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BIBTEX RIS

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

1999; Elsevier BV; Volume: 274; Issue: 42 Linguagem: Inglês

10.1074/jbc.274.42.29927

ISSN

1083-351X

Autores

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

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

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. 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. Stimulus-initiated arachidonic acid (AA) 1The abbreviations used are: AAarachidonic acidPLA2phospholipase A2sPLA2secretory PLA2cPLA2cytosolic PLA2PGprostaglandinCOXcyclooxygenaseGPIglycosylphosphatidylinositolGPI-PLCGPI-specific phospholipase CIL-1interleukin-1TNFtumor necrosis factorFCSfetal calf serumHSPGheparan sulfate proteoglycanFGFfibroblast growth factorHEKhuman embryonic kidneyFITCfluorescein isothiocyanatePBSphosphate-buffered saline1The abbreviations used are: AAarachidonic acidPLA2phospholipase A2sPLA2secretory PLA2cPLA2cytosolic PLA2PGprostaglandinCOXcyclooxygenaseGPIglycosylphosphatidylinositolGPI-PLCGPI-specific phospholipase CIL-1interleukin-1TNFtumor necrosis factorFCSfetal calf serumHSPGheparan sulfate proteoglycanFGFfibroblast growth factorHEKhuman embryonic kidneyFITCfluorescein isothiocyanatePBSphosphate-buffered saline 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) (1Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (754) Google Scholar). An expanding recognition of the structural and functional diversity of mammalian PLA2enzymes has revealed that the two major classes of Ca2+-dependent PLA2s, namely 85-kDa cytosolic PLA2 α (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 (2Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). 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 (4Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1453) Google Scholar, 5Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar). cPLA2undergoes Ca2+-dependent translocation from the cytosol to perinuclear and endoplasmic reticular membranes (6Glover S. Bayburt T. Jonas M. Chi E. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 7Schievella A.R. Regier M.K. Smith W.L. Lin L.-L. J. Biol. 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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) (12Ishizaki J. Hanasaki K. Higashino K. Kishino J. Kikuchi N. Ohara O. Arita H. J. Biol. Chem. 1994; 269: 5897-5904Abstract Full Text PDF PubMed Google Scholar, 13Nakazato Y. Simonson M.S. Herman W.H. Konieczkowski M. Sedor J.R. J. Biol. Chem. 1991; 266: 14119-14127Abstract Full Text PDF PubMed Google Scholar, 14Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 15Pfeilschifter J. Schalkwijk C. Briner V.A. van den Bosch H. J. Clin. Invest. 1993; 92: 2516-2523Crossref PubMed Scopus (208) Google Scholar), and is down-regulated by glucocorticoids (16Nakano T. Ohara O. Teraoka H. Arita H. J. Biol. Chem. 1990; 265: 12745-12748Abstract Full Text PDF PubMed Google Scholar). 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However, the molecular mechanisms whereby these sPLA2s regulate AA metabolism are still poorly understood. sPLA2s-IIA and -V have high affinities for heparanoids (2Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar), 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 (2Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 14Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 22Murakami M. Nakatani Y. Kudo I. J. Biol. 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Association of sPLA2-IIA with heparan or chondroitin sulfate chains increases the hydrolytic rate of phosphatidylcholine present in lipoprotein particles modestly (30Sartipy P. Johansen B. Camejo G. Rosengren B. Bondjers G. Hurt-Camejo E. J. Biol. Chem. 1996; 271: 26307-26314Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 31Sartipy P. Bondjers G. Hurt-Camejo E. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1934-1941Crossref PubMed Scopus (53) Google Scholar). 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 (32Koduri R.S. Baker S.F. Snitko Y. Han S.K. Cho W. Wilton D.C. Gelb M.H. J. Biol. Chem. 1998; 273: 32142-32153Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The cell surface HSPGs fall into two families of molecules that differ in their core protein domain structures (33David G. 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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 (36Mertens G. Van der Schueren B. Van den Berghe H. David G. J. Cell Biol. 1996; 132: 487-497Crossref PubMed Scopus (99) Google Scholar). Interestingly, recent immunohistochemical studies have revealed that significant portion of glypican translocates to the nucleus in cells undergoing cell division and activation (37Liang Y. Haring M. Roughley P.J. Margolis R.K. Margolis R.U. J. Cell Biol. 1997; 139: 851-864Crossref PubMed Scopus (119) Google Scholar). There is extensive literature concerning glycosaminoglycans in the nuclear compartment (38Ishihara M. Fedarko N.S. Conrad H.E. J. Biol. Chem. 1986; 261: 13575-13580Abstract Full Text PDF PubMed Google Scholar, 39Fedarko N.S. Conrad H.E. J. 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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 (45Smart E.J. Mineo C. Anderson R.G.W. J. Cell Biol. 1996; 134: 1169-1177Crossref PubMed Scopus (79) Google Scholar, 46Ilangumaran S. Robinson P.J. Hoessli D.C. Trends Cell Biol. 1996; 6: 163-167Abstract Full Text PDF PubMed Scopus (67) Google Scholar, 47Mayor S. Rothberg K.G. Maxfield F.R. Science. 1994; 264: 1948-1951Crossref PubMed Scopus (428) Google Scholar, 48Friedrichson T. Kurzchalia T.V. Nature. 1998; 394: 802-805Crossref PubMed Scopus (475) Google Scholar, 53Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274 (in press)Google Scholar), 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 (49Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1336) Google Scholar, 50Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1711) Google Scholar), 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. 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 (2Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 14Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 27Suga H. Murakami M. Kudo I. Inoue K. Eur. J. Biochem. 1993; 218: 807-813Crossref PubMed Scopus (70) Google Scholar). The cDNAs for mouse sPLA2-IIA and its heparin non-binding mutant KE4 (22Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), mouse cPLA2, human COX-1, and human COX-2 were described previously (2Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). 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 (51Murakami M. Kudo I. Natori Y. Inoue K. Biochim. Biophys. Acta. 1989; 1043: 34-42Crossref Scopus (50) Google Scholar). 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 TNFα was provided by Dr. H. Ishimaru (Asahi Chemical Industry). Human and mouse IL-1βs 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 heparinumheparinase 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 fromZymed Laboratories Inc. Cy3-conjugated donkey anti-rabbit IgG antibody was from Chemicon. Establishment of 293 cell transformants that stably expressed sPLA2-IIA, cPLA2, COX-1, and COX-2 was described previously (2Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). 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 theEcoRI 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. PLA2activity was assayed by measuring the amounts of free radiolabeled fatty acids released from the substrate 1-palmitoyl-2-[14 C]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 [14 C]fatty acids released were extracted and the radioactivity was counted as described previously (22Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). 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 [32 P]dCTP (Amersham Pharmacia Biotech) by random priming (Takara Shuzo). All hybridizations were carried out as described previously (22Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). 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 (22Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). 293 cells (5 × 104/ml) were seeded into each well of 24- or 48-well plates. To assess AA release, 0.1 μCi/ml [3 H]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-1β and/or 10% FCS was added to each well and the amount of free [3 H]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 [3 H]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 PGF2α. Therefore it is likely that the radioactivity released into the supernatants largely reflects [3 H]AA release. Culture and cytokine stimulation of 3Y1 (14Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) and BRL-3A (27Suga H. Murakami M. Kudo I. Inoue K. Eur. J. Biochem. 1993; 218: 807-813Crossref PubMed Scopus (70) Google Scholar) 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-1β and 100 units/ml human TNFα for 24 h in the medium containing 10% FCS. 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 (52Oishi T. Tamiya-Koizumi K. Kudo I. Iino S. Takagi K. Yoshida S. FEBS Lett. 1996; 394: 55-60Crossref PubMed Scopus (9) Google Scholar). 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. 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-1β (for 293 cells) or with 100 units/ml human TNFα and 1 ng/ml mouse IL-1β (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 sequ

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