Regulation of Delayed Prostaglandin Production in Activated P388D1 Macrophages by Group IV Cytosolic and Group V Secretory Phospholipase A2s*

Hiroyuki Shinohara, María A. Balboa, Christina A. Johnson, Jesús Balsinde, and Edward A. DennisDagger

From the Department of Chemistry and Biochemistry, School of Medicine and Revelle College, University of California at San Diego, La Jolla, California 92093-0601

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Group V secretory phospholipase A2 (sPLA2) rather than Group IIA sPLA2 is involved in short term, immediate arachidonic acid mobilization and prostaglandin E2 (PGE2) production in the macrophage-like cell line P388D1. When a new clone of these cells, P388D1/MAB, selected on the basis of high responsivity to lipopolysaccharide plus platelet-activating factor, was studied, delayed PGE2 production (6-24 h) in response to lipopolysaccharide alone occurred in parallel with the induction of Group V sPLA2 and cyclooxygenase-2 (COX-2). No changes in the level of cytosolic phospholipase A2 (cPLA2) or COX-1 were observed, and Group IIA sPLA2 was not detectable. Use of a potent and selective sPLA2 inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), and an antisense oligonucleotide specific for Group V sPLA2 revealed that delayed PGE2 was largely dependent on the induction of Group V sPLA2. Also, COX-2, not COX-1, was found to mediate delayed PGE2 production because the response was completely blocked by the specific COX-2 inhibitor NS-398. Delayed PGE2 production and Group V sPLA2 expression were also found to be blunted by the inhibitor methylarachidonyl fluorophosphonate. Because inhibition of Ca2+-independent PLA2 by an antisense technique did not have any effect on the arachidonic acid release, the data using methylarachidonyl fluorophosphonate suggest a key role for the cPLA2 in the response as well. Collectively, the results suggest a model whereby cPLA2 activation regulates Group V sPLA2 expression, which in turn is responsible for delayed PGE2 production via COX-2.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Arachidonic acid (AA)1 mobilization and the generation of prostaglandins by major immunoinflammatory cells such as macrophages and mast cells usually occur in two phases. The immediate phase, which takes minutes and is elicited by Ca2+-mobilizing agonists such as platelet-activating factor (PAF), is characterized by a burst of AA liberation. In some cells such as P388D1 macrophages (1, 2) and MMC-34 mast cells (3), this burst is mainly produced by a secretory phospholipase A2 (sPLA2) but is strikingly regulated by the cytosolic Group IV phospholipase A2 (cPLA2).

The delayed phase of prostaglandin production is accompanied by the continuous supply of AA over long incubation periods spanning several hours. There is some discrepancy about the identity of the PLA2 isoform(s) involved in the delayed phase. Despite this phase being independent of a Ca2+ increase, the cPLA2 has often been suggested to be critically involved (3-5). However, other studies have suggested the quantitatively more important role of the sPLA2, an enzyme that is dramatically induced during long term incubation of the cells with a variety of stimuli (4-6). There is, however, agreement that COX-2, another enzyme whose expression is augmented dramatically after long term stimulation, is absolutely required for long term PGE2 production, irrespective of the constitutive presence of COX-1 (7-9).

Using a new clone of the P388D1 macrophage-like cells termed P388D1/MAB, we provide herein evidence for the involvement of Group V sPLA2 in delayed PGE2 production. Furthermore, our results suggest that Group V sPLA2 expression is dependent upon the activation of Group IV cPLA2.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Mouse P388D1 macrophage-like cells were obtained from the American Type Culture Collection (Rockville, MD). Iscove's modified Dulbecco's medium (endotoxin <0.05 ng/ml) was from Whittaker Bioproducts (Walkersville, MD). Fetal bovine serum was from Hyclone Laboratories (Logan, UT). Nonessential amino acids were from Irvine Scientific (Santa Ana, CA). [5,6,8,9,11,12,14,15-3H]Arachidonic acid (specific activity, 100 Ci/mmol) was from NEN Life Science Products, and 1-palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine (specific activity, 54 mCi/mmol) was from Amersham Pharmacia Biotech. PAF, LPS (Escherichia coli 0111:B4), and actinomycin D were from Sigma. Methylarachidonyl fluorophosphonate (MAFP) and NS-398 were from Biomol (Plymouth Meeting, PA). Antibodies against murine COX isoforms were generously provided by Dr. W. L. Smith (Department of Biochemistry, Michigan State University, East Lansing, MI). The antibody against Group IV cPLA2 was generously provided by Dr. Ruth Kramer (Lilly). The sPLA2 inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), was generously provided by Dr. Edward Mihelich (Lilly). cDNA probes for Groups V and IIA sPLA2s were synthesized as described previously (11). cDNA probes for murine glyceraldehyde-3-phosphate dehydrogenase were from Cayman (Ann Arbor, MI).

Cell Culture and Labeling Conditions-- P388D1 cells were maintained at 37 °C in a humidified atmosphere at 90% air and 10% CO2 in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and nonessential amino acids. P388D1 cells were plated at 106/well, allowed to adhere overnight, and used for experiments the following day. All experiments were conducted in serum-free Iscove's modified Dulbecco's medium. When required, radiolabeling of the P388D1 cells with [3H]AA was achieved by including 0.5 µCi/ml [3H]AA during the overnight adherence period (20 h). Labeled AA that had not been incorporated into cellular lipids was removed by washing the cells four times with serum-free medium containing 1 mg/ml albumin.

Measurement of PGE2 Production and Extracellular [3H]AA Release-- The cells were placed in serum-free medium for 30 min before the addition of LPS for different periods of time. Afterward, the supernatants were removed and cleared of detached cells by centrifugation, and PGE2 was quantitated using a specific radioimmunoassay (PersPective Biosystems, Framingham, MA). For [3H]AA release experiments, cells labeled with [3H]AA were used, and the incubations were performed in the presence of 0.5 mg/ml bovine serum albumin. The supernatants were removed, cleared of detached cells by centrifugation, and assayed for radioactivity by liquid scintillation counting. The standard LPS/PAF stimulation protocol for immediate responses has been described previously (1). Briefly, the cells were incubated for 1 h with 200 ng/ml LPS followed by a 10-min incubation with 100 nM PAF.

Western Blot Analyses-- The cells were overlaid with a buffer consisting of 10 mM Hepes, 0.5% Triton X-100, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, 20 µM aprotinin, pH 7.5. Samples from cell extracts (10 µg for cPLA2, 200 µg for COX) were separated by SDS-polyacrylamide gel electrophoresis (10% acrylamide gel) and transferred to Immobilon-P (Millipore). For cPLA2 mobility shift studies, 24-cm acrylamide gels were run. Nonspecific binding was blocked by incubating the membranes with 5% nonfat milk in phosphate-buffered saline for 1 h. Membranes were then incubated with anti-cPLA2, anti-COX-1, or anti-COX-2 antisera and treated with horseradish peroxidase-conjugated protein A (Amersham Pharmacia Biotech). Bands were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).

Northern Blot Analyses-- Total RNA was isolated from unstimulated or LPS-stimulated cells by the TriZOL reagent method (Life Technologies, Inc.), exactly as indicated by the manufacturer. Fifteen µg of RNA were electrophoresed in a 1% formaldehyde/agarose gel and transferred to nylon filters (Hybond, Amersham Pharmacia Biotech) in 10× SSC buffer. Hybridizations were performed in QuickHyb solution (Stratagene) following the manufacturer's instructions. 32P-Labeled probes for Group IIA or glyceraldehyde-3-phosphate dehydrogenase were co-incubated with the filters for 1 h at 66 °C followed by three washes with 2× SSC containing 0.1% SDS at room temperature for 30 min. A final wash was carried out at 60 °C for 30 min with 1× SSC containing 0.1% SDS. For Group V sPLA2, hybridizations were performed in ExpressHyb solution (CLONTECH) following the manufacturer's instructions. The 32P-labeled probes were co-incubated with the filters for 1 h at 66 °C followed by two washes with 2× SSC containing 0.05% SDS for 15 min: the first at room temperature and the second at 40 °C. Afterward the filters were washed twice more with 0.1× SSC containing 0.1% SDS for 15 min at room temperature. Bands were visualized by autoradiography.

Phospholipase A2 Assay-- Aliquots (50-100 µl) of supernatants from LPS-treated cells were assayed for PLA2 activity as follows. The assay mixture (500 µl) consisted of 100 µM 1-palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine substrate (2000 cpm/nmol), 10 mM CaCl2, 100 mM KCl, 25 mM Tris-HCl, pH 8.5. Reactions proceeded at 40 °C for 30 min, after which [14C]palmitate release was determined by a modified Dole procedure (10).

Antisense Inhibition Studies in P388D1 Cells-- Transient transfection of P388D1 cells with antisense oligonucleotide, ASGV-2, or its sense counterpart, SGV-2, plus LipofectAMINE was carried out as described (11). Briefly, P388D1 cells were transfected with oligonucleotide (125 nM) in the presence of 5 µg/ml LipofectAMINE (Life Technologies, Inc.) under serum-free conditions for 8 h prior to treating the cells with or without 100 ng/ml LPS for 10 h after transfection (11). Antisense oligonucleotide ASGV-2 (5'-GGA CUU GAG UUC UAG CAA GCC-3') is complementary to nucleotides 64-84 of the mouse Group V PLA2 gene. SGV-2 (5'-GGC UUG CUA GAA CUC AAG UCC-3') is the sense complement of ASGV-2.

For Group VI iPLA2 antisense experiments, a protocol identical to that reported elsewhere was followed (12).

Data Presentation-- Assays were carried out in duplicate or triplicate. Each set of experiments was repeated three times with similar results. Unless otherwise indicated, the data presented are from representative experiments.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

AA Release in a Novel P388D1 Cell Clone (MAB)-- Stimulation of murine P388D1 macrophages with nanomolar amounts of the receptor agonist PAF results in very little AA mobilization. However, preincubation of the cells with LPS prior to stimulation with PAF increases the release of AA by these cells well above unstimulated levels, the relative magnitude of the response being dependent on the cell batch (13, 14). We have now selected by limit dilution a clone of the P388D1 cells termed MAB, which shows a remarkably higher [3H]AA release response to LPS/PAF when compared with the ATCC batch of P388D1 cells from which the MAB clone was obtained (Fig. 1). More interestingly, in addition to an immediate response to LPS/PAF (Fig. 2A), cells from the MAB clone also exhibited a delayed [3H]AA release response, spanning several hours, to LPS alone (Fig. 2A). The dose response of the effect of LPS on long term [3H]AA release is shown in Fig. 2B. The maximal effect was observed at a LPS dose as low as 10 ng/ml.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1.   AA release in a new P388D1 cell clone, MAB. [3H]AA release in LPS/PAF-treated (closed bars) or untreated (open bars) was assayed in cells from the ATCC or the MAB clone as indicated. The cells were incubated with 200 ng/ml LPS for 1 h followed by a 10-min incubation with 100 nM PAF.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   LPS-stimulated long term [3H]AA metabolism in P388D1 macrophages. A, time course of [3H]AA release in response to LPS/PAF (the latter was added 1 h after the former, and the incubations proceeded for the time indicated) (closed triangles), LPS alone (closed circles), or neither (open circles). B, dose response of the LPS effect (20-h incubation). C, the time course of PGE2 production by cells treated with (closed circles) or without (open circles) 100 ng/ml LPS.

Prostaglandin Production by P388D1/MAB Cells-- Fig. 2C shows the time course of PGE2 production by LPS in these cells as measured by radioimmunoassay, which corresponded well with the [3H]AA mobilization response. Thus, LPS-induced PGE2 barely increased above controls within the first 3 h of treatment, rising afterward, and reaching a plateau after 12 h.

The effect of LPS on the protein levels of the two COX isoenzymes these cells express (2) was assessed by immunoblot. Expression of COX-1 did not change along the time course of LPS activation (data not shown), whereas COX-2 levels noticeably increased with maximal expression between 12 and 18 h (Fig. 3A). Interestingly, LPS-induced COX-2 expression almost parallels PGE2 generation (cf. Figs. 2C and 3A), suggesting that COX-2 is the enzyme responsible for LPS-induced PGE2 synthesis. Indeed, the COX-2-specific inhibitor NS-398 (15) completely blocked LPS-induced PGE2 production (Fig. 3B). Therefore, LPS-delayed PGE2 generation depends exclusively on COX-2, irrespective of the continued presence of COX-1.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   LPS-stimulated long term PGE2 production. A, the effect of 100 ng/ml LPS on COX-2 protein levels at the indicated times (h) as measured by immunoblot. B, the effect of NS-398 on LPS-induced PGE2 production. The cells were treated with (closed bars) or without (open bars) 5 µM NS-398 for 20 min before the addition of LPS for 18 h.

cPLA2 Involvement in LPS-induced Long Term Responses-- Expression of the Group IV cPLA2 protein in P388D1/MAB cells was constitutive and did not change after exposure to LPS. To address the possible involvement of cPLA2 in LPS-induced AA mobilization in P388D1/MAB cells, experiments were conducted with MAFP, an inhibitor that has previously been shown to block the immediate, cPLA2-dependent [3H]AA release in LPS/PAF-treated macrophages (1). As shown in Fig. 4, MAFP strongly blocked the LPS-induced long term [3H]AA release response. MAFP has recently been observed to inhibit the Group VI iPLA2 in addition to the cPLA2 (10). Therefore, it could be possible that part of the MAFP effects reported herein resulted from inhibition of the iPLA2 in addition to any effect on the cPLA2. We have recently described the inhibition of iPLA2 expression in P388D1 cells by antisense RNA oligonucleotides (12). Using this technique, we have been able to significantly inhibit iPLA2 expression, assayed both by protein content by immunoblot and activity by a specific in vitro assay (12). Antisense RNA inhibition of the iPLA2 under identical conditions as those shown previously (12) showed no reduction in AA release in response to LPS (not shown). Therefore these data make it likely that the above reported effects of MAFP on the response are because of the inhibition of the cPLA2.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of MAFP and LY311727 on LPS-induced AA release. The cells were treated with 25 µM MAFP (closed triangles), 25 µM LY311727 (closed squares), or neither (closed circles) for 20 min before the addition of LPS. Open circles denote control incubations, i.e. those that received neither LPS nor inhibitors. The inhibitors alone did not change the control release.

Role of sPLA2-- LPS-induced long term [3H]AA release was also noticeably blocked by the selective sPLA2 inhibitor LY311727 (17), indicating that in addition to the cPLA2, a sPLA2 is also involved in the process (Fig. 4). PGE2 production by LPS was also inhibited by LY311727 by about 90%. Although originally described as a selective Group II sPLA2 inhibitor (17), we have recently shown that this compound is also a potent Group V sPLA2 inhibitor (18).

Our previous work (11) has demonstrated that P388D1 cells express measurable message levels for Group V sPLA2, both under unstimulated and LPS/PAF-stimulated conditions. However, message levels for Groups IIA sPLA2 or IIC sPLA2 were undetectable even by reverse transcriptase-polymerase chain reaction (11). As shown in Fig. 5A, an antisense oligonucleotide specific for Group V sPLA2 (ASGV-2) strongly blocked PGE2 production in LPS-treated cells, whereas its sense control (SGV-2) had no effect. Moreover, mRNA analyses by Northern blot at long times of stimulation with LPS confirmed the presence of mRNA for Group V sPLA2 (Fig. 5, B and C) but not for Group IIA sPLA2 (data not shown). The signal for Group V sPLA2 markedly increased after LPS stimulation, reaching a peak at approximately 10 h.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5.   Group V sPLA2 involvement in AA release. A, the effect of a specific Group V antisense oligonucleotide (AGV-2) or its sense control (SGV-2) on PGE2 production in cells treated without (open bars) or with (closed bars) 100 ng/ml LPS for 10 h. None denotes incubations that received no oligonucleotide. B, the effect of LPS on Group V sPLA2 message levels as determined by Northern blot. Total RNA from cells incubated with (+) or without (-) 100 ng/ml LPS for the indicated periods of time was isolated and analyzed by Northern blot with probes specific for Group V sPLA2 or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). C, densitometric analysis of the Group V sPLA2 signals normalized for the glyceraldehyde-3-phosphate dehydrogenase signal in each lane.

PLA2 activity measurements in the supernatants of LPS-stimulated cells revealed a time-dependent increase in activity (Fig. 6), which correlated well with the time course of Group V sPLA2 mRNA induction (cf. Figs. 5B and 6). Extracellular PLA2 activity was decreased if the experiments were conducted in the presence of the RNA synthesis inhibitor actinomycin D (Fig. 7A). This increased extracellular activity was found to correspond to that of Group V sPLA2 by the following criteria: (i) it was completely inhibited by the sPLA2 inhibitor LY311727 (Fig. 7B) and (ii) it was decreased in supernatants from cells treated with an antisense RNA oligonucleotide specific for Group V sPLA2, ASGV-2 (11) (Fig. 7C).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of MAFP on the appearance of sPLA2 activity in the supernatants of P388D1 cells and the effect of MAFP. The cells, pretreated with (closed triangles) or without (closed circles) 25 µM MAFP for 20 min, were challenged with (closed symbols) or without (open circles) 100 ng/ml LPS for the indicated times. Afterward, supernatants were collected and assayed for PLA2 activity. The amount of PLA2 activity detected in supernatants of cells not treated with LPS (open circles) was not changed whether the cells were pretreated or not with MAFP.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of different treatments on the appearance of PLA2 activity in supernatants from LPS-treated cells. A, the effect of actinomycin D is shown. PLA2 activity in the supernatants from cells treated with LPS plus the indicated concentrations of actinomycin D for 18 h is indicated. Control denotes incubations carried out without either LPS or actinomycin D. B, blockage by LY311727 of the PLA2 activity of supernatants from LPS-treated or untreated cells. An aliquot of the culture medium of cells treated without (Control) or with LPS for 18 h was incubated with or without 25 µM LY311727 for 20 min at 40 °C and then assayed for PLA2 activity. C, the effect of a specific Group V antisense oligonucleotide (AGV-2) or its sense control (SGV-2) on PLA2 activity in the supernatants from LPS-treated or untreated cells.

Role of cPLA2 in sPLA2 Activation-- Our previous studies have indicated that the immediate AA release triggered by LPS/PAF in these cells involves the sequential action of both cPLA2 and sPLA2, with the activity of the latter being dependent on previous activation of the former (1, 2). Thus we sought to investigate if a similar cross-talk exists between the two enzymes during long term stimulation conditions. We found that no increased PLA2 activity beyond what was observed in the basal state could be found in supernatants from cells treated with MAFP (Fig. 6). In addition, the cPLA2 inhibitor markedly decreased the LPS-induced expression of Group V sPLA2 mRNA (Fig. 8).


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 8.   The effect of 25 µM MAFP on Group V sPLA2 expression from LPS-treated (100 ng/ml, 18 h) or untreated cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A striking hallmark of the immunoinflammatory response is the generation of oxygenated derivatives of AA such as the prostaglandins. The response of major prostaglandin-secreting cells such as macrophages and mast cells to proinflammatory stimuli is generally biphasic (4). The first phase is completed within minutes after the addition of the stimulus, whereas the second phase usually takes several hours (4). Using the murine macrophage-like cell line P388D1, we have been studying the molecular mechanisms responsible for AA mobilization and prostaglandin production in response to LPS/PAF. When primed by LPS, these cells will respond to Ca2+-mobilizing stimuli such as PAF by generating a rapid burst of free AA, part of which is converted to prostaglandins such as PGE2. Strikingly, this process is completed within a few minutes after the addition of PAF (19). No free AA or prostaglandins are produced after the immediate phase is completed, not even after several hours of cell exposure to LPS/PAF (13). Such a behavior, which is abnormal for a macrophage cell, has prevented us from studying the molecular mechanisms responsible for delayed prostaglandin production in macrophages. In an attempt to overcome this problem, we subcloned the P388D1 cells by limit dilution, and selecting on the basis of high responsivity to LPS/PAF, we obtained a clone termed MAB, which shows enhanced sensitivity to LPS/PAF in the immediate phase (min) and exhibits a delayed response (h) to LPS alone.

Using the MAB clone, we have characterized the LPS-induced delayed prostaglandin production in terms of the role played by distinct PLA2 enzymes and their coupling with downstream COX enzymes during LPS signaling. Our previous work on the immediate response of the cells to LPS/PAF highlighted the very important role played by the novel Group V sPLA2 as the provider of most of the free AA directed to PGE2 biosynthesis (11). Herein, several lines of evidence suggest that Group V sPLA2 also behaves as a major provider of AA for the delayed phase of PGE2 production in LPS-treated cells. First, delayed [3H]AA release and PGE2 production correspond with the induction of Group V sPLA2 mRNA and enhanced secretion of a sPLA2-like activity to the supernatants, with no change in the constitutive levels of cPLA2 and no detectable induction of Group IIA sPLA2. Second, delayed PGE2 production is strongly blunted by LY311727, a selective sPLA2 inhibitor. Third, an antisense oligonucleotide specific for Group V sPLA2 (11) suppresses Group V sPLA2 activity and inhibits delayed PGE2 production. Our conclusions in this regard fully agree with recent works by Kudo and co-workers (20, 21) that were published while this manuscript was under review. By using transfection techniques, Kudo and co-workers (20, 21) have also documented the importance of Group V sPLA2 in delayed AA release and PGE2 production.

Our data have also implicated the cPLA2 as an important step in LPS signaling by enabling the subsequent action of the sPLA2. Thus the cPLA2 inhibitor MAFP (1) markedly blocked both long term [3H]AA release and Group V sPLA2 mRNA induction. Collectively, these results suggest an intriguing cross-talk between the cPLA2 and the Group V sPLA2 for the delayed phase of prostaglandin production in macrophages. This is a very interesting concept because cross-talk appears to exist as well between these two enzymes during the immediate phase of prostaglandin production (1, 2). In the immediate phase, cPLA2 activation generates a rapid and early burst of free AA inside the cell that enables sPLA2 activation by an as yet unidentified mechanism (1, 2). In the delayed phase, cPLA2 activation influences sPLA2 apparently by regulating sPLA2 mRNA levels.

Cross-talk between cPLA2 and sPLA2 in the immediate phase of prostaglandin production was also found to take place in mast cells (3) when the same protocol originally used in macrophages (1) was employed. Furthermore, a recent study by Kuwata et al. (22) about fibroblasts suggests that cross-talk between cPLA2 and sPLA2 in the delayed phase could also constitute a general mechanism of activation. Using a different cPLA2 inhibitor, arachidonyl trifluoromethyl ketone, Kuwata et al. (22) found that cPLA2 inhibition blocked sPLA2 expression in fibroblasts, leading to reduced PGE2 generation. The study by Kuwata et al. (22) is interesting not only because it supports the possible universality of cross-talk between cPLA2 and sPLA2 but because the sPLA2 expressed by rat fibroblasts is a Group IIA enzyme, not Group V. This lends further support to the emerging notion that Group IIA and Group V sPLA2 may be functionally redundant (23). In addition, Kuwata et al. (22) were able to show that overcoming cPLA2 inhibition by exogenous AA partially restored the Group IIA sPLA2 expression. These results suggest that the AA mobilized by cPLA2 is responsible for cross-talk between cPLA2 and sPLA2 (22). This is again reminiscent of what happens in the immediate phase of activation, wherein the cPLA2-derived AA is also responsible for cross-talk between cPLA2 and sPLA2, albeit by different mechanisms (1, 2). Unfortunately, inhibition by MAFP of Group V sPLA2 expression and activity could not be reversed in our macrophage studies with LPS alone by supplementing the medium with exogenous AA (up to 100 µM). This was not unexpected because P388D1 cells manifest an extraordinarily high capacity to import free AA from exogenous sources and incorporate it into membranes (19, 24, 25), which is much higher than that of most other cells (26). Thus, the half-life of the free AA in the cell would be too short to adequately mimic the low but continued production of AA-derived cPLA2 upon long term LPS exposure.

A model has recently emerged suggesting differential actions of COX-1 and COX-2 by virtue of differential coupling to distinct PLA2s (2, 3, 6, 8, 20, 21, 27). Thus, depending on whether cPLA2 or sPLA2 is the provider of free AA, either COX-1 or COX-2 would be responsible for PGE2 release. However, which PLA2 form couples to which COX isoform appears to depend strongly on cell type. We have recently demonstrated that the immediate, PAF receptor-mediated phase of PGE2 production in LPS-primed macrophages involves sPLA2 coupling to COX-2 (2). The current results support a similar kind of coupling for the delayed PGE2 production in LPS-treated cells. Identical coupling has been suggested by Arm and co-workers (6) for the delayed phase of PGE2 generation in mast cells. These results raise another interesting concept regarding the regulation of PGE2 during both phases of activation. As is the case for AA release (Fig. 2A), we have observed that the amount of PGE2 generated during the Ca2+-dependent short term stimulation is comparable to the amount produced in the late phase. It follows from this comparison that although the effector enzymes involved in the response are the same (i.e. cPLA2, sPLA2, COX-2), the regulatory mechanisms differ. Thus, in the short phase at low levels of COX-2, it appears that the dramatic burst in AA release is what determines the amount of PGE2 produced. In contrast, in the delayed phase at comparably lower AA availability, it appears that both the induction of large amounts of COX-2 protein and of the AA provider, Group V sPLA2, determine the amount of PGE2 produced.

It is important to note, however, that our results have not excluded that a minor fraction of the long term PGE2 produced in response to LPS could arise from the AA generated by the cPLA2. Should this be the case, some cPLA2/COX-2 coupling may exist as well, similar to what has been suggested by Reddy and Herschman (3) for delayed PGD2 production in mast cells and by Murakami et al. (5) in cells derived from Group IIA-deficient mice. The striking feature of the current work is that although COX-1 is present in active form in the P388D1 cells (2), it appears to be spared from the process of long term PGE2 production. This finding remains unexplained but has recently been recognized in other cell types as well (6, 8, 22). Recent work by Spencer et al. (16) showed no differences in the distribution of COX-1 versus COX-2 among subcellular fractions in a variety of cells. Thus subcellular compartmentalization may not be the cause for COX-1 not being utilized during LPS signaling. Other putative explanations may include the existence of COX-selective regulatory components, selective coupling to terminal PG synthases, or kinetic differences in AA utilization by the two isoforms.

In summary, we have established a subclone of P388D1 cells, MAB, that displays long term responsiveness to LPS in terms of PGE2 generation. We have confirmed (11) that these cells express Group V sPLA2, not Group IIA sPLA2, and found that (i) Group V sPLA2 is a key enzyme in long term AA mobilization as well and (ii) Group V sPLA2 is functionally coupled to COX-2. Furthermore, our results have suggested that cPLA2 plays a key role in long term AA mobilization, at least partly by regulating the expression of Group V sPLA2.

    FOOTNOTES

* This work was supported by Grants HD 26,171 and GM 20,501 from the National Institutes of Health. This work was presented at the Keystone Symposium on Signal Transduction and Lipid Second Messengers held in Taos, New Mexico on March 1-6, 1998.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.: 619-534-3055; Fax: 619-534-7390.

    ABBREVIATIONS

The abbreviations used are: AA, arachidonic acid; PAF, platelet-activating factor; LPS, bacterial lipopolysaccharide; cPLA2, Group IV cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; COX, cyclooxygenase (prostaglandin H2 synthase); MAFP, methylarachidonyl fluorophosphonate; PGE2, prostaglandin E2; iPLA2, Ca2+-independent phospholipase A2.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Balsinde, J., and Dennis, E. A. (1996) J. Biol. Chem. 271, 6758-6765[Abstract/Free Full Text]
  2. Balsinde, J., Balboa, M. A., and Dennis, E. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7951-7956[Abstract/Free Full Text]
  3. Reddy, S. T., and Herschman, H. R. (1997) J. Biol. Chem. 272, 3231-3237[Abstract/Free Full Text]
  4. Murakami, M., Nakatani, Y., Atsumi, G., Inoue, K., and Kudo, I. (1997) Crit. Rev. Immunol. 17, 225-284[Medline] [Order article via Infotrieve]
  5. Murakami, M., Kuwata, H., Amakasu, Y., Shimbara, S., Nakatani, Y., Atsumi, G., and Kudo, I. (1997) J. Biol. Chem. 272, 19891-19897[Abstract/Free Full Text]
  6. Bingham, C. O., III, Murakami, M., Fujishima, H., Hunt, J. E., Austen, K. F., and Arm, J. P. (1996) J. Biol. Chem. 271, 25936-25944[Abstract/Free Full Text]
  7. Herschman, H. R. (1996) Biochim. Biophys. Acta 1299, 125-140[Medline] [Order article via Infotrieve]
  8. Murakami, M., Matsumoto, R., Urade, Y., Austen, K. F., and Arm, J. P. (1995) J. Biol. Chem. 270, 3239-3246[Abstract/Free Full Text]
  9. Langenbach, R., Morham, S. G., Tiano, H. F., Loftin, C. D., Ghanayem, B. I., Chulada, P. C., Mahler, J. F., Lee, C. A., Goulding, E. H., Kluckman, K. D., Kim, H. S., and Smithies, O. (1995) Cell 83, 483-492[Medline] [Order article via Infotrieve]
  10. Lio, Y. C., Reynolds, L. J., Balsinde, J., and Dennis, E. A. (1996) Biochim. Biophys. Acta 1302, 55-60[Medline] [Order article via Infotrieve]
  11. Balboa, M. A., Balsinde, J., Winstead, M. V., Tischfield, J. A., and Dennis, E. A. (1996) J. Biol. Chem. 271, 32381-32384[Abstract/Free Full Text]
  12. Balsinde, J., Balboa, M. A., and Dennis, E. A. (1997) J. Biol. Chem. 272, 29317-29321[Abstract/Free Full Text]
  13. Glaser, K. B., Asmis, R., and Dennis, E. A. (1990) J. Biol. Chem. 265, 8658-8664[Abstract/Free Full Text]
  14. Balsinde, J., Balboa, M. A., Insel, P. A., and Dennis, E. A. (1997) Biochem. J. 321, 805-809[Medline] [Order article via Infotrieve]
  15. Futaki, N., Takahashi, S., Yokoyama, M., Arai, I., Higuchi, S., and Otomo, S. (1994) Prostaglandins 47, 55-59[CrossRef][Medline] [Order article via Infotrieve]
  16. Spencer, A. G., Woods, J. W., Arakawa, T., Singer, I. I., and Smith, W. L. (1998) J. Biol. Chem. 273, 9886-9893[Abstract/Free Full Text]
  17. Schevitz, R. W., Bach, N. J., Carlson, D. G., Chirgadze, N. Y., Clawson, D. K., Dillard, R. D., Draheim, S. E., Hartley, R. W., Jones, N. D., Mihelich, E. D., Olkowski, J. L., Snyder, D. W., Sommers, C., and Wery, J. P. (1996) Nat. Struct. Biol. 2, 458-464
  18. Chen, Y., and Dennis, E. A. (1998) Biochim. Biophys. Acta 1394, 57-64[Medline] [Order article via Infotrieve]
  19. Balsinde, J., Barbour, S. E., Bianco, I. D., and Dennis, E. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11060-11064[Abstract/Free Full Text]
  20. Murakami, M., Shimbara, S., Kambe, T., Kuwata, H., Winstead, M. V., Tischfield, J. A., and Kudo, I. (1998) J. Biol. Chem. 273, 14411-14423[Abstract/Free Full Text]
  21. Murakami, M., Kambe, T., Shimbara, S., and Kudo, I. (1999) J. Biol. Chem. 274, 3103-3115[Abstract/Free Full Text]
  22. Kuwata, H., Nakatani, Y., Murakami, M., and Kudo, I. (1998) J. Biol. Chem. 273, 1733-1740[Abstract/Free Full Text]
  23. Tischfield, J. A. (1997) J. Biol. Chem. 272, 17247-17250[Free Full Text]
  24. Balsinde, J., Bianco, I. D., Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8527-8531[Abstract]
  25. Balsinde, J., and Dennis, E. A. (1996) Eur. J. Biochem. 235, 480-485[Abstract]
  26. Surette, M. E., and Chilton, F. H. (1998) Biochem. J. 330, 915-921[Medline] [Order article via Infotrieve]
  27. Murakami, M., Nakatani, Y., and Kudo, I. (1996) J. Biol. Chem. 271, 30041-30051[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.