Dichotomy of Ca2+ Signals Triggered by Different Phospholipid Pathways in Antigen Stimulation of Human Mast Cells*

Mast cell activation triggers Ca2+ signals and the release of enzyme-containing granules, events that play a major role in allergic/hypersensitivity reactions. However, the precise molecular mechanisms that regulate antigen-triggered degranulation and Ca2+ fluxes in human mast cells are still poorly understood. Here we show, for the first time, that a receptor can trigger Ca2+ via two separate molecular mechanisms. Using an antisense approach, we show that IgE-antigen stimulation of human bone marrow-derived mast cells triggers a sphingosine kinase (SPHK) 1-mediated fast and transient Ca2+ release from intracellular stores. However, phospholipase C (PLC) γ1 triggers a second (slower) wave of calcium release from intracellular stores, and it is this PLCγ1-generated signal that is responsible for Ca2+entry. Surprisingly, FcεRI (a high affinity receptor for IgE)-triggered mast cell degranulation depends on the first, sphingosine kinase-mediated Ca2+ signal. These two pathways act independently because antisense knock down of either enzyme does not interfere with the activity of the other enzyme. Of interest, similar to PLCγ1, SPHK1 translocates rapidly to the membrane after FcεRI cross-linking. Here we also show that SPHK1 activity depends on phospholipase D1 and that FcεRI-triggered mast cell degranulation depends primarily on the activation of both phospholipase D1 and SPHK1.

Aggregation of the high affinity receptor for IgE (Fc⑀RI) on mast cells triggers the Ca 2ϩ -dependent release and production of a wide range of mediators responsible for the major symptoms of immediate hypersensitivity reactions (1)(2)(3). Although some of the signaling cascades triggered by Fc⑀RI have been characterized, the regulatory mechanisms governing mast cell degranulation and calcium release from internal stores are only partially understood. Fc⑀RI is a heterotrimeric receptor complex (␣␤␥ 2 ) that contains immunoreceptor tyrosine-based activation motifs in both the ␤ and ␥ subunit cytoplasmic domains (4). The protein-tyrosine kinase Lyn is associated with the ␤ subunit in resting cells (5), and its activation is promoted by Fc⑀RI cross-linking (6). Activated Lyn phosphorylates immunoreceptor tyrosine-based activation motifs of the ␤ and ␥ subunits, resulting in the recruitment of other Src-like as well as Syk protein tyrosine kinases through Src homology 2 do-main-mediated interactions with phosphotyrosine residues (7,8). Activation of these newly recruited protein tyrosine kinases, in turn, facilitates the translocation and phosphorylation of multiple signaling molecules, including phospholipase C (PLC) 1 ␥ isoforms and phosphoinositide 3-kinases (9). Activated PLC␥ hydrolyses phosphatidylinositol 4,5-bisphosphate to D-myo-inositol 1,4,5-trisphosphate and diacylglycerol, which induce the release of Ca 2ϩ from intracellular stores and the activation of protein kinase C isoforms, respectively. The amplitude and duration of the Ca 2ϩ response potentially modulate the activation of different transcription factors (10), regulating different gene expression. Ca 2ϩ signals are also indispensable for the release of histamine-containing granules (1), the synthesis of arachidonic acid-derived mediators, and the release and generation of various cytokines (2), which together are responsible for the major symptoms of immediate hypersensitivity reactions. Thus, an understanding of mast cell activation and Ca 2ϩ signaling therefore has obvious therapeutic implications. It has previously been shown that Fc⑀RI, on the rat mast cell line RBL-2H, triggers Ca 2ϩ signals via a novel pathway potentially involving sphingosine kinase activity (11) and not phospholipase C␥, even though IP 3 production was observed (11). We have recently shown that a similar receptor, Fc␥RI in monocytes, triggers intracellular Ca 2ϩ via the sequential activation of phospholipase D and sphingosine kinase (12); however, no IP 3 generation was observed in this case (12). Phospholipase D hydrolyses phosphatidylcholine to yield phosphatidic acid and choline (13); phosphatidic acid has been shown to have many intracellular signaling functions (13,14), including the activation of sphingosine kinase (SPHK) (14 -16). SPHK phosphorylates sphingosine to generate sphingosine-1phosphate (15)(16)(17). Sphingosine-1-phosphate has been demonstrated to act as an alternative second messenger to inositol 1,4,5-trisphosphate in the release of Ca 2ϩ from intracellular stores (11,12). In this study, we show for the first time a dual molecular mechanism responsible for triggering different calcium signals. Firstly, a rapid rise in internal calcium is triggered by the sequential activation of phospholipase D (PLD) 1 and SPHK1. Secondly, a prolonged calcium response is triggered by phospholipase C␥1. Furthermore, mast cell degranulation is triggered by the combined action of PLD1 and SPHK1. However, the PLC␥1 activation is necessary to trigger calcium entry into the cells.
Understanding the intracellular signaling pathways coupling Fc⑀RI activation, by IgE-antigen, to physiological responses triggered by mast cell activation has profound therapeutic implications for allergic/inflammatory diseases.

MATERIALS AND METHODS
Unless stated otherwise, all chemicals and reagents were obtained from Sigma.
BMMC Generation and Cell Culture-Bone marrow was collected from human donors following the protocol approved by the FDA Committee for Research Involving Human Subjects. Normal donor eligibility criteria included healthy males and nonpregnant females between the ages of 18 and 45 years. The donors were required to have a negative medical history for all major diseases. Bone marrow was withdrawn by board-certified physicians from two separate sites of the posterior pelvic bone into syringes containing preservative-free heparin sodium injection (20 -50 units heparin/ml bone marrow). BMMCs were generated using the previously described (18) protocol as follows: fresh human bone marrow cells were cultured in complete RPMI 1640 medium supplemented with 10 ng/ml interleukin 3 (Calbiochem) and 100 ng/ml stem cell factor (Calbiochem) for 2 weeks. BMMCs were characterized by flow cytometry as CD45ϩ, CD117ϩ, CD9ϩ, fluorescein isothiocyanate-labeled human IgE-positive, CD4Ϫ, CD8Ϫ, CD45Ϫ, CD11bϪ, CD11cϪ, and major histocompatibility complex class IIϪ. Purity was estimated at Ͼ95%. All antibodies were fluorescein isothiocyanate-or biotin-labeled (Serotec).
Antisense oligonucleotides were purchased from Oswell DNA Services; 20-mers were synthesized, capped at either end by the phosphorothiorate linkages (the first two and last two linkages), and corresponded to the reverse complement of the first 20 coding nucleotides for PLD1, SPHK1, PLC␥1, and a scrambled oligonucleotide for control. The sequences of the oligonucleotides were as follows: 5Ј-CCGTGGCTCG-TTTTTCAGTG-3Ј for PLD1, 5Ј-CCCGCAGGATCCATAACCTC-3Ј for SPHK1, 5Ј-GGGGACGCGGCGCCCGCCAT-3Ј for PLC␥1, and 5Ј-CT-GGTGGAAGAAGAGGACGT-3Ј for the scrambled oligonucleotide control.
Cells were incubated/transfected with oligonucleotides (1 M) mixed with transfection reagent (FuGENE 6; Roche Molecular Biochemicals) for a total of 48 h (for 36 h prior to sensitization and for the duration of sensitization).
Reverse Transcription-PCR-mRNA from BMMCs was isolated using the Qiagen midi kit for mRNA extraction. Specific forward (TGAAC-CCGCGCGGCAAGGGC) and reverse (GGTCAGCCGGCGCCATC-CACG) primers were designed for human SPHK1 to yield a 570-bp fragment.
Peptide-derived Polyclonal Antibody Specific for Human SPHK1-A peptide sequence specific for human SPHK1 was selected for its apparent hydrophobic properties and synthesized.
The peptide used was FIADVDLESEKYRRLGEMRFTLGT. Two rabbits were immunized, giving rise to two peptide-derived antisera. The polyclonal antibodies were purified using protein A-agarose affinity columns. The polyclonal antibodies only recognized one band in Western blots for the correct molecular weight of endogenous or recombinant human SPHK1. The antibody was also successfully used as primary antibody for immunostaining for confocal microscopy analysis.
Fc⑀RI Aggregation-BMMCs were sensitized with 1 g/ml human dinitrophenol-specific IgE overnight. Then cells were collected, washed, resuspended in RPMI 1640 medium-1% fetal bovine serum, and activated by the antigen dinitrophenol-bovine serum albumin (1 g/ml), and activation was stopped at the times indicated in the figures.
Cell Lysates and Subcellular Fractionation-For translocation experiments, cell lysates and subcellular fractions were prepared following the method described previously (19). Briefly, cells were harvested and resuspended in cold nuclear preparation lysis buffer (10 mM Tris-HCl, pH 7.4, 2 mM magnesium chloride, 140 mM sodium chloride, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium orthovanadate, 10 g/ml chymostatin, 10 g/ml leupeptin, 10 g/ml antipain, and 10 g/ml pepstatin). After lysis by three cycles of freeze-thawing (in liquid nitrogen), the nuclei and cell debris (containing the cytoskeleton) were removed from the total cell lysates by centrifugation at 15,000 ϫ g for 5 min. The supernatant was centrifuged at 100,000 ϫ g and 4°C for 60 min. The pellet containing the nuclear-free membrane fraction was resuspended in 200 l of nuclear preparation buffer (without detergents) and stored at Ϫ20°C. The amount of protein recovered in each fraction was quantified using the Bradford reagent system (Bio-Rad).
Gel Electrophoresis and Western Blots-Unless stated otherwise, 40 g of lysate for each sample was resolved on 10% polyacrylamide gels (SDS-PAGE) under denaturing conditions and then transferred to 0.45-m nitrocellulose membranes. For translocation experiments, 40 g of lysate for each sample was fractionated as mentioned above, and the supernatant and the membrane fractions for each sample were resolved separately on 10% polyacrylamide gels (SDS-PAGE) under denaturing conditions and then transferred to 0.45-m nitrocellulose membranes. After blocking overnight at 4°C with 5% nonfat milk in Tris-buffered saline/0.1% Tween 20 and washing, the membranes were incubated with the relevant antibodies for 4 h at room temperature. The membranes were washed extensively in Tris-buffered saline/0.1% Tween 20 (washing buffer). The blots were probed using specific monoclonal (anti-PLC␥1; Santa Cruz Biotechnology) or polyclonal (anti-PLD1; Quality Control Biolabs) (anti-SPHK1; made in house as described) primary antibodies. Blots were stripped and reprobed with polyclonal antibodies: an anti-PDGFR␣ antibody (against the ␣ subunit of the PDGFR; Santa Cruz Biotechnology) for membrane loading control or an anti-HSP 90 antibody (H-114; against heat shock protein 90; Santa Cruz Biotechnology) for cytosol loading control. The anti-PDGFR␣ antibody was also used as a loading control for blots containing whole cell lysates. Bands were visualized using the appropriate horseradish peroxidase-conjugated secondary antibody and the ECL Western blotting detection system (Amersham Biosciences). Phosphatidylcholine-PLD Activity-PLD activity was measured as described previously (12) using the transphosphatidylation assay. Briefly, BMMCs were labeled (10 6 cells/ml) with [ 3 H]palmitic acid (5 Ci/ml; Amersham Biosciences) in the cell culture medium for 16 h. After washing, the cells were incubated at 37°C for 15 min in RPMI 1640 ϩ 1% BSA medium containing butan-1-ol (0.3%, final concentration). After Fc⑀RI aggregation, cells were incubated for an additional 30 min and then extracted by Bligh-Dyer phase separation. The accumulated phosphatidyl butanol was assayed as described previously (12).
Inositol 1,4,5-Trisphosphate-IP 3 was measured as described previously (20), using the BIOTRAK TRK 1000 kit (Amersham Biosciences). Briefly, this is a competition binding assay in which cellular generated (unlabeled) IP 3 competes with a fixed, known amount of [ 3 H]IP 3 for binding to the IP 3 receptor present in homogenates from bovine adrenal glands, which has a high affinity and specificity for IP 3 . Fc⑀RI aggregation was carried out described above, and activation was stopped at the times indicated in the figures.
Cytosolic Ca 2ϩ -Cytosolic calcium was measured as described previously (12,21), except that for some experiments the buffer was supplemented with Ca 2ϩ (final concentration, 1.5 mM Ca 2ϩ ). Briefly, sensitized cells were loaded with 1 g/ml Fura-2/AM (Molecular Probes, Leiden, The Netherlands) in phosphate-buffered saline, 1.5 mM Ca 2ϩ , and 1% bovine serum albumin. After removal of excess reagents by dilution and centrifugation, the cells were resuspended in 1.5 mM Ca 2ϩsupplemented phosphate-buffered saline and warmed to 37°C in the cuvette. Fc⑀RI was aggregated as described above. Fluorescence was measured at 340 and 380 nm, and the background-corrected 340:380 ratio was calibrated as described previously (12).
Confocal Microscopy-After receptor aggregation, suspended cells were fixed in 4% paraformaldehyde, deposited on microscope slides in a cytospin centrifuge, and then permeabilized for 5 min in 0.1% Triton X-100 in phosphate-buffered saline. Fluorescence labeling was performed as described previously (22), using anti-PLC␥1 monoclonal antibody (Santa Cruz Biotechnology) or anti-SPHK1 polyclonal antibody (made in house as described) as primary antibody. Stainings were analyzed in horizontal confocal microscopy sections (50 -100 sections of 0.2 m) and recorded by a Leica TCS NT, and images were deconvoluted. Signals were projected into one image as an extended focus view.
␤-Hexosaminidase Release-Degranulation was measured using a previously described (23) colorimetric assay to assess the release of ␤-hexosaminidase. Briefly, 50 l of the sample supernatant was incubated with 200 l of 1 mM p-nitrophenyl N-acetyl-␤-D-glucosiaminide for 1 h at 37°C. The total ␤-hexosaminidase concentration was determined by a 1:1 extraction of the remaining buffer and cells with 1% Triton X-100; a 50-l aliquot was removed and analyzed as described. Reactions were quenched by the addition of 500 l of 0.1 M sodium carbonate buffer. The enzyme concentration was determined by measuring the absorbance at 400 nm. ␤-Hexosaminidase release was represented as a percentage of total enzyme.

RESULTS
In this study, we explored the molecular mechanisms regulating receptor coupling to various lipid-modifying enzymes and relate these to the triggering of Ca 2ϩ signals and degranulation.
Because SPHK had been shown to play a potential role in triggering Ca 2ϩ release from intracellular stores, we decided to investigate whether SPHK is indeed involved in the Fc⑀RI triggering of Ca 2ϩ signals in human mast cells. Two human sphingosine kinases have recently been cloned and characterized, namely, SPHK1 (15) and SPHK2 (17). First, the presence of specific SPHK isozymes present in the cells was examined. In bone marrow-derived mast cells, only SPHK1 was found by reverse transcription-PCR (Fig. 1A), and by Western blot (Fig.  1B). Western blot analysis showed that SPHK1, in resting cells, is found primarily in the cytosolic fraction of the cells (Fig. 1B,  top panel). However, aggregation of Fc⑀RI resulted in the rapid translocation of SPHK1 from the cytosol to the nuclear-free membrane fraction (Fig. 1B, bottom panel). In agreement with this, confocal microscopy also shows that SPHK1 is primarily cytosolic in resting cells, but after receptor engagement, it translocates rapidly to the cell periphery (Fig. 1C).
Sphingosine kinase activity and its product, sphingosine-1phosphate, have been shown to be involved in many cellular processes, including Ca 2ϩ signals (11,12), suppression of ceramide-mediated apoptosis (24), and cell survival and proliferation (24). However, the regulation of SPHK activity is still poorly understood. We have previously shown that a similar receptor, Fc␥RI, in monocytic cells triggers sphingosine kinase activity dependent on PLD activation (11,21). There is also in vitro evidence for the regulation of SPHK activity by acidic phospholipids (such as phosphatidic acid, the direct product of PLD activity) (16). Here we show that in human bone marrowderived mast cells, Fc⑀RI couples to PLD1 to activate SPHK1. Antisense oligonucleotide to PLD1 blocks Fc⑀RI-triggered PLD activity ( Fig. 2A, top panel) and considerably reduces endogenous PLD1 expression levels to only 18 Ϯ 5% of the PLD1 expressed in control cells, which was taken as 100% ( Fig. 2A,  bottom panel). In resting cells, very little SPHK activity is observed; however, after Fc⑀RI cross-linking, SPHK activity increases very rapidly (Fig. 2B). In agreement with the translocation experiments (Fig. 1, A and C), very little SPHK activity is observed in the membrane fraction of resting cells; however, after Fc⑀RI cross-linking, SPHK activity increases very rapidly in the membrane fraction to levels higher than that observed in the cytosolic fraction (Fig. 2B, right and left panels, respectively). Moreover, antisense oligonucleotide to PLD1 blocked Fc⑀RI-triggered SPHK1 activity (Fig. 2B, top panel) but had no effect on SPHK1 expression (Fig. 2B, bottom panel). Furthermore, an antisense oligonucleotide to SPHK1 blocked Fc⑀RItriggered sphingosine kinase activity (Fig. 2B, top panel) and had no effect on PLD activity ( Fig. 2A, top panel) or PLD1 expression levels ( Fig. 2A, bottom panel) but considerably reduced endogenous SPHK1 expression levels to only 15 Ϯ 5% of the SPHK1 expressed in the control cells, which was taken as 100% (Fig. 2B, bottom panel). The scrambled oligonucleotide used as a control had no effect on the level of expression of either protein. These data suggest that SPHK1 is downstream of PLD1 activity in the Fc⑀RI-triggered signal transduction pathways.
In the rat mast cell line RBL-2H, Choi et al. (11) showed that F⑀RI-triggered Ca 2ϩ release from intracellular stores was dependent on sphingosine kinase activity by addition of the nonselective sphingosine kinase inhibitor dihydrosphingosine, which reduced the increase in Ca 2ϩ in response to antigen, whereas the antigen-induced production of IP 3 was unimpaired. However, IP 3 is widely known to trigger the release of Ca 2ϩ from intracellular stores by activating specific receptors on the membranes of these stores (25,26), and PLC␥ has been shown to be phosphorylated and to translocate after Fc⑀RI triggering in rat mast cells (27,28). To determine whether Fc⑀RI triggers IP 3 generation and IP 3 -mediated Ca 2ϩ release in human mast cells, IP 3 generation was monitored over time. We found that in the human mast cells, PLC is activated by Fc⑀RI, as shown by the generation of IP 3 (Fig. 3A). Moreover, PLC␥1 is the PLC isoform that translocates to the membrane after Fc⑀RI engagement (Fig. 3B, top panel). Furthermore, antisense oligonucleotide to PLC␥1 inhibited IP 3 generation triggered by Fc⑀RI (Fig. 3A) and down-regulated the endogenous PLC␥1 expression levels (Fig. 3B, bottom panel). Antisense oligonucleotide to SPHK1 had no effect on IP 3 production (Fig. 3A) or on PLC␥1 expression levels (Fig. 3B, bottom panel).
For all experiments, the antisense transfection efficiency was very even; an average 85% of the cells treated with any of the antisense oligonucleotides used showed complete downregulation in expression levels of the targeted protein (fluorescence-activated cell-sorting analysis; data not shown).
To clarify the roles of SPHK1 and/or PLC␥1 in the Ca 2ϩ signals generated after Fc⑀RI engagement, we used the antisense oligonucleotides against human SPHK1 and PLC␥1 to demonstrate which pathway was responsible for Ca 2ϩ triggering in the human mast cells. It was found that antisense down-regulation of SPHK1 substantially inhibited the initial rise in Ca 2ϩ release from intracellular stores (Fig. 4A); however, Ca 2ϩ entry was unaffected (Fig. 4A). In cells pretreated with antisense oligonucleotide to PLC␥1, the first peak in Ca 2ϩ was unaffected (Fig. 4B); however, calcium entry was reduced (Fig. 4B). Experiments without extracellular Ca 2ϩ showed that the initial rise in intracellular Ca 2ϩ is due to SPHK1 (Fig. 4C), whereas a second, smaller increase in Ca 2ϩ release from internal stores was due to PLC␥1 (Fig. 4C). A combination of both antisense oligonucleotide to SPHK1 and antisense oligonucleotide to PLC␥1 completely blocked the calcium response triggered by Fc⑀RI (Fig. 4D). The use of specific inhibitors for SPHK (N,N-dimethyl-sphingosine) or PLC (ET-18-OCH 3 ) generated results similar to those observed with the antisense oligonucleotides (data not shown).
These results show that Fc⑀RI triggers Ca 2ϩ signals by two different pathways: (a) by a novel pathway that uses SPHK1 and is responsible for the initial strong Ca 2ϩ release from internal stores, and (b) by a more classical pathway that triggers IP 3 generation via PLC␥, which triggers a second but smaller peak in Ca 2ϩ release from intracellular stores and is responsible for triggering Ca 2ϩ entry.
In contrast to previous studies in rat mast cells (11), the nonselective tyrosine kinase inhibitor genistein completely blocked SPHK activity and IP 3 generation (Fig. 5, A and B) as well as Fc⑀RI-induced PLD activity but had no effect on phorbol 12-myristate 13-acetate-induced PLD activity, suggesting that the tyrosine kinase inhibitor does not directly inhibit PLD activity (Fig. 5C). The Fc⑀RI-triggered membrane translocation of SPHK1 and PLC␥1 was also inhibited by the tyrosine kinase inhibitor (Fig. 5, D and E, respectively). Moreover, the Ca 2ϩ signals triggered by Fc⑀RI were also completely blocked in cells pretreated with genistein (Fig. 5D). These data show that SPHK1 and PLC␥1 activities, as well as all the Ca 2ϩ signals triggered by Fc⑀RI, are completely tyrosine kinase-dependent.
Mast cell degranulation has been shown to be Ca 2ϩ -dependent (2,3), and linked to phospholipase D activity (23,29,30). Antisense down-regulation of different PLD isoforms is proving to be a very useful tool in dissecting the functions of each particular isoenzyme (31). Antisense down-regulation of PLD1 substantially inhibits Fc⑀RI-triggered mast cell degranulation (Fig. 6A) but has no effect on IP 3 generation (Fig. 6B). Similarly, antisense oligonucleotide to SPHK1 also inhibited enzyme release (Fig. 6A), and a combination of antisense oligonucleotide to PLD1 and antisense oligonucleotide to SPHK1 almost completely inhibited Fc⑀RI-triggered degranulation ( Fig. 6A) but had no effect on IP 3 production. Antisense oligonucleotide to PLC␥1 had no effect on enzyme release (Fig. 6A) but significantly reduced IP 3 generation (Fig. 6B).
These results show that both PLD1 and SPHK1 are necessary for Fc⑀RI to trigger mast cell degranulation. DISCUSSION Taken together, the data presented here demonstrate that the activation of Fc⑀RI by surface IgE-antigen complexes on human bone marrow-derived mast cells stimulates two different pathways to trigger Ca 2ϩ release from internal stores. A novel pathway, which couples PLD1 to SPHK1 activation, is responsible for the initial peak in the Fc⑀RI-generated Ca 2ϩ signals as well as the mast cell degranulation, and a more classical pathway triggers PLC␥1 activity and an IP 3 -dependent second wave of Ca 2ϩ release from internal stores, as well as Ca 2ϩ entry into the cells.
The activation of sphingosine kinase and the generation of sphingosine-1-phosphate have been previously proposed to play a role in mobilizing calcium from intracellular stores (11,12,(32)(33)(34). However, this proposal has proven highly controversial due to the presence of extracellular G protein-coupled receptors for sphingosine-1-phosphate (35,36), which are able to mobilize calcium through conventional IP 3 receptor-dependent pathways. However, the resent cloning of the SCaMPER receptor (37) provides additional evidence that sphingoid de-rivatives are able to engage intracellular receptors and effect calcium release from intracellular stores independently of IP 3 generation. The data presented here provide evidence for specific immune receptor triggering of this pathway in mast cells. Thus, aggregation of Fc⑀RI resulted in the rapid membrane translocation and activation of SPHK1. The results presented in this report demonstrate that the initial peak in Ca 2ϩ release from intracellular stores, triggered by Fc⑀RI, is dependent on sphingosine kinase activity. In this respect, aggregation of Fc⑀RI in human mast cells is behaving like in the rat mast cell line RBL-2H (11) and like the high affinity IgG receptor, Fc␥RI, in human myeloid cells (12). Of interest, both these receptors use the same signal-transducing molecule (␥-chain) to recruit soluble tyrosine kinases (38,39). However, unlike the study in the RBL-2H cells (11) and that of Fc␥RI in human myeloid cells (12), a second peak in Ca 2ϩ release from internal stores as well as Ca 2ϩ influx to the cells triggered by Fc⑀RI in mast cells was dependent on PLC␥1 activation. The mechanism of coupling of tyrosine kinases to sphingosine kinase activation after Fc⑀RI aggregation in the RBL-2H cells was unclear (11). Here, we demonstrate that PLD1 is activated after Fc⑀RI aggregation in human mast cells and that SPHK1 activation is dependent on PLD1 activation. The immediate product of phosphatidylcholine-PLD is phosphatidic acid, and this is subsequently converted to diacylglycerol through the action of phosphatidic acid phosphohydrolases (14). Previous studies have shown that sphingosine kinase is activated by phosphatidic acid (16,40) and not by diacylglycerol (16,40), a product of both phospholipase D and phospholipase C. Our finding that sphingosine kinase is downstream of PLD is therefore consistent with this in vitro work. Moreover, both components of this novel Fc⑀RIcoupled intracellular signaling pathway involving the sequential activation of PLD and sphingosine kinase depend on tyrosine kinase. This finding is consistent with previous in vitro studies demonstrating that v-Src can activate PLD (41).
Aggregation of Fc⑀RI in mast cells triggers a number of effector functions. The novel intracellular signaling pathway demonstrated here appears to be functionally associated with these. Thus, previous studies have implicated phospholipase D in modulation of neutrophil, monocyte, and macrophage function, in particular by influencing the respiratory burst/NADPH oxidase cascade (42), vesicular trafficking (6), and phagocytosis (43). In the study reported here, inhibiting this pathway at either the PLD1 or SPHK1 level reduced the ability of this receptor to mobilize Ca 2ϩ from intracellular stores. In addition, the inhibition of PLD and/or sphingosine kinase significantly reduced enzyme release/degranulation. Of interest, ADP-ribosylation factor plays a major role in regulating vesicular trafficking (44 -46), and this low molecular weight G protein has also been demonstrated to regulate phospholipase D activity (31,(45)(46)(47).
This potential diversity of phospholipid signaling pathways offers the opportunity within the cell to very tightly regulate different physiological events of the cell effector mechanisms. The finding that Fc⑀RI is coupled to the release of calcium from intracellular stores and enzyme release/degranulation via a novel pathway has profound implications for the development of strategies for therapeutic intervention against different allergic and inflammatory responses.