Roles of GR Isoforms and Hsp90-binding Immunophilins in the Modulation of Glucocorticoid Biological Responses.

Glucocorticoid steroids play cardinal roles during the life span of an individual, modulating almost all aspects of the physiology, including the metabolism of carbohydrates, lipids and amino acids, as well as the immune response, neurological biology, stress adaptation, apoptosis, cell division, cell fate, inflammatory responses, etc. Glucocorticoids exert their biological effects by activation of the glucocorticoid receptor (GR), a bona fide ligand-activated transcription factor belonging to the nuclear receptor superfamily. The GR is expressed in virtually all cells of the human body showing isoformic versions and also transcription variants. GR forms oligomeric heterocomplexes that include the 90-kDa heat-shock protein (Hsp90) as an essential hub of the chaperone oligomer. The nature of chaperones associated with this heterocomplex is responsible for the modulation of the subcellular localization of the GR and its biological actions in a given tissue or cell type. In this sense, the discovery that immunophilins containing tetratricopeptide repeats (TPR) domains are responsible for the GR cytoplasmic transport mechanism and the nuclear retention half-time of the receptor opened new trends in our understanding of its complex mechanism of action. Because the properties of GR ligands influence these protein-protein interactions, specific steroid•receptor complexes may confer the GR different features providing new therapeutic opportunities to manage the disease. In this article, we analyze multiple aspects of the GR mechanism of action, some properties of the GR isoforms, and the latest findings revealing the roles of Hsp90-binding immunophilins to manage the glucocorticoid biological response.

Group 3 innate lymphoid cells secret neutrophil chemoattractants and are insensitive to glucocorticoid via aberrant GR phosphorylation.

BACKGROUND: Patients with neutrophil-mediated asthma have poor response to glucocorticoids. The roles and mechanisms of group 3 innate lymphoid cells (ILC3s) in inducing neutrophilic airway inflammation and glucocorticoid resistance in asthma have not been fully clarified. METHODS: ILC3s in peripheral blood were measured by flow cytometry in patients with eosinophilic asthma (EA) and non-eosinophilic asthma (NEA). ILC3s were sorted and cultured in vitro for RNA sequencing. Cytokines production and signaling pathways in ILC3s after IL-1ß stimulation and dexamethasone treatment were determined by real-time PCR, flow cytometry, ELISA and western blot. RESULTS: The percentage and numbers of ILC3s in peripheral blood was higher in patients with NEA compared with EA, and negatively correlated with blood eosinophils. IL-1ß stimulation significantly enhanced CXCL8 and CXCL1 production in ILC3s via activation of p65 NF-κB and p38/JNK MAPK signaling pathways. The expression of neutrophil chemoattractants from ILC3s was insensitive to dexamethasone treatment. Dexamethasone significantly increased phosphorylation of glucocorticoid receptor (GR) at Ser226 but only with a weak induction at Ser211 residues in ILC3s. Compared to human bronchial epithelial cell line (16HBE cells), the ratio of p-GR S226 to p-GR S211 (p-GR S226/S211) was significantly higher in ILC3s at baseline and after dexamethasone treatment. In addition, IL-1ß could induce Ser226 phosphorylation and had a crosstalk effect to dexamethasone via NF-κB pathway. CONCLUSIONS: ILC3s were elevated in patients with NEA, and associated with neutrophil inflammation by release of neutrophil chemoattractants and were glucocorticoid (GC) resistant. This paper provides a novel cellular and molecular mechanisms of neutrophil inflammation and GC-resistance in asthma. Trial registration The study has been prospectively registered in the World Health Organization International Clinical Trials Registry Platform (ChiCTR1900027125).

The adrenal stress response is an essential host response against therapy-induced lethal immune activation.

Cytokine release syndrome (CRS) is a systemic inflammatory syndrome associated with infection- or drug-induced T cell activation and can cause multiple organ failure and even death. Because current treatments are ineffective in some patients with severe CRS, we set out to identify risk factors and mechanisms behind severe CRS that might lead to preventive therapies and better clinical outcomes in patients. In mice, we found that deficiency in the adrenal stress response-with similarities to such in patients called relative adrenal insufficiency (RAI)-conferred a high risk for lethal CRS. Mice treated with CD3 antibodies were protected against lethal CRS by the production of glucocorticoids (GC) induced by the adrenal stress response in a manner dependent on the scavenger receptor B1 (SR-BI), a receptor for high-density lipoprotein (HDL). Mice with whole-body or adrenal gland-specific SR-BI deficiency exhibited impaired GC production, more severe CRS, and increased mortality in response to CD3 antibodies. Pretreatment with a low dose of GC effectively suppressed the development of CRS and rescued survival in SR-BI-deficient mice without compromising T cell function through apoptosis. Our findings suggest that RAI may be a risk factor for therapy-induced CRS and that pretreating RAI patients with GC may prevent lethal CRS.

Linking the relation between gut microbiota and glucocorticoid-induced osteoporosis.

Osteoporosis (OP) is the most prevalent metabolic bone disease, characterized by the low bone mass and microarchitectural deterioration of bone tissue. Glucocorticoid (GC) clinically acts as one of the anti-inflammatory, immune-modulating, and therapeutic drugs, whereas the long-term use of GC may cause rapid bone resorption, followed by prolonged and profound suppression of bone formation, resulting in the GC-induced OP (GIOP). GIOP ranks the first among secondary OP and is a pivotal risk for fracture, as well as high disability rate and mortality, at both societal and personal levels, vital costs. Gut microbiota (GM), known as the "second gene pool" of human body, is highly correlated with maintaining the bone mass and bone quality, and the relation between GM and bone metabolism has gradually become a research hotspot. Herein, combined with recent studies and based on the cross-linking relationship between GM and OP, this review is aimed to discuss the potential mechanisms of GM and its metabolites on the OP, as well as the moderating effects of GC on GM, thereby providing an emerging thought for prevention and treatment of GIOP.

Polymorphism in interferon alpha/beta receptor contributes to glucocorticoid response and outcome of ARDS and COVID-19.

BACKGROUND: The use of glucocorticoids has given contradictory results for treating acute respiratory distress syndrome (ARDS). The use of intravenous Interferon beta (IFN ß) for the treatment of ARDS was recently tested in a phase III ARDS trial (INTEREST), in which more than half of the patients simultaneously received glucocorticoids. Trial results showed deleterious effects of glucocorticoids when administered together with IFN ß, and therefore, we aimed at finding the reason behind this. METHODS: We first sequenced the genes encoding the IFN α/ß receptor of the patients, who participated in the INTEREST study (ClinicalTrials.gov Identifier:  NCT02622724 , November 24, 2015) in which the patients were randomized to receive an intravenous injection of IFN ß-1a (144 patients) or placebo (152 patients). Genetic background was analyzed against clinical outcome, concomitant medication, and pro-inflammatory cytokine levels. Thereafter, we tested the influence of the genetic background on IFN α/ß receptor expression in lung organ cultures and whether, it has any effect on transcription factors STAT1 and STAT2 involved in IFN signaling. RESULTS: We found a novel disease association of a SNP rs9984273, which is situated in the interferon α/ß receptor subunit 2 (IFNAR2) gene in an area corresponding to a binding motif of the glucocorticoid receptor (GR). The minor allele of SNP rs9984273 associates with higher IFNAR expression, more rapid decrease of IFN γ and interleukin-6 (IL-6) levels and better outcome in IFN ß treated patients with ARDS, while the major allele associates with a poor outcome especially under concomitant IFN ß and glucocorticoid treatment. Moreover, the minor allele of rs9984273 associates with a less severe form of coronavirus diseases (COVID-19) according to the COVID-19 Host Genetics Initiative database. CONCLUSIONS: The distribution of this SNP within clinical study arms may explain the contradictory results of multiple ARDS studies and outcomes in COVID-19 concerning type I IFN signaling and glucocorticoids.

Context-Dependent Role of Glucocorticoid Receptor Alpha and Beta in Breast Cancer Cell Behaviour.

Background. The dual role of GCs has been observed in breast cancer; however, due to many concomitant factors, GR action in cancer biology is still ambiguous. In this study, we aimed to unravel the context-dependent action of GR in breast cancer. Methods. GR expression was characterized in multiple cohorts: (1) 24,256 breast cancer specimens on the RNA level, 220 samples on the protein level and correlated with clinicopathological data; (2) oestrogen receptor (ER)-positive and -negative cell lines were used to test for the presence of ER and ligand, and the effect of the GRß isoform following GRα and GRß overexpression on GR action, by in vitro functional assays. Results. We found that GR expression was higher in ER- breast cancer cells compared to ER+ ones, and GR-transactivated genes were implicated mainly in cell migration. Immunohistochemistry showed mostly cytoplasmic but heterogenous staining irrespective of ER status. GRα increased cell proliferation, viability, and the migration of ER- cells. GRß had a similar effect on breast cancer cell viability, proliferation, and migration. However, the GRß isoform had the opposite effect depending on the presence of ER: an increased dead cell ratio was found in ER+ breast cancer cells compared to ER- ones. Interestingly, GRα and GRß action did not depend on the presence of the ligand, suggesting the role of the "intrinsic", ligand-independent action of GR in breast cancer. Conclusions. Staining differences using different GR antibodies may be the reason behind controversial findings in the literature regarding the expression of GR protein and clinicopathological data. Therefore, caution in the interpretation of immunohistochemistry should be applied. By dissecting the effects of GRα and GRß, we found that the presence of the GR in the context of ER had a different effect on cancer cell behaviour, but independently of ligand availability. Additionally, GR-transactivated genes are mostly involved in cell migration, which raises GR's importance in disease progression.

11ß-HSD1 determines the extent of muscle atrophy in a model of acute exacerbation of COPD.

Muscle atrophy is an extrapulmonary complication of acute exacerbations (AE) in chronic obstructive pulmonary disease (COPD). The endogenous production and therapeutic application of glucocorticoids (GCs) have been implicated as drivers of muscle loss in AE-COPD. The enzyme 11 ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) activates GCs and contributes toward GC-induced muscle wasting. To explore the potential of 11ßHSD1 inhibition to prevent muscle wasting here, the objective of this study was to ascertain the contribution of endogenous GC activation and amplification by 11ßHSD1 in skeletal muscle wasting during AE-COPD. Emphysema was induced by intratracheal (IT) instillation of elastase to model COPD in WT and 11ßHSD1/KO mice, followed by vehicle or IT-LPS administration to mimic AE. µCT scans were obtained prior and at study endpoint 48 h following IT-LPS, to assess emphysema development and muscle mass changes, respectively. Plasma cytokine and GC profiles were determined by ELISA. In vitro, myonuclear accretion and cellular response to plasma and GCs were determined in C2C12 and human primary myotubes. Muscle wasting was exacerbated in LPS-11ßHSD1/KO animals compared with WT controls. RT-qPCR and western blot analysis showed elevated catabolic and suppressed anabolic pathways in muscle of LPS-11ßHSD1/KO animals relative to WTs. Plasma corticosterone levels were higher in LPS-11ßHSD1/KO animals, whereas C2C12 myotubes treated with LPS-11ßHSD1/KO plasma or exogenous GCs displayed reduced myonuclear accretion relative to WT counterparts. This study reveals that 11ß-HSD1 inhibition aggravates muscle wasting in a model of AE-COPD, suggesting that therapeutic inhibition of 11ß-HSD1 may not be appropriate to prevent muscle wasting in this setting.

Mycobacterium vaccae protects against glucocorticoid resistance resulting from combined physical and psychosocial trauma in mice.

Stress-related somatic and psychiatric disorders are often associated with a decline in regulatory T cell (Treg) counts and chronic low-grade inflammation. Recent preclinical evidence suggests that the latter is at least partly mediated by stress-induced upregulation of toll-like receptor (TLR)2 in newly generated neutrophils and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), as well as glucocorticoid (GC) resistance in predominantly PMN-MDSCs following stress-induced upregulation of TLR4 expression. Here we show in mice exposed to the chronic subordinate colony housing (CSC) paradigm that repeated intragastric (i.g.) administrations of a heat-killed preparation of Mycobacterium vaccae NCTC 11659, a saprophytic microorganism with immunoregulatory properties, protected against the stress-induced reduction in systemic Tregs, increase in basal and LPS-induced in vitro splenocyte viability, as well as splenic in vitro GC resistance. Our findings further support the hypothesis that i.g. M. vaccae protects against CSC-associated splenic GC resistance via directly affecting the myeloid compartment, thereby preventing the CSC-induced upregulation of TLR4 in newly generated PMN-MDSCs. In contrast, the protective effects of i.g. M. vaccae on the CSC-induced upregulation of TLR2 in neutrophils and the subsequent increase in basal and LPS-induced in vitro splenocyte viability seems to be indirectly mediated via the Treg compartment. These data highlight the potential for use of oral administration of M. vaccae NCTC 11659 to prevent stress-induced exaggeration of inflammation, a risk factor for development of stress-related psychiatric disorders.

Allolobophora caliginosa coelomic fluid and extract alleviate glucocorticoid-induced osteoporosis in mice by suppressing oxidative stress and regulating osteoblastic/osteoclastic-related markers.

Allolobophora calignosa (Ac) is a folk medicine for millennia, as it possesses many biological activities. This study aimed to investigate the chemo-preventive activity of A.calignosa coelomic fluid (AcCF) and A.calignosa extract (AcE) on glucocorticoid-induced osteoporosis (GIOP) in mice. Characterization and in vitro biological activity of AcE and AcCF has been assessed. Male CD-1 mice were subcutaneously received dexamethasone (DEX) (1 mg/kg, 5 times/week) and concurrently intraperitoneally treated with either AcCF (20 mg/kg) or AcE (45 mg/kg) every other day for 28 days. Serum and bone homogenates were subjected for qPCR and biochemical analysis. AcE and AcCF treatment significantly increased bone mineral density (BMD), bone mineral content (BMC), calcium (Ca), phosphorus (P), and calcitonin levels, whereas activity of serum alkaline phosphatase (ALP), bone alkaline phosphatase (BALP), serum acidic phosphatase (ACP), bone acidic phosphatase (BACP) and parathyroid hormone (PTH) levels were significantly reduced compare with untreated GIOP mice. Treatment with AcE and AcCF modulates oxidative stress and downregulated Rank and Mmp9 expression, as well as increased glycosaminoglycan content in the organic bone matrix, resulting in osteoclastogenesis inhibition. Overall, AcCF and AcE show a chemo-preventive activity against GIOP by inhibiting oxidative stress and regulating expression and/or activity of osteoblast/osteoclast-related markers.

Adrenalectomy prevents the effects of social defeat stress on PER2 rhythms in some peripheral tissues in male mice.

While stress does not affect the phase or period of the central pacemaker in the suprachiasmatic nucleus, it can shift clocks in peripheral tissues. Our previous studies showed significant delays of the PER2 rhythms in lung and kidney following social defeat stress. The mechanism underlying these effects is not fully understood, but might involve glucocorticoids (GC) released during the stressor. In the present study, we performed social defeat stress in adrenalectomized (ADX) mice to see if the induction of endogenous GC is necessary for the stress-induced phase shifts of peripheral clocks. We used mice that carry a luciferase reporter gene fused to the circadian clock gene Period2 (PER2::LUC) to examine daily rhythms of PER2 expression in various peripheral tissues. Mice were exposed to 5 consecutive daily social defeat stress in the late dark phase (ZT21-22). Running wheel rotations were recorded during 7 baseline and 5 social defeat days, which showed that social defeat stress suppressed locomotor activity without affecting the phase of the rhythm. This suppression of activity was not prevented by ADX. One hour after the last stressor, tissue samples from the liver, kidney and lung were collected and cultured for ex vivo bioluminescence recordings. In the liver, PER2 rhythms were not affected by social defeat stress or ADX. In the kidney, social defeat stress caused a > 4 h phase delay of the PER2 rhythm, which was prevented by ADX, supporting the hypothesis of a crucial role of GC in this stress effect. In the lung, social defeat stress caused an 8 h phase delay, but, surprisingly, a similar phase delay was seen in ADX animals independent of defeat. The latter indicates complex effects of stress and stress hormones on the lung clock. In conclusion, the findings show that repeated social defeat stress in the dark phase can shift PER2 rhythms in some tissues (lung, kidney) and not others (liver). Moreover, the social defeat stress effect in some tissues appears to be mediated by glucocorticoids (kidney) whereas the mechanism in other tissues is more complex (lung).

Dexamethasone activates c-Jun NH2-terminal kinase (JNK) which interacts with GR and protects it from ubiquitin-mediated degradation in NSCLC cells.

Dexamethasone-mediated pharmacological activation of the glucocorticoid receptor (GR) is widely used in the treatment regimen of hematological malignancies and solid cancers. However, DEX sensitivity towards patients primarily depends on the endogenous protein levels of GR. We observed that DEX treatment leads to an increase in GR protein levels despite inhibition of neo-protein synthesis in non-small cell lung cancer (NSCLC) cells. Mechanistically, DEX-stimulation concomitantly increased the JNK phosphorylation and GR protein levels, however the JNK stimulation preceds GR upregulation. Moreover, we also observed that DEX-mediated phosphorylation is partially mediated by upregulation in MEKK1 phosphorylation. Further, GR protein levels were significantly decreased in JNK inhibitor (JNKi, SP600125) treated cells whereas MG132 treatment restored GR levels indicating that DEX induced JNK activity regulated the GR protein levels through proteasomal-degradation pathway. Next, we showed that DEX led to JNK activation which physically interacts with GR and protects it from ubiquitination-mediated degradation. Furthermore, at basal level GR interacts with JNK in cytoplasm whereas upon DEX stimulation GR and pJNK both localized to nucleus and interact with each other. Next, we show that JNK-mediated GR stabilization affects its nuclear transcriptional functional activity in NSCLC cells. In line with these in vitro data, patient dataset analysis also shows that increased levels of both JNK and GR contributes towards better prognosis of NSCLC patients. Taken together, our data shows that DEX treatment may lead to positive feedback regulation of GR by activating JNK and thus highlights importance of GR-JNK crosstalk in NSCLC.

Glypican-4 regulated actin cytoskeletal reorganization in glucocorticoid treated trabecular meshwork cells and involvement of Wnt/PCP signaling.

A common adverse response to the clinical use of glucocorticoids (GCs) is elevated intraocular pressure (IOP) which is a major risk factor for glaucoma. Elevated IOP arises due to impaired outflow of aqueous humor (AH) through the trabecular meshwork (TM). Although GC-induced changes in actin cytoskeletal dynamics, contractile characteristics, and cell adhesive interactions of TM cells are believed to influence AH outflow and IOP, the molecular mechanisms mediating changes in these cellular characteristics are poorly understood. Our studies focused on evaluating changes in the cytoskeletal and cytoskeletal-associated protein (cytoskeletome) profile of human TM cells treated with dexamethasone (Dex) using label-free mass spectrometric quantification, identified elevated levels of specific proteins known to regulate actin stress fiber formation, contraction, actin networks crosslinking, cell adhesion, and Wnt signaling, including LIMCH1, ArgBP2, CNN3, ITGBL1, CTGF, palladin, FAT1, DIAPH2, EPHA4, SIPA1L1, and GPC4. Several of these proteins colocalized with the actin cytoskeleton and underwent alterations in distribution profile in TM cells treated with Dex, and an inhibitor of Abl/Src kinases. Wnt/Planar Cell Polarity (PCP) signaling agonists-Wnt5a and 5b were detected prominently in the cytoskeletome fraction of TM cells, and studies using siRNA to suppress expression of glypican-4 (GPC4), a known modulator of the Wnt/PCP pathway revealed that GPC4 deficiency impairs Dex induced actin stress fiber formation, and activation of c-Jun N-terminal Kinase (JNK) and Rho kinase. Additionally, while Dex augmented, GPC4 deficiency suppressed the formation of actin stress fibers in TM cells in the presence of Dex and Wnt5a. Taken together, these results identify the GPC4-dependent Wnt/PCP signaling pathway as one of the crucial upstream regulators of Dex induced actin cytoskeletal reorganization and cell adhesion in TM cells, opening an opportunity to target the GPC4/Wnt/PCP pathway for treatment of ocular hypertension in glaucoma.

Targeting Glutaminolysis Shows Efficacy in Both Prednisolone-Sensitive and in Metabolically Rewired Prednisolone-Resistant B-Cell Childhood Acute Lymphoblastic Leukaemia Cells.

The prognosis for patients with relapsed childhood acute lymphoblastic leukaemia (cALL) remains poor. The main reason for treatment failure is drug resistance, most commonly to glucocorticoids (GCs). The molecular differences between prednisolone-sensitive and -resistant lymphoblasts are not well-studied, thereby precluding the development of novel and targeted therapies. Therefore, the aim of this work was to elucidate at least some aspects of the molecular differences between matched pairs of GC-sensitive and -resistant cell lines. To address this, we carried out an integrated transcriptomic and metabolomic analysis, which revealed that lack of response to prednisolone may be underpinned by alterations in oxidative phosphorylation, glycolysis, amino acid, pyruvate and nucleotide biosynthesis, as well as activation of mTORC1 and MYC signalling, which are also known to control cell metabolism. In an attempt to explore the potential therapeutic effect of inhibiting one of the hits from our analysis, we targeted the glutamine-glutamate-α-ketoglutarate axis by three different strategies, all of which impaired mitochondrial respiration and ATP production and induced apoptosis. Thereby, we report that prednisolone resistance may be accompanied by considerable rewiring of transcriptional and biosynthesis programs. Among other druggable targets that were identified in this study, inhibition of glutamine metabolism presents a potential therapeutic approach in GC-sensitive, but more importantly, in GC-resistant cALL cells. Lastly, these findings may be clinically relevant in the context of relapse-in publicly available datasets, we found gene expression patterns suggesting that in vivo drug resistance is characterised by similar metabolic dysregulation to what we found in our in vitro model.

Melatonin-mediated FKBP4 downregulation protects against stress-induced neuronal mitochondria dysfunctions by blocking nuclear translocation of GR.

The physiological crosstalk between glucocorticoid and melatonin maintains neuronal homeostasis in regulating circadian rhythms. However, the stress-inducing level of glucocorticoid triggers mitochondrial dysfunction including defective mitophagy by increasing the activity of glucocorticoid receptors (GRs), leading to neuronal cell death. Melatonin then suppresses glucocorticoid-induced stress-responsive neurodegeneration; however, the regulatory mechanism of melatonin, i.e., associated proteins involved in GR activity, has not been elucidated. Therefore, we investigated how melatonin regulates chaperone proteins related to GR trafficking into the nucleus to suppress glucocorticoid action. In this study, the effects of glucocorticoid on suppressing NIX-mediated mitophagy, followed by mitochondrial dysfunction, neuronal cell apoptosis, and cognitive deficits were reversed by melatonin treatment by inhibiting the nuclear translocation of GRs in both SH-SY5Y cells and mouse hippocampal tissue. Moreover, melatonin selectively suppressed the expression of FKBP prolyl isomerase 4 (FKBP4), which is a co-chaperone protein that works with dynein, to reduce the nuclear translocation of GRs among the chaperone proteins and nuclear trafficking proteins. In both cells and hippocampal tissue, melatonin upregulated melatonin receptor 1 (MT1) bound to Gαq, which triggered the phosphorylation of ERK1. The activated ERK then enhanced DNA methyltransferase 1 (DNMT1)-mediated hypermethylation of FKBP52 promoter, reducing GR-mediated mitochondrial dysfunction and cell apoptosis, the effects of which were reversed by knocking down DNMT1. Taken together, melatonin has a protective effect against glucocorticoid-induced defective mitophagy and neurodegeneration by enhancing DNMT1-mediated FKBP4 downregulation that reduced the nuclear translocation of GRs.

Survival Mechanisms of Metastatic Melanoma Cells: The Link between Glucocorticoids and the Nrf2-Dependent Antioxidant Defense System.

Circulating glucocorticoids increase during stress. Chronic stress, characterized by a sustained increase in serum levels of cortisol, has been associated in different cases with an increased risk of cancer and a worse prognosis. Glucocorticoids can promote gluconeogenesis, mobilization of amino acids, fat breakdown, and impair the body's immune response. Therefore, conditions that may favor cancer growth and the acquisition of radio- and chemo-resistance. We found that glucocorticoid receptor knockdown diminishes the antioxidant protection of murine B16-F10 (highly metastatic) melanoma cells, thus leading to a drastic decrease in their survival during interaction with the vascular endothelium. The BRAFV600E mutation is the most commonly observed in melanoma patients. Recent studies revealed that VMF/PLX40-32 (vemurafenib, a selective inhibitor of mutant BRAFV600E) increases mitochondrial respiration and reactive oxygen species (ROS) production in BRAFV600E human melanoma cell lines. Early-stage cancer cells lacking Nrf2 generate high ROS levels and exhibit a senescence-like growth arrest. Thus, it is likely that a glucocorticoid receptor antagonist (RU486) could increase the efficacy of BRAF-related therapy in BRAFV600E-mutated melanoma. In fact, during early progression of skin melanoma metastases, RU486 and VMF induced metastases regression. However, treatment at an advanced stage of growth found resistance to RU486 and VMF. This resistance was mechanistically linked to overexpression of proteins of the Bcl-2 family (Bcl-xL and Mcl-1 in different human models). Moreover, melanoma resistance was decreased if AKT and NF-κB signaling pathways were blocked. These findings highlight mechanisms by which metastatic melanoma cells adapt to survive and could help in the development of most effective therapeutic strategies.

Role of tPA in Corticosterone-Induced Apoptosis of Mouse Mural Granulosa and Oviductal Epithelial Cells.

Although studies indicate that female stress-increased secretion of glucocorticoids impairs oocyte competence and embryo development, by inducing apoptosis of ovarian and oviductal cells, respectively, the mechanisms by which glucocorticoids induce apoptosis of ovarian and oviductal cells are largely unclear. Tissue plasminogen activator (tPA) has been involved in apoptosis of different cell types. However, while some studies indicate that tPA is proapoptotic, others demonstrate its antiapoptotic effects. This study has explored the role and action mechanisms of tPA in corticosterone-induced apoptosis of mouse mural granulosa cells (MGCs) and oviductal epithelial cells (OECs). The results demonstrate that culture with corticosterone significantly increased apoptosis, while decreasing levels of tPA (Plat) mRNA and tPA protein in both MGCs and OECs. Culture with tPA ameliorated corticosterone-induced apoptosis of MGCs and OECs. Furthermore, while tPA protected MGCs from corticosterone-induced apoptosis by interacting with low-density lipoprotein receptor-related protein 1 (LRP1), it protected OECs from the apoptosis by acting on Annexin 2 (ANXA2). In conclusion, tPA is antiapoptotic in both MGCs and OECs, and it protects MGCs and OECs from corticosterone-induced apoptosis by interacting with LRP1 and ANXA2, respectively, suggesting that tPA may use different receptors to inhibit apoptosis in different cell types.

ß-adrenoceptors of the infra-limbic cortex mediate corticosterone-induced enhancement of acquisition and consolidation of fear memory extinction in rats.

The extinction of auditory fear conditioning (AFC) refers to reducing the fear responses induced following repeated presentation of a conditioned stimulus (tone) in the absence of an unconditioned stimulus (electric foot shock). Glucocorticoid receptors (GRs) play an important role in extinction, but the underlying neurobiological mechanisms are unclear. This study aimed to investigate the interaction between glucocorticoids and ß-adrenoceptors of the infra-limbic cortex (IL) in regulating the acquisition and consolidation of fear memory extinction in rats. Male rats were trained to AFC and received three trial tones (30 s, 4 kHz, 80 dB) co-terminated with a footshock (0.8 mA, 1 s; unconditioned stimulus). Extinction trials were conducted over 3 days after training (Ext 1-3). In experiment 1, rats received clenbuterol (0.25 mg/kg/2 ml, IP) as a ß2-adrenoceptor agonist or propranolol (2.5 mg/kg/2 ml, IP) as a ß-adrenoceptors antagonist before Ext 1 and immediately after Ext 1 and Ext 2 followed by systemic injection of corticosterone (3 mg/kg/2 ml, IP). In Experiment 2, separate groups of rats received a bilateral intra-IL injection of clenbuterol (50 ng/0.5 µl/side) or propranolol (500 ng/0.5 µl/side) followed by a systemic injection of corticosterone (3 mg/kg/2 ml) before Ext 1 and immediately after Ext 1 and Ext 2. Results indicated that systemic and intra-IL injections of clenbuterol and propranolol inhibited and increased the facilitative effects of corticosterone on fear memory extinction, respectively. These findings show that activating ß-adrenergic receptors in the IL mediates glucocorticoid effects on the acquisition and consolidation of auditory-conditioned fear memory extinction.

Peptide-guided delivery improves the therapeutic efficacy and safety of glucocorticoid drugs for treating acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening conditions with excessive inflammation in the lung. Glucocorticoids had been widely used for ALI/ARDS, but their clinical benefit remains unclear. Here, we tackled the problem by conjugating prednisolone (PSL) with a targeting peptide termed CRV. Systemically administered CRV selectively homes to the inflamed lung of a murine ALI model, but not healthy organs or the lung of healthy mice. The expression of the CRV receptor, retinoid X receptor ß, was elevated in the lung of ALI mice and patients with interstitial lung diseases, which may be the basis of CRV targeting. We then covalently conjugated PSL and CRV with a reactive oxygen species (ROS)-responsive linker in the middle. While being intact in blood, the ROS linker was cleaved intracellularly to release PSL for action. In vitro, CRV-PSL showed an anti-inflammatory effect similar to that of PSL. In vivo, CRV conjugation increased the amount of PSL in the inflamed lung but reduced its accumulation in healthy organs. Accordingly, CRV-PSL significantly reduced lung injury and immune-related side effects elsewhere. Taken together, our peptide-based strategy for targeted delivery of glucocorticoids for ALI may have great potential for clinical translation.

Dexamethasone leads to Zn2+ accumulation and increased unbound Zn2+ in C2C12 muscle and 3T3-L1 adipose cells.

Skeletal muscle atrophy is associated with increases in circulating glucocorticoid levels and insulin resistance. Zinc accumulates in atrophic muscle, but the relationship between atrophy, insulin resistance, and Zn2+ homeostasis remains unclear. In this study, the effect of the glucocorticoid dexamethasone (DEX) on insulin and Zn2+ homeostasis was explored. Treatment of differentiated C2C12 skeletal myotubes and 3T3-L1 adipocytes with DEX significantly increased mRNA expression of the metal-binding proteins Mt1 and 2 and altered energy storage as shown by the increased size of lipid droplets in 3T3-L1 cells. In C2C12 cells the total cellular Zn2+ was higher after DEX treatment, and in both C2C12 and 3T3-L1 adipocytes, free unbound Zn2+ was increased. Insulin treatment led to a gradual increase in free Zn2+ in C2C12 cells, and no significant change in DEX-treated cells such that concentrations were similar 10 min after insulin treatment. These data demonstrate that DEX disturbs Zn2+ homeostasis in muscle and fat cells. Further study of the molecular pathways involved to identify novel therapeutic targets for treatment of skeletal muscle atrophy is warranted.

The resilience of adolescent male rats to acute stress-induced delayed anxiety is age-related and glucocorticoid release-dependent.

Studies investigated how stressful experiences modulate physiological and behavioral responses and the consequences of stress-induced corticosterone release in anxiety-like behavior. Adolescence is crucial to brain maturation, and several neurobiological changes in this period lead individuals to increased susceptibility or resilience to aversive situations. Despite the effects of stress in adults, information about adolescents' responses to acute stress is lacking. We aimed to understand how adolescence affects acute stress responses. Male adolescent rats (30 days old) were 2 h restrained, and anxiety-like behaviors were measured immediately or 10 days after stress in the elevated plus-maze (EPM) and the light-dark box (LDB) tests. To verify the importance of CORT modulation in stress-induced anxiety, another group of rats was treated, 30 min before restraint, with metyrapone to blunt the stress-induced CORT peak and tested immediately after stress. To show that stress effects on behavior were age-dependent, another set of rats was tested in two different periods - early adolescence (30 days old) and mid-adolescence (40 days old) and were treated or not with metyrapone before the stress session and tested immediately or ten days later in the LDB test. Only early adolescent male rats were resilient to delayed anxiety-like behavior in EPM and LDB tests. Metyrapone treatment increased the rats' exploration immediately and ten days after stress. These data suggest a specific age at which adolescent rats are resilient to the delayed effects of acute restraint stress and that the metyrapone treatment has long-term behavioral consequences.