Differential Lyn-dependence of the SHIP1-deficient mast cell phenotype

Abstract

BackgroundAntigen (Ag)/IgE-mediated mast cell (MC) responses play detrimental roles in allergic diseases. MC activation via the high-affinity receptor for IgE (FcεRI) is controlled by the Src family kinase Lyn. Lyn-deficient (-/-) bone marrow-derived MCs (BMMCs) have been shown by various laboratories to exert stronger activation of the PI3K pathway, degranulation, and production of pro-inflammatory cytokines compared to wild-type (wt) cells. This mimics the phenotype of BMMCs deficient for the SH2-containing inositol-5’-phosphatase 1 (SHIP1). In this line, Lyn has been demonstrated to tyrosine-phosphorylate and activate SHIP1, thereby constituting a negative feedback control of PI3K-mediated signals. However, several groups have also reported on Lyn-/- BMMCs degranulating weaker than wt BMMCs.ResultsLyn-/- BMMCs, which show a suppressed degranulation response, were found to exhibit abrogated tyrosine phosphorylation of SHIP1 as well. This indicated that even in the presence of reduced SHIP1 function MC degranulation is dependent on Lyn function. In contrast to the reduced immediate secretory response, pro-inflammatory cytokine production was augmented in Lyn-/- BMMCs. For closer analysis, Lyn/SHIP1-double-deficient (dko) BMMCs were generated. In support of the dominance of Lyn deficiency, dko BMMCs degranulated significantly weaker than SHIP1-/- BMMCs. This coincided with reduced LAT1 and PLC-γ1 phosphorylation as well as Ca2+ mobilization in those cells. Interestingly, activation of the NFκB pathway followed the same pattern as measured by IκBα phosphorylation/degradation as well as induction of NFκB target genes. This suggested that Ag-triggered NFκB activation involves a Ca2+-dependent step. Indeed, IκBα phosphorylation/degradation and NFκB target gene induction were controlled by the Ca2+-dependent phosphatase calcineurin.ConclusionsLyn deficiency is dominant over SHIP1 deficiency in MCs with respect to Ag-triggered degranulation and preceding signaling events. Moreover, the NFκB pathway and respective targets are activated in a Lyn- and Ca2+-dependent manner, reinforcing the importance of Lyn for MC activation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0135-0) contains supplementary material, which is available to authorized users.

Stimulus strength determines the BTK-dependence of the SHIP1-deficient phenotype in IgE/antigen-triggered mast cells

“…This indicated that effects on degranulation and Ca 2+ mobilisation were quantitatively uncoupled in DKO BMMCs. Such uncoupling has also been reported, amongst others, in Lyn -deficient as well as in Ship1/Lyn double-deficient BMMCs 27 , 28 . The exact mechanism has not been elucidated yet.…”

“…1b , Y-P of PLCγ1 and PLCγ2 was reduced in Btk −/− BMMCs compared to WT cells; moreover, Y-P of PLCγ1 and PLCγ2 in Ship1 −/− BMMCs was drastically augmented (Fig. 2e ) 28 . This enhancement was reduced in DKO BMMCs to Y-P levels found in WT cells (Fig.…”

Lyn prevents aberrant inflammatory responses to Pseudomonas infection in mammalian systems by repressing a SHIP-1-associated signaling cluster

“…The cytosolic phosphatase SHIP-1, SH2-containing inositol-5′-phosphatase, has critical roles in microphages, including generation of alternatively activated macrophages, 39 regulation of LPS-induced macrophage inflammatory responses 40 and modulation of myeloproliferation. 11 Diverse cellular functions have been previously investigated in association of Lyn and SHIP-1, 41 , 42 but whether Lyn is involved in SHIP-1 expression or membrane translation 43 remains unclear. To investigate it, we measured SHIP-1 expression in Lyn-silenced and control MH-S cells at 0, 30 and 60 min post infection of PAO1.…”

“…Diverse cellular functions have been previously investigated in association with both Lyn and SHIP-1. 41 , 42 SHIP-1 and Lyn are involved in regulation of integrin alpha IIb-β3 signaling in platelets, 41 and in respect to Ag-triggered degranulation in mast cells. 42 In macrophages, Lyn and SHIP-1 negatively regulate macrophage colony-stimulating factor-induced Akt activity.…”

“… 41 , 42 SHIP-1 and Lyn are involved in regulation of integrin alpha IIb-β3 signaling in platelets, 41 and in respect to Ag-triggered degranulation in mast cells. 42 In macrophages, Lyn and SHIP-1 negatively regulate macrophage colony-stimulating factor-induced Akt activity. 24 A previous study demonstrated that activation of macrophages by the stimulation of LPS is negatively regulated by a Lyn/PI3K axis and promoted by SHIP-1.…”