Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding.

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and Kd values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.

Reprogramming of glucocorticoid receptor function by hypoxia.

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

ZBTB32 performs crosstalk with the glucocorticoid receptor and is crucial in glucocorticoid responses to starvation.

The hypothalamic-pituitary-adrenal (HPA) axis forms a complex neuroendocrine system that regulates the body's response to stress such as starvation. In contrast with the glucocorticoid receptor (GR), Zinc finger and BTB domain containing 32 (ZBTB32) is a transcription factor with poorly described functional relevance in physiology. This study shows that ZBTB32 is essential for the production of glucocorticoids (GCs) in response to starvation, since ZBTB32-/- mice fail to increase their GC production in the absence of nutrients. In terms of mechanism, GR-mediated upregulation of adrenal Scarb1 gene expression was absent in ZBTB32-/- mice, implicating defective cholesterol import as the cause of the poor GC synthesis. These lower GC levels are further associated with aberrations in the metabolic adaptation to starvation, which could explain the progressive weight gain of ZBTB32-/- mice. In conclusion, ZBTB32 performs a crosstalk with the GR in the metabolic adaptation to starvation via regulation of adrenal GC production.

Combined glucocorticoid resistance and hyperlactatemia contributes to lethal shock in sepsis.

Sepsis is a potentially lethal syndrome resulting from a maladaptive response to infection. Upon infection, glucocorticoids are produced as a part of the compensatory response to tolerate sepsis. This tolerance is, however, mitigated in sepsis due to a quickly induced glucocorticoid resistance at the level of the glucocorticoid receptor. Here, we show that defects in the glucocorticoid receptor signaling pathway aggravate sepsis pathophysiology by lowering lactate clearance and sensitizing mice to lactate-induced toxicity. The latter is exerted via an uncontrolled production of vascular endothelial growth factor, resulting in vascular leakage and collapse with severe hypotension, organ damage, and death, all being typical features of a lethal form of sepsis. In conclusion, sepsis leads to glucocorticoid receptor failure and hyperlactatemia, which collectively leads to a lethal vascular collapse.

Zinc inhibits lethal inflammatory shock by preventing microbe-induced interferon signature in intestinal epithelium.

The cytokine TNF drives inflammatory diseases, e.g., Crohn's disease. In a mouse model of TNF-induced systemic inflammatory response syndrome (SIRS), severe impact on intestinal epithelial cells (IECs) is observed. Zinc confers complete protection in this model. We found that zinc no longer protects in animals which lack glucocorticoids (GCs), or express mutant versions of their receptor GR in IECs, nor in mice which lack gut microbiota. RNA-seq studies in IECs showed that zinc caused reduction in expression of constitutive (STAT1-induced) interferon-stimulated response (ISRE) genes and interferon regulatory factor (IRF) genes. Since some of these genes are involved in TNF-induced cell death in intestinal crypt Paneth cells, and since zinc has direct effects on the composition of the gut microbiota (such as several Staphylococcus species) and on TNF-induced Paneth cell death, we postulate a new zinc-related anti-inflammatory mechanism. Zinc modulates the gut microbiota, causing less induction of ISRE/IRF genes in crypt cells, less TNF-induced necroptosis in Paneth cells, and less fatal evasion of gut bacteria into the system.

Glucocorticoids limit lipopolysaccharide-induced lethal inflammation by a double control system.

Lipopolysaccharides (LPS) can lead to a lethal endotoxemia, which is a systemic inflammatory response syndrome (SIRS) characterized by a systemic release of cytokines, such as TNF. Endotoxemia is studied intensely, as a model system of Gram-negative infections. LPS- and TNF-induced SIRS involve a strong induction of interferon-stimulated genes (ISGs), some of which cause cell death in the intestinal epithelium cells (IECs). It is well known that glucocorticoids (GCs) protect against endotoxemia. By applying numerous mutant mouse lines, our data support a model whereby GCs, via their glucocorticoid receptor (GR), apply two key mechanisms to control endotoxemia, (i) at the level of suppression of TNF production in a GR monomer-dependent way in macrophages and (ii) at the level of inhibition of TNFR1-induced ISG gene expression and necroptotic cell death mediators in IECs in a GR dimer-dependent way. Our data add new important insights to the understanding of the role of TNF in endotoxemia and the two separate key roles of GCs in suppressing TNF production and activity.

Potential of glucocorticoids to treat intestinal inflammation during sepsis.

Glucocorticoids (GCs) are steroid hormones characterized by their anti-inflammatory and immunosuppressive nature. Although GCs are very commonly prescribed, in several diseases, including sepsis, their clinical treatment is hampered by side effects and by the occurrence of glucocorticoid resistance (GCR). Sepsis is defined as a life-threatening organ dysfunction, initiated by a dysregulated systemic host response to infections. With at least 19 million cases per year and a lethality rate of about 25%, sepsis is one of the most urgent unmet medical needs. The gut is critically affected during sepsis and is considered as a driving force in this disease. Despite there is no effective treatment for sepsis, pre-clinical studies show promising results by preserving or restoring gut integrity. Since GC treatment reveals therapeutic effects in Crohn's disease (CD) and in pre-clinical sepsis models, we hypothesize that targeting GCs to the gut or stimulating local GC production in the gut forms an interesting strategy for sepsis treatment. According to recent findings that show that dimerization of the glucocorticoid receptor (GR) is essential in inducing anti-inflammatory effects in pre-clinical sepsis models, we predict that new generation GCs that selectively dimerize the GR, can therefore positively affect the outcome of sepsis treatment.

Hepatic PPARα function and lipid metabolic pathways are dysregulated in polymicrobial sepsis.

Despite intensive research and constant medical progress, sepsis remains one of the most urgent unmet medical needs of today. Most studies have been focused on the inflammatory component of the disease; however, recent advances support the notion that sepsis is accompanied by extensive metabolic perturbations. During times of limited caloric intake and high energy needs, the liver acts as the central metabolic hub in which PPARα is crucial to coordinate the breakdown of fatty acids. The role of hepatic PPARα in liver dysfunction during sepsis has hardly been explored. We demonstrate that sepsis leads to a starvation response that is hindered by the rapid decline of hepatic PPARα levels, causing excess free fatty acids, leading to lipotoxicity, and glycerol. In addition, treatment of mice with the PPARα agonist pemafibrate protects against bacterial sepsis by improving hepatic PPARα function, reducing lipotoxicity and tissue damage. Since lipolysis is also increased in sepsis patients and pemafibrate protects after the onset of sepsis, these findings may point toward new therapeutic leads in sepsis.

An extracellular microRNA can rescue lives in sepsis.

Sepsis, or blood poisoning, is a savage response of the body to infection. It can lead to organ failure, blood pressure decline, heart failure, and coma. Between 20 and 30 million people suffer from sepsis each year, leading to 8 million deaths. Although certain people are more at risk than others (young children, elderly), anyone can develop sepsis. Patients are resuscitated and treated with antibiotics, and their organ functions are supported. Despite the investment in sepsis research during the previous decades, successful clinical trials are scarce and sepsis remains one of the most difficult and deadly unmet medical needs of today. A study in this issue now provides new insight into sepsis and points to a therapeutic future [1 ].

Cognitive dysfunction in mice lacking proper glucocorticoid receptor dimerization.

Stress is a major risk factor for depression and anxiety. One of the effects of stress is the (over-) activation of the hypothalamic-pituitary-adrenal (HPA) axis and the release of stress hormones such as glucocorticoids (GCs). Chronically increased stress hormone levels have been shown to have detrimental effects on neuronal networks by inhibiting neurotrophic processes particularly in the hippocampus proper. Centrally, GCs modulate metabolic as well as behavioural processes by activating two classes of corticoid receptors, high-affinity mineralocorticoid receptors (MR) and low-affinity glucocorticoid receptors (GR). Upon activation, GR can modulate gene transcription either as a monomeric protein, or as a dimer interacting directly with DNA. GR can also modulate cellular processes via non-genomic mechanisms, for example via a GPCR-protein interaction. We evaluated the behavioral phenotype in mice with a targeted mutation in the GR in a FVB/NJ background. In GRdim/dim mice, GR proteins form poor homodimers, while the GR monomer remains intact. We evaluated the effect of poor GR dimerization on hippocampus-dependent cognition as well as on exploration and emotional behavior under baseline and chronically increased stress hormone levels. We found that GRdim/dim mice did not behave differently from GRwt/wt littermates under baseline conditions. However, after chronic elevation of stress hormone levels, GRdim/dim mice displayed a significant impairment in hippocampus-dependent memory compared to GRwt/wt mice, which correlated with differential expression of hippocampal Bdnf/TrkB and Fkbp5.

TNF-α inhibits glucocorticoid receptor-induced gene expression by reshaping the GR nuclear cofactor profile.

Glucocorticoid resistance (GCR) is defined as an unresponsiveness to the therapeutic effects, including the antiinflammatory ones of glucocorticoids (GCs) and their receptor, the glucocorticoid receptor (GR). It is a problem in the management of inflammatory diseases and can be congenital as well as acquired. The strong proinflammatory cytokine TNF-alpha (TNF) induces an acute form of GCR, not only in mice, but also in several cell lines: e.g., in the hepatoma cell line BWTG3, as evidenced by impaired Dexamethasone (Dex)-stimulated direct GR-dependent gene up- and down-regulation. We report that TNF has a significant and broad impact on this transcriptional performance of GR, but no impact on nuclear translocation, dimerization, or DNA binding capacity of GR. Proteome-wide proximity-mapping (BioID), however, revealed that the GR interactome was strongly modulated by TNF. One GR cofactor that interacted significantly less with the receptor under GCR conditions is p300. NFκB activation and p300 knockdown both reduced direct transcriptional output of GR whereas p300 overexpression and NFκB inhibition reverted TNF-induced GCR, which is in support of a cofactor reshuffle model. This hypothesis was supported by FRET studies. This mechanism of GCR opens avenues for therapeutic interventions in GCR diseases.

Should we target TNF receptors in the intestinal epithelium with glucocorticoids during systemic inflammation?

INTRODUCTION: Reverting Systemic inflammatory response syndromes (SIRS), particularly sepsis, is a huge challenge of contemporary medicine. Inhibition of the cytokine tumor necrosis factor alpha (TNFα), originally considered as a mediator in sepsis, has led to frustrating results. Equally so, glucocorticoids (GCs), renowned for their role in numerous inflammatory diseases, remain controversial in sepsis. Areas covered: We discuss how, in SIRS, the intestinal epithelium is a critical TNF-responsive target. Inhibition of TNF receptor 1 (TNFR1), rather than TNF, may be a more targeted and safe therapeutic approach. In intestinal epithelial cells (IECs), a strong interplay between GCs and TNF exists. Addressing GCs in these cells is crucial in SIRS and sepsis and would avoid dose-limiting off-target effects, for example on immune cells and phagocytes. Expert opinion: The targeting of TNFR1 specifically at the level of IECs, potentially combined with IEC-specific stimulation of GR, could lead to a more safe and targeted treatment for SIRS and sepsis.

A screening assay for Selective Dimerizing Glucocorticoid Receptor Agonists and Modulators (SEDIGRAM) that are effective against acute inflammation.

It has been suggested that glucocorticoid receptor (GR) agonists that promote GR homodimerization more than standard glucocorticoids such as Dexamethasone could be more effective anti-inflammatory molecules against acute and life-threatening inflammatory conditions. To test this hypothesis, we set up a screening pipeline aimed at discovering such Selective Dimerizing GR Agonists and Modulators (SEDIGRAM). The pipeline consists of a reporter gene assay based on a palindromic glucocorticoid responsive element (GRE). This assay represents GR dimerization in human A549 lung epithelial cells. In the pipeline, this is followed by analysis of endogenous GRE-driven gene expression, a FRET assay confirming dimerization, and monitoring of in vitro and in vivo anti-inflammatory activity. In a proof of principle experiment, starting from seven candidate compounds, we identified two potentially interesting compounds (Cortivazol and AZD2906) that confer strong protection in a mouse model of aggressive TNF-induced lethal inflammation. A screening pipeline for SEDIGRAM may assist the search for compounds that promote GR dimerization and limit overwhelming acute inflammatory responses.

Using the inbred mouse strain SPRET/EiJ to provide novel insights in inflammation and infection research.

Inbred mouse strains derived from the species Mus spretus have been very informative in the study of certain gene polymorphisms in inflammation and infection. Based on our interest in sepsis, we used SPRET/EiJ mice and mapped several critical loci that are linked to sensitivity to cytokine-induced inflammation and endotoxemia. These studies were based on prominent phenotypes that have never been observed in strains derived from Mus musculus and we mapped them at a resolution that enables us to draw conclusions on the mechanisms. Now that the genome of SPRET/EiJ has been sequenced, and other tools have become available, it is time to revisit this strain and emphasize its advantages and disadvantages as a research tool and a discovery platform.

Glucocorticoid receptor dimers control intestinal STAT1 and TNF-induced inflammation in mice.

TNF is an important mediator in numerous inflammatory diseases, e.g., in inflammatory bowel diseases (IBDs). In IBD, acute increases in TNF production can lead to disease flares. Glucocorticoids (GCs), which are steroids that bind and activate the glucocorticoid receptor (GR), are able to protect animals and humans against acute TNF-induced inflammatory symptoms. Mice with a poor transcriptional response of GR dimer-dependent target genes were studied in a model of TNF-induced lethal inflammation. In contrast to the GRWT/WT mice, these GRdim/dim mice displayed a substantial increase in TNF sensitivity and a lack of protection by the GC dexamethasone (DEX). Unchallenged GRdim/dim mice had a strong IFN-stimulated gene (ISG) signature, along with STAT1 upregulation and phosphorylation. This ISG signature was gut specific and, based on our studies with antibiotics, depended on the gut microbiota. GR dimers directly bound to short DNA sequences in the STAT1 promoter known as inverted repeat negative GRE (IR-nGRE) elements. Poor control of STAT1 in GRdim/dim mice led to failure to repress ISG genes, resulting in excessive necroptosis induction by TNF. Our findings support a critical interplay among gut microbiota, IFNs, necroptosis, and GR in both the basal response to acute inflammatory challenges and pharmacological intervention by GCs.

The nature of the GRE influences the screening for GR-activity enhancing modulators.

Glucocorticoid resistance (GCR), i.e. unresponsiveness to the beneficial anti-inflammatory activities of the glucocorticoid receptor (GR), poses a serious problem in the treatment of inflammatory diseases. One possible solution to try and overcome GCR, is to identify molecules that prevent or revert GCR by hyper-stimulating the biological activity of the GR. To this purpose, we screened for compounds that potentiate the dexamethasone (Dex)-induced transcriptional activity of GR. To monitor GR transcriptional activity, the screen was performed using the lung epithelial cell line A549 in which a glucocorticoid responsive element (GRE) coupled to a luciferase reporter gene construct was stably integrated. Histone deacetylase inhibitors (HDACi) such as Vorinostat and Belinostat are two broad-spectrum HDACi that strongly increased the Dex-induced luciferase expression in our screening system. In sharp contrast herewith, results from a genome-wide transcriptome analysis of Dex-induced transcripts using RNAseq, revealed that Belinostat impairs the ability of GR to transactivate target genes. The stimulatory effect of Belinostat in the luciferase screen further depends on the nature of the reporter construct. In conclusion, a profound discrepancy was observed between HDACi effects on two different synthetic promoter-luciferase reporter systems. The favorable effect of HDACi on gene expression should be evaluated with care, when considering them as potential therapeutic agents. GEO accession number GSE96649.