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Originally published In Press as doi:10.1074/jbc.M204190200 on October 22, 2002

J. Biol. Chem., Vol. 277, Issue 52, 50431-50438, December 27, 2002
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Overexpression of Phospholipid Hydroperoxide Glutathione Peroxidase Modulates Acetyl-CoA, 1-O-Alkyl-2-lyso-sn-glycero-3-phosphocholine Acetyltransferase Activity*

Hikaru Sakamoto, Takaki Tosaki, and Yasuhito NakagawaDagger

From the School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan

Received for publication, April 30, 2002, and in revised form, October 21, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 upon A23187 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 in A23187-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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1-4). 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 (5-7), human neutrophils (8), guinea pig parotid glands (9), and mouse mast cells (10) 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 (8, 9) and the calcium/calmodulin-dependent protein kinase (9) 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 (11).

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 (12, 13). Of these enzymes, PHGPx is capable of directly reducing peroxidized lipids that have been produced in cell membranes and lipoproteins (14-16). PHGPx exists both as a mitochondrial and a non-mitochondrial enzyme (17, 18). We have conducted a series of experiments to clarify the roles of the two types of PHGPx (19, 20), 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 (21, 22). 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 (23-25), increases the production of PAF in cultured human umbilical vein endothelial cells (26). 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- 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 from Calbiochem. 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.

Cell Culture-- We used the previously established L9 cells, which overexpress non-mitochondrial PHGPx, together with the S1 control cell line (20, 27). 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.

Assaying Intracellular PAF Accumulation-- PAF accumulation was measured as the incorporation of [3H]acetic acid as described previously (28). 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 (29). 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.

Measurement of Acetyltransferase Activity-- Acetyltransferase activity was determined according to the method described by Nakagawa et al. (28). Briefly, cells (5 × 106 cells) 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 µM lyso-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.

Liberation of [1-14C]Arachidonic Acid from A23187-stimulated Cells-- 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 µM A23187 for 10 min. The release of radiolabeled arachidonic acid was determined as described previously (30).

Immunoblotting Analysis-- Cells (5 × 106 cells) 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 (31), 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 (22). 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 (Amersham Biosciences).

In Vitro Activation of Acetyltransferase by p38 Kinase-- 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. (19). 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 µM lyso-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.

Quantitation of Proteins-- Concentrations of proteins were determined with Protein Assay Reagent (Bio-Rad) with bovine serum albumin as the standard.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PAF Synthesis Is Suppressed in PHGPx-overexpressing RBL-2H3 Cells-- 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. 1A). 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. 1A). To verify that PAF synthesis is suppressed in these cells because of the overexpression of PHGPx, we examined the effect of adding DEM (Table I). 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 (30). 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 A23187 stimulation. 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.


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  		Fig. 1.   Platelet-activating factor synthesis by cells that overexpress non-mitochondrial PHGPx. A, rate of PAF synthesis. Control cells (S1) and cells that overexpress non-mitochondrial PHGPx (L9) were labeled with [3H]acetic acid for 10 min and then stimulated with A23187 for 10 min. The cells with culture media were collected, and total lipids were extracted, concentrated, and applied to a TLC plate. After the plate had been developed, the radioactivity of the products was determined by liquid scintillation counting. B, rate of [1-14C]arachidonic acid release. Cells were labeled with [1-14C]arachidonic acid for 24 h and then stimulated with A23187 for 10 min. The rate of radioactive arachidonic acid release into the incubation medium was determined. Open bars and hatched bars represent unstimulated cells and A23187-stimulated cells, respectively. Values are means ± S.D. of results from three independent experiments.

                              
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Table I
Effects of diethyl maleate on PAF synthesis by PHGPx-overexpressing cells
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.

Acetyl-CoA:Lyso-PAF Acetyltransferase Activity Is Suppressed by PHGPx Overexpression-- 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 PLA2 activity, 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. 1B). 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. 2A). 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. 2A). 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 µM A23187 but that the acetyltransferase activity remained profoundly suppressed in the L9 cell lysates regardless of the A23187 concentrations we used (Fig. 2B). Thus, acetyl-CoA:lyso-PAF acetyltransferase activity is inhibited in PHGPx-overexpressing cells.


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  		Fig. 2.   Lyso-PAF acetyltransferase activity in PHGPx-overexpressing cells. A, effect on lyso-PAF acetyltransferase activity over time for A23187-stimulated control cells (S1; open circles) and PHGPx-overexpressing cells (L9; closed circles). Cells were stimulated with A23187 for the indicated period before cell lysate preparation, after which acetyltransferase activity was determined. B, effect of A23187 dose on lyso-PAF acetyltransferase activity in control cells (S1; open circles) and PHGPx-overexpressing cells (L9; closed circles). Cells were stimulated with various concentrations of A23187 for 5 min before cell lysate preparation, after which acetyltransferase activity was determined. Values are means ± S.D. of results from three independent experiments.

Supplementation of Lipid Hydroperoxides in PHGPx-overexpressing Cells Reverses the Defect in PAF Synthesis-- 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, and F). Thus, inadequate levels of hydroperoxides in PHGPx-overexpressing cells inhibit PAF synthesis, apparently by blocking acetyltransferase activity.


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  		Fig. 3.   Effects of hydroperoxides on PAF synthesis in PHGPx-overexpressing cells. Control cells (A, C, and E; S1) and PHGPx-overexpressing cells (B, D, and F; L9), which had been labeled with [3H]acetic acid, were stimulated with A23187 for 10 min after preincubation with various concentrations of 12-HpETE (A and B), 15-HpETE (C and D), or H2O2 (E and F) for 10 min. The rate of PAF synthesis was then determined. Values are means ± S.D. of results from three independent experiments. Asterisk represents significant difference (p < 0.05) from the peroxide non-treated value.

p38 Kinase Inhibitors Block PAF Synthesis and Acetyltransferase Activation in Stimulated RBL-2H3 Cells-- To determine the enzyme that is responsible for modulating acetyltransferase activity in A23187-stimulated RBL-2H3 cells, we examined the effect on PAF synthesis of pretreating RBL-2H3 cells with kinase inhibitors before A23187 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. 4A). 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. 4B). Thus, p38 kinase is responsible for the activation of acetyltransferase in A23187-stimulated RBL-2H3 cells.


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  		Fig. 4.   Effect of protein kinase inhibitors on PAF synthesis and acetyltransferase activity in RBL-2H3 cells. A, effect of protein kinase inhibitors on PAF synthesis. RBL-2H3 cells were incubated with 50 µM of either KN-93, H-7, PD98059, U0126, SB203580, or SB202190 for 20 min and then labeled with [3H]acetic acid for 10 min. The labeled cells were stimulated with A23187 for 10 min, and PAF synthesis was determined. B, effects of inhibitors on the activity of acetyltransferase. RBL-2H3 cells were stimulated with A23187 for 5 min after incubation with 50 µM of a MAPK inhibitor (PD98059, U0126, SB203580, or SB202190) for 20 min. The cells were sonicated and subsequently assayed for acetyltransferase activity. Data represent the means ± S.D. of three independent experiments.

p38 Kinase Phosphorylation Is Suppressed by PHGPx Overexpression-- 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.


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  		Fig. 5.   Cellular levels of phosphorylated MAPKs in PHGPx-overexpressing cells. A, phosphorylation of ERK in cells stimulated with A23187. Control cells (S1) and PHGPx-overexpressing cells (L9) were stimulated with A23187 for 5 min, after which the cells were collected and lysed. The proteins in the lysates were subjected to SDS-PAGE and then blotted onto a PVDF membrane. Immunoblotting was performed using mouse monoclonal antibodies against phosphorylated ERK. B, phosphorylation of p38 in cells stimulated with A23187. Lysates of stimulated S1 and L9 cells were subjected to SDS-PAGE, and the proteins were blotted onto a PVDF membrane. Immunoblotting was performed using mouse monoclonal antibodies against p38 kinase, rabbit polyclonal antibodies against phosphorylated p38 kinase, or mouse monoclonal antibodies against phosphorylated MKK3/6.

Activated Recombinant p38 Kinase Activates Acetyltransferase-- 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. 6A). 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. 6B). 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.


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  		Fig. 6.   Activation of acetyltransferase with recombinant p38 kinase. A, subcellular localization of acetyltransferase in RBL-2H3 cells. Cells were fractionated by differential centrifugation into nuclear (Nuc), mitochondrial (Mit), microsomal (Mic), and cytosolic (Cyt) fractions. Acetyltransferase activity in the fractions was determined. B, effect of adding recombinant p38 kinase on the activity of acetyltransferase. Mitochondrial and microsomal proteins were incubated with (hatched bars) and without (open bars) GST-tagged recombinant p38 kinase. Acetyltransferase activity was then assayed. Data represent the means ± S.D. of three independent experiments.

Alkaline Phosphatase Treatment Inactivates Acetyltransferase in the Mitochondrial Fraction-- 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 of A23187-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 activity in quiescent cells.


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  		Fig. 7.   Inactivation of acetyltransferase by alkaline phosphatase treatment in vitro. The mitochondrial proteins prepared from S1 cells were incubated with GST-tagged recombinant p38 kinase. The kinase reaction mixture was then treated with (+) or without (-) 5 units of alkaline phosphatase in assay buffer (pH 9.0) for 15 min at 25 °C, and then acetyltransferase activity was assayed. Data represent the means ± S.D. of three independent experiments.


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  		Fig. 8.   Effect of alkaline phosphatase treatment on the activity of acetyltransferase activated in vivo. S1 cells were stimulated with A23187 for 5 min before the preparation of the mitochondrial fraction. Mitochondrial protein was treated with (+) or without (-) 5 units of alkaline phosphatase in assay buffer (pH 9.0) for 15 min at 25 °C, and then acetyltransferase activity was assayed. Open bars and hatched bars represent unstimulated and A23187-stimulated cells, respectively. Values are means ± S.D. of results from three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is known that PAF is synthesized in response to stimulants by a variety of cells, including mast cells, basophils, endothelial cells, neutrophils, eosinophils, macrophages, and platelets (32). To investigate the regulatory mechanisms involved in PAF synthesis, we have examined the effect on PAF synthesis of overexpressing the non-mitochondrial form of PHGPx in the rat basophilic leukemia cell line RBL-2H3. The overexpression of non-mitochondrial PHGPx dramatically suppressed the PAF that was generated in response to A23187 stimulation, indicating that PHGPx is involved in regulating PAF. It is known that RBL-2H3 cells predominantly synthesize PAF via the remodeling pathway that involves PLA2 and acetyltransferase, and we have shown that A23187 stimulates RBL-2H3 cells to activate cytosolic PLA2 (cPLA2) (21). It appears, however, that cPLA2 is not affected by the overexpression of PHGPx as PHGPx-overexpressing cells and normal cells did not differ in their rates of arachidonic acid release. In contrast, the acetyltransferase activity in PHGPx-overexpressing cells was much lower than in control cells, suggesting that the target of PHGPx is the acetyltransferase rather than cPLA2.

Stimulation with A23187 provokes the production of intracellular peroxides (21). PHGPx acts to reduce such molecules, and thus their levels in PHGPx-overexpressing cells are low. It is known that the addition of the fatty acid hydroperoxide 12-HpETE dramatically increases the intracellular hydroperoxide levels in PHGPx-overexpressing cells (21). When we incubated PHGPx-overexpressing cells with hydroperoxide, 12-HpETE, 15-HpETE, or H2O2, prior to A23187 stimulation, we found these cells increased their production of PAF. This indicates that lipid hydroperoxide levels participate in regulating PAF synthesis. To determine whether other GPx besides PHGPx could suppress PAF synthesis, we examined the effect of ebselen. Ebselen is a seleno-organic compound existing mainly in cytosol (33) and exhibiting glutathione peroxidase activity (34). PAF synthesis in stimulated L9 cells was hardly altered by treatment with ebselen, but in S1 cells, treatment with ebselen suppressed 60% of untreated cells in response to A23187 stimulation (data not shown). This indicates that other GPx besides PHGPx may also be involved in the regulation of PAF synthesis via modulating intracellular hydroperoxide levels.

ROS, like H2O2, appear to be involved in regulating multiple cellular processes by their interactions with various protein and lipid species (35). These observations have led to the notion that ROS act as cofactors in the regulation of many intracellular signal transduction cascades. One such cascade is the MAPK cascade, which is a major signaling system by which cells transduce extracellular stimuli into intracellular signals. A key molecule in the MAPK cascade is p38 MAPK, which appears to be an important mediator of cytokine production (36), transcriptional regulation (37), cytoskeletal reorganization (38), and proapoptotic program in various cells (39, 40). p38 kinase, like c-Jun N-terminal protein kinase (JNK), is considered to be a stress-activated protein kinase in mammalian cells, and several reports (41-43) indicate that it is activated in cells exposed to hyperosmotic stress, UV irradiation, endotoxin, and oxidative stress induced by ROS. The specific molecular targets that are critical for peroxide-mediated p38 kinase activation are largely unknown. However, Saitoh et al. (44) reported that a redox regulatory protein thioredoxin associated with apoptosis signal-regulation kinase (Ask) 1 acts as an oxidative stress sensor for the p38 signaling pathway. Ask1 is a MAP kinase kinase kinase that can activate MKK3/6 leading to p38 activation (45). Upon oxidative stress, oxidation of thioredoxin provokes the dissociation from Ask1 permitting the activation of the Ask1 and subsequent signaling pathway (44). With regard to PAF synthesis in response to oxidative stress, p38 kinase has been implicated in its regulation by the following lines of evidence. First, oxidative stress is known to induce the activation of signal transduction systems and the production of PAF (46). Second, Chiu et al. (47) found that activation of p38 MAPK induced by transforming growth factor-beta is mediated by the intracellular generation of ROS. Third, lipid peroxidation induced by acute short term UVB irradiation and pro-oxidant treatment resulted in the activation of the ERK1/2 and p38 signaling pathway (48) and the subsequent production of PAF and related 1-acyl species (49, 50). Supporting the role of p38 in oxidative stress-driven PAF synthesis, we found in the study reported here that the addition of either of two selective p38 kinase inhibitors (SB203580 and SB202190) blocked both PAF synthesis and acetyltransferase activity in A23187-stimulated RBL-2H3 cells, indicating the involvement of this MAPK in regulating acetyltransferase activity and thereby PAF synthesis.

Unlike the p38 inhibitors, ERK inhibitors had no effect on acetyltransferase activity and PAF synthesis in stimulated RBL-2H3 cells. This is significant because there is some controversy about the roles of ERK and p38 in the steps that regulate PAF synthesis. When cells are stimulated with A23187, it is known that cPLA2 becomes activated before PAF is synthesized. ERK1/2 are associated with this activation of cPLA2, but how they interact with p38 is unclear. In platelets, thrombin induces a rapid and robust activation of p38 MAPK which then phosphorylates cPLA2. In human eosinophils (51), macrophages (52), and Fc-gamma RIIa- or Fc-gamma RIIIb-stimulated neutrophils (53), however, both ERK1/2 and p38 MAPK are necessary for the onset and the maintenance of cPLA2 activation. In the study reported here, we observed no difference between PHGPx-overexpressing and normal cells in the A23187-stimulated release of arachidonic acid or the phosphorylation of ERK. Thus, overexpression of PHGPx does not affect the activation of cPLA2 but does suppress the phosphorylation of p38 MAPK, indicating that p38 MAPK is not involved in the A23187-stimulated phosphorylation of cPLA2 in PHGPx-overexpressing RBL-2H3 cells.

The p38 family of MAPK includes the p38, beta , beta 2, gamma , and delta  isoforms. These p38 kinases are activated by being phosphorylated on the Thr and Tyr residues within the Thr-Gly-Tyr motif that is located in kinase subdomain VIII. The pyridinylimidazole inhibitors that we used, SB203580 and SB202190, inhibit in vitro the p38, p38beta 2, and p38beta isoforms but not p38gamma or p38delta . Kumar et al. (54) have suggested that in vivo SB203580 inhibits p38 and p38beta . However, SB203580 has been reported to affect 3-phosphoinositide-dependent protein kinase-1 activity (55), and thus, it cannot be excluded that SB203580 targets an enzyme other than p38 MAPK. However, SB202190 also suppressed the activation of acetyltransferase which suggests that p38 and/or p38beta 2 are involved in acetyltransferase activation during PAF biosynthesis.

The activation of p38 kinase is dependent on upstream tyrosine/threonine kinases, the MKKs. Although MKK4, which can phosphorylate p38 MAPK, stimulates JNK and p38 MAPK (56-58), MKK3 and MKK6 are known to specifically activate p38 MAPK. We showed that A23187 stimulation of RBL-2H3 cells induced the phosphorylation of both MKK3 and MKK6. Thus, A23187 stimulation may result in the phosphorylation and activation of MKK3/6, which then go on to phosphorylate and activate p38 MAPK, which subsequently activates acetyltransferase.

Although the intracellular compartment that contains acetyltransferase has not been unequivocally determined, it is known that acetyltransferase frequently occurs in the microsomal fraction of a variety of tissues (59) and blood cells (60). It is also distributed in the plasma membrane and the endoplasmic reticulum of neutrophils (61). We fractionated RBL-2H3 cells and examined each fraction for acetyltransferase activity (Fig. 6). We found that acetyltransferase activity is predominantly localized in the mitochondrial fraction of RBL-2H3 cells. Treatment of this fraction with recombinant, constitutively activated p38 MAPK increased acetyltransferase activity, whereas treatment with phosphatase after the kinase reaction reversed this. Thus, phosphorylation of acetyltransferase by p38 activates it. Supporting this is that when RBL-2H3 cells were treated with alkaline phosphatase, both the acetyltransferase activity induced by A23187 stimulation and the basal level of acetyltransferase activity were reduced. Thus, the activity of acetyltransferase is directly regulated by its phosphorylation by p38.

It is well known that the MAPKs ERK1/2, p38, and JNK are deeply involved with the regulation of inflammation. At inflammatory sites, proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor, and other mediators, such as PAF, a highly potent phospholipid (62, 63), play crucial roles in cell-cell interactions and activate the MAPKs p38 and ERK1/2 in neutrophils (64). These inflammatory mediators also provoke endothelial cells to synthesize PAF in a regulated fashion, which then activates polymorphonuclear leukocytes, the key effector cells in the acute inflammatory response. Activated leukocytes generate ROS, such as superoxide and hydrogen peroxide, and release them. ROS production by phorbol 12-myristate 13-acetate-stimulated leukocytes is known to be suppressed by antioxidative enzymes and antioxidants (65, 66). Thus, antioxidant effectors could influence the multiple intracellular events that involve ROS production. Supporting this is that Schnurr et al. (67) reported that IL-4- and IL-13-treated A549 cells up-regulate 12/15-lipoxygenase and down-regulate PHGPx, resulting markedly increased levels of endogenous lipid peroxides. They suggested that the generation of hydroperoxides and peroxide-reducing enzymes is inversely regulated. Our observations support this, indicating that small variations in the levels of intracellular lipid hydroperoxides can modulate the production of PAF by altering the degree with which p38 kinase activates the acetyltransferase that is responsible for PAF biosynthesis. As PHGPx regulates intracellular hydroperoxide concentrations and thus can intercept the intracellular signal transduction system involving p38 MAPK, it may play an important role in regulating inflammatory reactions that involve the release of ROS. We have reported previously that PHGPx may also be important in regulating the metabolism of arachidonic acid, the synthesis of leukotrienes, and the synthesis of prostanoids (21, 22). As it appears that lipid hydroperoxide levels controlled by PHGPx are important in the regulation of PAF biosynthesis (although the precise mechanism remains to be elucidated), it may be that PHGPx also regulates the biosyntheses of fundamental lipid mediators, leukotrienes, and prostanoids using the same hydroperoxide triggers.

Thus, in summary, our present study suggests that PHGPx may regulate cellular function and signal transduction by modulating intracellular levels of lipid hydroperoxides.

    ACKNOWLEDGEMENTS

We thank Yoko Kuratsuji and Satomi Tsuyuki for their expert technical assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel./Fax: 81-3-3444-4943; E-mail: nakagaway@pharm.kitasato-u.ac.jp.

Published, JBC Papers in Press, October 22, 2002, DOI 10.1074/jbc.M204190200

    ABBREVIATIONS

The abbreviations used are: PAF, platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; DEM, diethyl maleate; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular-signal regulated kinase; HpETE, hydroperoxyeicosatetraenoic acid; IL, interleukin; JNK, c-Jun N-terminal protein kinase; lyso-PAF, 1-O-alkyl-2-lyso-glycero-3-phosphocholine; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; PBS, phosphate-buffered saline; PHGPx, phospholipid hydroperoxide glutathione peroxidase; PLA2, phospholipase A2; PVDF, polyvinylidene difluoride; RBL, rat basophilic leukemia cells; ROS, reactive oxygen species; cPLA2, cytosolic PLA2; GST, glutathione S-transferase; HBSS, Hanks' balanced salt solution.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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