RESUMEN
Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (beta-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response.
Asunto(s)
Animales , Humanos , Ratones , Factor de Transcripción Activador 2/metabolismo , Separación Celular , Quimiocinas/biosíntesis , Quimiotaxis/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , FN-kappa B/metabolismo , Neutrófilos/citología , Toxina del Pertussis/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Wnt/metabolismo , Fosfolipasas de Tipo C/metabolismo , Proteínas Wnt/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-gamma1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-gamma1, abolished interactions with translational elongation factor 1-alpha. Here, we report that the Y509A/F510A mutant PLC-gamma1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-gamma1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-gamma1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-gamma1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-gamma1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-gamma1 activation in vivo.