The glucocorticoid receptor modulator relacorilant reverses the immunosuppressive effects of cortisol.

Cortisol, an endogenous glucocorticoid receptor (GR) agonist, controls a broad transcriptional program that affects T-cell activation, pro-inflammatory cytokine secretion, apoptosis, and immune-cell trafficking. The degree to which endogenous cortisol blunts the anti-tumor immune response checkpoint inhibitors stimulate had not been assessed. We addressed this question using relacorilant, a selective GR modulator (SGRM) that competitively antagonizes the effects of cortisol activity. GR expression in human tumor and immune cells positively correlated with PD-L1 expression and tumor infiltration of Th2 and Treg cells, and negatively correlated with Th1-cell infiltration. In vitro, cortisol inhibited, and relacorilant restored, T-cell activation and pro-inflammatory cytokine secretion in human peripheral blood mononuclear cells. In the ovalbumin-expressing EG7 and MC38 immune-competent tumor models, relacorilant significantly improved anti-PD-1 antibody efficacy and showed favorable effects on antigen-specific T-cells and systemic TNFα and IL-10. These data characterize the broad immunosuppressive effects of endogenous cortisol and highlight the potential of combining an SGRM with an immune checkpoint inhibitor.

Discovery of PPARγ and glucocorticoid receptor dual agonists to promote the adiponectin and leptin biosynthesis in human bone marrow mesenchymal stem cells.

Adiponectin and leptin are major adipocytokines that control crosstalk between adipose tissue and other organ systems. Hypoadiponectinemia and hypoleptinemia are associated with human metabolic diseases. Compounds with adipocytokine biosynthesis-stimulating activities could be developed as therapeutics against diverse metabolic conditions. In phenotypic screening with human bone marrow mesenchymal stem cells (hBM-MSCs), (E)-4-hydroxy-3-(3-(4-hydroxy-3-methoxyphenyl)acryloyl)-6-methyl-2H-pyran-2-one (1) was identified to increase adiponectin biosynthesis during adipogenesis and simultaneously to stimulate leptin production. Using the compound 1 structure, the structure-activity relationship study was performed to discover more potent compounds stimulating both adiponectin and leptin production. (E)-3-(3-(2-fluoropyridin-4-yl)acryloyl)-4-hydroxy-6-methyl-2H-pyran-2-one (11) exhibited the most potent adiponectin (EC50, 2.87 µM) and leptin (EC50, 2.82 µM) biosynthesis-stimulating activities in hBM-MSCs. In a target identification study, compound 11 was characterized as a dual modulator binding to both peroxisome proliferator-activated receptor (PPAR) γ and glucocorticoid receptor (GR). This study provides a novel pharmacophore for PPARγ/GR dual modulators with therapeutic potential against human metabolic diseases.

Multimodal regulatory elements within a hormone-specific super enhancer control a heterogeneous transcriptional response.

The hormone-stimulated glucocorticoid receptor (GR) modulates transcription by interacting with thousands of enhancers and GR binding sites (GBSs) throughout the genome. Here, we examined the effects of GR binding on enhancer dynamics and investigated the contributions of individual GBSs to the hormone response. Hormone treatment resulted in genome-wide reorganization of the enhancer landscape in breast cancer cells. Upstream of the DDIT4 oncogene, GR bound to four sites constituting a hormone-dependent super enhancer. Three GBSs were required as hormone-dependent enhancers that differentially promoted histone acetylation, transcription frequency, and burst size. Conversely, the fourth site suppressed transcription and hormone treatment alleviated this suppression. GR binding within the super enhancer promoted a loop-switching mechanism that allowed interaction of the DDIT4 TSS with the active GBSs. The unique functions of each GR binding site contribute to hormone-induced transcriptional heterogeneity and demonstrate the potential for targeted modulation of oncogene expression.

Corticosterone-mediated regulation and functions of miR-218-5p in rat brain.

Chronic stress is one of the key precipitating factors in major depressive disorder (MDD). Stress associated studies have underscored the mechanistic role of epigenetic master players like microRNAs (miRNAs) in depression pathophysiology at both preclinical and clinical levels. Previously, we had reported changes in miR-218-5p expression in response to corticosterone (CORT) induced chronic stress. MiR-218-5p was one of the most significantly induced miRNAs in the prefrontal cortex (PFC) of rats under chronic stress. In the present report, we have investigated how chronic CORT exposure mechanistically affected miR-218-5p expression in the rat brain and how miR-218 could trigger molecular changes on its downstream regulatory pathways. Elevated expression of miR-218-5p was found in the PFC of CORT-treated rats. A glucocorticoid receptor (GR) targeted Chromatin-Immunoprecipitation (ChIP) assay revealed high GR occupancy on the promoter region of Slit3 gene hosting miR-218-2 in its 3rd intron. RNA-sequencing data based on RNA Induced silencing Complex Immunoprecipitation (RISC-IP) with AGO2 in SH-SY5Y cells detected six consistent target genes of miR-218-5p (APOL4, DTWD1, BNIP1, METTL22, SNAPC1, and HDAC6). The expression of all five genes, except APOL4, was successfully validated with qPCR in CORT-treated rat PFC. Further, Hdac6-based ChIP-seq experiment helped in mapping major genomic loci enriched for intergenic regions in the PFC of CORT-treated rat. A proximity-based gene ontology (GO) analysis revealed a majority of the intergenic sites to be part of key genes implicated in central nervous system functions, notably synapse organization, neuron projection morphogenesis, and axonogenesis. Our results suggest that the upregulation of miR-218-5p in PFC of CORT-treated rats possibly resulted from GR biding in the promoter region of Slit3 gene. Interestingly, Hdac6 was one of the consistent target genes potentially found to regulate CNS related genes by chromatin modification. Collectively, these findings establish the role of miR-218-5p in chronic stress and the epigenetic function it plays to induce chromatin-based transcriptional changes of several CNS genes in triggering stress-induced disorders, including depression. This also opens up the scope to understand the role of miR-218-5p as a potential target for noncoding RNA therapeutics in clinical depression.

Aldosterone and dexamethasone activate African lungfish mineralocorticoid receptor: Increased activation after removal of the amino-terminal domain.

Aldosterone, the main physiological mineralocorticoid in humans and other terrestrial vertebrates, first appears in lungfish, which are lobe-finned fish that are forerunners of terrestrial vertebrates. Aldosterone activation of the MR regulates internal homeostasis of water, sodium and potassium, which was critical in the conquest of land by vertebrates. We studied transcriptional activation of the slender African lungfish MR by aldosterone, other corticosteroids and progesterone and find that aldosterone, 11-deoxycorticosterone, 11-deoxycortisol and progesterone have half-maximal responses (EC50 s) below 1 nM and are potential physiological mineralocorticoids. In contrast, EC50 s for corticosterone and cortisol were 23 nM and 66 nM, respectively. Unexpectedly, truncated lungfish MR, consisting of the DNA-binding, hinge and steroid-binding domains, had a stronger response to corticosteroids and progesterone than full-length lungfish MR, indicating that the N-terminal domain represses steroid activation of lungfish MR, unlike human MR in which the N-terminal domain contains an activation function. BLAST searches of GenBank did not retrieve a GR ortholog, leading us to test dexamethasone and triamcinolone for activation of lungfish MR. At 10 nM, both synthetic glucocorticoids are about 4-fold stronger than 10 nM aldosterone in activating full-length lungfish MR, leading us to propose that lungfish MR also functions as a GR.

Impaired glucocorticoid receptor expression in liver disrupts feeding-induced gene expression, glucose uptake, and glycogen storage.

The transition from a fasted to a fed state is associated with extensive transcriptional remodeling in hepatocytes facilitated by hormonal- and nutritional-regulated transcription factors. Here, we use a liver-specific glucocorticoid receptor (GR) knockout (L-GRKO) model to investigate the temporal hepatic expression of GR target genes in response to feeding. Interestingly, in addition to the well-described fasting-regulated genes, we identify a subset of hepatic feeding-induced genes that requires GR for full expression. This includes Gck, which is important for hepatic glucose uptake, utilization, and storage. We show that insulin and glucocorticoids cooperatively regulate hepatic Gck expression in a direct GR-dependent manner by a 4.6 kb upstream GR binding site operating as a Gck enhancer. L-GRKO blunts preprandial and early postprandial Gck expression, which ultimately affects early postprandial hepatic glucose uptake, phosphorylation, and glycogen storage. Thus, GR is positively involved in feeding-induced gene expression and important for postprandial glucose metabolism in the liver.

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

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

Rapid Screening of Glucocorticoid Receptor (GR) Effectors Using Cortisol-Detecting Sensor Cells.

Cortisol, a stress hormone, plays key roles in mediating stress and anti-inflammatory responses. As abnormal cortisol levels can induce various adverse effects, screening cortisol and cortisol analogues is important for monitoring stress levels and for identifying drug candidates. A novel cell-based sensing system was adopted for rapid screening of cortisol and its functional analogues under complex cellular regulation. We used glucocorticoid receptor (GR) fused to a split intein which reconstituted with the counterpart to trigger conditional protein splicing (CPS) in the presence of targets. CPS generates functional signal peptides which promptly translocate the fluorescent cargo. The sensor cells exhibited exceptional performance in discriminating between the functional and structural analogues of cortisol with improved sensitivity. Essential oil extracts with stress relief activity were screened using the sensor cells to identify GR effectors. The sensor cells responded to peppermint oil, and L-limonene and L-menthol were identified as potential GR effectors from the major components of peppermint oil. Further analysis indicated L-limonene as a selective GR agonist (SEGRA) which is a potential anti-inflammatory agent as it attenuates proinflammatory responses without causing notable adverse effects of GR agonists.

Glucocorticoid induced transcript 1 represses airway remodeling of asthmatic mouse via inhibiting IL-13/periostin/TGF-ß1 signaling.

Asthma is characterized by airway remodeling. Glucocorticoid induced transcript 1 (GLCCI1) was reported to be associated with the development of asthma, while its exact mechanism is still not clear. In our study, ovalbumin (OVA) combined with aluminum hydroxide were used to establish asthmatic mouse model. ELISA assay was fulfilled to ensure the concentration of inflammatory factors in both bronchoalveolar lavage fluid and serum. The pathological changes and collagen deposition in lung tissues were analyzed using H&E staining and Masson staining, respectively. The expression of proteins was measured using western blot, and the expression of GLCCI1 mRNA was ensured by qRT-PCR. Here, we demonstrated that OVA-induced inflammation, lung structural remodeling and collagen deposition in asthmatic mice was notably improved by hydroprednisone treatment or GLCCI1 overexpressing. The expression of GLCCI1 was decreased, while IL-13, periostin and TGF-ß1 were increased in the lung tissue of asthmatic mice. Importantly, upregulation of GLCCI1 suppressed the expression of IL-13, periostin and TGF-ß1, phosphorylation of Smad2 and Smad3, and extracellular matrix (ECM) deposition-related proteins expression. IL-13-induced upregulation of periostin and TGF-ß1 expression, phosphorylation of Smad2 and Smad3, and ECM deposition in airway epithelial cells (AECs) was repressed by GLCCI1 increasing. Furthermore, our results showed that overexpression of GLCCI1 repressed the effect of IL-13 on AECs via inhibiting periostin expression. Overall, our data revealed that GLCCI1 limited the airway remodeling in mice with asthma through inhibiting IL-13/periostin/TGF-ß1 signaling pathway. Our data provided a novel target for asthma treatment.

Repression of transcription by the glucocorticoid receptor: A parsimonious model for the genomics era.

Glucocorticoids are potent anti-inflammatory drugs that are used to treat an extraordinary range of human disease, including COVID-19, underscoring the ongoing importance of understanding their molecular mechanisms. Early studies of GR signaling led to broad acceptance of models in which glucocorticoid receptor (GR) monomers tether repressively to inflammatory transcription factors, thus abrogating inflammatory gene expression. However, newer data challenge this core concept and present an exciting opportunity to reframe our understanding of GR signaling. Here, we present an alternate, two-part model for transcriptional repression by glucocorticoids. First, widespread GR-mediated induction of transcription results in rapid, primary repression of inflammatory gene transcription and associated enhancers through competition-based mechanisms. Second, a subset of GR-induced genes, including targets that are regulated in coordination with inflammatory transcription factors such as NF-κB, exerts secondary repressive effects on inflammatory gene expression. Within this framework, emerging data indicate that the gene set regulated through the cooperative convergence of GR and NF-κB signaling is central to the broad clinical effectiveness of glucocorticoids in terminating inflammation and promoting tissue repair.

The glucocorticoid receptor specific modulator CORT108297 reduces brain pathology following status epilepticus.

OBJECTIVE: Glucocorticoid levels rise rapidly following status epilepticus and remain elevated for weeks after the injury. To determine whether glucocorticoid receptor activation contributes to the pathological sequelae of status epilepticus, mice were treated with a novel glucocorticoid receptor modulator, C108297. METHODS: Mice were treated with either C108297 or vehicle for 10 days beginning one day after pilocarpine-induced status epilepticus. Baseline and stress-induced glucocorticoid secretion were assessed to determine whether hypothalamic-pituitary-adrenal axis hyperreactivity could be controlled. Status epilepticus-induced pathology was assessed by quantifying ectopic hippocampal granule cell density, microglial density, astrocyte density and mossy cell loss. Neuronal network function was examined indirectly by determining the density of Fos immunoreactive neurons following restraint stress. RESULTS: Treatment with C108297 attenuated corticosterone hypersecretion after status epilepticus. Treatment also decreased the density of hilar ectopic granule cells and reduced microglial proliferation. Mossy cell loss, on the other hand, was not prevented in treated mice. C108297 altered the cellular distribution of Fos protein but did not restore the normal pattern of expression. INTERPRETATION: Results demonstrate that baseline corticosterone levels can be normalized with C108297, and implicate glucocorticoid signaling in the development of structural changes following status epilepticus. These findings support the further development of glucocorticoid receptor modulators as novel therapeutics for the prevention of brain pathology following status epilepticus.

Activation of the glucocorticoid receptor rapidly triggers calcium-dependent serotonin release in vitro.

AIMS: Glucocorticoids rapidly provoke serotonin (5-HT) release in vivo. We aimed to investigate molecular mechanisms of glucocorticoid receptor (GR)-triggered 5-HT release. METHODS: Employing 1C11 cells to model 5-HT neurotransmission, immunofluorescence and Pearson's Correlation Coefficient were used to analyze colocalization of GR, 5-HT, vesicle membrane protein synaptotagmin 1 and vesicle dye FM4-64FX. FFN511 and FM4-64FX dyes as well as calcium imaging were used to visualize vesicular 5-HT release upon application of GR agonist dexamethasone, GR antagonist mifepristone and voltage-gated calcium channel (VGCC) inhibitors. RESULTS: GR, 5-HT, synaptotagmin 1 and FM4-64FX showed overlapping staining patterns, with Pearson's Correlation Coefficient indicating colocalization. Similarly to potassium chloride, dexamethasone caused a release of FFN511 and uptake of FM4-64FX, indicating vesicular 5-HT release. Mifepristone, calcium depletion and inhibition of L-type VGCC significantly diminished dexamethasone-induced vesicular 5-HT release. CONCLUSIONS: In close proximity to 5-HT releasing sites, activated GR rapidly triggers L-type VGCC-dependent vesicular 5-HT release. These findings provide a better understanding of the interrelationship between glucocorticoids and 5-HT release.

JTP-117968, a novel selective glucocorticoid receptor modulator, exhibits significant anti-inflammatory effect while maintaining bone mineral density in mice.

Classic glucocorticoids have been prescribed for various inflammatory diseases, such as rheumatoid arthritis, due to their outstanding anti-inflammatory effects. However, glucocorticoids cause numerous unwanted side effects, including osteoporosis and diabetes. Hence, selective glucocorticoid receptor modulators (SGRMs), which retain anti-inflammatory effects with minimized side effects, are among the most anticipated drugs in the clinical field. The assumption is that there are two major mechanisms of action via glucocorticoid receptors, transrepression (TR) and transactivation (TA). In general, anti-inflammatory effects of glucocorticoids are largely due to TR, while the side effects associated with glucocorticoids are mostly mediated through TA. We previously reported that JTP-117968, a novel SGRM, maintained partial TR activity while remarkably reducing the TA activity. In this study, we investigated the anti-inflammatory effect of JTP-117968 on a lipopolysaccharide (LPS) challenge model and collagen-induced arthritis (CIA) model in mice. Meanwhile, we tested the effect of JTP-117968 on the bone mineral density (BMD) in mouse femur to evaluate the side effect. Based on the evaluation, JTP-117968 reduced the plasma levels of tumor necrosis factor α induced by LPS challenge in mice significantly. Remarkably, CIA development was suppressed by JTP-117968 comparably with prednisolone and PF-802, an active form of fosdagrocorat that has been developed clinically as an orally available SGRM. Strikingly, the side effect of JTP-117968 on mouse femoral BMD was much lower than those of PF-802 and prednisolone. Therefore, JTP-117968 has attractive potential as a new therapeutic option against inflammatory diseases with minimized side effects compared to classic glucocorticoids.

GR/NF-κB signaling pathway regulates hippocampal inflammatory responses in diabetic rats with chronic unpredictable mild stress.

Clinical studies have shown that diabetes can present with underlying depression, and a combination of the two can lead to emotional, memory and cognitive disorders, closely associated with hippocampal neuroinflammation. However, the mechanism underlying the development of hippocampal neuroinflammation under the above condition remains elusive. The aims of this study were to explore the pathogenesis of diabetes combined with depression, and the effect of dexamethasone (Dex), a glucocorticoid receptor (GR) agonist, on hippocampal neuroinflammation in diabetic rats with chronic unpredictable mild stress (CUMS). Therefore, rats were intragastrically fed on a high-fat diet (10% cholesterol 10 ml/kg) for 14 days and thereafter injected with 38 mg/kg of streptozotocin on the 15th day to induce diabetes. Dex treatment of the diabetic and CUMS rats ameliorated the depression-associated behavior in the respective rats. Apart from enhanced depressive behavior, diabetes-depressed condition also up-regulated the expression of hippocampus microglia chemokine Ⅰ receptor (CX3CR1) and secretion of several pro-inflammatory factors, in particular, interleukin 1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor - α (TNF-α). Hematoxylin-eosin staining revealed inflammatory damages in the hippocampus. Western blot analysis further revealed repression of GR proteins converse to the nuclear factor kappa-B (NF-κB) proteins, which were up-regulated. Intriguingly, Dex reversed the above events by inhibiting inflammatory reactions in the hippocampus. Consequently, played an antidepressant effect in diabetic and CUMS model rats. Overall, findings of this research suggest that the physiopathology of diabetes with stress cormobity are mediated by inflammatory reactions in the hippocampus. In particular, the responses are associated with regulation of GR/NF-κB signaling pathway.

Clinical stage molecule PT150 is a modulator of glucocorticoid and androgen receptors with antiviral activity against SARS-CoV-2.

PT150 is a clinical-stage molecule, taken orally, with a strong safety profile having completed Phase 1 and Phase 2 clinical trials for its original use as an antidepressant. It has an active IND for COVID-19. Antiviral activities have been found for PT150 and other members of its class in a variety of virus families; thus, it was now tested against SARS-CoV-2 in human bronchial epithelial lining cells and showed effective 90% inhibitory antiviral concentration (EC90) of 5.55 µM. PT150 is a member of an extended platform of novel glucocorticoid receptor (GR) and androgen receptor (AR) modulating molecules. In vivo, their predominant net effect is one of systemic glucocorticoid antagonism, but they also show direct downregulation of AR and minor GR agonism at the cellular level. We hypothesize that anti-SARS-CoV-2 activity depends in part on this AR downregulation through diminished TMPRSS2 expression and modulation of ACE2 activity. Given that hypercortisolemia is now suggested to be a significant co-factor for COVID-19 progression, we also postulate an additive role for its potent immunomodulatory effects through systemic antagonism of cortisol.

Comparative efficacy of glucocorticoid receptor agonists on Th2 cell function and attenuation by progesterone.

BACKGROUND: Corticosteroids (CS)s suppress cytokine production and induce apoptosis of inflammatory cells. Prednisone and dexamethasone are oral CSs prescribed for treating asthma exacerbations. While prednisone is more commonly prescribed, dexamethasone is long acting and a more potent glucocorticoid receptor (GR) agonist. It can be administered as a one or two dose regime, unlike the five to seven days required for prednisone, a feature that increases compliance. We compared the relative ability of these two oral CSs to suppress type 2 inflammation. Since progesterone has affinity for the GR and women are more likely to relapse following an asthma exacerbation, we assessed its influence on CS action. RESULTS: Dexamethasone suppressed the level of IL-5 and IL-13 mRNA within Th2 cells with ~ 10-fold higher potency than prednisolone (the active form of prednisone). Dexamethasone induced a higher proportion of apoptotic and dying cells than prednisolone, at all concentrations examined. Addition of progesterone reduced the capacity of both CS to drive cell death, though dexamethasone maintained significantly more killing activity. Progesterone blunted dexamethasone-induction of FKBP5 mRNA, indicating that the mechanism of action was by interference of the CS:GR complex. CONCLUSIONS: Dexamethasone is both more potent and effective than prednisolone in suppressing type 2 cytokine levels and mediating apoptosis. Progesterone attenuated these anti-inflammatory effects, indicating its potential influence on CS responses in vivo. Collectively, our data suggest that when oral CS is required, dexamethasone may be better able to control type 2 inflammation, eliminate Th2 cells and ultimately lead to improved long-term outcomes. Further research in asthmatics is needed.

Crosstalk between Hedgehog pathway and the glucocorticoid receptor pathway as a basis for combination therapy in T-cell acute lymphoblastic leukemia.

Notwithstanding intensified therapy, a considerable fraction of T-cell acute lymphoblastic leukemia (T-ALL) patients face a dismal prognosis due to primary resistance to treatment and relapse, raising the need for more efficient and targeted therapies. Hedgehog (HH) signaling is a major developmental pathway frequently deregulated in cancer, for which a role in T-ALL is emerging. Mounting evidence suggests that ligand-independent activation of HH pathway occurs in cancer including T-ALL, emphasizing the necessity of dissecting the complex interplay between HH and other signaling pathways regulating activation. In this work, we present a therapeutically relevant crosstalk between HH signaling and the glucocorticoid receptor (NR3C1) pathway acting at the level of GLI1 transcription factor. GLI inhibitor GANT61 and dexamethasone were shown to exert a synergistic anti-leukemic effect in vitro in T-ALL cell lines and patient-derived xenografts. Mechanistically, dexamethasone-activated NR3C1 impaired GLI1 function by dynamically modulating the recruitment of PCAF acetyltransferase and HDAC1 deacetylase. Increased GLI1 acetylation was associated with compromised transcriptional activity and reduced protein stability. In summary, our study identifies a novel crosstalk between GLI1 and NR3C1 signaling pathway which could be exploited in HH-dependent malignancies to increase therapeutic efficacy.

Cell-specific expression of ENACα gene by FOXA1 in the glucocorticoid receptor pathway.

INTRODUCTION: The glucocorticoid receptor (GR) is one of the most widely studied ligand-dependent nuclear receptors. The combination of transcriptional regulatory factors required for the expression of individual genes targeted by GR varies across cell types; however, the mechanisms underlying this cell type-specific regulation of gene expression are not yet clear. METHODS: Here, we investigated genes regulated by GR in two different cell lines, A549 and ARPE-19, and examined how gene expression varied according to the effect of pioneer factors using RNA-seq and RT-qPCR. RESULTS: Our RNA-seq results identified 19 and 63 genes regulated by GR that are ARPE-19-specific and A549-specific, respectively, suggesting that GR induces the expression of different sets of genes in a cell type-specific manner. RT-qPCR confirmed that the epithelial sodium channel (ENACα) gene is an ARPE-19 cell-specific GR target gene, whereas the FK506 binding protein 5 (FKBP5) gene was A549 cell-specific. There was a significant decrease in ENACα expression in FOXA1-deficient ARPE-19 cells, suggesting that FOXA1 might function as a pioneer factor enabling the selective expression of ENACα in ARPE-19 cells but not in A549 cells. CONCLUSION: These findings indicate that ENACα expression in ARPE-19 cells is regulated by FOXA1 and provide insights into the molecular mechanisms of cell type-specific expression of GR-regulated genes.

Application of generalized concentration addition to predict mixture effects of glucocorticoid receptor ligands.

Environmental exposures often occur in complex mixtures and at low concentrations. Generalized concentration addition (GCA) is a method used to estimate the joint effect of receptor ligands that vary in efficacy. GCA models have been successfully applied to mixtures of aryl hydrocarbon receptor (AhR) and peroxisome proliferator-activated receptor gamma (PPARγ) ligands, each of which can be modeled as a receptor with a single binding site. Here, we evaluated whether GCA could be applied to homodimer nuclear receptors, which have two binding sites, to predict the combined effect of full glucocorticoid receptor (GR) agonists with partial agonists. We measured transcriptional activation of GR using a cell-based bioassay. Individual concentration-response curves for dexamethasone (full agonist), prednisolone (full agonist), and medroxyprogesterone 17-acetate (partial agonist) were generated and applied in three additivity models, GCA, effect summation (ES), and relative potency factor (RPF), to generate response surfaces. GCA and RPF yielded adequate predictions of the experimental data for two full agonists. However, GCA fit experimental data significantly better than ES and RPF for all other binary mixtures. This work extends the application of GCA to homodimer nuclear receptors and improves prediction accuracy of mixture effects of GR agonists.

Nephrotic syndrome-associated hypercoagulopathy is alleviated by both pioglitazone and glucocorticoid which target two different nuclear receptors.

BACKGROUND: Thrombosis is a potentially life-threatening nephrotic syndrome (NS) complication. We have previously demonstrated that hypercoagulopathy is proportional to NS severity in rat models and that pioglitazone (Pio) reduces proteinuria both independently and in combination with methylprednisolone (MP), a glucocorticoid (GC). However, the effect of these treatments on NS-associated hypercoagulopathy remains unknown. We thus sought to determine the ability of Pio and GC to alleviate NS-associated hypercoagulopathy. METHODS: Puromycin aminonucleoside-induced rat NS was treated with sham, Low- or High-dose MP, Pio, or combination (Pio + Low-MP) and plasma was collected at day 11. Plasma samples were collected from children with steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS) upon presentation and after 7 weeks of GC therapy. Plasma endogenous thrombin potential (ETP), antithrombin (AT) activity, and albumin (Alb) were measured using thrombin generation, amidolytic, and colorimetric assays, respectively. RESULTS: In a rat model of NS, both High-MP and Pio improved proteinuria and corrected hypoalbuminemia, ETP and AT activity (p < .05). Proteinuria (p = .005) and hypoalbuminemia (p < .001) were correlated with ETP. In childhood NS, while ETP was not different at presentation, GC therapy improved proteinuria, hypoalbuminemia, and ETP in children with SSNS (p < .001) but not SRNS (p = .330). CONCLUSIONS: Both Pio and GC diminish proteinuria and significantly alleviate hypercoagulopathy. Both Pio and MP improved hypercoagulopathy in rats, and successful GC therapy (SSNS) also improved hypercoagulopathy in childhood NS. These data suggest that even a partial reduction in proteinuria may reduce NS-associated thrombotic risk.