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• W2011209855 abstract "Oxidant stress and phospholipase A2 (PLA2) activation have been implicated in numerous proinflammatory responses of the mesangial cell (MC). We investigated the cross-talk between group IVα cytosolic PLA2 (cPLA2α) and secretory PLA2s (sPLA2s) during H2O2-induced arachidonic acid (AA) release using two types of murine MC: (i) MC+/+, which lack group IIa and V PLA2s, and (ii) MC–/–, which lack groups IIa, V, and IVα PLA2s. H2O2-induced AA release was greater in MC+/+ compared with MC–/–. It has been argued that cPLA2α plays a regulatory role enhancing the activity of sPLA2s, which act on phospholipids to release fatty acid. Group IIa, V, or IVα PLA2s were expressed in MC–/– or MC+/+ using recombinant adenovirus vectors. Expression of cPLA2α in H2O2-treated MC–/– increased AA release to a level approaching that of H2O2-treated MC+/+. Expression of either group IIa PLA2 or V PLA2 enhanced AA release in MC+/+ but had no effect on AA release in MC–/–. When sPLA2 and cPLA2α are both present, the effect of H2O2 is manifested by preferential release of AA compared with oleic acid. Inhibition of the ERK and protein kinase C signaling pathways with the MEK-1 inhibitor, U0126, and protein kinase C inhibitor, GF 1092030x, respectively, and chelating intracellular free calcium with 1,2-bis(2-aminophenoyl)ethane-N,N,N′,N′-tetraacetic acid-AM, which also reduced ERK1/2 activation, significantly reduced H2O2-induced AA release in MC+/+ expressing either group IIa or V PLA2s. By contrast, H2O2-induced AA release was not enhanced when ERK1/2 was activated by infection of MC+/+ with constitutively active MEK1-DD. We conclude that the effect of group IIa and V PLA2s on H2O2-induced AA release is dependent upon the presence of cPLA2α and the activation of PKC and ERK1/2. Group IIa and V PLA2s are regulatory and cPLA2α is responsible for AA release. Oxidant stress and phospholipase A2 (PLA2) activation have been implicated in numerous proinflammatory responses of the mesangial cell (MC). We investigated the cross-talk between group IVα cytosolic PLA2 (cPLA2α) and secretory PLA2s (sPLA2s) during H2O2-induced arachidonic acid (AA) release using two types of murine MC: (i) MC+/+, which lack group IIa and V PLA2s, and (ii) MC–/–, which lack groups IIa, V, and IVα PLA2s. H2O2-induced AA release was greater in MC+/+ compared with MC–/–. It has been argued that cPLA2α plays a regulatory role enhancing the activity of sPLA2s, which act on phospholipids to release fatty acid. Group IIa, V, or IVα PLA2s were expressed in MC–/– or MC+/+ using recombinant adenovirus vectors. Expression of cPLA2α in H2O2-treated MC–/– increased AA release to a level approaching that of H2O2-treated MC+/+. Expression of either group IIa PLA2 or V PLA2 enhanced AA release in MC+/+ but had no effect on AA release in MC–/–. When sPLA2 and cPLA2α are both present, the effect of H2O2 is manifested by preferential release of AA compared with oleic acid. Inhibition of the ERK and protein kinase C signaling pathways with the MEK-1 inhibitor, U0126, and protein kinase C inhibitor, GF 1092030x, respectively, and chelating intracellular free calcium with 1,2-bis(2-aminophenoyl)ethane-N,N,N′,N′-tetraacetic acid-AM, which also reduced ERK1/2 activation, significantly reduced H2O2-induced AA release in MC+/+ expressing either group IIa or V PLA2s. By contrast, H2O2-induced AA release was not enhanced when ERK1/2 was activated by infection of MC+/+ with constitutively active MEK1-DD. We conclude that the effect of group IIa and V PLA2s on H2O2-induced AA release is dependent upon the presence of cPLA2α and the activation of PKC and ERK1/2. Group IIa and V PLA2s are regulatory and cPLA2α is responsible for AA release. Phospholipase A2s (PLA2s) 1The abbreviations used are: PLA2, phospholipase A2; cPLA2α, cytosolic phospholipase A2α; sPLA2, secretory phospholipase A2; ERK1/2, extracellular signal-regulated kinases 1/2; MAPK, mitogen-activated protein kinase; MEK-1, ERK kinase; PKC, protein kinase C; MC, mesangial cells; m.o.i., multiplicity of infection; BAPTA, 1,2-bis(2-aminophenoyl)ethane-N,N,N′,N′-tetraacetic acid; [14C]PE, 1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphoethanolamine; OA, oleic acid; iPLA2, Ca2+-independent phospholipase A2; GFP, green fluorescent protein; [Ca2+]i, intracellular Ca2+; AA, arachidonic acid; BEL, bromoenol lactone. are a family of enzymes that liberate free fatty acids, including arachidonic acid (AA), and lysophospholipid from glycerophospholipids. Several mammalian intracellular and small molecular weight (13,000–16,000) secretory PLA2s (sPLA2) have been characterized and classified into different groups (1Kudo I. Murakami M. Hara S. Inoue K. Biochim. Biophys. Acta. 1993; 117: 217-231Google Scholar, 2Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Google Scholar, 3Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Google Scholar, 4Murakami M. Kudo I. J. Biochem. (Tokyo). 2002; 131: 285-292Google Scholar). Non-secretory PLA2s include the Ca2+-sensitive arachidonoyl-selective 85-kDa group IVα cytosolic PLA2 (cPLA2α) (5Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Google Scholar, 6Bonventre J.V. J. Am. Soc. Nephrol. 1999; 10: 404-412Google Scholar), paralogs of this enzyme (7Pickard R.T. Strifler B.A. Kramer R.M. Sharp J.D. J. Biol. Chem. 1999; 274: 8823-8831Google Scholar), and several Ca2+-independent PLA2s (iPLA2s) (8Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Google Scholar). A number of mammalian sPLA2s have been identified to date (groups IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII) and they display distinct yet partially overlapping tissue distributions (4Murakami M. Kudo I. J. Biochem. (Tokyo). 2002; 131: 285-292Google Scholar, 9Valentin E. Ghomashchi F. Gelb M.H. Lazdunski M. Lambeau G. J. Biol. Chem. 1999; 274: 31195-31202Google Scholar, 10Bezzine S. Koduri R.S. Valentin E. Murakami M. Kudo I. Ghomashchi F. Sadilek M. Lambeau G. Gelb M.H. J. Biol. Chem. 2000; 275: 3179-3191Google Scholar). Whereas cPLA2α has a preferential effect on AA-containing membrane phospholipids as compared with those containing other fatty acids, sPLA2s do not exhibit acyl chain specificity. The cPLA2α, group IIa, and group V PLA2s have each been implicated as the primary PLA2 responsible for production of AA and its metabolites in fibroblastic, endothelial, mast and macrophage mammalian cell lines (1Kudo I. Murakami M. Hara S. Inoue K. Biochim. Biophys. Acta. 1993; 117: 217-231Google Scholar, 11Murakami M. Kudo I. Inoue K. J. Biol. Chem. 1993; 268: 839-844Google Scholar, 12Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Google Scholar, 13Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Google Scholar, 14Pruzanski W. Stefanski E. Vadas P. Kennedy B.P. Bosch H.V.D. Biochem. Biophys. Acta. 1998; 1403: 47-56Google Scholar, 15Balsinde J. Balboa M.A. Dennis E.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Google Scholar). Most investigators have concluded that cPLA2α plays a regulatory role whereas sPLA2 provides most of the AA release that occurs in response to agonists. Both cPLA2α and the sPLA2s have been implicated in various physiological and pathological functions including lipid digestion, release of proinflammatory mediators, cell proliferation, ischemic injury, inflammatory disease, cancer, and anti-bacterial defense (6Bonventre J.V. J. Am. Soc. Nephrol. 1999; 10: 404-412Google Scholar, 9Valentin E. Ghomashchi F. Gelb M.H. Lazdunski M. Lambeau G. J. Biol. Chem. 1999; 274: 31195-31202Google Scholar, 16Sapirstein A. Bonventre J.V. Biochim. Biophys. Acta. 2000; 1488: 139-148Google Scholar). Oxidant stress has been implicated in numerous proinflammatory responses in mammalian cells (17Weigel G. Griesmacher A. Toma C.D. Baecker C. Heinzl H. Mueller M.M. Thromb. Res. 1997; 87: 363-375Google Scholar, 18Roberts M.L. Cowsert L.M. Biochem. Biophys. Res. Commun. 1998; 251: 166-172Google Scholar, 19Terry C.M. Clikeman J.A. Hoidal J.R. Callahan K.S. Am. J. Physiol. 1999; 276: H1493-H1501Google Scholar, 20Shin E.A. Kim K.H. Han S.I. Ha K.S. Kim J.H. Kang K.I. Kim H.D. Kang H.S. FEBS Lett. 1999; 452: 355-359Google Scholar). Hydrogen peroxide (H2O2) triggers AA release and metabolism in various cell types (21Boyer S.C. Bannenberg G.L. Neve E.P.A. Ryrfeldt A. Moldeus P. Biochem. Pharmacol. 1995; 50: 753-761Google Scholar, 22Meyer T.N. Gloy J. Hug M.J. Greger R. Schollmeyer P. Pavenstadt H. Kidney Int. 1996; 49: 388-395Google Scholar, 23Tournier C. Thomas G. Pierre J. Jacquemin C. Pierre M. Saunie B. Eur. J. Biochem. 1997; 244: 587-595Google Scholar, 24Cane A. Breton M. Koumanov K. Bereziat G. Colard O. Am. J. Physiol. 1998; 274: C1040-C1046Google Scholar). The understanding of which forms of PLA2 are important for AA release and how multiple forms may interact has been hampered by the fact that mammalian cells generally contain more than one form of PLA2. Furthermore, various PLA2 inhibitors and antisense approaches lack specificity and/or efficacy even though much useful information has been derived from these approaches (15Balsinde J. Balboa M.A. Dennis E.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Google Scholar, 25Balsinde J. Barbour S.E. Bianco I. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11060-11064Google Scholar, 26Reddy S.T. Winstead M.V. Tischfield J.A. Herschman H.R. J. Biol. Chem. 1997; 272: 13591-13596Google Scholar, 27Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Google Scholar, 28Xu G.Y. McDonagh T. Yu H.A. Nalefski E.A. Clark J.D. Cumming D.A. J. Mol. Biol. 1998; 280: 485-500Google Scholar). Thus the understanding of specific interactions of PLA2 enzymes contributing to AA release and metabolite production is complex and has been difficult to clarify. Cellular kinase signal transduction pathways have been implicated in PLA2 activation and its downstream effects. cPLA2α is regulated post-translationally by phosphorylation and by calcium (5Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Google Scholar). cPLA2α contains an N-terminal calcium-dependent phospholipid binding domain. Mitogen-activated protein kinase (MAPK) cascades and protein kinase C (PKC) have been implicated in the phosphorylation and activation of cPLA2α (29Nemenoff R.A. Winitz S. Qian N.X. Van Putten V. Johnson G.L. Heasley L.E. J. Biol. Chem. 1993; 268: 1960-1964Google Scholar, 30Lin L.L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Google Scholar, 31Qiu Z.H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Google Scholar, 32Kramer R.M. Roberts E.F. Um S.L. Borsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Google Scholar, 33Geijsen N. Dijkers P.F. Lammers J.W.J. Koenderman L. Coffer P.J. FEBS Lett. 2000; 471: 83-88Google Scholar). Balsinde and Dennis (34Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Google Scholar) and Hernandez et al. (35Hernandez M. Burillo S.L. Crespo M.S. Nieto M.L. J. Biol. Chem. 1998; 273: 606-612Google Scholar) reported the involvement of extracellular signal-regulated kinases (ERKs) as mediators of cross-talk between sPLA2s and cPLA2α in polymorphonuclear leukocytes and macrophages. Others have reported that sPLA2s can activate MAPK cascades and PKC, which subsequently activate cPLA2α (30Lin L.L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Google Scholar, 32Kramer R.M. Roberts E.F. Um S.L. Borsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Google Scholar, 36Huwiler A. Staudt G. Kramer R.M. Pfeilschifter J. Biochim. Biophys. Acta. 1997; 1348: 257-272Google Scholar). H2O2 has been reported to increase the activity of MAPK cascades (23Tournier C. Thomas G. Pierre J. Jacquemin C. Pierre M. Saunie B. Eur. J. Biochem. 1997; 244: 587-595Google Scholar) and stimulate cPLA2α activity in smooth muscle cells (37Rao G.N. Runge M.S. Alexander R.W. Biochim. Biophys. Acta. 1995; 1265: 67-72Google Scholar). Renal mesangial cells are an important source of both eicosanoid and reactive oxygen species generation in the kidney during normal and pathological states. Reactive oxygen species have been implicated in the response of mesangial cells to hyperglycemia (38Catherwood M.A. Powell L.A. Anderson P. McMaster D. Sharpe P.C. Trimble E.R. Kidney Int. 2002; 61: 599-608Google Scholar) and increased levels of prostaglandins are characteristic of diabetic and other glomerulopathies (39Mahadevan P. Larkins R.G. Fraser J.R. Dunlop M.E. Diabetes. 1996; 45: 44-50Google Scholar, 40Lianos E.A. Bresnahan B.A. J. Lab. Clin. Med. 1999; 134: 478-482Google Scholar). The role of PLA2s in the generation of eicosanoids and propagation of inflammation has been extensively studied by several groups using mesangial cells derived from rats. Our laboratory has produced a mouse line with the cPLA2α gene mutated (41Bonventre J.V. Huang Z. Taheri M.R. O'Leary E. Li E. Moskowitz M.A. Sapirstein A. Nature. 1997; 390: 622-625Google Scholar). The mouse strains (C57b/6 and SV/129) used to construct the cPLA2α–/– strain have spontaneous null mutations in the gene encoding group IIa PLA2 (42Kennedy B.P. Payette P. Mudgett J. Vadas P. Pruzanski W. Kwan M. Tang C. Rancourt D.E. Cromlish W.A. J. Biol. Chem. 1995; 270: 22378-22385Google Scholar). In addition to group IIa PLA2, our murine mesangial cells do not express group V PLA2 under quiescent or stimulated conditions, unlike rat mesangial cells, which are known to synthesize group IIa and V PLA2s upon stimulation of the cells with cytokines (43van der Helm H.A. Aarsman A.J. Janssen M.J.W. Neys F.W. van den Bosch H. Biochim. Biophys. Acta. 2000; 1484: 215-224Google Scholar). Two types of MC were used: (i) cPLA2α+/+ MC (MC+/+), which lack group IIa and V PLA2s and (ii) cPLA2α–/– MC (MC–/–), which lack group IIa, V, and IVα PLA2s. We expressed group IVα, IIa, and V PLA2 proteins in MC–/– and MC+/+ with recombinant adenoviral vectors. Using this approach we dissect the specific roles played by cPLA2α and the sPLA2s in the mediation of H2O2-induced AA release in mesangial cells. To better define the cross-talk between cPLA2α and sPLA2s during oxidant stress, we examined the effect of expression of various forms of PLA2 on H2O2-induced AA release in murine mesangial cells. This is the first time that the cross-talk between cPLA2α and sPLA2s in a mammalian cell has been studied by utilizing recombinant adenovirus and cPLA2α knockout MC, which lack group IIa, V, and IVα PLA2s. We report here that group IIa and V PLA2s potentiate H2O2-induced AA release in a cPLA2α-, PKC-, and ERK1/2-dependent manner. Activation of ERK1/2 is necessary but not sufficient for H2O2-mediated AA release. We conclude that in murine mesangial cells, cPLA2α is the major enzyme responsible for AA release whereas sPLA2 serves an amplifying role. Materials—[5,6,8,9,11,12,4,15-3H]Arachidonic acid ([3H] AA, 98.6 Ci/mmol; 1 Ci = 37 GBq) and [1-14C]oleic acid ([14C]OA, 50 mCi/mmol; 1 Ci = 38 GBq) were from PerkinElmer Life Sciences. 1-Palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphoethanolamine ([14C]PE, 56 mCi/mmol; 1 Ci = 37 GBq) was from Amersham Biosciences. H2O2, CsCl, probenecid, BAPTA-AM, and EGTA were from Sigma. Bovine serum albumin fraction V was from Roche Diagnostics. U0126 was from Promega Corp. (Madison, WI). SB203580 and bisindolylmaleimide 1 (GF 1092030x) were from Calbiochem (San Diego, CA). Bromoenol lactone (BEL), group IIa and V PLA2 polyclonal antibodies were from Cayman Chemical (Ann Arbor, MI). Acetoxymethyl ester of Fura-2 (Fura-2AM) and pluronic F-127 were from Molecular Probes (Eugene, OR). Immobilon-P (polyvinylidene difluoride) was from Millipore (Bedford, MA). Mouse group V PLA2 and human group IIa PLA2 were provided by Dr. Jonathan Arm. LY311727 was provided by Eli Lilly. cPLA2α polyclonal antibody was provided by Dr. Andrey Cybulsky. Peroxidase-conjugated, goat anti-rabbit immunoglobulin was from DAKO (Carpinteria, CA). Total ERK1/2 and p38 kinase antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phospho-specific (Ser505) cPLA2α polyclonal antibody and phospho-specific ERK1/2 and p38 kinase antibodies were from Cell Signaling Technology (Beverly, MA). Protein measurements based on Bradford's assay were performed with reagents from Bio-Rad. Generation of Primary Murine Mesangial Cells—Primary murine MC were cultivated from wild type and cPLA2α knockout mice. Cell lines were generated from kidneys taken from 3 cPLA2α+/+ and 3 cPLA2α–/– littermates. The cortices of each mouse were dissected under sterile conditions. The glomeruli were isolated by mechanical disruption, passaged through 140 μm and then collected on a 46-μm sieve, followed by centrifugation. Following isolation the glomeruli were treated with 1 mg/ml collagenase IV for 30 min at 37 °C. Clones with apparent MC morphology were used for further processing. The cells were grown in RPMI 1640 (Cellgro) supplemented with 10% (v/v) fetal bovine serum (Invitrogen) at 37 °C in 95% air, 5% CO2. MC exhibit the typical stellate morphology. Moreover, they stain positively for the intermediate filaments desmin and vimentin, which are considered to be specific for myogenic cells. Passages 6–10 of MC were used for the reported experiments. Analysis of mRNA Expression of Group V PLA2in MC—Total RNA was extracted from mesangial cells by using Ultraspec (Biotecx, Houston, TX) according to the manufacturer's instructions. Primers derived from the 5′ (5′-CAGGGGGCTTGCTAGAACTCAA-3′) and 3′ (5′-AAGAGGGTTGTAAGTCCAGAGG-3′) ends of the coding region of the mouse group V PLA2 were used in a reverse transcriptase-polymerase chain reaction to clone the mouse group V PLA2 cDNA. The reverse transcriptase reaction was carried out after 10 min incubation at 70 °C of denatured template and dNTP with 10 pmol of reverse primer and then incubated with Moloney murine leukemia virus reverse transcriptase (Stratagene, La Jolla, CA) for 1 h at 42 °C to generate a cDNA template for PCR. The PCR was carried out for 40 cycles of 95 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min in buffer with 0.5 units of Taq polymerase. The amplified products were resolved in 2% agarose gels and visualized with ethidium bromide. The expression of glyceraldehyde-3-phosphate dehydrogenase was used as internal control. Total PLA2and iPLA2Assay—MC were washed with phosphate-buffered saline and lysed by sonication in a buffer containing 150 mm NaCl, 1 mm EDTA, and 50 mm Tris/HCl at pH 9. The lysate was centrifuged at 3,000 rpm for 30 min at 4 °C. Total PLA2 activity was assayed by measuring the amounts of free fatty acid released from the substrate [14C]PE. Each reaction mixture consisted of an aliquot of the required sample, 75 mm Tris-HCl, pH 7.4, 5 mm CaCl2, and 2 μm substrate. After incubation for 30 min at 37 °C, the [14C]PE released was extracted by Dole's method and the radioactivity was counted (44Sapirstein A. Spech R.A. Witzgall R. Bonventre J.V. J. Biol. Chem. 1996; 271: 21505-21513Google Scholar). For the iPLA2 assay, lysate was preincubated with various concentrations of BEL (iPLA2 inhibitor) for 10 min and then iPLA2 activity was assayed by measuring the amounts of free fatty acid released from the substrate [14C]PE. Each reaction mixture consisted of an aliquot of the sample, 75 mm Tris-HCl, pH 7.4, 5 mm EGTA, and 2 μm substrate. After incubation for 30 min at 37 °C, the [14C]PE released was extracted by Dole's method and the radioactivity was counted. Arachidonic and Oleic Acid Release—Confluent MC in 12-well plates were labeled for 14–16 h either with 0.15 μCi of [3H]AA (specific activity: 1 Ci = 37 GBq) or 0.15 μCi of [14C]OA (specific activity: 1 Ci = 38 GBq) in 1 ml of RPMI 1640 with 0.2% (v/v) fetal bovine serum. After labeling, the medium was removed, and cells were washed three times with RPMI containing 0.2% bovine serum albumin. To measure H2O2-induced AA or OA release, cells were exposed to 75 μm H2O2 or vehicle in RPMI, 0.2% bovine serum albumin for 3 to6hat37 °C in 95% air, 5% CO2. The medium was removed and centrifuged to remove detached cells. The cells were solubilized with 1 ml of 0.5% NaOH. The radioactivity in 800 μl of supernatant and cells was measured in a liquid scintillation counter. The amount of [3H]AA or [14C]OA released into the medium was expressed as a percentage of the total (cell-associated plus released). SDS-PAGE and Western Blot Analysis—Total MC extracts were harvested with lysis buffer (20 mm HEPES, pH 7.4, 2 mm EGTA, 1 mm dithiothreitol, 1 mm NaVO4, 1% Triton X-100, 10% glycerol, 2 μm leupeptin, 400 μm phenylmethylsulfonyl fluoride, 50 mm β-glycerophosphate) and mixed with 6× sample buffer. Fifteen micrograms of cell extracts were subjected to SDS-PAGE (10% acrylamide gel), and proteins were transferred onto Immobilon-P membranes for 1 h at 400 mA using a Bio-Rad transblot apparatus. The transfer buffer used was 50 mm Tris-HCl, pH 7.4, 384 mm glycine, 0.01% SDS, and 20% methanol. After the transfer, the membrane was blocked with a buffer containing: 1× phosphate-buffered saline, 5% nonfat dry milk, and 0.5% Tween 20. The membrane was incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibody, respectively. Proteins were visualized with an enhanced chemiluminescence detection system (PerkinElmer Life Sciences). Measurement of Intracellular Free Calcium Concentration—Intracellular free Ca2+ concentration ([Ca2+]i) was determined with the Ca2+-sensitive fluorescent dye Fura-2 according to Cheung et al. (45Cheung J.Y. Constantine J.M. Bonventre J.V. Am. J. Physiol. 1986; 251: F690-F701Google Scholar) with modification. Cells grown on coverslips coated with bovine collagen type I were rinsed with phosphate-buffered saline and loaded with 3 μm Fura-2AM in Earle's balanced salt solution. Pluronic F-127 (20%) at 1:1000 (v/v) dilution was added to Fura-2AM to facilitate cell loading. In addition, 2 mm probenecid was added to minimize intracellular compartment transport or extrusion of Fura-2-free acid. Cells were loaded with Fura-2AM for 1 h at 37 °C and washed 2–3 times with Earle's balanced salt solution containing probenecid. The coverslips were positioned in a quartz cuvette containing 3.5 ml of Earle's balanced salt solution with probencid for fluorescence analysis using a Shimadzu RF-5000 spectrofluorophotometer (Shimadzu, Columbia, MD). [Ca2+]i was calculated as equal to Kd (224 nm) × (R – Rmin)/(Rmax – R) according to Grykiewicz et al. (46Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Google Scholar) as described previously. Fluorescence emission was monitored at 505 nm. R is the ratio (F1/F2) of the fluorescence at excitation 340 nm to that at excitation 380 nm. Construction of Recombinant Adenovirus Vectors Carrying the cDNA of Group IIa and V PLA2—The system for generation of recombinant adenoviruses has been described previously (47He T.C. Zhou S.S. Costa L.T.D. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Google Scholar). Briefly the mouse V PLA2 cDNA (500-base pair HindIII/XbaI restriction fragment) and human IIa PLA2 cDNA (780-base pair KPN1/XhoI restriction fragment) were subcloned into a shuttle vector (pAdTrack-CMV). The shuttle vector plasmid was linearized with PmeI (restriction endonuclease) and transformed together with supercoiled adenoviral backbone vector (pAdEasy-1) into Escherichia coli strain BJ5183 by electroporation in a Bio-Rad Gene Pulser electroporator. Recombinants were selected with kanamycin (50 μg/ml) and screened by restriction endonuclease digestions (PacI, SpeI, and BamHI). The recombinant adenoviral construct was transformed into DH 5α cells for large scale amplification by electroporation and was purified by CsCl banding. For production of adenoviruses in mammalian cells, the recombinant adenoviral construct linearized by PacI was transfected into 293 cells using LipofectAMINE and Opti-MEM (Invitrogen). The process of viral production was monitored by visualization of the green fluorescent protein (GFP) expression, which is incorporated into the viral backbone. Viruses were harvested after 7–10 days. The viruses were purified by CsCl banding and viral particles were measured by optical density (OD) (viral particles = OD × dilution factor (usually 10) × 1012). The recombinant adenoviral vectors carrying the cDNA of cPLA2α (Ad-cPLA2α), cDNA of GFP/E. coli LacZ gene encoding β-galactosidase (Ad-GFP/LacZ) as an adenovirus control, and cDNA of constitutively active MEK1-DD (Ad-MEK1-DD) were constructed as previously described in our laboratory (48Choukroun G.J. Marshansky V. Gustafson C.E. Mckee M. Hajjar R.J. Rosenzweig A. Brown D. Bonventre J.V. J. Clin. Invest. 2000; 106: 983-993Google Scholar, 49Choukroun G. Hajjar R. Kyriakis J.M. Bonventre J.V. Rosenzweig A. Force T. J. Clin. Invest. 1998; 102: 1311-1320Google Scholar). Introduction of PLA2Enzymes into Primary Murine Mesangial Cells—Subconfluent MC were infected with adenoviral vectors at varying levels of infection, as reflected by plaque forming units/cell, for 48 h in RPMI 1640 with 2% (v/v) fetal bovine serum. The adenovirus-mediated gene transfers were followed by the expression of GFP under UV light for Ad-IIa and VPLA2s and Ad-GFP/LacZ, and by Western blot analysis of cell lysate for Ad-cPLA2α and Ad-MEK1-DD, respectively. After confirming the infection, cells were used for experiments. Statistical Analysis—Data are expressed as the mean ± S.E. Statistical differences among the groups were calculated on raw data using the analysis of variance test. Significance was tested using Student's t test between groups. A value of p < 0.05 was chosen to determine statistical significance. Each experiment was performed in triplicate and independently three to five times. Expression of cPLA2α and Its Effect on H2O2-induced AA Release in MC–/–—MC–/– were infected with Ad-cPLA2α. Seventy-two hours after infection, total cell extracts were collected to confirm expression of the cPLA2α in MC–/– by Western blotting. Fig. 1A shows the expression of cPLA2α in a dose-dependent manner after Ad-cPLA2α infection at different multiplicity of infections (m.o.i.) in MC–/–. At the sites of inflammation, H2O2 levels can reach relatively high local concentrations (range from 0.1 to 1.0 mm) in the presence of activated polymorphonuclear leukocytes (50Homan-Muller J.W. Weening R.S. Roos D. J. Lab. Clin. Med. 1975; 85: 198-207Google Scholar, 51Nathan C.F. J. Clin. Invest. 1987; 80: 1550-1560Google Scholar, 52Negri M. Bellavite P. Lauciello C. Guzzo P. Zatti M. Clin. Chim. Acta. 1991; 199: 305-310Google Scholar). To study the effect of cPLA2α on H2O2-induced AA release from MC, MC–/– infected with varying m.o.i. of Ad-cPLA2α, were stimulated with 75 μm H2O2 for periods of 3 and 6 h and AA released into media was measured (Fig. 1B). The elevation of AA release was dependent on Ad-cPLA2α m.o.i. and exposure time to H2O2. Expression of cPLA2α in H2O2-treated MC–/– significantly increased AA release at 3 and 6 h. H2O2 at a concentration of 75 μm did not result in cellular injury as monitored by trypan blue staining and lactate dehydrogenase release (data not shown). Expression of Group IIa and V PLA2s in MC—The mouse strains (C57b/6 and SV/129) used to construct the cPLA2α–/– strain have spontaneous null mutations in the gene encoding group IIa PLA2. Furthermore, MC derived from cPLA2α+/+ (MC+/+) and cPLA2α–/– (MC–/–) mice, which have mixed C57b/6 and SV/129 backgrounds, do not express group V PLA2 mRNA under quiescent or stimulated conditions (Fig. 2A). MC+/+ and MC–/– were infected with Ad-sPLA2. Seventy-two hours after infection, total cell extracts were collected to confirm expression of the sPLA2s in MC by Western blotting. Fig. 2B shows the dose-dependent expression of either group IIa or V PLA2s after Ad-IIa PLA2 or Ad-V PLA2 infection at different m.o.i. in MC+/+. Infection with adenovirus expressing the GFP/LacZ enzyme had no effect upon the levels of either of the PLA2s. This indicates that the infection process is not associated with endogenous sPLA2 expression. Fig. 2C shows that there is an increase in total PLA2 activity in Ad-IIaPLA2, Ad-V PLA2-infected unstimulated MC. No increase in activity is seen in cells infected with Ad-GFP/LacZ compared with uninfected cells. LY311727, which selectively inhibits group IIa (53Schevitz 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: 458-465Google Scholar) and group V PLA2 activity (54Yang H.C. Mosior M. Johnson C.A. Chen Y. Dennis E.A. Anal. Biochem. 1999; 269: 278-288Google Scholar), completely inhibited the increased total PLA2 activity in the Ad-IIa- or Ad-V PLA2-infected cells. Role of Cellular Group IIa and V PLA2s in the H2O2-induced AA Release—To evaluate potential interactions between sPLA2s and cPLA2α during the H2O2-induced AA release, sPLA2s enzymes were expressed in MC+/+ and MC–/– using recombinant adenoviral vectors encoding group IIa PLA2 (Ad-IIa PLA2) or V PLA2 (Ad-V PLA2). Fig. 3A shows that H2O2-induced AA release is significantly increased when MC+/+ are infected with either Ad-IIa PLA2 (m.o.i. = 50) or Ad-V PLA2 (m.o.i. = 50). Ad-IIa or Ad-V PLA2s has no effect, however, on AA release in MC–/– (Fig. 3B). Neither group IIa nor V PLA2s have any effect on unstimulated AA release levels in MC+/+ and MC–/–. Fig. 3C shows that Ad-GFP/LacZ (m.o.i. = 40) infection had no effect on H2O2-induced AA release when compared with non-infected groups. Thus adenovirus infection itself had no effect upon AA release. Role of Cellular sPLA2in the H2O2-induced OA Release— Because, unlike cPLA2α, sPLA2 does not have a preferential effect on AA-containing membrane phospholipid" @default.
• W2011209855 created "2016-06-24" @default.
• W2011209855 creator A5012031480 @default.
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• W2011209855 creator A5015447566 @default.
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• W2011209855 creator A5090218080 @default.
• W2011209855 date "2003-06-01" @default.
• W2011209855 modified "2023-10-16" @default.
• W2011209855 title "Cross-talk between Cytosolic Phospholipase A2α (cPLA2α) and Secretory Phospholipase A2 (sPLA2) in Hydrogen Peroxide-induced Arachidonic Acid Release in Murine Mesangial Cells" @default.
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• W2011209855 doi "https://doi.org/10.1074/jbc.m300424200" @default.
• W2011209855 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12676927" @default.

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