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J. Biol. Chem., Vol. 279, Issue 21, 22505-22513, May 21, 2004
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RI-triggered Generation of Arachidonic Acid and Eicosanoids Requires iPLA2 but Not cPLA2 in Human Monocytic Cells*
From the Department of Physiology, Faculty of Medicine, National University of Singapore, Singapore 117597
Received for publication, August 8, 2003 , and in revised form, February 12, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rs) on myeloid cells activates a series of events that are key in the inflammatory response and that can ultimately lead to targeted cell killing by antibody-directed cellular cytotoxicity. Generation of lipid-derived proinflammatory mediators is an important component of the integrated cellular response mediated by receptors for the constant region of immunoglobulins (Fc). We have demonstrated previously that, in interferon--primed U937 cells, the high affinity receptor for IgG, FcRI, is coupled to a novel intracellular signaling pathway that involves the sequential activation of phospholipase D, sphingosine kinase, calcium transients, and protein kinase C isoforms, leading to the activation of the NADPH-oxidative burst. Here, we investigate the nature of the phospholipase that regulates arachidonic acid and eicosanoid production. Our data show that FcRI couples to iPLA2
for the release of arachidonic acid and the generation of leukotriene B4 and prostaglandin E2. Activation of iPLA2 was protein kinase C-dependent; on the other hand, platelet-activating factor triggered cPLA2
by means of the mitogen-activated protein kinase pathway. These studies demonstrate that intracellular PLA2s can be selectively regulated by different stimuli and suggest a critical role for iPLA2 in the intracellular signaling cascades initiated by FcRI and its functional role in the generation of key inflammatory mediators. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RI) activates, through non-receptor tyrosine kinases, a novel signaling pathway that involves the sequential activation of phosphatidylinositol 3-kinase, phospholipase D, and sphingosine kinase (5-7). This pathway is necessary for efficient intracellular trafficking of FcRI-internalized immune complexes to lysosomes for degradation, the release of calcium from intracellular stores, and the activation of the NADPH oxidative burst (6-8).
Eicosanoids (such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4)) are important mediators of inflammation. Eicosanoids generation originates from arachidonic acid (AA), a 20-carbon, unsaturated fatty acid that is hydrolyzed from membrane phospholipids by phospholipase A2 (PLA2) (9).
At present, 14 different PLA2 groups have been identified (10, 11). These include 10 groups of enzymes utilizing a catalytic histidine, which show millimolar requirements for Ca2+ and are collectively referred to as the secreted PLA2s (Groups I, II, III, V, IX, X, XI, XII, XIII, and XIV) (10, 11), and two groups of intracellular, high molecular mass enzymes, which utilize a catalytic serine (Groups IV and VI). Group IV comprise IVA, IVB and IVC PLA2, also known as cytosolic PLA2 (cPLA2, cPLA2, and cPLA2, respectively), which are highly regulated, Ca2+-dependent enzymes (10, 11). Whereas Group VI PLA2, or iPLA2, are Ca2+-independent enzymes (10, 11), also possessing a catalytic serine, yet its structure is far distant from that of the cPLA2 family. iPLA2 occurs in multiple alternative splicing variants, the majority of which are enzymatically functional (12, 13). Mammalian iPLA2s are classified as groups VIA and VIB, (iPLA2 and iPLA2, respectively) (10).
The best studied PLA2s are Groups IIA, V, and IVA, which for a long time have been shown to be responsible for AA release and prostaglandin generation in different systems (14-16). iPLA2 has been shown to be implicated in many cellular functions ranging from basal fatty acid reacylation reactions (17), to playing major roles in intracellular signaling cascades, including its involvement in agonist-induced eicosanoid production (18, 19), stimulation of smooth muscle (20) and endothelial cells (21), in lymphocyte proliferation (22), and in endothelium-dependent vascular relaxation (21). Very recently, it has been reported that myocardial ischemia activates iPLA2 in intact myocardium, and that this iPLA2 activation is sufficient to induce malignant ventricular arrhythmias (23), and also that functional iPLA2 is required for activation of store-operated channels and capacitative Ca2+ influx in several cell types (24).
Here, we demonstrate that coupling of FcRI to AA generation and production of LTB4 and PGE2 absolutely requires iPLA2 activation. Although both intracellular forms of PLA2 (cPLA2 and iPLA2) are present in U937 cells, only iPLA2 functionally couples FcRI to trigger physiological responses, such as the generation of AA and the production of LTB4 and PGE2. Moreover, only iPLA2 translocates to the plasma membrane and triggers the generation of AA and eicosanoids after FcRI activation. Furthermore, by using specific antisense oligonucleotides against iPLA2 and cPLA2, we found that both isoforms can be activated independently by different receptors, because the addition of platelet-activating factor (PAF) triggers cPLA2-dependent generation of AA without activating iPLA2. Thus, these studies demonstrate that both intracellular PLA2s can be selectively regulated by different stimuli and suggest a critical role for iPLA2 in the intracellular signaling cascades initiated by immune-receptors and its functional role in the generation of key inflammatory mediators.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(Bender Wien Ltd, Vienna, Austria) for 16 h. Antisense oligonucleotides were purchased from Oswell DNA Services; 24-mers were synthesized, capped at either end by the phosphorothioate linkages (first two and last two linkages), and corresponded to the reverse complement of the first eight amino acids for either iPLA2 or cPLA2. The sequences of the oligonucleotides were 5'-CAGGCGGCCAAAGAACTGCATCTT-3' for iPLA2 and 5'-GGTAAGGATCTATAAATGACAT-3' for cPLA2.
Cells were incubated in 1 µM oligonucleotide mixed with 20 µl of Superfect (Qiagen) for a total of 36 h (20 h prior to the addition of INF, and then incubated for the duration of culture with IFN-).
Receptor Stimulation—FcRI aggregation was carried out as described previously (6-8). Briefly, cells were harvested by centrifugation and then incubated at 4 °C for 45 min with 1 µM human monomeric IgG (Serotec UK) to occupy surface FcRI in the presence or absence of inhibitors or alcohols. Excess unbound ligand was removed by dilution and centrifugation of the cells. Cells were resuspended in ice-cold RHB medium (RPMI 1640 medium, 10 mM HEPES, 0.1% BSA) and surface immune complexes formed by incubating with cross-linking antibody (sheep anti-human IgG; 1:50), without or in the continued presence of inhibitors. Cells were then warmed to 37 °C for the times specified in each assay.
PAF Stimulation—Cells were harvested by centrifugation and resuspended in ice-cold RHB medium, and surface platelet-activating factor receptor was stimulated by the addition of 10 µM platelet-activating factor (PAF) (Calbiochem), without or in the continued presence of inhibitors. Cells were then warmed to 37 °C for the times specified in each assay.
For protein kinase C (PKC) inhibition, cells were pretreated for 20 min prior to receptor stimulation with 50 nM of bisindolylmaleimide I (Bis) (Sigma). For mitogen-activated protein kinase (MAPK) inhibition, cells were pretreated for 20 min prior to receptor stimulation with 10 µM of SB2038580 (Sigma). For chelating intracellular calcium, cells were preincubated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM; Calbiochem) for 30 min at 37 °C prior to receptor stimulation.
Immunoprecipitations—iPLA2 and cPLA2 were immunoprecipitated from cell lysates stimulated by FcRI for the indicated times. Specific goat-polyclonal anti-iPLA2 or rabbit-polyclonal anti-cPLA2 (Santa Cruz Biotechnology, Inc) were incubated with protein A-agarose (50% slurry from Amersham Biosciences) at 4 °C, with rocking for 2 h to form precipitating complexes. Cell lysates were precleared with protein A-agarose (incubated for 30 min under rocking conditions); after the removal of the protein A-agarose, the precleared cell extracts were incubated with either anti-iPLA2 or anti-cPLA2 precipitating complexes and placed in a tumbler at 4 °C for 4 h, after which the precipitates were washed 3x in ice-cold phosphate-buffered saline to discard unbound material. The precipitated proteins were resolved by SDS-PAGE.
Gel Electrophoresis and Western Blotting—Proteins were resolved as described previously (6). Briefly, immunoprecipitates were resolved on 10% polyacrylamide gels (SDS-PAGE) under denaturing conditions and then transferred to 0.45 µm 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 (rabbit-polyclonal anti-phosphoserine, Chemicon International; goat-polyclonal anti-iPLA2, Santa Cruz Biotechnology; or rabbit-polyclonal anti-cPLA2, Santa Cruz Biotechnology) for 4 h at room temperature. The membranes were washed extensively in the washing buffer and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (anti-goat IgG-peroxidase conjugate, Sigma) or anti-rabbit IgG-peroxidase conjugate (Sigma) for 3 h at room temperature. The membranes were washed extensively in the washing buffer, and bands were visualized using ECL Western blotting detection system (Amersham Biosciences). On separate experiments, the eluted proteins from the immunoprecipitation were resolved as above, and the gels were subjected to silver staining.
Measurement of Arachidonic Acid Release—AA release was measured as described previously (25). Briefly, cells were labeled (2 x 106 cells/ml) with [3H]AA (1 µCi/ml, Amersham Biosciences) in the cell culture medium for 16 h. After washing, the cells were incubated at 37 °C for 30 min in RPMI 1640 medium, 1% fetal calf serum containing or not the specific inhibitors. FcRI was stimulated and reactions were stopped at the indicated times. After stimulation, the cells were spun down at 4 °C and supernatants were removed to measure the released [3H]AA, whereas the cell pellet was resuspended to measure total cellular [3H]AA incorporation. AA release was measured as the percentage of the total [3H]AA incorporated into the cell membranes.
Measurement of LTB4 Generation—LTB4 production was measured after receptor stimulation by the BiotrakTM leukotriene B4 enzyme immunoassay system from Amersham Biosciences. Briefly, the assay is based on the competition between unlabelled LTB4 and a fixed quantity of peroxidase-labeled LTB4 for a limited number of binding sites on an LTB4-specific antibody. The amount of LTB4 in the experimental sample will be inversely proportional to the signal generated by the fixed amount of peroxidase-labeled LTB4.
Measurement of PGE2 Synthesis—PGE2 production was measured after receptor stimulation by the BiotrakTM PGE2 system from Amersham Biosciences. Briefly, the assay is based on the competition between unlabelled PGE2 and a fixed quantity of peroxidase-labeled PGE2 for a limited number of binding sites on a PGE2-specific antibody. The amount of PGE2 in the experimental sample will be inversely proportional to the signal generated by the fixed amount of peroxidase-labeled PGE2.
Fluorescence Microscopy—After receptor aggregation, suspended cells were fixed in 4% paraformaldehyde and deposited on microscope slides using a cytospin centrifuge; they were then permeabilized for 5 min in 0.1% Triton X-100 in phosphate-buffered saline. The permeabilized cells were blocked for nonspecific binding with 5% fetal calf serum for 10 min at room temperature. Fluorescent labeling was performed by incubating the cells with goat-polyclonal anti-iPLA2 or rabbit-polyclonal anti-cPLA2 (Santa Cruz Biotechnology) and primary antibodies for 1 h at room temperature. The cells were washed with phosphate-buffered saline and secondary antibodies (anti-goat IgG-TRITC conjugate or anti-rabbit-FITC conjugate, Sigma) were added. To a set of cells, only the secondary antibodies were added for control. Stainings were analyzed with an inverted fluorescence Leica DM IRB microscope and recorded by a Leica DC 300F digital camera; pictures were analyzed with the Leica IM500 Image Manager software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and cPLA2 Are Both Endogenously Expressed in the Monocytes—U937 cells express cPLA2 and iPLA2 but not sPLA2 (26-28). To ascertain whether the expression patterns of either cytosolic form of PLA2 changed with the cytokine differentiation of U937 to human monocytes, we performed Western blotting analysis. Relative levels of protein expression were compared in untreated cells and in cytokine (IFN-) differentiated cells.
Total cell-extracts from untreated or IFN--U937 cells revealed that both iPLA2 and cPLA2 proteins were readily detectable. Western blot analysis revealed immunoreactive bands corresponding to the predicted molecular weights for iPLA2 and cPLA2. The intracellular PLA2 expression profiles did not alter after IFN- differentiation (Fig. 1a).
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RI Aggregation Triggers AA Release—FcRI aggregation triggers a quick and sustained increase of AA generation over time (Fig. 1b). This FcRI-triggered AA generation was almost completely inhibited in cells pretreated with 30 µM of methyl arachidonyl fluorophosphate (MAF), an inhibitor of cPLA2 and iPLA2 (16), suggesting the participation of cPLA2 and/or iPLA2 in the AA generation (Fig. 1b). To discern which of the two isoforms was activated by FcRI, we examined the effect of E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2-H-pyran-2-one (BEL) (29), a relatively selective inhibitor for iPLA2. The FcRI-triggered AA release was inhibited in cells pretreated with 10 µM of BEL (Fig. 1c).
Even though the quantity of BEL used has not been shown to inhibit cPLA2 activity (30), we investigated the role of BEL in the AA release triggered by PAF, a stimulant that we knew activates cPLA2 (31). Although 30 µM of MAF inhibited PAF-triggered AA generation from the IFN-primed cells (Fig. 2a), 10 µM of BEL did not have an effect at all (Fig. 2b). Taken together, these data suggest that PAF indeed activates cPLA2, whereas FcRI couples to iPLA2.
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RI Aggregation Specifically Stimulates iPLA2—To gain further proof of the nature of PLA2, which plays a major role in the signaling pathways triggered by FcRI, we designed specific antisense oligonucleotides against iPLA2 and cPLA2 to knockdown specifically the expression of each enzyme. We have shown previously that U937 cells are sensitive to antisense manipulation (7, 8). IFN--primed cells were treated with antisense oligonucleotide; AA generation was assayed either in unstimulated cells to measure basal levels of activity or after stimulation with FcRI activation either by immune complexes or with PAF (PAF was used as control). The specificity of the antisense oligonucleotides on relative PLA2 isozyme expression was checked by Western blot analysis (Fig. 3a). Thus, in cells treated with antisense to iPLA2, there was a substantial reduction in iPLA2 immunoreactivity (80% reduction measured by densitometry), whereas cPLA2 immunoreactivity was unaffected. Conversely, in cells treated with antisense to cPLA2, there was a reduction in cPLA2 immunoreactivity (85% reduction measured by densitometry), whereas iPLA2 immunoreactivity remained unchanged. Each antisense oligonucleotide, therefore, acted as an internal control for the other.
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, the increase in AA generation, observed after FcRI aggregation was significantly reduced, compared with the control cells (Fig. 3b). The reduction in the increase after FcRI activation was about 80% in cells treated with antisense iPLA2 compared with control cells and was proportional to the observed reduction in protein expression by Western blot analysis (Fig. 3a). In contrast, treatment of cells with the antisense oligonucleotide to cPLA2 had no effect on the FcRI-mediated generation of AA (Fig. 3b). Contrary to the AA generation triggered by FcRI, AA generation stimulated by PAF was significantly reduced by about 80% in cells pretreated with the antisense oligonucleotide to cPLA2 but not in cells treated with the antisense against iPLA2 (Fig. 3c).
Moreover, by immunoprecipitation and Western blotting, we found that in FcRI-stimulated cells, iPLA2 is phosphorylated on serine residues (Fig. 4a, upper left panel), whereas cPLA2 is not phosphorylated by FcRI engagement (Fig. 4b, top panels). Equal protein loading is shown by stripping the blots and reprobing for iPLA2 or cPLA2 (Fig. 4a, upper right panel; Fig. 4b, upper right and lower right panels). As a control for the iPLA2 immunoprecipitation, a Western blot of cell extracts depleted of iPLA2 is shown (Fig. 4a, lower left panel), as well as a silver-stained gel showing the elution of a single band after immunoprecipitation with the anti-iPLA2 antibody (Fig. 4a, lower right panel). To further establish the specificity of the system, we show that PAF stimulation causes the serine-phosphorylation of cPLA2 (Fig. 4b, lower panels), whereas PAF stimulation does not cause iPLA2 phosphorylation (data not shown). Furthermore, microscopy analysis of the subcellular localization of the different intracellular PLA2 revealed that, after FcRI aggregation, iPLA2 translocates to the plasma membrane (Fig. 4c), whereas the cytosolic localization of cPLA2 remained unchanged (Fig. 4d). For all fluorescence microscopy experiments, controls were carried out by adding the secondary antibodies to the cells without giving any signals; the antisense treatment did not influence the levels either of FcRI- or PAF-receptor expression (data not shown). These data strongly suggest that only iPLA2 is coupled to FcRI activation
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Couples FcRI to the Generation of LTB4 and PGE2—As coupling of FcRI to arachidonic acid release requires iPLA2 activation, the role of this enzyme in coupling FcRI to other signaling pathways, such as the production of eicosanoids, was investigated. Reduction in the expression of iPLA2 by pre-treatment of cells with the antisense oligonucleotide to iPLA2 resulted in a substantial inhibition of peak LTB4 and PGE2 observed after aggregation of FcRI (Fig. 5, a and b, respectively). However, the antisense to cPLA2 had no effect on the FcRI-triggered eicosanoids production (Fig. 5, a and b).
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RI activation in cells treated with the antisense oligonucleotide to iPLA2 was a feature of the loss of coupling of the receptor and not some direct effect of the iPLA2 antisense oligonucleotide on other members of the signaling pathways (such as cyclooxygenases), LTB4 and PGE2 were measured after activation of cells with PAF. Addition of PAF to control cells or cells treated with the antisense iPLA2 resulted in an identical increase in LTB4 and PGE2 production (Fig. 5c); on the other hand, in cells pretreated with the antisense to cPLA2, eicosanoid production was substantially inhibited (Fig. 5d). These data indicate that, in cells pretreated with antisense oligonucleotides, the reduction in LTB4 and PGE2 after FcRI activation reflects role of iPLA2 in the generation of eicosanoids.
Role of PKC in Triggering iPLA2 after FcRI Aggregation—It has been shown that iPLA2 activation requires PKC activity (18). Here we show that Bis, a selective PKC inhibitor, inhibits AA generation triggered by FcRI aggregation (Fig. 6a), whereas a MAPK inhibitor (SB203580) did not have any effect on the FcRI-triggered AA generation (Fig. 6b). In contrast, the AA generation triggered by PAF was not affected by the PKC inhibitor (Fig. 6a), but it was substantially reduced by the MAPK inhibitor (Fig. 6b). These findings indicate that PKC activity indeed may be involved in the FcRI-triggered stimulation of iPLA2, and confirm that the PAF-triggered activation of cPLA2 is MAPK-dependent.
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activation, we examined the effect of Bis on the FcRI-triggered iPLA2 translocation and phosphorylation patterns. Here we also show that pretreatment of cells with Bis markedly suppressed the FcRI-triggered iPLA2 translocation to the cell membranes (Fig. 6c). Furthermore, Bis also completely inhibited the phosphorylation of iPLA2 triggered by FcRI (Fig. 6d).
FcRI-triggered iPLA2 Activation Is Calcium-independent—It is well established that in immune cells, antigen receptor-induced AA release is, in most cases, calcium-dependent (18, 30, 31) and even, at least in one case (where iPLA2 was indeed activated), intracellular calcium depletion prevented the generation of AA (although in this case (18), the authors suggested that this result was due to the inhibition of a calcium-dependent PKC). Here we show that chelating intracellular calcium with BAPTA had no significant effect on iPLA2 translocation (Fig. 7a), iPLA2 phosphorylation (Fig. 7b), or on AA release triggered by FcRI (Fig. 7c), whereas the BAPTA treatment did indeed block the PAF-induced AA release in the same cells.
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RI triggers calcium-independent PKC activities (32, 33) and suggest that calcium-independent PKC(s) may be involved in triggering iPLA2 after FcRI aggregation in human monocytes. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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does not significantly alter the expression levels of either of the two enzymes. Our aim was to find out which PLA2 was involved in the FcRI intracellular signaling cascades leading to the generation of eicosanoids. In this study, we demonstrated that FcRI is functionally coupled to iPLA2, and that this enzyme is required for FcRI-mediated generation of arachidonic acid and the formation of leukotrienes and prostaglandins.
iPLA2 contains a calmodulin (CaM)-binding domain near the C terminus which binds calcium-activated CaM and regulates enzyme activity (34). The binding of CaM to iPLA2 results in the inhibition of iPLA2 activity, which is reversible through the removal of Ca+2, and subsequent dissociation of CaM from the C terminus of iPLA2 (34). Thus, in some models, it is possible for iPLA2 to be regulated through alterations in cellular calcium ion homeostasis and become activated after dissociation from its complex with Ca+2/CaM when intracellular calcium stores are depleted (e.g. by sarco/endoplasmic reticulum calcium ATPase inhibitors, calcium-ionophores, or agonist stimulation; ref. 35).
Here we report that the FcRI-triggered AA generation was almost completely inhibited in cells pretreated with MAF, an inhibitor of both cPLA2 and iPLA2 (36), suggesting the participation of cPLA2 and/or iPLA2 in the AA generation. To discern which of the two isoforms was activated by FcRI, we examined the effect of BEL, a relatively selective inhibitor for iPLA2 (29). The FcRI-triggered AA release was inhibited in cells pretreated with BEL. As a control for the specificity of BEL, we investigated the role of BEL in the AA release triggered by PAF, a stimulant known to activate cPLA2 (31). We found that, although MAF inhibited PAF-triggered AA generation, treatment of the cells with BEL did not have an effect on the AA release triggered by PAF, showing the selectivity of BEL and suggesting to us the possibility that iPLA2 was the enzyme involved in the FcRI-triggered AA release.
To be more specific, we designed antisense oligonucleotides against iPLA2 and cPLA2 to selectively down-modulate the protein levels of these enzymes. Our data show that the iPLA2 antisense substantially decreased AA release and LTB4 and PGE2 generation induced by FcRI aggregation, whereas the antisense against cPLA2 had no effect on the FcRI pathway. Moreover, the antisense to iPLA2 did not affect PAF-induced AA release or LTB4 and PGE2 generation, whereas the antisense against cPLA2 inhibited the PAF-triggered generation of AA, showing that both pathways utilize different phospholipases as well as the selectivity of each antisense oligonucleotide.
Furthermore, our data demonstrate that aggregation of FcRI triggers the serine phosphorylation and membrane translocation of iPLA2 but not of cPLA2. We found that the selective PKC inhibitor Bis substantially decreased the FcRI-triggered AA generation, whereas the MAPK inhibitor (BS203580) did not. In contrast, the PAF-triggered AA generation was inhibited by the MAPK inhibitor but not by the PKC inhibitor. These results suggest that FcRI triggers iPLA2 activation by means of PKC, whereas PAF-triggers cPLA2 via the activation of MAP-kinases. Moreover, the PKC inhibitor also blocked iPLA2 translocation to the cell periphery and completely blocked the phosphorylation of iPLA2 that follows FcRI aggregation. Taking these data together, we suggest that PKC is involved in triggering the activation of iPLA2 in the FcRI signaling cascade by phosphorylating and thus promoting the translocation of iPLA2 to the cell's plasma membrane.
Different stimuli induce AA release in monocytes and macrophages in a Ca+2-dependent and phosphorylation-dependent manner because of the activation of cPLA2 (37, 38). However, PGE2 generation by zymosan-stimulated macrophages is significantly attenuated by BEL or iPLA2 antisense (30). Paradoxically, in these cells, iPLA2 activation seems to be regulated by protein kinase C and is Ca+2-dependent, although in this case, the authors (18) suggested that this result was due to a calcium-dependent PKC, which, in turn, activated iPLA2. In contrast, other studies have shown ligand-stimulated eicosanoid production in cells that have been treated with calcium chelators such as BAPTA and EDTA (35). In agreement with the latter, we show here that chelating intracellular calcium with BAPTA had no significant effect on iPLA2 translocation, phosphorylation, or AA release triggered by FcRI, whereas the same BAPTA-AM treatment completely blocked the PAF-induced AA release.
Based upon the effects of BEL, it has been suggested for many years that iPLA2 mediates AA in different cells stimulated by various agonists (29, 39-42), including during IgG-mediated phagocytosis of human monocytes, where AA release was shown to be triggered in a calcium-independent manner (41, 42). For iPLA2, several important signaling functions have been suggested, including its role in agonist-induced stimulation of smooth muscle (20) and endothelial cells (21), in lymphocyte proliferation (22), and in endothelium-dependent vascular relaxation (21). Very recently, it was reported that myocardial ischemia activates iPLA2 in intact myocardium, and that iPLA2 activation is sufficient to induce malignant ventricular arrhythmias (23). Another recent study shows that functional iPLA2 is required for activation of store-operated channels and capacitative Ca2+ influx in several cell types (24). We show here that in a human monocytic cell line, iPLA2 plays a critical role in the intracellular signaling cascades initiated by the high affinity receptor for IgG (FcRI) and in its functional role to coordinate the response to antigen stimulation for the production of lipid-derived proinflammatory mediators such as leukotrienes and prostaglandins. These observations strongly suggest iPLA2 as a potential therapeutic candidate for treating human conditions ranging from ischemia to antigen-mediated inflammatory diseases.
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FOOTNOTES |
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Supported with a scholarship by the Singapore Millennium Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiology, Faculty of Medicine, National University of Singapore, Singapore 117597. Tel.: 65-6874-1697; Fax: 65-6778-8161; E-mail: phsmraj{at}nus.edu.sg.
1 The abbreviations used are: Fc, immunoglobulin constant region receptors; FcRI, high affinity receptor for IgG; PGE2, prostaglandin E2; LTB4, leukotriene B4; AA, arachidonic acid; PLA2, phospholipase A2; PAF, platelet-activating factor; IFN, interferon; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; TBS, Tris-buffered saline; BEL, E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2-H-pyran-2-one; MAF, methyl arachidonyl fluorophosphate; Bis, bisindolylmaleimide I; CaM, calmodulin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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