RESUMEN
CD28 superagonistic antibodies (CD28SAb) can preferentially activate and expand immunosuppressive regulatory T cells (Treg) in mice. However, pre-clinical trials assessing CD28SAbs for the therapy of autoimmune diseases reveal severe systemic inflammatory response syndrome in humans, thereby implying the existence of distinct signalling abilities between human and mouse CD28. Here, we show that a single amino acid variant within the C-terminal proline-rich motif of human and mouse CD28 (P212 in human vs. A210 in mouse) regulates CD28-induced NF-κB activation and pro-inflammatory cytokine gene expression. Moreover, this Y209APP212 sequence in humans is crucial for the association of CD28 with the Nck adaptor protein for actin cytoskeleton reorganisation events necessary for CD28 autonomous signalling. This study thus unveils different outcomes between human and mouse CD28 signalling to underscore the importance of species difference when transferring results from preclinical models to the bedside.
Asunto(s)
Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/metabolismo , Antígenos CD28/genética , Antígenos CD28/metabolismo , Humanos , Activación de Linfocitos/genética , Activación de Linfocitos/fisiología , Ratones , FN-kappa B/metabolismo , Proteínas Oncogénicas/metabolismo , Unión Proteica , Transducción de Señal/genética , Linfocitos T Reguladores/metabolismoRESUMEN
Mitochondrial activity and metabolic reprogramming influence the phenotype of cancer cells and resistance to targeted therapy. We previously established that an insulin-like growth factor 1 (IGF-1)-inducible mitochondrial UTP carrier (PNC1/SLC25A33) promotes cell growth. This prompted us to investigate whether IGF signaling is essential for mitochondrial maintenance in cancer cells and whether this contributes to therapy resistance. Here we show that IGF-1 stimulates mitochondrial biogenesis in a range of cell lines. In MCF-7 and ZR75.1 breast cancer cells, IGF-1 induces peroxisome proliferator-activated receptor γ coactivator 1ß (PGC-1ß) and PGC-1α-related coactivator (PRC). Suppression of PGC-1ß and PRC with siRNA reverses the effects of IGF-1 and disrupts mitochondrial morphology and membrane potential. IGF-1 also induced expression of the redox regulator nuclear factor-erythroid-derived 2-like 2 (NFE2L2 alias NRF-2). Of note, MCF-7 cells with acquired resistance to an IGF-1 receptor (IGF-1R) tyrosine kinase inhibitor exhibited reduced expression of PGC-1ß, PRC, and mitochondrial biogenesis. Interestingly, these cells exhibited mitochondrial dysfunction, indicated by reactive oxygen species expression, reduced expression of the mitophagy mediators BNIP3 and BNIP3L, and impaired mitophagy. In agreement with this, IGF-1 robustly induced BNIP3 accumulation in mitochondria. Other active receptor tyrosine kinases could not compensate for reduced IGF-1R activity in mitochondrial protection, and MCF-7 cells with suppressed IGF-1R activity became highly dependent on glycolysis for survival. We conclude that IGF-1 signaling is essential for sustaining cancer cell viability by stimulating both mitochondrial biogenesis and turnover through BNIP3 induction. This core mitochondrial protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Mitofagia , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Transducción de Señal , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Supervivencia Celular/genética , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Células MCF-7 , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Unión al ARN , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Using antibodies against intracellular phosphoproteins, flow cytometry can be used to monitor simultaneously multiple signaling pathways. Here, we tested a recently released procedure to analyze phosphorylation events in human monocytes upon different types of stimulation. METHODS: Whole blood was treated by lipopolysaccharide (LPS) or granulocyte-macrophage colony-stimulating factor (GM-CSF), then cells were labeled by antibodies recognizing cell surface and cytosolic proteins. Human monocytes were identified by a CD14 - CD45 staining and three phosphorylated proteins such as AKT, ERK-1/2, and STAT5, were simultaneously detected by multicolor phosphoflow analysis. RESULTS: By this rapid method, we are able to detect directly from a blood sample several signaling events in human monocytes where LPS stimulation induces preferentially ERK-1/2 phosphorylation where as GM-CSF stimulation induces STAT5 phosphorylation. CONCLUSIONS: This procedure provides a simultaneous measurement of multiple activated signaling molecules using a simplified and rapid protocol. © 2015 International Clinical Cytometry Society.
Asunto(s)
Citometría de Flujo , Antígenos Comunes de Leucocito/genética , Receptores de Lipopolisacáridos/genética , Fosfoproteínas/aislamiento & purificación , Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Lipopolisacáridos/farmacología , Proteína Quinasa 3 Activada por Mitógenos/genética , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Factor de Transcripción STAT5/genética , Transducción de Señal/efectos de los fármacosRESUMEN
The CD28 costimulatory receptor has a pivotal role in T cell biology as this molecule amplifies T cell receptor (TCR) signals to provide an efficient immune T cell response. There is a large debate about how CD28 mediates these signals. Here, we designed a CD28 gene-targeted knock-in mouse strain lacking the cytoplasmic tail of CD28. As is the case in CD28-deficient (CD28 knock-out) mice, regulatory T cell homeostasis and T cell activation are altered in these CD28 knock-in mice. Unexpectedly, the presence of a CD28 molecule deprived of its cytoplasmic tail could partially induce some early activation events in T cells such as signaling events or expression of early activation markers. These results unravel a new mechanism of T cell costimulation by CD28, independent of its cytoplasmic tail.
Asunto(s)
Antígenos CD28/fisiología , Activación de Linfocitos/fisiología , Linfocitos T/inmunología , Animales , Antígenos CD28/química , Antígenos CD28/genética , Técnicas de Sustitución del Gen , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Estructura Terciaria de Proteína , Fiebre Q/inmunología , Transducción de SeñalRESUMEN
Downstream of tyrosine kinase (Dok) proteins Dok-1 and Dok-2 are involved in T cell homeostasis maintenance. Dok protein tyrosine phosphorylation plays a key role in establishing negative feedback loops of T cell signaling. These structurally related adapter molecules contain a pleckstrin homology (PH) domain generally acting as a lipid/protein-interacting module. We show that the presence of this PH domain is necessary for the tyrosine phosphorylation of Dok proteins and their negative functions in T cells. We find that Dok-1/Dok-2 PH domains bind in vitro to the rare phosphoinositide species, phosphatidylinositol 5-phosphate (PtdIns5P). Dok tyrosine phosphorylation correlates with PtdIns5P production in T cells upon TCR triggering. Furthermore, we demonstrate that PtdIns5P increase regulates Dok tyrosine phosphorylation in vivo. Together, our data identify a novel lipid mediator in T cell signaling and suggest that PH-PtdIns5P interactions regulate T cell responses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas de Unión al ADN/inmunología , Activación de Linfocitos/inmunología , Fosfatos de Fosfatidilinositol/biosíntesis , Fosfoproteínas/inmunología , Proteínas de Unión al ARN/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Células Jurkat , Fosfatos de Fosfatidilinositol/inmunología , Fosfoproteínas/metabolismo , Fosforilación , Proteínas de Unión al ARN/metabolismo , Resonancia por Plasmón de Superficie , Linfocitos T/metabolismoRESUMEN
Zanolimumab is a human IgG1 antibody against CD4, which is in clinical development for the treatment of cutaneous and nodal T-cell lymphomas. Here, we report on its mechanisms of action. Zanolimumab was found to inhibit CD4+ T cells by combining signaling inhibition with the induction of Fc-dependent effector mechanisms. First, T-cell receptor (TCR) signal transduction is inhibited by zanolimumab through a fast, dual mechanism, which is activated within minutes. Ligation of CD4 by zanolimumab effectively inhibits early TCR signaling events but, interestingly, activates signaling through the CD4-associated tyrosine kinase p56lck. An uncoupling of p56lck from the TCR by anti-CD4 allows the kinase to transmit direct inhibitory signals via the inhibitory adaptor molecules Dok-1 and SHIP-1. Second, CD4+ T cells are killed by induction of antibody-dependent cell-mediated cytotoxicity, to which CD45RO+ cells are more sensitive than CD45RA+ cells. Finally, zanolimumab induces down-modulation of CD4 from cell surfaces via a slow Fc-dependent mechanism. In conclusion, zanolimumab rapidly inhibits T-cell signaling via a dual mechanism of action combined with potent Fc-dependent lysis of CD4+ T cells and may act long-term by down-regulating CD4.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfoma Cutáneo de Células T/inmunología , Linfoma Cutáneo de Células T/terapia , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Citotoxicidad Celular Dependiente de Anticuerpos , Complejo CD3/inmunología , Antígenos CD4/biosíntesis , Antígenos CD4/genética , Antígenos CD4/inmunología , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/inmunología , Método Doble Ciego , Regulación hacia Abajo , Humanos , Inositol Polifosfato 5-Fosfatasas , Activación de Linfocitos/efectos de los fármacos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Psoriasis/inmunología , Psoriasis/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The insulin/insulin-like growth factor (IGF) signaling pathway to mTOR is essential for the survival and growth of normal cells and also contributes to the genesis and progression of cancer. This signaling pathway is linked with regulation of mitochondrial function, but how is incompletely understood. Here we show that IGF-I and insulin induce rapid transcription of the mitochondrial pyrimidine nucleotide carrier PNC1, which shares significant identity with the essential yeast mitochondrial carrier Rim2p. PNC1 expression is dependent on PI-3 kinase and mTOR activity and is higher in transformed fibroblasts, cancer cell lines, and primary prostate cancers than in normal tissues. Overexpression of PNC1 enhances cell size, whereas suppression of PNC1 expression causes reduced cell size and retarded cell cycle progression and proliferation. Cells with reduced PNC1 expression have reduced mitochondrial UTP levels, but while mitochondrial membrane potential and cellular ATP are not altered, cellular ROS levels are increased. Overall the data indicate that PNC1 is a target of the IGF-I/mTOR pathway that is essential for mitochondrial activity in regulating cell growth and proliferation.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Transporte de Nucleótidos/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular Transformada , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Proteínas de Transporte de Nucleótidos/química , Proteínas de Transporte de Nucleótidos/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor IGF Tipo 1/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR , Uridina Trifosfato/metabolismoRESUMEN
The Dok adaptor family of proteins binding to RasGAP, consisting of Dok-1 and Dok-2, are critical regulators in cell proliferation. These molecules are partners and/or substrates of different protein tyrosine kinases considered as oncoproteins. Here, we show that Dok-1 and Dok-2 are the major tyrosine-phosphorylated proteins associated to Tec, a protein tyrosine kinase expressed in T cells. Furthermore, we evaluate the effect of Dok-1 or Dok-2 on Tec-mediated signalling pathways in T cells. Here, we provide evidence that Dok-1 and Dok-2 proteins are involved in a negative feedback regulation of Tec via a downregulation of its tyrosine phosphorylation and downstream signalling pathways including the Ras pathway. Either Dok-1 or Dok-2 therefore represents a mean of potent retrograde control for protein tyrosine kinase signalling, and then possibly of tumor development.