ABSTRACT
The clinical course of COVID-19 is variable and often unpredictable. To test the hypothesis that disease progression and inflammatory responses associate with alterations in the microbiome and metabolome, we analyzed metagenome, metabolome, cytokine, and transcriptome profiles of repeated samples from hospitalized COVID-19 patients and uninfected controls, and leveraged clinical information and post-hoc confounder analysis. Severe COVID-19 was associated with a depletion of beneficial intestinal microbes, whereas oropharyngeal microbiota disturbance was mainly linked to antibiotic use. COVID-19 severity was also associated with enhanced plasma concentrations of kynurenine and reduced levels of several other tryptophan metabolites, lysophosphatidylcholines, and secondary bile acids. Moreover, reduced concentrations of various tryptophan metabolites were associated with depletion of Faecalibacterium, and tryptophan decrease and kynurenine increase were linked to enhanced production of inflammatory cytokines. Collectively, our study identifies correlated microbiome and metabolome alterations as a potential contributor to inflammatory dysregulation in severe COVID-19.
Subject(s)
COVID-19 , Cytokines , Dysbiosis , Gastrointestinal Microbiome , SARS-CoV-2 , Tryptophan , Humans , COVID-19/microbiology , COVID-19/immunology , Tryptophan/metabolism , Male , Female , Middle Aged , Cytokines/blood , Cytokines/metabolism , Metabolome , Inflammation , Kynurenine/metabolism , Kynurenine/blood , Aged , AdultABSTRACT
Hospital-acquired pneumonia (HAP) is associated with high mortality and costs, and frequently caused by multidrug-resistant (MDR) bacteria. Although prior antimicrobial therapy is a major risk factor for HAP, the underlying mechanism remains incompletely understood. Here, we demonstrate that antibiotic therapy in hospitalized patients is associated with decreased diversity of the gut microbiome and depletion of short-chain fatty acid (SCFA) producers. Infection experiments with mice transplanted with patient fecal material reveal that these antibiotic-induced microbiota perturbations impair pulmonary defense against MDR Klebsiella pneumoniae. This is dependent on inflammatory monocytes (IMs), whose fatty acid receptor (FFAR)2/3-controlled and phagolysosome-dependent antibacterial activity is compromized in mice transplanted with antibiotic-associated patient microbiota. Collectively, we characterize how clinically relevant antibiotics affect antimicrobial defense in the context of human microbiota, and reveal a critical impairment of IM´s antimicrobial activity. Our study provides additional arguments for the rational use of antibiotics and offers mechanistic insights for the development of novel prophylactic strategies to protect high-risk patients from HAP.
Subject(s)
Anti-Bacterial Agents , Anti-Infective Agents , Humans , Mice , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Monocytes , Anti-Infective Agents/pharmacology , Klebsiella pneumoniae , LungABSTRACT
AIMS: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15â kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown. METHODS AND RESULTS: Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system. CONCLUSION: Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.
Subject(s)
Coxsackievirus Infections , Cytokines , Energy Metabolism , Glycolysis , Mitochondria, Heart , Myocytes, Cardiac , Ubiquitins , Animals , Humans , Male , Coxsackievirus Infections/metabolism , Coxsackievirus Infections/virology , Coxsackievirus Infections/genetics , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Enterovirus B, Human/pathogenicity , Enterovirus B, Human/metabolism , Host-Pathogen Interactions , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Heart/metabolism , Mitochondria, Heart/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/virology , Myocytes, Cardiac/pathology , Protein Processing, Post-Translational , Signal Transduction , Ubiquitins/metabolism , Ubiquitins/geneticsABSTRACT
BACKGROUND: CKD is characterized by a sustained proinflammatory response of the immune system, promoting hypertension and cardiovascular disease. The underlying mechanisms are incompletely understood but may be linked to gut dysbiosis. Dysbiosis has been described in adults with CKD; however, comorbidities limit CKD-specific conclusions. METHODS: We analyzed the fecal microbiome, metabolites, and immune phenotypes in 48 children (with normal kidney function, CKD stage G3-G4, G5 treated by hemodialysis [HD], or kidney transplantation) with a mean±SD age of 10.6±3.8 years. RESULTS: Serum TNF-α and sCD14 were stage-dependently elevated, indicating inflammation, gut barrier dysfunction, and endotoxemia. We observed compositional and functional alterations of the microbiome, including diminished production of short-chain fatty acids. Plasma metabolite analysis revealed a stage-dependent increase of tryptophan metabolites of bacterial origin. Serum from patients on HD activated the aryl hydrocarbon receptor and stimulated TNF-α production in monocytes, corresponding to a proinflammatory shift from classic to nonclassic and intermediate monocytes. Unsupervised analysis of T cells revealed a loss of mucosa-associated invariant T (MAIT) cells and regulatory T cell subtypes in patients on HD. CONCLUSIONS: Gut barrier dysfunction and microbial metabolite imbalance apparently mediate the proinflammatory immune phenotype, thereby driving the susceptibility to cardiovascular disease. The data highlight the importance of the microbiota-immune axis in CKD, irrespective of confounding comorbidities.
Subject(s)
Cardiovascular Diseases , Gastrointestinal Microbiome , Renal Insufficiency, Chronic , Humans , Dysbiosis/microbiology , Gastrointestinal Microbiome/physiology , Inflammation , Renal Insufficiency, Chronic/complications , Renal Insufficiency, Chronic/therapy , Renal Insufficiency, Chronic/metabolism , Tumor Necrosis Factor-alpha , Child , AdolescentABSTRACT
Recently, there has been growing interest in short-chain fatty acids (SCFA) and ketone bodies (KB) due to their potential use as biomarkers of health and disease. For instance, these diet-related metabolites can be used to monitor and reduce the risk of immune response, diabetes, or cardiovascular diseases. Given the interest in these metabolites, different targeted metabolomic methods based on UPLC-MS/MS have been developed in recent years to detect and quantify SCFA and KB. In this case study, we discovered that applying an existing validated, targeted UPLC-MS/MS method to mouse plasma, resulted in a fragment ion (194 m/z) being originally misidentified as acetic acid (a SCFA), when its original source was 3-hydroxybutyric acid (a KB). Therefore, we report a modified, optimized LC method that can separate both signals. In addition, the metabolite coverage was expanded in this method to detect up to eight SCFA: acetic, propanoic, butyric, isobutyric, 2-methylbutyric, valeric, isovaleric, and hexanoic acids, two KB: 3-hydroxybutyric, and acetoacetic acids, and one related metabolite: 3-hydroxy-3-methylbutyric acid. The optimization of this method increased the selectivity of the UPLC-MS/MS method towards the misidentified compound. These findings encourage the scientific community to increase efforts in validating the original precursor of small molecule fragments in targeted methods.
Subject(s)
Fatty Acids, Volatile , Tandem Mass Spectrometry , Animals , Chromatography, Liquid/methods , Fatty Acids, Volatile/metabolism , Ketone Bodies , Mice , Plasma , Tandem Mass Spectrometry/methodsABSTRACT
Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.
Subject(s)
Endothelial Cells/enzymology , Endothelial Cells/pathology , Glutamate-Ammonia Ligase/metabolism , Glutamine/biosynthesis , Neovascularization, Pathologic , Actins/metabolism , Animals , Cell Movement , Endothelial Cells/metabolism , Female , Glutamate-Ammonia Ligase/deficiency , Glutamate-Ammonia Ligase/genetics , Glutamate-Ammonia Ligase/physiology , HEK293 Cells , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/enzymology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Lipoylation , Mice , Palmitic Acid/metabolism , Protein Processing, Post-Translational , Stress Fibers/metabolism , rho GTP-Binding Proteins/chemistry , rho GTP-Binding Proteins/metabolism , rho-Associated Kinases/metabolismABSTRACT
The role of fatty acid synthesis in endothelial cells (ECs) remains incompletely characterized. We report that fatty acid synthase knockdown (FASNKD) in ECs impedes vessel sprouting by reducing proliferation. Endothelial loss of FASN impaired angiogenesis in vivo, while FASN blockade reduced pathological ocular neovascularization, at >10-fold lower doses than used for anti-cancer treatment. Impaired angiogenesis was not due to energy stress, redox imbalance, or palmitate depletion. Rather, FASNKD elevated malonyl-CoA levels, causing malonylation (a post-translational modification) of mTOR at lysine 1218 (K1218). mTOR K-1218 malonylation impaired mTOR complex 1 (mTORC1) kinase activity, thereby reducing phosphorylation of downstream targets (p70S6K/4EBP1). Silencing acetyl-CoA carboxylase 1 (an enzyme producing malonyl-CoA) normalized malonyl-CoA levels and reactivated mTOR in FASNKD ECs. Mutagenesis unveiled the importance of mTOR K1218 malonylation for angiogenesis. This study unveils a novel role of FASN in metabolite signaling that contributes to explaining the anti-angiogenic effect of FASN blockade.
Subject(s)
Fatty Acid Synthase, Type I/physiology , Human Umbilical Vein Endothelial Cells/metabolism , Malonyl Coenzyme A/metabolism , Retinal Neovascularization/pathology , TOR Serine-Threonine Kinases/metabolism , Acetyl-CoA Carboxylase/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell Proliferation , Fatty Acid Synthase, Type I/antagonists & inhibitors , Fatty Acid Synthase, Type I/genetics , Human Umbilical Vein Endothelial Cells/cytology , Humans , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Orlistat/therapeutic use , Protein Processing, Post-Translational , Retinal Neovascularization/drug therapyABSTRACT
Little is known about the metabolism of quiescent endothelial cells (QECs). Nonetheless, when dysfunctional, QECs contribute to multiple diseases. Previously, we demonstrated that proliferating endothelial cells (PECs) use fatty acid ß-oxidation (FAO) for de novo dNTP synthesis. We report now that QECs are not hypometabolic, but upregulate FAO >3-fold higher than PECs, not to support biomass or energy production but to sustain the tricarboxylic acid cycle for redox homeostasis through NADPH regeneration. Hence, endothelial loss of FAO-controlling CPT1A in CPT1AΔEC mice promotes EC dysfunction (leukocyte infiltration, barrier disruption) by increasing endothelial oxidative stress, rendering CPT1AΔEC mice more susceptible to LPS and inflammatory bowel disease. Mechanistically, Notch1 orchestrates the use of FAO for redox balance in QECs. Supplementation of acetate (metabolized to acetyl-coenzyme A) restores endothelial quiescence and counters oxidative stress-mediated EC dysfunction in CPT1AΔEC mice, offering therapeutic opportunities. Thus, QECs use FAO for vasculoprotection against oxidative stress-prone exposure.
Subject(s)
Carnitine O-Palmitoyltransferase/metabolism , Energy Metabolism , Fatty Acids/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , NADP/metabolism , Receptor, Notch1/metabolism , Animals , Cell Proliferation , HEK293 Cells , Homeostasis , Humans , Mice , Mice, Inbred C57BL , Oxidation-Reduction , Oxidative StressABSTRACT
Blockade of the glycolytic activator PFKFB3 in cancer cells (using a maximum tolerable dose of 70 mg/kg of the PFKFB3 blocker 3PO) inhibits tumor growth in preclinical models and is currently being tested as a novel anticancer treatment in phase I clinical trials. However, a detailed preclinical analysis of the effects of such maximum tolerable dose of a PFKFB3 blocker on the tumor vasculature is lacking, even though tumor endothelial cells are hyper-glycolytic. We report here that a high dose of 3PO (70 mg/kg), which inhibits cancer cell proliferation and reduces primary tumor growth, causes tumor vessel disintegration, suppresses endothelial cell growth for protracted periods, (model-dependently) aggravates tumor hypoxia, and compromises vascular barrier integrity, thereby rendering tumor vessels more leaky and facilitating cancer cell intravasation and dissemination. These findings contrast to the effects of a low dose of 3PO (25 mg/kg), which induces tumor vessel normalization, characterized by vascular barrier tightening and maturation, but reduces cancer cell intravasation and metastasis. Our findings highlight the importance of adequately dosing a glycolytic inhibitor for anticancer treatment.
Subject(s)
Neoplasms/blood supply , Neoplasms/drug therapy , Neovascularization, Pathologic/drug therapy , Phosphofructokinase-2/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Melanoma, Experimental/blood supply , Melanoma, Experimental/pathology , Melanoma, Experimental/ultrastructure , Mice, Inbred C57BL , Neoplasm Metastasis , Neoplasms/pathology , Neovascularization, Pathologic/pathology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Phosphofructokinase-2/metabolism , Pyridines/pharmacologyABSTRACT
Endothelial cell (EC) metabolism is emerging as a regulator of angiogenesis, but the precise role of glutamine metabolism in ECs is unknown. Here, we show that depriving ECs of glutamine or inhibiting glutaminase 1 (GLS1) caused vessel sprouting defects due to impaired proliferation and migration, and reduced pathological ocular angiogenesis. Inhibition of glutamine metabolism in ECs did not cause energy distress, but impaired tricarboxylic acid (TCA) cycle anaplerosis, macromolecule production, and redox homeostasis. Only the combination of TCA cycle replenishment plus asparagine supplementation restored the metabolic aberrations and proliferation defect caused by glutamine deprivation. Mechanistically, glutamine provided nitrogen for asparagine synthesis to sustain cellular homeostasis. While ECs can take up asparagine, silencing asparagine synthetase (ASNS, which converts glutamine-derived nitrogen and aspartate to asparagine) impaired EC sprouting even in the presence of glutamine and asparagine. Asparagine further proved crucial in glutamine-deprived ECs to restore protein synthesis, suppress ER stress, and reactivate mTOR signaling. These findings reveal a novel link between endothelial glutamine and asparagine metabolism in vessel sprouting.
Subject(s)
Asparagine/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Endothelial Cells/drug effects , Endothelial Cells/physiology , Glutamine/metabolism , Neovascularization, Physiologic/drug effects , Culture Media/chemistry , Endothelial Cells/metabolism , Glutaminase/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Metabolic Networks and Pathways , Neovascularization, PathologicABSTRACT
Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid ß-oxidation, impairs lymphatic development. LECs use fatty acid ß-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid ß-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1-p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo.
Subject(s)
Fatty Acids/chemistry , Fatty Acids/metabolism , Lymphangiogenesis , Lymphatic Vessels/cytology , Lymphatic Vessels/metabolism , Acetates/pharmacology , Acetyl Coenzyme A/metabolism , Acetylation/drug effects , Animals , Carnitine O-Palmitoyltransferase/antagonists & inhibitors , Carnitine O-Palmitoyltransferase/genetics , Carnitine O-Palmitoyltransferase/metabolism , Cell Differentiation/drug effects , Cell Differentiation/genetics , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Epigenesis, Genetic , Female , Histones/metabolism , Homeodomain Proteins/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Lymphangiogenesis/drug effects , Lymphangiogenesis/genetics , Lymphatic Vessels/drug effects , Mice , Mice, Inbred C57BL , Oxidation-Reduction/drug effects , Protein Biosynthesis , Transcription, Genetic , Tumor Suppressor Proteins/metabolism , Umbilical Arteries/cytology , Up-RegulationABSTRACT
Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1(-/-) hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease.
Subject(s)
Apoptosis/physiology , Hepatocytes/cytology , Hepatocytes/physiology , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , NF-kappa B/metabolism , Procollagen-Proline Dioxygenase/metabolism , Animals , Cell Hypoxia/physiology , Cell Line , Gene Expression Regulation, Enzymologic/physiology , HEK293 Cells , Humans , MiceABSTRACT
Several questions about the role of the oxygen sensor prolyl-hydroxylase 2 (PHD2) in cancer have not been addressed. First, the role of PHD2 in metastasis has not been studied in a spontaneous tumor model. Here, we show that global PHD2 haplodeficiency reduced metastasis without affecting tumor growth. Second, it is unknown whether PHD2 regulates cancer by affecting cancer-associated fibroblasts (CAFs). We show that PHD2 haplodeficiency reduced metastasis via two mechanisms: (1) by decreasing CAF activation, matrix production, and contraction by CAFs, an effect that surprisingly relied on PHD2 deletion in cancer cells, but not in CAFs; and (2) by improving tumor vessel normalization. Third, the effect of concomitant PHD2 inhibition in malignant and stromal cells (mimicking PHD2 inhibitor treatment) is unknown. We show that global PHD2 haplodeficiency, induced not only before but also after tumor onset, impaired metastasis. These findings warrant investigation of PHD2's therapeutic potential.
Subject(s)
Fibroblasts/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Neoplasms/metabolism , Animals , Cell Line, Tumor , Female , Humans , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Immunoblotting , Immunohistochemistry , Male , Mice , Models, Biological , Neoplasm Metastasis , Neoplasms/pathology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis.
Subject(s)
Carbon/metabolism , Endothelial Cells/metabolism , Fatty Acids/chemistry , Fatty Acids/metabolism , Nucleotides/biosynthesis , Acetic Acid/pharmacology , Adenosine Triphosphate/metabolism , Animals , Blood Vessels/cytology , Blood Vessels/drug effects , Blood Vessels/metabolism , Blood Vessels/pathology , Carnitine O-Palmitoyltransferase/antagonists & inhibitors , Carnitine O-Palmitoyltransferase/deficiency , Carnitine O-Palmitoyltransferase/genetics , Carnitine O-Palmitoyltransferase/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Citric Acid Cycle , DNA/biosynthesis , Disease Models, Animal , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Gene Silencing , Glucose/metabolism , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/pathology , Humans , Mice , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Nucleotides/chemistry , Nucleotides/pharmacology , Oxidation-Reduction/drug effects , Retinopathy of Prematurity/drug therapy , Retinopathy of Prematurity/metabolism , Retinopathy of Prematurity/pathologyABSTRACT
Hypoxia is a prominent feature of chronically inflamed tissues. Oxygen-sensing hydroxylases control transcriptional adaptation to hypoxia through the regulation of hypoxia-inducible factor (HIF) and nuclear factor κB (NF-κB), both of which can regulate the inflammatory response. Furthermore, pharmacologic hydroxylase inhibitors reduce inflammation in multiple animal models. However, the underlying mechanism(s) linking hydroxylase activity to inflammatory signaling remains unclear. IL-1ß, a major proinflammatory cytokine that regulates NF-κB, is associated with multiple inflammatory pathologies. We demonstrate that a combination of prolyl hydroxylase 1 and factor inhibiting HIF hydroxylase isoforms regulates IL-1ß-induced NF-κB at the level of (or downstream of) the tumor necrosis factor receptor-associated factor 6 complex. Multiple proteins of the distal IL-1ß-signaling pathway are subject to hydroxylation and form complexes with either prolyl hydroxylase 1 or factor inhibiting HIF. Thus, we hypothesize that hydroxylases regulate IL-1ß signaling and subsequent inflammatory gene expression. Furthermore, hydroxylase inhibition represents a unique approach to the inhibition of IL-1ß-dependent inflammatory signaling.
Subject(s)
Gene Expression Regulation/physiology , Hypoxia/physiopathology , Inflammation/physiopathology , Mixed Function Oxygenases/metabolism , NF-kappa B/metabolism , Signal Transduction/physiology , Analysis of Variance , Blotting, Western , HeLa Cells , Humans , Hydroxylation , Hypoxia/metabolism , Immunoprecipitation , Inflammation/metabolism , Interleukin-1beta/metabolism , Luciferases , Mass Spectrometry , Prolyl Hydroxylases/metabolism , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/metabolismABSTRACT
Activation of the hypoxia-inducible factor (HIF) pathway is a critical step in the transcriptional response to hypoxia. Although many of the key proteins involved have been characterised, the dynamics of their interactions in generating this response remain unclear. In the present study, we have generated a comprehensive mathematical model of the HIF-1α pathway based on core validated components and dynamic experimental data, and confirm the previously described connections within the predicted network topology. Our model confirms previous work demonstrating that the steps leading to optimal HIF-1α transcriptional activity require sequential inhibition of both prolyl- and asparaginyl-hydroxylases. We predict from our model (and confirm experimentally) that there is residual activity of the asparaginyl-hydroxylase FIH (factor inhibiting HIF) at low oxygen tension. Furthermore, silencing FIH under conditions where prolyl-hydroxylases are inhibited results in increased HIF-1α transcriptional activity, but paradoxically decreases HIF-1α stability. Using a core module of the HIF network and mathematical proof supported by experimental data, we propose that asparaginyl hydroxylation confers a degree of resistance upon HIF-1α to proteosomal degradation. Thus, through in vitro experimental data and in silico predictions, we provide a comprehensive model of the dynamic regulation of HIF-1α transcriptional activity by hydroxylases and use its predictive and adaptive properties to explain counter-intuitive biological observations.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mixed Function Oxygenases/metabolism , Models, Biological , Repressor Proteins/metabolism , Computational Biology , HEK293 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/pharmacology , Oxygen/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Stability , Proteolysis , RNA, Small Interfering/genetics , Repressor Proteins/genetics , Repressor Proteins/pharmacology , Signal Transduction , Transcriptional Activation/geneticsABSTRACT
Oxygen-sensing prolyl hydroxylase domain enzymes (PHDs) target hypoxia-inducible factor (HIF)-α subunits for proteasomal degradation in normoxia through hydroxylation. Recently, novel mechanisms of PHD activation and function have been unveiled. Interestingly, PHD3 can unexpectedly amplify HIF signaling through hydroxylation of the glycolytic enzyme pyruvate kinase (PK) muscle isoform 2 (PKM2). Recent studies have also yielded insight into HIF-independent PHD functions, including the control of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking in synaptic transmission and the activation of transient receptor potential cation channel member A1 (TRPA1) ion channels by oxygen levels in sensory nerves. Finally, PHD activation has been shown to involve the iron chaperoning function of poly(rC) binding protein (PCBP)1 and the (R)-enantiomer of 2-hydroxyglutarate (2-HG). The intersection of these regulatory pathways and interactions highlight the complexity of PHD regulation and function.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia/metabolism , Oxygen/metabolism , Procollagen-Proline Dioxygenase/metabolism , Signal Transduction , Animals , HumansABSTRACT
Carbon dioxide (CO(2)) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO(2) (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO(2) or associated acidosis to this response remain poorly understood. Recently, a role for the non-canonical NF-κB pathway has been postulated to be important in signaling the cellular transcriptional response to CO(2). In this study, we demonstrate that in cells exposed to elevated CO(2), the NF-κB family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3ß or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO(2)-sensitive NF-κB family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung.