ABSTRACT
Gene expression signatures associated with breast cancer metastases suggest that metabolic re-wiring is important for metastatic growth in lungs, bones, and other organs. However, since pathway fluxes depend on additional factors such as ATP demand, allosteric effects, and post-translational modification, flux analysis is necessary to conclusively establish phenotypes. In this study, the metabolic phenotypes of breast cancer cell lines with low (T47D) or high (MDA-MB-231) metastatic potential, as well as lung (LM)- and bone (BoM)-homing lines derived from MDA-MB-231 cells, were assessed by 13C metabolite labeling from [1,2-13C] glucose or [5-13C] glutamine and the rates of nutrient and oxygen consumption and lactate production. MDA-MB-231 and T47D cells produced 55 and 63%, respectively, of ATP from oxidative phosphorylation, whereas LM and BoM cells were more glycolytic, deriving only 20-25% of their ATP from mitochondria. ATP demand by BoM and LM cells was approximately half the rate of the parent cells. Of the anabolic fluxes assessed, nucleotide synthesis was the major ATP consumer for all cell lines. Glycolytic NADH production by LM cells exceeded the rate at which it could be oxidized by mitochondria, suggesting that the malate-aspartate shuttle was not involved in re-oxidation of these reducing equivalents. Serine synthesis was undetectable in MDA-MB-231 cells, whereas 3-5% of glucose was shunted to serine by LM and BoM lines. Proliferation rates of T47D, BoM, and LM lines tightly correlated with their respiration-normalized NADPH production rates. In contrast, MDA-MB-231 cells produced NADPH and GSH at higher rates, suggesting this line is more oxidatively stressed. Approximately half to two-thirds of NADPH produced by T47D, MDA-MB-231, and BoM cells was from the oxidative PPP, whereas the majority in LM cells was from the folate cycle. All four cell lines used the non-oxidative PPP to produce pentose phosphates, although this was most prominent for LM cells. Taken together, the metabolic phenotypes of LM and BoM lines differed from the parent line and from each other, supporting the metabolic re-wiring hypothesis as a feature of metastasis to lung and bone.
ABSTRACT
Histone lysine demethylases facilitate the activity of oncogenic transcription factors, including possibly MYC. Here we show that multiple histone demethylases influence the viability and poor prognosis of neuroblastoma cells, where MYC is often overexpressed. We also identified the approved small-molecule antifungal agent ciclopirox as a novel pan-histone demethylase inhibitor. Ciclopirox targeted several histone demethylases, including KDM4B implicated in MYC function. Accordingly, ciclopirox inhibited Myc signaling in parallel with mitochondrial oxidative phosphorylation, resulting in suppression of neuroblastoma cell viability and inhibition of tumor growth associated with an induction of differentiation. Our findings provide new insights into epigenetic regulation of MYC function and suggest a novel pharmacologic basis to target histone demethylases as an indirect MYC-targeting approach for cancer therapy. Cancer Res; 77(17); 4626-38. ©2017 AACR.
Subject(s)
Antifungal Agents/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Histone Demethylases/antagonists & inhibitors , Neuroblastoma/drug therapy , Proto-Oncogene Proteins c-myc/metabolism , Pyridones/pharmacology , Animals , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Ciclopirox , Epigenesis, Genetic , Histones/metabolism , Humans , Mice , Mice, SCID , Neuroblastoma/enzymology , Neuroblastoma/pathology , Oxidative Phosphorylation/drug effects , Proto-Oncogene Proteins c-myc/genetics , RNA, Small Interfering/genetics , Transcription, Genetic/drug effects , Tumor Cells, CulturedABSTRACT
Influenza is a worldwide health and financial burden posing a significant risk to the immune-compromised, obese, diabetic, elderly, and pediatric populations. We identified increases in glucose metabolism in the lungs of pediatric patients infected with respiratory pathogens. Using quantitative mass spectrometry, we found metabolic changes occurring after influenza infection in primary human respiratory cells and validated infection-associated increases in c-Myc, glycolysis, and glutaminolysis. We confirmed these findings with a metabolic drug screen that identified the PI3K/mTOR inhibitor BEZ235 as a regulator of infectious virus production. BEZ235 treatment ablated the transient induction of c-Myc, restored PI3K/mTOR pathway homeostasis measured by 4E-BP1 and p85 phosphorylation, and reversed infection-induced changes in metabolism. Importantly, BEZ235 reduced infectious progeny but had no effect on the early stages of viral replication. BEZ235 significantly increased survival in mice, while reducing viral titer. We show metabolic reprogramming of host cells by influenza virus exposes targets for therapeutic intervention.
Subject(s)
Influenza, Human/metabolism , Influenza, Human/therapy , Animals , Cell Survival/drug effects , Drug Evaluation, Preclinical , Female , Glucose/metabolism , Glutamine/metabolism , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Influenza, Human/virology , Lung/drug effects , Lung/metabolism , Lung/virology , Metabolic Flux Analysis , Mice, Inbred C57BL , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/metabolism , Orthomyxoviridae Infections/virology , Proteome/metabolism , Quinolines/pharmacology , Quinolines/therapeutic use , Toll-Like Receptors/metabolismABSTRACT
T cell activation results in a rapidly proliferating T cell endowed with a metabolic phenotype necessary for growth and division. However, before the cell can proceed towards this burst of cell division a phase of quiescence occurs, during which the basic mechanisms governing regulation of metabolic reprograming are established. This review focuses on key cellular processes controlling early metabolic regulation and how these circuits of metabolic control dictate distinct cellular fates upon the first asymmetric division.
Subject(s)
Asymmetric Cell Division , Energy Metabolism , Lymphocyte Activation/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Asymmetric Cell Division/genetics , Asymmetric Cell Division/immunology , Biomarkers , Gene Expression Regulation , Humans , Lymphocyte Activation/genetics , Signal TransductionABSTRACT
Asymmetric cell division, the partitioning of cellular components in response to polarizing cues during mitosis, has roles in differentiation and development. It is important for the self-renewal of fertilized zygotes in Caenorhabditis elegans and neuroblasts in Drosophila, and in the development of mammalian nervous and digestive systems. T lymphocytes, upon activation by antigen-presenting cells (APCs), can undergo asymmetric cell division, wherein the daughter cell proximal to the APC is more likely to differentiate into an effector-like T cell and the distal daughter is more likely to differentiate into a memory-like T cell. Upon activation and before cell division, expression of the transcription factor c-Myc drives metabolic reprogramming, necessary for the subsequent proliferative burst. Here we find that during the first division of an activated T cell in mice, c-Myc can sort asymmetrically. Asymmetric distribution of amino acid transporters, amino acid content, and activity of mammalian target of rapamycin complex 1 (mTORC1) is correlated with c-Myc expression, and both amino acids and mTORC1 activity sustain the differences in c-Myc expression in one daughter cell compared to the other. Asymmetric c-Myc levels in daughter T cells affect proliferation, metabolism, and differentiation, and these effects are altered by experimental manipulation of mTORC1 activity or c-Myc expression. Therefore, metabolic signalling pathways cooperate with transcription programs to maintain differential cell fates following asymmetric T-cell division.
Subject(s)
CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Cell Division , Cell Polarity , Lymphocyte Activation , Amino Acid Transport Systems/metabolism , Amino Acids/metabolism , Animals , Cell Differentiation/genetics , Cell Polarity/genetics , Female , Male , Mechanistic Target of Rapamycin Complex 1 , Mice , Multiprotein Complexes/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction/genetics , TOR Serine-Threonine Kinases/metabolism , Transcription, GeneticABSTRACT
The role of apoptosis inducing factor (AIF) in promoting cell death versus survival remains controversial. We report that the loss of AIF in fibroblasts led to mitochondrial electron transport chain defects and loss of proliferation that could be restored by ectopic expression of the yeast NADH dehydrogenase Ndi1. Aif-deficiency in T cells led to decreased peripheral T cell numbers and defective homeostatic proliferation, but thymic T cell development was unaffected. In contrast, Aif-deficient B cells developed and functioned normally. The difference in the dependency of T cells versus B cells on AIF for function and survival correlated with their metabolic requirements. Ectopic Ndi1 expression rescued homeostatic proliferation of Aif-deficient T cells. Despite its reported roles in cell death, fibroblasts, thymocytes and B cells lacking AIF underwent normal death. These studies suggest that the primary role of AIF relates to complex I function, with differential effects on T and B cells.
Subject(s)
Apoptosis Inducing Factor/metabolism , B-Lymphocytes/metabolism , Mitochondria/physiology , T-Lymphocytes/metabolism , Animals , Apoptosis , Cell Respiration/physiology , Electron Transport Complex I/metabolism , Fibroblasts/metabolism , Glycolysis/physiology , Mice , Mice, Knockout , Mice, Mutant StrainsABSTRACT
As a phenotypically plastic cellular population, macrophages change their physiology in response to environmental signals. Emerging evidence suggests that macrophages are capable of tightly coordinating their metabolic programs to adjust their immunological and bioenergetic functional properties, as needed. Upon mitogenic stimulation, quiescent macrophages enter the cell cycle, increasing their bioenergetic and biosynthetic activity to meet the demands of cell growth. Proinflammatory stimulation, however, suppresses cell proliferation, while maintaining a heightened metabolic activity imposed by the production of bactericidal factors. Here, we report that the mitogenic stimulus, colony-stimulating factor 1 (CSF-1), engages a myelocytomatosis viral oncogen (Myc)-dependent transcriptional program that is responsible for cell cycle entry and the up-regulation of glucose and glutamine catabolism in bone marrow-derived macrophages (BMDMs). However, the proinflammatory stimulus, lipopolysaccharide (LPS), suppresses Myc expression and cell proliferation and engages a hypoxia-inducible factor alpha (HIF1α)-dependent transcriptional program that is responsible for heightened glycolysis. The acute deletion of Myc or HIF1α selectively impaired the CSF-1- or LPS-driven metabolic activities in BMDM, respectively. Finally, inhibition of glycolysis by 2-deoxyglucose (2-DG) or genetic deletion of HIF1α suppressed LPS-induced inflammation in vivo. Our studies indicate that a switch from a Myc-dependent to a HIF1α-dependent transcriptional program may regulate the robust bioenergetic support for an inflammatory response, while sparing Myc-dependent proliferation.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Inflammation/metabolism , Inflammation/pathology , Macrophages/metabolism , Macrophages/pathology , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction , Animals , Cell Cycle/drug effects , Cell Polarity/drug effects , Cell Proliferation/drug effects , Disease Models, Animal , Gene Deletion , Glycolysis/drug effects , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Interferon-gamma/pharmacology , Lipopolysaccharides/pharmacology , Macrophage Colony-Stimulating Factor/pharmacology , Macrophages/drug effects , Metabolic Networks and Pathways/drug effects , Metabolic Networks and Pathways/genetics , Mice, Inbred C57BL , Mitogens/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sepsis/metabolism , Sepsis/pathology , Signal Transduction/drug effects , Transcriptome/drug effects , Transcriptome/geneticsABSTRACT
Somatic mitochondrial DNA (mtDNA) mutations contribute to the pathogenesis of age-related disorders, including myelodysplastic syndromes (MDS). The accumulation of mitochondria harboring mtDNA mutations in patients with these disorders suggests a failure of normal mitochondrial quality-control systems. The mtDNA-mutator mice acquire somatic mtDNA mutations via a targeted defect in the proofreading function of the mtDNA polymerase, PolgA, and develop macrocytic anemia similar to that of patients with MDS. We observed an unexpected defect in clearance of dysfunctional mitochondria at specific stages during erythroid maturation in hematopoietic cells from aged mtDNA-mutator mice. Mechanistically, aberrant activation of mechanistic target of rapamycin signaling and phosphorylation of uncoordinated 51-like kinase (ULK) 1 in mtDNA-mutator mice resulted in proteasome-mediated degradation of ULK1 and inhibition of autophagy in erythroid cells. To directly evaluate the consequence of inhibiting autophagy on mitochondrial function in erythroid cells harboring mtDNA mutations in vivo, we deleted Atg7 from erythroid progenitors of wild-type and mtDNA-mutator mice. Genetic disruption of autophagy did not cause anemia in wild-type mice but accelerated the decline in mitochondrial respiration and development of macrocytic anemia in mtDNA-mutator mice. These findings highlight a pathological feedback loop that explains how dysfunctional mitochondria can escape autophagy-mediated degradation and propagate in cells predisposed to somatic mtDNA mutations, leading to disease.
Subject(s)
Anemia/genetics , Autophagy/genetics , DNA, Mitochondrial/genetics , Erythrocytes/cytology , TOR Serine-Threonine Kinases/metabolism , Aging , Animals , Cell Separation , DNA Polymerase gamma , DNA-Directed DNA Polymerase/metabolism , Erythroid Cells/metabolism , Flow Cytometry , Heterozygote , Mice , Mitochondria/metabolism , Mutation , Myelodysplastic Syndromes/genetics , Oxygen Consumption , Phenotype , Phosphorylation , Proteasome Endopeptidase Complex/metabolism , Ribosomes/metabolismABSTRACT
B cell activation leads to proliferation and Ab production that can protect from pathogens or promote autoimmunity. Regulation of cell metabolism is essential to support the demands of lymphocyte growth and effector function and may regulate tolerance. In this study, we tested the regulation and role of glucose uptake and metabolism in the proliferation and Ab production of control, anergic, and autoimmune-prone B cells. Control B cells had a balanced increase in lactate production and oxygen consumption following activation, with proportionally increased glucose transporter Glut1 expression and mitochondrial mass upon either LPS or BCR stimulation. This contrasted with metabolic reprogramming of T cells, which had lower glycolytic flux when resting but disproportionately increased this pathway upon activation. Importantly, tolerance greatly affected B cell metabolic reprogramming. Anergic B cells remained metabolically quiescent, with only a modest increase in glycolysis and oxygen consumption with LPS stimulation. B cells chronically stimulated with elevated BAFF, however, rapidly increased glycolysis and Ab production upon stimulation. Induction of glycolysis was critical for Ab production, as glycolytic inhibition with the pyruvate dehydrogenase kinase inhibitor dichloroacetate sharply suppressed B cell proliferation and Ab secretion in vitro and in vivo. Furthermore, B cell-specific deletion of Glut1 led to reduced B cell numbers and impaired Ab production in vivo. Together, these data show that activated B cells require Glut1-dependent metabolic reprogramming to support proliferation and Ab production that is distinct from T cells and that this glycolytic reprogramming is regulated in tolerance.
Subject(s)
Antibody Formation , B-Cell Activating Factor/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Clonal Anergy/immunology , Animals , B-Cell Activating Factor/genetics , Dichloroacetic Acid/pharmacology , Glucose/metabolism , Glucose Transporter Type 1/metabolism , Glycolysis/drug effects , Homeostasis , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lymphocyte Activation/immunology , Mice , Mice, Transgenic , Mitochondria/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-myc/metabolism , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Programmed necrosis (or necroptosis) is a form of cell death triggered by the activation of receptor interacting protein kinase-3 (RIPK3). Several reports have implicated mitochondria and mitochondrial reactive oxygen species (ROS) generation as effectors of RIPK3-dependent cell death. Here, we directly test this idea by employing a method for the specific removal of mitochondria via mitophagy. Mitochondria-deficient cells were resistant to the mitochondrial pathway of apoptosis, but efficiently died via tumor necrosis factor (TNF)-induced, RIPK3-dependent programmed necrosis or as a result of direct oligomerization of RIPK3. Although the ROS scavenger butylated hydroxyanisole (BHA) delayed TNF-induced necroptosis, it had no effect on necroptosis induced by RIPK3 oligomerization. Furthermore, although TNF-induced ROS production was dependent on mitochondria, the inhibition of TNF-induced necroptosis by BHA was observed in mitochondria-depleted cells. Our data indicate that mitochondrial ROS production accompanies, but does not cause, RIPK3-dependent necroptotic cell death.
Subject(s)
Apoptosis/drug effects , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , 3T3 Cells , Animals , Butylated Hydroxyanisole/pharmacology , Caspase 8/genetics , Caspase 8/metabolism , Cell Line , Mice , Mitophagy/drug effects , Necrosis/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Caspase-8 or cellular FLICE-like inhibitor protein (cFLIP) deficiency leads to embryonic lethality in mice due to defects in endothelial tissues. Caspase-8(-/-) and receptor-interacting protein kinase-3 (RIPK3)(-/-), but not cFLIP(-/-) and RIPK3(-/-), double-knockout animals develop normally, indicating that caspase-8 antagonizes the lethal effects of RIPK3 during development. Here, we show that the acute deletion of caspase-8 in the gut of adult mice induces enterocyte death, disruption of tissue homeostasis, and inflammation, resulting in sepsis and mortality. Likewise, acute deletion of caspase-8 in a focal region of the skin induces local keratinocyte death, tissue disruption, and inflammation. Strikingly, RIPK3 ablation rescues both phenotypes. However, acute loss of cFLIP in the skin produces a similar phenotype that is not rescued by RIPK3 ablation. TNF neutralization protects from either acute loss of caspase-8 or cFLIP. These results demonstrate that caspase-8-mediated suppression of RIPK3-induced death is required not only during development but also for adult homeostasis. Furthermore, RIPK3-dependent inflammation is dispensable for the skin phenotype.
Subject(s)
CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Caspase 8/metabolism , Animals , Apoptosis , CASP8 and FADD-Like Apoptosis Regulating Protein/deficiency , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , Caspase 8/genetics , Enterocytes/cytology , Homeostasis/drug effects , Mice , Mice, Knockout , Neutrophils/metabolism , Neutrophils/pathology , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Skin/drug effects , Skin/metabolism , Tamoxifen/pharmacology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
NOD2 receptor and the cytosolic protein kinase RIPK2 regulate NF-κB and MAP kinase signaling during bacterial infections, but the role of this immune axis during viral infections has not been addressed. We demonstrate that Nod2(-/-) and Ripk2(-/-) mice are hypersusceptible to infection with influenza A virus. Ripk2(-/-) cells exhibited defective autophagy of mitochondria (mitophagy), leading to enhanced mitochondrial production of superoxide and accumulation of damaged mitochondria, which resulted in greater activation of the NLRP3 inflammasome and production of IL-18. RIPK2 regulated mitophagy in a kinase-dependent manner by phosphorylating the mitophagy inducer ULK1. Accordingly, Ulk1(-/-) cells exhibited enhanced mitochondrial production of superoxide and activation of caspase-1. These results demonstrate a role for NOD2-RIPK2 signaling in protection against virally triggered immunopathology by negatively regulating activation of the NLRP3 inflammasome and production of IL-18 via ULK1-dependent mitophagy.
Subject(s)
Alphainfluenzavirus/immunology , Carrier Proteins/metabolism , Inflammasomes/metabolism , Mitochondria/physiology , Mitophagy , Orthomyxoviridae Infections/immunology , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Animals , Autophagy-Related Protein-1 Homolog , Caspase 1/metabolism , Cells, Cultured , Immunity, Active/genetics , Interleukin-18/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein , Nod2 Signaling Adaptor Protein/genetics , Nod2 Signaling Adaptor Protein/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , Signal Transduction/genetics , Superoxides/metabolism , Virulence Factors/geneticsABSTRACT
MCL-1, an anti-apoptotic BCL-2 family member that is essential for the survival of multiple cell lineages, is also among the most highly amplified genes in cancer. Although MCL-1 is known to oppose cell death, precisely how it functions to promote survival of normal and malignant cells is poorly understood. Here, we report that different forms of MCL-1 reside in distinct mitochondrial locations and exhibit separable functions. On the outer mitochondrial membrane, an MCL-1 isoform acts like other anti-apoptotic BCL-2 molecules to antagonize apoptosis, whereas an amino-terminally truncated isoform of MCL-1 that is imported into the mitochondrial matrix is necessary to facilitate normal mitochondrial fusion, ATP production, membrane potential, respiration, cristae ultrastructure and maintenance of oligomeric ATP synthase. Our results provide insight into how the surprisingly diverse salutary functions of MCL-1 may control the survival of both normal and cancer cells.
Subject(s)
Mitochondria/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Adenosine Triphosphate/biosynthesis , Animals , Apoptosis , Cell Respiration , Cells, Cultured , Humans , Membrane Potentials , Mice , Microscopy, Electron, Transmission , Mitochondria/ultrastructure , Myeloid Cell Leukemia Sequence 1 Protein , Proto-Oncogene Proteins c-bcl-2/genetics , Proton-Translocating ATPases/metabolismABSTRACT
Ataxia-telangiectasia mutated (ATM) plays a central role in DNA damage responses, and its loss leads to development of T-cell malignancies. Here, we show that ATM loss also leads to intrinsic mitochondrial abnormalities in thymocytes, including elevated reactive oxygen species, increased aberrant mitochondria, high cellular respiratory capacity, and decreased mitophagy. A fraction of ATM protein is localized in mitochondria, and it is rapidly activated by mitochondrial dysfunction. Unexpectedly, allelic loss of the autophagy regulator Beclin-1 significantly delayed tumor development in ATM-null mice. This effect was not associated with rescue of DNA damage signaling but rather with a significant reversal of the mitochondrial abnormalities. These data support a model in which ATM plays direct roles in modulating mitochondrial homeostasis and suggest that mitochondrial dysfunction and associated increases in mitochondrial reactive oxygen species contribute to the cancer-prone phenotype observed in organisms lacking ATM. Thus, ataxia-telangiectasia should be considered, at least in part, as a mitochondrial disease.
Subject(s)
Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Mitochondria/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Adenosine Triphosphate/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Ataxia Telangiectasia/genetics , Ataxia Telangiectasia/metabolism , Ataxia Telangiectasia/physiopathology , Ataxia Telangiectasia Mutated Proteins , Autophagy , Beclin-1 , Cell Cycle Proteins/genetics , Cells, Cultured , DNA-Binding Proteins/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression , Humans , Immunoblotting , Kaplan-Meier Estimate , Lymphoma, T-Cell/genetics , Lymphoma, T-Cell/metabolism , Membrane Potential, Mitochondrial , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron, Transmission , Mitochondria/genetics , Mitochondria/physiology , Oxygen Consumption , Protein Serine-Threonine Kinases/genetics , RNA Interference , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Thymocytes/metabolism , Thymocytes/ultrastructure , Tumor Suppressor Proteins/geneticsABSTRACT
To fulfill the bioenergetic and biosynthetic demand of proliferation, T cells reprogram their metabolic pathways from fatty acid ß-oxidation and pyruvate oxidation via the TCA cycle to the glycolytic, pentose-phosphate, and glutaminolytic pathways. Two of the top-ranked candidate transcription factors potentially responsible for the activation-induced T cell metabolic transcriptome, HIF1α and Myc, were induced upon T cell activation, but only the acute deletion of Myc markedly inhibited activation-induced glycolysis and glutaminolysis in T cells. Glutamine deprivation compromised activation-induced T cell growth and proliferation, and this was partially replaced by nucleotides and polyamines, implicating glutamine as an important source for biosynthetic precursors in active T cells. Metabolic tracer analysis revealed a Myc-dependent metabolic pathway linking glutaminolysis to the biosynthesis of polyamines. Therefore, a Myc-dependent global metabolic transcriptome drives metabolic reprogramming in activated, primary T lymphocytes. This may represent a general mechanism for metabolic reprogramming under patho-physiological conditions.
Subject(s)
Lymphocyte Activation , Proto-Oncogene Proteins c-myc/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Gene Expression Regulation , Glucose/metabolism , Glutamine/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lymphocyte Activation/genetics , Metabolic Networks and Pathways/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Ornithine/metabolism , Polyamines/metabolism , Proto-Oncogene Proteins c-myc/genetics , TOR Serine-Threonine Kinases/metabolism , TranscriptomeABSTRACT
The lysosomal pathway digests material received by two main routes, phagocytosis and autophagy. Cells use phagocytosis to ingest extracellular particles by invaginations of the plasma membrane. In autophagy, a double membrane structure isolates portions of the cytoplasm to target it for degradation. During infection, phagocytes use both of these cellular functions to restrict microbial replication and at the same time to orchestrate an appropriate response against the invader. Toll-like receptor recognition of a pathogen initiates an innate immune response against the pathogen that includes production of inflammatory cytokines, upregulation of costimulatory molecules to prime an adaptive immune response, and activation of phagocytosis and autophagy. Signaling through this family of receptors also produces a hybrid response in which proteins that participate in autophagy are recruited to phagosomes, resulting in expedited microbial elimination. In this review, we discuss recent views on how Toll-like receptors direct microbes to final destruction by regulating the different pathways that lead to the lysosome.
Subject(s)
Autophagy/immunology , Lysosomes/metabolism , Phagosomes/metabolism , Signal Transduction/immunology , Toll-Like Receptors/immunology , Toll-Like Receptors/metabolism , Animals , Antigen Presentation/immunology , Autophagy/genetics , Autophagy-Related Protein 12 , Homeostasis , Humans , Infections/immunology , Lysosomes/immunology , Membrane Fusion/immunology , Microtubule-Associated Proteins/immunology , Microtubule-Associated Proteins/metabolism , Phagosomes/immunology , Protein Kinases/immunology , Protein Transport/immunology , Small Ubiquitin-Related Modifier Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/immunology , Small Ubiquitin-Related Modifier Proteins/metabolism , TOR Serine-Threonine Kinases , Toll-Like Receptors/geneticsABSTRACT
D6 is a heptahelical receptor that suppresses inflammation and tumorigenesis by scavenging extracellular pro-inflammatory CC chemokines. Previous studies suggested this is dependent on constitutive trafficking of stable D6 protein to and from the cell surface via recycling endosomes. By internalizing chemokine each time it transits the cell surface, D6 can, over time, remove large quantities of these inflammatory mediators. We have investigated the role of the conserved 58-amino acid C terminus of human D6, which, unlike the rest of the protein, shows no clear homology to other heptahelical receptors. We show that, in human HEK293 cells, a serine cluster in this region controls the constitutive phosphorylation, high stability, and intracellular trafficking itinerary of the receptor and drives green fluorescent protein-tagged beta-arrestins to membranes at, and near, the cell surface. Unexpectedly, however, these properties, and the last 44 amino acids of the C terminus, are dispensable for D6 internalization and effective scavenging of the chemokine CCL3. Even in the absence of the last 58 amino acids, D6 still initially internalizes CCL3 but, surprisingly, exposure to ligand inhibits subsequent CCL3 uptake by this mutant. Progressive scavenging is therefore abrogated. We conclude that the heptahelical body of D6 on its own can engage the endocytotic machinery of HEK293 cells but that the C terminus is indispensable for scavenging because it prevents initial chemokine engagement of D6 from inhibiting subsequent chemokine uptake.
Subject(s)
Chemokine CCL3/metabolism , Endosomes/metabolism , Receptors, CCR10/metabolism , Animals , Arrestins/metabolism , Cell Line , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Humans , Inflammation/genetics , Inflammation/metabolism , Phosphorylation , Protein Structure, Secondary/genetics , Protein Structure, Tertiary/genetics , Protein Transport/genetics , Receptors, CCR10/genetics , beta-Arrestins , Chemokine Receptor D6ABSTRACT
In cells undergoing apoptosis, mitochondrial outer-membrane permeabilization (MOMP) is followed by caspase activation promoted by released cytochrome c. Although caspases mediate the apoptotic phenotype, caspase inhibition is generally not sufficient for survival following MOMP; instead cells undergo a "caspase-independent cell death" (CICD). Thus, MOMP may represent a point of commitment to cell death. Here, we identify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a critical regulator of CICD. GAPDH-expressing cells preserved their clonogenic potential following MOMP, provided that caspase activation was blocked. GAPDH-mediated protection of cells from CICD involved an elevation in glycolysis and a nuclear function that correlated with and was replaced by an increase in Atg12 expression. Consistent with this, protection from CICD reflected an increase in and a dependence upon autophagy, associated with a transient decrease in mitochondrial mass. Therefore, GAPDH mediates an elevation in glycolysis and enhanced autophagy that cooperate to protect cells from CICD.
Subject(s)
Apoptosis , Autophagy , Cell Survival/physiology , Glyceraldehyde-3-Phosphate Dehydrogenases/physiology , Caspases/metabolism , Cytochromes c/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , HeLa Cells , Humans , Jurkat Cells , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , RNA InterferenceABSTRACT
Following inducible expression in HEK293 cells, the human orexin-1 receptor was targeted to the cell surface but became internalized following exposure to the peptide agonist orexin A. By contrast, constitutive expression of the human cannabinoid CB1 receptor resulted in a predominantly punctate, intracellular distribution pattern consistent with spontaneous, agonist-independent internalization. Expression of the orexin-1 receptor in the presence of the CB1 receptor resulted in both receptors displaying the spontaneous internalization phenotype. Single cell fluorescence resonance energy transfer imaging indicated the two receptors were present as heterodimers/oligomers in intracellular vesicles. Addition of the CB1 receptor antagonist SR-141716A to cells expressing only the CB1 receptor resulted in re-localization of the receptor to the cell surface. Although SR-141716A has no significant affinity for the orexin-1 receptor, in cells co-expressing the CB1 receptor, the orexin-1 receptor was also re-localized to the cell surface by treatment with SR-141716A. Treatment of cells co-expressing the orexin-1 and CB1 receptors with the orexin-1 receptor antagonist SB-674042 also resulted in re-localization of both receptors to the cell surface. Treatment with SR-141716A resulted in decreased potency of orexin A to activate the mitogen-activated protein kinases ERK1/2 only in cells co-expressing the two receptors. Treatment with SB-674042 also reduced the potency of a CB1 receptor agonist to phosphorylate ERK1/2 only when the two receptors were co-expressed. These studies introduce an entirely novel pharmacological paradigm, whereby ligands modulate the function of receptors for which they have no significant inherent affinity by acting as regulators of receptor heterodimers.
Subject(s)
Receptor, Cannabinoid, CB1/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/metabolism , Base Sequence , Cell Line , DNA Primers , Dimerization , Humans , Ligands , Orexin Receptors , Pyrrolidines/pharmacology , Receptor, Cannabinoid, CB1/chemistry , Receptor, Cannabinoid, CB1/physiology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/physiology , Receptors, Neuropeptide/antagonists & inhibitors , Receptors, Neuropeptide/chemistry , Receptors, Neuropeptide/physiology , Thiazoles/pharmacologyABSTRACT
The chemokines CCL19, CCL21 and CCL25, by signalling through the receptors CCR7 or CCR9, play critical roles in leukocyte homing. They also bind another heptahelical surface protein, CCX-CKR. CCX-CKR cannot couple to typical chemokine receptor signalling pathways or mediate chemotaxis, and its function remains unclear. We have proposed that it controls chemokine bioavailability. Here, using transfected HEK293 cells, we have shown that both CCX-CKR and CCR7 mediate rapid CCL19 internalisation upon initial chemokine exposure. However, internalised CCL19 was more efficiently retained and degraded after uptake via CCX-CKR. More importantly, CCR7 rapidly became refractory for CCL19 uptake, but the sequestration activity of CCX-CKR was enhanced. These properties endowed CCX-CKR with an impressive ability to mediate progressive sequestration and degradation of large quantities of CCL19, and conversely, prevented CCR7-expressing cells from extensively altering their chemokine environment. These differences may be linked to the routes of endocytosis used by these receptors. CCX-CKR, unlike CCR7, was not critically dependent on beta-arrestins or clathrin-coated pits. However, over-expression of caveolin-1, which stabilises caveolae, blocked CCL19 uptake by CCX-CKR while having no impact on other chemokine receptors, including CCR7. These data predict that CCX-CKR scavenges extracellular chemokines in vivo to modify responses through CCR7.