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J. Biol. Chem., Vol. 275, Issue 26, 20146-20156, June 30, 2000

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Cytosolic Phospholipase A₂ Is Required for Macrophage Arachidonic Acid Release by Agonists That Do and Do Not Mobilize Calcium

NOVEL ROLE OF MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS IN CYTOSOLIC PHOSPHOLIPASE A₂ REGULATION^*

Miguel A. Gijón^§, Diane M. Spencer, Abdur R. Siddiqi, Joseph V. Bonventre^¶, and Christina C. Leslie^**

From the  Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, the  Department of Pathology, University of Colorado School of Medicine, Denver, Colorado 80262, and the ^¶ Department of Medicine, Harvard Medical School, Massachusetts General Hospital East, Charlestown, Massachusetts 02129

Received for publication, November 5, 1999, and in revised form, April 26, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 85-kDa cytosolic phospholipase A₂ (cPLA₂) mediates agonist-induced arachidonic acid release and eicosanoid production. Calcium and phosphorylation on Ser-505 by mitogen-activated protein kinases (MAPKs) regulate cPLA₂. Arachidonic acid release and eicosanoid production induced by stimuli that do (A23187, zymosan) or do not (phorbol myristate acetate (PMA), okadaic acid) mobilize calcium were quantitatively suppressed in cPLA₂-deficient mouse peritoneal macrophages. The contribution of MAPKs to cPLA₂-mediated arachidonic acid release was investigated. Both extracellular signal-regulated kinases (ERKs) and p38 contributed to cPLA₂ phosphorylation on Ser-505. However, although ERK inhibition did not affect A23187-induced arachidonic acid release, it suppressed zymosan-, PMA-, and okadaic acid-induced arachidonic acid release under conditions where phosphorylation of cPLA₂ on Ser-505 was unaffected. This indicates an additional regulatory mechanism for the ERK pathway. A role for transcriptional regulation is suggested by data showing that cycloheximide and actinomycin D inhibited arachidonic acid release induced by zymosan, PMA and, okadaic acid but not by A23187. Our results show that MAPK pathways contribute to arachidonic acid release in macrophages through alternative mechanisms in addition to their ability to phosphorylate cPLA₂ on Ser-505 and suggest a role for new protein synthesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 85-kDa cytosolic phospholipase A₂ (cPLA₂)¹ plays a central role in the release of arachidonic acid that occurs in many types of cells in response to a wide variety of stimuli (1, 2). Recent evidence obtained with cPLA₂-deficient mice demonstrates that cPLA₂ plays an essential role in eicosanoid production and allergic responses (3, 4). Increases in the cytosolic concentration of calcium and phosphorylation by mitogen-activated protein kinases (MAPKs) have been shown to play important roles in cPLA₂ activation (1, 2). Activation of cPLA₂ involves its translocation from cytosol and its stable association with membrane (5-7). Calcium is required for promoting the binding of cPLA₂ to membrane through a calcium-dependent lipid binding domain that shares homology with C2 domains present in a variety of proteins including phospholipase C and protein kinase C (6, 8). The C2 domain of cPLA₂ is necessary and sufficient for translocation of cPLA₂ in response to calcium-mobilizing agonists, and it also contains hydrophobic residues that are essential for penetration of cPLA₂ into the membrane bilayer (9-11). Upon elevation of intracellular calcium, cPLA₂ translocates primarily to the nuclear envelope and the perinuclear region (7, 12-16). However, translocation of cPLA₂ can occur without increased levels of calcium, suggesting alternative regulatory mechanisms (16).

Extracellular signal-regulated kinases (ERKs) phosphorylate cPLA₂ on Ser-505, which modestly increases its catalytic activity and results in a characteristic retardation of its electrophoretic mobility or gel shift (13, 17, 18). Although ERKs have been implicated in Ser-505 phosphorylation of cPLA₂ in different models, recent reports show that p38 is the MAPK responsible for cPLA₂ phosphorylation in thrombin- and collagen-activated platelets and tumor necrosis factor--stimulated neutrophils (19-21). Phosphorylation of Ser-505 has been shown to be essential for agonist-induced arachidonic acid release in CHO cells overexpressing human cPLA₂, and we have recently reported that arachidonic acid release in Sf9 cells expressing a S505A mutant of cPLA₂ is partially suppressed compared with cells expressing the wild type enzyme (13, 16). However, phosphorylation of cPLA₂ on Ser-505 is not required for arachidonic acid release in thrombin-stimulated platelets, but it may be involved in the platelet response to collagen (19, 20). More data are required to clarify the role of MAPK pathways and cPLA₂ phosphorylation in the regulation of arachidonic acid release in response to different agonists.

We have used mouse peritoneal macrophages as a model system to study the mechanisms involved in cPLA₂ activation and the regulation of arachidonic acid release (22). A variety of agonists, such as the calcium ionophore A23187, the phosphatase inhibitor okadaic acid, the protein kinase C activator phorbol myristate acetate (PMA), and the phagocytic particle zymosan, induce arachidonic acid release in these cells. These agonists also trigger phosphorylation of cPLA₂ and activation of ERKs by either protein kinase C-dependent (PMA, zymosan) or protein kinase C-independent (A23187, okadaic acid) pathways (23, 24). A23187 and zymosan increase intracellular calcium, but PMA and okadaic acid induce arachidonic acid release by unknown mechanisms that do not involve calcium mobilization (25). cPLA₂ has been shown to be essential for the immediate arachidonic acid release induced in mouse peritoneal macrophages by A23187 and the combination of A23187 and PMA (3, 4). The results presented in this study demonstrate that cPLA₂ is necessary for arachidonic acid release induced in macrophages by calcium-mobilizing agonists such as A23187 and zymosan but also by PMA and okadaic acid, implicating mechanisms for cPLA₂ regulation that do not require an increase in calcium. In evaluating the role of Ser-505 phosphorylation in regulation of cPLA₂ in response to PMA and okadaic acid, we found that MAPK pathways play an essential role in cPLA₂-mediated arachidonic acid release by an alternative mechanism in addition to their role in Ser-505 phosphorylation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Mice in which the 85-kDa cPLA₂ gene has been disrupted were generated using 129 embryonic stem cells in a C57BL/6 strain background (4, 26). Macrophages from these mice and strain-matched wild type littermates were used for the experiments shown in Figs. 1 and 2. Pathogen-free ICR mice were obtained from Harlan Sprague-Dawley and used for all other experiments. [5,6,8,9,11,12,14,15-³H]Arachidonic acid (specific activity 100 Ci/mmol), [³H]myristic acid (49 Ci/mmol), and 1-hexadecyl-2-[³H]arachidonoyl-sn-phosphatidylcholine (200 Ci/mmol) were from NEN Life Science Products. Anti-rabbit IgG horseradish peroxidase-linked F(ab')₂ fragment and the reagents for enhanced chemiluminescence detection on immunoblots were from Amersham Pharmacia Biotech. Phospho-specific antibodies against threonine- and tyrosine-phosphorylated p42 and p44 ERKs, p46 and p54 cJun-N-terminal kinases (JNKs), and p38 MAPK were purchased from New England Biolabs. Calcium ionophore A23187, zymosan, anisomycin, cycloheximide, actinomycin D, and protein A-Sepharose CL-4B beads were obtained from Sigma. Before use as a stimulus, zymosan was prepared as described previously (23). PD98059, SB203580, and SB202190 were from Calbiochem. The MAPK/ERK kinase (MEK) inhibitor U0126 was kindly provided by DuPont. PMA and the ammonium salt of okadaic acid were from LC Services Co. Dulbecco's modified Eagle's medium and 10× Hanks' balanced salt solution were from BioWhittaker, Inc. Fetal bovine serum was from Irvine Scientific. Human serum albumin was purchased from Intergen. Reagents for protein determination by the bicinchoninic acid method were obtained from Pierce.

Arachidonic Acid Release and Eicosanoid Production-- Murine resident peritoneal macrophages were isolated as detailed elsewhere (23). Macrophages were plated at a density of 0.5 × 10⁶ cells/cm² (in 24- or 48-well plates) and incubated overnight in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, 0.29 mg/ml glutamine, and 0.1 µCi/ml [³H]arachidonic acid. Cells were then washed three times in serum-free Dulbecco's modified Eagle's medium containing 0.1% human serum albumin and stimulated at 37 °C in a humidified atmosphere of 5% CO₂ in air. Unless otherwise specified, 0.5 µg/ml A23187, 1 µM okadaic acid, 32 nM PMA, or 10-30 particles of zymosan/cell were used. The amount of radioactive arachidonic acid released into the medium was measured by scintillation counting, and the results are expressed as percent of the total radioactivity incorporated (cell-associated plus medium). Background release (4-5%) from unstimulated cells treated with vehicle (0.1% Me₂SO) is subtracted from each experimental point. In the experiments with cPLA₂-deficient macrophages shown in Fig. 1, [³H]arachidonic acid was extracted from the culture medium and separated by thin-layer chromatography using hexane/diethyl ether/acetic acid 80:20:1 (27). The amounts of prostaglandin E₂ (PGE₂) and leukotriene C₄ (LTC₄) in the medium were determined by displacement enzyme-linked immunosorbent assay as described previously (28). All arachidonic acid release and eicosanoid production experiments were performed in triplicate.

Measurement of cPLA₂ Enzymatic Activity-- Macrophages (11 × 10⁶ cells/60-mm dish) were incubated with vehicle or 10 µM SB203580 for 15 min, then stimulated with anisomycin (25 ng/ml) for 30 min. Cell homogenates were prepared by sonication, and cPLA₂ activity was determined in the 100,000 × g cytosolic fraction (10 µg protein) using [³H]phosphatidylcholine as described previously (23).

Immunoblotting of Phosphorylated MAPKs and cPLA₂-- Macrophages were scraped into ice-cold lysis buffer: 50 mM Hepes, pH 7.4, 150 mM sodium chloride, 1.5 mM magnesium chloride, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 200 µM sodium vanadate, 10 mM tetrasodium pyrophosphate, 100 mM sodium fluoride, 300 nM p-nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Lysates were centrifuged at 15,000 × g for 15 min, and protein concentration was determined by the bicinchoninic acid method. Laemmli electrophoresis sample buffer (5×) was added to the lysates, and SDS-polyacrylamide gel electrophoresis and immunoblotting were performed using 10-20 µg of lysate protein (25). For analysis of cPLA₂ gel shift, samples (5-10 µg) were resolved on 10% polyacrylamide gels (16-cm long, pH 8.3) with 1% cross-linking (acrylamide:bis-acrylamide ratio 99:1).

NADPH Oxidase Activity-- Macrophages (0.5 × 10⁶ cells/well on 48-well tissue culture plates) were activated with PMA (320 nM) or zymosan (30 particles/cell) for 90 min, then washed three times with warm (37 °C) Hanks' balanced salt solution. Superoxide anion production was determined by measuring cytochrome c reduction at 550 nm as described previously (29).

Phospholipase D Activity-- Macrophages (1 × 10⁶ cells/well on 24-well plates) were labeled with [³H]myristic acid and stimulated with PMA (32 nM) or zymosan (30 particles/cell) for 30 min in the presence of 1% ethanol. After stimulation, lipids were extracted, and [³H]phosphatidylethanol formation was measured after thin-layer chromatography as described previously (30).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Role of cPLA₂ in Agonist-induced Arachidonic Acid Release in Macrophages-- Previous studies have implicated cPLA₂ in mediating arachidonic acid release from mouse peritoneal macrophages in response to a variety of stimuli (23-25, 31). However, since macrophages contain different types of PLA₂ enzymes, experiments were carried out to conclusively determine the role of cPLA₂, particularly with stimuli that do not mobilize calcium. Resident peritoneal macrophages from cPLA₂-deficient mice were used to evaluate the role of cPLA₂ in mediating arachidonic acid release and eicosanoid production induced by A23187, PMA, okadaic acid, and zymosan. As shown in Fig. 1, arachidonic acid release induced by all these agonists was quantitatively suppressed in macrophages derived from cPLA₂-deficient mice compared with cells from wild type mice. PMA was the only agonist that induced a small but consistent release of arachidonic acid in cPLA₂-deficient cells, approximately 20% of the response observed in wild type cells. PGE₂ production was nearly completely abolished in cPLA₂-deficient macrophages in response to A23187, PMA, zymosan, and okadaic acid. Macrophages from wild type mice produced significant amounts of LTC₄ in response to the calcium-mobilizing agonists A23187 and zymosan, and this was quantitatively suppressed in cPLA₂-deficient cells. Only a small amount of LTC₄ was produced in response to PMA, and none was produced in response to okadaic acid, which is not unexpected because of the requirement of 5-lipoxygenase for calcium (32, 33). However, when A23187 was used together with PMA, a synergistic effect on LTC₄ production was observed. These results show that cPLA₂ is required for arachidonic acid release and eicosanoid production induced by A23187, PMA, zymosan, and okadaic acid in macrophages and confirm that cPLA₂-mediated arachidonic acid release can be induced in cells without an increase in intracellular calcium.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Arachidonic acid release and eicosanoid production from wild type and cPLA2-deficient mouse peritoneal macrophages. [3H]Arachidonic acid-labeled macrophages obtained from wild type (cPLA2 +/+) or cPLA2-deficient (cPLA2 /) mice were incubated for 60 min with A23187, PMA, or zymosan, for 90 min with okadaic acid (O.A.), or with vehicle (unstimulated (US)). [3H]Arachidonic acid (A), PGE2 (B), and LTC4 (C) were measured as described under "Experimental Procedures." Results are the average ± S.D. of a representative experiment and were verified in two independent experiments.

Other responses known to be triggered by PMA and zymosan in wild type macrophages were measured in cPLA₂-deficient macrophages (Fig. 2). The activation of NADPH oxidase (superoxide anion production) and phospholipase D (phosphatidylethanol production) induced by PMA and zymosan were similar in cells from cPLA₂-deficient mice as compared with wild type mice, indicating that cPLA₂-deficient macrophages are responsive to these two agonists. It has been suggested that cPLA₂ is required for the activation of NADPH oxidase in human myeloid PLB-985 cells (34). It has also been proposed that activation of phospholipase D2 in mouse lymphocytic leukemia L1210 cells is dependent on cPLA₂ activation (35). However, the results presented here demonstrate that cPLA₂ is not involved in the activation of these enzymes by PMA or zymosan in mouse peritoneal macrophages.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   NADPH oxidase and phospholipase D activation in wild type and cPLA2-deficient macrophages. Macrophages from wild type (cPLA2 +/+) or cPLA2-deficient (cPLA2 /) mice were treated with vehicle (unstimulated, US) or stimulated with PMA or zymosan. A, activation of NADPH oxidase was determined by superoxide anion production and its ability to reduce cytochrome C. N.D., not detected above background levels. B, phospholipase D activation was measured in [3H]myristic acid-labeled macrophages by the production of [3H]phosphatidylethanol. Results (average ± S.D.) are representative of two independent experiments.

Role of ERK Activation in cPLA₂ Phosphorylation and Arachidonic Acid Release-- PMA, zymosan, and okadaic acid have previously been shown to induce sustained ERK activation and phosphorylation of cPLA₂ on Ser-505 (24, 25). This phosphorylation event in itself is not sufficient for arachidonic acid release, although agonists that activate ERKs can act synergistically with agonists that transiently increase intracellular calcium (17, 25, 31, 36). However, the role of Ser-505 phosphorylation in the activation of cPLA₂ by agonists that do not mobilize calcium is not known. The role of the ERK pathway was investigated using MAPK/ERK kinase (MEK) inhibitors, which prevent activation of ERKs. The various agonists used in this study act by different mechanisms, and the times required for their activation of ERK and arachidonic acid release vary considerably (23-25). Therefore, different times of stimulation were used for each agonist in these experiments to ensure maximal activation of ERKs and maximal Ser-505 phosphorylation: 15 min for A23187 and PMA, 30 min for zymosan, and 90 min for okadaic acid. In preliminary experiments comparing PD98059 and U0126, only the latter was found to be effective at quantitatively inhibiting the strong ERK activation induced by PMA, zymosan, and okadaic acid in mouse peritoneal macrophages (Fig. 3A and data not shown). This is consistent with the fact that U0126 has a 100-fold higher affinity than PD98059 for MEK1 (37). U0126 specifically inhibits MEK1 and MEK2, the kinases that phosphorylate and activate ERKs, with very little effect on other MAPK kinases (37). As shown in Fig. 3A, U0126 at concentrations of 1-10 µM suppressed ERK activation in response to A23187, PMA, zymosan, and okadaic acid.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Dose-dependent effect of the MEK inhibitor U0126 on agonist-induced ERK activation, cPLA2 gel shift, and arachidonic acid release. Macrophages were preincubated with the indicated concentrations of U0126 for 15 min and then treated with vehicle (unstimulated (US)), A23187 (15 min), PMA (15 min), zymosan (30 min), or okadaic acid (90 min). Activated ERKs (A) or cPLA2 gel shift (B) in whole cell lysates were analyzed by immunoblotting. Results are representative of three independent experiments. C, [3H]arachidonic acid-labeled macrophages were treated as indicated above, and the amount of arachidonic acid released into the medium was determined by scintillation counting. Results are expressed as the percentage of control arachidonic acid release induced by each agonist from control cells not preincubated with U0126. Control values, expressed as the percentage of total radioactivity incorporated that was released into the medium, were: A23187, 6.1%; PMA, 11.0%; zymosan, 14.2%; okadaic acid, 20.4%. Results (average ± S.D.) are representative of three independent experiments.

The effect of U0126 on Ser-505 phosphorylation of cPLA₂ was evaluated by determining the retardation in the electrophoretic mobility (gel shift) of cPLA₂. Of the four known phosphorylation sites on cPLA₂, only phosphorylation on Ser-505 leads to a gel shift (16, 17, 38). PMA, zymosan, and okadaic acid induced stoichiometric phosphorylation of cPLA₂ on Ser-505, whereas the gel shift induced by A23187 was only partial, consistent with a weak activation of ERKs (Fig. 3B). Despite the significant inhibition of ERK activation, zymosan-induced phosphorylation of cPLA₂ on Ser-505 was only slightly prevented by 0.1-10 µM U0126. The cPLA₂ gel shift induced by PMA and okadaic acid was unaffected by 0.1-1 µM U0126 and only slightly inhibited by 10 µM U0126. The partial A23187-induced cPLA₂ gel shift was suppressed by 0.1-10 µM U0126. The effect of U0126 on agonist-induced arachidonic acid release is shown in Fig. 3C. Inhibiting the ERK pathway had no effect on A23187-induced arachidonic acid release, as described previously (25). In contrast, in macrophages stimulated with zymosan, PMA, or okadaic acid, 10 µM U0126 significantly inhibited arachidonic acid release (by 65%, 90% and 99%, respectively). There was a direct correlation between the concentrations of U0126 that inhibited ERK activation and arachidonic acid release, suggesting that activation of the MEK/ERK pathway plays a critical role in regulating arachidonic acid release in macrophages in response to zymosan, PMA, and okadaic acid. Surprisingly, inhibition of arachidonic acid release occurred at concentrations of U0126 that had very little or no effect on the phosphorylation of cPLA₂ on Ser-505. These data indicate that the MEK/ERK pathway regulates cPLA₂-mediated arachidonic acid release through an unknown mechanism in addition to phosphorylation of Ser-505.

The effects of MEK inhibition shown in Fig. 3 were investigated using macrophages from ICR mice, whereas the wild type and cPLA₂-deficient macrophages used in the experiments shown in Figs. 1 and 2 were obtained from C57BL/6 mice, which are deficient in secreted PLA₂ IIA. Although unlikely, it is possible that MEK inhibition may be affecting secreted PLA₂ IIA activity in the ICR-derived macrophages. To investigate this possibility, the effect of U0126 on agonist-induced cPLA₂ gel shift and arachidonic acid release were tested in macrophages from wild type C57BL/6 mice. As observed in macrophages from ICR mice, arachidonic acid release was inhibited by 1-10 µM U0126, with very little or no effect on cPLA₂ gel shift (data not shown). Since cPLA₂ is essential for arachidonic acid release in macrophages from C57BL/6 mice, these results confirm that inhibiting the MEK/ERK pathway suppresses arachidonic acid release by affecting an alternative cPLA₂ regulatory mechanism.

Role of JNK and p38 in Agonist-induced cPLA₂ Phosphorylation and Arachidonic Acid Release-- As shown above, significant phosphorylation of cPLA₂ on Ser-505 was evident under conditions in which ERK activation was quantitatively inhibited, indicating the involvement of some other kinase, such as the MAPK homologues JNK or p38. Since okadaic acid is known to activate JNK and p38 in other cell types, the possible role of the different MAPK pathways in cPLA₂ phosphorylation and arachidonic acid release in macrophages was investigated (39, 40). As shown in Fig. 4, okadaic acid and zymosan triggered activation of both JNK and p38. PMA transiently activated p38 but did not activate JNK.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Time course of JNK and p38 MAPK activation. Macrophages were treated for the times indicated with vehicle (unstimulated (US)), PMA, zymosan, or okadaic acid. Activated p38 (top panel) and JNKs (bottom panel) were analyzed by immunoblotting using phospho-specific antibodies. Results shown are representative of three independent experiments.

The role of p38 and the relative contribution of the ERK and p38 pathways on agonist-induced cPLA₂ phosphorylation and arachidonic acid release in macrophages was tested using the p38 inhibitor SB202190 alone or in combination with U0126. Zymosan-induced arachidonic acid release was partially inhibited (by 45-50%) by SB202190 (Fig. 5A). Since U0126 also resulted in partial inhibition (by 60-65%), we tested the effect of combining the two inhibitors, and this resulted in quantitative inhibition of zymosan-induced arachidonic acid release. The effect of the inhibitors on the time course of phosphorylation of cPLA₂ on Ser-505 (gel shift) was also determined. As shown in Fig. 5B, zymosan induced a complete cPLA₂ gel shift between 30 and 60 min. U0126 and SB202190, when tested alone, delayed the zymosan-induced gel shift, which was clearly observed 15 min after agonist treatment. However, SB202190 had no effect on the gel shift observed at longer stimulation times (30-60 min), and the effect of U0126 was minimal. The combination of both inhibitors almost completely prevented Ser-505 phosphorylation in response to zymosan. As shown in Fig. 5C, SB202190 had no effect on ERK activation. These results suggest that both ERKs and p38 contribute to zymosan-induced phosphorylation of cPLA₂ on Ser-505 and arachidonic acid release.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Effect of U0126 and SB202190 on zymosan-induced arachidonic acid release, ERK activation, and cPLA2 gel shift. A, [3H]arachidonic acid-labeled macrophages were preincubated with vehicle (None), 10 µM U0126 for 15 min, 10 µM SB202190 for 60 min, or both inhibitors and then incubated for 60 min with 10 particles zymosan/cell. Arachidonic acid released to the medium was determined by scintillation counting as described under "Experimental Procedures." Results (average ± S.D.) are representative of three independent experiments. cPLA2 gel shift (B) and activated ERKs (C) were analyzed by immunoblotting of lysates from macrophages that were preincubated with inhibitors as above and then incubated for the times indicated with vehicle (unstimulated (US)) or with zymosan (10 particles/cell). Results are representative of three independent experiments.

PMA-induced arachidonic acid release was significantly inhibited (70%) by SB202190 and quantitatively suppressed by U0126, demonstrating that both the MEK/ERK and the p38 MAPK pathways are involved in this response (Fig. 6A). U0126 significantly delayed the cPLA₂ gel shift induced by PMA, and the combination of SB202190 and U0126 completely prevented it, suggesting that both pathways can contribute to PMA-induced Ser-505 phosphorylation (Fig. 6B). However, SB202190 alone had no effect on the gel shift, indicating that p38 can regulate arachidonic acid release by an alternative mechanism in addition to Ser-505 phosphorylation. As expected, U0126 prevented activation of ERKs by PMA at 5 min. However, weak and transient activation of ERKs was observed at 15 min even in U0126-treated cells. SB202190 had little effect on ERK activation, but the combination of U0126 and SB20190 resulted in complete suppression of PMA-induced ERK activation at all time points observed (Fig. 6C). This suggests that there may be some influence of the p38 pathway on PMA-induced ERK activation in macrophages. p38 has recently been shown to positively regulate the ERK pathway in arsenite-stimulated human embryonic kidney cells (41).

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Effect of U0126 and SB202190 on PMA-induced arachidonic acid release, ERK activation and cPLA2 gel shift. A, [3H]arachidonic acid-labeled macrophages were preincubated with vehicle (None), 10 µM U0126 for 15 min, 10 µM SB202190 for 60 min, or both inhibitors and then incubated for 60 min with 32 nM PMA. Arachidonic acid released to the medium was determined by scintillation counting as described under "Experimental Procedures." Results (average ± S.D.) are representative of three independent experiments. N.D., not detectable. cPLA2 gel shift (B) and activated ERKs (C) were analyzed by immunoblotting of lysates from macrophages that were preincubated with inhibitors as above and then incubated for the times indicated with vehicle (unstimulated (US)) or with PMA (32 nM). Results are representative of three independent experiments.

The effect of ERK and p38 inhibition on okadaic acid-induced responses was also evaluated. Although okadaic acid is a strong activator of p38, SB202190 had no effect on okadaic acid-induced arachidonic acid release, cPLA₂ gel shift, or ERK activation (Fig. 7). The responses to okadaic acid in macrophages are delayed, as previously reported (23). Only weak activation of ERKs and partial phosphorylation of cPLA₂ on Ser-505 were observed at 30 min, but these responses increased at 60 and 90 min (Fig. 7, B and C). U0126 had little effect on the time course of cPLA₂ phosphorylation on Ser-505 but completely inhibited ERK activation and arachidonic acid release, indicating that the MEK/ERK pathway regulates okadaic acid-induced arachidonic acid release by an alternative mechanism. The combination of U0126 and SB202190 almost completely prevented the cPLA₂ gel shift at 30 and 60 min, suggesting that both ERKs and p38 phosphorylate cPLA₂ on Ser-505 in response to okadaic acid.

View larger version (50K): [in this window] [in a new window]   Fig. 7.   Effect of U0126 and SB202190 on okadaic acid-induced arachidonic acid release, ERK activation and cPLA2 gel shift. A, [3H]arachidonic acid-labeled macrophages were preincubated with vehicle (None), 10 µM U0126 for 15 min, 10 µM SB202190 for 60 min, or both inhibitors and then incubated for 90 min with 1 µM okadaic acid. Arachidonic acid released to the medium was determined by scintillation counting as described under "Experimental Procedures." Results (average ± S.D.) are representative of three independent experiments. cPLA2 gel shift (B) and activated ERKs (C) were analyzed by immunoblotting of lysates from macrophages that were preincubated with inhibitors as above and then incubated for the times indicated with vehicle (unstimulated (US)) or with 1 µM okadaic acid (O.A.). Results are representative of three independent experiments.

The MAPK inhibitors U0126 and SB202190 decreased zymosan-induced phosphorylation of cPLA₂ on Ser-505 at early times but had no effect at later times. A similar delaying effect of PMA-induced cPLA₂ gel shift was observed with U0126. If the inhibitory effects of these compounds on PMA- and zymosan-induced arachidonic acid release was simply due to the delayed phosphorylation of Ser-505, it would be expected that arachidonic acid release would resume at similar rates and reach similar levels in inhibitor-treated cells after complete phosphorylation of cPLA₂ was attained. To determine if delayed phosphorylation of cPLA₂ correlated with delayed arachidonic acid release, the effect of U0126 and SB202190 on the time course of zymosan-, PMA-, and okadaic acid-induced arachidonic acid release was evaluated. Arachidonic acid release in cells treated with U0126 or SB202190 did not reach the same levels as in control cells between 1 and 3 h of zymosan treatment, when complete phosphorylation of cPLA₂ was observed (Fig. 8A). This indicates that the MEK/ERK and p38 pathways can affect arachidonic acid release through some alternative mechanism. Combining the two inhibitors resulted in almost complete inhibition of zymosan-induced arachidonic acid release at all times tested. As shown in Fig. 8B, PMA-induced arachidonic acid release was quantitatively inhibited by U0126 even between 1 and 3 h, demonstrating that inhibition is not due to the delay in cPLA₂ phosphorylation, since by 30 min a complete gel shift was observed in the presence of the inhibitor. Arachidonic acid release remained low in SB202190-treated macrophages in response to PMA, and this was not due to effects on Ser-505 phosphorylation, which was unaffected by SB202190.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Effect of U0126 and SB202190 on the time course of agonist-induced arachidonic acid release in macrophages. [3H]Arachidonic acid-labeled macrophages were preincubated with vehicle (Control), 10 µM U0126 for 15 min, 10 µM SB202190 for 60 min, or both inhibitors and then incubated for the times indicated with 10 particles of zymosan/cell (A), 32 nM PMA (B), or 1 µM okadaic acid (C). Arachidonic acid release was determined by scintillation counting as described under "Experimental Procedures." Results (average ± S.D.) are representative of three independent experiments.

Okadaic acid-induced arachidonic acid release was markedly delayed in comparison to PMA or zymosan (Fig. 8C). Preincubation with U0126 quantitatively suppressed okadaic acid-induced arachidonic acid release at all times tested, but as shown above (Fig. 7B), the stoichiometric phosphorylation of cPLA₂ on Ser-505 was unaffected. No effect on arachidonic acid release was observed with SB202190, demonstrating that the p38 pathway, although it plays a role in Ser-505 phosphorylation, does not contribute to okadaic acid-induced arachidonic acid release. A small but reproducible increase in arachidonic acid release was observed in cells pretreated with U0126 and SB202190 as compared with U0126 alone. The mechanism or the possible relevance of this effect is not known.

Effect of Anisomycin on MAPKs, cPLA₂ Activation, and Arachidonic Acid Release-- The role of p38 in regulating arachidonic acid release in macrophages was further investigated using anisomycin, an agonist that selectively activates JNKs and p38 and not ERKs (42). Anisomycin is an inhibitor of protein synthesis at high concentrations, but it has been shown to activate protein kinases in cells independently of translational arrest at concentrations as low as 25 ng/ml (42). The time courses of JNK and p38 activation in macrophages induced by anisomycin were determined (Fig. 9A). Activation of both MAPK homologues occurred by 5 min, peaked at 15-30 min, and decreased partially by 60 min. However, anisomycin did not significantly activate ERKs compared with okadaic acid, as determined both by phospho-specific antibodies (Fig. 9A) and a more sensitive method involving immunoprecipitation of ERKs followed by in vitro kinase assay (data not shown). Anisomycin induced a complete cPLA₂ gel shift by 15 min, which was sustained for 30 min and only slightly reversed after 60 min (Fig. 9B). Anisomycin alone had little effect on arachidonic acid release but did induce a modest increase in arachidonic acid release together with the calcium-mobilizing agonist ATP (Fig. 9C). Anisomycin also enhanced the catalytic activity of cPLA₂ in the cytosol of macrophages, from 17.9 ± 0.7 pmol of [³H]phosphatidylcholine hydrolyzed in 10 min by 10 µg of cytosolic protein in unstimulated cells (average ± S.D. of an experiment representative of three) to 32.3 ± 0.5 pmol/10 min/10 µg, comparable with the activity in cytosols from cells treated with zymosan (36.8 ± 3.1 pmol/10 min/10 µg). This increase in activity was reversed when macrophages were preincubated with the p38 inhibitor SB203580 (17.4 ± 3.3 pmol/10 min/10 µg), which also inhibited the cPLA₂ gel shift and the ATP/anisomycin-induced arachidonic acid release (Figs. 9, C and D). These results demonstrate that p38 can phosphorylate cPLA₂ on Ser-505 and that this phosphorylation is necessary for arachidonic acid release induced by the combination of ATP and anisomycin. However, despite stoichiometric phosphorylation of cPLA₂ on Ser-505 and an increase in the levels of calcium, ATP and anisomycin only weakly induced arachidonic acid release compared with PMA, zymosan, okadaic acid, or the combination of colony-stimulating factor 1 and ATP (25, 31).

View larger version (41K): [in this window] [in a new window]   Fig. 9.   Effect of anisomycin on activation of MAPKs, cPLA2 gel shift, and arachidonic acid release in macrophages. Macrophages were treated for the times indicated with vehicle (unstimulated (US)), 25 ng/ml anisomycin, or 1 µM okadaic acid (O.A.). Lysates were used for SDS-polyacrylamide gel electrophoresis and immunoblotting using phospho-specific antibodies against JNKs, p38, and ERKs (A) or against cPLA2 (B). C, [3H]arachidonic acid-labeled macrophages were preincubated with vehicle or 10 µM SB203580 for 15 min, after which anisomycin (25 ng/ml) was added, and 15 min later, 0.1 mM ATP was added. Cells were incubated for an additional 60 min. Arachidonic acid release (average ± S.D.) was determined by scintillation counting as described under "Experimental Procedures." D, macrophages were incubated for 15 min with vehicle or 10 µM SB203580, followed by 25 ng/ml anisomycin for 15 min. cPLA2 gel shift was analyzed by immunoblotting of cell lysates. Results are representative of three independent experiments.

Effect of Protein Synthesis Inhibition on Arachidonic Acid Release-- Activation of ERKs results in the activation of a variety of transcription factors and subsequent gene expression in many cells (43). It has previously been shown that transcriptional regulation can play a role in arachidonic acid release in mouse peritoneal macrophages induced by PMA and zymosan but not by A23187 (44, 45). Since this inhibitory effect paralleled that observed with U0126 and the responses to okadaic-acid were the most sensitive to U0126 inhibition, we investigated the effect of actinomycin D and cycloheximide on okadaic acid-induced arachidonic acid release compared with other agonists. A23187-induced arachidonic acid release was unaffected by inhibition of transcription or translation (Fig. 10A). In contrast, actinomycin D and cycloheximide decreased arachidonic acid release induced by okadaic acid, PMA, and zymosan to different extents. Okadaic acid-induced arachidonic acid release was quantitatively inhibited by actinomycin D and cycloheximide, and PMA-induced arachidonic acid release was inhibited by 45-55% with cycloheximide and 73-79% with actinomycin D. The inhibitory effect on the zymosan-induced response was less pronounced, 30-40% for cycloheximide and 42-50% for actinomycin D. The inhibitors were effective even when added just 30 min before agonist treatment. Interestingly, the relative inhibitory effects of cycloheximide and actinomycin D correlate highly with the effects of U0126, showing that PMA and okadaic acid are particularly dependent on both a MEK/ERK-dependent pathway and transcriptional events for arachidonic acid release. Testing concentrations of these inhibitors on okadaic acid-induced arachidonic acid release revealed that actinomycin D was more effective than cycloheximide by at least one order of magnitude, which is consistent with the effects previously reported for mouse peritoneal macrophages treated with PMA or zymosan (Fig. 10B) (44, 45). Cycloheximide and actinomycin D had no effect on the expression of cPLA₂ (not shown).

View larger version (35K): [in this window] [in a new window]   Fig. 10.   Effect of actinomycin D and cycloheximide on agonist-induced arachidonic acid release in macrophages. A, [3H]arachidonic acid-labeled macrophages were preincubated with 10 µg/ml actinomycin D (Act. D) or 10 µg/ml cycloheximide (CH) for 30 min and then stimulated for 60 min (A23187, PMA, and zymosan) or 90 min (okadaic acid). Arachidonic acid released into the medium (average ± S.D.) was determined and is expressed as the percentage of the release measured from control cells not treated with inhibitor. Control values, expressed as the percentage of total radioactivity incorporated that was released into the medium, were: A23187 (8.8%), okadaic acid (19.4%), PMA (14.9%), and zymosan (27.3%). B, [3H]arachidonic acid-labeled macrophages were preincubated with the indicated doses of actinomycin D or cycloheximide for 30 min and then stimulated with 1 µM okadaic acid for 90 min. Arachidonic acid release (average ± S.D.) was determined and is expressed as percentage of the release measured from control cells stimulated with okadaic acid (16.4%). Results shown are representative of three independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 85-kDa sn-2 arachidonoyl-specific cPLA₂ is implicated in regulating arachidonic acid release and eicosanoid production during cell activation by a wide variety of stimuli (1, 2). In many cases, evidence for the involvement of cPLA₂ in agonist-stimulated arachidonic acid release is correlative and based on cPLA₂ phosphorylation, as measured by its gel shift, and elevated cPLA₂ activity. However, there is increasing evidence that phosphorylation of cPLA₂ is not sufficient to induce arachidonic acid release in several cell types and, thus, does not provide definitive proof for a role for cPLA₂. In addition, many cells also contain other types of PLA₂ enzymes, including secretory PLA₂s IIA and V as well as the calcium-independent PLA₂, or iPLA₂, all of which have been shown to mediate arachidonic acid release in some systems (46-48). Furthermore, two paralogs of cPLA₂ have recently been identified, cPLA₂ and cPLA₂, with the name cPLA₂ being assigned to the well known 85-kDa cPLA₂ (49-51). This complicates the interpretation of some of the data obtained on the role of specific PLA₂ isoforms in the regulation of arachidonic acid release. Studies with chemical inhibitors and antisense oligonucleotides directed at PLA₂ enzymes can be informative if appropriately controlled and have provided useful data, but they are sometimes difficult to interpret due to evidence suggesting a lack of specificity by these reagents in cellular systems (52-55). Recently, cPLA₂-deficient mice have been generated, making it possible to assign an essential role for cPLA₂ in arachidonic acid release and eicosanoid production by agonist-treated primary mouse cells (3, 4, 26). In mouse peritoneal macrophages, cPLA₂ is essential for arachidonic acid release induced by A23187 or the combination of PMA and A23187 (3, 4). The role of cPLA₂ in regulating arachidonic acid release at basal calcium levels, however, has not been addressed. It has been proposed that arachidonic acid release in PMA-stimulated rat macrophages does not depend on a phospholipase A₂ but on the combined action of phospholipase C and diacylglycerol lipase (56). We now provide evidence that cPLA₂ is essential for arachidonic acid release and eicosanoid production induced by PMA and okadaic acid, which do not mobilize calcium in macrophages. The small arachidonic acid release observed in PMA-treated cPLA₂-deficient macrophages, however, indicates that another phospholipase contributes partially to the PMA response. cPLA₂-deficient mice were generated using two strains that have a natural disruption in the secreted PLA₂ IIA gene (57). Therefore, some other isoform of PLA₂ is contributing partially to PMA-induced arachidonic acid release in macrophages. Whether this release is due to an isoform of secreted PLA₂, calcium-independent PLA₂ or cPLA₂ remains to be determined.

We also show that cPLA₂ is essential for arachidonic acid release and eicosanoid production in response to zymosan. A recent report suggests that calcium-independent PLA₂ rather than cPLA₂ is partially responsible for the zymosan-induced arachidonic acid release and PGE₂ production in the macrophage-like cell line P388D₁ (58). This conclusion is based on the observations that zymosan-induced arachidonic acid release and PGE₂ production are partially inhibited (40-45%) by bromoenol lactone and by a calcium-independent PLA₂ antisense oligonucleotide. However, it has been recently pointed out that P388D₁ cells exhibit some atypical properties and features of arachidonate metabolism compared with primary macrophages (48). Most notably, these transformed cells are actively dividing and contain very low levels of esterified arachidonate (3% of the total esterified fatty acid mass as compared with about 25% in primary mouse macrophages) and relatively high levels of non-esterified arachidonic acid (48, 59, 60). This could explain the much lower levels of PGE₂ produced in response to zymosan by P388D₁ cells (less than 7 ng/mg of protein) compared with our measurements in mouse peritoneal macrophages (50 ng/10⁶ cells, equivalent to more than 1 µg PGE₂/mg protein) (58). Thus, results obtained in P388D₁ cells concerning arachidonic acid metabolism are not representative of primary macrophages. In this report, we present definitive evidence that cPLA₂ is required for zymosan-induced arachidonic acid release and LTC₄ and PGE₂ production in primary resident mouse peritoneal macrophages.

Translocation of cPLA₂ from the cytosol to membrane is an essential regulatory step and can be triggered by increased intracellular calcium (7, 12-16). However, our results demonstrating an essential role for cPLA₂ in arachidonic acid release induced by agonists that do not mobilize calcium indicate that translocation of cPLA₂ can occur by alternative mechanisms. This is supported by recent data showing that cPLA₂ expressed in Sf9 cells translocates to the perinuclear region and mediates arachidonic acid release in response to okadaic acid, which does not mobilize calcium (16). Arachidonic acid release under conditions of basal cytosolic calcium have also been reported in different mammalian systems, although the role of cPLA₂ has not been definitively established (21, 25, 61). In addition, cPLA₂ has been implicated in delayed arachidonic acid release, observed hours after stimulation, when calcium levels would not be expected to remain elevated (3, 4, 17, 26, 56). The ability of cPLA₂ to mediate arachidonic acid release without an increase in calcium levels cannot be explained by increased phosphorylation on Ser-505, which is not sufficient for cPLA₂ to mediate arachidonic acid release (17, 25, 31, 36). However, other phosphorylation sites have been identified in cPLA₂ (38). Okadaic acid induces phosphorylation primarily of Ser-727 on cPLA₂ in Sf9 cells, but this site does not to play a functional role in cPLA₂-mediated arachidonic acid release in the insect cell model (16, 38). Phosphorylation of cPLA₂ on Ser-727 has also been observed in agonist-treated human platelets and HeLa cells, where its role remains to be determined (62).

Our results demonstrate that the MEK/ERK and p38 MAPK pathways contribute to phosphorylation of cPLA₂ on Ser-505 induced by zymosan, PMA, and okadaic acid, since both U0126 and SB202190 are required to prevent the gel shift induced by these agonists. However, we could not determine conclusively whether phosphorylation of cPLA₂ on Ser-505 is required for arachidonic acid release in macrophages, since we unexpectedly found that MAPK pathways play a role in regulating agonist-induced arachidonic acid release by another unknown mechanism. Consequently, it is possible that multiple regulatory steps, including phosphorylation of Ser-505 and a novel MAPK-dependent mechanism, are required. Okadaic acid-induced arachidonic acid release was found to be dependent on the MEK/ERK pathway but completely independent of p38, whereas the response to PMA is dependent on both pathways. Even in response to the calcium-mobilizing agonist zymosan, both MAPK pathways appear to play a role in regulating arachidonic acid release by alternative mechanisms to phosphorylation of cPLA₂ on Ser-505. This is consistent with a recent report showing that arachidonic acid release in neutrophils stimulated with the calcium-mobilizing agonist formylmethionylleucylphenylalanine (fMLP) is inhibited by the combination of PD98059 and SB203580, whereas formylmethionylleucylphenylalanine-induced cPLA₂ gel shift is unaffected, also suggesting alternative regulatory mechanisms for the MEK/ERK and p38 pathways (21). Our data showing differences in the extent of macrophage responses to anisomycin/ATP compared with zymosan also suggest that zymosan regulates arachidonic acid release through alternative mechanisms. Arachidonic acid release induced by the combination of anisomycin and ATP is very small as compared with the zymosan response, despite increased levels of calcium, stoichiometric phosphorylation of cPLA₂ on Ser-505, and increased cPLA₂ catalytic activity. Although both calcium and phosphorylation appear to be required for arachidonic acid release induced by zymosan and the combination of anisomycin and ATP, the different magnitudes of the responses suggest that zymosan triggers other regulatory steps that potentiate cPLA₂ activation. A transient increase in calcium has been recently shown not to be sufficient to promote stable association of cPLA₂ with membrane, which is required for arachidonic acid release (7). It is possible that additional MAPK-dependent regulatory steps induced by zymosan may promote cPLA₂-mediated arachidonic acid release by stabilizing its association with membrane.

The results obtained with the protein synthesis inhibitors actinomycin D and cycloheximide suggest that transcriptional regulation could be at least one of the mechanisms by which MAPKs affect agonist-induced arachidonic acid release. It is known that protein synthesis inhibition diminishes arachidonic acid release in mouse peritoneal macrophages in response to PMA and zymosan, and a requirement for a rapidly turning over protein has been proposed (44, 45). It was also suggested that an increase in intracellular calcium was sufficient to overcome that requirement, since A23187-induced arachidonic acid release was unaffected by cycloheximide or actinomycin D (45). Our results are in agreement with this hypothesis, and we found in addition that the arachidonic acid release response to okadaic acid was particularly sensitive to inhibition of protein synthesis. There is a strong correlation between the inhibitory effects of actinomycin D or cycloheximide and those of U0126: A23187-induced arachidonic acid release is unaffected, zymosan-induced arachidonic acid is partially inhibited, and the responses to PMA and okadaic acid are more effectively suppressed. These data strongly suggest that an ERK-dependent and transcription-dependent mechanism contributes to arachidonic acid release in macrophages and is particularly important in the absence of increased intracellular calcium. It has been shown that ERK and p38 activation induce the expression of immediate early genes within minutes of cell stimulation (63). Importantly, cycloheximide does not inhibit agonist-induced cPLA₂ phosphorylation on Ser-505. In fact, it induces a complete gel shift of cPLA₂,² consistent with the ability of protein synthesis inhibitors to activate stress kinases (40, 64). Therefore, these results suggest that MAPKs may be inducing the rapid synthesis of one or more proteins that contribute to cPLA₂-induced arachidonic acid release.

In summary, evidence is presented that cPLA₂ is required for arachidonic acid release induced in macrophages both by agonists that do and do not mobilize calcium. Although calcium and phosphorylation on Ser-505 can regulate cPLA₂ activation, our results demonstrate alternative mechanisms and a more complex role for MAPK pathways, which may involve regulation of gene expression. An important corollary to our results is that suppression of agonist-induced arachidonic acid release by inhibitors of the MEK/ERK pathway does not necessarily implicate a role for Ser-505 phosphorylation of cPLA₂. However, even though MAPKs can influence cPLA₂ by multiple mechanisms, this in itself does not fully explain the ability of okadaic acid and PMA to induce cPLA₂-mediated arachidonic acid release in the absence of increased calcium levels. Our previous work has shown that colony-stimulating factor 1 strongly activates ERKs but is unable to induce arachidonic acid release in macrophages. Consequently, additional mechanisms triggered through protein kinase C activation by PMA or phosphatase inhibition by okadaic acid act together with activation of specific MAPK pathways to fully activate cPLA₂ and induce arachidonic acid release.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HL34303 (to C. C. L.), HL61378 (to C. C. L.), DK38452 (to J. V. B.), and NS10828 (to J. V. B.).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.

^§ Supported in part by the Andrew Goodman Fellowship in Pediatrics from the National Jewish Medical and Research Center.

^** To whom correspondence should be addressed: Dept. of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1214; Fax: 303-270-2155; E-mail: lesliec@njc.org.

Published, JBC Papers in Press, April 28, 2000, DOI 10.1074/jbc.M908941199

² M. A. Gijón and C. C. Leslie, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: cPLA₂, cytosolic phospholipase A₂; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; JNK, Jun kinase, cJun-N-terminal kinase; PGE₂, prostaglandin E₂; LTC₄, leukotriene C₄; PMA, phorbol myristate acetate; CHO, Chinese hamster ovary..

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES