RESUMEN
Serotonin is a critical neurotransmitter in the regulation of emotional behavior. Although emotion processing is known to engage a corticolimbic circuit, including the amygdala and prefrontal cortex, exactly how this brain system is modulated by serotonin remains unclear. Here, we hypothesized that serotonin modulates variability in excitability and functional connectivity within this circuit. We tested whether this modulation contributes to inter-individual differences in emotion processing. Using a multimodal neuroimaging approach with a simultaneous PET-3T fMRI scanner, we simultaneously acquired BOLD signal while participants viewed emotional faces depicting fear and anger, while also measuring serotonin transporter (SERT) levels, regulating serotonin functions. Individuals with higher activity of the medial amygdala BOLD in response to fearful or angry facial expressions, who were temperamentally more anxious, also exhibited lower SERT availability in the dorsal raphe nucleus (DRN). Moreover, higher connectivity of the medial amygdala with the left dorsolateral prefrontal and the anterior cingulate cortex was associated with lower levels of SERT availability in the DRN. These results demonstrate the association between the serotonin transporter level and emotion processing through changes in functional interactions between the amygdala and the prefrontal areas in healthy humans.
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Proteínas de Transporte de Serotonina en la Membrana Plasmática , Serotonina , Humanos , Tomografía Computarizada por Rayos X , Miedo/fisiología , Emociones/fisiología , Corteza Prefrontal/diagnóstico por imagen , Imagen por Resonancia Magnética , Expresión FacialRESUMEN
Nowadays, exome sequencing is a robust and cost-efficient genetic diagnostic tool already implemented in many clinical laboratories. Despite it has undoubtedly improved our diagnostic capacity and has allowed the discovery of many new Mendelian-disease genes, it only provides a molecular diagnosis in up to 25-30% of cases. Here, we comprehensively evaluate the results of a large sample set of 4974 clinical exomes performed in our laboratory over a period of 5 years, showing a global diagnostic rate of 24.62% (1391/4974). For the evaluation we establish different groups of diseases and demonstrate how the diagnostic rate is not only dependent on the analyzed group of diseases (43.12% in ophthalmological cases vs 16.61% in neurological cases) but on the specific disorder (47.49% in retinal dystrophies vs 24.02% in optic atrophy; 18.88% in neuropathies/paraparesias vs 11.43% in dementias). We also detail the most frequent mutated genes within each group of disorders and discuss, on our experience, further investigations and directions needed for the benefit of patients.
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Atrofia Óptica , Distrofias Retinianas , Humanos , Exoma/genética , Secuenciación del Exoma , Distrofias Retinianas/genética , Atrofia Óptica/genéticaRESUMEN
Syndromic retinal diseases (SRDs) are a group of complex inherited systemic disorders, with challenging molecular underpinnings and clinical management. Our main goal is to improve clinical and molecular SRDs diagnosis, by applying a structured phenotypic ontology and next-generation sequencing (NGS)-based pipelines. A prospective and retrospective cohort study was performed on 100 probands with an a priori diagnosis of non-Usher SRDs, using available clinical data, including Human Phenotype Ontology annotation, and further classification into seven clinical categories (ciliopathies, specific syndromes and five others). Retrospective molecular diagnosis was assessed using different molecular and bioinformatic methods depending on availability. Subsequently, uncharacterized probands were prospectively screened using other NGS approaches to extend the number of analyzed genes. After phenotypic classification, ciliopathies were the most common SRD (35%). A global characterization rate of 52% was obtained, with six cases incompletely characterized for a gene that partially explained the phenotype. An improved characterization rate was achieved addressing prospective cases (83%) and well-recognizable syndrome (62%) subgroups. The 27% of the fully characterized cases were reclassified into a different clinical category after identification of the disease-causing gene. Clinical-exome sequencing is the most appropriate first-tier approach for prospective cases, whereas whole-exome sequencing and bioinformatic reanalysis increases the diagnosis of uncharacterized retrospective cases to 45%, mostly those with unspecific symptoms. Our study describes a comprehensive approach to SRDs in daily clinical practice and the importance of thorough clinical assessment and selection of the most appropriate molecular test to be used to solve these complex cases and elucidate novel associations.
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Enfermedades Hereditarias del Ojo/diagnóstico , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Enfermedades de la Retina/diagnóstico , Ciliopatías/genética , Estudios de Cohortes , Enfermedades Hereditarias del Ojo/genética , Femenino , Estudios de Asociación Genética , Pruebas Genéticas , Humanos , Masculino , Técnicas de Diagnóstico Molecular , Mutación , Fenotipo , Estudios Prospectivos , Enfermedades de la Retina/genética , Estudios Retrospectivos , SíndromeRESUMEN
An amendment to this paper has been published and can be accessed via the original article.
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BACKGROUND: Despite the advent of clinical PET-MR imaging for routine use in 2011 and the development of several methods to address the problem of attenuation correction, some challenges remain. We have identified and investigated several issues that might affect the reliability and accuracy of current attenuation correction methods when these are implemented for clinical and research studies of the brain. These are (1) the accuracy of converting CT Hounsfield units, obtained from an independently acquired CT scan, to 511 keV linear attenuation coefficients; (2) the effect of padding used in the MR head coil; (3) the presence of close-packed hair; (4) the effect of headphones. For each of these, we have examined the effect on reconstructed PET images and evaluated practical mitigating measures. RESULTS: Our major findings were (1) for both Siemens and GE PET-MR systems, CT data from either a Siemens or a GE PET-CT scanner may be used, provided the conversion to 511 keV µ-map is performed by the PET-MR vendor's own method, as implemented on their PET-CT scanner; (2) the effect of the head coil pads is minimal; (3) the effect of dense hair in the field of view is marked (> 10% error in reconstructed PET images); and (4) using headphones and not including them in the attenuation map causes significant errors in reconstructed PET images, but the risk of scanning without them may be acceptable following sound level measurements. CONCLUSIONS: It is important that the limitations of attenuation correction in PET-MR are considered when designing research and clinical PET-MR protocols in order to enable accurate quantification of brain PET scans. Whilst the effect of pads is not significant, dense hair, the use of headphones and the use of an independently acquired CT-scan can all lead to non-negligible effects on PET quantification. Although seemingly trivial, these effects add complications to setting up protocols for clinical and research PET-MR studies that do not occur with PET-CT. In the absence of more sophisticated PET-MR brain attenuation correction, the effect of all of the issues above can be minimised if the pragmatic approaches presented in this work are followed.
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Sorting nexin 27 (SNX27) recycles PSD-95, Dlg1, ZO-1 (PDZ) domain-interacting membrane proteins and is essential to sustain adequate brain functions. Here we define a fundamental SNX27 function in T lymphocytes controlling antigen-induced transcriptional activation and metabolic reprogramming. SNX27 limits the activation of diacylglycerol (DAG)-based signals through its high affinity PDZ-interacting cargo DAG kinase ζ (DGKζ). SNX27 silencing in human T cells enhanced T cell receptor (TCR)-stimulated activator protein 1 (AP-1)- and nuclear factor κB (NF-κB)-mediated transcription. Transcription did not increase upon DGKζ silencing, suggesting that DGKζ function is dependent on SNX27. The enhanced transcriptional activation in SNX27-silenced cells contrasted with defective activation of the mammalian target of rapamycin (mTOR) pathway. The analysis of Snx27 -/- mice supported a role for SNX27 in the control of T cell growth. This study broadens our understanding of SNX27 as an integrator of lipid-based signals with the control of transcription and metabolic pathways.
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Diacilglicerol Quinasa/metabolismo , Metabolismo Energético , Nexinas de Clasificación/metabolismo , Linfocitos T/metabolismo , Transcripción Genética , Animales , Antígenos CD28/metabolismo , Movimiento Celular/genética , Movimiento Celular/inmunología , Silenciador del Gen , Humanos , Interleucina-2/biosíntesis , Células Jurkat , Activación de Linfocitos , Ratones Noqueados , Proteína Quinasa C-alfa/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Nexinas de Clasificación/genética , Linfocitos T/inmunologíaRESUMEN
Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as 'lethal exosomes' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic.
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Muerte Celular/genética , Diacilglicerol Quinasa/genética , Proteína Quinasa C/genética , Proteínas Quinasas/genética , Animales , Linfocitos B/metabolismo , Diacilglicerol Quinasa/metabolismo , Exosomas/genética , Exosomas/metabolismo , Proteína Ligando Fas , Ratones , Cuerpos Multivesiculares/genética , Cuerpos Multivesiculares/metabolismo , Proteína Quinasa C/metabolismo , Proteína Quinasa D2 , Proteínas Quinasas/metabolismo , Linfocitos T/metabolismoRESUMEN
Diacylglycerol kinases (DGKs) transform diacylglycerol (DAG) into phosphatidic acid (PA), balancing the levels of these key metabolic and signaling lipids. We previously showed that PA derived from the DGKζ isoform promotes mammalian target of rapamycin complex 1 (mTORC1) activation. This function might be crucial for the growth and survival of cancer cells, especially for those resistant to the allosteric mTOR inhibitor rapamycin. How this positive function of DGKζ coordinates with DAG metabolism and signaling is unknown. In this study, we used a rapamycin-resistant colon cancer cell line as a model to address the role of DGKζ in tumor cells. We found that DGKζ predominated over other PA sources such as DGKα or phospholipase D to activate mTORC1, and that its activity was a component of the rapamycin-induced feedback loops. We show that the DGKζ DAG-consuming function is central to cell homeostasis, as DAG negatively regulates levels of the lipogenic transcription factor SREBP-1. Our findings suggest a model in which simultaneous regulation of DAG and PA levels by DGKζ is integrated with mTOR function to maintain tumor cell homeostasis; we provide new evidence of the crosstalk between mTOR and lipid metabolism that will be advantageous in the design of drug therapies.
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Diacylglycerol (DAG) metabolism has a critical function in Ras-regulated functions in mature T cells, but causal data linking defects in DAG-based signals with altered thymus development are missing. To study the effect of increased DAG metabolism in T-cell development, we engineered a membrane-targeted constitutive active version of DAG kinase-α (DGKα). We show that transgenic expression of constitutive active DGK leads to developmental defects in T cells, with a marked accumulation of immature CD8 thymocytes and a reduction in positive selected populations. These alterations are reflected in the periphery by a CD4/CD8 cell imbalance and general T-cell lymphopenia. The results link DAG metabolism to T-cell homeostasis, and show that correctly controlled generation and consumption of this lipid at the plasma membrane ensure T-cell passage through quality-control checkpoints during differentiation.
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Diglicéridos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Timocitos/citología , Timocitos/metabolismo , Animales , Southern Blotting , Western Blotting , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Diacilglicerol Quinasa/genética , Diacilglicerol Quinasa/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Células Jurkat , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/fisiología , Timocitos/enzimologíaRESUMEN
Multivesicular bodies (MVBs) are endocytic compartments that contain intraluminal vesicles formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these vesicles contain pro-apoptotic Fas ligand (FasL), which may be secreted as 'lethal exosomes' upon fusion of MVBs with the plasma membrane. Diacylglycerol kinase α (DGKα) regulate the secretion of exosomes, but it is unclear how this control is mediated. T-lymphocyte activation increases the number of MVBs that contain FasL. DGKα is recruited to MVBs and to exosomes in which it has a double function. DGKα kinase activity exerts a negative role in the formation of mature MVBs, as we demonstrate by the use of an inhibitor. Downmodulation of DGKα protein resulted in inhibition of both the polarisation of MVBs towards immune synapse and exosome secretion. The subcellular location of DGKα together with its complex role in the formation and polarised traffic of MVBs support the notion that DGKα is a key regulator of the polarised secretion of exosomes.
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Diacilglicerol Quinasa/metabolismo , Exosomas/metabolismo , Proteína Ligando Fas/metabolismo , Cuerpos Multivesiculares/fisiología , Linfocitos T/metabolismo , Línea Celular , Diacilglicerol Quinasa/análisis , Diacilglicerol Quinasa/antagonistas & inhibidores , Humanos , Cuerpos Multivesiculares/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Linfocitos T/enzimología , Linfocitos T/inmunologíaRESUMEN
Mammalian cells depend on extracellular input for the regulation of growth, proliferation and survival. Cancer cells evade these requirements, and are able to take up nutrients in a cell-autonomous fashion, which allows continuous growth and proliferation. To fulfill the high bioenergetic demands imposed by transformation, tumors must develop alternative mechanisms of energy production. Accordingly, the biochemical signature of cancer cells involves a shift to aerobic glycolysis, also known as the "Warburg effect". This property of cancer cells has resulted of great utility in modern medicine for detection of early tumors by positron-emission scanning. Nonetheless, the underlying mechanisms and contribution of the Warburg effect to the malignant phenotype have remained obscure. Thanks to recent advances in cancer research, we are beginning to understand the link between cancer genetics and the abnormal use of glucose by tumors. A new scenario is thus emerging, in which bioenergetics would contribute to and sustain malignant transformation. These findings are not only important for a better understanding of tumorigenesis; tumor reliance on glycolysis can be exploited in the search for novel, more potent therapeutic approaches to cancer treatment (AU)
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Humanos , Animales , Aerobiosis , Células/metabolismo , Metabolismo Energético , Glucólisis , Mutación , Neoplasias/etiología , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias , Neoplasias/terapia , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Tomografía de Emisión de Positrones , Biosíntesis de Proteínas , Serina-Treonina Quinasas TOR , Factores de TiempoRESUMEN
Interleukin-2 (IL-2) regulates the proliferation and homeostasis of lymphocytes through the coordinated activation of distinct signaling pathways. Deletion of the acidic-rich domain of the IL-2 receptor beta chain (IL-2Rbeta) prevents association of Src tyrosine kinases to the receptor, as well as IL-2-induced Akt activation. Cells bearing this deletion (BafbetaDeltaA) maintain full proliferation in response to IL-2 both in vivo and in vitro, suggesting that those pathways are dispensable for this important function of IL-2. In this study, we re-examined phosphatidylinositol-3 kinase (PI3K) activation in BafbetaDeltaA cells and found that, in BaF/3 IL-2RbetaDeltaA cells, deletion of the acidic domain induced constitutive activation of the receptor-associated PI3K activity. This, in turn, was responsible for the higher basal Akt activity observed in cells expressing this deletion. Based on these data, and since pharmacological abrogation of PI3K activity prevented IL-2-driven cell proliferation of BafbetaDeltaA cells, we conclude that the PI3K/Akt pathway is still functionally relevant in cells bearing this mutation. Moreover, we show that the PI3K-induced signals are, at least in part, responsible for c-myc expression. In conclusion, we have used this model to better identify those signals that are integral components of the molecular mechanisms responsible for IL-2-regulated cell proliferation.
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Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Receptores de Interleucina-2/metabolismo , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Interleucina-2/metabolismo , Interleucina-2/fisiología , Ratones , Inhibidores de las Quinasa Fosfoinosítidos-3 , Pruebas de Precipitina , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Receptores de Interleucina-2/química , Transducción de Señal , Transfección , Familia-src Quinasas/metabolismoRESUMEN
Diacylglycerol kinase (DGK) is suggested to attenuate diacylglycerol-induced cell responses through the phosphorylation of this second messenger to phosphatidic acid. Here, we show that DGKalpha, an isoform highly expressed in T lymphocytes, translocates from cytosol to the plasma membrane in response to two different receptors known to elicit T cell activation responses: an ectopically expressed muscarinic type I receptor and the endogenous T cell receptor. Translocation in response to receptor stimulation is rapid, transient, and requires calcium and tyrosine kinase activation. DGKalpha-mediated phosphatidic acid generation allows dissociation of the enzyme from the plasma membrane and return to the cytosol, as demonstrated using a pharmacological inhibitor and a catalytically inactive version of the enzyme. The NH(2)-terminal domain of the protein is shown to be responsible for receptor-induced translocation and phosphatidic acid-mediated membrane dissociation. After examining induction of the T cell activation marker CD69 in cells expressing a constitutively active form of the enzyme, we present evidence of the negative regulation that DGKalpha exerts on diacylglycerol-derived cell responses. This study is the first to describe DGKalpha as an integral component of the signaling cascades that link plasma membrane receptors to nuclear responses.
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Diacilglicerol Quinasa/fisiología , Receptores Muscarínicos/metabolismo , Transducción de Señal/fisiología , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Sitios de Unión , Transporte Biológico , Biomarcadores , Antígenos CD28/metabolismo , Células COS , Carbacol/metabolismo , Carbacol/farmacología , Membrana Celular/metabolismo , Chlorocebus aethiops , Agonistas Colinérgicos/metabolismo , Agonistas Colinérgicos/farmacología , Reactivos de Enlaces Cruzados , Diacilglicerol Quinasa/metabolismo , Diglicéridos/farmacología , Proteínas Fluorescentes Verdes , Humanos , Células Jurkat , Lectinas Tipo C , Proteínas Luminiscentes , Activación de Linfocitos , Ácidos Fosfatidicos/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Receptor Muscarínico M1 , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Receptores Muscarínicos/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
CD28 is a costimulatory receptor expressed in most CD4(+) T cells. Despite the long-standing evidence for up- and downregulation of surface CD28 expression in vitro, and the key regulatory role assigned to the upregulation of CD28 counterreceptor [the CD152 (CTLA-4) molecule], in vivo CD28 induction has attracted little attention. We studied CD28 and CD152 expression and function in 33 rheumatoid arthritis (RA) patients, 20 clinically active and 13 inactive, and in 24 healthy donors. Four subsets of CD28(-), CD28(low), CD28(int), and CD28(high) peripheral blood human CD4(+) T cells were defined using three-color flow cytometry. The three CD28(+) subsets displayed a one-, two-, or threefold quantitative difference in their relative number of CD28 antibody binding sites, respectively (P < 0.01). RA patients, whether active or inactive, showed a distinct phenotype when compared to healthy donors: (i) the percentage of CD4(+)CD28(high) cells was increased twofold and the CD4(+)CD28(low) subset was reduced twofold (P < 0.01) and (ii) the CD4(+)CD28(high) cells from RA patients showed an in vivo activated phenotype, CD45RO(+)CD5(high)IL-2Ralpha(+) (P < 0.01). Active RA patients were different from inactive patients. They showed a twofold increase in mean CD28 expression (P < 0.05), whereas each of the CD28(+) subsets in the inactive RA patients showed reduced expression when compared to healthy donors. Notably, both active and inactive RA patients showed abnormal CD28 upregulation when T cells were activated in vitro with CD3 antibodies, but only inactive RA patients showed a hypoproliferative response to TCR/CD3 triggering when compared to healthy donors (P < 0.01). This defective proliferation was normalized by concurrent crosslinking with CD28 antibody. No differences were noted in the expression of CD152 or CD80, a CD28 and CD152 shared ligand. The disregulated in vivo expression of CD28 was related to the RA patients' disease activity and suggests that modulation of CD28 surface levels may be an additional mechanism to finely tune the delicate responsiveness/tolerance balance.
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Artritis Reumatoide/inmunología , Antígenos CD28/metabolismo , Linfocitos T CD4-Positivos/inmunología , Subgrupos de Linfocitos T/inmunología , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Secuencia de Bases , Antígenos CD28/genética , Linfocitos T CD4-Positivos/patología , Estudios de Casos y Controles , Cartilla de ADN/genética , Humanos , Tolerancia Inmunológica , Técnicas In Vitro , Interleucina-2/genética , Antígenos Comunes de Leucocito/metabolismo , Activación de Linfocitos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina-2/metabolismo , Membrana Sinovial/inmunología , Membrana Sinovial/patología , Subgrupos de Linfocitos T/patología , Regulación hacia ArribaRESUMEN
Hypoxia-inducible factor 1 (HIF-1) induces a gene expression program essential for the cellular adaptation to lowered oxygen environments. The intracellular mechanisms by which hypoxia induces HIF-1 remain poorly understood. Here we show that exposure of various cell types to hypoxia raises the intracellular level of phosphatidic acid primarily through the action of diacylglycerol kinase (DGK). Pharmacological inhibition of DGK activity through use of the specific DGK inhibitors and abrogated specifically HIF-1-dependent transcription analyzed with a HIF-1-responsive reporter plasmid. A more detailed analysis revealed that pharmacological inhibition of DGK activity prevented the hypoxia-dependent accumulation of the HIF-1alpha subunit and the subsequent HIF-1-DNA complex formation as well as hypoxia-induced activity of the HIF-1 transactivation domains localized to amino acids 530-582 and 775-826 of the HIF-1alpha subunit. Our results demonstrate for the first time that accumulation of phosphatidic acid through DGK underlines oxygen sensing and provide evidence for the involvement of this lipid kinase in the intracellular signaling that leads to HIF-1 activation.
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Proteínas de Unión al ADN/metabolismo , Diacilglicerol Quinasa/metabolismo , Hipoxia , Proteínas Nucleares/metabolismo , Oxígeno/metabolismo , Factores de Transcripción , Núcleo Celular/metabolismo , ADN/metabolismo , Diacilglicerol Quinasa/antagonistas & inhibidores , Diglicéridos/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Immunoblotting , Luciferasas/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfolípidos/metabolismo , Piperidinas/farmacología , Plásmidos/metabolismo , Pirimidinonas/farmacología , Quinazolinas/farmacología , Quinazolinonas , Proteínas Recombinantes/metabolismo , Transducción de Señal , Tiazoles/farmacología , Transcripción Genética , Activación Transcripcional , TransfecciónRESUMEN
Control of lymphocyte cell survival and proliferation is critical for both the immune response and for the prevention of autoimmune and infectious diseases. The actions of interleukin-2, the major T-cell regulatory cytokine, are mediated by the complex network of divergent signalling pathways controlled by its high-affinity receptor. Various studies have indicated that the generation of certain lipid second messengers is an important mechanism in the control of proliferation and cell death. We have examined the relationship between diacylglycerol and ceramide and the levels of the lipids phosphatidylcholine and sphingomyelin, their potential precursors, in the human T-cell line Kit 225 cultured in three distinct conditions to favor apoptosis, cell arrest, and proliferation. Our data show that, in proliferating cells, the ratios of diacylglycerol/ceramide and phosphatidylcholine/sphingomyelin are higher than those found in arrested cells and increase with time in culture. These ratios are rapidly reversed in apoptotic cells. Further experiments reveal that de novo synthesis of both diacylglycerol and phosphatidylcholine is greatest in proliferating cells, whereas sphingomyelin synthase activity is increased in cells undergoing apoptosis. In summary, our results demonstrate for the first time that the ratio of mitogenic/antimitogenic lipids changes dramatically during T-cell proliferation and cell death. These results indicate that lipid second messengers and the enzymes that are responsible for their generation may provide targets for novel therapeutic interventions in the clinical management of immunosuppression and autoimmune disease.
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Apoptosis , División Celular , Metabolismo de los Lípidos , Sistemas de Mensajero Secundario , Linfocitos T/metabolismo , Línea Celular , Ceramidas/metabolismo , Diglicéridos/metabolismo , Inhibidores de Crecimiento/metabolismo , Humanos , Mitógenos/metabolismo , Modelos Biológicos , Fosfatidilcolinas/metabolismo , Esfingomielinas/metabolismo , Linfocitos T/citología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
Despite the fact that phosphatidic acid (PtdOH) has been implicated as a lipid second messenger for nearly a decade, its intracellular targets have remained unclear. We sought to investigate how an increase in the level of PtdOH could modulate phosphatidylinositol 4-phosphate 5-kinase (PIPkin), an enzyme involved in phosphatidylinositol 4,5-bisphosphate synthesis. Transfection of porcine aortic endothelial (PAE) cells with haemagglutinin (HA)-tagged type Ialpha PIPkin followed by immunofluorescence confocal microscopy revealed the enzyme to be localised to the plasma membrane. When the transfected PAE cells were stimulated with lyso-PtdOH, increased PIPkin activity was found to be associated with HA immunoprecipitates in an in vitro assay. This PIPkin activation was found to be greatly reduced by prior treatment of the cells with 1-butanol, thereby implicating phospholipase D (PLD) as the in vivo generator of PtdOH. In order to determine if the PtdOH-dependent activation of type Ialpha PIPkin was dictated by a specific molecular composition of PtdOH, the wild type murine and porcine alpha isoforms of diacylglycerol kinase (DGK) were individually co-transfected along with type Ialpha PIPkin. Under these conditions an increase in type Ialpha PIPkin lipid kinase activity was found in HA immunoprecipitates in an in vitro assay. No increases in lipid kinase activity were observed when type Ialpha PIPkin was co-transfected with either the human DGKepsilon isoform or a kinase-dead mutant of the murine DGKalpha isoform. These results provide the first direct evidence for the unification of the production of saturated/monounsaturated PtdOH (through two different routes, PLD and DGK) and the in vivo activation of type Ialpha PIPkin by this lipid second messenger.
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Ácidos Fosfatidicos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Células Cultivadas , Diacilglicerol Quinasa/metabolismo , Endotelio Vascular/metabolismo , Activación Enzimática , Humanos , Líquido Intracelular/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Fosfolipasa D/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero Secundario , Porcinos , TransfecciónRESUMEN
The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.
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Antígenos CD , Antígenos de Diferenciación , Polaridad Celular/inmunología , Quimiocinas CXC/fisiología , Quimiotaxis de Leucocito/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Linfocitos T/enzimología , Linfocitos T/inmunología , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Movimiento Celular/inmunología , Polaridad Celular/genética , Quimiocina CXCL12 , Quimiotaxis de Leucocito/genética , Citoesqueleto/enzimología , Citoesqueleto/inmunología , Citoesqueleto/metabolismo , Inducción Enzimática/inmunología , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/metabolismo , Fosfatidilinositol 3-Quinasas/biosíntesis , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Receptores CXCR4/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Células del Estroma/enzimología , Células del Estroma/inmunología , Linfocitos T/citología , TransfecciónRESUMEN
Stimulation via IL-2R ligation causes T lymphocytes to transit through the cell cycle. Previous experiments by our group have demonstrated that, in human T cells, IL-2 binding induces phosphatidic acid production through activation of the alpha isoform of diacylglycerol kinase. In this study, using the IL-2-dependent mouse T cell line CTLL-2, we demonstrate that pharmacological inhibition of IL-2-induced diacylglycerol kinase activation is found to block IL-2-induced late G1 to S transition without affecting cell viability. Herein, we demonstrate that diacylglycerol kinase inhibition has a profound effect on the induction of the protooncogenes c-myc, c-fos, and c-raf by IL-2, whereas expression of bcl-2 and bcl-xL are not affected. When the IL-2-regulated cell cycle control checkpoints are examined in detail, we demonstrate that inhibition of diacylglycerol kinase activation prevents IL-2 induction of cyclin D3 without affecting p27 down-regulation. The strict control of cell proliferation exerted by phosphatidic acid through activation of diacylglycerol kinase is independent of other well-characterized IL-2R-derived signals, such as the phosphatidylinositol-3 kinase/Akt pathway, indicating the existence of a different and important mechanism to control cell division.
Asunto(s)
Proteínas de Ciclo Celular , Diacilglicerol Quinasa/antagonistas & inhibidores , Fase G1/inmunología , Interleucina-2/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Serina-Treonina Quinasas , Fase S/inmunología , Linfocitos T Citotóxicos/enzimología , Proteínas Supresoras de Tumor , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/inmunología , Línea Celular , Medio de Cultivo Libre de Suero , Ciclina D3 , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Diacilglicerol Quinasa/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Interleucina-4/antagonistas & inhibidores , Interleucina-4/fisiología , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Piperidinas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proto-Oncogenes/efectos de los fármacos , Proto-Oncogenes/inmunología , Quinazolinas/farmacología , Quinazolinonas , Proteína de Retinoblastoma/metabolismo , Fase S/efectos de los fármacos , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/metabolismoRESUMEN
In recent times 3-phosphoinositides have emerged as important regulators of cell metabolism, survival, and proliferation. During the last year, the phospholipid phosphatidylinositol 3, 5-bisphosphate (PtdIns3,5P2) was identified in yeast, fibroblasts, SV40-transformed kidney (COS-7) cells, and platelets. The discovery of this novel phospholipid has increased the complexity of the metabolism relating to the generation of biologically active inositol-containing lipids. We describe here the identification of PtdIns3,5P2 in the CTLL-2 mouse T-lymphocyte cell line using two in vivo radiolabeling protocols. Treatment of the cells with UV radiation led to an increase in the cellular content of PtdIns3,5P2. In contrast, preincubation of the cells with wortmannin or treatment with hypertonic medium (high concentration sorbitol) led to the opposite effect. Herein we demonstrate that interleukin-2 (IL-2), the growth factor required for CTLL-2 cell proliferation, was able to increase the level of PtdIns3,5P2 with similar kinetics to that of the formation of phosphatidylinositol 3,4-bisphosphate (PtdIns3, 4P2). An increase in this novel 3-phosphorylated lipid in response to IL-2 seems to be a general property of this cytokine because a similar result was obtained when the pre-B cell line BaF/3 expressing the high affinity IL-2 receptor was used. Using a constitutively active regulatory subunit of type I phosphatidylinositol 3-kinase and cells expressing a deletion of the serine-rich domain of the IL-2 receptor beta chain, which is required for IL-2-stimulated type I phosphatidylinositol 3-kinase activation, we demonstrate that IL-2-induced generation of PtdIns3, 5P2 is related to the activation of this enzyme. The results show for the first time the identification of PtdIns3,5P2 in both T- and B-lymphocytes and indicate its positive regulation by the mitogen IL-2.