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Department of Health Chemistry, Showa University School of Pharmaceutical Sciences, Hatanodai, Shinagawa-ku, Tokyo, Japan;
Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195; and
Institut de Pharmacologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique-Unité Propre de Recherche 411, Sophia Antipolis, Valbonne, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(cPLA2)-transfected cells after stimulation with IgE and
Ag. Site-directed mutagenesis of sPLA2-IIA and -V revealed
that both the catalytic and heparin-binding domains are essential for
this function. Confocal laser and electron microscopic analyses
revealed that sPLA2-IIA, which was stored in secretory
granules in unstimulated cells, accumulated on the membranous sites
where fusion between the plasma membrane and granule membranes occurred
in activated cells. These results suggest that the heparin-binding
sPLA2s bind to the perigranular membranes through their
heparin-binding domain, and lysophospholipids produced in situ by their
enzymatic action may facilitate the ongoing membrane fusion. In
contrast to the redundant role of sPLA2-IIA, -IID, and -V
in the regulation of degranulation, only sPLA2-V had the
ability to markedly augment IgE/Ag-stimulated immediate
PGD2 production, which reached a level comparable to that
elicited by cPLA2. The latter observation reveals an
unexplored functional segregation among the three related isozymes
expressed in the same cell population. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI) on mast cells by IgE and
multivalent Ag (IgE/Ag) or cytokine receptors by their cognate
ligands elicits a biphasic mediator release response that is thought to
promote allergic and chronic inflammatory diseases (1, 2).
The immediate response, which occurs within a few minutes of cell
activation, is accompanied by exocytosis of preformed mediators (such
as histamine, serotonin, proteoglycans, and proteases) stored in
secretory granules and generation of lipid mediators (such as
PGD2, leukotriene C4, and
platelet-activating factor). The immediate response is followed by
induction of the immediate-early genes and various cytokines and
prolonged production of PGD2, which constitute
the delayed phase of mast cell activation.
Phospholipase A2
(PLA2),3
which liberates free fatty acids, including arachidonic acid (AA), from
membrane phospholipids, represents a critical rate-limiting step for
the biosynthesis of eicosanoids. To date, >10
PLA2 gene products have been identified in
mammals (3, 4). Of these, 85-kDa group IVA cytosolic
PLA2 (cPLA2) and
several 14-kDa secretory PLA2
(sPLA2) enzymes have been shown to be involved in
the regulation of eicosanoid biosynthesis (5, 6, 7, 8, 9, 10, 11, 12).
Analyses using bone marrow-derived mast cells (BMMC) obtained from
cPLA2-knockout mice have provided definitive
evidence that cPLA2 is essential for both the
immediate and the delayed phases of eicosanoid biosynthetic responses
(13). Of the sPLA2 isozymes
identified, the genes for groups IIA, V, IIC, IID, IIE, and IIF are
clustered at the same chromosome locus and are classified as the group
II subfamily because of their structural similarities (4, 5, 6, 9, 10, 14, 15, 16, 17). Secretory PLA2-IIA and V are
two major sPLA2s that have been implicated in
PGD2 generation in mast cells, although their
expression and functional profiles before and after cell activation
differ as a function of mast cell phenotype (18, 19, 20, 21).
Several lines of evidence suggest the participation of sPLA2 in the exocytosis of several endocrine cells, including mast cells (22, 23, 24, 25, 26). As the lysophospholipid, another product of the PLA2 reaction, perturbs the structure of bilayer membranes (27, 28), its production has been postulated to promote membrane fusion, thereby facilitating the exocytotic process further. Some snake venom PLA2s and rat sPLA2-IIA added exogenously at very high concentrations directly elicit degranulation of rat serosal mast cells (25, 26). Several chemicals that inhibit the in vitro enzymatic activity of sPLA2-IIA reduce histamine release from IgE/Ag-activated rat serosal mast cells (23, 24). Although these pharmacological studies have led to the hypothesis that sPLA2-IIA stored in secretory granules may promote the exocytosis of mast cells, nonspecific effects of these agents cannot be ruled out. Moreover, recent advances in the sPLA2 field have led to the identification of several new members of this family (4, 14, 15, 16, 17, 29), thus complicating our understanding of redundant and/or segregated functions among sPLA2 enzymes.
To better understand the role of sPLA2s in mast cell activation, we have conducted gain-of-function studies by transfecting rat mastocytoma cell line RBL-2H3 with various mammalian sPLA2s. We show that, among sPLA2s normally expressed in mast cells, the heparin-binding group II subfamily of sPLA2s exhibits a redundant function in enhancing degranulation. In contrast, only sPLA2-V has the ability to enhance immediate PGD2 biosynthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To obtain mouse BMMC, bone marrow cells from male BALB/cJ and C57BL/6J mice were cultured for up to 10 wk in 50% enriched medium (RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan) containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 10% FCS) and 50% WEHI-3 cell-conditioned medium as a source of IL-3. After 3 wk, >98% of the cells in the culture were BMMC (1, 2). Rat mastocytoma RBL-2H3 cells (Japanese Cancer Resources Bank) were cultured in RPMI 1640 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 10% FCS.
Materials and methods
The cDNAs for mouse sPLA2-IIA (mIIA) and
rat sPLA2-V (rV) were subcloned into
pCXN2 (30); rat
sPLA2-IIA (rIIA), mouse
sPLA2-IIA mutants IIA-G30S and IIA-KE4
(31), rat sPLA2-V mutants V-G30S and
V-RS2 (5), and rat sPLA2-IIC (rIIC)
into pCR3.1 (Invitrogen, San Diego, CA); mouse
sPLA2-IID (mIID) into pCDNA3.1 (Invitrogen);
human sPLA2-IIA (hIIA) and human
sPLA2-V (hV) into pCI-neo (Pharmacia, Piscataway,
NJ); and mouse cPLA2 into pBK/CMV (Stratagene,
La Jolla, CA). The cDNA probes for hamster cytosolic group VI
Ca2+-independent PLA2
(iPLA2) (5), human
sPLA2-X (8), mouse
sPLA2-IIE, and mouse
sPLA2-IIF (4) were described
previously. Rabbit anti-rat sPLA2-IIA Ab,
which recognizes rat and mouse sPLA2-IIAs but not
other isozymes, was described previously (5, 7, 9). Rabbit
anti-human cPLA2 Ab was purchased from
Cayman Chemicals (Ann Arbor, MI). Rabbit Abs against mouse
cyclo-oxygenase-1 (COX-1) and COX-2 were described previously
(9). Rabbit Ab against rat hemopoietic
PGD2 synthase was provided by Dr. Urade (Osaka
Bioscience Institute, Osaka, Japan). An RIA kit for
PGD2 was purchased from Amersham (Arlington
Heights, IL). The histamine ELISA kit was obtained from Immunotech
(Marseille, France). Opti-MEM medium and TRIzol reagent were obtained
from Life Technologies (Grand Island, NY). FITC-conjugated goat
anti-rabbit IgG Ab was purchased from Zymed (South San Francisco,
CA). IgE anti-trinitrophenyl (anti-TNP) and TNP-conjugated BSA
were provided by Dr. H. Katz (Harvard Medical School, Boston,
MA).
Establishment of transfectants
RBL-2H3 cells were seeded into 150-mm diameter dishes and cultured for 2–3 days to subconfluence. The cells (107 cells) were harvested, washed twice with Opti-MEM, and suspended in 400 µl of Opti-MEM. The cells were mixed with each cDNA (2–5 µg) and subjected to electroporation (BTX electroporator ECM600; Bio Medical Equipment, Tokyo, Japan) with a 200-V pulse amplitude and a 900-µF capacitance. After culture for 2 days, cells were resuspended in 10 ml of culture medium containing 800 µg/ml geneticin (Life Technologies) and seeded into 96-well plates (100 µl/well). After culture for 2 wk, single colonies were expanded into 12-well plates. After reaching confluence, the expression of each PLA2 was assessed by RNA blotting or immunoblotting. Because mediator release by cells transfected with the empty vectors was comparable to that by parental cells (data not shown), we used a clone transfected with the empty pCDNA3.1 vector as a control for subsequent cell activation studies.
RNA blotting
All procedures were performed as described previously (9, 31). Briefly, equal amounts (5 µg) of total RNA, purified using TRIzol reagent, were applied to each lane of 1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore, Bedford, MA). The resulting blots were then sequentially probed with appropriate cDNA probes that had been labeled with [32P]dCTP (NEN Life Science Products, Boston, MA) by random priming (Takara Biomedical, Ohtsu, Japan). All hybridizations were conducted at 42°C overnight in a solution comprising 50% (v/v) formamide, 0.75 M NaCl, 75 mM sodium citrate, 0.1% (w/v) SDS, 1 mM EDTA, 10 mM sodium phosphate (pH 6.8), 5x Denhardt’s solution (Nacalai Tesque, Kyoto, Japan), 10% (w/v) dextran sulfate (Sigma, St. Louis, MO), and 100 µg/ml salmon sperm DNA (Sigma).
RT-PCR/Southern blotting
Synthesis of cDNA was performed using 0.5 µg of total RNA from
BMMC and avian myeloblastosis virus reverse transcriptase, according to
the manufacturer’s instructions supplied with the RNA PCR kit (Takara
Biomedical). Subsequent amplifications of the cDNA fragments for
PLA2 isozymes were performed using 1 µl of the
reverse transcribed mixture as a template with specific oligonucleotide
primers (Greiner Japan, Tokyo, Japan) as follows:
sPLA2-IB: sense, 5'-CCT CAC TCC TTC TGA AGA
TG-3'; and antisense, 5'-CTG ACA GCA GGT ACT TTA TTA G-3';
sPLA2-IIA: sense, 5'-TCA GCA TTT GGG CTT CTT-3';
and antisense, 5'-CCA TCC AAG AGA GCT GAC AGC-3';
sPLA2-IIC: sense, 5'-ATG GAC CTC CTG GTC TCC TCA
GG-3'; and antisense, 5'-CTA GCA ATG AGT TTG TCC CTG C-3';
sPLA2-IID: sense, 5'-ATT TTT GAG ACT TGC CCT GCT
GTG TG-3'; and antisense, 5'-TTA GCA TGC TGG AGT CTT GCC-3';
sPLA2-IIE: sense, 5'-ATG AAA CCT CCC ATT GCC
CTG-3'; and antisense, 5'-TCA GCA GGG TGG GGT GGG CCC AG-3';
sPLA2-IIF: sense, 5'-ATG AAG AAA TTC TTT GCC
ATC-3'; and antisense, 5'-CTA GCT TGA GAC AGG GGT CGC-3';
sPLA2-V: sense, 5'-CAG GGG GCT TGC AAC TCA A-3';
and antisense, 5'-AAG AGG GTT GTA AGT CCA GAG G-3';
sPLA2-X: sense, 5'-CTG GCA GGG ACC TTG GAT TGT
G-3'; and antisense, 5'-GAG GTA TTT CAG GTG GTA CTC-3';
cPLA2: sense, 5'-ATG TCA TTT ATA GAT CCT TAC
C-3'; and antisense, 5'-TCA AAG TTC AAG AGA CAT TTC AG-3'; and
iPLA2: sense, 5'-TAC GTG AAG AAG CCT GC GG-3';
and antisense, 5'-GAA GCT GTT GTT TGC TGA TCT TGG A-3'. The PCR
condition was 94°C for 30 s and then 33 cycles of amplification
at 94°C for 5 s and 68°C for 4 min, using the Advantage cDNA
polymerase mix (Clontech, Palo Alto, CA). The PCR products were
analyzed by 1% agarose gel electrophoresis with ethidium bromide. The
gels were further subjected to Southern blot hybridization using
specific PLA2 probes as described previously
(20, 22).
SDS-PAGE/immunoblotting
Cell lysates (105 cell equivalents) or culture supernatants were subjected to SDS-PAGE using 15% (w/v) gels for sPLA2s under nonreducing conditions, and 7.5% gels for cPLA2 and 10% gels for COX-1, COX-2, and hemopoietic PGD2 synthase under reducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH) using a semidry blotter (MilliBlot-SDE system, Millipore). The membranes were probed with the respective Abs and visualized using the ECL Western blot system (NEN Life Science Products) as described previously (9, 31).
Activation of RBL-2H3 cells
The cells (5 x 104 cells/ml) were seeded into 24-well plates and cultured for 2 days in 1 ml of culture medium. Then the cells were sensitized with 100 ng/ml IgE anti-TNP for 30 min, washed twice with culture medium, and activated for 10 min at 37°C with 10 ng/ml TNP-conjugated BSA in culture medium. This condition provided optimal activation of the cells. After harvesting the supernatants, the remaining cells were collected and disrupted by two freeze-thawing cycles. ß-Hexosaminidase (ß-HEX) release was assessed spectrophotometrically using p-nitrophenyl-ß-D-2-acetamido-2-deoxyglucopyranoside (Sigma) as a substrate, as described previously (2). The release of histamine and PGD2 was assessed by ELISA and RIA, respectively. The percent release of ß-HEX and histamine was calculated by the formula [S/(S + P)] x 100, where S and P are the amounts of ß-HEX and histamine present in the supernatant and pellet, respectively.
Immunocytostaining (confocal laser microscopy)
Cells grown on collagen-coated coverglasses (Iwaki Glass, Tokyo, Japan) were fixed with 3% paraformaldehyde for 30 min in PBS. After three washes with PBS, the fixed cells were sequentially treated with 3% BSA (for blocking) and 1% saponin (for permeabilization) in PBS for 1 h, with an anti-sPLA2-IIA Ab (1/500 dilution) for 1 h, and then with FITC-goat anti-rabbit IgG (1/100 dilution) for 1 h. After six washes with PBS, the cells were mounted on glass slips using Perma Fluor (Japan Tanner, Suita, Japan), and the sPLA2-IIA signal was visualized using a laser scanning confocal microscope (IX70, Olympus, New Hyde Park, NY).
Immunocytostaining (electron microscopy)
The cells grown on chamber slide glasses (Iwaki Glass) were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde for 30 min in PBS. After three washes with PBS, the fixed cells were sequentially treated with 10% calf serum and 0.1% Triton X-100 in PBS for 30 min, with an anti-sPLA2-IIA Ab (1/500 dilution) in PBS containing 10% calf serum overnight with biotinylated goat anti-rabbit IgG (1/100 dilution) for 1.5 h and then with HRP-conjugated streptavidin (1/500 dilution), for 1 h. After three washes with PBS, the cells were incubated with PBS containing 0.2% diaminobenzidine and 0.01% H2O2 for viewing. After three washes with PBS, the cells were treated for 30 min with 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), washed three times, and incubated with 2% OsC4 in 0.1 M cacodylate buffer. After six washes with cacodylate buffer, the cells were dehydrated with graded concentrations of ethanol and embedded in Epon epoxy resin. After incubating for 2 days at 60°C, ultrathin sections were prepared and examined with an electron microscope.
Statistical analysis
Data were analyzed by Student’s t test. Results are expressed as the mean ± SD, with p = 0.05 as the limit of significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated that mast cells express
sPLA2-IIA, -IIC, and -V, but not -IB, although
sPLA2 expression profiles differ in mast cells
with different phenotypes and from different animal species
(18, 19, 20, 21, 32). To determine whether mast cells express
recently discovered sPLA2 enzymes, we conducted
RT-PCR analysis using mouse BMMC RNA as a template and a set of primers
specific for each mouse sPLA2 and for mouse
cPLA2 and iPLA2. As
shown in Fig. 1
, transcripts for
sPLA2-IIC, -IID, -IIE, -IIF, and –V;
cPLA2; and iPLA2, but
not for sPLA2-IB and -X, were detected in BMMC
prepared from BALB/cJ and C57BL/6J mice. Under the same conditions,
sPLA2-IB and -X were readily detected in rat
stomach and lung (data not shown), the tissues where these enzymes are
expressed (15, 16, 17). Secretory
PLA2-IIA was detected in BALB/cJ BMMC, but not in
C57BL/6J BMMC (Fig. 1), consistent with the fact that the latter mouse
strain has a natural disruption of the sPLA2-IIA
gene (33). Thus, mast cells express transcripts for all
known group II sPLA2 subfamily members. Direct
comparison of the relative expression levels of the different
PLA2s was difficult, because they were varied
among BMMC prepared from different mice.
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cDNAs for sPLA2-IIA (mIIA, rIIA, and hIIA),
sPLA2-V (rV and hV), and mouse
cPLA2 were each subcloned into mammalian
expression vectors and transfected into rat mastocytoma RBL-2H3 cells
to establish drug-resistant stable transfectants. Expression of each
PLA2 in the established transfectants was
assessed by RNA blotting (sPLA2s) or
immunoblotting (cPLA2; Fig. 2, inset). Endogenous
expression of cPLA2 was detectable, whereas
that of sPLA2 was below detection, in control
RBL-2H3 cells under the conditions employed here.
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A), rIIA (Fig. 2B), hIIA (Fig. 2C),
rV(Fig. 2D), and hV (Fig. 2E) than in replicate
mock-transfected cells. The release of histamine, another
granule-derived mediator, was also enhanced markedly in cells
transfected with mIIA and rV (Fig. 2G). No significant
augmentation of ß-HEX release was observed in
cPLA2-transfected cells (Fig. 2F).
To determine the functional domains responsible for the
degranulation-enhancing effect of sPLA2-IIA and
-V, we transfected the catalytically inactive mutants (mIIA-G30S and
rV-G30S, in which Gly (31) essential for
Ca2+ binding was replaced by Ser
(5)) and those with significantly reduced heparanoid
affinity (mIIA-KE4 and rV-RS2, in which Lys and Arg clusters in the
C-terminal domain were replaced by Glu and Ser, respectively
(5)) into RBL-2H3 cells. As shown in Fig. 3, neither the catalytically inactive
mutants mIIA-G30S and rV-G30S nor the heparin-nonbinding mutants
mIIA-KE4 and rV-RS2 enhanced IgE-dependent release of ß-HEX, even
though their expression levels were even higher than those in cells
transfected with the respective wild-type (WT) enzymes (Fig. 3, insets). These results imply that both the catalytic and
heparanoid-binding domains are essential for enhancing
degranulation.
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50 and 7% of the total
sPLA2 activity, respectively, with remaining
activities being associated with the cells. Secretory
PLA2 activity was detected only minimally in the
supernatants of unstimulated cells. These results suggest that mIIA-WT
is retained on the outer surface of cells through its heparin-binding
domain even after cell activation (see below) and that a large fraction
of secreted mIIA-KE4 is released into the culture medium.
To further ascertain the importance of the heparanoid binding capacity
in the degranulation-enhancing effect, we transfected RBL-2H3 cells
with mIID, another heparin-binding sPLA2
(15, 16), and rIIC, a heparin-nonbinding isozyme
(5). Cells transfected with mIID (Fig. 4B), but not with rIIC (Fig. 4A), exhibited enhanced ß-HEX release following
IgE/Ag-directed activation. Thus, the three heparin-binding
group II subfamily sPLA2s, namely IIA, V, and
IID, but not the heparin-nonbinding sPLA2-IIC,
play a redundant degranulation-enhancing role in mast cells.
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Confocal microscopic immunocytostaining analyses of permeabilized
RBL-2H3 cells expressing mIIA-WT, using Ab specific for rodent
sPLA2-IIA, showed that the overexpressed enzyme
accumulated in punctate domains, most likely vesicular compartments,
that were distributed throughout the cytoplasm of unstimulated cells
(Fig. 5). These signals for mIIA-WT moved
in close proximity to the plasma membrane after IgE/Ag
activation. Although cells expressing mIIA-KE4 gave a cytoplasmic
punctate signal similar to that observed with mIIA-WT in the resting
state, staining beneath the plasma membrane, which was obvious in
activated mIIA-WT-expressing cells, was barely detected after IgE/Ag
stimulation (Fig. 5). Moreover, the overall intensity of the signals
for IIA-KE4 weakened after cell activation (Fig. 5), consistent with
its secretion into the extracellular fluids.
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A). After
FcRI cross-linking, mIIA-containing granules
were fused with the plasma membrane, where positive mIIA signals were
detected on the perigranular membranes, the area of fusion between the
plasma and granule membranes in particular, and in the matrix
components probably containing the granule proteoglycan serglycin (Fig. 6, B and C). The surface of the plasma membrane,
apart from the opening granules, was not stained by the Ab.
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PGD2 generation by the
sPLA2 and cPLA2
transfectants 10 min after stimulation with IgE/Ag was examined next.
Surprisingly, PGD2 generated by the transfectants
expressing mIIA (Fig. 7A),
rIIA (Fig. 7B), and hIIA (Fig. 7C) was almost
equal to that by mock-transfected cells, whereas
PGD2 generation by cells expressing rV (Fig. 7D) and hV (Fig. 7E) was markedly augmented,
reaching a level comparable to that produced by
cPLA2-transfected cells (Fig. 7F).
The rIIC failed to affect PGD2 generation (Fig. 7G), and mIID increased PGD2
generation modestly (Fig. 7H). Thus, among the group II
subfamily of sPLA2s examined, only
sPLA2-V elicited an efficient
PGD2 biosynthetic response. The expression of
endogenous COX-1 and hemopoietic PGD2 synthase
did not differ significantly, and that of endogenous COX-2 was
undetectable among the transfectants used as assessed by immunoblotting
(data not shown), indicating that the PGD2
biosynthetic effect of sPLA2-V was not due to
alteration in the expression of downstream enzymes in the COX pathway
as a result of forcible sPLA2-V expression. As
shown in Fig. 8, both rV-G30S and rV-RS2
failed to augment this PGD2 generation,
indicating that both the catalytic and heparanoid-binding domains are
crucial for PGD2 production, as in the case of
the degranulation-enhancing effect (Fig. 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Although the expression of their functional proteins
will need to be addressed in a future study using isozyme-specific Abs,
our results nonetheless imply that mast cells have the capacity to
express all known group II sPLA2 subfamily
members, the genes for which are clustered on mouse chromosome 4
(4, 14). This is reminiscent of the expression of
chromosome 14-associated serine proteases (known as mouse mast cell
protease MMCPs) in mouse BMMC (34, 35). Thus, it would be
of particular importance to know the functional roles of each
sPLA2 in mast cells.
In an attempt to clarify the roles of the group II subfamily of
sPLA2s in mast cell activation, we have conducted
transfection analyses using the rat mastocytoma cell line RBL-2H3 and
demonstrated that those group II sPLA2 subfamily
members with heparin-binding capacity play a redundant role in
enhancing the stimulus-induced exocytotic response. Cells transfected
with the heparin-binding sPLA2-IIA, -IID, and -V
enzymes released more secretory granule-associated mediators, ß-HEX
and histamine, than control cells after FcRI
cross-linking (
Figs. 2–4). On the other hand, cells transfected with
the heparin-nonbinding enzymes sPLA2-IIC,
sPLA2-IIA-KE4, and
sPLA2-V-RS2 did not show enhanced degranulation.
Catalytically inactive sPLA2-IIA and -V mutants
were without effect, implying that membrane phospholipid hydrolysis is
an absolute requirement for augmenting degranulation. Even though
overexpression studies do not always reflect the true functions of
endogenous enzymes, our results are in line with previous
pharmacological observations that sPLA2-IIA
inhibitors or a neutralizing Ab against sPLA2-IIA
blocked histamine release from rat serosal mast cells (23, 24). Although Fonteh et al. (36) have proposed that
the action of sPLA2 on mast cells depends on the
M-type sPLA2 receptor (37), our
results argue against this hypothesis, because the rat
sPLA2 M-type receptor does not bind to rat and
human sPLA2-IIAs (38, 39).
Confocal laser and electron microscopic analyses revealed that
sPLA2-IIA, which resides in secretory granules in
unstimulated cells, as has been observed by previous studies (32, 40), accumulates on the membranes where the granular membranes
are fused with the plasma membrane (
Figs. 4–6). This localization in a
limited compartment was not observed with the heparin-nonbinding
sPLA2-IIA mutant KE4, which was largely released
into the extracellular medium. Considering that lysophospholipids are
fusogenic (27), an attractive model is that the
heparin-binding sPLA2s are associated with the
particular membranous sites that are undergoing fusion through binding
to certain heparan sulfate proteoglycans or other anionic components,
and that this localization leads to spatially segregated
lysophospholipid production, which enhances membrane fusion. This
speculation is supported by the observation that the overexpression of
cPLA2, which acts on the perinuclear membrane
in IgE/Ag-stimulated RBL-2H3 cells (41), did not affect
degranulation (Fig. 2G). Chernomordik et al.
(42) have reviewed the mechanisms by which
lysophosphatidylcholine promotes fusion depending on which side of the
membrane this lipid is produced. It is possible that
lysophosphatidylcholine may be released by the heparin-binding
sPLA2s to promote degranulation on one side of
the perigranular membranes. More recently, Schmidt et al.
(26) have shown that the dynamic state between
phosphatidic acid and lysophosphatidic acid alters membrane curvature
and thereby influences granule invagination from the plasma membrane.
This indicates that lysophospholipids are generally involved in both
exocytosis and endocytosis processes. Moreover, hydrolysis of
phosphatidylserine leads to the production of lysophosphatidylserine, a
potent enhancer of mast cell degranulation (23, 24, 43).
As heparin-binding group II sPLA2s bind to
anionic phosphatidic acid and phosphatidylserine vesicles in marked
preference to zwitterionic phosphatidylcholine vesicles
(44, 45, 46), these sPLA2s may encounter
these anionic phospholipids around the perigranular membranes of
activated cells.
In contrast to the seemingly compensatory functions of the three
heparin-binding sPLA2s in degranulation, they
displayed distinct roles in the regulation of immediate
PGD2 biosynthesis. Among them,
sPLA2-V exerted a potent enhancing effect on
stimulus-dependent immediate production of PGD2
(Fig. 7). This result is reminiscent of the previous observation that
introduction of sPLA2-V antisense into MMC-34
mast cells reduced immediate PGD2 generation
(18). Secretory PLA2-IID also showed
a modest effect, whereas sPLA2-IIA was largely
ineffective (Fig. 7). These observations are in marked contrast to
PGE2 generation observed in several adherent
cells, where both sPLA2-IIA and -V expression
leads to AA release and PG formation (5, 6, 7, 8, 9, 10).
Site-directed mutagenesis strongly argues that the
PGD2-enhancing function of
sPLA2-V also depends on both catalytic and
heparanoid-binding activities of this enzyme (Fig. 7).
In this context it is tempting to speculate that sPLA2-V and, to a lesser extent, sPLA2-IID cause membrane hydrolysis more efficiently than sPLA2-IIA at the perigranular and plasma membrane fusion sites, where a large amount of AA, enough to reach the perinuclear COX-1, which is the dominant COX isozyme mediating the immediate phase of PGD2 generation in mast cells (1, 2), may be liberated. The amounts of AA released by sPLA2-IIA may be below the threshold necessary to promote PGD2 biosynthesis, even though lysophospholipids produced locally at the same time may be enough to accelerate the degranulation process. Another possibility is that sPLA2-IIA would preferentially hydrolyze phospholipids that do not contain AA, whereas sPLA2-V can act on those with AA, in the perigranular membranes. Alternatively, the ability of sPLA2-V to elicit PGD2 generation in RBL-2H3 cells may reflect the action of sPLA2-V on phosphatidylcholine in the outer plasma membrane, because sPLA2-V binds much more tightly to phosphatidylcholine-rich vesicles than does sPLA2-IIA in vitro (47). In this model, sPLA2-V may trigger degranulation at the fusion sites with the plasma membrane by locally producing lysophospholipids and is then secreted outside the cells and binds to the plasma membrane to produce AA from phosphatidylcholine for PGD2 generation. In support of these speculations, sPLA2-V is able to release AA far more efficiently than sPLA2-IIA when added exogenously to human neutrophils (48) and to the mouse macrophage-like cell line P388D1 (49). In fibroblastic cell lines HEK293 and 3Y1, expression of sPLA2-IIA and -V leads to enhanced AA release and PGE2 generation, probably not via action on the plasma membrane but on the caveolae-directed compartmentalized membranes (7). Nevertheless, additional studies are required to determine the mechanisms for different PGD2 biosynthetic functions among sPLA2s in mast cells.
Our present study has provided strong evidence that the heparin-binding group II sPLA2 subfamily members, including IIA, IID, and V, function as enhancers of mast cell degranulation. This particular function of these sPLA2s may not be limited to mast cells, as the involvement of an sPLA2-IIA-like enzyme(s) in the exocytotic process has been reported in other cell types, such as neuroendocrine cells (22). In contrast to the redundant degranulation-enhancing role of these three sPLA2s, sPLA2-V (and, to a lesser extent, sPLA2-IID) has the ability to elicit the PGD2 biosynthetic response in mast cells. Although molecular mechanisms for the functional segregation of the related sPLA2s in the PG biosynthetic response remain to be elucidated, these results together with our recent studies (5, 6, 7, 8, 9, 20, 31) imply that the actions of each sPLA2 differ according to cell type and state of cell activation.
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2 Address correspondence and reprint requests to Dr. Ichiro Kudo, Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
3 Abbreviations used in this paper: PLA, phospholipase A2; sPLA2, secretory PLA2; cPLA2, cytosolic PLA2; iPLA2, Ca2+-independent PLA2; AA, arachidonic acid; COX, cyclo-oxygenase; BMMC, bone marrow-derived mast cells; ß-HEX, ß-hexosaminidase; TNP, trinitrophenyl; WT, wild type.
Received for publication April 24, 2000. Accepted for publication June 21, 2000.
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) or without
(
) Ag. After collecting the culture supernatants, the release of
ß-HEX (A–F) and histamine (G) was
assessed. Values shown in A–F are the mean ± SD
of three to six experiments (p < 0.05 between
unstimulated and stimulated cells in A–G, and
p < 0.05 between sPLA2-transfected and
control cells after activation in A–E and
G). A representative result of two reproducible
experiments is shown in G. Expression of
sPLA2s (A–E, inset) and
cPLA2 (F, inset) was assessed
by RNA blotting and immunoblotting, respectively.
