Phosphoinositide 3-Kinase Facilitates Antigen-stimulated Ca2+ Influx in RBL-2H3 Mast Cells via a Phosphatidylinositol 3,4,5-Trisphosphate-sensitive Ca2+ Entry Mechanism*

Tsui-Ting Ching, Ao-Lin Hsu, Amy J. Johnson, and Ching-Shih ChenDagger

From the Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082

Received for publication, October 27, 2000, and in revised form, January 11, 2001

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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This study presents evidence that phosphoinositide 3-kinase (PI3K) plays a concerted role with phospholipase Cgamma in initiating antigen-mediated Ca2+ signaling in mast cells via a phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3)-sensitive Ca2+ entry pathway. Exogenous PI(3,4,5)P3 at concentrations close to its physiological level induces instantaneous Ca2+ influx into RBL-2H3 cells. This PI(3,4,5)P3-induced intracellular Ca2+ increase is independent of phospholipase C activity or the depletion of internal stores. Moreover, inhibition of PI3K by LY294002 or by overexpression of the dominant negative inhibitor Delta p85 suppresses the Ca2+ response to the cross-linking of the high affinity receptor for IgE (Fcepsilon RI). Concomitant treatment of RBL-2H3 cells with LY294002 or Delta p85 and 2-aminoethyl diphenylborate, a cell-permeant antagonist of D-myo-inositol 1,4,5-trisphosphate receptors, abrogates antigen-induced Ca2+ signals, whereas either treatment alone gives rise to partial inhibition. Conceivably, PI(3,4,5)P3-sensitive Ca2+ entry and capacitative Ca2+ entry represent major Ca2+ influx pathways that sustain elevated [Ca2+]i to achieve optimal physiological responses. This study also refutes the second messenger role of D-myo-inositol 1,3,4,5-tetrakisphosphate in regulating Fcepsilon RI-mediated Ca2+ response. Considering the underlying mechanism, our data suggest that PI(3,4,5)P3 directly stimulates a Ca2+ transport system in plasma membranes. Together, these data provide a molecular basis to account for the role of PI3K in the regulation of Fcepsilon RI-mediated degranulation in mast cells.

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ABSTRACT
INTRODUCTION
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Activation of mast cells by the cross-linking of the high affinity receptor for IgE (Fcepsilon RI)1 leads to the secretion and generation of an array of mediators that induce immediate allergic inflammation (for review, see Ref. 1). Although the Fcepsilon RI-mediated signaling cascade has been characterized, the regulatory mechanism governing mast cell degranulation is only partially understood. Fcepsilon RI is a heterotrimeric protein complex (alpha beta gamma 2) that contains immunoreceptor tyrosine-based activation motifs (ITAMs) in both the beta  and gamma  subunit cytoplasmic domains (2). The protein-tyrosine kinase (PTK) Lyn is associated with the beta  subunit at the resting state (3), and its action is promoted by Fcepsilon RI cross-linking (4). Lyn phosphorylates ITAMs of the beta  and gamma  subunits, resulting in the recruitment of Lyn and Syk, respectively, through Src homology-2 (SH2) domain-mediated interactions with phosphotyrosine residues (5, 6). Activation of these newly recruited PTKs, in turn, facilitates the translocation and phosphorylation of multiple substrates, including phospholipase Cgamma (PLCgamma ) isozymes and phosphoinositide 3-kinase (PI3K) (ref review, see Ref. 1). The activated PLCgamma hydrolyzes phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol, which induce the release of Ca2+ from intracellular stores and the activation of protein kinase C, respectively. On the other hand, stimulation of PI3K results in transient accumulation of micromolar levels of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Although the involvement of PI3K in antigen-induced degranulation is established (7-11), a clear consensus regarding how lipid products derived from the action of PI3K mediate the cellular responses has yet to emerge. Putative downstream targets for PI(3,4,5)P3 and PI(3,4)P2 include SH2- or pleckstrin homology (PH) domain-containing signaling enzymes such as PLC-gamma , Akt, and Bruton's tyrosine kinase (Btk) (12, 13), which transduce the signal to the respective signaling pathways. In addition, data from several laboratories suggest a role of PI3K in the regulation of intracellular Ca2+ ([Ca2+]i) increase in mast cells (9, 11, 14). Evidence suggests that PI3K may mediate [Ca2+]i elevation in mast cells via two distinct mechanisms. First, in vitro data indicate that PI(3,4,5)P3 stimulates Ins(1,4,5)P3 production by activating PLCgamma isozymes (15, 16). This PLCgamma activation may be attained directly by facilitating membrane translocation (15, 16) or indirectly via Btk (14, 17). Second, PI3K may increase [Ca2+]i by facilitating Ca2+ mobilization across plasma membranes (11). This premise was prompted by data showing the inhibitory effect of PI3K inhibitors on antigen-induced Ca2+ response (9, 11, 14), and is in line with the notion that two distinct Ca2+ influx pathways (capacitative versus non-capacitative) are operative in antigen-stimulated RBL-2H3 cells (18).

It is noteworthy that the proposed involvement of PI3K in the regulation of a non-capacitative Ca2+ influx pathway is reminiscent of our finding of a PI(3,4,5)P3-sensitive Ca2+ entry mechanism in platelets (19) and Jurkat T cells (20). Consequently, we hypothesize that this PI(3,4,5)P3-sensitive Ca2+ entry is a conserved mechanism that plays a crucial role in the regulation of receptor-mediated Ca2+ signaling among these hematopoietic cells. This hypothesis is corroborated by the present data showing the involvement of this PI(3,4,5)P3-mediated Ca2+ influx in Fcepsilon RI-mediated Ca2+ response in RBL-2H3 mast cells. Considering the crucial role of Ca2+ influx in the secretion of inflammatory mediators, this mechanism provides a molecular basis whereby PI3K regulates mast cell function. Moreover, given that PI3K and PLCgamma are the downstream effectors of the Fcepsilon RI-mediated tyrosine kinase cascade, we propose that these two enzymes act in a concerted manner to initiate Ca2+ response to antigen stimulation. In stimulated cells, PI3K and PLCgamma act on the mutual substrate PI(4,5)P2 to generate PI(3,4,5)P3 and Ins(1,4,5)P3, respectively, which activate Ca2+ channels at different cellular compartments to provide the elevated [Ca2+]i required for optimal physiological responses.

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Materials-- D-myo-Inositol 1,3,4,5-tetrakisphosphate, potassium salt (Ins(1,3,4,5)P4) 1-O-(1,2-di-O-palmitoyl-sn-glycero-3-O-phosphoryl)-D-myo-inositol 3,4,5-trisphosphate, protonated form (PI(3,4,5)P3), 1-O-(1,2-di-O-octanoyl-sn-glycero-3-O-phosphoryl)-D-myo-inositol 3,4,5-trisphosphate, protonated form (di-C8-PI(3,4,5)P3), 1-O-(1,2-di-O-palmitoyl-sn-glycero-3-O-phosphoryl)-D-myo-inositol 3,4-bisphosphate, protonated form (PI(3,4)P2), 1-O-(1,2-di-O-palmitoyl-sn-glycero- 3-O-phosphoryl)-D-myo-inositol 4,5-bisphosphate, protonated form (PI(4,5)P2), and 1-O-(1,2-di-O-palmitoyl-sn-glycero-3-O-phosphoryl)-D-myo-inositol 3-monophosphate, protonated form (PI(3)P) were synthesized as previously reported (21, 22). The identity and purity of all inositol phosphates and inositol lipids were verified by 1H and 31P NMR and high resolution mass spectrometry. Published data from this and other laboratories have shown that PI(3,4,5)P3 and other inositol lipids are cell-permeant and can readily fuse with cell membranes to exert cellular responses in different types of cells, including platelets (19), NIH3T3 cells (23), adipocytes (24), and Jurkat T cells (20). Fura-2 acetoxymethyl ester (fura-2 AM), fluo-3 acetoxymethyl ester (fluo-3 AM), and 2-aminoethyoxy diphenylborate (2-APB) were purchased from Calbiochem. Leupeptin, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and the antigen dinitrophenol-conjugated human serum albumin (DNP-HSA) were products from Sigma. [3H]Inositol was purchased from PerkinElmer Life Sciences. DNP-specific monoclonal IgE was a kind gift from Dr. Henry Metzger (Chemical Immunology Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health). The construct expressing hemagglutinin (HA)-tagged Delta p85 (HA·Delta p85) was provided by Professor Alex Toker (Harvard Medical School). Monoclonal anti-HA and rabbit anti-Btk antibodies were purchased from Roche Molecular Biochemicals and BD Biosciences, respectively.

Cell Culture-- RBL-2H3 cells (from ATCC) were maintained in monolayer culture in 75-cm3 plastic tissue culture flasks containing 15 ml of Eagle's minimum essential medium with 10% fetal bovine serum and 0.1% gentamicin at 37 °C in the presence of 5% CO2.

Fluorescence Spectrophotometric Measurement of PI(3,4,5)P3-Induced [Ca2+]i Response-- [Ca2+]i was monitored by change in the fluorescence intensity of fura-2-loaded cells. RBL-2H3 cells (1 × 107 cells/ml) were suspended in 1 ml of assay buffer consisting of 4.3 mM Na2HPO4, 24.3 mM NaH2PO4, 4.3 mM K2HPO4, 113 mM NaCl, 5 mM glucose, pH 7.4. fura-2 loading was achieved by exposing the cells to 10 µM fura-2 AM in the presence of 0.5% bovine serum albumin and 2 mM probenacid in the dark for 1 h at 37 °C. The cells were then pelleted by centrifugation at 1000 × g for 10 min, washed with assay buffer twice, and resuspended at 8 × 105 cells/ml in the same buffer containing 1 mM Ca2+. The effect of various inositol lipids on [Ca2+]i was examined by fura-2 fluorescence in a Hitachi F-2000 spectrofluorimeter at 37 °C with excitation and emission wavelengths at 340 and 510 nm, respectively, as described in the literature (25, 26). The maximum fura-2 fluorescence intensity (Fmax) in RBL-2H3 cells was determined by adding 4-bromo-A23187 (1 µM), and the minimum fluorescence (Fmin) was determined following depletion of external Ca2+ by 5 mM EGTA. The [Ca2+]i was calculated according to the equation [Ca2+]i = Kd(F - Fmin)/(Fmax - F), where Kd denotes the apparent dissociation constant (Kd = 224 nM) of the fluorescence dye-Ca2+ complex (27).

Fluorescence Spectrophotometric Measurement of Antigen-induced [Ca2+]i Response-- RBL-2H3 cells were sensitized with 1 µg/ml mouse DNP-specific IgE overnight. Cells were collected, suspended in assay buffer, loaded with fura-2, and activated by the antigen DNP-HSA (1 µg/ml). Changes in [Ca2+]i were monitored by fura-2 fluorimetry as described above.

All the above fura-2 experiments were also repeated with fluo-3-loaded cells in a similar manner. fluo-3 displays a lower Kd (864 nM at 37 °C) and a longer excitation wavelength (505 nm) of the dye-Ca2+ complex vis à vis fura-2 (28). Consistent results were obtained with both fluorescence dyes.

Transient Transfection-- Delta p85 is a deletion mutant that lacks a region required for tight association with p110 but is still able to bind to appropriate phosphotyrosine targets. Thus, Delta p85 can compete with native p85 for binding to essential signaling proteins and behaves as a dominant negative mutant. Transient transfection of HA·Delta p85 was carried out using the LipofectAMINE Plus reagent according to the protocol supplied by the manufacturer. In brief, Opti-MEM medium (1.5 ml) containing the indicated amount of the HA·Delta p85 expression vector (3-12 µg) was incubated with 120 µl of the Plus reagent at 25 °C for 15 min, and 12 µl of the LipofectAMINE reagent in 1.5 ml of Opti-MEM was added. In parallel, an empty pCMV/blue plasmid (12 µg) was subjected to the same treatment as a negative control. The mixture was incubated at 25 °C for 15 min and added to 9 ml of serum-free Opti-MEM (Life Technologies). RBL-2H3 cells in a T-75 flask were washed with serum-free Opti-MEM and were added to the aforementioned transfection medium. After 3 h at 37 °C, the transfection media were replaced with 15 ml of Eagle's minimum essential medium containing 10% fetal bovine serum. The transfected cells were allowed to grow for 2 days to express foreign DNA. The collected cells were analyzed for antigen-induced Ca2+ response by fluorescence spectrometry and for HA·Delta p85 expression by Western blot analysis using anti-HA antibody.

beta -Hexosaminidase Secretion-- The release of mast cell mediators by exocytosis was monitored by the beta -hexosaminidase assay. RBL-2H3 cells were grown in 12-well plates and passively sensitized with DNP-specific IgE. The IgE-sensitized cells were washed twice with Tyrode's buffer consisting of 10 mM Hepes, pH 7.4, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% bovine serum albumin. Secretion was initiated by exposing cells to the antigen DNP-HSA (1 µg/ml). After 1 h, the reaction was terminated by placing the plate on ice. The enzyme activities of beta -hexosaminidase in 50 µl of supernatants and attached cells solubilized with 0.5% Triton X-100 were measured with 200 µl of 1 mM p-nitrophenyl N-acetyl-beta -D-glucosaminide in 0.1 M sodium citrate, pH 4.5, at 37 °C for 1 h. The reaction was stopped by the addition of 500 µl of 0.1 M NaHCO3. The release of the product p-nitrophenol was measured by monitoring the absorbance at 400 nm. The percentage of degranulation was calculated by dividing the absorbance of the supernatant over the combined absorbance of the supernatant and cell lysate.

[3H]Inositol Phosphate Turnover Analysis-- The examination of phosphoinositol turnover was carried out according to a modification of the procedure described previously (20). In brief, RBL-2H3 cells were incubated with myo-[2-3H]inositol (10 µCi/106 cells/ml) in Eagle's minimum essential medium supplemented with 10% fetal bovine serum. The cells were then washed with the medium, sensitized with DNP-specific IgE, and washed with 20 mM Hepes, pH 7.4, containing 285 mM NaCl, 11 mM KCl, 1.3 mM Na2HPO4, 1 mM KH2PO4, 8.3 mM NaHCO3, 1.6 mM MgSO4, 2.2 mM MgCl2, 2.2 mM CaCl2, and 5.6 mM glucose. Aliquots containing 1 × 106 cells were each resuspended in 0.3 ml of the aforementioned assay buffer and transferred to 1.5-ml microcentrifuge tubes. Each sample was incubated with 0.3 µg of DNP-HSA for the indicated times and quenched by adding 0.25 ml of 6% trichloroacetic acid. The tubes were centrifuged for 2 min at 12,000 × g. The supernatant (200 µl) was analyzed by high pressure liquid chromatography on a 5-µm Adsorbosphere Sax column (4.6 × 200 mm) equilibrated with H2O. The [3H]inositol phosphates were eluted with a linear gradient of 0-0.9 M NH4H2PO4 in 60 min at a flow rate of 1 ml/min. Fractions were collected every 1 ml, and their radioactivity was measured by liquid scintillation. Synthetic [3H]Ins(1,3,4,5)P4, [3H]Ins(1,4,5)P3, [3H]Ins(4,5)P2, and Ins(4)P were used as standards. The respective retention times were 60, 48, 43, and 31 min.

Preparation of RBL-2H3 Cell Plasma Membrane Vesicles-- For the Ca2+ release assay, the plasma membrane was purified as previously described (20). In brief, RBL-2H3 cells (4 × 108 cells) were washed with phosphate-buffered saline and suspended in 5 ml of PM buffer consisting of 20 mM Hepes, pH 7.2, 110 mM KCl, 10 mM NaCl, 2 mM MgCl2, 5 mM KH2PO4, 1 mM dithiothreitol, 1 mM EGTA, 1 mM AEBSF, and 20 µg/ml leupeptin. The cell suspension was homogenized in a Dounce homogenizer using a loose pestle with five strokes up and down. The homogenate was centrifuged at 1500 × g for 10 min. The pellet was suspended in 3.125 ml of PM buffer and mixed with 5.5 ml of 69% (w/w) sucrose to make a final 44% (w/w) sucrose-membrane mixture. The mixture was overlaid with 42.3% (w/w) sucrose, and the two-phase suspension was subjected to centrifugation at 90,000 × g for 2 h in a swinging bucket rotor. The membrane material at the interface of the phases contained the greatest enrichment in plasma membranes based on the activity of (Na+-K+)-ATPase. This fraction was collected, suspended in 5 ml of 10 mM Hepes, pH 7.5, containing 1 mM dithiothreitol, 1 mM AEBSF, and 20 µg/ml leupeptin, and centrifuged at 25,000 × g for 10 min. The pellet was suspended in 1 ml of the same buffer.

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PI(3,4,5)P3 Induces [Ca2+]i Increase in RBL-2H3 Mast cells by Stimulating Ca2+ Influx-- To investigate the involvement of PI3K in antigen-stimulated Ca2+ response in mast cells, we first examined the effect of exogenous PI(3,4,5)P3 on [Ca2+]i in RBL-2H3 cells. Exposure of fura-2-loaded RBL-2H3 cells to PI(3,4,5)P3, ranging from 1 to 20 µM, elicited an instantaneous [Ca2+]i increase in a dose-dependent manner (Fig. 1A, upper panel).


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Fig. 1.   A, upper panel, time course and dose dependence of the effect of PI(3,4,5)P3 on [Ca2+]i in RBL-2H3 cells; lower panel, PI(3,4,5)P3-induced [Ca2+]i increase was abrogated in a Ca2+-depleted medium, indicating the increase was due to Ca2+ influx. External Ca2+ was depleted by treating medium with 5 mM EGTA for 10 min. B, phosphoinositide specificity in eliciting [Ca2+]i increase in RBL-2H3 cells. Upper panel, PI(3,4)P2; lower panel, PI(4,5)P2 and PI(3)P. [Ca2+]i was analyzed by fura-2 fluorimetry as described under "Experimental Procedures." The arrows indicate the time of addition of individual compounds. Traces are representative of three independent experiments. Consistent results were obtained when another fluorescence dye fluo-3 was used in the above experiments.

This PI(3,4,5)P3 effect became saturated at 20 µM, beyond which no significant enhancement in the amplitude of Ca2+ response was noted (data not shown). The PI(3,4,5)P3-induced [Ca2+]i rise was largely attributable to Ca2+ influx, because the increase could be abolished by Ca2+ depletion with 5 mM EGTA (Fig. 1A, lower panel). With regard to other phosphoinositides examined, PI(3,4)P2 at high doses (20 µM) could also elicit [Ca2+]i increase but to a much lesser extent than PI(3,4,5)P3 (Fig. 1B, upper panel), however. The potency was ~10% of that of PI(3,4,5)P3. On the other hand, PI(4,5)P2 and PI(3)P did not show any appreciable effect on [Ca2+]i (lower panel). These data suggest the presence of a PI(3,4,5)P3-sensitive Ca2+ influx mechanism in RBL-2H3 mast cells.

PI(3,4,5)P3 Does Not Disturb Internal Ca2+ Stores in RBL-2H3 Cells-- Data from several groups suggest that PI(3,4,5)P3 might stimulate Ins(1,4,5)P3 production by activating PLCgamma isozymes via discrete mechanisms (9, 11, 14). This raised a concern that PI(3,4,5)P3-induced [Ca2+]i increase might, in part, be due to Ca2+ release from endoplasmic reticulum stores. To refute this possibility, we first examined the effect of two distinct pharmacological inhibitors that blocked Ins(1,4,5)P3-induced Ca2+ mobilization. These included the PLC inhibitor U73122 (10 µM) and the cell-permeant antagonist of Ins(1,4,5)P3 receptors 2-APB (40 µM) (29, 30). Although these inhibitors were effective in attenuating Fcepsilon RI-mediated Ca2+ response in RBL-2H3 cells (Figs. 3 and 4), they did not display significant effect on PI(3,4,5)P3-induced [Ca2+]i response. The extents of the [Ca2+]i increase elicited by 20 µM PI(3,4,5)P3 in RBL-2H3 cells pretreated with U73122 and 2-APB were 95 ± 2% (n = 3) and 98 ± 2% (n = 3), respectively, of that of the untreated control.

Furthermore, we obtained evidence that thapsigargin-sensitive Ca2+ pools were not disturbed by PI(3,4,5)P3. RBL-2H3 cells were exposed to 20 µM PI(3,4,5)P3 in a Ca2+-depleted medium, followed by 1 µM thapsigargin. As shown in Fig. 2, PI(3,4,5)P3 did not affect the extent of thapsigargin-induced Ca2+ response vis à vis that without PI(3,4,5)P3 pretreatment.


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Fig. 2.   PI(3,4,5)P3 does not affect thapsigargin (TG)-sensitive internal Ca2+ pools. In the presence of 5 mM EGTA, fura-2-loaded RBL-2H3 cells were treated with, in tandem, 20 µM PI(3,4,5)P3 and 1 µM thapsigargin. Inset, in the presence of 5 mM EGTA, fura-2-loaded cells were treated with 1 µM thapsigargin alone. The arrows indicate the time of addition of individual compounds. Traces are representative of three independent experiments.

Together, these results bore out the premise that PI(3,4,5)P3-induced Ca2+ response was independent of PLC activity and was solely attributable to Ca2+ influx from the medium.

Role of PI3K in Fcepsilon RI-induced Ca2+ Response in RBL-2H3 Cells-- The existence of a PI(3,4,5)P3-sensitive Ca2+ entry mechanism suggests a link between PI3K and receptor-activated Ca2+ signaling in RBL-2H3 cells. Because both PI3K and PLCgamma are downstream effectors in Fcepsilon RI-mediated tyrosine kinase cascades, we hypothesized that PI3K acted in concert with PLCgamma in initiating the Ca2+ response to Fcepsilon RI cross-linking. To test this hypothesis, a combination of pharmacological and molecular genetic approaches was employed to characterize the role of PI3K in Fcepsilon RI-induced Ca2+ response.

First, the effect of the PI3K inhibitor LY294002 on antigen-stimulated Ca2+ response in fura-2-loaded RBL-2H3 cells was assessed. In this study, LY294002 was used in lieu of wortmannin to inhibit PI3K in cells in light of the nonspecific effect of wortmannin on myosin light-chain kinase, one of the key regulatory enzymes in mast cells (8). RBL-2H3 cells were sensitized with DNP-specific IgE overnight, loaded with the fluorescence indicator, and pretreated with varying concentrations of LY294002. After stimulation with the antigen DNP-HSA (1 µg/ml), changes in [Ca2+]i were monitored by fluorimetry. In line with the earlier reports that the antigen-stimulated Ca2+ response was largely attributable to Ca2+ influx (11, 31), depletion of external Ca2+ by EGTA substantially diminished the Ca2+ signal following antigen stimulation (Fig. 3A, inset).


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Fig. 3.   A, inhibitory effect of LY294002 on Fcepsilon RI-mediated Ca2+ response in RBL-2H3 cells. Cells were sensitized with DNP-specific IgE, loaded with fura-2, and activated with the antigen DNP-HSA (Ag). Traces a-d represent Ca2+ responses in the presence of 0, 40, 100, and 200 µM LY294002, respectively. The maximum inhibitory effect was attained at 200 µM. The inset depicts the effect of Ca2+ depletion on FceRI-mediated Ca2+ response. Traces a and b denote the Ca2+ responses of the control and in the presence of 5 mM EGTA, respectively. B, inhibitory effect of 2-APB (40 µM) alone (trace b) and of the combination of 2-APB (40 µM) and LY294002 (200 µM) (trace c). Trace a depicts the Ca2+ response of the control. [Ca2+]i was analyzed as described under "Experimental Procedures." Traces are representative of three independent experiments. Consistent results were obtained when another fluorescence dye fluo-3 was used in the above experiments.

Fig. 3 indicates that LY294002 pretreatment attenuated the amplitude of antigen-induced Ca2+ response in a dose-dependent manner. The effect of LY294002 on the Ca2+ response attained maximum at 200 µM, beyond which no further decrease was noticed (data not shown). Presumably, the residual Ca2+ response following LY294002 treatment was attributed to Ins(1,4,5)P3-induced Ca2+ release and the consequent capacitative Ca2+ entry. This premise was corroborated by the concomitant treatment of IgE-sensitized RBL-2H3 cells with LY294002 (200 µM) and 2-APB (40 µM) (Fig. 3B, trace c). For 2-APB (40 µM) alone, a 55% reduction in the Ca2+ response was noted (trace b), which was due to the inhibition of Ins(1,4,5)P3-induced Ca2+ release and the accompanied capacitative Ca2+ entry.

Second, an independent biochemical approach was taken to confirm the above results, in which RBL-2H3 cells were transiently transfected with a vector expressing HA epitope-tagged Delta p85 (HA·Delta p85). It is well documented that deletion of the binding motif for the catalytic p110 subunit of p85 confers PI3K dominant negative activity (32). We have applied this dominant negative inhibitor in Jurkat T cells as part of our effort to demonstrate the role of PI3K in T cell receptor-mediated Ca2+ signaling (20). Western analysis using anti-HA antibody verified the expression of HA·Delta p85 in transiently transfected RBL-2H3 cells (Fig. 4A).


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Fig. 4.   Overexpression of Delta p85 inhibits Fcepsilon RI-mediated Ca2+ response and secretion in RBL-2H3 cells. A, expression levels of HA·Delta p85 in RBL-2H3 cells that had been transfected with 1 µg/ml of a control pCMV/blue plasmid (a) or with increasing amounts of the HA·Delta p85-expressing plasmid (b, 0.25 µg/ml; c, 0.5 µg/ml; d, 0.75 µg/ml; e, 1 µg/ml) for 2 days. The Western analysis was carried out using antibodies against the HA tag. The loading amounts of individual samples were calibrated in reference to actin as internal standard (data not shown). Data are representative of three independent experiments. B, Delta p85 inhibited Fcepsilon RI-mediated Ca2+ response in a dose-dependent manner. C, Delta p85 inhibited Fcepsilon RI-mediated secretion of beta -hexosaminidase in a dose-dependent manner. Cells expressing different levels of Delta p85 (a-e, as indicated above) were collected after overnight sensitization with DNP-specific IgE. The collected cells were loaded with fura-2 and stimulated with DNP-HSA (Ag) to test for Ca2+ response and beta -hexosaminidase secretion (a-e in accordance with the above designations). In addition, cells expressing the highest level of Delta p85 were treated with 2-APB (40 µM) for 10 min before antigen stimulation. As shown, the concerted action of Delta p85 and 2-APB abrogated the Ca2+ signal (trace f in B) and beta -hexosaminidase secretion (vertical bar f in C). Traces are representative of three independent experiments. Vertical bars shown are means ± S.D. (n = 3). Consistent results were obtained when another fluorescence dye fluo-3 was used in the above experiments.

As shown, the level of Delta p85 expression displayed a direct correlation with the amount of cDNA used in the transient transfection. Accordingly, transfected RBL-2H3 cells expressing varying levels of HA·Delta p85 were tested for Ca2+ response to Fcepsilon RI cross-linking. As shown, the expression of Delta p85 attenuated the Ca2+ signal, ranging from 10 to 50%, in a dose-dependent manner (Fig. 4B, traces a-e). In line with the data shown in Fig. 3B, the antigen-induced Ca2+ response was nearly abrogated when the cells expressing the highest level of HA·Delta p85 were treated with 2-APB (40 µM) (trace f).

Moreover, the extent of inhibition on the Ca2+ response displayed a direct correlation with that of the antigen-stimulated release of beta -hexosaminidase (Fig. 4C). This observation reaffirms the close relationship among PI3K activity, [Ca2+]i increase, and mast cell degranulation.

Ins(1,3,4,5)P4 Is Not Physiologically Relevant in Antigen-induced Ca2+ Response in RBL-2H3 Cells-- In cells, the 3-phosphorylation of Ins(1,4,5)P3 by Ins(1,4,5)P3-specific kinase accounts for a major pathway for the formation of Ins(1,3,4,5)P4 (33). Consequently, Ins(1,4,5)P3 accumulation in response to antigen stimulation in RBL-2H3 cells was accompanied by an increase in Ins(1,3,4,5)P4 (Fig. 5A, left panel). Structurally, Ins(1,3,4,5)P4 represents the head group of PI(3,4,5)P3, and displays in vitro cross-reactivity with PI(3,4,5)P3 in many aspects of biochemical functions (19, 20). Moreover, Ins(1,3,4,5)P4 has been implicated as a second messenger in facilitating Ca2+ entry into non-excitable cells (34, 35). Consequently, a crucial issue that warranted investigation was whether Ins(1,3,4,5)P4 played a role in the regulation of Fcepsilon RI-mediated Ca2+ response in RBL-2H3 cells.


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Fig. 5.   Evidence that Ins(1,3,4,5)P4 is not involved in Fcepsilon RI-mediated Ca2+ response in RBL-2H3 cells. A, left panel, kinetics of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4,5)P4 production in [3H]inositol-labeled RBL-2H3 cells in response to Fcepsilon RI cross-linking as described under "Experimental Procedures." Results are given as means of three independent experiments. A, right panel, Fcepsilon RI-mediated Ca2+ response in RBL-2H3 cells. Cells were sensitized with DNP-specific IgE, loaded with fura-2, and activated with the antigen DNP-HSA as described under "Experimental Procedures." B, left panel, effect of adriamycin on kinetics of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4,5)P4 production in [3H]inositol-labeled RBL-2H3 cells in response to Fcepsilon RI cross-linking. [3H]Inositol-labeled RBL-2H3 cells were pretreated with 10 µM adriamycin for 1 h before Fcepsilon RI cross-linking. Results are given as means of three independent experiments. As shown, the Ins(1,4,5)P3 3-kinase inhibitor completely blocked the formation of Ins(1,3,4,5)P4 without affecting Ins(1,4,5)P3 production. B, right panel, effect of adriamycin (10 µM) on Fcepsilon RI-mediated Ca2+ response in RBL-2H3 cells. No appreciable difference was noted in the Ca2+ response between the control and adriamycin-treated cells, even though the inhibitor completely inhibited Ins(1,3,4,5)P4 synthesis. Traces are representative of three independent experiments.

In this study, RBL-2H3 cells were pretreated with the Ins(1,4,5)P3 3-kinase inhibitor adriamycin (10 µM) (25), which completely blocked Ins(1,3,4,5)P4 synthesis while not interfering with Ins(1,4,5)P3 formation (Fig. 5B, left panel). It is noteworthy that the complete inhibition of Ins(1,3,4,5)P4 production had no appreciable effect on antigen-induced Ca2+ response vis à vis the control (Fig. 5, A and B, right panels). Together, these data argue against any second-messenger role of Ins(1,3,4,5)P4 in regulating receptor-mediated Ca2+ entry in RBL-2H3 mast cells.

PI(3,4,5)P3 Directly Elicits Ca2+ Efflux from Ca2+-loaded Plasma Membrane Vesicles-- The above observations also prompted a question of how PI(3,4,5)P3 facilitated Ca2+ entry across plasma membranes. In principle, PI(3,4,5)P3 could directly sensitize a Ca2+-entry mechanism or it might mediate the action through the stimulation of other signaling components. For example, data from several laboratories suggest the involvement of Btk in PI(3,4,5)P3-regulated Ca2+ entry in platelets and B cells (36, 37). This premise is consistent with the notion that Fcepsilon RI-induced Ca2+ influx was impaired in Btk-deficient mast cells (38). Btk, a member of the Tec non-receptor PTK family, contains a pleckstrin (PH) domain that selectively binds PI(3,4,5)P3 in vitro (39, 40). Consequently, PI(3,4,5)P3 promotes the translocation and subsequent phosphorylation of Btk by Src family kinases (41). Once activated, Btk can phosphorylate and activate a number of protein substrates such as PLCgamma (42, 43) and c-Jun N-terminal kinases (44). However, the mechanism by which Btk regulates receptor-mediated [Ca2+]i increase remains enigmatic, because it has been reported to be independent of increased PLCgamma activity (36, 37).

To address whether PI(3,4,5)P3-mediated Ca2+ entry required any cytoplasmic component such as Btk, we examined the Ca2+-transporting activity of PI(3,4,5)P3 in plasma membrane vesicles prepared from RBL-2H3 cells. Western blot analysis confirmed that this plasma membrane preparation was devoid of Btk (Fig. 6A).


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Fig. 6.   Evidence that PI(3,4,5)P3-induced Ca2+ response does not require any cytoplasmic component such as Btk. A, Western blot analysis of Btk in the plasma membrane vesicle and cell lysate of RBL-2H3 cells. No appreciable amounts of Btk were detectable in the membrane vesicles. The blots are representative of three independent experiments. B, Ca2+ release from plasma membrane vesicles by di-C8-PI(3,4,5)P3 (20 µM). RBL-2H3 cell plasma membranes were prepared as described under "Material and Methods" and were treated with 30 µM Mg2+-ATP, 1 mM CaCl2, 1 µM thapsigargin, and 2.5 µg/ml oligomycin on ice for 10 min. The membrane vesicles were washed with 10 mM Hepes, pH 7.0, four times, and suspended in the same buffer. The assay medium consisted of 0.2-0.25 mg of membrane proteins and 1 µM fura-2 in 2 ml of 10 mM Hepes, pH 7.0. After the external Ca2+ concentration returned to a near-base level, the membrane vesicles were stimulated with 20 µM di-C8-PI(3,4,5)P3 as indicated. The inset indicates the Ca2+ response following the sequential additions of 25 µM PI(3,4,5)P3 and 1 µM 4-bromo-A23187. Traces are representative of three experiments.

The inside-out plasma membrane of RBL-2H3 cells displayed ATP-dependent Ca2+ sequestering activity. These membrane vesicles were loaded with Ca2+ by exposing them to ATP in the presence of thapsigargin (1 µM) and oligomycin (5 µg/ml). The Ca2+-loaded membrane vesicles were washed and analyzed for Ca2+ release induced by PI(3,4,5)P3, which was equivalent to Ca2+ influx in intact cells. Fura-2 fluorescence indicates that addition of di-C8-PI(3,4,5)P3 (20 µM) caused immediate Ca2+ release followed by slow re-uptake, which was presumably due to small quantities of residual ATP in the milieu (Fig. 6B). This finding suggests that PI(3,4,5)P3 might activate a Ca2+ entry mechanism on plasma membranes independent of cytoplasmic components. It is noteworthy that the Ca2+ release was not augmented by the subsequent challenge with PI(3,4,5)P3, which is in line with our observation found in the membrane vesicles prepared from Jurkat T cells (19). The lack of Ca2+ response was likely due to desensitization or saturation of the binding site instead of the depletion of Ca2+, since the addition of 1 µM 4-bromo-A23187 following PI(3,4,5)P3 treatment triggered the release of large amounts of Ca2+ (Fig. 6B, inset).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study presents pharmacological and biochemical evidence that PI3K plays a concerted role with PLCgamma in the regulation of Ca2+ influx in RBL-2H3 mast cells via a PI(3,4,5)P3-sensitive Ca2+ entry pathway. First, exogenous PI(3,4,5)P3 at concentrations close to its physiological levels induces instantaneous Ca2+ influx into RBL-2H3 cells. Second, inhibition of PI3K by LY294002 or by overexpression of the dominant negative inhibitor Delta p85 suppressed Fcepsilon RI-mediated Ca2+ response. Third, PI(3,4,5)P3 was capable of stimulating Ca2+ efflux from Ca2+-loaded plasma membrane vesicles, suggesting that PI(3,4,5)P3 interacts with a Ca2+-entry system on plasma membranes independent of cytoplasmic components. This unique Ca2+ influx mechanism has also been demonstrated in platelets (19) and Jurkat T cells (20) in our laboratory. In addition, a recent report indicates that the Ca2+ influx induced by the cross-linking of IgG-receptor (Fcgamma R) in human neutrophils was inhibited by PI3K inhibitors (45). Together, these data underscore a novel function of PI3K in the regulation of receptor-mediated Ca2+ responses in these hematopoietic cells. This premise is in line with the notion that receptor-stimulated tyrosine kinase cascades that lead to the translocation and activation of PI3K of PLCgamma bear a high degree of similarity in the signaling mechanism among these cell types.

Ca2+ signaling in response to Fcepsilon RI cross-linking is essential to the antigen-induced secretion of inflammatory mediators in RBL-2H3 cells (31, 46), and therefore is one of the key early events in mast-cell signal transduction. The present data support our hypothesis that PI3K and PLCgamma play a concerted role in initiating antigen-induced Ca2+ signaling (Fig. 7).


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Fig. 7.   A working hypothesis to account for the concerted action of PLCgamma and PI3K in initiating Ca2+ response following Fcepsilon RI cross-linking in mast cells.

PI3K and PLCgamma act on the mutual substrate PI(4,5)P2 to generate two important second messengers, PI(3,4,5)P3 and Ins(1,4,5)P3, respectively, that initiate the Ca2+ response to Fcepsilon RI cross-linking by activating Ca2+ transport systems in different cell compartments. It is well understood that the major part of Fcepsilon RI-mediated Ca2+ signals in mast cells is attributable to Ca2+ influx. According to our hypothesis, this Ca2+ influx is regulated by two discrete Ca2+ entry mechanisms on plasma membranes: 1) the capacitative Ca2+ entry that is secondary to the depletion of the intracellular Ca2+ store by Ins(1,4,5)P3, and 2) the PI(3,4,5)P3-sensitive Ca2+ entry that is independent of the filling state of internal Ca2+ stores. Considering the temporal relationship between these two mechanisms, PI(3,4,5)P3-senitive Ca2+ entry should precede capacitative Ca2+ entry, because it is independent of signals from store depletion. Consequently, the concerted action of these two discrete pathways allows the cells to sustain elevated [Ca2+]i to achieve optimal physiological responses in mast cells as well as other types of hematopoietic cells.

In the literature, Ins(1,3,4,5)P4, an immediate metabolite of Ins(1,4,5)P3, has been implicated in mediating Ca2+ entry in many non-excitable cells, including sea urchin eggs (34) and Xenopus oocytes (35). It was found that microinjection of Ins(1,3,4,5)P4 into the whole cells caused [Ca2+]i increase through a mechanism dependent on external Ca2+. In addition, several research groups have isolated an Ins(1,3,4,5)P4-binding protein, GAP1IP4BP, from platelet plasma membranes (47-50). However, the present study refutes the second messenger role of Ins(1,3,4,5)P4 in regulating FceRI-mediated Ca2+ inflow in mast cells. This finding is consistent with earlier data in mouse lacrimal acinar cells (51) and Jurkat T cells (20, 25) that have ruled out the involvement of Ins(1,3,4,5)P4 in receptor-activated Ca2+ influx. Considering the largely shared structural motifs, it is plausible that the microinjected Ins(1,3,4,5)P4 in sea urchin eggs and Xenopus oocytes might mimic PI(3,4,5)P3 to activate the PI(3,4,5)P3-sensitive Ca2+ entry mechanism, resulting in increased [Ca2+]i.

The involvement of PI3K and Btk in receptor-dependent Ca2+ signals in various hematopoietic cells has been the subject of many recent investigations (36-38). However, the link between Btk and PI(3,4,5)P3-sensitive Ca2+ entry remains inconclusive. Although Btk is an essential component for the activating phosphorylation of PLCgamma -2 (43), a recent study by Watson and colleagues indicates that the Ca2+ entry pathway mediated by PI(3,4,5)P3 and Btk in platelets and megakaryotes was independent of increased PLC activity (36). Moreover, data from Kinet and co-workers (37) suggest that Btk exerted its regulation on receptor-mediated Ca2+ signals by protecting PI(3,4,5)P3 from degradation by endogenous SH-2 containing inositol phosphatase. Therefore, Btk may play an auxiliary role by binding PI(3,4,5)P3 via its PH domain, thereby enhancing PI(3,4,5)P3 accumulation. This PI(3,4,5)P3 sequestration hypothesis is consistent with our observation that PI(3,4,5)P3 could directly stimulate Ca2+ transport in plasma membrane vesicles in the absence of Btk or any other cytoplasmic component. Nevertheless, the present data could not rule out the possibility that Btk might play the role as a regulator of the Ca2+ channel activity, which is under investigation.

In summary, this PI(3,4,5)P3-sensitive Ca2+ entry pathway not only provides molecular insights into the role of PI3K in the regulation of mast cell degranulation but also represents a potential target for the modulation of cell function in mast cells. However, questions remain outstanding regarding the regulatory mechanism underlying PI(3,4,5)P3-mediated Ca2+ entry and its relationship with Ca2+-release activated Ca2+ channels located on plasma membranes. Studies are currently under way in this laboratory to further understand these issues.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Henry Metzger (NIAMS/National Institutes of Health) for providing DNP-specific IgE and helpful comments on the manuscript, and to Dr. Alex Toker (Harvard Medical School) for the construct expressing HA-tagged Delta p85.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM53448.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: College of Pharmacy, The Ohio State University, 500 West 12th Ave., Columbus, OH 43210-1291. Tel.: 614-688-4008; Fax: 614-688-8556; E-mail: chen@dendrite.pharmacy.ohio-state.edu.

Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M009851200

    ABBREVIATIONS

The abbreviations used are: Fcepsilon RI, the high affinity receptor for IgE; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; PI(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PI(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PI(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3)P, phosphatidylinositol 3-monophosphate; Ins(1, 4,5)P3, D-myo-inositol 1,4,5-trisphosphate; Ins(1, 3,4,5)P4, D- myo-inositol 1,3,4,5-tetrakisphosphate; ITAM, immunoreceptor tyrosine-based activation motif; PTK, protein-tyrosine kinase; SH2, Src homology-2; Btk, Bruton's tyrosine kinase; DNP-HSA, dinitrophenol-conjugated human serum albumin; 2-APB, 2-aminoethoxy diphenylborate; HA·Delta p85, hemagglutinin epitope-tagged Delta p85; PH, pleckstrin homology; fura-2 AM, fura-2 acetoxymethyl ester; fluo-3 AM, fluo-3 acetoxymethyl ester; CMV, cytomegalovirus; di-C8-PI(3, 4,5)P3, 1-O-(1,2-di-O-octanoyl-sn-glycero-3-O-phosphoryl)-D-myo-inositol 3,4,5- trisphosphate.

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