Distinct Arachidonate-releasing Functions of Mammalian Secreted Phospholipase A2s in Human Embryonic Kidney 293 and Rat Mastocytoma RBL-2H3 Cells through Heparan Sulfate Shuttling and External Plasma Membrane Mechanisms
2001; Elsevier BV; Volume: 276; Issue: 13 Linguagem: Inglês
10.1074/jbc.m007877200
ISSN1083-351X
AutoresMakoto Murakami, Rao S. Koduri, Ayako Enomoto, Satoko Shimbara, Mimie Seki, Kumiko Yoshihara, Alan G. Singer, Emmanuel Di Valentin, Farideh Ghomashchi, Gérard Lambeau, Michael H. Gelb, Ichiro Kudo,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoWe analyzed the ability of a diverse set of mammalian secreted phospholipase A2(sPLA2) to release arachidonate for lipid mediator generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA2-IIA, -IID, and -V promote stimulus-dependent arachidonic acid release and prostaglandin E2 production in a manner dependent on the heparan sulfate proteoglycan glypican. In contrast, sPLA2-IB, -IIC, and -IIE, which bind weakly or not at all to heparanoids, fail to elicit arachidonate release, and addition of a heparin binding site to sPLA2-IIC allows it to release arachidonate. Heparin nonbinding sPLA2-X liberates arachidonic acid most likely from the phosphatidylcholine-rich outer plasma membrane in a glypican-independent manner. In rat mastocytoma RBL-2H3 cells that lack glypican, sPLA2-V and -X, which are unique among sPLA2s in being able to hydrolyze phosphatidylcholine-rich membranes, act most likely on the extracellular face of the plasma membrane to markedly augment IgE-dependent immediate production of leukotriene C4 and platelet-activating factor. sPLA2-IB, -IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells. These results are also supported by studies with sPLA2mutants and immunocytostaining and reveal that sPLA2-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent) mechanisms in HEK293 and RBL-2H3 cells. We analyzed the ability of a diverse set of mammalian secreted phospholipase A2(sPLA2) to release arachidonate for lipid mediator generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA2-IIA, -IID, and -V promote stimulus-dependent arachidonic acid release and prostaglandin E2 production in a manner dependent on the heparan sulfate proteoglycan glypican. In contrast, sPLA2-IB, -IIC, and -IIE, which bind weakly or not at all to heparanoids, fail to elicit arachidonate release, and addition of a heparin binding site to sPLA2-IIC allows it to release arachidonate. Heparin nonbinding sPLA2-X liberates arachidonic acid most likely from the phosphatidylcholine-rich outer plasma membrane in a glypican-independent manner. In rat mastocytoma RBL-2H3 cells that lack glypican, sPLA2-V and -X, which are unique among sPLA2s in being able to hydrolyze phosphatidylcholine-rich membranes, act most likely on the extracellular face of the plasma membrane to markedly augment IgE-dependent immediate production of leukotriene C4 and platelet-activating factor. sPLA2-IB, -IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells. These results are also supported by studies with sPLA2mutants and immunocytostaining and reveal that sPLA2-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent) mechanisms in HEK293 and RBL-2H3 cells. phospholipase A2 arachidonic acid antigen cyclooxygenase cytosolic PLA2 fetal calf serum interleukin-1 leukotriene platelet-activating factor phosphate-buffered saline phosphatidylcholine prostaglandin secreted PLA2 5-dimethylaminonaphthalene-1-sulfonyl Phospholipase A2(PLA2),1 which catalyzes the hydrolysis of membrane glycerophospholipids to produce free fatty acids and lysophospholipids, are a family of intracellular and extracellular enzymes (1Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar, 2Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 3Balsinde J. Dennis E.A. J. Biol. 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Acta. 2000; 1488: 59-70Crossref PubMed Scopus (313) Google Scholar, 27Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 23018-23024Abstract Full Text PDF PubMed Google Scholar). Group V sPLA2 (sPLA2-V) is expressed mainly in rat and human heart (5Valentin E. Lambeau G. Biochim. Biophys. Acta. 2000; 1488: 59-70Crossref PubMed Scopus (313) Google Scholar, 28Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 2365-2368Abstract Full Text PDF PubMed Google Scholar) and may in part compensate for sPLA2-IIA particularly in the mouse, in which sPLA2-V is inducibly expressed in many tissues by pro-inflammatory agents, whereas sPLA2-IIA expression is largely restricted to mouse intestine (29Sawada H. Murakami M. Enomoto A. Shimbara S. Kudo I. Eur. J. Biochem. 1999; 263: 826-835Crossref PubMed Scopus (83) Google Scholar, 30Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. 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Group IID (sPLA2-IID) and IIE (sPLA2-IIE) sPLA2s are structurally most related to sPLA2-IIA, and the genes for these three isozymes as well as those for group IIC, IIF, and V sPLA2s map to the same chromosome locus (4Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 5Valentin E. Lambeau G. Biochim. Biophys. Acta. 2000; 1488: 59-70Crossref PubMed Scopus (313) Google Scholar, 6Ishizaki J. Suzuki N. Higashino K. Yokota Y. Ono T. Kawamoto K. Fujii N. Arita H. Hanasaki K. J. Biol. Chem. 1999; 274: 24973-24979Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 7Valentin E. Koduri R.S. Scimeca J.C. Carle G. Gelb M.H. Lazdunski M Lambeau G. J. Biol. Chem. 1999; 274: 19152-19160Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 8Valentin E. Ghomashchi F. Gelb M.H. Lazdunski M. Lambeau G. J. Biol. 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Cellular functions of these novel sPLA2s remain to be elucidated. Because the sPLA2 family is diverse and the tissue distribution of each enzyme is unique, these enzymes are likely to have distinct physiological functions. In an effort to clarify the role of sPLA2s in the regulation of arachidonic acid (AA) release from membrane phospholipids, we have found that sustained expression of sPLA2-IIA or sPLA2-V by forcible gene transfer or by de novo induction following cytokine stimulation leads to efficient stimulus-dependent but not spontaneous AA release (24Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 33Murakami M. Kudo I. Inoue K. J. Biol. Chem. 1993; 268: 839-844Abstract Full Text PDF PubMed Google Scholar, 34Suga H. Murakami M. Kudo I. Inoue K. Eur. J. Biochem. 1993; 218: 807-813Crossref PubMed Scopus (70) Google Scholar, 35Murakami M. Nakatani Y. Kudo I. J. Biol. 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This liberated AA is functionally linked to cyclooxygenase (COX)-mediated prostaglandin (PG) production in several adherent cells (24Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 33Murakami M. Kudo I. Inoue K. J. Biol. Chem. 1993; 268: 839-844Abstract Full Text PDF PubMed Google Scholar, 34Suga H. Murakami M. Kudo I. Inoue K. Eur. J. Biochem. 1993; 218: 807-813Crossref PubMed Scopus (70) Google Scholar, 35Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 36Tada K. Murakami M. Kambe T. Kudo I. J. Immunol. 1998; 161: 5008-5015PubMed Google Scholar, 37Murakami 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, 38Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. 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To better understand the regulatory functions of sPLA2s in lipid mediator biosynthesis, we have extended our gain-of-function studies by transfecting human embryonic kidney 293 (HEK293) cells and rat mastocytoma RBL-2H3 cells with a variety of sPLA2s. Studies using sPLA2 mutants with altered heparanoid and interfacial binding properties provide additional data that help us to formulate models for the mechanisms of action of sPLA2s in these mammalian cells. Moreover, using RBL-2H3 cells, we have demonstrated, for the first time, the functional coupling between specific sPLA2s and the leukotriene (LT) and platelet-activating factor (PAF) biosynthetic pathways. HEK293 cells (Human Science Research Resources Bank) and RBL-2H3 cells (Riken Cell Bank) were cultured in RPMI 1640 (Nissui Pharmaceutical Co.) containing 10% fetal calf serum (Bioserum) as described previously (18Enomoto A. Murakami M. Valentin E. Lambeau G. Gelb M.H. Kudo I. J. 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Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), rat sPLA2-V and its mutant V-G30S (37Murakami 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), rat sPLA2-IIC, human sPLA2-X and its mutant X-G30S (40Murakami 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: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), rat glypican-1 (39Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), human COX-1 and -2 (38Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), all of which were subcloned into pcDNA3.1 (Invitrogen), were described previously. The cDNAs for mouse sPLA2-IID (7Valentin E. Koduri R.S. Scimeca J.C. Carle G. Gelb M.H. Lazdunski M Lambeau G. J. Biol. Chem. 1999; 274: 19152-19160Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), human sPLA2-IIA, and its mutants IIA-V3W and R7E/K10E/K16E (43Koduri 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,47Baker S.F. Othman R. Wilton D.C. Biochemistry. 1998; 37: 13203-13211Crossref PubMed Scopus (85) Google Scholar), and human sPLA2-V and its mutant V-W31A (44Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton W. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) were subcloned into pCI-neo (Promega). Mouse sPLA2-IIE cDNA (8Valentin E. Ghomashchi F. Gelb M.H. Lazdunski M. Lambeau G. J. Biol. Chem. 1999; 274: 31195-31202Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) was subcloned into pcDNA3.1(+)/hygro (Invitrogen). Rat sPLA2-IB cDNA was obtained by polymerase chain reaction using rat stomach cRNA as a template with a set of 23-base pair oligonucleotide primers corresponding to 5′- and 3′-nucleotide sequences of the open reading frame and subcloned into pCR3.1 (Invitrogen). C-terminally FLAG-tagged rat sPLA2-V, which was subcloned into pCR3.1, was described previously (29Sawada H. Murakami M. Enomoto A. Shimbara S. Kudo I. Eur. J. Biochem. 1999; 263: 826-835Crossref PubMed Scopus (83) Google Scholar). Mouse cytosolic PLA2 (cPLA2) cDNA was subcloned into pBK-CMV (Stratagene) (37Murakami 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, 52Nakatani Y. Tanioka Y. Sunaga S. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 1161-1168Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Site-directed mutagenesis was carried out directly on the mammalian expression plasmids using the QuickChange kit (Stratagene), and all plasmids were submitted to DNA sequencing of the full sPLA2 insert to confirm their sequences. Rabbit anti-human sPLA2-IIA antibody and the enzyme immunoassay kits for PGE2 and LTC4 were purchased from Cayman Chemicals. The rabbit anti-human COX-1, rabbit anti-human cPLA2, and goat anti-human COX-2 antibodies were purchased from Santa Cruz. Human IL-1β was 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 Pharmaceuticals. Heparin and Flavobacterium heparinum heparinase III were purchased from Sigma. Fluorescein isothiocyanate-conjugated goat anti-rabbit and -mouse IgG antibodies were purchased from Zymed Laboratories Inc. Mouse monoclonal anti-FLAG antibody was from Sigma. Mouse IgE anti-trinitrophenyl and trinitrophenyl-conjugated bovine serum albumin were provided by Dr. H. Katz (Harvard Medical School). Recombinant human sPLA2-IIA, mouse sPLA2-IID, and human sPLA2-X were produced in Escherichia coli as described (7Valentin E. Koduri R.S. Scimeca J.C. Carle G. Gelb M.H. Lazdunski M Lambeau G. J. Biol. Chem. 1999; 274: 19152-19160Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 43Koduri R.S. Baker S.F. Snitko Y. Han S.-K. Cho W. Wilton D.C. Gelb M.H. J. Biol. Chem. 1998; 273: 32142-32153Abst
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