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Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI (high affinity receptor for IgE) cross-linking.
Treatment of rat serosal connective tissue mast cells (CTMC) with nerve
growth factor (NGF) induced only the delayed phase of PGD2
generation that depended on inducible cyclooxygenase-2 (COX-2), but not
constitutive COX-1, even though the subcellular distributions of these
isoforms were similar. Experiments using several phospholipase
A2 (PLA2) isozyme-specific probes and
inhibitors suggested that both constitutive cytosolic PLA2
and inducible type IIA secretory PLA2 (sPLA2)
are involved in NGF-initiated, COX-2-dependent, delayed
PGD2 generation in rat CTMC. A type IIA sPLA2
inhibitor, but neither cytosolic PLA2 nor COX inhibitors,
reduced, while adding exogenous type IIA sPLA2 augmented,
NGF-induced COX-2 expression and its attendant PGD2
generation, indicating that the sPLA2-mediated increase in
delayed PGD2 generation was attributable mainly to enhanced
COX-2 expression. Type IIA sPLA2 and its close relative
type V sPLA2 associated with fibroblastic cell surfaces
increased NGF-induced COX-2 expression more efficiently than the
soluble enzymes, revealing a particular juxtacrine sPLA2
presentation route. Surprisingly, catalytically inactive type IIA
sPLA2 mutants, which were incapable of promoting
arachidonic acid release from cytokine-primed cells, retained the
ability to enhance COX-2 expression in CTMC, indicating that the
COX-2-inducing activities of sPLA2 are independent of their
catalytic functions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI)3 by IgE and
multivalent Ag (IgE/Ag) or cytokine receptors by their cognate ligands
elicits a biphasic response that is thought to be involved in 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 stored in the secretory granules
(such as histamine, serotonin, proteoglycans, and proteases) 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 that constitute
the delayed phase of mast cell activation.
Prostanoid biosynthesis in mammalian cells is regulated by three
sequential steps: liberation of arachidonic acid (AA) from membrane
phospholipids by cytosolic phospholipase A2
(cPLA2) or secretory PLA2 (sPLA2)
isozymes, conversion of AA to PGH2 by the two
cyclooxygenase (COX) isoforms, COX-1 and COX-2, and terminal conversion
of PGH2 to bioactive prostanoids by various terminal PG
synthases. The molecular basis of biphasic PGD2
biosynthesis by mast cells has been studied extensively using mouse
bone marrow-derived mast cells (BMMC) as a model system. IgE-dependent
or c-kit ligand-initiated activation of BMMC induces
immediate PGD2 generation, which is absolutely dependent
upon COX-1 (3, 4). BMMC activated with c-kit ligand or
IgE/Ag combined with the accessory cytokines IL-10 and IL-1ß elicit
cytokine-initiated delayed PGD2 generation over several
hours, a process entirely dependent upon the de novo induction of COX-2
(4, 5, 6). However, which PLA2 isozymes contribute to the
immediate and delayed PGD2 biosynthetic phases is rather
controversial. The importance of cPLA2 in the immediate
response was demonstrated by its Ca2+-dependent
translocation to the perinuclear envelope and mitogen-activated protein
kinase-mediated phosphorylation immediately after FcRI or
c-kit signaling (7, 8, 9). The observation that the acute
allergic response was considerably reduced in
cPLA2-deficient mice (10) lends support to this. With
respect to the delayed phase, Bingham et al. (11) demonstrated that a
heparin-sensitive PLA2 isozyme immunochemically related to
type IIA sPLA2 was functionally linked to COX-2, a finding
consistent with our observation that an Ab raised against mouse type
IIA sPLA2 attenuated IgE-dependent delayed PGD2
generation (12). However, Reddy et al. (13, 14) reported that the
immediate and delayed phases of PGD2 generation in murine
mast cell line MMC-34 were regulated by sPLA2 and
cPLA2, respectively, and the sPLA2 isozyme
involved was type V, not type IIA. Our recent studies, however,
revealed that sPLA2 isozyme expression by various mast cell
phenotypes derived from different animal species differs and also
depends on the culture systems in which the mast cells are maintained
(15, 16).
As the features of in vitro-derived cultured mast cells are relatively immature, we asked whether in vivo-derived mature mast cell phenotypes would elicit similar biphasic PGD2 biosynthetic responses to particular stimuli. We found that the stromal cell-derived, mast cell-poietic cytokine nerve growth factor (NGF) is a potent inducer of COX-2-dependent delayed PGD2 generation that is not accompanied by the immediate response in rat serosal connective tissue mast cells (CTMC) (17). Therefore, this system appears useful for analyzing the mechanisms that regulate the delayed response with no influence of the mediators released during the immediate response. We found that both cPLA2 and type IIA sPLA2 were functionally linked to COX-2, the expression of which was enhanced by the latter. This property of sPLA2 was markedly accelerated by contact of CTMC with fibroblasts, revealing a novel juxtacrine sPLA2-presenting route from fibroblasts to CTMC. Moreover, site-directed mutagenesis analyses yielded the important finding that sPLA2 appears to induce COX-2 by acting as a kind of ligand, rather than as an enzyme, on CTMC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse NGF 2.5S was purchased from Chemicon International (Temecula, CA). Human IL-1ß was purchased from Genzyme (Boston, MA). Rat type V sPLA2 cDNA was provided by Dr. J. A. Tischfield (Indiana University, Indianapolis, IN), rabbit anti-human cPLA2 antiserum and the sPLA2 inhibitor LY311727 (18) were provided by R. M. Kramer (Lilly Research Laboratory, Indianapolis, IN), rabbit anti-mouse COX-1 antiserum and the COX-1 inhibitor valeryl salicylate (19) were provided by W. L. Smith (Michigan State University, East Lansing, MI), and the COX-2 inhibitor NS-398 (20) was supplied by J. Trzaskos (Merck-DuPont, Wilmington, DE). Rabbit anti-mouse COX-2 antiserum and the cPLA2 inhibitor methyl arachidonylfluorophosphate (MAFP) (21) were purchased from Cayman Chemical (Ann Arbor, MI). Rat type IIA sPLA2 was purified, and the rabbit polyclonal Ab against rat type IIA sPLA2 was prepared as described previously (16). The cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3) (22) was purchased from Calbiochem (La Jolla, CA). A PGD2 RIA kit and enhanced chemiluminescence Western blotting kit were purchased from Amersham (Arlington Heights, IL). Recombinant sPLA2 isozymes were expressed by HEK293 cells transfected with the appropriate cDNA, as described previously (23). Porcine pancreatic type IB sPLA2 was purchased from Boehringer Mannheim (Mannheim, Germany).
Preparation and activation of rat peritoneal CTMC
CTMC were obtained from the peritoneal cavities of Wistar rats (Nippon Bio-Supply Center, Tokyo, Japan) as described previously (17). Briefly, rats (male, weighing >350 g) were injected i.p. with 50 ml of HBSS containing 0.1% (w/v) BSA, and the peritoneal cells were harvested. After centrifuging these cells with HBSS containing 38% BSA, CTMC were collected from the bottom of the tube. The purity and viability of the cells were assessed by staining with toluidine blue and trypan blue, respectively, and CTMC with >95% purity and viability were used for the subsequent studies.
In typical experiments, the cells were activated for various periods at 37°C by mouse NGF 2.5S in enriched medium (RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 0.1 mM nonessential amino acids). After incubation for the appropriate times, PGD2 generation was assessed by RIA, as described previously (17).
SDS-PAGE/immunoblot analysis
CTMC were washed once with 10 mM phosphate buffer, pH 7.4, containing 150 mM NaCl (PBS) and lysed in PBS containing 0.1% SDS at 1 x 107 cells/ml. Each lysate was applied to SDS-polyacrylamide gels and electrophoresed under reducing (10% gels for cPLA2, COX-1, and COX-2) or nonreducing (15% gels for sPLA2) conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) using a semidry blotter (MilliBlot-SDE system, Millipore, Bedford, MA) according to the manufacturer’s instructions. Then, membranes were washed once with Tris-buffered saline (TBS), pH 7.2, containing 0.1% Tween-20 (TBS-T) and blocked for 1 h with TBS-T containing 3% skimmed milk. After washing with TBS-T, Abs against cPLA2, COX-1, COX-2, or type IIA sPLA2 were added to produce final dilutions of 1/5,000, 1/7,000, 1/3,000, and 1/3,000, respectively, in TBS-T. The membranes were incubated for 2 h, washed three times with TBS-T, and treated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed, South San Francisco, CA; 1/10,000 dilution) in TBS-T. Finally, after six washes with TBS-T, the protein bands were visualized using an enhanced chemiluminescence Western blot analysis system.
Immunohistochemical staining
Cytospun CTMC were stained with toluidine blue or with Alcian blue and safranin, as described previously (24), and replicate cells were fixed with 2% paraformaldehyde in PBS for 30 min at 4°C. The cells were permeabilized by treating them for 20 min with PBS containing 1% saponin and 1% BSA, washed three washes with PBS, treated with the anti-COX-1 or anti-COX-2 Ab (1/1000 dilution in PBS containing 1% BSA) for 2 h at room temperature, washed five times with PBS, incubated with FITC-conjugated anti-rabbit IgG (Zymed; 1/200 dilution in PBS) for 1 h at room temperature, and after five washes with PBS were examined by fluorescence microscopy.
RT-PCR
Specific primers for the PCR, based on the sPLA2 sequences reported previously (25, 26, 27), were synthesized. The type IIA sPLA2 primers used were 5'-ATG AAG GTC CTC CTC CTG CTA G-3' and 5'-TCA GCA TTT GGG CTT CTT CC-3' (25), the type IIC sPLA2 primers were 5'-ATG GAC CTC CTG GTC TCC TCA GG-3' and 5'-CTA GCA ATG AGT TTG TCC CTG C-3' (26), and the type V sPLA2 primers were 5'-CAG GGG GCT TGC TAG AAC TCA A-3' and 5'-AAG AGG GTT GTA AGT CCA GAG G-3' (27). The RT-PCR was conducted using an RNA PCR kit (AMV, version 2, Takara Biomedicals, Ohtsu, Japan), according to the manufacturer’s instructions, with 0.2 µg of total RNA as a template. Equal amounts of each RT product were amplified by PCR with Ex Taq polymerase (Takara Biomedicals) by 35 cycles consisting of 30 s each at 94, 55, and 72°C. The amplified cDNA fragments were resolved electrophoretically on 1.5% (w/v) agarose gels, visualized by ethidium bromide staining, transferred onto Immobilon-N membranes, and probed with [32P]dCTP-labeled cDNAs for types IIA, IIC, and V sPLA2. Hybridization and subsequent washing were conducted as described previously (16).
Coculture of CTMC with HEK293 cells
HEK293 cells overexpressing type IIA sPLA2, its mutants lacking catalytic function (G30S, H48E), type V sPLA2, or type IIC sPLA2 were established as described previously (23). Control and sPLA2-expressing HEK293 cells (105 cells/well) in enriched medium were seeded into 24-well plates and cultured for 3 days, when they reached confluence. Then, the conditioned media were harvested, and 106 CTMC were added to these media and cultured for 24 h in the presence or the absence of 200 ng/ml NGF. The same numbers of CTMC were seeded onto the remaining HEK293 cells and cocultured with or without NGF in the same manner. Aliquots of HEK 293 cell monolayers and their culture supernatants were taken for sPLA2 assay or immunoblotting with the anti-type IIA sPLA2 Ab to ensure the expression of sPLA2 enzymes.
AA release from sPLA2-expressing HEK293 cells
Transfectants stably expressing native or mutant sPLA2 (5 x 104 cells in 1 ml of culture medium) were seeded into 24-well plates. To assess AA release, 0.1 µCi/ml [3H]AA (New England Nuclear, Boston, MA) was added to the cells on day 3, when they had nearly reached confluence, and culture was continued for another day. After three washes with fresh medium, 250 µl of enriched medium containing 1 ng/ml IL-1ß was added to each well, and the amount of free [3H]AA released into each supernatant after culture for 4 h was measured (23). The amount of AA release, as a percentage, was calculated using the formula [S/(S + P)] x 100, where S and P are the radioactivities of equal portions of the supernatant and cell pellet, respectively.
Measurement of sPLA2 activity
PLA2 activity was assayed by measuring the amounts of free [14C]AA released from 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoethanolamine (Amersham), as described previously (16). Each reaction mixture consisted of an aliquot of the required sample, 100 mM Tris-HCl (pH 7.4), 4 mM CaCl2, and 2 µM substrate. After incubation for 20 min at 37°C, the [14C]AA released was extracted by Dole’s method (28), and the radioactivity was counted.
Other procedures
The protein contents of samples were quantified using a BCA protein assay kit (Pierce, Rockford, IL) with BSA as the standard. The data were expressed as the mean ± SE and were analyzed using Student’s t test. Differences at p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PGD2 generation was induced by culturing rat serosal
CTMC with NGF for 5 to 24 h (Fig. 1
A) and occurred in parallel
with de novo induction of COX-2 expression, without changes in the
constitutive expression of COX-1 or cPLA2 (data not shown)
(17). No immediate PGD2 generation occurred during the
NGF-initiated response (Fig. 1A). NS-398, a COX-2 inhibitor,
suppressed this delayed PGD2 generation completely,
confirming that it was dependent upon COX-2 (17). By
immunohistochemical staining of NGF-treated CTMC with Abs against COX-1
and COX-2, both COX isozymes appeared to be located in the perinuclear
membranes and in some punctured structures in the cytoplasm (Fig. 1B).
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). Expression of the type IIA
sPLA2 transcript was barely detectable in unstimulated
cells, but it was markedly induced in NGF-stimulated cells. On the
other hand, the transcripts for types IIC and V sPLA2 were
not detected in CTMC even after NGF stimulation. Both transcripts were
readily detectable in the rat mastocytoma RBL-2H3 cells and BALB/cJ
mouse-derived BMMC used as positive controls (15).
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A). Similar inhibition was
observed with MAFP, another cPLA2-selective inhibitor (data
not shown). Delayed PGD2 generation was also suppressed by
the sPLA2 inhibitor LY311727 with an IC50 of
approximately 1 µM (Fig. 3B), a concentration comparable
to that at which LY311727 inhibited the activity of recombinant type
IIA sPLA2 (Fig. 3C). Under the same assay
conditions, type V sPLA2 was less sensitive
(IC50 >10 µM), whereas type IIC sPLA2 was
virtually insensitive, to LY311727. These results suggest that
constitutive cPLA2 and inducible type IIA sPLA2
function cooperatively in the regulation of COX-2-dependent delayed
PGD2 generation in NGF-stimulated rat CTMC.
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We found that LY311727 partially, but significantly, reduced
NGF-induced expression of COX-2, whereas COX-1 expression was
unaffected (Fig. 4A). In
contrast, inhibitors of other PG-biosynthetic enzymes, including
AACOCF3 and MAFP (cPLA2 inhibitors), valeryl
salicylate (COX-1 inhibitor), and NS-398 (COX-2 inhibitor), had no
appreciable effect on COX-2 expression (Fig. 4B). These
results suggest that endogenous type IIA sPLA2 plays a role
in augmenting COX-2 expression, for which endogenous eicosanoids are
not essential. Although exogenous lysophosphatidylserine augmented the
induction of COX-2 expression by NGF (17), endogenous
lysophosphatidylserine was unlikely to have participated in NGF-induced
COX-2 expression, as its level in CTMC was minimal and did not change
appreciably before or after NGF stimulation (data not shown).
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A) and dose-dependent (Fig. 5B) manner. Enhancement of COX-2 expression by type IIA
sPLA2 was evident during culture for 5 to 24 h (Fig. 5A) with sPLA2 at concentrations ranging from 1
ng/ml to 10 µg/ml (Fig. 5B), which were comparable to
those detected at inflamed sites (29). In the absence of NGF,
sPLA2 had no effect on COX-2 expression (Fig. 5B). Type IB sPLA2 was less active than type IIA
sPLA2 (Fig. 5B). NGF-induced AA release was
increased only 1.3-fold, whereas PGD2 generation was
increased >4-fold, by exogenous type IIA sPLA2 (17),
indicating that sPLA2-augmented PGD2 generation
was attributable mainly to increased COX-2 expression, rather than
increased AA release.
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In tissue microenvironments, CTMC interact spatially and
functionally with fibroblasts (30, 31). Since type IIA
sPLA2 expression is often induced in fibroblasts in
response to proinflammatory stimuli, we considered it worthwhile to
examine whether interaction with fibroblasts altered the sensitivity of
CTMC to sPLA2. To address this issue, we first established
an HEK293 cell transfectant that stably overexpressed type IIA
sPLA2. This cell line, designated 293(IIA), released
approximately 50% of the sPLA2 it produced into the
medium, and the rest was present on the cell surfaces in a heparan
sulfate proteoglycan-associated form (23). The sPLA2
concentrations in the supernatants of 293(IIA) after 3 days of culture
consistently reached 100 to 300 ng/ml, which appeared to be sufficient
to increase COX-2 expression in CTMC (Fig. 5
B). Then, CTMC
added to the conditioned medium of parental 293 or 293(IIA) cells and
those seeded onto 293 or 293(IIA) monolayers were cultured for 24
h with or without NGF (the procedure is illustrated in Fig. 6A). We found that although
the amounts of type IIA sPLA2 in both systems (i.e., that
released into the conditioned medium and that associated with the cell
surfaces) were comparable (Fig. 6B), the COX-2 expression
level in CTMC cocultured with 293(IIA) was higher than that in
replicate cells cultured with soluble sPLA2 (Fig. 6C). COX-2 expression was not augmented when CTMC were
cocultured with parental 293 cells (Fig. 6C). These results
indicate that type IIA sPLA2 associated with fibroblastic
cell surfaces may be presented to CTMC more efficiently than the
soluble enzyme and/or that fibroblasts may produce some factor(s) that
acts in synergy with NGF and sPLA2 to induce COX-2
expression through a direct cell-cell interaction.
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We constructed a type IIA sPLA2 mutant, G30S, in which
Gly30, which is essential for Ca2+ binding, was
replaced by Ser, resulting in a lack of catalytic activity (23).
IL-1ß-stimulated AA release by 293(IIA) cells was augmented
significantly, whereas that by the transformant cells expressing the
catalytically inactive mutant enzyme (293(IIAG30S)) was comparable to
that by parental 293 cells (Fig. 7A), confirming that enzyme
function is an absolute requirement for cytokine-induced AA release
(23). When CTMC were cocultured with these transformants, in which the
expression levels of native and mutant sPLA2 were
equivalent, we unexpectedly found that the COX-2 expression levels in
CTMC cocultured with 293(IIAG30S) and those in replicate cells
cocultured with 293(IIA) were comparable (Fig. 7B). Similar
results were obtained with another catalytically inactive
sPLA2 mutant, H48E, in which the catalytic center His was
replaced by Glu (data not shown). Thus, these mutational analyses
clearly demonstrated that the COX-2-inducing activity of
sPLA2, unlike its capacity to release AA from
agonist-primed cells, does not depend on its catalytic function.
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The mast cells of some mouse strains do not express type IIA
sPLA2 due to an intrinsic gene mutation, but instead
express type V sPLA2 as a major sPLA2 isoform
(14). Recently, we conducted overexpression analysis of HEK293 cells
and showed that type V sPLA2 behaved in a manner similar to
type IIA sPLA2 in terms of proteoglycan-dependent membrane
association and mediation of agonist-induced AA release (23). To
establish whether type V sPLA2, another cell
surface-binding sPLA2, could also participate in COX-2
induction in CTMC, we conducted coculture experiments using 293 cells
expressing type V sPLA2 (293(V)), about half of which were
distributed in the cell surface-associated fraction (Fig. 8A). NGF-dependent COX-2
expression increased only modestly when CTMC were cultured with
conditioned medium containing the soluble type V sPLA2, but
it increased markedly when replicate cells were cocultured with 293(V)
(Fig. 8B).
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), was
exclusively secreted into the medium when overexpressed in 293 cells
(293(IIC); Fig. 8A). Unlike the cases of types IIA and V
sPLA2, NGF-induced COX-2 expression in CTMC was augmented
by the conditioned medium containing secreted type IIC
sPLA2 more efficiently than that in replicate cells
cocultured with 293(IIC) (Fig. 8B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunohistochemical staining of NGF-treated CTMC revealed that both
COX-1 and COX-2 displayed perinuclear localization (Fig. 1B), as has been reported in other cells (34, 35). The
perinuclear membrane, to which cPLA2, an enzyme that
supplies AA to COXs, translocates from the cytosol in response to
various transmembrane stimuli, has recently been suggested to be the
central site for eicosanoid biosynthesis. Indeed, the complete
suppression of delayed PGD2 synthesis by the two
cPLA2 inhibitors we used (Fig. 3) implies that this
PLA2 isozyme plays an important role in this biosynthetic
process. Certain punctured structures distributed throughout the
cytoplasm were also stained intensely by the anti-COX Abs,
particularly anti-COX-2 Ab. In this regard, Dvorak et al. (36)
observed COX-1 in the cytoplasmic lipid bodies of human lung mast cells
and other hemopoietic cells and proposed that these lipid-rich
organelles could be nonmembrane sites of eicosanoid formation.
Nonetheless, whether the subtle differences between the intracellular
distributions of the two COX isoforms in NGF-stimulated CTMC account
for the selective utilization of COX-2 over COX-1 during the delayed
phase of PGD2 biosynthesis needs further study.
RT-PCR analysis revealed that of the sPLA2 isozymes
studied, type IIA sPLA2 is the only isozyme expressed in
CTMC, and its expression is dramatically induced following NGF
stimulation (Fig. 2). The other two sPLA2 isozymes
belonging to the group II subfamily of sPLA2, types IIC and
V, were not detected in rat CTMC, although they were detected in rat
RBL-2H3 cells, a mucosal-type mastocytoma, and mouse BMMC, a progenitor
population of mast cells. These results also imply that the
sPLA2 expression profiles differ among mast cell
phenotypes. The strong inducibility of type IIA sPLA2 (Fig. 2) and the pharmacologic inhibition of PGD2 generation by
the type IIA sPLA2 inhibitor LY311727 (Fig. 3) strongly
suggest that type IIA sPLA2 is also coupled with COX-2. A
requirement for both cPLA2 and type IIA sPLA2
in the delayed PG biosynthetic responses of several other cell systems
has been reported recently (16, 23, 32).
However, detailed analysis of the inhibitory actions of LY311727 on
PGD2 synthesis showed that this agent attenuated the
induction of COX-2 expression (Fig. 4). This was unexpected, because
treatment of other cell types, such as macrophages and fibroblasts,
with LY311727 did not alter the COX-2 expression level (data not
shown). This result raised the intriguing possibility that type IIA
sPLA2 plays an augmentative role in COX-2 expression in
CTMC during the delayed response through an as yet unidentified
mechanism. Support for this hypothesis was provided by our experiments
on exogenous type IIA sPLA2, which augmented NGF-dependent
delayed PGD2 generation in CTMC mainly by increasing COX-2
expression (Fig. 5), with only a modest effect on AA release (17). The
presence of an autocrine amplification loop of the eicosanoid
biosynthesis by endogenous eicosanoids or lysophospholipids, like that
observed in osteoblasts (33), fibroblasts (16), and mesangial cells
(37), is unlikely, because none of the cPLA2 or COX
inhibitors we used, the specificities of which had been confirmed in
vitro (19, 20, 21, 22) and subsequently used in several cellular studies (4, 5, 6, 16, 21, 22, 23, 32, 33), reduced COX-2 expression, and NGF-stimulated CTMC
did not accumulate lysophosphatidylserine, an activation cofactor of
the NGF-dependent response (17).
Conclusive evidence for the dissociation of the COX-2-inducing ability
of type IIA sPLA2 from its enzyme action was provided by
our experiments on sPLA2 mutants, which lack the enzyme
function as a result of replacing the critical residues for catalytic
activity (Gly30 or His48) with other amino
acids. Although introduction of these mutations completely disrupted
the AA-releasing activity of type IIA sPLA2 in
cytokine-stimulated cells (Fig. 7A), implying that its
enzymatic function is essential in this situation (23), the
COX-2-inducing effect on rat CTMC was virtually unaffected (Fig. 7).
Therefore, the action of type IIA sPLA2 on rat CTMC
probably depends on a catalytic activity-independent, ligand-like
process. The observations described above are reminiscent of our
earlier finding that triggering of mast cell degranulation by exogenous
type IIA sPLA2 was independent of its enzymatic activity
(38). Therefore, the observed inhibitory effect of LY311727, which
tightly binds to type IIA sPLA2 (18), might be due to
inhibition of sPLA2 association with cells rather than
inhibition of enzyme activity itself, the possibility supported by our
preliminary study (unpublished observation).
Whether sPLA2 receptors are present on mast cells is
obscure. It is known that several pharmacologic actions of snake venom
sPLA2s occur through the noncatalytic processes, some of
which are believe to be mediated by sPLA2-specific binding
sites (39, 40). Arita et al. (41, 42), who cloned the cDNA for the
M-type sPLA2 receptor that displays >1000 times higher
affinity for type IB sPLA2 (and several venom
sPLA2) than type IIA sPLA2, conducted a ligand
binding assay and RNA blotting and reported that this receptor was not
expressed in rat CTMC. We also failed to detect M-type receptor
expression in rat CTMC by RNA blotting (data not shown). Moreover, type
IIA sPLA2 was a more potent inducer of COX-2 expression in
CTMC than type IB sPLA2 (Fig. 5B), arguing
against the ligand specificity of the M-type receptor. Therefore, it
seems reasonable to suggest that a novel sPLA2 receptor,
which exhibits sPLA2 isozyme specificity different from
that of the cloned M-type receptor, is present on rat CTMC. Recently,
Nieto and colleagues (43) showed that type IIA sPLA2
activated mitogen-activated protein kinases in human astrocytomas
through a pathway independent of the catalytic activity. Interestingly,
COX-2 induction by NGF in rat CTMC also requires the activation of
mitogen-activated protein kinase (unpublished observation). It is,
therefore, possible that the putative sPLA2 receptors on
CTMC and astrocytomas are similar. However, our attempts to identify
the specific type IIA sPLA2-binding site(s) on rat CTMC
have been unsuccessful to date, probably due to the presence of large
amounts of heparin, for which type IIA sPLA2 has a
remarkably high affinity, in CTMC.
Plasma membrane association via heparan sulfate proteoglycan is a
characteristic of type IIA sPLA2 (23, 44). About half of
type IIA sPLA2 forcibly expressed in HEK293 cells was
present on the cell surfaces (Figs. 6B). Coculture of rat
CTMC with HEK293 cells overexpressing type IIA sPLA2
resulted in far more efficient induction of COX-2 than that observed
with replicate CTMC cultured with the soluble enzyme (Fig. 6C). The observation that type V sPLA2, another
membrane-associated sPLA2 (Fig. 8A), could
substitute for type IIA sPLA2 (Fig. 8B) is
important, as it is the dominant sPLA2 isoform expressed in
certain mouse mast cells (14). These results suggest that
sPLA2s expressed ectopically on fibroblasts may facilitate
the delayed response of CTMC through direct CTMC-fibroblast contact.
Clustering of these sPLA2s on fibroblastic cell surfaces
via proteoglycans would increase their local concentrations, leading to
more efficient presentation of these molecules to neighboring CTMC.
Alternatively, fibroblasts may produce a membrane-bound factor(s) that
increases the sensitivity of CTMC to the membrane-anchored
sPLA2s. Indeed, fibroblasts are known to promote sustained
survival, differentiation, and maturation and increase some effector
functions of mast cells (30); conversely, mast cells are known to alter
the functions of fibroblasts (31). Nevertheless, the juxtacrine pathway
of sPLA2 presentation to target cells we observed in this
study represents a previously undescribed mode of sPLA2
action. This system may also operate in other pathophysiologic
situations, such as endothelial cell-leukocyte interactions in the
circulation during inflammation, when sPLA2 accumulated on
endothelial cell surfaces might contribute to activation of adherent
leukocytes. This route may be compatible with the presentation of
chemokines from vascular endothelium to leukocytes, in which
proteoglycans capture chemokines on the surfaces of endothelial cells,
establishing a local concentration gradient from the point source of
chemokine secretion (45).
COX-2 induction in CTMC was enhanced by the condition medium of, but
not by coculture with, HEK293 cells overexpressing type IIC
sPLA2 (Fig. 8B). This observation is consistent
with the findings that type IIC sPLA2 has no
heparin-binding domain (23) and is secreted into the medium without
appreciable cell surface association (Fig. 8A). Our recent
analysis demonstrated that type IIC sPLA2 failed to promote
AA release by cytokine-primed fibroblasts because of its poor
membrane-binding ability (23). Therefore, to the best of our knowledge,
the COX-2-inducing property in CTMC is the first biologic activity of
type IIC sPLA2 to be demonstrated. Moreover, our results
provide tentative evidence that the putative sPLA2 receptor
expressed on rat CTMC can recognize at least three (types IIA, IIC, and
V) of sPLA2.
In conclusion, NGF-dependent delayed PGD2 generation, which is mediated by inducible COX-2, is regulated cooperatively by two PLA2 enzymes with different roles: cPLA2 contributes to the initiation of AA release, whereas type IIA sPLA2, of both endogenous and exogenous origins, augments COX-2 expression, thereby acting as an amplifier of the delayed response. The two discrete actions of sPLA2s on target cells, COX-2 induction through a ligand-like action and AA release as a result of enzymatic activity, imply bifunctional aspects to their roles in the control of cellular and tissue homeostasis.
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Footnotes |
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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, Tokyo 142, Japan. E-mail address:
3 Abbreviations used in this paper: FcRI, high affinity IgE receptor; AA, arachidonic acid; cPLA2, cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; COX, cyclooxygenase; BMMC, bone marrow-derived mast cells; NGF, nerve growth factor; CTMC, connective tissue mast cells; MAFP, methyl arachidonylfluorophosphate; AACOCF3, arachidonoyl trifluoromethyl ketone.
Received for publication April 10, 1998. Accepted for publication June 25, 1998.
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