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J Biol Chem, Vol. 274, Issue 42, 29927-29936, October 15, 1999

Functional Association of Type IIA Secretory Phospholipase A₂ with the Glycosylphosphatidylinositol-anchored Heparan Sulfate Proteoglycan in the Cyclooxygenase-2-mediated Delayed Prostanoid-biosynthetic Pathway^*

Makoto Murakami, Terumi Kambe, Satoko Shimbara, Shinji Yamamoto, Hiroshi Kuwata, and Ichiro Kudo

From the Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

An emerging body of evidence suggests that type IIA secretory phospholipase A₂ (sPLA₂-IIA) participates in the amplification of the stimulus-induced cyclooxygenase (COX)-2-dependent delayed prostaglandin (PG)-biosynthetic response in several cell types. However, the biological importance of the ability of sPLA₂-IIA to bind to heparan sulfate proteoglycan (HSPG) on cell surfaces has remained controversial. Here we show that glypican, a glycosylphosphatidylinositol (GPI)-anchored HSPG, acts as a physical and functional adaptor for sPLA₂-IIA. sPLA₂-IIA-dependent PGE₂ generation by interleukin-1-stimulated cells was markedly attenuated by treatment of the cells with heparin, heparinase or GPI-specific phospholipase C, which solubilized the cell surface-associated sPLA₂-IIA. Overexpression of glypican-1 increased the association of sPLA₂-IIA with the cell membrane, and glypican-1 was coimmunoprecipitated by the antibody against sPLA₂-IIA. Glypican-1 overexpression led to marked augmentation of sPLA₂-IIA-mediated arachidonic acid release, PGE₂ generation, and COX-2 induction in interleukin-1-stimulated cells, particularly when the sPLA₂-IIA expression level was suboptimal. Immunofluorescent microscopic analyses of cytokine-stimulated cells revealed that sPLA₂-IIA was present in the caveolae, a microdomain in which GPI-anchored proteins reside, and also appeared in the perinuclear area in proximity to COX-2. We therefore propose that a GPI-anchored HSPG glypican facilitates the trafficking of sPLA₂-IIA into particular subcellular compartments, and arachidonic acid thus released from the compartments may link efficiently to the downstream COX-2-mediated PG biosynthesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stimulus-initiated arachidonic acid (AA)¹ release, which is linked with the downstream cyclooxygenase (COX) and lipoxygenase pathways for eicosanoid biosynthesis, is a highly regulated cellular response that requires gene induction and/or posttranslational modification of a group of regulatory enzymes, namely phospholipase A₂ (PLA₂) (1). An expanding recognition of the structural and functional diversity of mammalian PLA₂ enzymes has revealed that the two major classes of Ca²⁺-dependent PLA₂s, namely 85-kDa cytosolic PLA₂  (cPLA₂; type IV) and 14-kDa secretory PLA₂ (sPLA₂) isozymes (types IIA and V), act as "signaling" PLA₂s, which contribute to the release of AA from agonist-stimulated cells, depending upon the phase of cell activation (2, 3). Among them, cPLA₂ has received much attention as a key regulator of stimulus-initiated eicosanoid biosynthesis, because it selectively releases AA, shows submicromolar Ca²⁺ sensitivity, and is activated by mitogen-activated protein kinase-directed phosphorylation (4, 5). cPLA₂ undergoes Ca²⁺-dependent translocation from the cytosol to perinuclear and endoplasmic reticular membranes (6, 7), where several downstream eicosanoid-generating enzymes, including two COX isozymes, are localized (8). Studies on cPLA₂-deficient mice have confirmed its critical role in lipid mediator generation during the acute allergic response, parturition, and postischemic brain injury (9, 10).

Among several members of the sPLA₂ family, sPLA₂-IIA is the most widely distributed isozyme in humans and rats (11). The expression of sPLA₂-IIA is often dramatically up-regulated by proinflammatory stimuli, such as bacterial endotoxin, interleukin (IL)-1, and tumor necrosis factor (TNF) (12-15), and is down-regulated by glucocorticoids (16). Raised sPLA₂-IIA levels at inflamed sites suggest that it plays a crucial role in the propagation of inflammatory responses (17-19), which has been further supported by recent in vivo studies (20, 21). Current in vitro studies suggest that sPLA₂-IIA can amplify stimulus-initiated AA metabolism, particularly the delayed prostaglandin (PG)-biosynthetic response, which is accompanied by de novo synthesis of sPLA₂-IIA and COX-2 (2, 3, 12, 14, 22, 23). In the mouse, sPLA₂-V, a close relative of sPLA₂-IIA, may replace sPLA₂-IIA under certain conditions (2, 3, 24, 25). However, the molecular mechanisms whereby these sPLA₂s regulate AA metabolism are still poorly understood.

sPLA₂s-IIA and -V have high affinities for heparanoids (2), and significant portions of these isozymes are associated with the cell surface, most likely through binding to heparan sulfate proteoglycans (HSPGs), which are expressed in most mammalian cells. We (2, 14, 22, 23, 26, 27) and others (28, 29) have shown that some of the cellular functions of these heparin-binding sPLA₂s depend on their cell surface HSPG-binding abilities. Association of sPLA₂-IIA with heparan or chondroitin sulfate chains increases the hydrolytic rate of phosphatidylcholine present in lipoprotein particles modestly (30, 31). On the other hand, some reports have indicated that the actions of exogenous sPLA₂-IIA on cells depend only on its interfacial interaction with substrate phospholipids rather than on its association with HSPGs (32).

The cell surface HSPGs fall into two families of molecules that differ in their core protein domain structures (33). The syndecans have core proteins with a transmembrane and a cytoplasmic domain, and they possess heparan and/or chondroitin sulfate chains near the N terminus distal to the plasma membrane (34). The glypicans, by contrast, lack a membrane-spanning domain, are anchored to the external surface of the plasma membrane via glycosylphosphatidylinositol (GPI), and have three heparan sulfate chains near the C terminus, which are close to the plasma membrane (35). Consistent with a GPI-anchored moiety, glypicans are mobile in the cell membrane and exhibit both apical and basolateral distributions, whereas syndecans are distributed basolaterally to be attached to extracellular matrix proteins (36). Interestingly, recent immunohistochemical studies have revealed that significant portion of glypican translocates to the nucleus in cells undergoing cell division and activation (37). There is extensive literature concerning glycosaminoglycans in the nuclear compartment (38-40). These findings appear to be compatible with the observations that several extracellular heparin-binding growth factors, such as fibroblast growth factor (FGF) and angiogenin, translocate into the nucleus via a HSPG-dependent route (41-44).

In an effort to clarify the role of sPLA₂-IIA in the regulation of the PG-biosynthetic pathway, we have identified the cellular component that is functionally associated with sPLA₂-IIA. We found that a GPI-anchored HSPG glypican acts as a cellular sPLA₂-IIA-binding partner that contributed to enhancement of the sPLA₂-IIA-mediated, cytokine-induced, delayed PG-biosynthetic response. In agreement with the emerging notion that GPI-anchored proteins reside in the microdomains called caveolae (45-48, 53), which are vesicular invaginations of the plasma membrane that are rich in signal-transducing molecules and implicated in vesicular transport and potocytosis between the plasma and intracellular membranes (49, 50), sPLA₂-IIA was found to accumulate in the caveolae and perinuclear sites, rather than being distributed uniformly on the cell surface as we had previously thought.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Human embryonic kidney (HEK) 293 cells were obtained from the Health Science Research Resources Bank, rat liver-derived BRL-3A cells were from RIKEN Cell Bank, and rat fibroblastic 3Y1 cells were from Dr. Y. Uehara (National Institute of Health, Tokyo). The culture conditions for these cell lines have been described previously (2, 3, 14, 27). The cDNAs for mouse sPLA₂-IIA and its heparin non-binding mutant KE4 (22), mouse cPLA₂, human COX-1, and human COX-2 were described previously (2, 3). The cDNA for rat glypican-1 was provided by Dr. R. Margolis (New York University Medical Center, New York, NY). The rabbit anti-human cPLA₂ antibody was provided by Dr. R. M. Kramer (Lilly Research). Preparation of the rabbit anti-rat sPLA₂-IIA antibody and its conjugation with cyanogen bromide-activated Sepharose (Amersham Pharmacia Biotech) were described previously (51). The goat anti-human COX-2 antibody and rabbit anti-human caveolin-2 antibody were purchased from Santa Cruz Biotechnology. The rabbit anti-human COX-1 antibody was provided by Dr. W. L. Smith (Michigan State University, Ann Arbor, MI). The PGE₂ enzyme immunoassay kit was purchased from Cayman Chemical. Human TNF was provided by Dr. H. Ishimaru (Asahi Chemical Industry). Human and mouse IL-1s were purchased from Genzyme. LipofectAMINE PLUS reagent, Opti-MEM medium, and TRIzol reagent were obtained from Life Technologies, Inc. RPMI 1640 medium was purchased from Nissui Pharmaceutical. Bacillus cereus GPI-specific phospholipase C (GPI-PLC) was purchased from Roche Molecular Biochemicals. Heparin and Flavobacterium heparinum heparinase III were purchased from Sigma. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG, FITC-rabbit anti-goat IgG ,and FITC-goat anti-rabbit IgG antibodies were purchased from Zymed Laboratories Inc. Cy3-conjugated donkey anti-rabbit IgG antibody was from Chemicon.

Establishment of Transfectants-- Establishment of 293 cell transformants that stably expressed sPLA₂-IIA, cPLA₂, COX-1, and COX-2 was described previously (2, 3). Briefly, 1 µg of each cDNA subcloned into pcDNA3.1 (Invitrogen) was mixed with 5 µl of LipofectAMINE PLUS in 200 µl of Opti-MEM medium for 30 min and then added to cells that had attained 40-60% confluence in six-well plates (Iwaki) containing 1 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 2 ml of fresh culture medium comprising RPMI 1640 containing 10% (v/v) fetal calf serum (FCS). After overnight culture, the medium was replaced again with 2 ml of fresh medium and culture was continued at 37 °C in a CO₂ incubator flushed with 5% CO₂ in humidified air. In order to establish stable transfectants, cells transfected with each cDNA were cloned by limiting dilution in 96-well plates in culture medium supplemented with 800 µg/ml Geneticin (Life Technologies, Inc.). After culture for 3-4 weeks, wells containing a single colony were chosen and the expression of each protein was assessed by immunoblotting. The established clones were expanded and used for the experiments as described below.

In order to establish sPLA₂-IIA/glypican-1 double transformants, 293 transformants expressing sPLA₂-IIA were subjected to a second transfection with glypican-1 cDNA, which had been subcloned into pcDNA3.1/Zeo(+) (Invitrogen) at the EcoRI site. Three days after transfection, the cells were used for the experiments or seeded into 96-well plates to be cloned by culture in the presence of 50 µg/ml zeocin (Invitrogen) in order to establish stable transformants overexpressing both sPLA₂-IIA and glypican-1. The expression of each was examined by immunoblotting, RNA blotting, and, in the case of sPLA₂-IIA, by measuring the PLA₂ activity of the supernatants.

Measurement of sPLA₂ Activity-- PLA₂ activity was assayed by measuring the amounts of free radiolabeled fatty acids released from the substrate 1-palmitoyl-2-[¹⁴C]arachidonoyl-sn-glycero-3-phosphoethanolamine (Amersham Pharmacia Biotech). Each reaction mixture (total volume 250 µl) consisted of a 10-µl aliquot of the required sample, 100 mM Tris-HCl (pH 7.4), 4 mM CaCl₂, and 2 µM substrate. After incubation for 10-30 min at 37 °C, the [¹⁴C]fatty acids released were extracted and the radioactivity was counted as described previously (22).

RNA Blotting-- Approximately equal amounts (~10 µg) of the total RNAs obtained from the transfected cells were applied to separate lanes of 1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore). The resulting blots were then probed with the respective cDNA probes that had been labeled with [³²P]dCTP (Amersham Pharmacia Biotech) by random priming (Takara Shuzo). All hybridizations were carried out as described previously (22).

SDS-Polyacrylamide Gel Electrophoresis/Immunoblotting-- Cell lysates (10⁵ cell eq) or culture supernatants were subjected to SDS-polyacrylamide gel electrophoresis using 15% (w/v) gels for sPLA₂-IIA and 10% gels for cPLA₂, COX-1, COX-2, and glypican-1 under non-reducing and reducing conditions, respectively. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell) using a semidry blotter (MilliBlot-SDE system; Millipore), according to the manufacturer's instructions. The membranes were probed with the respective antibodies and visualized using the ECL Western blot system (Amersham Pharmacia Biotech), as described previously (22).

Cell Activation-- 293 cells (5 × 10⁴/ml) were seeded into each well of 24- or 48-well plates. To assess AA release, 0.1 µCi/ml [³H]AA (Amersham Pharmacia Biotech) was added to the cells in each well on day 3, when they had nearly reached confluence, and culture was continued for another day. After three washes with fresh medium, 250 µl (24-well plate) or 100 µl (48-well plate) of RPMI 1640 with or without 1 ng/ml IL-1 and/or 10% FCS was added to each well and the amount of free [³H]AA released into the supernatant during culture for 4 h was measured. The percentage release of AA was calculated using the formula [S/(S + P)] × 100, where S and P are the radioactivities measured in equal portions of the supernatant and cell pellet, respectively. The supernatants from replicate cells were subjected to the PGE₂ enzyme immunoassay. AA release and PG generation by [³H]AA-prelabeled cells were also assessed by thin layer chromatography. Among the radiolabeled products released, >90% were AA and the rest (<10%) corresponded to PGs. Among PGs, PGE₂ was the major product, followed by modest production of PGD₂ and PGF₂. Therefore it is likely that the radioactivity released into the supernatants largely reflects [³H]AA release.

Culture and cytokine stimulation of 3Y1 (14) and BRL-3A (27) cells were performed according to our previous studies with slight modifications. In brief, 3Y1 or BRL-3A cells that had attained 60-80% confluence in 12-well plates (Iwaki) were replaced with Dulbecco's modified Eagle's medium (Nissui) containing 2% FCS. After overnight culture, the cells were stimulated with 1 ng/ml mouse IL-1 and 100 units/ml human TNF for 24 h in the medium containing 10% FCS.

Immunoaffinity Column Chromatography Using Anti-sPLA₂-IIA Antibody-- HEK293 cells coexpressing sPLA₂-IIA and glypican-1 were grown in a 150-mm diameter dish, washed once with phosphate-buffered saline (PBS), and lysed in 10 ml of PBS containing 1% Nonidet P-40 (Nakalai Tesque), 50 µg/ml leupeptin (Sigma), 1.5 µM pepstatin (Peptide Institute), 1 mM phenylmethanesulfonyl fluoride (Wako), and 5 mM EDTA (cell lysis buffer). After incubation for 30 min at 4 °C, the crude nuclear fraction was obtained by low spin centrifugation at 450 × g, as reported previously (52). The remaining supernatants were centrifuged for 1 h at 100,000 × g at 4 °C, and the resulting supernatants were applied to a rabbit anti-sPLA₂-IIA antibody-conjugated Sepharose column. After applying the samples, the column was washed with the cell lysis buffer. The bound proteins were eluted with glycine-HCl buffer (pH 2). In separate experiments, the column was washed with the cell lysis buffer containing 1 M NaCl, followed by elution with glycine-HCl buffer.

Immunocytostaining-- 293 cells expressing sPLA₂-IIA, 3Y1 cells, and BRL-3A cells were seeded onto collagen-coated cover glasses (Iwaki Glass) at 2.5 × 10⁴ cells/ml, cultured for 2 days, and activated with 1 ng/ml human IL-1 (for 293 cells) or with 100 units/ml human TNF and 1 ng/ml mouse IL-1 (for 3Y1 and BRL-3A cells) for appropriate periods. In some samples, 1 mg/ml heparin was added temporally as required for the experiments. After removing the supernatants, the cells were fixed with 2% (w/v) paraformaldehyde in PBS for 30 min at 4 °C. Then the cells were treated sequentially at room temperature with 1% (w/v) bovine serum albumin with or without 1% (w/v) saponin for 30 min in PBS to block nonspecific binding and to permeabilize the membranes, appropriate first antibodies against sPLA₂-IIA (1:500 dilution), cPLA₂ (1:500), COX-1 (1:1,000), COX-2 (1:200), or caveolin-2 (1:200) in PBS containing 1% albumin for 2 h, and FITC- and/or Cy3-conjugated second antibodies (1:100 dilution for each) in PBS containing 1% albumin for 1 h. The coverslips were mounted on glass slides using Perma Fluor (Japan Tanner) and examined using a FLUOVIEW laser fluorescence microscope (Olympus).

Statistical Analysis-- Data were analyzed by Student's t test. Results are expressed as the mean ± S.E., with p = 0.05 as the limit of significance.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Binding of sPLA₂-IIA to GPI-Anchored HSPG Is Essential for Its PG-Biosynthetic Activity-- We have previously shown that HEK293 cells transfected with sPLA₂-IIA cDNA mainly express a cell membrane-associated form of sPLA₂-IIA, which appears to play an important role in the promotion of IL-1-induced, COX-2-dependent delayed PGE₂ generation (2, 3). To verify whether sPLA₂-IIA is indeed associated with the HSPG moiety on 293 cell surfaces, we treated the cells with heparinase or exogenous heparin and then looked for the appearance of sPLA₂-IIA in their supernatants (Fig. 1, A and B). There was modest sPLA₂-IIA release, which we detected by enzyme assay (Fig. 1A) and very faintly by immunoblotting (Fig. 1B), from the sPLA₂-IIA-transfected, but not control, cells. sPLA₂-IIA activity in the supernatant increased time-dependently when the sPLA₂-IIA-expressing, but not control, cells were cultured in the presence of heparinase, which degrades heparan sulfate chains (27, 28, 35-37) (Fig. 1A). Pretreatment of the sPLA₂-IIA-expressing, but not control, cells with heparin, which competes with cell surface HSPG for heparin-binding proteins (14, 22, 26-29), also caused an increase in sPLA₂-IIA activity in the culture media (Fig. 1A), the result essentially consistent with our previous observations (2). This heparinase (data not shown) or heparin (see Fig. 5) treatment solubilized the cell-associated sPLA₂-IIA almost completely. The increased release of sPLA₂-IIA into the supernatants of the sPLA₂-IIA-expressing cells after treatment with heparinase or heparin was confirmed by immunoblotting (Fig. 1B). sPLA₂-IIA mRNA expression in the sPLA₂-IIA-transfected cells was unaffected after treatment with heparinase or heparin (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effects of heparinase and heparin on sPLA2-IIA release and IL-1-induced PGE2 generation by sPLA2-IIA-expressing HEK293 cells. A, control and sPLA2-IIA-expressing 293 cells grown in 48-well plates were cultured for the indicated periods with 0.4 unit/ml heparinase or 1 mg/ml heparin, and sPLA2 activity released into the supernatants was measured. B, aliquots of the supernatants of control and sPLA2-IIA-expressing 293 cells cultured with or without heparinase (for 48 h) or heparin (for 24 h) were analyzed by immunoblotting using an anti-sPLA2-IIA antibody. C, control and sPLA2-IIA-expressing 293 cells preincubated for the indicated periods with heparinase or heparin were stimulated for an additional 4 h with IL-1, and PGE2 generation was assessed. Representative results from three independent experiments are shown.

sPLA₂-IIA-expressing, but not control, cells treated with IL-1 produced a significant amount of PGE₂ (Fig. 1C), the production of which depended upon inducible COX-2, as reported previously (2, 3). Treatment of the sPLA₂-IIA-expressing cells with heparinase suppressed this PGE₂ generation markedly; the kinetics indicated an inverse relationship between augmented sPLA₂-IIA release (Fig. 1A) and inhibition of PGE₂ generation (Fig. 1C). Treatment of replicate cells with heparin also led to a nearly 80% reduction of IL-1-induced PGE₂ generation. These results, together with our previous findings that a heparin-nonbinding sPLA₂-IIA mutant KE4, in which a cluster of four lysine residues in the C-terminal domain is replaced by glutamic acid, is not associated with the cell surface and does not augment IL-1-induced AA metabolism (2, 3, 22), strongly suggest that cellular HSPG is required for the full action of sPLA₂-IIA in this experimental system.

As there are two families of cellular HSPGs, the integral syndecans and the GPI-anchored glypicans (33-35), we wanted to know which HSPG species is the major sPLA₂-IIA-binding target. To address this issue, we used GPI-PLC, which cleaves the GPI linkage and is thereby capable of solubilizing GPI-anchored plasma membrane proteins and their associated proteins. We found that treatment of the sPLA₂-IIA-expressing, but not control, 293 cells with GPI-PLC markedly increased the amount of soluble sPLA₂-IIA, as assessed by both enzyme assay (Fig. 2A) and immunoblotting (Fig. 2A, inset), and reduced sPLA₂-IIA-mediated PGE₂ generation in response to IL-1 by nearly 80% (Fig. 2B). These results suggest that binding to a GPI-anchored HSPG glypican is required for sPLA₂-IIA to exert its PG-biosynthetic function.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effects of GPI-PLC on both sPLA2-IIA release and IL-1-induced PGE2 generation by sPLA2-IIA-expressing HEK293 cells. A, control and sPLA2-IIA-expressing 293 cells grown in 48-well plates were cultured for 24 h with 0.5 unit/ml GPI-PLC, and sPLA2 activity released into the supernatants was measured. Inset, aliquots of the supernatants of sPLA2-IIA-expressing 293 cells cultured with or without GPI-PLC for 24 h were subjected to immunoblotting using an anti-sPLA2-IIA antibody. B, cells used in A were cultured for an additional 4 h with IL-1, and PGE2 generation was assessed. Values are the means ± S.E. for three independent experiments.

Binding of sPLA₂-IIA to Glypican-- To obtain more convincing evidence for the interaction between sPLA₂-IIA and the GPI-anchored HSPG glypican, we attempted to establish sPLA₂-IIA/glypican-1 double transfectants. Fig. 3A depicts the expression levels of transcripts for sPLA₂-IIA and glypican-1 in HEK293 cells transfected with their cDNAs alone or in combination. Endogenous glypican-1 protein was faintly detected in 293 cells (Fig. 3A, clones 1, 3, and 5), and was significantly elevated in the glypican-1 transfectants (clones 2, 4, and 6). Several clones with different expression levels of sPLA₂-IIA were selected, including those expressing sPLA₂-IIA at a low level (clones 3 and 4), a very high level (clones 5 and 6), and without expression (clones 1 and 2).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Glypican enhances cellular functions of sPLA2-IIA. A, expression of glypican-1 and sPLA2-IIA in HEK293 transfectants (clones 1-6) was assessed by immunoblotting and RNA blotting, respectively. B, AA release (upper panel) and PGE2 generation (lower panel) by the sPLA2-IIA or glypican-1 transfectants (clones 1-6) after stimulation for 4 h with IL-1. Values are the means ± S.E. for five independent experiments. C, COX-2 expression in the sPLA2-IIA- or glypican-1 transfectants was assessed by RNA blotting after culture for 4 h with or without IL-1. A representative result from three independent experiments is shown.

The culture supernatants of 293 clones with high sPLA₂-IIA expression (clones 5 and 6) were collected after 3 days of culture, and the cells were then washed for 15 min with culture medium containing 1 M NaCl to solubilize the cell surface-associated sPLA₂-IIA, by a procedure that was reported earlier (2). The ratio of sPLA₂-IIA released into the supernatants to that associated with the cell surface increased from 1:3 in cells overexpressing sPLA₂-IIA alone (clone 5) to 1:10 in cells overexpressing both sPLA₂-IIA and glypican (clone 6), as assessed by enzyme assay. Hence, glypican-1 overexpression revealed a more than 3-fold increase in the cells' capacity to capture sPLA₂-IIA.

The 100,000 × g supernatant of Nonidet P-40-solubilized cells coexpressing sPLA₂-IIA and glypican-1 (clone 6) was analyzed by immunoprecipitation using an anti-sPLA₂-IIA antibody-conjugated Sepharose column, followed by immunoblotting with anti-sPLA₂-IIA and anti-glypican-1 antibodies (Fig. 4A). After washing the column with cell lysis buffer, proteins bound to the column were eluted with an acidic buffer (pH 2). As anticipated, a 14-kDa protein band corresponding to sPLA₂-IIA was detected in fractions eluted from the column, but not in the flow-through and washing fractions (Fig. 4A). Notably, a 64-kDa glypican-1 protein band was detected in the acid-eluted fractions in which sPLA₂-IIA was also recovered, whereas the flow-through and washing fractions contained only a trace level of glypican-1 (Fig. 4A). When the replicate immunoprecipitate was washed with the cell lysis buffer containing 1 M NaCl, which was expected to facilitate the dissociation of sPLA₂-IIA from the heparan sulfate chains of glypican-1 without disturbing the interaction between sPLA₂-IIA and the anti-sPLA₂-IIA antibody, and then the antibody-bound protein was eluted with the acidic buffer, most of the glypican-1 was recovered from the NaCl-eluted fraction with only a minor portion being retained until elution with the acidic buffer, whereas sPLA₂-IIA was detected exclusively in the acidic buffer fractions (Fig. 4B). These results support the idea that sPLA₂-IIA physically interacts with glypican-1, most likely through its heparan sulfate chains, in 293 cells.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Glypican-1 binding to sPLA2-IIA. A, the 100,000 × g supernatant of Nonidet P-40-solubilized 293 cells that coexpressed sPLA2-IIA and glypican-1 was applied to an anti-sPLA2-IIA antibody affinity column. Bound proteins were eluted with an acidic buffer (pH 2), and each fraction was analyzed by immunoblotting using antibodies against glypican-1 or sPLA2-IIA. Right, a crude nuclear fraction obtained from 293 cells that coexpressed sPLA2-IIA and glypican-1 was immunoblotted using the same antibodies. B, Anti-sPLA2-IIA antibody-conjugated beads were incubated with the high speed supernatant of Nonidet P-40-lyzed 293 cells that coexpressed sPLA2-IIA and glypican-1 and then washed with a buffer containing 1 M NaCl followed by an acidic buffer. Fractions were analyzed by immunoblotting using the same antibodies. A result representative of three independent experiments is shown.

Functional Interaction between sPLA₂-IIA and Glypican-- To assess the functional significance of the sPLA₂-IIA/glypican interaction, the established transfectants (Fig. 3A, clones 1-6) were prelabeled with [³H]AA and delayed [³H]AA release in response to IL-1 was investigated (Fig. 3B, upper panel). Cells expressing a high level of sPLA₂-IIA showed increased AA release up to 2.7% (clone 5) relative to the control cells, which released no more than 0.7% AA (clone 1). AA release by the sPLA₂-IIA high expression cells (clone 5) was augmented to 4.4% by glypican-1 coexpression (clone 6), whereas the expression of glypican-1 alone increased AA release only minimally (clone 2), revealing a synergistic action between the two. The augmentative role of glypican-1 in sPLA₂-IIA-mediated AA release was more obvious when the sPLA₂-IIA expression level was low (clone 3); AA release by these cells was comparable to that of the control cells (clone 1), indicating that this level of sPLA₂-IIA was insufficient to modulate IL-1-induced AA release, yet introduction of glypican-1 into these cells dramatically enhanced the AA release to a maximal and saturable level (clone 4).

The role of glypican-1 in enhancing the functions of sPLA₂-IIA was further pronounced when IL-1-induced PGE₂ biosynthesis was assessed (Fig. 3B, lower panel). Cells expressing a high level of sPLA₂-IIA produced 2.5 ng of PGE₂/10⁶ cells (clone 5), which was augmented up to 10 ng/10⁶ cells by coexpression with glypican-1 (clone 6), whereas glypican-1 alone increased PGE₂ generation only modestly (clone 2) relative to the control cells (clone 1). Although PGE₂ generation by cells expressing a suboptimal level of sPLA₂-IIA was minimal (clone 3), it reached nearly 10 ng/10⁶ cells when glypican-1 was coexpressed in these cells (clone 4).

Comparison of the increases in AA release and PGE₂ generation in the clones with high sPLA₂-IIA expression (clones 5 and 6) revealed an approximately 1.8-fold increase in AA and a 4-fold increase in PGE₂ following glypican-1 coexpression, reflecting greater sensitivity of PGE₂ generation than of AA release to the action of sPLA₂-IIA. This fact led us to examine whether overexpression of sPLA₂-IIA and/or glypican affected the expression of endogenous COX-2, a COX isozyme predominantly involved in the conversion of AA released by sPLA₂-IIA to PGE₂ during the delayed response (2, 3, 14, 22). COX-2 expression, which was induced modestly in the control cells by IL-1 stimulation, was significantly augmented in cells transfected with sPLA₂-IIA alone (Fig. 3C). Cells transfected with glypican-1 alone expressed COX-2 even before IL-1 stimulation at a level comparable to that observed in IL-1-stimulated control 293 cells, and IL-1 stimulation increased its expression further in the glypican-1-expressing cells. Most importantly, coexpression of sPLA₂-IIA and glypican-1 increased COX-2 expression in a synergistic manner in the presence, but not in the absence, of IL-1 stimulation (Fig. 3C). These results are reminiscent of our recent finding that sPLA₂-V, another heparin-binding sPLA₂ isozyme, augments IL-1 induction of COX-2 expression in a heparin-sensitive manner, whereas the heparin-nonbinding sPLA₂-IIA mutant KE4 and the heparin-nonbinding isozyme sPLA₂-X fail to do so (53).

Collectively, these results suggest that (i) glypican acts as an adaptor for sPLA₂-IIA, probably by bringing it close to the cellular membrane through heparan sulfate chains near the C terminus; (ii) glypican facilitates sPLA₂-IIA-mediated AA release from IL-1-stimulated cells; (iii) glypican and sPLA₂-IIA act in synergy to enhance IL-1-induced COX-2 expression; and (iv) increased AA release and COX-2 induction by the coordinated actions of glypican and sPLA₂-IIA lead to marked elevation of PGE₂ generation.

Subcellular Localization of sPLA₂-IIA Overexpressed in HEK293 Cells-- -Immunostaining of permeabilized sPLA₂-IIA-expressing 293 cells showed that sPLA₂-IIA accumulated in punctate domains and in the perinuclear area (Fig. 5, A and B). The punctate domains were evident, whereas the perinuclear signal was barely found, in non-permeabilized cells (Fig. 5C). Incubation of sPLA₂-IIA-expressing cells with heparin abolished the punctate sPLA₂-IIA signal, whereas some perinuclear staining remained in permeabilized (Fig. 5D), but not in non-permeabilized (Fig. 5E), cells. Cells expressing the heparin-nonbinding sPLA₂-IIA mutant KE4, which was exclusively released into the supernatant (2, 22), were not stained appreciably by the anti-sPLA₂-IIA antibody (Fig. 5F), although RNA blotting showed that the expression level of KE4 was comparable with that of the native enzyme in the respective transfectants (2). Based on these observations, we hypothesize that sPLA₂-IIA is sorted predominantly into punctate microdomains on the plasma membrane that are sensitive to washing with exogenous heparin, and that a small portion is transported into the perinuclear area, possibly by the vesicular transport machinery. Importantly, double antibody staining of sPLA₂-IIA-expressing cells revealed that sPLA₂-IIA was colocalized with caveolin-2 (Fig. 5G). This result implies that the punctate domains in which sPLA₂-IIA resided are caveolae, organella that contain GPI-anchored proteins and may be responsible for potocytotic vesicular transport (49, 50). In support of this speculation, subcellular fractionation studies showed the presence of both immunoreactive sPLA₂-IIA and glypican-1 in the nucleus-enriched fraction (Fig. 3A).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Subcellular distributions of sPLA2-IIA and other PG-biosynthetic enzymes in HEK293 transfectants. Immunostaining of IL-1-stimulated 293 cells expressing native sPLA2-IIA (A-E) or its heparin-nonbinding mutant KE4 (F) with anti-sPLA2-IIA antibody. After fixation with paraformaldehyde, cells were treated with (A, B, D, and F) or without (C and E) saponin, followed by rabbit anti-sPLA2-IIA antibody and FITC-conjugated anti-rabbit IgG antibody. In D and E, the cells were cultured in the presence of 1 mg/ml heparin before fixation. G, double staining of sPLA2-IIA-expressing cells with rabbit anti-sPLA2-IIA and mouse anti-caveolin-2 antibodies, which were visualized using Cy3-conjugated anti-rabbit IgG and FITC-conjugated anti-mouse IgG antibodies, respectively. H and I, immunostaining for cPLA2. Cells incubated with (I) or without (H) IL-1 for 10 min were fixed, permeabilized, and then treated with rabbit anti-cPLA2 antibody. J and K, immunostaining for two COX isoforms. Transfectants expressing either COX-1 or COX-2 were fixed, permeabilized, and then treated with rabbit anti-COX-1 antibody and goat anti-COX-2 antibody, respectively. cPLA2 and COX-1 were each visualized using FITC-conjugated anti-rabbit IgG antibody and COX-2 by FITC-conjugated anti-goat IgG antibody.

The subcellular localization of cPLA₂ and two COX isoforms, COX-1 and COX-2, in the respective HEK293 transfectants was also investigated. cPLA₂ was detected throughout the cytoplasm in unstimulated cells (Fig. 5H), and IL-1 stimulation promoted the translocation of cPLA₂ to the perinuclear region within 10 min (Fig. 5I). This perinuclear redistribution of cPLA₂ continued over 4 h of culture with IL-1 (data not shown), during which cPLA₂-mediated AA release continued to take place (2, 3). Both COX-1 (Fig. 5J) and COX-2 (Fig. 5K) were found in the perinuclear envelope and endoplasmic reticulum. These staining patterns were essentially consistent with current reports (6-8).

Subcellular Localization of Cytokine-inducible Endogenous sPLA₂-IIA-- To ensure that the preferential distribution of sPLA₂-IIA in the caveolae and perinuclear location was not a peculiarity of the 293 transfectants, we next examined the subcellular distribution of endogenous sPLA₂-IIA, which was induced by proinflammatory stimuli in several cell types. For this purpose, we used rat fibroblastic 3Y1 cells because the expression of sPLA₂-IIA and COX-2 and attendant delayed generation of PGE₂ are known to be strongly induced by stimulation with IL-1 and TNF in these cells (14). Whereas sPLA₂-IIA immunoreactivity was weak in the punctate domains in unstimulated 3Y1 cells (Fig. 6, A and B), it increased markedly in the punctate and perinuclear compartments in replicate cells activated with IL-1 and TNF for 24 h (Fig. 6C), the period in which delayed PGE₂ generation occurs at the maximal rate (14). sPLA₂-IIA staining in IL-1/TNF-stimulated cells was markedly reduced when the cells were cultured in the presence of heparin (Fig. 6D), which solubilizes the cell surface-associated sPLA₂-IIA without affecting its expression level and eventually causes a reduction in PGE₂ generation (14). COX-2 was distributed in the perinuclear envelope and endoplasmic reticulum in IL-1/TNF-stimulated cells (Fig. 6E), but not in control cells (data not shown). Double antibody staining for sPLA₂-IIA and caveolin-2 demonstrated their colocalization (Fig. 6F), providing further support for the accumulation of endogenous sPLA₂-IIA in the caveolae of cytokine-stimulated 3Y1 cells.

View larger version (63K): [in this window] [in a new window]   Fig. 6.   Subcellular distribution of endogenous sPLA2-IIA in rat 3Y1 fibroblasts. 3Y1 cells were cultured for 24 h with (A and C-F) or without (B) both IL-1 and TNF, fixed, permeabilized, and incubated with control rabbit antibody (A), rabbit anti-sPLA2-IIA antibody (B-D), or goat anti-COX-2 antibody (E). In D, cells were cultured in the presence of 1 mg/ml heparin. sPLA2-IIA and COX-2 were visualized using FITC-conjugated anti-rabbit and anti-goat IgG antibodies, respectively. F, double staining of 3Y1 cells stimulated for 24 h with IL-1 and TNF with rabbit anti-sPLA2-IIA and mouse anti-caveolin-2 antibodies, which were visualized using Cy3-conjugated anti-rabbit IgG and FITC-conjugated anti-mouse IgG antibodies, respectively.

The other cell type we examined was rat liver-derived BRL-3A cells, in which a heparin-sensitive, membrane-associated, cytokine-inducible sPLA₂-IIA has been reported to be involved in cytokine-stimulated delayed PGE₂ generation (27). Immunocytostaining of BRL-3A cells revealed again that sPLA₂-IIA was localized in the punctate and perinuclear domains, in which caveolin-2 coexisted, in IL-1/TNF-stimulated, but not in unstimulated, cells (Fig. 7). As in the 3Y1 cells, COX-2 was found in the perinuclear envelope and endoplasmic reticulum in IL-1/TNF-stimulated BRL-3A cells (data not shown).

View larger version (53K): [in this window] [in a new window]   Fig. 7.   Subcellular distribution of endogenous sPLA2-IIA in rat liver BRL-3A cells. BRL-3A cells cultured for 24 h with (B-D) or without (A) IL-1 and TNF were fixed, permeabilized, and double-immunostained using a rabbit anti-sPLA2-IIA antibody (A and B), a mouse anti-caveolin-2 antibody (C), or both (D). sPLA2-IIA and caveolin-2 were visualized using Cy3-conjugated anti-rabbit IgG and FITC-conjugated anti-mouse IgG antibodies, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Several previous studies have demonstrated the dependence of cellular functions of sPLA₂-IIA on HSPGs, particularly when delayed PG generation is accompanied by its concomitant expression in cells exposed to proinflammatory stimuli (2, 3, 14, 22, 26, 27). In a proposed model of this, sPLA₂-IIA released from cytokine-stimulated cells is captured by the heparan sulfate chains of a HSPG and thus accumulates on the plasma membrane, from which the enzyme liberates AA. sPLA₂-IIA bound to a HSPG on the surface of fibroblasts greatly enhances the PG-biosythetic response in adjacent mast cells through a juxtacrine route (23). Furthermore, deposition of sPLA₂-IIA into the matrix proteoglycan biglycan increases its hydrolytic efficiency of lipoprotein particles severalfold, an event that is implicated in the exacerbation of atherosclerosis (30, 31). As opposed to these positive regulatory effects of HSPG on sPLA₂-IIA functions, however, there are also some contradictory reports that sPLA₂-IIA acts independently of HSPG (32), which suggests that the relation between proteoglycans and sPLA₂-IIA action is more complicated than previously thought. To reconcile this discrepancy, it seemed necessary to clarify what kinds of HSPG bind sPLA₂-IIA and how they affect sPLA₂-IIA functions in cells undergoing delayed PG generation.

The findings of the present study suggest that endogenously expressed sPLA₂-IIA associates with the GPI-anchored form of HSPG, glypican. Removal of GPI-anchored HSPG from cell surfaces markedly attenuated sPLA₂-IIA-mediated PGE₂ production (Figs. 1 and 2). sPLA₂-IIA and glypican were coimmunoprecipitated from the cells, implying their association in vivo (Fig. 4). Moreover, the ability of sPLA₂-IIA to enhance IL-1-induced AA metabolism was markedly enhanced by coexpression with glypican (Fig. 3). This effect was particularly evident when the expression of sPLA₂-IIA was suboptimal. Importantly, the maximal response was produced even by low concentrations of sPLA₂-IIA (ng/ml as estimated by enzymatic activity and the intensity of immunoblot bands) when combined with overexpressed glypican that were about 1,000 times lower than that required for exogenously added sPLA₂-IIA to exhibit AA-releasing function (23, 28, 32, 54, 55). It is therefore likely that the role of glypican is to amplify the function of sPLA₂-IIA expressed at physiological levels. As demonstrated by Gelb and co-workers (32), high concentrations of exogenous sPLA₂-IIA could act on cells independently of HSPG to elicit rapid and transient AA release.

GPI-anchored proteins generally occur in microdomains of the cell membrane called caveolae or the caveolae-related domain (45-48). It has been shown recently that there are dynamic changes in the subcellular distribution of glypican, which moves to the nucleus and punctate caveolae-like domains, depending upon the cell cycle (37). Based on our present finding that sPLA₂-IIA is functionally associated with the GPI-anchored HSPG glypican, it is tempting to speculate that glypican plays a specific role in the sorting of sPLA₂-IIA into specific subcellular compartments; AA released by sPLA₂-IIA in these compartments will be more accessible to the COX-2-dependent PGE₂-biosynthetic pathway than if it was released randomly from the plasma membrane surface as was thought previously. Indeed, sPLA₂-IIA appears to be localized in the caveolae in three different cell lines that exhibit cytokine-stimulated delayed PGE₂ generation (Figs. 5-7), although final conclusion for this localization must be awaited until more detailed electron microscopic studies will be perfomed. In this regard, an earlier work using electron microscopic analysis showed a punctate staining pattern of sPLA₂-IIA in TNF-stimulated rat vascular smooth muscle cells, where the surface of the cave-shaped compartments on the plasma membrane gave a strong signal for sPLA₂-IIA (56). We expect that the cave-shaped compartments observed in the vascular smooth muscle cells also correspond to caveolae.

Caveolae are known to form a unique endocytic and exocytic compartment at the surface of most cells, capable of importing molecules, delivering them to specific locations within the cell, and compartmentalizing a variety of signaling activities (49, 50). They are not simply an endocytic device with a peculiar membrane shape but constitute an entire membrane system with multiple functions essential for the cell. Our detection of both sPLA₂-IIA and caveolin-2 in the perinuclear region (Fig. 6) suggests that the caveolae-mediated endocytotic event called potocytosis occurs in cytokine-stimulated cells. Several previous subcellular fractionation studies demonstrated the presence of significant sPLA₂-IIA in some intracellular organelle fractions including the nucleus (52, 57). The perinuclear localization of sPLA₂-IIA is also supported by the fact that glypican-1, an sPLA₂-IIA-adaptor protein, has a bipartite motif for nuclear localization signals (37). Since considerable evidence has indicated that caveolae are a site of Ca²⁺ storage and entry into the cell (50), it is likely that sPLA₂-IIA, a Ca²⁺-dependent enzyme, present inside caveola particles retains its enzyme activity even after internalization and translocation to the perinuclear domain. Moreover, sPLA₂-IIA has been reported to be active in the presence of only micromolar concentrations of Ca²⁺ under certain assay conditions (58), raising the possibility that it is active even within the cell. The caveolae membrane is enriched in sphingomyelin, which inhibits the enzymatic activity of sPLA₂-IIA in vitro (59). The high packing density of the bilayer leaflet enriched in this lipid hinders the penetration of sPLA₂-IIA into the plasma membrane, which may explain why quiescent cells are fairly resistant to sPLA₂-IIA. In keeping with the present finding that sPLA₂-IIA resides in caveolae, a sphingomyelin-rich microdomain (49, 50), a decrease in the cellular sphingomyelin content caused by sphingomyelinase in response to cytokines (60) may allow the otherwise silent sPLA₂-IIA to become active toward the caveola membrane. Cholesterol, which is also abundant in caveolae (49, 50, 61), counteracts the effect of sphingomyelin-based inhibition of sPLA₂-IIA (62) and may contribute to the temporal and spatial regulation of this enzyme during cell activation. Rather selective release of [³H]AA by sPLA₂-IIA, which we observed in sPLA₂-transfected cells (2), may be a reflection of the fact that [³H]AA is preferentially incorporated into the caveola and perinuclear membranes (63). It should be noted, however, that we have not yet conclusively shown that AA release by sPLA₂-IIA for PG production is due to enzyme on the inside of the cell. The enhanced AA metabolism by glypican expression could be due to enhanced internalization of sPLA₂-IIA or due to enhanced capture on the outside of the cell for AA release from caveolae on the plasma membrane. This point should be clarified in a future study.

Enhancement of cytokine-induced COX-2 expression is another intriguing feature of sPLA₂-IIA, which was synergistically augmented by coexpression with glypican (Fig. 3C). This result is consistent with our recent finding that COX-2 induction by sPLA₂-IIA and sPLA₂-V in 293 cells depends on their binding to HSPG as well as on their enzymatic activities (53). Thus, increased production of AA metabolites by the concerted actions of sPLA₂-IIA and glypican may lead to further amplification of COX-2 expression through a positive feedback route in this occasion. Alternatively, the binding of sPLA₂-IIA to the heparan sulfate chains of glypican facilitates its presentation to the putative sPLA₂-IIA receptor, which transduces signals leading to increased COX-2 expression. In line with this hypothesis, COX-2 induction by sPLA₂-IIA in rat serosal mast cells appears to involve a receptor-mediated pathway (23). This type of receptor system, utilizing both HSPG and a signal-transducing receptor subunit, has been described for the FGF receptor system in which the ligand, its tyrosine kinase receptor, and HSPG form a stable ternary complex on the cell surface, thereby potentiating transmembrane signals (41, 42, 64). The participation of a receptor-like molecule(s) in this sPLA₂-IIA action should be examined because many receptor molecules as well as intracellular signal-transducing molecules are known to be associated with the caveolae (49, 50). Note that the M-type receptor, the only sPLA₂ receptor cloned to date, possesses a sequence stretch for receptor internalization in its short cytoplasmic domain (65, 66), and that sPLA₂-IB, a ligand for the M-type receptor, translocates to the perinuclear compartment through the receptor-dependent process (67).

It is likely that sPLA₂-IIA is able to associate with proteoglycans other than glypican. The binding of sPLA₂-IIA to extracellular matrix HSPGs, such as perlecan and biglycan, may prevent its movement to the plasma membrane, eventually leading to inhibition of its cellular functions. This situation is reminiscent of FGF, which is stored in the extracellular matrix as a latent form; active FGF is released by the degradation of matrix HSPG by heparinase and proteases, and then interacts with a signal-transducing receptor complex composed of the FGF receptor and HSPG (glypican or syndecan) on the target cells (64). sPLA₂-IIA associated with matrix HSPG has a specific role in the development of atherosclerosis (19). Whether the syndecans, integral cellular HSPGs, regulate sPLA₂-IIA functions remains to be elucidated. In hematopoietic cells, such as platelets and mast cells, sPLA₂-IIA is stored in secretory granules rather than binding to a cell surface HSPG (68-70), implying that the subcellular distribution of sPLA₂-IIA varies according to the cell type and HSPG molecular species. The secretory granules of mast cells contain serglycin, a soluble proteoglycan (71), which may trap sPLA₂-IIA inside the proteoglycan-formed particles and may inhibit its activity to prevent unregulated hydrolysis of granule membranes. Activation of mast cells causes rapid exocytosis of sPLA₂-IIA, which may be released from serglycin to become active on target cells.

In summary, we have provided new insight into the mechanisms that regulate delayed PG biosynthesis via sPLA₂-IIA. sPLA₂-IIA produced by cytokine-stimulated cells (autocrine route) or from extracellular microenvironments (paracrine or juxtacrine route) binds to GPI-anchored HSPG, glypican, which plays a crucial role in sorting of sPLA₂-IIA into the caveola signalsomes. A significant portion of sPLA₂-IIA may enter the cells through potocytosis and reach the perinuclear area, where the upstream (cPLA₂) and downstream (COX-2) PG-biosynthetic enzymes are located. Considering that prior activation of cPLA₂ is crucial for sPLA₂ to act (72), cPLA₂ may play a role in the sensitization of the caveola and/or perinuclear membrane to sPLA₂-IIA. The AA thus released from the particular compartments by sPLA₂-IIA may be readily supplied to COX-2, allowing their efficient functional coupling. sPLA₂-IIA can also has the ability to increase COX-2 expression, which contributes to further amplification of PGE₂ production. sPLA₂-IIA secreted from the cells mediates the transcellular PG-biosynthetic response (3). Continuous production and supply of sPLA₂-IIA are crucial for it to function fully in the delayed PG-biosynthetic response. It is likely that sPLA₂-V utilizes the same regulatory machinery in some cells, because it also binds to a cellular HSPG and elicits delayed PG biosynthesis in a manner similar to sPLA₂-IIA under certain (2, 3, 72-74), if not all (75), conditions. Whether sPLA₂-IID, a recently identified sPLA₂-IIA homolog (76, 77), also utilizes the same machinery is now under investigation. A heparin-nonbinding isozyme, sPLA₂-X, may hydrolyze phosphatidylcholine in the outer leaflet of the plasma membrane randomly and thereby exhibit unique fatty acid-releasing properties different from sPLA₂-IIA (53).

	ACKNOWLEDGEMENTS

We thank Drs. R. U. Margolis, W. L. Smith, and R. M. Kramer for providing cDNAs and antibodies. We thank Drs. M. Suematsu, Y. Wakabayashi, and R. Takamiya (Keio University, Tokyo) for their helpful advice about immunohistochemical staining.

	FOOTNOTES

^* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan, the Human Science Foundation, and special coordination funds for promoting science and technology from the Science and Technology Agency.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-3-3784-8196; Fax: 81-3-3784-8245; E-mail: kudo@pharm.showa-u.ac.jp.

	ABBREVIATIONS

The abbreviations used are: AA, arachidonic acid; PLA₂, phospholipase A₂; sPLA₂, secretory PLA₂; cPLA₂, cytosolic PLA₂; PG, prostaglandin; COX, cyclooxygenase; GPI, glycosylphosphatidylinositol; GPI-PLC, GPI-specific phospholipase C; IL-1, interleukin-1; TNF, tumor necrosis factor; FCS, fetal calf serum; HSPG, heparan sulfate proteoglycan; FGF, fibroblast growth factor; HEK, human embryonic kidney; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES