Crosstalk interactions between transcription factors ERRα and PPARα assist PPARα-mediated gene expression.

OBJECTIVE: The peroxisome proliferator-activated receptor α (PPARα) is a transcription factor driving target genes involved in fatty acid ß-oxidation. To what extent various PPARα interacting proteins may assist its function as a transcription factor is incompletely understood. An ORFeome-wide unbiased mammalian protein-protein interaction trap (MAPPIT) using PPARα as bait revealed a PPARα-ligand-dependent interaction with the orphan nuclear receptor estrogen-related receptor α (ERRα). The goal of this study was to characterize the nature of the interaction in depth and to explore whether it was of physiological relevance. METHODS: We used orthogonal protein-protein interaction assays and pharmacological inhibitors of ERRα in various systems to confirm a functional interaction and study the impact of crosstalk mechanisms. To characterize the interaction surfaces and contact points we applied a random mutagenesis screen and structural overlays. We pinpointed the extent of reciprocal ligand effects of both nuclear receptors via coregulator peptide recruitment assays. On PPARα targets revealed from a genome-wide transcriptome analysis, we performed an ERRα chromatin immunoprecipitation analysis on both fast and fed mouse livers. RESULTS: Random mutagenesis scanning of PPARα's ligand-binding domain and coregulator profiling experiments supported the involvement of (a) bridging coregulator(s), while recapitulation of the interaction in vitro indicated the possibility of a trimeric interaction with RXRα. The PPARα·ERRα interaction depends on 3 C-terminal residues within helix 12 of ERRα and is strengthened by both PGC1α and serum deprivation. Pharmacological inhibition of ERRα decreased the interaction of ERRα to ligand-activated PPARα and revealed a transcriptome in line with enhanced mRNA expression of prototypical PPARα target genes, suggesting a role for ERRα as a transcriptional repressor. Strikingly, on other PPARα targets, including the isolated PDK4 enhancer, ERRα behaved oppositely. Chromatin immunoprecipitation analyses demonstrate a PPARα ligand-dependent ERRα recruitment onto chromatin at PPARα-binding regions, which is lost following ERRα inhibition in fed mouse livers. CONCLUSIONS: Our data support the coexistence of multiple layers of transcriptional crosstalk mechanisms between PPARα and ERRα, which may serve to finetune the activity of PPARα as a nutrient-sensing transcription factor.

Spns2/S1P: it takes two to tango with inflammation and metabolic rewiring during sepsis.

Sepsis is the result of a dysregulated host response to an infection and causes high morbidity and mortality at the intensive care units worldwide. Despite intensive research, the current management of sepsis is supportive rather than curative. Therefore, new therapeutic interventions for sepsis and septic shock patients are urgently needed. In this issue of EMBO Reports, Fang et al have used rat sepsis models to show that macrophage-expressed SPNS2, a major transporter of S1P, is a crucial mediator of metabolic reprogramming of macrophages during sepsis which regulates inflammation via the lactate-ROS axis.

The impact of hepatocyte-specific deletion of hypoxia-inducible factors on the development of polymicrobial sepsis with focus on GR and PPARα function.

Introduction: Polymicrobial sepsis causes acute anorexia (loss of appetite), leading to lipolysis in white adipose tissue and proteolysis in muscle, and thus release of free fatty acids (FFAs), glycerol and gluconeogenic amino acids. Since hepatic peroxisome proliferator-activated receptor alpha (PPARα) and glucocorticoid receptor (GR) quickly lose function in sepsis, these metabolites accumulate (causing toxicity) and fail to yield energy-rich molecules such as ketone bodies (KBs) and glucose. The mechanism of PPARα and GR dysfunction is not known. Methods & results: We investigated the hypothesis that hypoxia and/or activation of hypoxia inducible factors (HIFs) might play a role in these issues with PPARα and GR. After cecal ligation and puncture (CLP) in mice, leading to lethal polymicrobial sepsis, bulk liver RNA sequencing illustrated the induction of the genes encoding HIF1α and HIF2α, and an enrichment of HIF-dependent gene signatures. Therefore, we generated hepatocyte-specific knock-out mice for HIF1α, HIF2α or both, and a new HRE-luciferase reporter mouse line. After CLP, these HRE-luciferase reporter mice show signals in several tissues, including the liver. Hydrodynamic injection of an HRE-luciferase reporter plasmid also led to (liver-specific) signals in hypoxia and CLP. Despite these encouraging data, however, hepatocyte-specific HIF1α and/or HIF2α knock-out mice suggest that survival after CLP was not dependent on the hepatocyte-specific presence of HIF proteins, which was supported by measuring blood levels of glucose, FFAs, and KBs. The HIF proteins were also irrelevant in the CLP-induced glucocorticoid resistance, but we found indications that the absence of HIF1α in hepatocytes causes less inactivation of PPARα transcriptional function. Conclusion: We conclude that HIF1α and HIF2α are activated in hepatocytes in sepsis, but their contribution to the mechanisms leading to lethality are minimal.

Hepatic Peroxisome Proliferator-Activated Receptor Alpha Dysfunction in Porcine Septic Shock.

Despite decades of research, sepsis remains one of the most urgent unmet medical needs. Mechanistic investigations into sepsis have mainly focused on targeting inflammatory pathways; however, recent data indicate that sepsis should also be seen as a metabolic disease. Targeting metabolic dysregulations that take place in sepsis might uncover novel therapeutic opportunities. The role of peroxisome proliferator-activated receptor alpha (PPARɑ) in liver dysfunction during sepsis has recently been described, and restoring PPARɑ signaling has proven to be successful in mouse polymicrobial sepsis. To confirm that such therapy might be translated to septic patients, we analyzed metabolic perturbations in the liver of a porcine fecal peritonitis model. Resuscitation with fluids, vasopressor, antimicrobial therapy and abdominal lavage were applied to the pigs in order to mimic human clinical care. By using RNA-seq, we detected downregulated PPARɑ signaling in the livers of septic pigs and that reduced PPARɑ levels correlated well with disease severity. As PPARɑ regulates the expression of many genes involved in fatty acid oxidation, the reduced expression of these target genes, concomitant with increased free fatty acids in plasma and ectopic lipid deposition in the liver, was observed. The results obtained with pigs are in agreement with earlier observations seen in mice and support the potential of targeting defective PPARɑ signaling in clinical research.

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.

Bidirectional Crosstalk Between Hypoxia Inducible Factors and Glucocorticoid Signalling in Health and Disease.

Glucocorticoid-induced (GC) and hypoxia-induced transcriptional responses play an important role in tissue homeostasis and in the regulation of cellular responses to stress and inflammation. Evidence exists that there is an important crosstalk between both GC and hypoxia effects. Hypoxia is a pathophysiological condition to which cells respond quickly in order to prevent metabolic shutdown and death. The hypoxia inducible factors (HIFs) are the master regulators of oxygen homeostasis and are responsible for the ability of cells to cope with low oxygen levels. Maladaptive responses of HIFs contribute to a variety of pathological conditions including acute mountain sickness (AMS), inflammation and neonatal hypoxia-induced brain injury. Synthetic GCs which are analogous to the naturally occurring steroid hormones (cortisol in humans, corticosterone in rodents), have been used for decades as anti-inflammatory drugs for treating pathological conditions which are linked to hypoxia (i.e. asthma, ischemic injury). In this review, we investigate the crosstalk between the glucocorticoid receptor (GR), and HIFs. We discuss possible mechanisms by which GR and HIF influence one another, in vitro and in vivo, and the therapeutic effects of GCs on HIF-mediated diseases.

Turning a pathogen protein into a therapeutic tool for sepsis.

Sepsis causes unacceptably high amounts of deaths worldwide. It is a huge unmet medical need, and new therapeutic interventions for sepsis and septic shock are urgently needed. By studying the mechanism by which a bacterial protein undermines the inflammatory function of macrophages, Kim et al, in the last issue of EMBO Molecular Medicine, have developed a new therapeutic protein drug, which appears to have very promising protective activities in a well-validated and aggressive polymicrobial sepsis model in mice. The chimeric protein is thought to limit macrophage inflammation while activating phagocytosis, and so, it hits two macrophage pathways at once.

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.

Taking the STING Out of Sepsis?

In this issue of Cell Host & Microbe, Zhang et al. use a sepsis mouse model to show that macrophage-specific release of coagulation factor F3 depends on pathogen detection and responses mediated by TMEM173/STING. The therapeutic power of targeting TMEM173/STING-F3 is evident in mice, but will it penetrate the sepsis bedside?

Hypoxia-inducible factors in metabolic reprogramming during sepsis.

Sepsis is a highly heterogeneous syndrome that is caused by an imbalanced host response to infection. Despite huge investments, sepsis remains a contemporary threat with significant burden on health systems. Vascular dysfunction and elevated oxygen consumption by highly metabolically active immune cells result in tissue hypoxia during inflammation. The transcription factor hypoxia-inducible factor-1a (HIF1α), and its family members, plays an important role in cellular metabolism and adaptation to cellular stress caused by hypoxia. In this review, we discuss the role of HIF in sepsis. We show possible mechanisms by which the inflammatory response activated during sepsis affects the HIF pathway. The activated HIF pathway in turn changes the metabolism of both innate and adaptive immune cells. As HIF expression in leukocytes of septic patients can be directly linked with mortality, we discuss multiple ways of interfering with the HIF signaling pathway.

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.

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.

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.