Inhibitory effects of polymethoxy flavones isolated from Citrus reticulate on degranulation in rat basophilic leukemia RBL-2H3: enhanced inhibition by their combination

Polymethoxy flavones (PMFs) are present in fruit tissues of Citrus species. It has been reported that flavonoids isolated from several Citrus have been shown to suppress the degranulation as inferred by histamine release in rat basophilic leukemia RBL-2H3 cells. In this study, we examined the effect of PMFs (PMF-1: 6,7,4',5'-tetramethoxy-5-monohydroxyflavone, PMF-2: 5,6,8,3',6'-pentamethoxy flavone, PMF-3: 5,6,7,3',4',5'-hexamethoxy flavone) on the degranulation in RBL-2H3 cells. All the PMFs suppressed the degranulation from Ag-stimulated RBL-2H3 cells. Interestingly, PMF-combination (PMF-1+PMF-2; PMF-1+PMF-3) treatment enhanced the inhibition of degranulation compared with PMF-single treatment. In order to clarify the inhibitory mechanism of degranulation by PMFs, we examined the activation of intracellular signaling molecules such as Lyn, Syk, and PLCgammas. All the PMFs significantly suppressed the activation of Syk and PLCgammas. In Ag-mediated activation of Fc epsilonRI on mast cells, three major subfamilies of mitogen-activated protein kinases, especially ERK44/42, were activated. These PMFs reduced the level of phospho-ERKs. The intracellular free Ca(2+) concentration ([Ca(2+)]i) was elevated by Fc epsilonRI activation, and PMF treatment reduced the elevation of [Ca(2+)]i by suppressing Ca(2+) influx. Thus, it was suggested that the suppression of Ag-stimulated degranulation by these PMFs mainly is due to the Syk/PLCgammas/PKC pathway and Ca(2+) influx. Furthermore, to be noted in the PMF-combination treatment, inactivation of Syk was enhanced compared with PMF-single treatment. But the inhibitory effect of degranulation by PMF-combination treatment was not associated with the suppression of Ca(2+) influx.     

Phospholipid scramblase 1 modulates a selected set of IgE receptor-mediated mast cell responses through LAT-dependent pathway

Engagement of the IgE receptor (FcepsilonRI) on mast cells leads to the release of preformed and newly formed mediators as well as of cytokines. The signaling pathways responsible for these responses involve tyrosine phosphorylation of multiple proteins. We previously reported the phosphorylation on tyrosine of phospholipid scramblase 1 (PLSCR1) after FcepsilonRI aggregation. Here, PLSCR1 expression was knocked down in the RBL-2H3 mast cell line using short hairpin RNA. Knocking down PLSCR1 expression resulted in significantly impaired degranulation responses after FcepsilonRI aggregation and release of vascular endothelial growth factor, whereas release of MCP-1 was minimally affected. The release of neither leukotriene C4 nor prostaglandin D2 was altered by knocking down of PLSCR1. Analysis of FcepsilonRI-dependent signaling pathways revealed that whereas tyrosine phosphorylation of ERK and Akt was unaffected, tyrosine phosphorylation of LAT was significantly reduced in PLSCR1 knocked down cells. Tyrosine phosphorylation of phospholipase Cgamma1 and consequently the mobilization of calcium were also significantly reduced in these cells. In nonactivated mast cells, PLSCR1 was found in part in lipid rafts where it was further recruited after cell activation and was constitutively associated with Lyn and Syk but not with LAT or Fyn. Altogether, these data identify PLSCR1 as a novel amplifier of FcepsilonRI signaling that acts selectively on the Lyn-initiated LAT/phospholipase Cgamma1/calcium axis, resulting in potentiation of a selected set of mast cell responses.     

Effects of arachidonic acid analogs on FcepsilonRI-mediated activation of mast cells

Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) have been shown to modulate a number of inflammatory disorders. Mast cells play a critical role in the initiation and maintenance of inflammatory responses. However, the effects of PUFAs on mast cell functions have not been fully addressed. We here-in examined the effects of PUFAs on the high affinity IgE receptor (FcepsilonRI)-mediated mast cell activation using RBL-2H3 cells, a rat mast cell line, that were cultured in the medium containing palmitic acid (PA), AA, or the AA analogs mead acid (MA) and eicosapentaenoic acid (EPA). In AA-supplemented cells, the FcepsilonRI-mediated beta-hexosamidase and TNF-alpha release, calcium (Ca(2+)) influx, and some protein tyrosine phosphorylations including Syk and linker for activation of T cells (LAT) were enhanced, whereas, in MA- or PA-supplemented cells, they were not changed when compared with cells cultured in control medium. In EPA-supplemented cells, the enhancements of beta-hexosamidase release and protein tyrosine phosphorylations were observed. Furthermore, in AA- or EPA-supplemented cells, FcepsilonRI-mediated intracellular production of reactive oxygen species (ROS) that is required for the tyrosine phosphorylation of LAT and Ca(2+) influx were enhanced when compared with the other cells. Thus, preincubation of AA or EPA augmented FcepsilonRI-mediated degranulation in mast cells by affecting early events of FcepsilonRI signal transduction, which might be associated with the change of fatty acid composition of the cell membrane and enhanced production of ROS. The results suggest that some PUFAs can modulate FcepsilonRI-mediated mast cell activation and might affect FcepsilonRI/mast cell-mediated inflammation, such as allergic reaction.     

Cutting edge: dexamethasone negatively regulates Syk in mast cells by up-regulating SRC-like adaptor protein

We have identified Src-like adaptor protein (SLAP) as one of several dexamethasone-inducible inhibitory regulators in mast cells. SLAP is a known inhibitor of T cell signaling and interacts with the tyrosine kinase, Zap70. Exposure of RBL-2H3 mast cells to dexamethasone markedly increased expression of SLAP. Cells so exposed or made to overexpress SLAP exhibited reduced Ag-stimulated phosphorylation of Syk (a cognate of Zap70), linker for activation of T cells, phospholipase Cgamma, and ERK. Ca(2+) mobilization, Ca(2+)-dependent degranulation, and ERK-dependent release of arachidonic acid were suppressed as well. Small interfering RNA directed against SLAP blocked the induction of SLAP and reversed the inhibitory effects of dexamethasone on phosphorylation of Syk, linker for activation of T cells, and phospholipase Cgamma, but not downstream events, which are likely suppressed by up-regulation of downstream of tyrosine kinase-1 and MAPK phosphatase-1. The induction of these inhibitory regulators may contribute to the immunosuppressive activity of dexamethasone in mast cells.     

The Effects of Low-dose Ionizing Radiation in the Activated Rat Basophilic Leukemia (RBL-2H3) Mast Cells

Mast cells play important roles in many biological responses, such as those during allergic diseases and inflammatory disorders. Although laser and UV irradiation have immunosuppressive effects on inflammatory diseases by suppressing mast cells, little is known about the effects of γ-ionizing radiation on mast cells. In this study, we investigated the effects of γ-ionizing radiation on RBL-2H3 cells, a convenient model system for studying regulated secretion by mast cells. Low-dose radiation (<0.1 gray (Gy)) did not induce cell death, but high-dose radiation (>0.5 Gy) induced apoptosis. Low-dose ionizing radiation significantly suppressed the release of mediators (histamine, β-hexosaminidase, IL-4, and tumor necrosis factor-α) from immunoglobulin E (IgE)-sensitized RBL-2H3 cells. To determine the mechanism of mediator release inhibition by ionizing radiation, we examined the activation of intracellular signaling molecules such as Lyn, Syk, phospholipase Cγ, PKCs, and MAPK, and intracellular free calcium concentrations ([Ca(2+)](i)). The phosphorylation of signaling molecules following stimulation of high-affinity IgE receptor I (FcεRI) was specifically inhibited by low-dose ionizing radiation (0.01 Gy). These results were due to the suppression of FcεRI expression by the low-dose ionizing radiation. Therefore, low-dose ionizing radiation (0.01 Gy) may function as a novel inhibitor of mast cell activation.     

Cutting edge: extracellular signal-regulated kinase activates syk: a new potential feedback regulation of Fc epsilon receptor signaling.

The protein tyrosine kinase Syk is an essential element in several cascades coupling Ag receptors to cell responses. Syk and the mitogen-activated protein kinase extracellular signal-regulated kinase 1 (ERK1) were found to form a tight complex in both resting and Ag-stimulated rat mucosal-type mast cells (rat basophilic leukemia 2H3 cell line RBL-2H3). A direct serine phosphorylation and activation of Syk by ERK was observed in in vitro experiments. Moreover the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase (MEK) inhibitors markedly decreased the Ag-induced phosphorylation of the tyrosyl residues of Syk and its activation as well as suppressed the degranulation of the cells. These results suggest a positive feedback regulation of Syk by ERK in the cascade coupling the type 1 Fc epsilon receptor to the secretory response of mast cells; hence, the existence of a novel type of cross-talk between protein serine/threonine kinases and protein tyrosine kinases is suggested.     

Polygoni cuspidati radix inhibits the activation of Syk kinase in mast cells for antiallergic activity

The antiallergic activity of Polygoni cuspidati radix (PR) and the mechanism of action by which it functions were investigated in this study. The extract of PR exhibited potent inhibitory activity in mast cells; its IC50 values were 62 +/- 2.1 microg/ml for RBL-2H3 mast cells and 46 +/- 3.2 microg/m for bone marrow-derived mast cells by antigen stimulation, and it also suppressed the expression of tumor necrosis factor-alpha and interleukin-4 in RBL-2H3 cells. According to the in vivo animal allergy model, it inhibited a local allergic reaction, passive cutaneous anaphylaxis, in a dose-dependent manner. With regard to its mechanism of action, PR inhibited the activating phosphorylation of Syk, a key signaling protein for the activation of mast cells. It also suppressed Akt and the mitogen-activated protein kinases ERK1/2, p38, and JNK, which are critical for the production of various inflammatory cytokines in mast cells. The results of the study indicate that the antiallergic activity of PR is mediated through the inhibition of histamine release and allergic cytokine production by the inhibition of Syk activating phosphorylation in mast cells.     

Exposure of RBL-2H3 mast cells to Ag(+) induces cell degranulation and mediator release

There is a growing need to understand the impact of environmental sulfhydryl group-reactive heavy metals on the immune system. Here we show that Ag(+) induces mast cell degranulation, as does the aggregation of the high affinity immunoglobulin E receptor (FcepsilonRI). Micromolar quantities of Ag(+) specifically induced degranulation of mast cell model rat basophilic leukemia (RBL-2H3) cells without showing cytotoxicity. The Ag(+)-mediated degranulation could be observed as rapidly as 5 min after the addition of the ions. Ag(+) also induced a rapid change in tyrosine phosphorylation of multiple cellular proteins including the focal adhesion kinase but not Syk kinase. The Syk-selective inhibitor piceatannol and the Src family-selective tyrosine kinase inhibitor PP1 dose-dependently inhibited FcepsilonRI-mediated degranulation, whereas neither compound inhibited the Ag(+)-mediated degranulation. Furthermore, likewise FcepsilonRI aggregation, Ag(+) also induced leukotriene secretion. These results show that Ag(+) activates RBL-2H3 mast cells through a tyrosine phosphorylation-linked mechanism, which is distinct from that involved in FcepsilonRI-mediated activation.     

Protein-tyrosine kinases and adaptor proteins in FcepsilonRI-mediated signaling in mast cells

Mast cells function as the initiator of the allergic reaction and play a role in the innate immune system. Aggregation of the high affinity IgE receptor (FcepsilonRI) on mast cells triggers degranulation with the release of chemical mediators such as histamine, production of cytokines and leukotrienes. FcepsilonRI signals by activating proximal non-receptor type of protein-tyrosine kinases, Lyn, Syk, Btk and Fyn. Activated tyrosine kinases then phosphorylate their specific substrates which include other enzymes and adaptor proteins and assemble these cytoplasmic signaling molecules for cellular activation. The adaptor proteins have multiple domains that allow binding to effector molecules and therefore act as positive or negative regulators controlling FcepsilonRI signaling. Deletion of the adaptor proteins such as LAT, SLP-76 or Gab2 resulted in decreased FcepsilonRI-mediated anaphylactic reaction in vivo. Functional analysis of adaptor proteins has raised the possibility that they may be new targets for the discovery of anti-allergic drugs.     

Involvement of protein kinase C-alpha and -epsilon in extracellular Ca(2+) signalling mediated by the calcium sensing receptor

The sensing of extracellular Ca(2+) concentration ([Ca(2+)](o)) and modulation of cellular processes associated with acute or sustained changes in [Ca(2+)](o) are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca(2+)](o) signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca(2+)](o) activated PKC-alpha and PKC- in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca(2+)](o) required influx of Ca(2+)through Ni(2+)-sensitive Ca(2+)channels and phosphatidylinositol-dependent phospholipase C-beta activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-alpha or - with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca(2+)](o). Activation of ERK1/2 by high [Ca(2+)](o) was not necessary for the [Ca(2+)](o)-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca(2+)](o) signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.     

Phosphorylation of Syk activation loop tyrosines is essential for Syk function. An in vivo study using a specific anti-Syk activation loop phosphotyrosine antibody

Syk is an important protein-tyrosine kinase in immunoreceptor signaling. FcepsilonRI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of FcepsilonRI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation with its function, an antibody was developed specific for phosphorylated tyrosines in the activation loop of Syk. FcepsilonRI aggregation on mast cells induced the phosphorylation of both tyrosine residues of the activation loop. The kinase activity of Syk played the major role in phosphorylating its activation loop tyrosines both in vivo and in vitro. In FcepsilonRI-stimulated mast cells, the total Syk tyrosine phosphorylation paralleled the phosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast, the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosine phosphorylation of Syk and minimal histamine release and weak phosphorylation of activation loop tyrosines. These results demonstrate that phosphorylation of the activation loop tyrosines is important for mediating receptor signaling and is a better marker of Syk function than is total Syk tyrosine phosphorylation.     

Fc epsilon RI signaling of mast cells activates intracellular production of hydrogen peroxide: role in the regulation of calcium signals

Earlier studies, including our own, revealed that activation of mast cells is accompanied by production of reactive oxygen species (ROS) that help to mediate the release of the inflammatory mediators, including histamine and eicosanoids. However, little is known about the mechanisms of ROS production, including the species of oxidants produced. In this study we show that in both the RBL-2H3 mast cell line and bone marrow-derived mast cells, FcepsilonRI cross-linking stimulates intracellular oxidative burst, including hydrogen peroxide (H(2)O(2)) production, as defined with the oxidant-sensitive dyes dichlorofluorescein and scopoletin and the selective scavenger ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one). The oxidative burst was observed immediately after stimulation and was most likely due to an NAD(P)H oxidase. Experiments using selective pharmacological inhibitors demonstrated that activation of tyrosine kinases and phosphatidylinositol-3-kinase is required for induction of the oxidative burst. Blockade of the oxidative burst by diphenyleneiodonium impaired the release of preformed granular mediators, such as histamine and beta-hexosaminidase, and the secretion of newly synthesized leukotriene C(4), whereas selective scavenging H(2)O(2) by ebselen impaired leukotriene C(4) secretion, but not degranulation. Sustained elevation of cytosolic calcium through store-operated calcium entry was totally abolished when ROS production was blocked. In contrast, selective depletion of H(2)O(2) caused a considerable decrease and delay of the calcium response. Finally, tyrosine phosphorylation of phospholipase Cgamma and the linker for activation of T cells, an event required for calcium influx, was suppressed by diphenyleneiodonium and ebselen. These studies demonstrate that activation of the intracellular oxidative burst is an important regulatory mechanism of mast cell responses.     

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.     

Activation of Syk tyrosine kinase is required for c-Cbl-mediated ubiquitination of Fcepsilon RI and Syk in RBL cells

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells and basophils results in FcepsilonRI beta and gamma subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FcepsilonRI engagement on RBL-2H3 cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FcepsilonRI beta and gamma chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FcepsilonRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FcepsilonRI and activated Syk to the proteasome for degradation.     

Blunted IgE-mediated activation of mast cells in mice lacking the Ca2+-activated K+ channel KCa3.1

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.     

Critical role of protein kinase C betaII in activation of mast cells by monomeric IgE

Accumulating evidence suggests that IgE-mediated activation of mast cells occurs even in the absence of antigen, which is referred to as "monomeric IgE" responses. Although monomeric IgE was found to induce a wide variety of responses, such as up-regulation of the FcepsilonRI, survival, cytokine production, histamine synthesis, and adhesion to fibronectin, it remains to be clarified how mast cells are activated in the absence of antigen. It has been controversial whether monomeric IgE responses are mediated by a similar signaling mechanism to antigen stimulation, although recent studies suggest that IgE can induce the FcepsilonRI aggregation even in the absence of antigen. In this study, we focused on the role of conventional protein kinase C (cPKC), since this response is suppressed by a specific inhibitor for cPKC. Monomeric IgE-induced Ca(2+) influx was not observed in a mouse mastocytoma cell line, which lacks the expression of PKCbetaII, although Ca(2+) influx induced by cross-linking of the FcepsilonRI was intact. Transfection of PKCbetaII cDNA was found to restore the Ca(2+) influx induced by monomeric IgE in this cell line. Furthermore, the dominant negative form of PKCbetaII (PKCbetaII/T500V) significantly suppressed the Ca(2+) influx, histamine synthesis, and interleukin-6 production in another mouse mast cell line, which is highly sensitive to monomeric IgE. Expression of PKCbetaII/T500V was found not to affect the antigen-induced responses. These results suggest that PKCbetaII plays a critical role in monomeric IgE responses, but not in antigen responses.     

Cross-linking of FcepsilonRI causes Ca2+ mobilization via a sphingosine kinase pathway in a clathrin-dependent manner

Clathrin-coated pits are now recognized to be involved in cell signaling in addition to receptor down-regulation. Here we tried to identify signaling pathways that might be dependent on clathrin. Our initial data with pharmacological inhibitors of formation of clathrin-coated pits or lipid-rafts indicated that Ca(2+) response evoked by cross-linking of the high affinity receptors for IgE (FcepsilonRI) was dependent on clathrin. To confirm this finding, we created clathrin-knockdown cells by transfecting the mast cell line RBL-2H3 with a shRNA-clathrin heavy chain construct. In these cells, the FcepsilonRI-mediated Ca(2+) response was almost completely abolished, which was accompanied by the inhibition of sphingosine 1-phosphate (S1P) production with no changes in inositol 1,4,5-trisphosphate (IP(3)) production. This suggests that the Ca(2+) signaling pathway via a sphingosine kinase (SK) is dependent on clathrin. Furthermore, antigen-induced tyrosine phosphorylation of p85 and p110 subunits of PI3K was almost completely inhibited in clathrin-knockdown cells. In contrast, antigen-induced tyrosine phosphorylation of phospholipase Cgamma was not affected by clathrin-knockdown and tyrosine phosphorylation of Syk and degranulation were partially inhibited in clathrin-knockdown cells. The present study identifies the SK/Ca(2+) pathway to be dependent on clathrin.     

Distinct roles for the linker region tyrosines of Syk in FcepsilonRI signaling in primary mast cells

Stimulation of FcepsilonRI, the high affinity IgE receptor of mast cells results in the rapid binding of the Syk tyrosine kinase to cytoplasmic domains of FcepsilonRI and to its subsequent activation. Syk plays an essential role in signal transduction from FcepsilonRI as shown by Syk-deficient mast cells, which are defective in receptor-induced degranulation, cytokine synthesis, and intracellular pathways. However the mechanism by which Syk activates these pathways remains unclear. Activation of Syk is associated with its phosphorylation on several tyrosine residues, including the linker tyrosines Tyr317, Tyr342, and Tyr346. These residues have been proposed to play important roles in the transduction of signals by binding to other signaling proteins. To test these hypotheses in primary murine mast cells, we used retroviral infection of Syk-deficient mast cells to generate cells expressing Syk proteins bearing mutations in the linker tyrosines. We show that Tyr342 and Tyr346 contribute positively to the function of Syk and have both overlapping as well as distinct functions. Mutations in either Tyr342 or Tyr346 alone had no effect on FcepsilonRI-induced degranulation or calcium flux, whereas mutation of both residues caused a significant reduction in both pathways. In contrast, phosphorylation of PLCgamma1, PLCgamma2, and Vav1 was strongly decreased by a mutation in Tyr342 alone, whereas phosphorylation of ERK and Akt was more dependent on Tyr346. Finally we show that Tyr317 functions as a negative regulatory site and that its mutation can partially compensate for the loss of both Tyr342 and Tyr346.     

Co-aggregation of FcgammaRII with FcepsilonRI on human mast cells inhibits antigen-induced secretion and involves SHIP-Grb2-Dok complexes

Signaling through the high affinity IgE receptor FcepsilonRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcgammaRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcgammaRII and FcepsilonRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcgammaRII but not FcgammaRI or FcgammaRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcepsilonRI and FcgammaRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca(2+) mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62(dok); Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcepsilon-Fcgamma co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcepsilonRI-mediated responses can be inhibited by co-aggregation with FcgammaRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.