Résumé
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.
Sujets)
Animaux , Humains , Souris , Facteur de transcription ATF-2/métabolisme , Séparation cellulaire , Chimiokines/biosynthèse , Chimiotaxie/effets des médicaments et des substances chimiques , Milieux de culture conditionnés/pharmacologie , Extracellular Signal-Regulated MAP Kinases/métabolisme , Protéines G/métabolisme , JNK Mitogen-Activated Protein Kinases/métabolisme , Lipopolysaccharides/pharmacologie , Macrophages/effets des médicaments et des substances chimiques , Facteur de transcription NF-kappa B/métabolisme , Granulocytes neutrophiles/cytologie , Toxine pertussique/pharmacologie , Phosphatidylinositol 3-kinases/métabolisme , Protéines proto-oncogènes c-akt/métabolisme , Récepteurs Wnt/métabolisme , Type C Phospholipases/métabolisme , Protéines de type Wingless/pharmacologie , p38 Mitogen-Activated Protein Kinases/métabolismeRésumé
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.