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• W2038688567 abstract "Group IVA cytosolic phospholipase A2 (cPLA2α) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA2α–/– lung fibroblasts stimulated with A23187 or serum. cPLA2α+/+ fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA2α–/– fibroblasts nonspecifically released multiple fatty acids. Arachidonic acid release from cPLA2 α–/– fibroblasts was inhibited by the cPLA2α inhibitors pyrrolidine-2 (IC50, 0.03 μm) and Wyeth-1 (IC50, 0.1 μm), implicating another C2 domain-containing group IV PLA2. cPLA2 α–/– fibroblasts contain cPLA2β and cPLA2ζ but not cPLA2ϵ or cPLA2δ. Purified cPLA2ζ exhibited much higher lysophospholipase and PLA2 activity than cPLA2β and was potently inhibited by pyrrolidine-2 and Wyeth-1, which did not inhibit cPLA2β. In contrast to cPLA2β, cPLA2ζ expressed in Sf9 cells mediated A23187-induced arachidonic acid release, which was inhibited by pyrrolidine-2 and Wyeth-1. cPLA2ζ exhibits specific activity, inhibitor sensitivity, and low micromolar calcium dependence similar to cPLA2α and has been identified as the PLA2 responsible for calcium-induced fatty acid release and prostaglandin E2 production from cPLA2 α–/– lung fibroblasts. In response to ionomycin, EGFP-cPLA2ζ translocated to ruffles and dynamic vesicular structures, whereas EGFP-cPLA2α translocated to the Golgi and endoplasmic reticulum, suggesting distinct mechanisms of regulation for the two enzymes. Group IVA cytosolic phospholipase A2 (cPLA2α) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA2α–/– lung fibroblasts stimulated with A23187 or serum. cPLA2α+/+ fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA2α–/– fibroblasts nonspecifically released multiple fatty acids. Arachidonic acid release from cPLA2 α–/– fibroblasts was inhibited by the cPLA2α inhibitors pyrrolidine-2 (IC50, 0.03 μm) and Wyeth-1 (IC50, 0.1 μm), implicating another C2 domain-containing group IV PLA2. cPLA2 α–/– fibroblasts contain cPLA2β and cPLA2ζ but not cPLA2ϵ or cPLA2δ. Purified cPLA2ζ exhibited much higher lysophospholipase and PLA2 activity than cPLA2β and was potently inhibited by pyrrolidine-2 and Wyeth-1, which did not inhibit cPLA2β. In contrast to cPLA2β, cPLA2ζ expressed in Sf9 cells mediated A23187-induced arachidonic acid release, which was inhibited by pyrrolidine-2 and Wyeth-1. cPLA2ζ exhibits specific activity, inhibitor sensitivity, and low micromolar calcium dependence similar to cPLA2α and has been identified as the PLA2 responsible for calcium-induced fatty acid release and prostaglandin E2 production from cPLA2 α–/– lung fibroblasts. In response to ionomycin, EGFP-cPLA2ζ translocated to ruffles and dynamic vesicular structures, whereas EGFP-cPLA2α translocated to the Golgi and endoplasmic reticulum, suggesting distinct mechanisms of regulation for the two enzymes. Mammals contain a number of phospholipases A2 (PLA2) 2The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic PLA2; iPLA2, calcium-independent PLA2; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PGE2, prostaglandin E2; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; Sf9, Spodoptera frugiperda; MLF, mouse lung fibroblasts; IMLF, immortalized MLF; EGFP, enhanced green fluorescent protein; GIV, group IV; RACE, rapid amplification of cDNA ends; MOPS, 4-morpholinepropanesulfonic acid.2The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic PLA2; iPLA2, calcium-independent PLA2; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PGE2, prostaglandin E2; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; Sf9, Spodoptera frugiperda; MLF, mouse lung fibroblasts; IMLF, immortalized MLF; EGFP, enhanced green fluorescent protein; GIV, group IV; RACE, rapid amplification of cDNA ends; MOPS, 4-morpholinepropanesulfonic acid. that include secreted forms, intracellular group IV cytosolic PLA2s (GIV cPLA2), and group VI calcium-independent PLA2s (GVI iPLA2) (1Kudo I. Murakami M. Prostaglandins Other Lipid Mediat. 2002; 68–69: 3-58Crossref PubMed Scopus (655) Google Scholar, 2Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1203) Google Scholar). The presence of diverse PLA2s provides cells with differentially regulated pathways for the hydrolysis of fatty acids from phospholipid. Intracellular PLA2s exhibit multiple enzymatic activities (PLA2, PLA1, lysophospholipase, transacylase) to varying degrees, which can potentially result in the formation of a diverse number of phospholipid breakdown products (3Leslie C.C. Biochem. Cell Biol. 2004; 82: 1-17Crossref PubMed Scopus (106) Google Scholar, 4Ghosh M. Tucker D.E. Burchett S.A. Leslie C.C. Prog. Lipid Res. 2006; 45: 487-510Crossref PubMed Scopus (304) Google Scholar). The direct products of PLA2 action, lysophospholipids and fatty acids, can themselves act as cellular mediators or serve as precursors for the formation of mediators such as platelet-activating factor and eicosanoids.There are six enzymes classified as GIV PLA2s: cPLA2α (GIVA), cPLA2β (GIVB), cPLA2γ (GIVC), cPLA2δ (GIVD), cPLA2ϵ (GIVE), and cPLA2ζ (GIVF) (4Ghosh M. Tucker D.E. Burchett S.A. Leslie C.C. Prog. Lipid Res. 2006; 45: 487-510Crossref PubMed Scopus (304) Google Scholar). These enzymes contain a conserved Ser/Asp active site dyad and an Arg residue, which are critical for catalytic activity. cPLA2α has been studied extensively because it is the only PLA2 that exhibits specificity for hydrolysis of sn-2 arachidonic acid from phospholipids (4Ghosh M. Tucker D.E. Burchett S.A. Leslie C.C. Prog. Lipid Res. 2006; 45: 487-510Crossref PubMed Scopus (304) Google Scholar, 5Clark J.D. Schievella A.R. Nalefski E.A. Lin L.-L. J. Lipid Mediat. Cell Signal. 1995; 12: 83-117Crossref PubMed Scopus (425) Google Scholar, 6Leslie C.C. Prostagl. Leukot. Essent. Fatty Acids. 2004; 70: 373-376Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Arachidonic acid is the precursor of a large number of biologically active oxygenated metabolites including prostaglandins and leukotrienes. cPLA2α is a highly regulated enzyme, which is important in controlling the availability of free arachidonic acid in cells for the production of eicosanoids (7Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (2997) Google Scholar). cPLA2α is regulated by phosphorylation and an increase in intracellular calcium. Calcium binds to the calcium- and phospholipid-binding C2 domain on cPLA2α, which promotes its translocation from the cytosol to the Golgi, endoplasmic reticulum, and nuclear envelope, where it can access substrate (4Ghosh M. Tucker D.E. Burchett S.A. Leslie C.C. Prog. Lipid Res. 2006; 45: 487-510Crossref PubMed Scopus (304) Google Scholar, 8Glover 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, 9Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar, 10Perisic O. Fong S. Lynch D.E. Bycroft M. Williams R.L. J. Biol. Chem. 1998; 273: 1596-1604Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 11Evans J.H. Spencer D.M. Zweifach A. Leslie C.C. J. Biol. Chem. 2001; 276: 30150-30160Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). cPLA2α is phosphorylated on serine residues in the catalytic domain. Phosphorylation of Ser-505 by mitogen-activated protein kinases occurs in response to diverse agonists and is required for cPLA2α-mediated release of arachidonic acid in stimulated cells (12Lin 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, 13Hefner Y. Borsch-Haubold A.G. Murakami M. Wilde J.I. Pasquet S. Schieltz D. Ghomashchi F. Yates 3rd, J.R. Armstrong C.G. Paterson A. Cohen P. Fukunaga R. Hunter T. Kudo I. Watson S.P. Gelb M.H. J. Biol. Chem. 2000; 275: 37542-37551Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar).Much less is known about the regulation and physiological function of the other GIV PLA2s (cPLA2β, -γ, -δ, -ϵ, -ζ) (4Ghosh M. Tucker D.E. Burchett S.A. Leslie C.C. Prog. Lipid Res. 2006; 45: 487-510Crossref PubMed Scopus (304) Google Scholar). cPLA2γ is the only GIV enzyme that does not contain a C2 domain (14Pickard R.T. Strifler B.A. Kramer R.M. Sharp J.D. J. Biol. Chem. 1999; 274: 8823-8831Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 15Underwood K.W. Song C. Kriz R.W. Chang X.J. Knopf J.L. Lin L.-L. J. Biol. Chem. 1998; 273: 21926-21932Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). It contains fatty acyl and C-terminal farnesyl groups, and is constitutively bound to membrane (16Jenkins C.M. Han X. Yang J. Mancuseo D.J. Sims H.G. Muslin A.J. Gross R.W. Biochemistry. 2003; 42: 11798-11807Crossref PubMed Scopus (30) Google Scholar, 17Tucker D.E. Stewart A. Nallan L. Bendale P. Ghomashchi F. Gelb M.H. Leslie C.C. J. Lipid Res. 2005; 46: 2122-2133Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Human cPLA2γ is expressed most abundantly in heart and skeletal muscle; however, its role in these tissues is unknown. In contrast, the mouse cPLA2γ homologue is only 50% homologous to human cPLA2γ and is expressed exclusively in oocytes (18Vitale A. Perlin J. Leonelli L. Herr J. Wright P. Digilio L. Coonrod S. Dev. Biol. 2005; 282: 374-384Crossref PubMed Scopus (20) Google Scholar). cPLA2δ, cPLA2ϵ, and cPLA2ζ form a gene cluster near cPLA2β in humans and mice and have more homology to cPLA2β than to cPLA2α (4Ghosh M. Tucker D.E. Burchett S.A. Leslie C.C. Prog. Lipid Res. 2006; 45: 487-510Crossref PubMed Scopus (304) Google Scholar, 19Chiba H. Michibata H. Wakimoto K. Seishima M. Kawasaki S. Okubo K. Mitsui H. Torii H. Imai Y. J. Biol. Chem. 2004; 279: 12890-12897Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 20Ohto T. Uozumi N. Hirabayashi T. Shimizu T. J. Biol. Chem. 2005; 280: 24576-24583Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Human cPLA2δ is associated with psoriatic lesions and is expressed in stratified squamous epithelium (19Chiba H. Michibata H. Wakimoto K. Seishima M. Kawasaki S. Okubo K. Mitsui H. Torii H. Imai Y. J. Biol. Chem. 2004; 279: 12890-12897Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Human cPLA2β is widely expressed and occurs as multiple splice variants (14Pickard R.T. Strifler B.A. Kramer R.M. Sharp J.D. J. Biol. Chem. 1999; 274: 8823-8831Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 21Song C. Chang X.J. Bean K.M. Proia M.S. Knopf J.L. Kriz R.W. J. Biol. Chem. 1999; 274: 17063-17067Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 22Ghosh M. Loper R. Gelb M.H. Leslie C.C. J. Biol. Chem. 2006; 281: 16615-16624Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). It contains a novel, N-terminal-truncated JmjC domain immediately upstream of the C2 domain. We have recently found that the principle form of cPLA2β translated in human cells is a novel splice variant (cPLA2β3) that contains an internal deletion in the catalytic domain (22Ghosh M. Loper R. Gelb M.H. Leslie C.C. J. Biol. Chem. 2006; 281: 16615-16624Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). cPLA2β3 exhibits calcium-dependent PLA2 activity but is constitutively bound to mitochondria and early endosomes in cells, suggesting a mechanism of regulation and function distinct from cPLA2α. cPLA2δ, cPLA2ϵ, and cPLA2ζ have been cloned from mouse tissues; however, only preliminary information is available about their biochemical properties, and nothing is known of their functional roles (20Ohto T. Uozumi N. Hirabayashi T. Shimizu T. J. Biol. Chem. 2005; 280: 24576-24583Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar).It is well documented that cPLA2α functions to release arachidonic acid for the production of eicosanoids. However, eicosanoid production is not completely ablated in the cPLA2α knock-out mouse indicating a role for other PLA2s in mediating arachidonic acid release (23Downey P. Sapirstein A. O'Leary E. Sun T. Brown D. Bonventre J.V. Am. J. Physiol. 2001; 280: F607-F619Crossref PubMed Google Scholar, 24Wong D.A. Kita Y. Uozumi N. Shimizu T. J. Exp. Med. 2002; 196: 349-357Crossref PubMed Scopus (50) Google Scholar). We previously isolated mouse lung fibroblasts (MLF) from cPLA2α wild type and knock-out mice and demonstrated a primary role for cPLA2α in mediating arachidonic acid release and prostaglandin E2 (PGE2) production (25Ghosh M. Stewart A. Tucker D.E. Bonventre J.V. Murphy R.C. Leslie C.C. Am. J. Respir. Cell Mol. Biol. 2004; 30: 91-100Crossref PubMed Scopus (44) Google Scholar). However, we found that cPLA2α–/– MLFs (MLF–/–) release lower, but significant, levels of arachidonic acid and produce PGE2 in response to calcium ionophore and serum (25Ghosh M. Stewart A. Tucker D.E. Bonventre J.V. Murphy R.C. Leslie C.C. Am. J. Respir. Cell Mol. Biol. 2004; 30: 91-100Crossref PubMed Scopus (44) Google Scholar). We have identified cPLA2β and cPLA2ζ in MLF–/– and provide evidence here that cPLA2ζ is the enzyme that mediates calcium-dependent arachidonic acid release.EXPERIMENTAL PROCEDURESMaterials—[5,6,8,9,11,12,14,15-3H]Arachidonic acid (100 Ci/mmol), 1-palmitoyl-2-[14C]arachidonyl-phosphatidylcholine (PC) (48 mCi/mmol), 1-[14C]palmitoyl-2-lyso-PC (55 mCi/mmol), 1-palmitoyl-2-[14C]arachidonyl-phosphatidylethanolamine (PE) (48 mCi/mmol), and 1-palmitoyl-2-[14C]oleoyl-PC (55 mCi/mmol) were from PerkinElmer Life Sciences. 1-Palmitoyl-[14C]linoleoyl-PE (55 mCi/mmol) was from Amersham Biosciences. 1-Palmitoyl-2-arachidonyl-PE, 1-palmitoyl-2-linoleoyl-PE, and 1-hexadecyl-2-arachidonyl-PC were from Avanti Polar Lipids. 1-Palmitoyl-2-arachidonyl-PC, dioleoylglycerol, bovine serum albumin (BSA), fatty acid-free BSA, pluronic acid, and anti-His6 monoclonal antibody were from Sigma. 1-Arachidonyl-2-hexadecyl-PC was prepared as described (26Hanel A.M. Gelb M.H. Biochemistry. 1995; 34: 7807-7818Crossref PubMed Scopus (42) Google Scholar). PLA2 inhibitors indoxam, pyrrolidine-2, and Wyeth-1 were synthesized as described previously (27Mounier C.M. Ghomashchi F. Lindsay M.R. James S. Singer A.G. Parton R.G. Gelb M.H. J. Biol. Chem. 2004; 279: 25024-25038Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 28Yokota Y. Hanasaki K. Ono T. Nakazato H. Kobayashi T. Arita H. Biochim. Biophys. Acta. 1999; 1438: 213-222Crossref PubMed Scopus (61) Google Scholar, 29Ni Z. Okeley N.M. Smart B.P. Gelb M.H. J. Biol. Chem. 2006; 281: 16245-16255Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 30Seno K. Okuno T. Nishi K. Murakami Y. Watanabe F. Matsuura T. Wada M. Fujii Y. Yamada M. Ogawa T. Okada T. Hashizume H. Kii M. Hara S. Hagishita S. Nakamoto S. Yamada K. Chikazawa Y. Ueno M. Teshirogi I. Ono T. Ohtani M. J. Med. Chem. 2000; 43: 1041-1044Crossref PubMed Scopus (145) Google Scholar). Dulbecco's modified Eagle's medium (DMEM) was from BioWhittaker. Penicillin-streptomycin-l-glutamine solution was from Invitrogen. Fetal bovine serum was from Irvine Scientific. Bromoenol lactone was purchased from Biomol. Silica gel LC-Si SPE columns were from Sigma-Supelco. Protease inhibitor mixture tablets were from Roche Applied Science. The Total RNA Isolation kit was purchased from Promega, and the Advantage reverse transcription-PCR kit, BD SMART RACE cDNA amplification kit, and EGFP vector were from Clontech. The TA cloning vector was from Invitrogen. The plasmid isolation kit, RNeasy mini kit, and nickel-nitrilotriacetic acid-agarose beads were from Qiagen. iScript cDNA synthesis kit was obtained from Bio-Rad.Culture of MLF and Assays for Fatty Acid Release and PGE2 Production—Lung fibroblasts were isolated from wild type (MLF+/+) and cPLA2α knock-out (MLF–/–) mice, and SV40 immortalized MLF (IMLF) were generated as described previously (31Stewart A. Ghosh M. Spencer D.M. Leslie C.C. J. Biol. Chem. 2002; 277: 29526-29536Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Fibroblasts were plated in 24-well tissue culture plates at a density of 2.5 × 104 cells/well in supplemented DMEM (10% fetal bovine serum, 0.1% nonessential amino acids, 1 mm sodium pyruvate, and 1% penicillin-streptomycin-l-glutamine solution) and incubated for 6 h at 37 °C with 5% CO2. Cells were washed twice with serum-free DMEM and incubated in serum-free DMEM containing 0.1% BSA and radiolabeled fatty acids (0.2 μCi of [3H]arachidonic acid, 0.5 μCi of [3H]palmitic acid, 0.25 μCi of [3H]oleic acid, and 0.5 μCi of [14C]linoleic acid/well). After incubation overnight, the cells were washed three times with DMEM containing 0.1% fatty acid-free BSA and stimulated with agonists in albumin-containing medium for the times indicated. For inhibitor experiments, cells were preincubated with inhibitors for 15 min prior to stimulation. The culture medium was removed and centrifuged at 15,000 rpm for 15 min. Cells were solubilized with 0.1% Triton-X-100. The level of radioactivity in the culture medium and in the cells was measured, and the amount released was calculated as a percentage of the total (released plus cellular) radioactivity.To measure the effect of inhibitors on PGE2 production, MLF–/– were incubated overnight in serum-free medium containing transforming growth factor β to up-regulate cyclooxygenase-2 as reported previously (25Ghosh M. Stewart A. Tucker D.E. Bonventre J.V. Murphy R.C. Leslie C.C. Am. J. Respir. Cell Mol. Biol. 2004; 30: 91-100Crossref PubMed Scopus (44) Google Scholar). The cells were incubated with and without pyrrolidine-2 and Wyeth-1 (both at 10 μm) for 15 min and then stimulated with A23187 (2 μg/ml) for 45 min. PGE2 in the culture medium was quantified by enzyme-linked immunosorbent assay (Elisa Tech, Aurora, CO).Quantitative Real-time PCR—The primers and probes used for real-time PCR of mouse cPLA2β, cPLA2ζ, cPLA2δ, and cPLA2ϵ were obtained from Applied Biosystems (premade Taqman gene expression assays). Assay IDs for mouse cPLA2β, cPLA2ζ, cPLA2δ, and cPLA2ϵ are Mm 01271073_g1, Mm 01338177_g1, Mm 01279782_m1, and Mm 00625711_m1, respectively. Total RNA was isolated from MLF and IMLF using a Qiagen RNeasy mini kit, and 1 μg of total RNA was used to make cDNA using iScript cDNA synthesis kit from Bio-Rad following the manufacturer's instructions. Each PCR reaction (25 μl) contained 500 ng of cDNA, PCR master mix, and premade Taqman gene expression assay components containing a 6-carboxyfluorescein reporter dye at the 5′-end of the Taqman probe and a nonfluorescent quencher at the 3′-end of the probe. Rodent glyceraldehyde-phosphate dehydrogenase was used as a control to verify the quality of cDNA template. Real-time PCR was performed and analyzed by the dual-labeled fluorogenic probe method using an ABI Prism 7000 sequence detector from Applied Biosystems.Cloning of Mouse cPLA2β and cPLA2ζ—To clone cPLA2β cDNA from IMLF–/–, cells were cultured in supplemented DMEM; total RNA was isolated, 1 μg of which was used to generate cDNA. PCR analysis was performed using 10 μl of cDNA for cPLA2β and 5 μl of cDNA for glyceraldehyde-phosphate dehydrogenase following the manufacturer's instructions (Clontech Advantage reverse transcription-PCR kit). Specific primers used for mouse cPLA2β were as follows: 5′-gtctacaagcttatgcaggcaaaggtg-3′, 5′-gccaactttggcggtaccggcaagagc-3′, 5′-gctcttgccggtaccgccaaagttggc-3′, and 5′-cagctgggatcctcactccggcctaaac-3′. The primers were designed based on the mouse cPLA2β genomic sequence available from NCBI (gi:211429) to amplify the full-length cDNA in two fragments. The PCR products were cloned into the TA cloning vector, and the fragments were sequenced and then assembled into the full-length clone using the internal KpnI site present in the PCR products.Mouse cPLA2ζ was cloned from IMLF–/– cells and from mouse thyroid using the following primer sets: 5′-ctgggacctgagctgctactgctgg-3′, 5′-gaatactactcccgggaaaagagag-3′, 5′-ctctcttttcccgggagtagtattc-3′, and 5′-gtttaaagtcttccctctccctcag-3′. These were designed based on the mouse cPLA2ζ sequence (NCBI NM_001024145) to amplify the full-length cDNA in two fragments. PCR products were cloned into the TA cloning vector, and the fragments were sequenced and then assembled into the full-length cPLA2ζ clone using the internal SmaI site present in the PCR products. For immunofluorescence microscopy, cPLA2ζ and cPLA2β cDNAs were cloned into the EGFP vector in the XhoI/HindIII and XhoI/BamHI sites, respectively.Production of Recombinant Baculoviruses and Expression in Sf9 Cells—Mouse cPLA2β cDNA was cloned into the baculovirus vector pAcHLT in the StyI/NotI sites and cPLA2ζ in the XhoI/SacI sites. Recombinant baculovirus was generated by co-transfection of Sf9 cells with cPLA2-containing constructs and linearized baculovirus DNA (Baculogold) following the manufacturer's instructions (BD Biosciences-Pharmingen), and amplified by standard protocols. To determine the expression of cPLA2s, Sf9 cells were plated in a 12-well tissue culture plate at a density of 0.5 × 106 cells/well and infected with recombinant viruses at different multiplicities of infection for 1 h. The virus-containing medium was replaced with fresh medium, and cells were incubated for 48 h. Expression of His6-cPLA2β and His6-cPLA2ζ was determined by Western blot analysis using anti-His6 monoclonal antibodies. His6-cPLA2β and His6-cPLA2ζ expressed in Sf9 cells were affinity-purified using nickel-agarose beads following the manufacturer's instructions (Qiagen). The concentration of the enzymes in eluted fractions was determined by comparing the intensity of Coomassie-stained bands on SDS-polyacrylamide gels with a standard curve made with BSA and also by the bicinchoninic acid method.Western Blotting—Cells were washed with phosphate-buffered saline and then scraped into ice-cold lysis buffer (50 mm Hepes, pH 7.4, 150 mm sodium chloride, 1.5 mm magnesium chloride, 10% glycerol, 1% Triton X-100, 1 mm EGTA, 200 μm sodium vanadate, 10 mm terasodium pyrophosphate, 100 mm sodium fluoride, 300 nm p-nitrophenyl phosphate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). After incubation on ice for 30 min, lysates were centrifuged at 15,000 rpm for 15 min, and protein concentration in the supernatant was determined. Lysates were boiled for 5 min in Laemmli electrophoresis sample buffer, and the proteins (15–25 μg total protein) were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membrane. After blocking with 5% milk for 1 h, membranes were incubated overnight at 4 °C with monoclonal anti-His6 antibody in 20 mm Tris, pH 7.6, 137 mm NaCl, and 0.05% Tween containing 5% milk and then incubated with anti-mouse IgG horseradish peroxidase antibody (1:5000 dilution) for 30 min at room temperature. The immunoreactive proteins were detected using the Amersham Biosciences ECL system.Assay for Arachidonic Acid Release from Sf9 Cells—Sf9 cells were plated in 24-well plates (2.5 × 105 cells/well) in 500 μl of TNM-FH medium containing 10% fetal bovine serum (complete medium) and incubated at 27 °C for 15 min. The medium was removed and baculoviruses added in 150 μl of complete medium. After incubation for 1 h, complete medium (350 μl/well) was added and the cells incubated 25–30 h followed by incubation overnight in complete medium (500 μl/well) containing 0.2 μCi [3H]arachidonic acid. The cells were washed three times with serum-free TNM-FH medium containing 0.1% human serum albumin and stimulated for 45 min with A23187 (2 μg/ml). The level of released [3H]arachidonic acid was determined as described above for fibroblasts. A portion of the Triton lysate was used for Western blotting to determine the relative level of expression of the cPLA2s.Enzyme Assays—PLA2 activity was assayed using 1-palmitoyl-2-[14C]arachidonyl-PC, 1-palmitoyl-2-[14C]oleoyl-PC, 1-palmitoyl-2-[14C]arachidonyl-PE, and 1-palmitoyl-2-[14C]linoleoyl-PE as substrates. To prepare substrate, solvents were evaporated from the lipid mixture under a stream of nitrogen, 50 mm Hepes buffer, pH 7.4, was added and the lipid mixture sonicated at 4 °C for 10 s on ice using a microprobe (Braun Instruments) to form small unilamellar vesicles. The reaction mixture (50 μl final volume) contained 30 μm phospholipid substrate (100,000 dpm), 9 μm dioleoylglycerol (which was co-sonicated with the substrate), 150 mm sodium chloride, 1 mg/ml fatty acid free BSA, 1 mm EGTA, and 5 mm CaCl2. For assays with palmitoyl-arachidonyl-PE, dioleoylglycerol was not added. Reactions were started by the addition of affinity-purified enzyme (50 ng-1 μg) and incubated at 37 °C for the times indicated. Free fatty acids were extracted using Dole reagent (propan-2-ol:heptane:1 n H2SO4, 20:5:1) and separated by silicic acid chromatography as described previously using unlabeled oleic acid (25 μg) as carrier lipid (32Leslie C.C. Gelb M.H. Methods Mol. Biol. 2004; 284: 229-242PubMed Google Scholar).The calcium dependence of the PLA2 activity of cPLA2α and cPLA2ζ was measured using 1-palmitoyl-2-[14C]arachidonyl-PC. Vesicles were made by extrusion through two 0.2-μm Nucleopore membranes as described previously (33Bayburt T. Gelb M.H. Biochemistry. 1997; 36: 3216-3231Crossref PubMed Scopus (61) Google Scholar) in buffer (10 mm MOPS, 100 mm KCl, 0.5 mm EGTA, pH 7.2) to give 4–4.5 mm total phospholipid at a final specific radioactivity of 2.7 Ci/mol. The phospholipid concentration in the stock solution after extrusion was calculated from the initial concentration and the yield of radioactivity. The same buffer containing various concentrations of free calcium from 0 to 20 μm was prepared by fluorimetric titration using fluo-3 and Calcium Green 5N as described previously (34Hixon M.S. Ball A. Gelb M.H. Biochemistry. 1998; 37: 8516-8526Crossref PubMed Scopus (61) Google Scholar). A small aliquot of extruded vesicle stock was added to give 200 μm total phospholipid in each assay. Reactions (80 μl) were carried out in various calcium buffers supplemented with 0.5 mg/ml fatty acid-free BSA for 2 min at 37 °C. Reactions were quenched and analyzed for radiolabeled free arachidonic acid as described earlier (35Ghomashchi F. Schuttel S. Jain M.K. Gelb M.H. Biochemistry. 1992; 31: 3814-3824Crossref PubMed Scopus (62) Google Scholar).Reactions to study the PLA2 and PLA1 activities were carried out as follows. 1-Hexadecyl-2-arachidonyl-PC or 1-arachidonyl-2-hexadecyl-PC was sonicated at 60 μm in 50 mm Hepes, pH 7.4, to form a stock solution of small unilamellar vesicles as described (36Jain M.K. Gelb M.H. Methods Enzymol. 1991; 197: 112-125Crossref PubMed Scopus (82) Google Scholar). Reaction mixtures contained 30 μm phospholipid in 250 μl of 50 mm Hepes, pH 7.4, 150 mm NaCl, 1 mg/ml fatty acid-free BSA, 1 mm EGTA, 5 mm CaCl2, and either cPLA2α (10 ng), cPLA2β (7.5 μg), or cPLA2ζ (20 ng). After 20 min at 37 °C, reactions were processed for arachidonic acid analysis using gas chromatography/mass spectrometry as described with d8-arachidonic acid (Cayman Chemicals, Inc.) as an internal standard (37Degousee N. Ghomashchi F. Stefanski E. Singer A. Smart B.P. Borregaard N. Reithmeier R. Lindsay T.F. Lichtenberger C. Reinisch W. Lambeau G. Arm J. Tischfield J. Gelb M.H. Rubin B.B. J. Biol. Chem. 2002; 277: 5061-5073Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar).Lysophospholipase activity was measured using 1-[14C] palmitoyl-2-lyso-PC sonicated in 50 mm Hepes, pH 7.4, to make micelles as described previously (38de Carvalho M.G.S. Garritano J. Leslie C.C. J. Biol. Chem. 1995; 270: 20439-20446Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Assays contained 50 μm substrate (120,000 dpm), 1 mm EGTA, and 5 mm CaCl2 in a final volume of 50 μl. Reactions were started by adding affinity-purified enzyme and incubated at 37 °C for the times indicated. Free fatty acid product was extracted using Dole reagent. After vortexing, the upper heptane phase was removed and dried under a stream of nitrogen, and 0.5 ml of heptane added. Radiolabeled free fatty acids were measured by liquid scintillation spectrometry. For inhibitor experiments, enzymes were preincubated for 2 min at 37 °C with inhibitors, and reactions were started by addition of substrate.Immunofluorescence Microscopy—IMLF–/– were transfected with 10 μg of EGFP-cPLA2ζ and EGFP-cPLA2α cDNA using nucleofection technology (Amaxa Biosystems), with solution T, following the manufacturer's instructions. Transfected fibroblasts were plated in 35-mm glass-bottom MatTek plates at a density of 0.5 × 106/cm2 and incubated for 48 h. Cells were then washed twice with serum-free DMEM and incubated in serum-free DMEM containing 0.1% BSA overnight. For live cell imaging, fibroblasts were washed with and incubated in Hanks' balanced salt solution buffered with 25 mm Hepes. Cells were stimulated with 0.5 μm ionomycin and imaged at 37 °C with an inverted Zeiss 200M microscope with a 175-watt xenon lamp using a ×63 oil immersion objective and GFP filters. Images were acquired every 5 s for a total of 10 min with a charge-coupled device camera from Sensicam, and data were analyzed using Intelligent Imaging Innovations Inc. (3I) software.RESULTSFatty Acid Release from IMLF+/+ and IMLF –/– and Effect of PLA2 Inhibitors—We previously reported that primary MLF–/– and immortalized MLF–/– release arachidonic acid and produce PGE2 in response to A23187 and serum indicating the presence of a calcium-regulated PLA2 that can initiate lipid mediator production (25Ghosh M. Stewart A. Tucker D.E. Bonventre J.V. Murphy R.C. Leslie C.C. Am. J. Respir" @default.
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