinase 2 (ERK2) are key regulators of hippocampal formation development, structure and functioning and, thus, are potential mediators of PAE's effects on this brain region. In the present studies, we employed a well-characterized mouse model of PAE to identify biochemical mechanisms that may underlie activity-dependent learning and memory deficits associated with PAE. Mouse dams consumed either 10% (w/v) ethanol in 0.066% (w/v) saccharin (SAC) or 0.066% (w/v) SAC alone using a limited (4-h) access, drinking-in-the-dark paradigm. Male and female offspring (∼180-days of age) were trained using a delay conditioning procedure and contextual fear responses (freezing behavior) were measured 24 h later. Hippocampal formation tissue and blood were collected from three behavioral groups of animals: 20 min following conditioning (conditioning only group), 20 min following the re-exposure to the context (conditioning plus re-exposure group), and behaviorally naïve (naïve group) mice. Plasma corticosterone levels were measured by enzyme immunoassay. Immunoblotting techniques were used to measure protein levels of the GR, MR, ERK1 and ERK2 in nuclear and membrane fractions prepared from the hippocampal formation. Adult SAC control male and female mice displayed similar levels of contextual fear. However, significant sex differences were observed in freezing exhibited during the conditioning session. Compared to same-sex SAC controls, male and female PAE mice demonstrated context fear deficits While plasma corticosterone concentrations were elevated in PAE males and females relative to their respective SAC naïve controls, plasma corticosterone concentrations in the conditioning only and conditioning plus re-exposure groups were similar in SAC and PAE animals. Relative to the respective naïve group, nuclear GR protein levels were increased in SAC, but not PAE, male hippocampal formation in the conditioning only group. In contrast, no difference was observed between nuclear GR levels in the naïve and conditioning plus re-exposure groups. In females, nuclear GR levels were significantly reduced by PAE but there was no effect of behavioral group or interaction between prenatal treatment and behavioral group. In males, nuclear MR levels were significantly elevated in the SAC conditioning plus re-exposure group compared to SAC naïve mice. In PAE females, nuclear MR levels were elevated in both the conditioning only and conditioning plus re-exposure groups relative to the naïve group. Levels of activated ERK2 (phospho-ERK2 expressed relative to total ERK2) protein were elevated in SAC, but not PAE, males following context re-exposure, and a significant interaction between prenatal exposure group and behavioral group was found. No main effects or interactions of behavioral group and prenatal treatment on nuclear ERK2 were found in female mice. These findings suggest a sex difference in which molecular pathways are activated during fear conditioning in mice. In PAE males, the deficits in contextual fear were associated with the loss of responsiveness of hippocampal formation nuclear GR, MR and ERK2 to signals generated by fear conditioning and context re-exposure. In contrast, the contextual fear deficit in PAE female mice does not appear to be associated with activity-dependent changes in GR and MR levels or ERK2 activation during training or memory recall, although an overall reduction in nuclear GR levels may play a role. These studies add to a growing body of literature demonstrating that, at least partially, different mechanisms underlie learning, memory formation and memory recall in males and females and that these pathways are differentially affected by PAE.

Fibroblast growth factor 2 is necessary for the antidepressant effects of fluoxetine.

Previous research has shown that fibroblast growth factor 2 protein (FGF2) can act as an anxiolytic and anti-depressive agent in rodents. Levels of hippocampal FGF2 and FGF2 receptors are decreased in post-mortem brains of individuals with mood disorders. No changes in FGF2 were noted in the post-mortem brains of individuals with mood disorders that were successfully treated with anti-depressant medication prior to death. Mutations in the FGF2 gene in humans have been shown to predict non-responsiveness to the therapeutic effects of selective serotonin reuptake inhibitors (SSRIs). These findings suggest that FGF2 may potentially be a target of and/or required for the therapeutic effects of antidepressant medications. To test this, we employed a rodent model of depressive behaviour, chronic variable stress (CVS) in conjunction with antidepressant treatment (fluoxetine) in wild-type (WT) and FGF2 knockout mice (FGF2KO) and examined depressive and anxiety behaviors. Results showed that fluoxetine reversed the effects of CVS on depressive and anxiety behaviours in wild-type mice only, suggesting that the FGF2 gene is indeed necessary for the therapeutic effects of fluoxetine. Interestingly, CVS decreased hippocampal FGF2 levels and fluoxetine partially reversed this effect. Because FGF2 has been previously shown to modify HPA activity through hippocampal glucocorticoid receptors (GR), we examined levels of glucocorticoid receptors and found a decrease in GR in response to CVS, with a further decrease in FGF2KO. No effect of fluoxetine on GR was observed in either WT or FGF2KO mice. This suggests that further changes in glucocorticoid receptors are not necessary for the anti-depressant effects of fluoxetine in WT mice, although decreased glucocorticoid receptors in response to FGF2 deletion may preclude the therapeutic actions of fluoxetine in FGF2KO. Whether astroglia, astroglial functions, or HPA changes are the downstream target of FGF2-mediated changes induced by fluoxetine remains to be determined, however, the current study reaffirms the potential of FGF2 as a novel therapeutic target in the treatment of depression and anxiety disorders.

The glucocorticoid receptor and cortisol levels in pediatric septic shock

BackgroundThere is controversy around the prescription of adjunct corticosteroids in patients with fluid-refractory septic shock, and studies provide mixed results, showing benefit, no benefit, and harm. Traditional means for evaluating whether a patient receives corticosteroids relied on anecdotal experience or measurement of serum cortisol production following stimulation. We set out to measure both serum cortisol and the intracellular signaling receptor for cortisol, the glucocorticoid receptor (GCR), in this group of patients.MethodsWe enrolled pediatric patients admitted to the pediatric intensive care unit with a diagnosis of systemic inflammatory response syndrome (SIRS), sepsis, or septic shock as well as healthy controls. We measured serum cortisol concentration and GCR expression by flow cytometry in peripheral blood leukocytes on the day of admission and day 3.ResultsWe enrolled 164 patients for analysis. There was no difference between GCR expression comparing SIRS, sepsis, and septic shock. When all patients with septic shock were compared, those patients with a complicated course, defined as two or more organ failures at day 7 or death by day 28, had lower expression of GCR in all peripheral blood leukocytes. Further analysis suggested that patients with the combination of low GCR and high serum cortisol had higher rates of complicated course (75%) compared with the other three possible combinations of GCR and cortisol levels: low GCR and low cortisol (33%), high GCR and high cortisol (33%), and high GCR and low cortisol (13%; P <0.05).ConclusionsWe show that decreased expression of the GCR correlated with poor outcome from septic shock, particularly in those patients with high serum cortisol. This is consistent with findings from transcriptional studies showing that downregulation of GCR signaling genes portends worse outcome.

Treatment of impaired wounds – Understanding the therapeutic mechanisms of umbilical cord-derived mesenchymal stem cells

Our previous studies suggest that prenatal arsenic exposure (50 ppb) modifies epigenetic control of the programming of the glucocorticoid receptor (GR) signaling system in the developing mouse brain. These deficits may lead to long-lasting consequences, including deficits in learning and memory, increased depressive-like behaviors, and an altered set-point of GR feedback throughout life. To understand the arsenic-induced changes within the GR system, we assessed the impact of in utero arsenic exposure on the levels of the GR and growth arrest-specific-5 (Gas5), a noncoding RNA, across a key gestational period for GR programming (gestational days, GD 14–18) in mice. Gas5 contains a glucocorticoid response element (GRE)-like sequence that binds the GR, thereby decreasing GR-GRE-dependent gene transcription and potentially altering GR programming. Prenatal arsenic exposure resulted in sex-dependent and age-dependent shifts in the levels of GR and Gas5 expression in fetal telencephalon. Nuclear GR levels were reduced in males, but unchanged in females, at all gestational time points tested. Total cellular Gas5 levels were lower in arsenic-exposed males with no changes seen in arsenic-exposed females at GD16 and 18. An increase in total cellular Gas-5 along with increased nuclear levels in GD14 arsenic-exposed females, suggests a differential regulation of cellular compartmentalization of Gas5. RIP assays revealed reduced Gas5 associated with the GR on GD14 in the nuclear fraction prepared from arsenic-exposed males and females. This decrease in levels of GR-Gas5 binding continued only in the females at GD18. Thus, nuclear GR signaling potential is decreased in prenatal arsenic-exposed males, while it is increased or maintained at levels approaching normal in prenatal arsenic-exposed females. These findings suggest that females, but not males, exposed to arsenic are able to regulate the levels of nuclear free GR by altering Gas5 levels, thereby keeping GR nuclear signaling closer to control (unexposed) levels.

Repression of the Glucocorticoid Receptor Aggravates Acute Ischemic Brain Injuries in Adult Mice.

Strokes are one of the leading causes of mortality and chronic morbidity in the world, yet with only limited successful interventions available at present. Our previous studies revealed the potential role of the glucocorticoid receptor (GR) in the pathogenesis of neonatal hypoxic-ischemic encephalopathy (HIE). In the present study, we investigate the effect of GR knockdown on acute ischemic brain injuries in a model of focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in adult male CD1 mice. GR siRNAs and the negative control were administered via intracerebroventricular (i.c.v.) injection 48 h prior to MCAO. The cerebral infarction volume and neurobehavioral deficits were determined 48 h after MCAO. RT-qPCR was employed to assess the inflammation-related gene expression profiles in the brain before and after MCAO. Western Blotting was used to evaluate the expression levels of GR, the mineralocorticoid receptor (MR) and the brain-derived neurotrophic factor/tropomyosin receptor kinase B (BDNF/TrkB) signaling. The siRNAs treatment decreased GR, but not MR, protein expression, and significantly enhanced expression levels of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) in the brain. Of interest, GR knockdown suppressed BDNF/TrkB signaling in adult mice brains. Importantly, GR siRNA pretreatment significantly increased the infarction size and exacerbated the neurobehavioral deficits induced by MCAO in comparison to the control group. Thus, the present study demonstrates the important role of GR in the regulation of the inflammatory responses and neurotrophic BDNF/TrkB signaling pathway in acute ischemic brain injuries in adult mice, revealing a new insight into the pathogenesis and therapeutic potential in acute ischemic strokes.