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• W2020165436 abstract "Fas-mediated apoptosis of human leukemic U937 cells was accompanied by increased arachidonic acid (AA) and oleic acid release from membrane glycerophospholipids, indicating phospholipase A2 (PLA2) activation. During apoptosis, type IV cytosolic PLA2 (cPLA2), a PLA2 isozyme with an apparent molecular mass of 110 kDa critical for stimulus-coupled AA release, was converted to a 78-kDa fragment with concomitant loss of catalytic activity. Cleavage of cPLA2 correlated with increased caspase-3-like protease activity in apoptotic cells and was abrogated by a caspase-3 inhibitor. A mutant cPLA2 protein in which Asp522 was replaced by Asn, which aligns with the consensus sequence of the caspase-3 cleavage site (DXXD↓X), was resistant to apo-ptosis-associated proteolysis. Moreover, a COOH-terminal deletion mutant of cPLA2 truncated at Asp522 comigrated with the 78-kDa fragment and exhibited no enzymatic activity. Thus, caspase-3-mediated cPLA2 cleavage eventually leads to destruction of a catalytic triad essential for cPLA2 activity, thereby terminating its AA-releasing function. In contrast, the activity of type VI Ca2+-independent PLA2 (iPLA2), a PLA2 isozyme implicated in phospholipid remodeling, remained intact during apoptosis. Inhibitors of iPLA2, but neither cPLA2 nor secretory PLA2 inhibitors, suppressed AA release markedly and, importantly, delayed cell death induced by Fas. Therefore, we conclude that iPLA2-mediated fatty acid release is facilitated in Fas-stimulated cells and plays a modifying although not essential role in the apoptotic cell death process. Fas-mediated apoptosis of human leukemic U937 cells was accompanied by increased arachidonic acid (AA) and oleic acid release from membrane glycerophospholipids, indicating phospholipase A2 (PLA2) activation. During apoptosis, type IV cytosolic PLA2 (cPLA2), a PLA2 isozyme with an apparent molecular mass of 110 kDa critical for stimulus-coupled AA release, was converted to a 78-kDa fragment with concomitant loss of catalytic activity. Cleavage of cPLA2 correlated with increased caspase-3-like protease activity in apoptotic cells and was abrogated by a caspase-3 inhibitor. A mutant cPLA2 protein in which Asp522 was replaced by Asn, which aligns with the consensus sequence of the caspase-3 cleavage site (DXXD↓X), was resistant to apo-ptosis-associated proteolysis. Moreover, a COOH-terminal deletion mutant of cPLA2 truncated at Asp522 comigrated with the 78-kDa fragment and exhibited no enzymatic activity. Thus, caspase-3-mediated cPLA2 cleavage eventually leads to destruction of a catalytic triad essential for cPLA2 activity, thereby terminating its AA-releasing function. In contrast, the activity of type VI Ca2+-independent PLA2 (iPLA2), a PLA2 isozyme implicated in phospholipid remodeling, remained intact during apoptosis. Inhibitors of iPLA2, but neither cPLA2 nor secretory PLA2 inhibitors, suppressed AA release markedly and, importantly, delayed cell death induced by Fas. Therefore, we conclude that iPLA2-mediated fatty acid release is facilitated in Fas-stimulated cells and plays a modifying although not essential role in the apoptotic cell death process. Release of free fatty acids from glycerophospholipids, a major component of cell membranes, is crucial for various cellular responses, such as signal transduction and membrane remodeling and is tightly regulated by a diverse family of phospholipase A2(PLA2) 1The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic phospholipase A2; sPLA2, secretory PLA2; iPLA2, Ca2+-independent PLA2; TNF, tumor necrosis factor; AA, arachidonic acid; AACOCF3, arachidonoyl trifluoromethyl ketone; MAFP, methyl arachidonylfluorophosphonate; PAGE, polyacrylamide gel electrophoresis; MAPK, mitogen-activated protein kinase; DAPI, 4′,6′-diamidino-2-phenylindole; PIPES, 1,4-piperazinediethanesulfonic acid. enzymes. Type IV cytosolic PLA2 (cPLA2), which exhibits strict substrate specificity for arachidonic acid (AA)-containing phospholipids in the presence of submicromolar concentrations of Ca2+, is a key enzyme that triggers stimulus-initiated AA metabolism, leading to production of bioactive lipid mediators in activated cells (1Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar, 2Clark 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 (1465) Google Scholar). Various Ca2+-mobilizing agonists and proinflammatory cytokines induce cPLA2 activation for immediate and delayed eicosanoid biosynthesis, respectively, through mechanisms involving Ca2+-dependent translocation to the perinuclear and endoplasmic reticular membranes and phosphorylation by kinases belonging to the mitogen-activated protein kinase (MAPK) family (3Schievella A.R. Regier M.K. Smith W.L. Lin L.-L. J. Biol. Chem. 1995; 270: 30749-30754Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 4Lin 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 (1659) Google Scholar, 5Glover S. de Carvalho M.S. Bayburt T. Jonas M. Chi E. Leslie C.C. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 6Roshak A. Sathe G. Marshall L.A. J. Biol. Chem. 1994; 269: 25999-26005Abstract Full Text PDF PubMed Google Scholar, 7Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Evidence is also accumulating that certain phases of stimulus-initiated AA release are regulated by types IIA or V secretory PLA2 (sPLA2) isozymes, which require millimolar concentrations of Ca2+ for catalytic activity (8Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 9Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 10Murakami, M., Shimbara, S., Kambe, T., Kuwata, H., Winstead, M. V., Tischfield, J. A., and Kudo, I. (1998) J. Biol. Chem.273, in pressGoogle Scholar, 11Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 12Reddy S.T. Winstead M.V. Tischfield J.A. Herschman H.R. J. Biol. Chem. 1997; 272: 13591-13596Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In contrast, type VI Ca2+-independent PLA2 (iPLA2) has been proposed to participate in fatty acid release associated with phospholipid remodeling and to play a minimal role in signal transduction (13Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 14Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 15Balsinde J. Balboa M.A. Dennis E.A. J. Biol. Chem. 1997; 272: 29317-29321Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The programmed cell death pathway called apoptosis plays a fundamental role in tissue homeostasis, development, and host defense. The discovery of increasing numbers of death factors (e.g. Fas ligand and tumor necrosis factor (TNF) α), their receptors, adapter molecules, and caspases have provided new insight into the molecular mechanisms leading to apoptosis that are highly conserved in eukaryotic cells (16Cleveland J.L. Ihle J.N. Cell. 1995; 81: 479-482Abstract Full Text PDF PubMed Scopus (323) Google Scholar, 17Liu Z.-G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar). Following ligation of these receptors by their cognate ligands, several cytoplasmic signal-transducing adapters are recruited to the receptors to form multimeric complexes, thereby leading to either apoptosis or cellular activation, depending on the adapters involved (18Hsu H. Shu H.-B. Pan M.-G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar, 19Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1749) Google Scholar). In the apoptotic pathway, these adapter molecules in turn recruit particular caspases (20Boldin M.P. Goncharav T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar, 21Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar, 22Duan H. Dixit V.M. Nature. 1997; 385: 86-89Crossref PubMed Scopus (469) Google Scholar), which trigger the protease cascade where multiple caspases are sequentially activated as a consequence of their proteolytic processing at a specific Asp residue, allowing them to become self-activated and activate one another. Activated caspases then cleave their respective substrates (23Enari M. Talanian R.V. Wong W.W. Nagata S. Nature. 1996; 380: 723-726Crossref PubMed Scopus (954) Google Scholar, 24Fraser A. Evan G. Cell. 1996; 85: 781-784Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, 25Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2165) Google Scholar, 26Martins L.M. Earnshaw W.C. Trends Cell Biol. 1997; 7: 111-114Abstract Full Text PDF PubMed Scopus (118) Google Scholar, 27Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar, 28Martin S.J. Green D.R. Cell. 1995; 82: 349-352Abstract Full Text PDF PubMed Scopus (1263) Google Scholar, 29Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar, 30Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (605) Google Scholar), which are key regulatory and structural proteins such as protein kinases and proteins involved in DNA repair and cytoskeleton integrity, thereby contributing to the demise of the cell. The changes in glycerophospholipid metabolism, unlike those in protein and DNA levels, during apoptosis are rather obscure. Although the involvement of particular PLA2 enzymes in stimulus-coupled eicosanoid biosynthesis has been studied extensively as discussed above, relatively little is known about their roles in fatty acid release associated with apoptotic signaling. Recently, we and others showed that the plasma membrane phospholipids of apoptotic cells are the preferred substrates for type IIA sPLA2 (31Atsumi G. Murakami M. Tajima M. Shimbara S. Hara N. Kudo I. Biochim. Biophys. Acta. 1997; 1349: 43-54Crossref PubMed Scopus (89) Google Scholar, 32Wang H. Harrison-Shostak D.C. Lemasters J.J. Herman B. FASEB J. 1996; 10: 1318-1325Crossref Scopus (23) Google Scholar), which is induced by proinflammatory stimuli and has been implicated in the pathogenesis of inflammation (10Murakami, M., Shimbara, S., Kambe, T., Kuwata, H., Winstead, M. V., Tischfield, J. A., and Kudo, I. (1998) J. Biol. Chem.273, in pressGoogle Scholar, 33Kudo I. Murakami M. Hara S. Inoue K. Biochim. Biophys. Acta. 1993; 1170: 217-231Crossref PubMed Scopus (372) Google Scholar). It has been speculated that the AA thus liberated is taken up by surrounding live cells to be metabolized to eicosanoids, representing a particular transcellular pathway for AA metabolism that contributes to propagation of inflammation. cPLA2 has also been implicated in AA release during certain cell death processes, such as TNFα-induced apoptosis (34Voelkel-Johnson C. Thorne T. Laster S.M. J. Immunol. 1996; 156: 201-207PubMed Google Scholar, 35Hayakawa M. Ishida N. Takeuchi K. Shibamoto S. Hori T. Oku N. Ito F. Tsujimoto M. J. Biol. Chem. 1993; 268: 11290-11295Abstract Full Text PDF PubMed Google Scholar, 36Wissing D. Mouritzen H. Egeblad M. Poirier G.G. Jaattela M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5073-5077Crossref PubMed Scopus (183) Google Scholar) and hydroperoxide-induced cytotoxicity (37Sapirstein A. Spech R.A. Witzgall R. Bonventre J.V. J. Biol. Chem. 1996; 271: 21505-21513Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). In these studies, the use of cPLA2 inhibitors, overexpression, and antisense technologies revealed significant correlations between the cPLA2 expression level, amounts of AA released, and cell death. However, the role of cPLA2 is uncertain in Fas-mediated apoptosis (38Enari M. Hug H. Hayakawa M. Ito F. Nishimura Y. Nagata S. Eur. J. Biochem. 1996; 236: 533-538Crossref PubMed Scopus (52) Google Scholar). In order to explore the role of particular PLA2 isozymes in fatty acid release and glycerophospholipid metabolism alterations associated with the apoptotic pathway, we chose the Fas system, which strongly promotes apoptosis but barely elicits cell activation signals. We now provide evidence that Fas-mediated apoptosis of human leukemic U937 cells is accompanied by gradual fatty acid release, which appears to be mediated by iPLA2 but not by cPLA2 or sPLA2. During apoptosis, cPLA2 is inactivated by caspase-3-dependent proteolytic cleavage at Asp522. Inhibition of iPLA2-evoked fatty acid release results in slight but significant retardation of cell death, suggesting that disturbed phospholipid turnover renders cells more susceptible to Fas-mediated apoptotic signaling. Mouse cPLA2 cDNA and a rabbit antiserum against human cPLA2 were provided by Drs. M. Tsujimoto (RIKEN Institute) and J. D. Clark (Genetics Institute), respectively. The agonistic anti-Fas antibody (CH-11) (39Yonehara S. Ishii A. Yonehara M. J. Exp. Med. 1989; 169: 1747-1756Crossref PubMed Scopus (1428) Google Scholar) was purchased from MBL. The cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3) (40Street I.P. Lin H.-K. Laliberte F. Ghomashchi F. Wang Z. Perrier H. Tremblay N.M. Huang Z. Weech P.K. Gelb M.H. Biochemistry. 1993; 32: 5935-5940Crossref PubMed Scopus (419) Google Scholar) was purchased from Calbiochem. Methyl arachidonylfluorophosphonate (MAFP), which inhibits both cPLA2 and iPLA2 (14Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar), and the iPLA2 inhibitor bromoenol lactone (13Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 14Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 15Balsinde J. Balboa M.A. Dennis E.A. J. Biol. Chem. 1997; 272: 29317-29321Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) were purchased from Cayman Chemical. The type IIA sPLA2 inhibitor LY311727 (41Schevitz R.W. Bach N.J. Carlson D.G. Chirgadze N.Y. Clawson D.K. Dillard R.D. Draheim S.E. Hartley L.W. Jones N.D. Mihelich E.D. Olkowski J.L. Snyder D.W. Sommers C. Wery J.-P. Nat. Struct. Biol. 1995; 2: 458-465Crossref PubMed Scopus (236) Google Scholar) was donated by Dr. R. M. Kramer (Lilly Research). Two caspase inhibitors (Ac-YVAD-CHO and Ac-DEVD-CHO) and chymostatin were obtained from the Peptide Institute, and caspase substrates were obtained from Takara Biomedicals. LipofectAMINE PLUS reagent, Opti-MEM medium, and TRIzol reagent were obtained from Life Technologies. Etoposide,p-bromophenacyl bromide, 4′,6′-diamidino-2-phenylindole (DAPI), leupeptin, antipain, pepstatin, cytochalasin B, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate andNα-benzoyl-arginine ethyl ester were purchased from Sigma. All the other reagents used were of analytical grade and purchased from Wako, unless stated otherwise. The human monocytic leukemia cell lines U937 and HL-60 and human embryonic kidney 293 cells (RIKEN Cell Bank) were maintained in RPMI 1640 medium (Nissui Pharmaceutical) supplemented with 10% (v/v) fetal calf serum, 2 mmglutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in humidified air with 5% CO2. In order to investigate AA and oleic acid release, U937 cells were preincubated with 0.1 and 0.5 μCi/ml [3H]AA and [3H]oleic acid (NEN Life Science Products), respectively, for 24 h. Then the cells were washed three times, resuspended in culture medium to produce 1 × 107 cells/ml, and incubated for various periods with or without the anti-Fas antibody. In the experiments to determine the effect of etoposide treatment, the cells were incubated with etoposide for 1 h, washed three times, and then incubated for a further 10 h in culture medium without etoposide. In some experiments, various PLA2 inhibitors were added to the cells during incubation. The process of cell death was monitored by observing the morphological changes, assessing the cell viability by trypan blue dye exclusion, and quantifying DNA fragmentation fluorometrically using DAPI as described previously (42Kizaki H. Tadakuma T. Odaka C. Muramatsu J. Ishimura Y. J. Immunol. 1989; 143: 1790-1794PubMed Google Scholar). The free 3H-labeled fatty acids released were extracted by the method of Dole and Meinertz (43Dole V.P. Meinertz H. J. Biol. Chem. 1960; 235: 2595-2599Abstract Full Text PDF PubMed Google Scholar), and the associated radioactivity was counted using a liquid β-scintillation counter (Aloka). The amount of each fatty acid released, expressed as a percentage, was calculated using the formula [S/(S +P)] × 100, where S and P are the radioactivities of equal portions of supernatant and cell pellet, respectively. Cells were washed with phosphate-buffered saline and then lysed in phosphate-buffered saline containing 100 μm p-4-(2-aminoethyl)-benzenesulfonyl fluoride, 5 μm iodoacetamide, 5 mm EDTA, 1 μm pepstatin, 1 mg/ml soybean trypsin inhibitor, and 100 μm leupeptin by sonication for 1 min with a Branson Sonifer (power 30, 50% pulse cycle). The samples (10 μg protein equivalents/lane) were subjected to 10% (w/v) SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions and electroblotted onto nitrocellulose membranes (Schleicher & Schuell), which were probed with the antibody against human cPLA2 and visualized with the ECL Western blot analysis system (Amersham Pharmacia Biotech), as described previously (8Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The protein contents were quantified using a BCA protein assay kit (Pierce). In order to assess cPLA2 activity, cells were washed once with phosphate-buffered saline, suspended in buffer comprising 10 mm Tris-HCl (pH 7.4) and 150 mm NaCl (Tris-buffered saline), and lysed by sonication as described above. The resulting lysates were incubated in 250 μl of buffer comprising 100 mm Tris-HCl (pH 9.0), 4 mm CaCl2, and 2 μm1-palmitoyl-2-[14C]arachidonyl-glycerophosphoethanolamine (NEN Life Science Products) as the substrate at 37 °C for 30 min. In order to assess iPLA2 activity, the lysates, prepared in 10 mm HEPES (pH 7.5) containing 1 mm EDTA, 1 mm dithiothreitol, and 0.34 m sucrose, were incubated in 250 μl of buffer comprising 100 mm HEPES (pH 7.5), 5 mm EDTA, 0.4 mm Triton X-100, 0.1 mm ATP, and 10 μm1-palmitoyl-2-[14C]arachidonyl-glycerophosphoethanolamine at 40 °C for 30 min (13Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 15Balsinde J. Balboa M.A. Dennis E.A. J. Biol. Chem. 1997; 272: 29317-29321Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The [14C]AA released was extracted by the method of Dole and Meinertz (43Dole V.P. Meinertz H. J. Biol. Chem. 1960; 235: 2595-2599Abstract Full Text PDF PubMed Google Scholar), and the associated radioactivity was counted. The total lipids were extracted from [3H]AA-labeled cells and their supernatants by the method of Bligh and Dyer (44Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-918Crossref PubMed Scopus (43132) Google Scholar) and developed by two-dimensional thin layer chromatography on silica 60 gel plates (Merck), according to a method described previously with a slight modification (31Atsumi G. Murakami M. Tajima M. Shimbara S. Hara N. Kudo I. Biochim. Biophys. Acta. 1997; 1349: 43-54Crossref PubMed Scopus (89) Google Scholar). The first solvent system consisted of chloroform/methanol/acetic acid/water (65/25/4/2, v/v/v), and the second comprised chloroform/methanol/formic acid (65/25/8.8, v/v/v). The zones on the silica gel corresponding to AA and phospholipids were identified by comparison with the mobilities of authentic standards and visualized with iodine vapor. Each zone was scraped into a vial, and its radioactivity was counted using a liquid β-scintillation counter. To separate free fatty acids and other neutral lipids, including triacylglyceride, the neutral lipid fraction was developed further on fresh plates with a solvent system of hexane/ether/acetic acid (80/30/1, v/v/v). Sf9 cells (Invitrogen) were transfected with mouse cPLA2 cDNA that had been subcloned into the pVL1392 baculovirus transfection vector (Pharmingen), as described previously (8Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The cells were harvested, suspended in a buffer comprising 250 mm sucrose, 10 mm Tris-HCl (pH 7.0), 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 mm Nα-benzoyl-arginine ethyl ester, 10 μg/ml leupeptin, and 10 μg/ml antipain, and disrupted by sonication for 1 min, as described above. The lysates were centrifuged at 100,000 × g for 1 h at 4 °C, and the resulting supernatants were applied to a DEAE-Sephacel column (Amersham Pharmacia Biotech) that had been pre-equilibrated with Tris-buffered saline. After washing the column with Tris-buffered saline, the bound proteins were eluted with a linear NaCl gradient from 0.15 to 0.5m in 10 mm Tris-HCl (pH 7.4). The eluted fractions containing cPLA2 activity were pooled, applied to a phenyl-Superose column (Amersham Pharmacia Biotech) that had been pre-equilibrated with 10 mm Tris-HCl (pH 7.4) containing 1m NaCl, and the bound cPLA2 was eluted stepwise with 10 mm Tris-HCl (pH 7.4). Cells were washed with phosphate-buffered saline, resuspended (108 cells/ml) in an extraction buffer comprising 50 mm PIPES-NaOH (pH 7.0), 50 mm KCl, 5 mm EGTA, 2 mmMgCl2, 1 mm dithiothreitol, 20 μmcytochalasin B, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 50 μg/ml antipain, and 10 mg/ml chymostatin, and disrupted by freeze-thawing twice. Aliquots (10 μg of protein equivalents) were incubated with 1 μmMca-YVADAPK(Dnp)-OH or Mca-DEVDAPK(Dnp)-OH, substrates for caspase-1 and caspase-3, respectively (45Enari M. Talanian R.V. Wong W.W. Nagata S. Nature. 1996; 380: 723-726Crossref PubMed Scopus (969) Google Scholar), in a buffer containing 100 mm HEPES (pH 7.5), 10% (w/v) sucrose, 0.1% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 mm dithiothreitol, and 0.1 mg/ml ovalbumin, at 30 °C for 30 min. Then the fluorescence of each cleaved substrate was determined using a spectrofluorometer (Hitachi) at excitation and emission wavelengths of 325 and 392 nm, respectively. Substitution of Asp522 of mouse cPLA2 with Asn (cPLA2-D522N) was performed by altering the codon GTC to GTT using the QuikChange™ site-directed mutagenesis kit (Stratagene) with two synthetic oligonucleotides for the sense and antisense strands: 5′-C CTT CGA TGA CGA GCT CAA CGC AGC GGT AGC AG-3′ and 5′-C TGC TAC CGC TGC GTT GAG CTC GTC ATC GAA GG-3′, respectively (altered codons are underlined). Briefly, mouse cPLA2 cDNA subcloned into pBK-CMV (Stratagene) at theEcoRI site was denatured and annealed with the two oligonucleotide primers containing the desired mutation. After incubation with Pfu DNA polymerase followed by treatment with DpnI, the resulting mutated plasmid was transfected into Escherichia coli XL1-Blue supercompetent cells (Stratagene). A cPLA2 deletion mutant, cPLA2(1–522), was constructed by polymerase chain reaction amplification of the cPLA2 coding sequence with ex Taq polymerase (Takara) using the oligonucleotide pair 5′-ATG TCA TTT ATA GAT CCT TAC-3′ and 5′-TCA GTC GAG CTC GTC ATC GAA-3′. The polymerase chain reaction product was ligated into pCR™3.1 (Invitrogen) and was transfected into Top10F′ supercompetent cells (Invitrogen). Colonies were picked up, and the plasmids were isolated and sequenced using a Taq cycle sequencing kit (Takara) and an auto-fluorometric DNA sequencer (DSQ-1000L, Shimadzu) to confirm the mutation. [35S]Methionine-labeled cPLA2 and its mutants were synthesized using a PROTEINscript™ kit (Ambion). Briefly, plasmids containing mouse cPLA2, cPLA2-D522N, or cPLA2(1–522) cDNA were transcribed using RNA polymerase and then incubated with [35S]methionine (NEN Life Science Products) and rabbit reticulocyte lysate. The products were subjected to SDS-PAGE and visualized autoradiographically. Aliquots of [35S]cPLA2 or baculovirus-derived recombinant cPLA2 proteins prepared as described above were incubated with the cytosolic fraction obtained from U937 cells that had been treated for 12 h with or without the anti-Fas antibody in the presence or absence of a caspase inhibitor (Ac-YVAD-CHO or Ac-DEVD-CHO) (45Enari M. Talanian R.V. Wong W.W. Nagata S. Nature. 1996; 380: 723-726Crossref PubMed Scopus (969) Google Scholar). The reaction products were analyzed by SDS-PAGE followed by autoradiography and immunoblotting, respectively. The cDNAs for mouse cPLA2 and its mutants cPLA2-D522N and cPLA2(1–522) that had been subcloned into pBK-CMV and PCR 3.1™, respectively, were each transfected into 293 cells using LipofectAMINE PLUS reagent according to the manufacturer's instructions as described previously (46Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar). The cells were harvested 48 h after transfection, lysed, and subjected to immunoblotting and PLA2 assay. Human monocytic leukemia U937 cells were prelabeled with [3H]AA for 24 h, washed, and then exposed to various concentrations of the agonistic anti-Fas antibody for various periods to assess the changes in the free [3H]AA levels (Fig.1 A). AA release by cells cultured without the anti-Fas antibody increased minimally over 24 h, whereas that by replicate cells treated with the anti-Fas antibody increased significantly after culture for 3–24 h in a concentration-dependent manner. AA release by U937 cells treated with 10, 50, and 100 ng/ml anti-Fas antibody for 24 h was about 3.5, 5.0, and 6.5 times higher, respectively, than that by the cells before anti-Fas antibody addition (Fig. 1 A). AA release was almost parallel to the reductions in cell viability and DNA fragmentation, assessed by trypan blue dye exclusion and fluorometric quantification with DAPI (33Kudo I. Murakami M. Hara S. Inoue K. Biochim. Biophys. Acta. 1993; 1170: 217-231Crossref PubMed Scopus (372) Google Scholar), respectively (Fig. 1, B andC), as well as DNA rudder formation, assessed by agarose gel electrophoresis (data not shown). After a 24-h preincubation of U937 cells with [3H]AA, nearly 95% of the radioactivity was incorporated into phospholipid pools, mainly into phosphatidylcholine and phosphatidylethanolamine, followed by phosphatidylinositol and phosphatidylserine (Fig.2). Up to 5% of the radioactivity was incorporated into the neutral lipid fraction, mainly into triacylglycerol (Fig. 2, right panel). After incubation with 50 ng/ml anti-Fas antibody, the free [3H]AA level increased significantly, accompanied by reductions in the percentages of the total [3H]AA remaining in phosphatidylethanolamine and phosphatidylcholine without appreciable changes of those in other phospholipids and triacylglycerol (Fig. 2). In terms of the total counts, the combined decrements in these two phospholipids roughly matched the net increase in free [3H]AA. Initially, we thought that the increased Fas-mediated AA release from apoptotic U937 cells was due to cPLA2activation. Unexpectedly, however, the cPLA2 catalytic activity toward exogenous substrate gradually declined during the Fas-mediated apoptotic process: the cPLA2 activities of cells after treatment with 10 and 100 ng/ml anti-Fas antibody for 12 h were about one-half and one-ninth, respectively, of that of untreated cells (Fig. 3 A). The cPLA2 inhibitor AACOCF3, at 1 μm, abolished the cPLA2 activity of U937 cells, whereas [3H]AA release by apoptotic cells incubated with and without AACOCF3 at 10 μm, the concentration at which it inhibits cPLA2 but not other PLA2s (Ref. 13Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar and data not shown), did not differ significantly (Fig.3 B). Moreover, net amounts of [3H]oleic acid comparable with those of [3H]AA were released by anti-Fas antibody-treated cells prelabeled with these fatty acids (Fig.3 C), arguing against the AA selective property of cPLA2. In order to elucidate the mechanisms responsible for the decrease in cPLA2 activity in cells undergoing Fas-mediated apoptosis, we examined cPLA2 protein expression by immunoblotting. We found that treating U937 cells with the anti-Fas antibody resulted in time- (Fig. 4 A) and dose-dependent (Fig. 4 B) cleavage of intact cPLA2 with an apparent molecular mass of 110 kDa, which was present in untreated cells, to a proteolytic fragment of approximately 78 kDa. Proteolysis of cPLA2 was detectable with" @default.
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• W2020165436 title "Fas-induced Arachidonic Acid Release Is Mediated by Ca2+-independent Phospholipase A2 but Not Cytosolic Phospholipase A2, Which Undergoes Proteolytic Inactivation" @default.
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