Protein tyrosine kinase Fyn regulates TLR4-elicited responses on mast cells controlling the function of a PP2A-PKC a/b signaling node leading to TNF secretion

Abstract Signaling pathways leading to cytokine production after TLR-4 triggering in mast cells (MC) are not well described. In this work, the participation of the Src family kinase Fyn on the production of TNF after stimulation with LPS was evaluated MCs from WT and Fyn-deficient mice. Fyn−/− cells showed higher LPS-induced secretion of preformed and de novo synthesized TNF and the processes underlying that phenotype were investigated. In both cell types, TNF co-localized with VAMP3-positive compartments. Although LPS addition provoked coalescence of VAMP3 and the interaction of VAMP3 with SNAP23 in WT and Fyn−/− cells, the apparent size of VAMP3-containing aggregates was larger in Fyn−/− cells and increased formation of VAMP3-SNAP23 complexes was observed compared to WT. LPS-addition to Fyn−/− cells lead to higher activation of TAK-1, ERK1/2 and IKK, which was reflected on increased nuclear translocation of transcription factors such as NFkB and CEBPd. Increased LPS responsiveness in Fyn−/− cells was associated to a lower activity of Protein Phosphatase 2A(PP2A) and augmented phosphorylation of key signaling kinases, such as PKCa/b, which controlled TLR4-induced VAMP3 coalescence. Reconstitution of MC-deficient Wsh mice with Fyn−/− MC produced an increased LPS-dependent production of TNF in peritoneal cavity. Our data show that Fyn kinase is activated after TLR-4 triggering and exerts an important negative control on LPS-dependent TNF production in MC controlling the inactivation of PP2Ac and PKC a/b phosphorylation necessary for the secretion of TNF by VAMP3-positive carriers. Supported by Grant ANR-Conacyt 188565 to UB, CGE and MMS.