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• W2029735110 abstract "The synthesis of platelet-activating factor (PAF) by A23187-stimulated RBL-2H3 cells was significantly suppressed by overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx). When the cells overexpressing PHGPx (L9 cells) were pretreated with diethyl maleate, which reduces PHGPx activity, PAF synthesis uponA23187 stimulation rose to levels seen in mock-transfected cells (S1 cells). Hydroperoxide levels, which are reduced in L9 cells, are involved in regulating PAF synthesis, because the addition of hydroperoxyeicosatetraenoic acid increased PAF production inA23187-stimulated L9 cells to control cell levels. The activity of acetyl-CoA:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase, which is involved in the last step of PAF synthesis, is also reduced in L9 cells. p38 kinase inhibitors block acetyltransferase activity in normal A23187-stimulated cells, suggesting that p38 kinase is involved in regulating acetyltransferase activity. Recombinant active p38 kinase activates acetyltransferase, whereas alkaline phosphatase reverses this, suggesting p38 kinase directly phosphorylates acetyltransferase. p38 kinase phosphorylation is blocked in L9 cells, indicating that high hydroperoxide levels are needed for the activation of p38 kinase. Thus, intracellular hydroperoxide levels participate in regulating p38 kinase phosphorylation, which in turn controls the activation of acetyltransferase and thus the synthesis of PAF. These observations suggest that PHGPx is an important component of the mechanisms regulating inflammation. The synthesis of platelet-activating factor (PAF) by A23187-stimulated RBL-2H3 cells was significantly suppressed by overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx). When the cells overexpressing PHGPx (L9 cells) were pretreated with diethyl maleate, which reduces PHGPx activity, PAF synthesis uponA23187 stimulation rose to levels seen in mock-transfected cells (S1 cells). Hydroperoxide levels, which are reduced in L9 cells, are involved in regulating PAF synthesis, because the addition of hydroperoxyeicosatetraenoic acid increased PAF production inA23187-stimulated L9 cells to control cell levels. The activity of acetyl-CoA:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase, which is involved in the last step of PAF synthesis, is also reduced in L9 cells. p38 kinase inhibitors block acetyltransferase activity in normal A23187-stimulated cells, suggesting that p38 kinase is involved in regulating acetyltransferase activity. Recombinant active p38 kinase activates acetyltransferase, whereas alkaline phosphatase reverses this, suggesting p38 kinase directly phosphorylates acetyltransferase. p38 kinase phosphorylation is blocked in L9 cells, indicating that high hydroperoxide levels are needed for the activation of p38 kinase. Thus, intracellular hydroperoxide levels participate in regulating p38 kinase phosphorylation, which in turn controls the activation of acetyltransferase and thus the synthesis of PAF. These observations suggest that PHGPx is an important component of the mechanisms regulating inflammation. platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine diethyl maleate Dulbecco's modified Eagle's medium extracellular-signal regulated kinase hydroperoxyeicosatetraenoic acid interleukin c-Jun N-terminal protein kinase 1-O-alkyl-2-lyso-glycero-3-phosphocholine mitogen-activated protein kinase MAPK kinase phosphate-buffered saline phospholipid hydroperoxide glutathione peroxidase phospholipase A2 polyvinylidene difluoride rat basophilic leukemia cells reactive oxygen species cytosolic PLA2 glutathione S-transferase Hanks' balanced salt solution Platelet-activating factor (PAF1; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipid that is synthesized by a variety of different cells and tissues in response to different stimuli. PAF is involved in numerous biological responses. Consequently, PAF is recognized as a major mediator that plays a central role in a variety of host defense system mechanisms and inflammatory diseases (1Patel K.D. Zimmerman G.A. Prescott S.M. McEver R.P. McIntyre T.M. J. Cell Biol. 1991; 112: 749-759Crossref PubMed Scopus (528) Google Scholar, 2Krull M. Dold C. Hippenstiel S. Rosseau S. Lohmeyer J. Suttorp N. J. Immunol. 1996; 157: 4133-4140PubMed Google Scholar, 3Guiney D.G. J. Clin. Invest. 1997; 99: 565-569Crossref PubMed Scopus (70) Google Scholar, 4Nagase T. Ishii S. Kume K. Uozumi N. Izumi T. Ouchi Y. Shimizu T. J. Clin. Invest. 1999; 104: 1071-1076Crossref PubMed Scopus (105) Google Scholar). In inflammatory cells, the remodeling pathway appears to be involved in synthesizing most of the PAF that is generated in response to a variety of stimuli. In this pathway, a phospholipase A2 (PLA2), such as cytosolic phospholipase A2, hydrolyzes 1-alkyl-2-arachidonyl-sn-glycero-3-phosphocholine to 1-alkyl-2-lyso-glycero-3-phosphocholine (lyso-PAF), thereby liberating arachidonic acid. Lyso-PAF is then acetylated by acetyl-CoA:lyso-PAF acetyltransferase to generate PAF. As acetyl-CoA:lyso-PAF acetyltransferase is unstable, it is difficult to purify, and consequently little is known about the enzyme. However, the use of rat spleen (5Lenihan D.J. Lee T.C. Biochem. Biophys. Res. Commun. 1984; 120: 834-839Crossref PubMed Scopus (75) Google Scholar, 6Gomez C.J. Velasco S. Mato J.M. Sanchez C.M. Biochim. Biophys. Acta. 1985; 845: 516-519Crossref PubMed Scopus (40) Google Scholar, 7Gomez C.J. Mato J.M. Vivanco F. Sanchez C.M. Biochem. J. 1987; 245: 893-897Crossref PubMed Scopus (21) Google Scholar), human neutrophils (8Nieto M.L. Velasco S. Sanchez C.M. J. Biol. Chem. 1988; 263: 4607-4611Abstract Full Text PDF PubMed Google Scholar), guinea pig parotid glands (9Domenech C. Machado-De D.E. Soling H.D. J. Biol. Chem. 1987; 262: 5671-5676Abstract Full Text PDF PubMed Google Scholar), and mouse mast cells (10Ninio E. Joly F. Hieblot C. Bessou G. Mencia H.J. Benveniste J. J. Immunol. 1987; 139: 154-160PubMed Google Scholar) as sources of the enzyme in in vitro assays has shown that the activity of the enzyme appears to be regulated by intracellular calcium levels and involves a phosphorylation-dephosphorylation mechanism. The kinase responsible for the phosphorylation is unknown, but catalytic subunits of the cyclic AMP-dependent protein kinase (8Nieto M.L. Velasco S. Sanchez C.M. J. Biol. Chem. 1988; 263: 4607-4611Abstract Full Text PDF PubMed Google Scholar, 9Domenech C. Machado-De D.E. Soling H.D. J. Biol. Chem. 1987; 262: 5671-5676Abstract Full Text PDF PubMed Google Scholar) and the calcium/calmodulin-dependent protein kinase (9Domenech C. Machado-De D.E. Soling H.D. J. Biol. Chem. 1987; 262: 5671-5676Abstract Full Text PDF PubMed Google Scholar) are capable of activating the acetyltransferase in vitro. Furthermore, it has also been shown that the p38 mitogen-activated protein kinase (MAPK) can lead to increased activation of acetyltransferase in neutrophils (11Nixon A.B. O'Flaherty J.T. Salyer J.K. Wykle R.L. J. Biol. Chem. 1999; 274: 5469-5473Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In aerobic cells, reactive oxygen species (ROS), such as the superoxide anion, hydrogen peroxide, and hydroxy radicals, are constantly formed as a result of mitochondrial respiration and reactions catalyzed by enzymes such as NADH/NADPH oxidase, xanthine oxidase, monooxidases, and cyclooxygenase. The ROS-mediated damage to intracellular molecules is limited by cellular antioxidant enzymes such as phospholipid hydroperoxide glutathione peroxidase (PHGPx), classical glutathione peroxidase, superoxide dismutase, and catalase. The glutathione peroxidases, which include four different selenoenzymes, are known for their ability to reduce organic and inorganic hydroperoxides (12Burk R.F. Hill K.E. Annu. Rev. Nutr. 1993; 13: 65-81Crossref PubMed Scopus (267) Google Scholar, 13Ursini F. Maiorino M. Brigelius F.R. Aumann K.D. Roveri A. Schomburg D. Flohe L. Methods Enzymol. 1995; 252: 38-53Crossref PubMed Scopus (666) Google Scholar). Of these enzymes, PHGPx is capable of directly reducing peroxidized lipids that have been produced in cell membranes and lipoproteins (14Ursini F. Maiorino M. Gregolin C. Biochim. Biophys. Acta. 1985; 839: 62-70Crossref PubMed Scopus (760) Google Scholar, 15Schnurr K. Belkner J. Ursini F. Schewe T. Kuhn H. J. Biol. Chem. 1996; 271: 4653-4658Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 16Thomas J.P. Maiorino M. Ursini F. Girotti A.W. J. Biol. Chem. 1990; 265: 454-461Abstract Full Text PDF PubMed Google Scholar). PHGPx exists both as a mitochondrial and a non-mitochondrial enzyme (17Imai H. Sumi D. Hanamoto A. Arai M. Sugiyama A. J. Biochem. (Tokyo). 1995; 118: 1061-1067Crossref PubMed Scopus (59) Google Scholar, 18Arai M. Imai H. Sumi D. Imanaka T. Takano T. Chiba N. Nakagawa Y. Biochem. Biophys. Res. Commun. 1996; 227: 433-439Crossref PubMed Scopus (96) Google Scholar). We have conducted a series of experiments to clarify the roles of the two types of PHGPx (19Arai M. Imai H. Koumura T. Yoshida M. Emoto K. Umeda M. Chiba N. Nakagawa Y. J. Biol. Chem. 1999; 274: 4924-4933Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 20Nomura K. Imai H. Koumura T. Kobayashi T. Nakagawa Y. Biochem. J. 2000; 351: 183-193Crossref PubMed Scopus (332) Google Scholar), and we have demonstrated recently that the non-mitochondrial type of PHGPx suppresses the production of bioactive eicosanoids such as prostaglandins and leukotrienes by lowering the intracellular peroxide level (21Imai H. Narashima K. Arai M. Sakamoto H. Chiba N. Nakagawa Y. J. Biol. Chem. 1998; 273: 1990-1997Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 22Sakamoto H. Imai H. Nakagawa Y. J. Biol. Chem. 2000; 275: 40028-40035Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Although direct evidence for the involvement of GPx in the production of PAF is still lacking, it has been shown that selenium deficiency, which causes a drop in GPx activity along with a corresponding rise in hydroperoxide levels (23Bryant R.W. Bailey J.M. Biochem. Biophys. Res. Commun. 1980; 92: 268-276Crossref PubMed Scopus (117) Google Scholar, 24Baker S.S. Cohen H.J. J. Immunol. 1983; 130: 2856-2860PubMed Google Scholar, 25Bryant R.W. Simon T.C. Bailey J.M. Biochem. Biophys. Res. Commun. 1983; 117: 183-189Crossref PubMed Scopus (43) Google Scholar), increases the production of PAF in cultured human umbilical vein endothelial cells (26Hampel G. Watanabe K. Weksler B.B. Jaffe E.A. Biochim. Biophys. Acta. 1989; 1006: 151-158Crossref PubMed Scopus (52) Google Scholar). These observations suggest that oxidative stress could modulate PAF synthesis and/or that scavenger enzymes could regulate the activity of the enzymes involved in the biosynthesis of PAF. Supporting the latter option is that PHGPx can interact with biomembrane in which the synthesis of PAF occurs. On the basis of these observations, we asked whether PHGPx can regulate PAF synthesis. To do this, we used RBL-2H3 cells that overexpress the non-mitochondrial form of PHGPx and show here that PAF synthesis in these cells is significantly suppressed. Intracellular hydroperoxide levels are reduced in PHGPx-overexpressing cells, as is acetyltransferase activity. We found p38 kinase was not activated in stimulated PHGPx-overexpressing cells, and experiments showed that this MAPK directly phosphorylates acetyltransferase. Thus, hydroperoxide levels affect the intracellular signal transduction system involving p38 MAPK and thereby modulate PAF synthesis. Mouse monoclonal antibodies specific for phosphorylated ERK, p38 kinase, and phosphorylated MKK3/6 were obtained from Santa Cruz Biotechnology. Phosphorylated p38 kinase-specific rabbit polyclonal antibodies were obtained from New England Biolabs Inc. [1-14C]Arachidonic acid (2.22 GBq/mmol) and [3H]acetic acid (5.55 GBq/mmol) were purchased from PerkinElmer Life Sciences. [3H]Acetyl coenzyme A was purchased from Amersham Biosciences. Diethyl maleate (DEM), KN-93, sodium salt of acetyl coenzyme A, fatty acid-free bovine serum albumin, and A23187 were obtained from Sigma. PD98059, U0126, SB203580, SB202190, and GST-tagged mouse recombinant p38 were obtained fromCalbiochem. H-7 was obtained from Seikagaku Co., Ltd. (Tokyo, Japan). 12-HpETE, 15-HpETE, PAF, and lyso-PAF were obtained from Funakoshi Co., Ltd. (Tokyo, Japan). The TLC plates were from Merck. We used the previously established L9 cells, which overexpress non-mitochondrial PHGPx, together with the S1 control cell line (20Nomura K. Imai H. Koumura T. Kobayashi T. Nakagawa Y. Biochem. J. 2000; 351: 183-193Crossref PubMed Scopus (332) Google Scholar, 27Imai H. Sumi D. Sakamoto H. Hanamoto A. Arai M. Chiba N. Nakagawa Y. Biochem. Biophys. Res. Commun. 1996; 222: 432-438Crossref PubMed Scopus (69) Google Scholar). Both lines were derived from the RBL-2H3 rat basophilic leukemia cell line and were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Aliquots of 1 × 106 cells/ml were transferred to fresh medium when the cells had reached semi-confluence. PAF accumulation was measured as the incorporation of [3H]acetic acid as described previously (28Nakagawa Y. Sugai M. Karasawa K. Tokumura A. Tsukatani H. Setaka M. Nojima S. Biochim. Biophys. Acta. 1992; 1126: 277-285Crossref PubMed Scopus (18) Google Scholar). Briefly, culture medium was removed from 1.7 × 106 cells in a 3.5-cm diameter dish and was replaced with 1 ml of Hanks' balanced salt solution (HBSS) (containing 1.3 mm Ca2+, 10 mm HEPES (pH 7.4) that contained 370 kBq/ml [3H]acetic acid. Following a 10-min preincubation at 37 °C, cells were incubated with A23187 for the indicated period. The concentration of A23187 was 5 μm unless otherwise indicated. The incubation was terminated by adding 1.5 ml of methanol containing 2% acetic acid. The cells were harvested, and total lipids were extracted according to the method of Bligh and Dyer (29Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42871) Google Scholar). Each extract was evaporated to dryness under reduced pressure, and the residues were then dissolved in a small amount of a 2:1 v/v mixture of chloroform and methanol and applied to a TLC plate (Silica Gel 60 F254). The plate was developed with a 65:35:6 v/v mixture of chloroform, methanol, and H2O. The products and standards were visualized with primulin reagent, and the products were identified by comparison with chromatographic standards. PAF was then scraped from TLC plate, and the radioactivity incorporated into PAF was determined by liquid scintillation counting. Acetyltransferase activity was determined according to the method described by Nakagawaet al. (28Nakagawa Y. Sugai M. Karasawa K. Tokumura A. Tsukatani H. Setaka M. Nojima S. Biochim. Biophys. Acta. 1992; 1126: 277-285Crossref PubMed Scopus (18) Google Scholar). Briefly, cells (5 × 106cells) were stimulated with A23187 in 2 ml of HBSS, and the reaction was terminated by adding 0.5 ml of ice-cold 10 mm HEPES buffer. The cells were harvested and sonicated in HEPES buffer with a probe sonicator (20 s, 50 watts, model 5202; Ohtaka Works, Tokyo, Japan). The cell lysates (200 μl, about 100 μg of total protein) were then incubated with 200 μm[3H]acetyl-CoA (0.25 GBq/mmol) and 350 μmlyso-PAF in a total volume of 1 ml. Reactions were carried out at 37 °C for 20 min and terminated by adding 1.75 ml of ethanol containing 2% acetic acid. As described above, total lipids were extracted and dried, and the residues were dissolved in a small amount of chloroform and methanol, after which they were applied to a TLC plate (Silica Gel 60 F254). The plate was developed and PAF scraped from the TLC plate, and its radioactivity was determined by liquid scintillation counting. Enzyme activities were calculated from the radioactivity of PAF. Cells (1.5 × 106 cells) were incubated with [1-14C]arachidonic acid (1.85 kBq, 0.42 μm) in 2 ml of culture medium for 24 h at 37 °C. Labeled cells were washed twice with phosphate-buffered saline (PBS) containing 1 mg/ml fatty acid-free bovine serum albumin. The cells were preincubated for 10 min in HBSS and then stimulated with 5 μmA23187 for 10 min. The release of radiolabeled arachidonic acid was determined as described previously (30Sakamoto H. Kitahara J. Nakagawa Y. J. Biochem. (Tokyo). 1999; 125: 90-95Crossref PubMed Scopus (8) Google Scholar). Cells (5 × 106cells) were activated with A23187, harvested, washed with PBS, and lysed with lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 0.2 trypsin inhibitor unit/ml aprotinin, and 1 mm sodium orthovanadate in PBS). Proteins (30 μg) from the lysate were separated by SDS-PAGE on a 12.5% polyacrylamide gel as described by Laemmli (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar), after which they were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane filter (ATTO Instruments, Tokyo, Japan) at 2 mA/cm2 for 1 h in 100 mm Tris, 192 mm glycine, and 5% (v/v) methanol in a protein-transfer system (ATTO), as described previously (22Sakamoto H. Imai H. Nakagawa Y. J. Biol. Chem. 2000; 275: 40028-40035Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The PVDF membrane bearing the blotted proteins was blocked for 2 h by incubation with 3% (w/v) defatted milk in 10 mm Tris-HCl (pH 7.4) that contained 150 mm NaCl and 0.1% Tween 20 (TBS-T). The membrane was then incubated for 1.5 h with rabbit polyclonal antibodies against phosphorylated p38 kinase or mouse monoclonal antibodies against phosphorylated ERK, p38 kinase, or phosphorylated MKK3/6 that had been diluted with TBS-T to an appropriate concentration. After the PVDF membrane had been washed twice with TBS-T, it was incubated with horseradish peroxidase-conjugated goat antibodies against rabbit or mouse IgG (Zymed Laboratories Inc., South San Francisco). The binding of the antibodies to antigens on the PVDF membrane was detected with an enhanced chemiluminescence Western blotting analysis system (AmershamBiosciences). Cells (3 × 107 cells) were washed twice with PBS, harvested, and centrifuged at 700 × g for 5 min at room temperature. The pellets were suspended in 2 ml of sucrose buffer (0.25 m sucrose, 1 mm EDTA, 3 mm imidazole, and 0.1% (v/v) ethanol that contained 10 μm leupeptin, 10 μm pepstatin A, 10 μm antipain, 10 μm chymostatin, and 100 μm phenylmethylsulfonyl fluoride (pH 7.2)) and homogenized in a Teflon/glass Potter-Elvehjem homogenizer. Subcellular fractions were obtained by differential centrifugation according to the method described by Arai et al. (19Arai M. Imai H. Koumura T. Yoshida M. Emoto K. Umeda M. Chiba N. Nakagawa Y. J. Biol. Chem. 1999; 274: 4924-4933Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Proteins (50 μg) in either the mitochondrial or the microsomal fractions from quiescent cells were immediately incubated at 37 °C for 30 min in a final volume of 40 μl containing 1 μm recombinant p38 kinase, 50 μm Mg2+, 50 μm ATP, and 25 mm HEPES (pH 7.5). Recombination p38 kinase was activated by the incubation with ATP, which induces autophosphorylation of p38 kinase. To measure acetyltransferase activity, this mixture (40 μl) was incubated with 200 μm[3H]acetyl-CoA (0.25 GBq/mmol) and 350 μmlyso-PAF in a total volume of 700 μl. Reactions were carried out at 37 °C for 20 min and terminated by the addition of 1.75 ml of ethanol containing 2% acetic acid. As described above, total lipids were extracted, and the residues were dissolved in a small amount of chloroform and methanol before they were applied to a TLC plate (Silica Gel 60 F254). The plate was developed and PAF scraped from the TLC plate, and PAF radioactivity was determined by liquid scintillation counting. Enzyme activities were calculated from the radioactivity of PAF. Concentrations of proteins were determined with Protein Assay Reagent (Bio-Rad) with bovine serum albumin as the standard. The production of PAF in RBL-2H3 cells that overexpress non-mitochondrial PHGPx (L9 cells) and in mock-transfected cells (S1 cells) was examined by incubating the cells with radioactive acetic acid and then determining the radioactivity of the resulting PAF (Fig. 1 A). S1 cells produced PAF when they were stimulated with A23187 as PAF levels were approximately four times higher in stimulated cells compared with unstimulated cells. In the cells overexpressing non-mitochondrial PHGPx, however, the production of PAF in response to A23187 stimulation was markedly inhibited (Fig. 1 A). To verify that PAF synthesis is suppressed in these cells because of the overexpression of PHGPx, we examined the effect of adding DEM (TableI). DEM reduces the activity of glutathione peroxidases such as classical glutathione peroxidase and PHGPx by lowering intracellular glutathione levels. In our experiments, DEM decreases intracellular glutathione levels in RBL-2H3 cells to about 5% that in untreated cells (30Sakamoto H. Kitahara J. Nakagawa Y. J. Biochem. (Tokyo). 1999; 125: 90-95Crossref PubMed Scopus (8) Google Scholar). PAF synthesis in stimulated S1 cells was not altered by treatment with DEM, but in L9 cells, treatment with DEM markedly increased the production of PAF in response to A23187stimulation. The levels of PAF in DEM-treated L9 cells were 94% that in S1 cells. Thus, reduced PAF synthesis in L9 cells is indeed due to the excess PHGPx activity.Table IEffects of diethyl maleate on PAF synthesis by PHGPx-overexpressing cellsControl line of cellsPHGPx-overexpressing cellsdpm/10 6 cellsControl242 ± 33332 ± 1945 μmA23187, 10 min719 ± 92307 ± 70DEM + A23187695 ± 48656 ± 59Control cells (S1) and PHGPx-overexpressing cells were incubated for 2 h at 37 °C with or without 1 mm DEM, after which they were labeled with [3H]acetic acid for 10 min. Labeled cells were stimulated with A23187 for 10 min, and PAF synthesis was determined. Data represent the means ± S.D. of three independent experiments. Open table in a new tab Control cells (S1) and PHGPx-overexpressing cells were incubated for 2 h at 37 °C with or without 1 mm DEM, after which they were labeled with [3H]acetic acid for 10 min. Labeled cells were stimulated with A23187 for 10 min, and PAF synthesis was determined. Data represent the means ± S.D. of three independent experiments. To identify which step(s) of PAF synthesis is inhibited in L9 cells, the rate at which arachidonic acid is released from the membrane lipids, which indicates PLA2activity, and the activity of acetyl-CoA:lyso-PAF acetyltransferase were determined. S1 and L9 cells did not differ significantly in the release of arachidonic acid (Fig. 1 B). To clarify the effect on acetyltransferase activity of overexpressing PHGPx, we measured the activity of acetyltransferase in a cell-free system using cell lysates prepared from quiescent and A23187-stimulated cells. The cell lysates were incubated with lyso-PAF, the substrate of the acetyltransferase, and [3H]acetyl-CoA, whose radioactive acetyl group is transferred to lyso-PAF by the acetyltransferase. The radioactivity of the resulting PAF was then measured. In lysates of quiescent cells, the acetyltransferase activity of S1 and L9 cells did not differ significantly (Fig. 2 A). In the stimulated cell lysates, however, L9 cells showed little acetyltransferase activation, whereas in S1 cells, acetyltransferase activity increased over time, a maximum being reached within 5 min after A23187 stimulation, after which the activity slowly declined (Fig. 2 A). When we examined the effect of A23187 dose on acetyltransferase activity, we found maximal activity was observed in the lysate prepared from S1 cells stimulated with 5 μmA23187 but that the acetyltransferase activity remained profoundly suppressed in the L9 cell lysates regardless of the A23187concentrations we used (Fig. 2 B). Thus, acetyl-CoA:lyso-PAF acetyltransferase activity is inhibited in PHGPx-overexpressing cells. To determine whether insufficient levels of hydroperoxides in PHGPx-overexpressing cells could account for the suppression of PAF synthesis in these cells, we determined PAF levels in A23187-stimulated cells that had been preincubated with various concentrations of the fatty acid hydroperoxide 12-hydroperoxyeicosatetraenoic acid (12-HpETE), 15-HpETE, or of H2O2. Each hydroperoxide had no effect on PAF synthesis by S1 cells (Fig. 3,A, C, and E). In contrast, the addition of hydroperoxides significantly increased PAF synthesis in L9 cells and at a concentration of 0.1 ng/ml 12-HpETE, 0.05 ng/ml 15-HpETE, or 100 μm H2O2, synthesis had recovered to 90, 98, or 90% of the levels in stimulated S1 cells, respectively (Fig. 3, B, D, andF). Thus, inadequate levels of hydroperoxides in PHGPx-overexpressing cells inhibit PAF synthesis, apparently by blocking acetyltransferase activity. To determine the enzyme that is responsible for modulating acetyltransferase activity inA23187-stimulated RBL-2H3 cells, we examined the effect on PAF synthesis of pretreating RBL-2H3 cells with kinase inhibitors beforeA23187 stimulation. The inhibitors we chose selectively inhibit Ca2+/calmodulin-dependent protein kinase II (KN-93), protein kinase C (H-7), MAPK/ERK kinase (PD98059 and U0126), and p38 kinase (SB203580 and SB202190). The PAF synthesis that occurs in RBL-2H3 cells after A23187 stimulation was abolished by treatment with the two p38 kinase blockers, but the other inhibitors had no effect (Fig. 4 A). The activation of acetyltransferase by A23187 treatment of RBL-2H3 cells was also almost totally suppressed by treatment with the two p38 kinase inhibitors but was unaffected by either of the ERK inhibitors (Fig. 4 B). Thus, p38 kinase is responsible for the activation of acetyltransferase in A23187-stimulated RBL-2H3 cells. To determine whether the lack of acetyltransferase activity in PHGPx-overexpressing cells is due to insufficient activation of p38 kinase, we monitored the phosphorylation of p38 kinase in A23187-stimulated L9 cells by immunoblotting analysis (Fig. 5). We could not detect the phosphorylated form of p38 kinase in unstimulated S1 cells or L9 cells. However, A23187 stimulation of S1 caused the apparent phosphorylation of p38 kinase and its upstream activator MAPK kinases 3 and 6 (MKK3 and MKK6). In contrast, no such phosphorylation was observed in stimulated L9 cells. The levels of p38 expression did not differ in S1 cells and L9 cells, regardless of whether they had been stimulated or not. With regard to ERK1/2, A23187 stimulation equally enhanced its phosphorylation in both S1 cells and L9 cells. To verify our observations made with the whole cells, we assessed the effect on acetyltransferase activation of adding activated recombinant p38 kinase. To perform this experiment, we first had to obtain a source of acetyltransferase. To do this, we determined the subcellular localization of acetyltransferase in RBL-2H3 cells by fractionating them by differential centrifugation. The acetyltransferase activity in the individual subfractions was then determined and was found to be largely concentrated in the mitochondrial and microsomal fractions (Fig. 6 A). The subcellular distribution of acetyltransferase activity did not differ in S1 cells and L9 cells (data not shown). Consequently, we used the mitochondrial and microsomal fractions prepared from quiescent S1 cells and L9 cells as acetyltransferase sources (Fig. 6 B). When activated recombinant p38 kinase was added to the mitochondrial fraction prepared from quiescent S1 cells, the acetyltransferase activity was doubled, whereas the activity in the microsomal fraction was only moderately enhanced. With regard to L9 cells, both the basal level of acetyltransferase activity in the mitochondrial fraction and the enhancement of this activity by adding activated p38 kinase were similar to that seen in the S1 cell fractions. Thus, reduction of acetyltransferase activity in PHGPx-overexpressing cells is due to the blocking of the intracellular signal transduction pathway that leads to the activation of p38 kinase. p38 kinase most likely activates acetyltransferase by phosphorylating it. To test this, the mitochondrial fraction of S1 cells was incubated with alkaline phosphatase after adding the activated recombinant p38 molecules and performing the kinase reaction (Fig. 7). The alkaline phosphatase treatment reduced the acetyltransferase activity in p38 kinase-treated mitochondrial fractions to almost the same levels seen in the control mitochondrial fraction, which had been treated with neither p38 nor alkaline phosphatase (Fig. 7). Thus, p38 kinase-mediated phosphorylation of acetyltransferase can activate the enzyme. To investigate whether the phosphorylation of acetyltransferase participates in the activation of the enzyme in A23187-stimulated RBL-2H3 cells, we examined the effect of alkaline phosphatase on the activity of acetyltransferase in the mitochondrial fraction prepared from control and A23187-stimulated S1 cells (Fig. 8). The acetyltransferase activity ofA23187-stimulated cells was twice as high as the activity of the unstimulated cells, but treatment with alkaline phosphatase reduced this to untreated control cell levels. Furthermore, when unstimulated cells were treated with alkaline phosphatase, the basal level of acetyltransferase activity was reduced by 30%. Thus, phosphorylation of acetyltransferase is essential for both the activation of acetyltransferase in stimulated cells and the expression of the basal a" @default.
• W2029735110 created "2016-06-24" @default.
• W2029735110 creator A5010831372 @default.
• W2029735110 creator A5079179398 @default.
• W2029735110 creator A5089796596 @default.
• W2029735110 date "2002-12-01" @default.
• W2029735110 modified "2023-09-30" @default.
• W2029735110 title "Overexpression of Phospholipid Hydroperoxide Glutathione Peroxidase Modulates Acetyl-CoA, 1-O-Alkyl-2-lyso-sn-glycero-3-phosphocholine Acetyltransferase Activity" @default.
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