RESUMEN
Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme ATP citrate lyase (ACLY). However, ablation of ACLY triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When ACLY is functional, ACSS2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, loss of ACLY renders CD8 T cells dependent on acetate (via ACSS2) to maintain acetyl-CoA production and effector function. Together, ACLY and ACSS2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.
Asunto(s)
ATP Citrato (pro-S)-Liasa , Acetatos , Acetilcoenzima A , Linfocitos T CD8-positivos , Cromatina , Ratones Endogámicos C57BL , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Animales , Cromatina/metabolismo , Acetilcoenzima A/metabolismo , ATP Citrato (pro-S)-Liasa/metabolismo , ATP Citrato (pro-S)-Liasa/genética , Ratones , Acetatos/metabolismo , Acetato CoA Ligasa/metabolismo , Acetato CoA Ligasa/genética , Acetilación , Ratones Noqueados , Citosol/metabolismo , Histonas/metabolismoRESUMEN
Infusion of 13C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes-infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)-which regulates de novo aspartate synthesis-for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.
Asunto(s)
Acetatos , Linfocitos T CD8-positivos , Isótopos de Carbono , Glutamina , Glutamina/metabolismo , Animales , Linfocitos T CD8-positivos/metabolismo , Acetatos/metabolismo , Ratones , Listeriosis/metabolismo , Listeriosis/inmunología , Listeriosis/microbiología , Listeria monocytogenes , Ciclo del Ácido Cítrico , Glucosa/metabolismo , Ratones Endogámicos C57BLRESUMEN
That uncoupling protein 1 (UCP1) is the sole mediator of adipocyte thermogenesis is a conventional viewpoint that has primarily been inferred from the attenuation of the thermogenic output of mice genetically lacking Ucp1 from birth (germline Ucp1-/-). However, germline Ucp1-/- mice harbor secondary changes within brown adipose tissue. To mitigate these potentially confounding ancillary changes, we constructed mice with inducible adipocyte-selective Ucp1 disruption. We find that, although germline Ucp1-/- mice succumb to cold-induced hypothermia with complete penetrance, most mice with the inducible deletion of Ucp1 maintain homeothermy in the cold. However, inducible adipocyte-selective co-deletion of Ucp1 and creatine kinase b (Ckb, an effector of UCP1-independent thermogenesis) exacerbates cold intolerance. Following UCP1 deletion or UCP1/CKB co-deletion from mature adipocytes, moderate cold exposure triggers the regeneration of mature brown adipocytes that coordinately restore UCP1 and CKB expression. Our findings suggest that thermogenic adipocytes utilize non-paralogous protein redundancy-through UCP1 and CKB-to promote cold-induced energy dissipation.
Asunto(s)
Adipocitos Marrones , Tejido Adiposo Pardo , Animales , Ratones , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Termogénesis , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Forma BB de la Creatina-Quinasa/metabolismoRESUMEN
Infusion of 13C-labeled metabolites provides a gold-standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, acetate) in Listeria monocytogenes (Lm)-infected mice, we demonstrate that CD8+ T effector (Teff) cells utilize metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily towards nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support ATP and de novo pyrimidine synthesis. Additionally, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)-which regulates de novo aspartate synthesis-for effector cell expansion in vivo. Importantly, Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with Teff cell function in vivo.
RESUMEN
Noradrenaline (NA) regulates cold-stimulated adipocyte thermogenesis1. Aside from cAMP signalling downstream of ß-adrenergic receptor activation, how NA promotes thermogenic output is still not fully understood. Here, we show that coordinated α1-adrenergic receptor (AR) and ß3-AR signalling induces the expression of thermogenic genes of the futile creatine cycle2,3, and that early B cell factors, oestrogen-related receptors and PGC1α are required for this response in vivo. NA triggers physical and functional coupling between the α1-AR subtype (ADRA1A) and Gαq to promote adipocyte thermogenesis in a manner that is dependent on the effector proteins of the futile creatine cycle, creatine kinase B and tissue-non-specific alkaline phosphatase. Combined Gαq and Gαs signalling selectively in adipocytes promotes a continual rise in whole-body energy expenditure, and creatine kinase B is required for this effect. Thus, the ADRA1A-Gαq-futile creatine cycle axis is a key regulator of facultative and adaptive thermogenesis.
Asunto(s)
Creatina , Termogénesis , Creatina/metabolismo , Termogénesis/genética , Adipocitos/metabolismo , Metabolismo Energético/genética , Creatina Quinasa/metabolismoRESUMEN
The factors that promote T cell expansion are not fully known. Creatine is an abundant circulating metabolite that has recently been implicated in T cell function; however, its cell-autonomous role in immune-cell function is unknown. Here, we show that creatine supports cell-intrinsic CD8+ T cell homeostasis. We further identify creatine kinase B (CKB) as the creatine kinase isoenzyme that supports these T cell properties. Loss of the creatine transporter (Slc6a8) or Ckb results in compromised CD8+ T cell expansion in response to infection without influencing adenylate energy charge. Rather, loss of Slc6a8 or Ckb disrupts naive T cell homeostasis and weakens TCR-mediated activation of mechanistic target of rapamycin complex 1 (mTORC1) signaling required for CD8+ T cell expansion. These data demonstrate a cell-intrinsic role for creatine transport and creatine transphosphorylation, independent of their effects on global cellular energy charge, in supporting CD8+ T cell homeostasis and effector function.
Asunto(s)
Linfocitos T CD8-positivos , Creatina , Creatina/metabolismo , Creatina Quinasa/metabolismo , Fosforilación , Transducción de SeñalRESUMEN
Obesity is a major risk factor for adverse outcomes in breast cancer; however, the underlying molecular mechanisms have not been elucidated. To investigate the role of crosstalk between mammary adipocytes and neoplastic cells in the tumor microenvironment (TME), we performed transcriptomic analysis of cancer cells and adjacent adipose tissue in a murine model of obesity-accelerated breast cancer and identified glycine amidinotransferase (Gatm) in adipocytes and Acsbg1 in cancer cells as required for obesity-driven tumor progression. Gatm is the rate-limiting enzyme in creatine biosynthesis, and deletion in adipocytes attenuated obesity-driven tumor growth. Similarly, genetic inhibition of creatine import into cancer cells reduced tumor growth in obesity. In parallel, breast cancer cells in obese animals upregulated the fatty acyl-CoA synthetase Acsbg1 to promote creatine-dependent tumor progression. These findings reveal key nodes in the crosstalk between adipocytes and cancer cells in the TME necessary for obesity-driven breast cancer progression.
Asunto(s)
Neoplasias de la Mama/patología , Comunicación Celular/fisiología , Creatina/metabolismo , Obesidad/patología , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Amidinotransferasas/deficiencia , Amidinotransferasas/genética , Amidinotransferasas/metabolismo , Animales , Línea Celular Tumoral , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Dieta Alta en Grasa , Femenino , Humanos , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Microambiente TumoralRESUMEN
Obesity increases the risk of mortality because of metabolic sequelae such as type 2 diabetes and cardiovascular disease1. Thermogenesis by adipocytes can counteract obesity and metabolic diseases2,3. In thermogenic fat, creatine liberates a molar excess of mitochondrial ADP-purportedly via a phosphorylation cycle4-to drive thermogenic respiration. However, the proteins that control this futile creatine cycle are unknown. Here we show that creatine kinase B (CKB) is indispensable for thermogenesis resulting from the futile creatine cycle, during which it traffics to mitochondria using an internal mitochondrial targeting sequence. CKB is powerfully induced by thermogenic stimuli in both mouse and human adipocytes. Adipocyte-selective inactivation of Ckb in mice diminishes thermogenic capacity, increases predisposition to obesity, and disrupts glucose homeostasis. CKB is therefore a key effector of the futile creatine cycle.
Asunto(s)
Tejido Adiposo/metabolismo , Forma BB de la Creatina-Quinasa/metabolismo , Creatina/metabolismo , Termogénesis , Adipocitos/metabolismo , Tejido Adiposo/citología , Tejido Adiposo/enzimología , Animales , Forma BB de la Creatina-Quinasa/deficiencia , Forma BB de la Creatina-Quinasa/genética , AMP Cíclico/metabolismo , Metabolismo Energético/genética , Femenino , Glucosa/metabolismo , Homeostasis , Humanos , Masculino , Ratones , Mitocondrias/metabolismo , Obesidad/enzimología , Obesidad/genética , Obesidad/metabolismo , Transducción de SeñalRESUMEN
Metastasis is the leading cause of cancer-related deaths, and obesity is associated with increased breast cancer (BC) metastasis. Preclinical studies have shown that obese adipose tissue induces lung neutrophilia associated with enhanced BC metastasis to this site. Here we show that obesity leads to neutrophil-dependent impairment of vascular integrity through loss of endothelial adhesions, enabling cancer cell extravasation into the lung. Mechanistically, neutrophil-produced reactive oxygen species in obese mice increase neutrophil extracellular DNA traps (NETs) and weaken endothelial junctions, facilitating the influx of tumor cells from the peripheral circulation. In vivo treatment with catalase, NET inhibitors or genetic deletion of Nos2 reversed this effect in preclinical models of obesity. Imaging mass cytometry of lung metastasis samples from patients with cancer revealed an enrichment in neutrophils with low catalase levels correlating with elevated body mass index. Our data provide insights into potentially targetable mechanisms that underlie the progression of BC in the obese population.
Asunto(s)
Neoplasias de la Mama , Neoplasias Pulmonares , Animales , Neoplasias de la Mama/metabolismo , Catalasa/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Neutrófilos/metabolismo , Obesidad/complicaciones , Estrés OxidativoRESUMEN
Epigenetic modifications on DNA and histones regulate gene expression by modulating chromatin accessibility to transcription machinery. Here we identify methionine as a key nutrient affecting epigenetic reprogramming in CD4+ T helper (Th) cells. Using metabolomics, we showed that methionine is rapidly taken up by activated T cells and serves as the major substrate for biosynthesis of the universal methyl donor S-adenosyl-L-methionine (SAM). Methionine was required to maintain intracellular SAM pools in T cells. Methionine restriction reduced histone H3K4 methylation (H3K4me3) at the promoter regions of key genes involved in Th17 cell proliferation and cytokine production. Applied to the mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis), dietary methionine restriction reduced the expansion of pathogenic Th17 cells in vivo, leading to reduced T cell-mediated neuroinflammation and disease onset. Our data identify methionine as a key nutritional factor shaping Th cell proliferation and function in part through regulation of histone methylation.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Epigénesis Genética/efectos de los fármacos , Histonas/metabolismo , Metionina , Esclerosis Múltiple , Células Th17/metabolismo , Animales , Proliferación Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Células HEK293 , Humanos , Metionina/metabolismo , Metionina/farmacología , Metilación , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/metabolismo , Células Th17/citologíaRESUMEN
Naive CD8+ T cells differentiating into effector T cells increase glucose uptake and shift from quiescent to anabolic metabolism. Although much is known about the metabolism of cultured T cells, how T cells use nutrients during immune responses in vivo is less well defined. Here, we combined bioenergetic profiling and 13C-glucose infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. In contrast to in vitro-activated T cells, which display hallmarks of Warburg metabolism, physiologically activated CD8+ T cells displayed greater rates of oxidative metabolism, higher bioenergetic capacity, differential use of pyruvate, and prominent flow of 13C-glucose carbon to anabolic pathways, including nucleotide and serine biosynthesis. Glucose-dependent serine biosynthesis mediated by the enzyme Phgdh was essential for CD8+ T cell expansion in vivo. Our data highlight fundamental differences in glucose use by pathogen-specific T cells in vivo, illustrating the impact of environment on T cell metabolic phenotypes.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Metabolismo Energético , Glucosa/metabolismo , Activación de Linfocitos/inmunología , Metaboloma , Metabolómica , Animales , Proliferación Celular , Cromatografía de Gases y Espectrometría de Masas , Glucólisis , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Activación de Linfocitos/genética , Metabolómica/métodos , Ratones , Estrés Oxidativo , Virosis/genética , Virosis/inmunología , Virosis/metabolismo , Virosis/virologíaAsunto(s)
Adipocitos/metabolismo , Creatina/metabolismo , Dieta Alta en Grasa , Obesidad/etiología , Termogénesis , Tejido Adiposo Pardo/metabolismo , Animales , Transporte Biológico , Metabolismo Energético , Quininógenos/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Noqueados , Obesidad/metabolismo , Grasa Subcutánea/metabolismoRESUMEN
T cell subsets including effector (Teff), regulatory (Treg), and memory (Tmem) cells are characterized by distinct metabolic profiles that influence their differentiation and function. Previous research suggests that engagement of long-chain fatty acid oxidation (LC-FAO) supports Foxp3+ Treg cell and Tmem cell survival. However, evidence for this is mostly based on inhibition of Cpt1a, the rate-limiting enzyme for LC-FAO, with the drug etomoxir. Using genetic models to target Cpt1a specifically in T cells, we dissected the role of LC-FAO in primary, memory, and regulatory T cell responses. Here we show that the ACC2/Cpt1a axis is largely dispensable for Teff, Tmem, or Treg cell formation, and that the effects of etomoxir on T cell differentiation and function are independent of Cpt1a expression. Together our data argue that metabolic pathways other than LC-FAO fuel Tmem or Treg differentiation and suggest alternative mechanisms for the effects of etomoxir that involve mitochondrial respiration.
Asunto(s)
Acetil-CoA Carboxilasa/fisiología , Linfocitos T CD8-positivos/metabolismo , Carnitina O-Palmitoiltransferasa/fisiología , Compuestos Epoxi/farmacología , Ácidos Grasos/metabolismo , Memoria Inmunológica/efectos de los fármacos , Mitocondrias/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Acetil-CoA Carboxilasa/genética , Animales , Carnitina O-Palmitoiltransferasa/genética , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Niño , Preescolar , Femenino , Técnicas de Inactivación de Genes , Humanos , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Linfocitos T Reguladores/metabolismoRESUMEN
Reactive oxygen species (ROS) are continuously produced as a by-product of mitochondrial metabolism and eliminated via antioxidant systems. Regulation of mitochondrially produced ROS is required for proper cellular function, adaptation to metabolic stress, and bypassing cellular senescence. Here, we report non-canonical regulation of the cellular energy sensor AMP-activated protein kinase (AMPK) by mitochondrial ROS (mROS) that functions to maintain cellular metabolic homeostasis. We demonstrate that mitochondrial ROS are a physiological activator of AMPK and that AMPK activation triggers a PGC-1α-dependent antioxidant response that limits mitochondrial ROS production. Cells lacking AMPK activity display increased mitochondrial ROS levels and undergo premature senescence. Finally, we show that AMPK-PGC-1α-dependent control of mitochondrial ROS regulates HIF-1α stabilization and that mitochondrial ROS promote the Warburg effect in cells lacking AMPK signaling. These data highlight a key function for AMPK in sensing and resolving mitochondrial ROS for stress resistance and maintaining cellular metabolic balance.
Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Homeostasis/genética , Redes y Vías Metabólicas/genética , Mitocondrias/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas Activadas por AMP/deficiencia , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Senescencia Celular/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Células HEK293 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Transgénicos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/deficiencia , Cultivo Primario de Células , Estabilidad Proteica , Transducción de Señal , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismo , Proteína Desacopladora 3/genética , Proteína Desacopladora 3/metabolismo , Glutatión Peroxidasa GPX1RESUMEN
During immune challenge, T lymphocytes engage pathways of anabolic metabolism to support clonal expansion and the development of effector functions. Here we report a critical role for the non-essential amino acid serine in effector T cell responses. Upon activation, T cells upregulate enzymes of the serine, glycine, one-carbon (SGOC) metabolic network, and rapidly increase processing of serine into one-carbon metabolism. We show that extracellular serine is required for optimal T cell expansion even in glucose concentrations sufficient to support T cell activation, bioenergetics, and effector function. Restricting dietary serine impairs pathogen-driven expansion of T cells in vivo, without affecting overall immune cell homeostasis. Mechanistically, serine supplies glycine and one-carbon units for de novo nucleotide biosynthesis in proliferating T cells, and one-carbon units from formate can rescue T cells from serine deprivation. Our data implicate serine as a key immunometabolite that directly modulates adaptive immunity by controlling T cell proliferative capacity.
Asunto(s)
Metaboloma , Serina/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Animales , Carbono/metabolismo , Puntos de Control del Ciclo Celular , Proliferación Celular , Dieta , Metabolismo Energético , Espacio Extracelular/metabolismo , Glicina , Listeria monocytogenes/inmunología , Redes y Vías Metabólicas , Ratones Endogámicos C57BL , Nucleótidos de Purina/biosíntesisRESUMEN
A central hallmark of cancer cells is the reprogramming of cellular metabolism to meet the bioenergetic and biosynthetic demands of malignant growth. Here, we report that the miR-17â¼92 microRNA (miRNA) cluster is an oncogenic driver of tumor metabolic reprogramming. Loss of miR-17â¼92 in Myc(+) tumor cells leads to a global decrease in tumor cell metabolism, affecting both glycolytic and mitochondrial metabolism, whereas increased miR-17â¼92 expression is sufficient to drive increased nutrient usage by tumor cells. We mapped the metabolic control element of miR-17â¼92 to the miR-17 seed family, which influences cellular metabolism and mammalian target of rapamycin complex 1 (mTORC1) signaling through negative regulation of the LKB1 tumor suppressor. miR-17-dependent tuning of LKB1 levels regulates both the metabolic potential of Myc(+) lymphomas and tumor growth in vivo. Our results establish metabolic reprogramming as a central function of the oncogenic miR-17â¼92 miRNA cluster that drives the progression of MYC-dependent tumors.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Regulación Neoplásica de la Expresión Génica , Linfocitos/metabolismo , Linfoma/metabolismo , MicroARNs/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Secuencia de Bases , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Glucólisis/genética , Xenoinjertos , Humanos , Transfusión de Linfocitos , Linfocitos/patología , Linfoma/genética , Linfoma/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , MicroARNs/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosforilación Oxidativa , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Podocytes are specialized epithelial cells of the kidney blood filtration barrier that contribute to permselectivity via a series of interdigitating actin-rich foot processes. Positioned between adjacent projections is a unique cell junction known as the slit diaphragm, which is physically connected to the actin cytoskeleton via the transmembrane protein nephrin. Evidence indicates that tyrosine phosphorylation of the intracellular tail of nephrin initiates signaling events, including recruitment of cytoplasmic adaptor proteins Nck1 and Nck2 that regulate actin cytoskeletal dynamics. Nephrin tyrosine phosphorylation is altered in human and experimental renal diseases characterized by pathologic foot process remodeling, prompting the hypothesis that phosphonephrin signaling directly influences podocyte morphology. To explore this possibility, we generated and analyzed knockin mice with mutations that disrupt nephrin tyrosine phosphorylation and Nck1/2 binding (nephrin(Y3F/Y3F) mice). Homozygous nephrin(Y3F/Y3F) mice developed progressive proteinuria accompanied by structural changes in the filtration barrier, including podocyte foot process effacement, irregular thickening of the glomerular basement membrane, and dilated capillary loops, with a similar but later onset phenotype in heterozygous animals. Furthermore, compared with wild-type mice, nephrin(Y3F/Y3F) mice displayed delayed recovery in podocyte injury models. Profiling of nephrin tyrosine phosphorylation dynamics in wild-type mice subjected to podocyte injury indicated site-specific differences in phosphorylation at baseline, injury, and recovery, which correlated with loss of nephrin-Nck1/2 association during foot process effacement. Our results define an essential requirement for nephrin tyrosine phosphorylation in stabilizing podocyte morphology and suggest a model in which dynamic changes in phosphotyrosine-based signaling confer plasticity to the podocyte actin cytoskeleton.
Asunto(s)
Podocitos/fisiología , Podocitos/ultraestructura , Tirosina/metabolismo , Animales , Femenino , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Fosforilación , Transducción de SeñalRESUMEN
Cancer cells adapt metabolically to proliferate under nutrient limitation. Here we used combined transcriptional-metabolomic network analysis to identify metabolic pathways that support glucose-independent tumor cell proliferation. We found that glucose deprivation stimulated re-wiring of the tricarboxylic acid (TCA) cycle and early steps of gluconeogenesis to promote glucose-independent cell proliferation. Glucose limitation promoted the production of phosphoenolpyruvate (PEP) from glutamine via the activity of mitochondrial PEP-carboxykinase (PCK2). Under these conditions, glutamine-derived PEP was used to fuel biosynthetic pathways normally sustained by glucose, including serine and purine biosynthesis. PCK2 expression was required to maintain tumor cell proliferation under limited-glucose conditions in vitro and tumor growth in vivo. Elevated PCK2 expression is observed in several human tumor types and enriched in tumor tissue from non-small-cell lung cancer (NSCLC) patients. Our results define a role for PCK2 in cancer cell metabolic reprogramming that promotes glucose-independent cell growth and metabolic stress resistance in human tumors.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Regulación Neoplásica de la Expresión Génica , Gluconeogénesis/genética , Neoplasias Pulmonares/metabolismo , Neoplasias/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Adaptación Fisiológica/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Ciclo del Ácido Cítrico/genética , Glucosa/deficiencia , Glutamina/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Metabolómica , Ratones , Ratones Desnudos , Mitocondrias/metabolismo , Neoplasias/genética , Neoplasias/patología , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Purinas/biosíntesis , Ácido Pirúvico/metabolismo , Serina/biosíntesisRESUMEN
One of the major metabolic changes associated with cellular transformation is enhanced nutrient utilization, which supports tumor progression by fueling both energy production and providing biosynthetic intermediates for growth. The liver kinase B1 (LKB1) is a serine/threonine kinase and tumor suppressor that couples bioenergetics to cell-growth control through regulation of mammalian target of rapamycin (mTOR) activity; however, the influence of LKB1 on tumor metabolism is not well defined. Here, we show that loss of LKB1 induces a progrowth metabolic program in proliferating cells. Cells lacking LKB1 display increased glucose and glutamine uptake and utilization, which support both cellular ATP levels and increased macromolecular biosynthesis. This LKB1-dependent reprogramming of cell metabolism is dependent on the hypoxia-inducible factor-1α (HIF-1α), which accumulates under normoxia in LKB1-deficient cells and is antagonized by inhibition of mTOR complex I signaling. Silencing HIF-1α reverses the metabolic advantages conferred by reduced LKB1 signaling and impairs the growth and survival of LKB1-deficient tumor cells under low-nutrient conditions. Together, our data implicate the tumor suppressor LKB1 as a central regulator of tumor metabolism and growth control through the regulation of HIF-1α-dependent metabolic reprogramming.