Protein Tyrosine Kinase Fyn Regulates TLR4-Elicited Responses on Mast Cells Controlling the Function of a PP2A-PKCα/β Signaling Node Leading to TNF Secretion

Abstract

Mast cells produce proinflammatory cytokines in response to TLR4 ligands, but the signaling pathways involved are not fully described. In this study, the participation of the Src family kinase Fyn in the production of TNF after stimulation with LPS was evaluated using bone marrow–derived mast cells from wild-type and Fyn-deficient mice. Fyn−/− cells showed higher LPS-induced secretion of preformed and de novo–synthesized TNF. In both cell types, TNF colocalized with vesicle-associated membrane protein (VAMP)3-positive compartments. Addition of LPS provoked coalescence of VAMP3 and its interaction with synaptosomal-associated protein 23; those events were increased in the absence of Fyn. Higher TNF mRNA levels were also observed in Fyn-deficient cells as a result of increased transcription and greater mRNA stability after LPS treatment. Fyn−/− cells also showed higher LPS-induced activation of TAK-1 and ERK1/2, whereas IκB kinase and IκB were phosphorylated, even in basal conditions. Increased responsiveness in Fyn−/− cells was associated with a lower activity of protein phosphatase 2A (PP2A) and augmented activity of protein kinase C (PKC)α/β, which was dissociated from PP2A and increased its association with the adapter protein neuroblast differentiation–associated protein (AHNAK, desmoyokin). LPS-induced PKCα/β activity was associated with VAMP3 coalescence in WT and Fyn-deficient cells. Reconstitution of MC-deficient Wsh mice with Fyn−/− MCs produced greater LPS-dependent production of TNF in the peritoneal cavity. Our data show that Fyn kinase is activated after TLR4 triggering and exerts an important negative control on LPS-dependent TNF production in MCs controlling the inactivation of PP2Ac and activation of PKCα/β necessary for the secretion of TNF by VAMP3+ carriers.

Orai and TRPC channel characterization in FcεRI‐mediated calcium signaling and mediator secretion in human mast cells

“…In late phase mast cell responses, however TNFα may be synthesized de novo and secreted along with other cytokines through an alternative pathway (Martin‐Avila et al. ), independent of calcium influx through Orai, such as shown here for Serpin E1. As with other de novo synthesized cytokines and chemokines, calcium‐sensitivity will be depend on the transcription factors driving their synthesis and integrated signaling with co‐stimulatory receptors (Gilfillan and Tkaczyk ; Klein et al.…”

Fyn kinase mediates cortical actin ring depolymerization required for mast cell migration in response to TGF‐β in mice

“…B and C). The increase on basal phosphorylation on tested proteins in Fyn‐deficient cells, together with our recent finding indicating that Fyn kinase controls the activity of PP2A in mast cells , led us to test if a defective activity of PP2A could be involved in the observed phenotype, since this phosphatase plays an important role in the migration of immune cells .…”

“…In particular, Fyn controls the Gab2/PI3K axis , leukotriene synthesis, NFκB activation and calcium mobilization , and the activity of sphingosine kinase 1 and 2 after FcεRI crosslinking. In response to TLR‐4 receptor triggering, Fyn positively controls the activity of PP2A phosphatase , modulating the activation/inactivation cycles of a number of signaling proteins involved in cytokine secretion.…”

Tumor necrosis factor alpha mediates neuromuscular synapse elimination

“…S1a). The intracellular TNFα puncta in muscle cells were reminiscent of vesicular TNFα signals during trafficking and secretion 42,43 ( Fig. 1b arrows and Supplementary Fig.…”