Glucocorticoid Receptor Antagonist RU486 Research Articles

Glucocorticoids alleviate particulate matter-induced COX-2 expression and mitochondrial dysfunction through the Bcl-2/GR complex in A549 cells

Exposure to particulate matter (PM) causes mitochondrial dysfunction and lung inflammation. The cyclooxygenase-2 (COX-2) pathway is important for inflammation and mitochondrial function. However, the mechanisms by which glucocorticoid receptors (GRs) suppress COX-2 expression during PM exposure have not been elucidated yet. Hence, we examined the mechanisms underlying the dexamethasone-mediated suppression of the PM-induced COX-2/prostaglandin E2 (PGE2) pathway in A549 cells. The PM-induced increase in COX-2 protein, mRNA, and promoter activity was suppressed by glucocorticoids; this effect of glucocorticoids was antagonized by the GR antagonist RU486. COX-2 induction was correlated with the ability of PM to increase reactive oxygen species (ROS) levels. Consistent with this, antioxidant treatment significantly abolished COX-2 induction, suggesting that ROS is involved in PM-mediated COX-2 induction. We also observed a low mitochondrial membrane potential in PM-treated A549 cells, which was reversed by dexamethasone. Moreover, glucocorticoids significantly enhanced Bcl-2/GR complex formation in PM-treated A549 cells. Glucocorticoids regulate the PM-exposed induction of COX-2 expression and mitochondrial dysfunction and increase the interaction between GR and Bcl-2. These findings suggest that the COX-2/PGE2 pathway and the interaction between GR and Bcl-2 are potential key therapeutic targets for the suppression of inflammation under PM exposure.

Voluntary wheel exercise ameliorates cognitive impairment, hippocampal neurodegeneration and microglial abnormalities preceded by demyelination in a male mouse model of noise-induced hearing loss

Acquired peripheral hearing loss (APHL) in midlife has been identified as the greatest modifiable risk factor for dementia; however, the pathophysiological neural mechanisms linking APHL with an increased risk of dementia remain to be elucidated. Here, in an adult male mouse model of noise-induced hearing loss (NIHL), one of the most common forms of APHL, we demonstrated accelerated age-related cognitive decline and hippocampal neurodegeneration during a 6-month follow-up period, accompanied by progressive hippocampal microglial aberrations preceded by immediate-onset transient elevation in serum glucocorticoids and delayed-onset sustained myelin disruption in the hippocampus. Pretreatment with the glucocorticoid receptor antagonist RU486 before stressful noise exposure partially mitigated the early activation of hippocampal microglia, which were present at 7 days post noise exposure (7DPN), but had no impact on later microglial aberrations, hippocampal neurodegeneration, or cognitive decline exhibited at 1 month post noise exposure (1MPN). One month of voluntary wheel exercise following noise exposure barely affected either the hearing threshold shift or hippocampal myelin changes but effectively countered cognitive impairment and the decline in hippocampal neurogenesis in NIHL mice at 1MPN, paralleled by the normalization of microglial morphology, which coincided with a reduction in microglial myelin inclusions and a restoration of microglial hypoxia-inducible factor-1α (HIF1α) expression. Our results indicated that accelerated cognitive deterioration and hippocampal neuroplastic decline following NIHL are most likely driven by the maladaptive response of hippocampal microglia to myelin damage secondary to hearing loss, and we also demonstrated the potential of voluntary physical exercise as a promising and cost-effective strategy to alleviate the detrimental impact of APHL on cognitive function and thus curtail the high and continuously increasing global burden of dementia. Furthermore, the findings of the present study highlight the contribution of myelin debris overload to microglial malfunction and identify the microglial HIF1α-related pathway as an attractive candidate for future comprehensive investigation to obtain a more definitive picture of the underlying mechanisms linking APHL and dementia.

Systemic corticosterone administration impairs the late fear memory reconsolidation via basolateral amygdala glucocorticoid receptors: Dependence on the time window and memory age

Glucocorticoid receptors (GRs) of the basolateral amygdala (BLA) play an important role in memory reconsolidation. The present study investigated the role of the BLA GRs in the late reconsolidation of fear memory using an inhibitory avoidance (IA) task in male Wistar rats. Stainless steel cannulae were implanted bilaterally into the BLA of the rats. After 7 days of recovery, the animals were trained in a one-trial IA task (1 mA, 3 s). In Experiment One, 48 h after the training session, the animals received 3 systemic doses of corticosterone (CORT; 1, 3, or 10 mg/kg, i.p.) followed by an intra-BLA microinjection of the vehicle (0.3 µl/side) at different time points (immediately, 12, or 24 h) after memory reactivation. Memory reactivation was performed by returning the animals to the light compartment while the sliding door was open. No shock was delivered during memory reactivation. CORT (10 mg/kg) injection 12 h after memory reactivation most effectively impaired the late memory reconsolidation (LMR). In the second part of Experiment One, immediately, 12, or 24 h after memory reactivation, GR antagonist RU38486 (RU; 1 ng/0.3 µl/side) was injected into BLA following a systemic injection of CORT (10 mg/kg) to examine whether it would block the CORT effect. RU inhibited the impairing effects of CORT on LMR. In Experiment Two, the animals received CORT (10 mg/kg) with time windows immediately, 3, 6, 12, and 24 h after memory reactivation. Again, CORT (10 mg/kg) injection 12 h after memory reactivation impaired LMR. Memory reactivation was performed in the third Experiment, 7, 14, 28, or 56 days after the training session. Injection of CORT (10 mg/kg) 12 h later had no significant effect on the LMR. The impairing effect of CORT was seen only in 2-day-old but not 7, 14, 28, and 56-day-old memories. GRs located in BLA seem to play an important role in the LMR of young memory, as with increasing the age of memories, they become less sensitive to manipulation.

Glucocorticoids regulate thrombopoiesis by remodeling the megakaryocyte transcriptome

BackgroundGlucocorticoids are widely known for their immunomodulatory action. Their synthetic analogs are used to treat several autoimmune diseases, including immune thrombocytopenia. However, their efficacy and mechanisms of action in immune thrombocytopenia are not fully understood. ObjectivesTo investigate the mechanism of glucocorticoid actions on platelet production. MethodsThe actions of glucocorticoids on platelet production were studied combining in vivo, ex vivo and in vitro approaches. ResultsDexamethasone reduced bleeding in mice and rapidly increased circulating young platelet counts. In vitro glucocorticoid treatment stimulated proplatelet formation by megakaryocytes and platelet-like particle release. This effect was blocked by glucocorticoid receptor antagonist RU486, indicating a glucocorticoid receptor–dependent mechanism. Genome-wide analysis revealed that dexamethasone regulates the expression of >1000 genes related to numerous cellular functions, including predominant cytoplasm and cytoskeleton reorganization. Dexamethasone and other glucocorticoids induced the expression of Gda (the gene encoding guanine deaminase), which has been reported to have a role in dendrite development. Inhibition of guanine deaminase enzymatic activity blocked dexamethasone stimulation of proplatelet formation, implicating a critical role for this enzyme in glucocorticoid-mediated platelet production. ConclusionOur findings identify glucocorticoids as new regulators of thrombopoiesis.

Corticosterone injection into the dorsal and ventral hippocampus impairs fear memory reconsolidation in a time-dependent manner in rats

Reconsolidation is an active process induced following the reactivation of previously consolidated memories. Recent studies suggest brain corticosteroid receptors may participate in the modulation of fear memory reconsolidation. Glucocorticoid receptors (GRs), with 10-fold lower affinity than mineralocorticoid receptors (MRs), are mainly occupied during the peak of the circadian rhythm, and after stress, so they probably have a more critical role than MRs in memory phases during stressful situations. This study investigated the role of dorsal and ventral hippocampal (DH and VH) GRs and MRs on fear memory reconsolidation in rats. Male Wistar rats with surgically implanted bilaterally cannulae at the DH and VH were trained and tested in an inhibitory avoidance task. The animals received bilateral microinjections of vehicle (0.3 µl/side), corticosterone (3 ng/0.3 µl/side), the GRs antagonist RU38486 (3 ng/0.3 µl/side), or the MRs antagonist spironolactone (3 ng/0.3 µl/side) immediately after memory reactivation. Moreover, drugs were injected into VH 90 min after memory reactivation. Memory tests were performed 2, 9, 11, and 13 days after memory reactivation. Results indicated that injection of corticosterone into the DH but not VH immediately after memory reactivation significantly impaired fear memory reconsolidation. Moreover, corticosterone injection into VH 90 min after memory reactivation impaired fear memory reconsolidation. RU38486, but not spironolactone reversed these effects. These findings indicate that corticosterone injection into the DH and VH via GRs activation impairs the reconsolidation of fear memory in a time-dependent manner.

Gastroprotective effect of electrical spinal cord stimulation may be mediated by glucocorticoids produced in response to this stimulation

We demonstrated previously that stress-induced glucocorticoids are gastroprotective hormones but not ulcerogenic ones (Filaretova et al., 2016). Recently electrical spinal cord stimulation began to be used for both experimental studies of motor functions regulation and rehabilitation of motor functions in patients with spinal cord injury (Gerasimenko et al., 2021; Moshonkina et al., 2016). The spinal cord stimulation directed to the activation of spinal locomotor related networks affected visceral systems as well. Recently in our teamwork we have shown that the electrical spinal cord stimulation can cause an increase in blood glucocorticoid level accompanied by a gastroprotective effect (Filaretova et al., 2022). The aim of the present work was to verify the hypothesis that the gastroprotective effect of the electrical spinal cord stimulation may be mediated by glucocorticoids produced in response to this stimulation. Gastric injury was induced in anesthetized rats by prolonged gastric ischemia/reperfusion (I/R, 30 min occlusion of celiac artery followed by 3 h of reperfusion) or indomethacin administration (IM, 35 mg/kg, sc). For spinal stimulation, stainless steel wire electrodes were fixed in fasciae between T11-12 and L1–L2 vertebrae of anesthetized animals. Motor evoked responses in hindlimbs muscles to spinal stimulation were recorded. Spinal stimulation at 30 Hz was carried out with a subthreshold current for inducing muscle contractions (80% of the threshold). Sham-stimulated (control) rats were subjected to the same manipulations except for the stimulation itself. To test the participation of glucocorticoids in the gastroprotective effect of the stimulation, two approaches were used: pretreatment by the inhibitor of glucocorticoid synthesis, metyrapone (30 mg/kg, i.p.) and the antagonist of glucocorticoid receptors RU-38486 (20 mg/kg, i.p.). The concentration of corticosterone in blood plasma was determined using commercial ELISA kits. In control rats the spinal cord stimulation resulted in an increase of blood corticosterone level 1 h later. The spinal stimulation, applied 1 h before the onset of ulcerogenic stimuli, also significantly attenuated gastric erosion formation induced by I/R and IM as well. Metyrapone injected shortly before the stimulation caused a fast inhibition of corticosterone response and reversed the gastroprotective effect stimulation. The gastroprotective effect of the stimulation was also prevented by the pretreatment rats with glucocorticoid receptor antagonist RU-38486. The obtained results indicate on contribution of glucocorticoids to gastroprotective effect of spinal cord stimulation. In conclusion, the findings support the hypothesis that the gastroprotective effect of the electrical spinal cord stimulation may be mediated by glucocorticoids produced in response to this stimulation. The study was supported by the Ministry of Education and Science of the Russian Federation (agreement No. 075-15-2020-921 for the creation and development of the world-class scientific center “Pavlov Center “Integrative Physiology - to medicine, high-tech healthcare and technologies of stress resistance”). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Stress Induces Prolonged Pain Recovery After Surgery: Involvement of Glucocorticoid-Related Pathway.

Increasing evidence suggests that chronic stress increases pain sensitivity and exacerbates existing pain. However, whether and how chronic unpredictable stress (CUS) affects surgical pain is unclear. A postsurgical pain model was performed by longitudinal incision from 0.3 cm of the proximal edge of the heel toward the toes. The skin was sutured, and the wound site was covered. Sham surgery groups underwent the same procedure without an incision. The short-term CUS procedure was conducted by exposure of mice to 2 different stressors each day for 7 days. The behavior tests were conducted between 9:00 am and 4:00 pm. Mice were killed on day 19, and the mouse bilateral L4/5 dorsal root ganglia, spinal cord, anterior cingulate and insular cortex, and amygdala were collected for immunoblot analyses. Presurgical exposure of mice to CUS every day for 1-7 days showed significant depression-like behavior as evidenced by reduced sucrose preference in the sucrose consumption test and prolonged immobility time in the forced swimming task. This short-term CUS procedure did not affect the basal nociceptive response to mechanical and cold stimuli in the Von Frey and acetone-induced allodynia tests, but it delayed pain recovery after surgery, as indicated by the prolonged hypersensitivity in mechanical and cold stimuli by 12 days. The subsequent studies demonstrated that this CUS caused an increase in adrenal gland index. The abnormalities in pain recovery and adrenal gland index after surgery were reversed by a glucocorticoid receptor (GR) antagonist RU38486. Moreover, the prolonged pain recovery after surgery induced by CUS seemed to involve an increase in GR expression and decreases in cyclic adenosine monophosphate, phosphorylated cAMP response element binding protein, and brain-derived neurotrophic factor levels in emotion-related brain regions, such as anterior cingulate and insular cortex, amygdala, dorsal horn, and dorsal root ganglion. This finding indicates that stress-induced GR change may result in dysfunction of GR-related neuroprotective pathway.

11β-Hydroxysteroid dehydrogenase type 1 amplifies inflammation in LPS-induced THP-1 cells.

The role of glucocorticoids as anti-inflammatory and immune-stimulatory drugs has been widely reported. However, the role of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which catalyzes the conversion of inactive cortisone into active cortisol, in inflammation remains unclear. This study aimed to examine the mechanism of actions of 11β-HSD1 in lipopolysaccharide (LPS)-induced THP-1 cells. The gene expression of 11β-HSD1 and pro-inflammatory cytokines was detected via RT-PCR. The protein expression of IL-1β in cell supernatants was detected via ELISA. Oxidative stress and mitochondrial membrane potential were assessed using a reactive oxygen species (ROS) kit and a mitochondrial membrane potential (MMP) kit, respectively. The expression of Nuclear Factor- Kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) was detected via western blotting. Elevated levels of 11β-HSD1 contributed to the expression of inflammatory cytokines, whereas BVT.2733, a selective 11β-HSD1 inhibitor, ameliorated inflammatory responses, ROS, and mitochondrial damage in LPS-stimulated THP-1 cells. Furthermore, cortisone and cortisol, which are the substrate and product of 11β-HSD1, respectively, showed biphasic responses and induced the expression of pro-inflammatory cytokines at a low concentration in both LPS-stimulated or untreated THP-1 cells. The enhanced inflammation was attenuated by co-treatment with BVT.2733 and the glucocorticoid receptor (GR) antagonist RU486, but not in those treated with the mineralocorticoid receptor (MR) antagonist spironolactone. Overall, the results indicate that 11β-HSD1 amplifies inflammatory responses by activating the NF-κB and MAPK signaling pathways. Inhibition of 11β-HSD1 may serve as a potential therapeutic target against the excessive activation of inflammation.

β-sitosterol targets glucocorticoid receptor to reduce airway inflammation and remodeling in allergic asthma

IntroductionIn most asthma patients, symptoms are controlled by treatment with glucocorticoid, but long-term or high-dose use can produce adverse effects. Therefore, it is crucial to find new therapeutic strategies. β-sitosterol could suppress type Ⅱ inflammation in ovalbumin (OVA)-induced mice, but its mechanisms have remained unclear. MethodsA binding activity of β-sitosterol with glucocorticoid receptor (GR) was analyzed by molecular docking. Human bronchial epithelial cells (BEAS-2B) and human bronchial smooth muscle cells (HBSMC) were treated with different concentrations (0, 1, 5, 10, 20, and 50 μg/mL) of β-sitosterol for suitable concentration selection. In transforming growth factor (TGF)-β1 treated BEAS-2B and HBSMC, cells were treated with 20 μg/mL β-sitosterol or dexamethasone (Dex) to analyze its possible mechanism. In OVA-induced mice, 2.5 mg/kg β-sitosterol or Dex administration was performed to analyze the therapeutic mechanism of β-sitosterol. A GR antagonist RU486 was used to confirm the mechanism of β-sitosterol in the treatment of asthma. ResultsA good binding of β-sitosterol to GR (score = −8.2 kcal/mol) was found, and the GR expression was upregulated with β-sitosterol dose increase in BEAS-2B and HBSMC. Interleukin (IL)-25 and IL-33 secretion was significantly decreased by β-sitosterol in the TGF-β1-induced BEAS-2B, and the levels of collagen 1A and α-smooth muscle actin (SMA) were reduced in the TGF-β1-induced HBSMC. In the OVA-challenged mice, β-sitosterol treatment improved airway inflammation and remodeling through suppressing type Ⅱ immune response and collagen deposition. The therapeutic effects of β-sitosterol were similar to Dex treatment in vitro and in vivo. RU486 treatment clearly hampered the therapeutic effects of β-sitosterol in the TGF-β1-induced cells and OVA-induced mice. ConclusionThis study identified that β-sitosterol binds GR to perform its functions in asthma treatment. β-sitosterol represent a potential therapeutic drug for allergic asthma.

ODP651 Abnormal Primary Ciliogenesis in the Developing Mouse Kidney of Glucocorticoid Receptor-Deficient Mice

Abstract Glucocorticoid (GC) hormones have well characterised regulatory roles for the development and maturation of several fetal organs, however, specific developmental roles of GCs in the fetal kidney have not been described (1). We have explored both the expression and localisation of the glucocorticoid receptor (GR) within the developing fetal mouse kidney during organogenesis and the effect on the transcriptome of eliminating GR expression in gene-targeted GR-deficient mice. Staining for the GR by immunohistochemistry at E14.5, E16.5 and E18.5 demonstrated widespread expression of the GR in the renal tubule and the interstitial renal mesenchyme, with strong staining in collecting ducts and increasing expression in mesenchyme up to E18.5. Loss of GR expression in GR-null mice had a profound effect on the renal transcriptome with altered expression (at an FDR of 0.5 and fold change >2. 0) for 454 genes in the GR-null mouse kidney at E18.5, with a greater number of genes showing significantly reduced mRNA levels (305) than increased mRNA levels (99) relative to wild-type controls. Interestingly these deregulated genes included key primary cilia-associated genes, such as centrosomal protein 290 (Cep290), with the largest fold change of -3.6, Cep97, Ccp110, and the primary cilia-associated kinesin Kif3A. In addition, two renal tubule markers, Kidney androgen regulated protein (Kap) (proximal tubule marker) and S100 calcium binding protein G (S100g) (distal tubule marker) were also strongly downregulated with fold changes of -8.61 and -2.5 respectively. Analysis of primary cilia in sections of E18.5 kidney using multicolour confocal microscopy demonstrated that primary cilium length was significantly decreased and abnormally projected from kidney proximal tubule cells in E18.5 GR-null mice (5.23 + 0.12, µm + SEM) compared with wild type controls (6.27+ 0.15, µm + SEM). Finally, in cultured IMCD3 mouse collecting duct cells dexamethasone treatment was shown to increase the length of primary cilia compared to vehicle-treated control cells (vehicle 2.49 + 0. 03, µm + SEM vs dexamethasone 3.17 + 0. 05 µm + SEM), an effect that could be blocked by pre-treatment with the GR antagonist RU486. Taken together these results indicate that GC signalling mediated via the GR contributes either directly or indirectly to the normal formation of primary cilia in the proximal tubule and is therefore required for normal kidney development. This also suggests that abnormalities in GR-regulation of ciliogenesis may underpin or contribute to causes of human ciliopathy conditions. 1. Cole TJ, Short KL, Hooper SB (2019) The Science of Steroids. Sem. Fetal and Neonatal Med. 24: 170-175. Presentation: No date and time listed

Loss of α7nAChR enhances endothelial‐to‐mesenchymal transition after myocardial infarction via NF-κB activation

The myocardial fibrosis in response to myocardial infarction (MI) is closely related to the dysbalance of endothelial-to-mesenchymal transition (EndMT). Although numerous reports indicate that α7 nicotinic acetylcholine receptor (α7nAChR) activates the cholinergic anti-inflammatory pathway (CAP) to regulate the magnitude of inflammatory responses, the role of α7nAChR in myocardial fibrosis, as well as the underlying mechanisms, have not been elucidated. In this study, we evaluated cardiac function, fibrosis, and EndMT signaling using a mouse model of MI and interleukin (IL)-1β-induced human cardiac microvascular endothelial cells (HCMECs). In vivo, α7nAChR deletion increased cardiac dysfunction, exacerbated the cardiac inflammatory response, and NF-κB activation, and enhanced EndMT, as shown by higher expression levels of fibroblast markers (FSP-1, α-SMA, collagen I, Snail) and decreased levels of the FGFR1, glucocorticoid receptor (GR) and endothelial marker (CD31) compared to wild-type mice. In vitro, the pharmacological activation of α7nAChR with PNU282987 significantly inhibited IL-1β-induced EndMT, as shown by a reduced transition to the fibroblast-like phenotype and the expression of fibrotic markers. Moreover, the IL-1β-mediated activation of NF-κB pathway was suppressed by PNU282987. This anti-EndMT effect of α7nAChR was associated with regulation of Snail. Furthermore, Western blot analysis further revealed that the GR antagonist RU38486 could partially counteract the effect of PNU282987 on NF-κB expression. In conclusion, our results show that α7nAChR is involved in cardiac fibrosis by inhibiting EndMT, providing a novel approach to the treatment of MI.

Application of a pharmacological transcriptome filter identifies a shortlist of mouse glucocorticoid receptor target genes associated with memory consolidation

Glucocorticoids regulate memory consolidation, facilitating long-term storage of relevant information to adequately respond to future stressors in similar conditions. This effect of glucocorticoids is well-established and is observed in multiple types of behaviour that depend on various brain regions. By and large, higher glucocorticoid levels strengthen event-related memory, while inhibition of glucocorticoid signalling impairs consolidation. The mechanism underlying this glucocorticoid effect remains unclear, but it likely involves the transcriptional effects of the glucocorticoid receptor (GR). We here used a powerful paradigm to investigate the transcriptional effects of GR in the dorsal hippocampus of mice after training in an auditory fear conditioning task, aiming to identify a shortlist of GR target genes associated to memory consolidation. Therefore, we utilized in an explorative study the properties of selective GR modulators (CORT108297 and CORT118335), alongside the endogenous agonist corticosterone and the classical GR antagonist RU486, to pinpoint GR-dependent transcriptional changes. First, we confirmed that glucocorticoids can modulate memory strength via GR activation. Subsequently, by assessing the specific effects of the available GR-ligands on memory strength, we established a pharmacological filter which we imposed on the hippocampal transcriptome data. This identified a manageable shortlist of eight genes by which glucocorticoids may modulate memory consolidation, warranting in-depth follow-up. Overall, we showcase the strength of the concept of pharmacological transcriptome filtering, which can be readily applied to other research topics with an established role of glucocorticoids.

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

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

Characteristics of the adipocytes differentiated by a glucocorticoid receptor antagonist RU486

Characteristics of the adipocytes differentiated by a glucocorticoid receptor antagonist RU486

Non-Invasive Remote Ischemic Preconditioning May Protect the Gastric Mucosa Against Ischemia-Reperfusion-Induced Injury Through Involvement of Glucocorticoids.

Remote ischemic preconditioning (RIPC) is one of the most effective approaches to attenuate tissue injury caused by severe ischemia-reperfusion (I/R). Experimental studies have demonstrated that RIPC is capable of producing a protective effect not only on heart, but also on brain, lungs, kidneys, liver, intestine, and stomach. We previously demonstrated that glucocorticoids participate in protective effect of local gastric ischemic preconditioning against I/R-induced gastric injury. In the present study we investigated whether RIPC may protect the gastric mucosa against I/R-induced injury through involvement of glucocorticoids. Anesthetized fasted Sprague Dawley male rats were exposed to prolonged gastric I/R (30 min occlusion of celiac artery followed by 3 h of reperfusion) alone or with preliminary brief RIPC (10 min non-invasive occlusion of right hind limb blood flow followed by reperfusion for 30 min). First, we investigated the effect of RIPC on I/R-induced injury by itself. Then to study the role of glucocorticoids similar experiments were carried out: 1) in rats pretreated with the inhibitor of glucocorticoid synthesis, metyrapone (30 mg/kg, i.p), and in control animals; 2) in adrenalectomized rats without or with corticosterone replacement (4 mg/kg, s.c.) and in sham-operated animals; 3) in rats pretreated with glucocorticoid receptor antagonist RU-38486 (20 mg/kg, s.c.) and in control animals. I/R induced corticosterone rise and resulted in the gastric erosion formation. RIPC significantly reduced the erosion area in control animals. Metyrapone injected shortly before RIPC caused a decrease in plasma corticosterone levels and prevented the gastroprotective effect of RIPC and, moreover, further aggravated the deleterious effect of I/R. Adrenalectomy performed 1 week before experiment created long-lasting corticosterone deficiency and had no effect on the gastroprotective effect of RIPC. Nevertheless, corticosterone replacement which mimics the corticosterone rise, similar to RIPS, significantly reduced erosion areas of gastric mucosa in adrenalectomized rats supporting the role of glucocorticoids in gastroprotection. RU-38486, which occupied glucocorticoid receptors, similar to metyrapone prevented the gastroprotective effect of RIPC and, moreover, further aggravated the deleterious effect of I/R. The results of the present study demonstrate for the first time that RIPC may protect the gastric mucosa against I/R-induced injury through involvement of glucocorticoids.

Stress hormones promote DNA damage in human oral keratinocytes

Chronic stress increases the systemic levels of stress hormones norepinephrine and cortisol. As well as tobacco-specific carcinogen NNK (4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone), they can induce expressive DNA damage contributing to the cancer development. However, it is unknown whether stress hormones have genotoxic effects in oral keratinocytes. This study investigated the effects of stress hormones on DNA damage in a human oral keratinocyte cell line (NOK-SI). NOK-SI cells stimulated with norepinephrine or cortisol showed higher DNA damage compared to untreated cells. Norepinephrine-induced DNA damage was reversed by pre-treatment with beta-adrenergic blocker propranolol. Cells treated with NNK combined to norepinephrine displayed reduced levels of caspases 3 and 7. Cortisol also reduced the activity of pro-apoptotic enzymes. NNK or norepinephrine promoted single-strand breaks and alkali-label side breaks in the DNA of NOK-SI cells. Pre-treatment of cells with propranolol abolished these effects. Carcinogen NNK in the presence or absence of cortisol also induced DNA damage of these cells. The genotoxic effects of cortisol alone and hormone combined with NNK were blocked partially and totally, respectively, by the glucocorticoid receptor antagonist RU486. DNA damage promoted by NNK or cortisol and carcinogen combined to the hormone led to intracellular γH2AX accumulation. The effects caused by NNK and cortisol were reversed by propranolol and glucocorticoid receptor antagonist RU486, respectively. Propranolol inhibited the oxidation of basis induced by NNK in the presence of DNA-formamidopyrimidine glycosylase. DNA breaks induced by norepinephrine in the presence or absence of NNK resulted in higher 8OHdG cellular levels. This effect was also induced through beta-adrenergic receptors. Together, these findings indicate that stress hormones induce DNA damage of oral keratinocytes and could contribute to oral carcinogenesis.

Age-Specific Modulation of Prefrontal Cortex LTP by Glucocorticoid Receptors Following Brief Exposure to HFD

The corticolimbic circuits in general and the medial prefrontal cortex in particular, undergo maturation during juvenility. It is thus expected that environmental challenges in forms of obesogenic diet can exert different effects in juvenile animals compared to adults. Further, the relationship between glucocorticoids and obesity has also been demonstrated in several studies. As a result, glucocorticoid receptor (GR) antagonists are currently being tested as potential anti-obesity agents. In the present study, we examined the effects of short-term exposure to high-fat diet (HFD) on prefrontal long-term potentiation (LTP) in both juvenile and adult rats, and the role of glucocorticoid receptors (GRs) in modulating these effects. We found HFD impaired prefrontal LTP in both juveniles and adults, but the effects of GR modulation were age- and diet-dependent. Specifically, GR antagonist RU-486 reversed the impairment of LTP in juvenile animals following HFD, and had no effect on control-diet animals. In adult animals, RU-486 has no effect on HFD-impaired LTP, but abolished LTP in control-diet animals. Furthermore, impairments in the prefrontal LTP following HFD are involved with an increase in the mPFC GR levels only in the juveniles. Further, we found that in vivo application of GR agonists into adult mPFC rescued HFD-induced impairment in LTP, suggesting that these receptors might represent strategic therapeutic targets to potentially combat obesity and metabolic related disorder.

Peripheral corticotropin-releasing hormone may protect the gastric musosa against indometacin-induced injury through involvement of glucocorticoids.

The hypothalamic-pituitary-adrenocortical (HPA) system is a key hormonal branch of the brain-gut axis in stress and corticotropin-releasing hormone (CRH) is a principal stimulator of the HPA system. According to our finding activation of the HPA system has gastroprotective role in stress and CRH may protect the gastric mucosa against stress-induced injury through involvement of glucocorticoids. To extend this idea to indomethacin-induced gastric injury in the present work we studied whether CRH may protect the gastric mucosa against ulcerogenic action of indomethacin (IM) through involvement of glucocorticoids. CRH administration (1.25 μg/kg and 2.5 μg/kg, i.p.) markedly, dose-dependently, increased plasma corticosterone level and significantly, dose-dependently, suppressed the occurrence of gastric erosion induced by IM (35 mg/kg, s.c.) in conscious rats. To estimate the role of glucocorticoids in CRH-induced gastroprotection, the effect of CRH (1.25 μg/kg) on the IM-induced gastric erosion was studied after acute reduction of corticosterone release by metyrapone (30 mg/kg, i.p., 30 min before CRH administration) or by CRH receptor type 1 antagonist NBI 27914 (10 mg/kg, i.p., 15 min before CRH administration) and also after occupation of glucocorticoid receptors by their antagonist RU-38486 (20 mg/kg, i.p., 2 h before CRH administration). The effects were compared with those in control rats without acute reduction of corticosterone release or occupation of glucocorticoid receptors. Both metyrapone and NBI 27914 injected shortly before CRH administration caused an inhibition of CRH-induced corticosterone response and prevented protective effect of CRH on the gastric mucosa against the IM-induced erosion. The gastroprotective effect of CRH was also eliminated by the pretreatment with glucocorticoid receptor antagonist RU-38486. The results obtained suggest that exogenous CRH may protect the gastric mucosa against IM-induced gastric injury through involvement of glucocorticoids.

Steroid sulfatase in the mouse NIH-3T3 fibroblast cell line: Characterization, and downregulation by glucocorticoids

Steroid hormones often circulate in the blood as inactive sulfated forms, such as estrone sulfate and dehydroepiandrosterone sulfate. The enzyme steroid sulfatase (STS) converts these steroids into active forms, mainly estrogens, in peripheral tissues. We have previously characterized STS activity in human and mouse breast and bone tissues, and we have shown that STS can provide estrogens to these tissues from circulating sulfated precursors. This study was designed to characterize STS activity in a mouse fibroblast cell line (NIH-3T3). Using a radioactive estrone sulfate (E1S) conversion assay, we detected STS activity in cultured NIH-3T3 cells. This activity was blocked by the STS inhibitors EMATE and STX-64, indicating authentic STS activity. We also found that microsomes prepared from NIH-3T3 cells had relatively high STS activity and that cytosols had low activity, consistent with the known distribution of this enzyme to the endoplasmic reticulum. Michaelis-Menten analysis of the NIH-3T3 microsomes indicated a Km of 10.9 µM using E1S as substrate. Primary fibroblasts prepared from mouse ears and tails also had measurable STS activity, as indicated by 3H-E1S conversion assay, further supporting the conclusion that fibroblasts possess STS. Furthermore, Western blotting confirmed the presence of immunoreactive STS in NIH-3T3 microsomes. With regard to regulation, treatments of cultured NIH-3T3 cells revealed that cortisol and the synthetic glucocorticoids dexamethasone and prednisolone decreased STS activity, as we have found for cell lines from other tissues. The effect of cortisol was seen at both 10 µM and 1.0 µM but not at 0.1 µM. Western blotting also indicated a decrease in STS immunoreactivity in cortisol-treated microsomes. The reduction in STS activity by dexamethasone in whole cells was reversed by the glucocorticoid receptor antagonist RU-486, indicating that glucocorticoid downregulation of STS activity is receptor mediated. An inhibition assay on NIH-3T3 microsomes revealed that STS activity was inhibited significantly by 10 µM estradiol-17β, a known substrate inhibitor of E1S for STS, but not by 10 µM cortisol. This is consistent with the idea that cortisol inhibits STS in NIH-3T3 cells through a regulatory mechanism rather than by substrate inhibition. Our results could have important implications regarding local estrogen production by STS in fibroblasts, which are the most common connective tissue cells in the body, and on possible regulation of local estrogen levels by cortisol.

Corticosterone in the dorsolateral striatum facilitates the extinction of stimulus-response memory

Glucocorticoid hormones are crucially involved in modulating mnemonic processing of stressful or emotionally arousing experiences. They are known to enhance the consolidation of new memories, including those that extinguish older memories. In this study, we investigated whether glucocorticoids facilitate the extinction of a striatum-dependent, and behaviorally more rigid, stimulus-response memory. For this, male rats were initially trained for six days on a stimulus-response task in a T-maze to obtain a reward after making an egocentric right-turn body response, regardless of the starting position in this maze. This training phase was followed by three extinction sessions in which right-turn body responses were not reinforced. Corticosterone administration into the dorsolateral region of the striatum after the first extinction session dose-dependently enhanced the consolidation of extinction memory: Rats administered the higher dose of corticosterone (30 ng), but not lower doses (5 or 10 ng), exhibited significantly fewer right-turn body responses and had longer latencies compared to vehicle-treated animals on the second and third extinction sessions. Co-administration of the glucocorticoid receptor antagonist RU 486 (10 ng) prevented the corticosterone effect, indicating that glucocorticoids enhance the extinction of stimulus-response memory via activation of the glucocorticoid receptor. Corticosterone administration into the dorsomedial striatum did not affect extinction memory. These findings indicate that stress-response mechanisms involving corticosterone actions in the dorsolateral striatum facilitate the extinction of stimulus-response memory that might allow for the development of an opportune behavioral strategy.