Functional Coupling Between Various Phospholipase A2s and Cyclooxygenases in Immediate and Delayed Prostanoid Biosynthetic Pathways*

Makoto Murakami, Terumi Kambe, Satoko Shimbara, and Ichiro KudoDagger

From the Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan

    ABSTRACT
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Abstract
Introduction
References

Several distinct phospholipase A2s (PLA2s) and two cyclooxygenases (COXs) were transfected, alone or in combination, into human embryonic kidney 293 cells, and their functional coupling during immediate and delayed prostaglandin (PG)-biosynthetic responses was reconstituted. Signaling PLA2s, i.e. cytosolic PLA2 (cPLA2) (type IV) and two secretory PLA2s (sPLA2), types IIA (sPLA2-IIA) and V (sPLA2-V), promoted arachidonic acid (AA) release from their respective transfectants after stimulation with calcium ionophore or, when bradykinin receptor was cotransfected, with bradykinin, which evoked the immediate response, and interleukin-1 plus serum, which induced the delayed response. Experiments on cells transfected with either COX alone revealed subtle differences between the PG-biosynthetic properties of the two isozymes in that COX-1 and COX-2 were favored over the other in the presence of high and low exogenous AA concentrations, respectively. Moreover, COX-2, but not COX-1, could turn on endogenous AA release, which was inhibited by a cPLA2 inhibitor. When PLA2 and COX were coexpressed, AA released by cPLA2, sPLA2-IIA and sPLA2-V was converted to PGE2 by both COX-1 and COX-2 during the immediate response and predominantly by COX-2 during the delayed response. Ca2+-independent PLA2 (iPLA2) (type VI), which plays a crucial role in phospholipid remodeling, failed to couple with COX-2 during the delayed response, whereas it was linked to ionophore-induced immediate PGE2 generation via COX-1 in marked preference to COX-2. Finally, coculture of PLA2 and COX transfectants revealed that extracellular sPLA2s-IIA and -V, but neither intracellular cPLA2 nor iPLA2, augmented PGE2 generation by neighboring COX-expressing cells, implying that the heparin-binding sPLA2s play a particular role as paracrine amplifiers of the PG-biosynthetic response signal from one cell to another.

    INTRODUCTION
Top
Abstract
Introduction
References

Phospholipase A2 (PLA2),1 which regulates the release of arachidonic acid (AA) from membrane phospholipids, and cyclooxygenase (COX), which converts AA to the intermediate prostaglandin (PG) precursor PGH2, represent the two crucial rate-limiting steps for the PG-biosynthetic pathway. To date, more than 10 PLA2 (1, 2) and 2 COX (3) isozymes have been identified in mammals. The existence of two kinetically distinct PG-biosynthetic responses, the immediate and delayed phases, implies the recruitment of different sets of biosynthetic enzymes to this pathway. Although several works have suggested that preferential coupling between these biosynthetic enzymes accounts for the differential regulation of the immediate and delayed responses, conflicting evidence has been yielded by different experimental systems (4-11).

A rapidly expanding body of evidence suggests that the two COXs, the constitutive COX-1 and inducible COX-2, play distinct roles in regulating AA metabolism (3). Several investigators have proposed that COX-1 and COX-2 respectively metabolize exogenous and endogenous arachidonic acid (AA) to PGs (12, 13). This statement, however, is an oversimplification, because in certain situations, COX-1 metabolizes endogenous AA to PGs, e.g. thromboxane generation by platelets (14) and macrophages (11) and PGD2 generation by mast cells (4-6), and COX-2 also utilizes exogenous AA (11). More generally, utilization of COX-1 is observed during the early phase of PG biosynthesis occurring within several minutes of stimulation, whereas COX-2-dependent PG generation proceeds over several hours in parallel with the induction of COX-2 expression (4-6, 10, 11, 15). Although subtle differences in the subcellular distributions of these two isozymes may be responsible for their separate functions, a recent electron microscopic analysis showed that their locations in the perinuclear and endoplasmic reticular membranes were indistinguishable (16).

Whether distinct PLA2s are utilized selectively in the different PG-biosynthetic phases and couple specifically with each COX isozyme is controversial. PLA2s are subdivided into several classes, among which cytosolic PLA2 (cPLA2; recently called cPLA2alpha after the recent discovery of two related isozymes cPLA2 beta  and gamma  (17)), a family of secretory PLA2s (sPLA2s), and Ca2+-independent PLA2 (iPLA2) have been paid considerable attention. The increased cytoplasmic Ca2+ concentration and activation of mitogen-activated protein kinases, which are prerequisite for cPLA2 activation, occur rapidly and are often transient, implying that cPLA2 plays a crucial role in immediate AA release (18-20). Evidence is accumulating that cPLA2 is also involved in the delayed response (9, 21, 22), although the mechanism whereby cPLA2 is activated under such a Ca2+-free condition is poorly understood. Some studies showed that sPLA2 couples selectively with COX-1 during the immediate phase (6), whereas others demonstrated that sPLA2, particularly type IIA (sPLA2-IIA), functions during the delayed phase, as its expression is often induced markedly in response to proinflammatory stimuli and correlates with ongoing PG biosynthesis (10, 23-25). Several types of cell express type V sPLA2 (sPLA2-V), which appears to mediate certain phases of PG biosynthesis (26, 27). Supportive (28, 29) and contradictory (30, 31) evidence for the involvement of iPLA2 in stimulus-dependent AA release has been reported. The conflicting observations reported so far may be due to the limitations of studies using chemical inhibitors, antibodies, and even antisense oligonucleotides, which often cannot gain access to certain cellular compartments and may cause cross-inhibition or undesirable side effects, thereby leading to misinterpretation.

Recently, in an attempt to elucidate the general functions of each PLA2 in AA release, we analyzed the functional effects of transfecting various PLA2 isozymes into mammalian cell lines, such as human embryonic kidney 293 cells and Chinese hamster ovary (CHO) cells. This approach enabled us to assess the overlapping and different functions of five distinct PLA2s, namely cPLA2, sPLA2s (IIA, IIC, and V), and iPLA2 (32). We demonstrated that cPLA2 and the two heparin-binding sPLA2s, sPLA2-IIA and sPLA2-V, act as "signaling" PLA2s that promote stimulus-dependent AA release during both the immediate and delayed responses, whereas iPLA2 mediates spontaneous fatty acid release during culture, consistent with its proposed role in "phospholipid remodeling" rather than signaling (30, 31).

In this study, we extended our previous study in order to investigate particular functional cooperation between PLA2 and COX enzymes during different phases of PG biosynthesis by cotransfecting each PLA2 and COX into 293 cells. Reconstitution of the immediate and delayed PGE2 biosynthetic responses of these transfectants confirmed that distinct functional coupling between different PLA2 and COX enzymes occurs during each phase.

    EXPERIMENTAL PROCEDURES

Materials----- The cDNAs for mouse cPLA2, mouse sPLA2-IIA, and its mutants (G30S, H48E, and KE4), rat sPLA2-V, rat sPLA2-IIC, and hamster iPLA2 were described previously (32). Human COX-1 and COX-2 cDNAs were provided by Dr. S. Nagata (Osaka University) and subcloned into pcDNA3.1 (Invitrogen). Rat bradykinin receptor (BKR) B2 cDNA (33), obtained from a rat genomic library (CLONTECH) by performing the reverse transcription-polymerase chain reaction using a RNA polymerase chain reaction kit (AMV) version-2 (Takara Shuzo), was subcloned into pCR3.1 (Invitrogen). Human embryonic kidney 293 cells were obtained from Riken Cell Bank, and CHO-K1 cells stably expressing human COX-1 and COX-2 were provided by Dr. M. Sugimoto (Chugai Pharmaceutical Co. Ltd.). The rabbit anti-human cPLA2 antibody and sPLA2 inhibitor LY311727 (34) were provided by Dr. R. M. Kramer (Lilly Research). The rabbit anti-rat sPLA2-IIA antibody was prepared as described previously (35). The goat anti-human COX-2 antibody was purchased from Santa Cruz. The rabbit anti-human COX-1 antibody and COX-1 inhibitor valeryl salicylate (36) were provided by Dr. W. L. Smith (Michigan State University). The COX-2 inhibitor NS-398 (37) was provided by Dr. J. Trzaskos (DuPont Merck Pharmaceutical Co.). The cPLA2 inhibitor methyl arachidonylfluorophosphate (MAFP) (38), the iPLA2 inhibitor bromoenol lactone (39), AA, and the PGE2 enzyme immunoassay kit were purchased from Cayman Chemical. A23187 was purchased from Calbiochem. BK was purchased from Sigma. Human and mouse interleukin (IL)-1beta s were purchased from Genzyme. LipofectAMINE PLUS reagent, Opti-MEM medium, and Trizol reagent were obtained from Life Technologies. RPMI 1640 medium was purchased from Nissui Pharmaceutical.

Establishment of PLA2 and COX Transfectants-- Transformants of 293 cells that stably expressed cPLA2, sPLA2-IIA, sPLA2-V, sPLA2-IIC, and iPLA2 were established as described previously (32). COX-1 and COX-2 cDNAs were transfected into 293 cells using LipofectAMINE PLUS, according to the manufacturer's instructions. Briefly, 1 µg of plasmid was mixed with 5 µl of LipofectAMINE PLUS in 200 µl of Opti-MEM medium for 30 min and then added to cells that had attained 40-60% confluence in 6-well plates (Iwaki) containing 1 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 2 ml of fresh culture medium comprising RPMI 1640 containing 10% (v/v) fetal calf serum (FCS). After overnight culture, the medium was replaced again with 2 ml of fresh medium, and culture was continued at 37 °C in a CO2 incubator flushed with 5% CO2 in humidified air. For transient expression analysis, the cells were harvested 3 days after transfection and used immediately. In order to establish stable transfectants, cells transfected with each cDNA were cloned by limiting dilution in 96-well plates in culture medium supplemented with 800 µg/ml G418 (Life Technologies). After culture for 2-4 weeks, wells containing a single colony were chosen, and the expression of each COX was assessed by immunoblotting. The established clones were expanded and used for the experiments as described below.

In order to establish PLA2/COX double transformants, 293 transformants expressing each COX were subjected to a second transfection with each PLA2 cDNA, which had been subcloned into pcDNA3.1/Zeo (+) (Invitrogen). Three days after transfection, the cells were used for the experiments or seeded into 96-well plates to be cloned by culture in the presence of 50 µg/ml zeocin (Invitrogen) in order to establish stable transformants overexpressing both PLA2 and COX. The expression of each PLA2 was examined by RNA blotting, and in the case of sPLA2s, by measuring PLA2 activities released into the supernatants. A similar strategy was employed to produce PLA2/BKR double transformants: after the second transfection with cPLA2 or sPLA2-IIA cDNA in pcDNA3.1/Zeo (+), G418-resistant 293 cells stably expressing rat B2-type BKR were selected with zeocin.

Measurement of sPLA2 Activity-- Cells in 1 ml of culture medium were seeded, at a density of 5 × 104 cells/ml, into each well of 24-well plates. After culture for 4 days, the supernatants were collected, and the cells were incubated for a further 15 min at 37 °C with 1 ml of culture medium containing M NaCl. This allowed cell surface-associated sPLA2s to be recovered quantitatively from the medium, as described previously (32). The PLA2 activity was assayed by measuring the amounts of free radiolabeled fatty acids released from the substrates 1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphoethanolamine (NEN Life Science Products) and 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoethanolamine (Amersham Pharmacia Biotech) for sPLA2-V and sPLA2-IIA, respectively. Each reaction mixture consisted of an aliquot of the required sample, 100 mM Tris-HCl, pH 6.0 (for sPLA2-V) or 7.4 (for sPLA2-IIA), 4 mM CaCl2, and 2 µM substrate. After incubation for 10-30 min at 37 °C, the [14C]fatty acids released were extracted by the method of Dole and Meinertz (40), and the radioactivity was counted.

RNA Blotting-- Approximately equal amounts (~10 µg) of the total RNAs obtained from transfected cells were applied to each lane of 1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore). The resulting blots were then probed with the respective cDNA probes that had been labeled with [32P]dCTP (Amersham Pharmacia Biotech) by random priming (Takara Shuzo). All hybridizations were carried out as described previously (10).

SDS-Polyacrylamide Gel Electrophoresis/Immunoblotting-- Cell lysates (105 cell equivalents) were subjected to SDS-polyacrylamide gel electrophoresis (7.5% (w/v) for cPLA2 and 10% for COX-1 and COX-2) under reducing conditions. In order to analyze sPLA2-IIA, the cells were treated for 15 min with culture medium containing 1 M NaCl, and aliquots of the resulting supernatants were subjected to 15% SDS-polyacrylamide gel electrophoresis under nonreducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell) using a semidry blotter (MilliBlot-SDE system; Millipore), according to the manufacturer's instructions. The membranes were probed with the respective antibodies and visualized using the ECL Western blot analysis system (Amersham Pharmacia Biotech), as described previously (10).

Cell Activation-- Cells (5 × 104 in 1 ml of culture medium) were seeded into each well of 24-well plates. In order to assess AA release, 0.1 µCi/ml [3H]AA (Amersham Pharmacia Biotech) was added to the cells in each well on day 3, when they had nearly reached confluence, and culture was continued for another day. After three washes with fresh medium, 250 µl of RPMI 1640 medium with or without 10 µM A23187, 10 µM BK, or 1 ng/ml IL-1beta and/or 10% FCS was added to each well and the amount of free [3H]AA released into each supernatant during culture (up to 30 min after the addition of A23187 and BK and up to 8 h after IL-1beta ) was measured. The percentage release of AA was calculated using the formula (S/(S + P)) × 100, where S and P are the radioactivities measured in equal portions of the supernatant and cell pellet, respectively. The supernatants from replicate cells cultured without added radiolabeled fatty acids were subjected to the PGE2 enzyme immunoassay. Conversion of exogenous AA to PGE2 was determined by treating cells with AA for 30 min and immunoassaying the PGE2 produced.

    RESULTS

PGE2 Generation by Transfectants Expressing Either COX Alone-- Human COX-1 and COX-2 cDNAs, subcloned into a mammalian expression vector with a neomycin-resistant marker, were individually transfected into 293 and CHO cells to establish stable transformants. Expression of COX-1 and COX-2 proteins was barely detectable in parental 293 cells but was detected clearly in the respective transformants (Fig. 1A). In order to assess immediate and delayed PGE2 generation from endogenous AA by each COX, we stimulated the transformants with 10 µM A23187 for 30 min and 1 ng/ml IL-1 in the presence of 10% FCS for 4 h, respectively (Fig. 1B). Whereas treatment of parental 293 cells with A23187 resulted in minimal PGE2 generation, consistent with undetectable expression levels of both COX isozymes (Fig. 1A), replicate cells expressing COX-1 and COX-2 produced approximately 15 and 35 times more PGE2 in response to A23187 than the control cells (Fig. 1B, top). When COX-2-expressing cells were stimulated with IL-1/FCS, delayed PGE2 generation increased markedly, reaching approximately 14 times the amount generated by the control cells (Fig. 1B, bottom). In contrast, COX-1-expressing cells produced only minimal amounts of PGE2 that did not differ significantly from the amounts produced by the control cells.


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Fig. 1.   Effects of COX overexpression. A, expression of COX-1 and COX-2 proteins, assessed by immunoblotting, in 293 cells transfected with each cDNA. B, PGE2 generation from endogenous AA by control 293 cells and COX-1 and COX-2 transfectants following stimulation with 10 µM A23187 for 30 min (top) and 1 ng/ml human IL-1beta in the presence of 10% FCS for 4 h (bottom). C, conversion of exogenous AA to PGE2 by COX-1 (circles) and COX-2 (closed squares) transfectants and control cells (open squares). The cells were incubated with various concentrations of AA in RPMI 1640 medium for 30 min. D, effects of the COX-1 inhibitor valeryl salicylate and the COX-2 inhibitor NS-398 on the conversion of exogenous AA to PGE2. The cells were preincubated for 5 h with each inhibitor, washed, and then incubated for 30 min with 50 µM AA in the continued presence of each inhibitor. E, COX-2 itself stimulated AA release. Control and COX-2-expressing 293 cells prelabeled with [3H]AA were treated for 2 h with 10 µM MAFP, 50 µM bromoenol lactone (BEL), or 5 ng/ml NS-398, washed, and then cultured for a further 4 h in RPMI 1640 medium in the continued presence of each inhibitor to assess [3H]AA release (top) and PGE2 generation (bottom). F, expression of COX-1 and COX-2 proteins, assessed by immunoblotting, in parental CHO cells and CHO cells transfected with each cDNA (top) and that of endogenous cPLA2 protein in parental 293 and CHO cells (bottom). G, PGE2 generation by control CHO cells and COX-1 or COX-2 transfectants after treatment with 10 µM A23187 for 30 min (left), 1 ng/ml mouse IL-1beta in the presence or absence of 10% FCS for 4 h (middle), or various concentrations of exogenous AA for 30 min (right). Means ± S.E. of three to five experiments are shown in B, C, and E, and representative results of three independent experiments are shown in A, D, F, and G.

To investigate the functional differences between COX-1 and COX-2 further, we measured the COX activities by supplying the transfectants with exogenous AA (Fig. 1C). In our assay system, COX-1 transfectants produced PGE2 when the exogenous AA concentration exceeded 10 µM, reaching nearly 100 ng of PGE2 in the presence of 50 µM AA, whereas conversion to PGE2 was undetectable when the AA concentration was below 10 µM. COX-2-expressing cells produced substantial amounts of PGE2 even when the AA concentration was below 10 µM. Approximately 20 ng of PGE2 was produced by COX-2-expressing cells treated with 50 µM AA, only <FR><NU>1</NU><DE>5</DE></FR> the amount produced by COX-1 transfectants. No appreciable conversion of AA to PGE2 by the control cells was observed. Conversion of exogenous AA to PGE2 by cells expressing COX-1 and COX-2 was inhibited by the COX-1-specific inhibitor valeryl salicylate and the COX-2-specific inhibitor NS-398, respectively, but not vice versa (Fig. 1D). Collectively, these data suggest that COX-1 is favored over COX-2 when excess AA is supplied, whereas COX-2 is more active than COX-1 when the supply of AA is limited.

We observed that some PGE2 was produced by cells expressing COX-2, but not COX-1, even in the absence of exogenous AA or any extracellular stimulus (Fig. 1, C and E), indicating that COX-2 could utilize endogenous AA in this situation. Interestingly, COX-2-expressing cells not only spontaneously produced more PGE2 but also released more AA than the control cells (Fig. 1E). This increased AA release induced by COX-2 transfection was significantly suppressed by the cPLA2 inhibitor MAFP but not by the iPLA2 inhibitor bromoenol lactone, the COX-2 inhibitor NS-398 (Fig. 1E), or the sPLA2 inhibitor LY311727 (data not shown), whereas PGE2 generation by COX-2 transfectants was suppressed by MAFP and NS-398 but not by bromoenol lactone. Immunoblot analysis revealed a trace amount of cPLA2 (Fig. 1F) but none of other PLA2s examined in this study (data not shown) in parental 293 cells. These observations suggest that COX-2 can activate AA release mediated by the MAFP-sensitive cPLA2 through a mechanism independent of COX activity.

In order to verify that the observations described above was also applicable to other cells, we carried out similar experiments using CHO cells transfected with COX-1 and COX-2 (Fig. 1, F and G). CHO cells expressing COX-1 and COX-2 produced 16 and 25 times more PGE2, respectively, than the parental cells during the immediate response to A23187 (Fig. 1G, left). Significant and comparable increases in PGE2 generation by parental and COX-1-expressing cells after stimulation with IL-1 plus FCS was observed (Fig. 1G, middle). This increase was suppressed almost completely by NS-398 (data not shown) (32), indicating its dependence upon endogenous COX-2, trace amounts of which were expressed in CHO cells (Fig. 1F). The COX-2 transfectants exhibited a 3-fold increase in delayed PGE2 generation compared with the control cells (Fig. 1G, middle). The experiment using exogenous AA revealed that, as in the experiments on 293 cells (Fig. 1, A-E), COX-2 was more active than COX-1 when the AA concentrations were below 10 µM, whereas COX-1 was active in the presence of high AA concentrations (Fig. 1G, right). Moreover, significant PGE2 generation by COX-2, but not COX-1, transfectants occurred even without extracellular stimuli or exogenous AA (Fig. 1G). CHO cells expressed more endogenous cPLA2 than 293 cells (Fig. 1F), and the spontaneous PGE2 generation by CHO COX-2 transfectants was inhibited by MAFP (data not shown).

PGE2 Generation by Transfectants Expressing Both cPLA2 and COX-- In order to assess the functional coupling between particular PLA2 enzymes and each COX, we attempted to establish PLA2/COX double transfectants. Fig. 2A depicts the expression levels of cPLA2, COX-1 and COX-2 in cells transfected with these cDNAs alone or in combination. Although the expression of cPLA2 alone was accompanied by a marked increase in A23187-induced AA release, as reported previously (32), no appreciable PGE2 generation occurred (Fig. 2B) due to the absence of constitutive expression of endogenous COXs (Fig. 2A). Although the expression of COX-1 alone increased PGE2 generation only minimally, coexpression of cPLA2 and COX-1 led to a dramatic increase in PGE2 generation (Fig. 2B). As described above, the expression of COX-2 alone evoked AA release, which was accompanied by a significant increase in PGE2 generation. When cPLA2 and COX-2 were coexpressed, they increased AA release and PGE2 generation in a synergistic manner (Fig. 2B). A23187-induced PGE2 generation by these double transformants, which was dependent upon the coexistence of cPLA2 and either COX, displayed the typical immediate response pattern, reaching a peak 5-10 min after stimulation and a plateau by 30 min (Fig. 2C).


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Fig. 2.   Coexpression of cPLA2 and COX-1 or COX-2. A, expression of cPLA2, COX-1 and COX-2 proteins, assessed by immunoblotting, in 293 cells transfected with each cDNA alone or in combination. B, AA release (top) and PGE2 generation (bottom) by cPLA2, COX-1, and COX-2 single transfectants and cotransfectants after stimulation for 30 min with 10 µM A23187. C, time course of A23187-induced PGE2 generation in cells expressing cPLA2/COX-1 (solid squares), cPLA2/COX-2 (solid circles), and parental cells (open circles). D, AA release (top) and PGE2 generation (bottom) by the cPLA2, COX-1, and COX-2 single transfectants and cotransfectants after stimulation for 4 h with 1 ng/ml IL-1beta plus 10% FCS. E, time course of IL-1/FCS-induced PGE2 generation in cells expressing cPLA2/COX-1 (solid triangles), cPLA2/COX-2 (solid circles), COX-2 (solid squares), and parental cells (open circles). F, dependence of delayed PGE2 generation on IL-1 and FCS. Control cells and transfectants expressing COX-2 alone or cPLA2/COX-2 were cultured for 4 h in the presence or absence of 1 ng/ml IL-1beta plus 10% FCS. G, effects of COX inhibitors. Cells expressing cPLA2/COX-1 and cPLA2/COX-2 were pretreated for 2 h with 1 µg/ml valeryl salicylate (VS) or 5 ng/ml NS-398 (NS), washed, and then activated for 30 min with A23187 (top) or for 4 h with IL-1/FCS (bottom) to assess PGE2 generation. Means ± S.E. of six to seven independent experiments are shown in B, D, and F, and representative results of three to four independent experiments are shown in A, C, E, and G.

As A23187 is a nonphysiologic stimulus, we wanted to establish whether cPLA2 could couple with COX-1 and COX-2 in the immediate response following a more physiologic stimulus. In order to address this issue, we introduced BKR, a G-protein-linked receptor with seven membrane-spanning regions, into 293 cells, cPLA2 cDNA was then transfected into these cells, and G-protein-dependent activation of cPLA2 was assessed (Fig. 3, A and B). Note that the expression levels of cPLA2 in transfectants expressing cPLA2 alone (Fig. 2A) and those expressing both cPLA2 and BKR (Fig. 3A) were comparable. After stimulation for 30 min with 10 µM BK, cells stably expressing both cPLA2 and BKR released approximately 3 times more AA than the replicate cells expressing BKR alone (Fig. 3B). Significant increases in BK-dependent PGE2 generation after transfecting either COX-1 or COX-2 into cPLA2/BKR, but not BKR alone, transformants were observed (Fig. 3C), confirming that cPLA2 can couple with both COX-1 and COX-2 in the immediate phase elicited by a physiologic stimulus.


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Fig. 3.   Immediate response induced by BK. A, expression of cPLA2 and BKR assessed by RNA blotting. B, AA release by BKR and cPLA2/BKR transfectants after stimulation with 10 µM BK for 30 min. C, PGE2 generation by cPLA2/COX/BKR triple transfectants. BKR- and cPLA2/BKR-expressing 293 cells were transfected transiently with either COX-1 or COX-2 after stimulation with BK for 30 min. Three days after COX transfection, the cells were stimulated with 10 µM BK for 30 min to assess immediate PGE2 generation. D, expression of sPLA2-IIA and BKR assessed by RNA blotting. E, AA release by BKR and sPLA2-IIA/BKR transfectants in response to BK. F, PGE2 generation by sPLA2-IIA/COX/BKR triple transfectants in response to BK. Representative results for two (A, C, D, and F) and four (B and E) independent experiments are shown.

Expression of cPLA2 alone increased IL-1/FCS-induced delayed AA release, which was accompanied by a modest increase in PGE2 generation that was dependent upon endogenously induced COX-2 (Fig. 2D), as demonstrated in our previous study (32). Expression of COX-1 had a minimal effect on delayed PGE2 generation, even when cPLA2 was coexpressed (Fig. 2D). The expression of COX-2 alone stimulated AA release, accompanied by a significant increase in PGE2 generation. When cPLA2 and COX-2 were coexpressed, they acted in synergy, resulting in marked augmentation of AA release and PGE2 generation (Fig. 2D). Kinetic experiments showed that IL-1/FCS-induced PGE2 generation, the typical delayed response, proceeded gradually and continuously during culture of cPLA2/COX-2 double transfectants for 8 h (Fig. 2E). IL-1 dependence of the delayed PGE2 generation, in which IL-1, in concert with FCS, played a prerequisite role in the induction of endogenous COX-2 expression (32), by cells stably expressing COX-2 appeared less obvious, although IL-1 exerted some augmentative effect on FCS-dependent PGE2 generation (Fig. 2F) likely through enhancing cPLA2-mediated AA release (32). A23187-induced immediate PGE2 generation by cPLA2/COX-1 and cPLA2/COX-2 transfectants was suppressed selectively by valeryl salicyate and NS-398, respectively, and IL-1/FCS-induced delayed PGE2 generation by cPLA2/COX-2 transfectants was suppressed almost completely by NS-398 (Fig. 2G).

PGE2 Generation by Transfectants Expressing sPLA2 and COX-- Next, we established transformants coexpressing sPLA2-IIA and either COX-1 or COX-2, and their expression levels in each transfectant were verified by immunoblotting (Fig. 4A, top). sPLA2-IIA expression was also checked by determining the enzymatic activities in the supernatants (Fig. 4A, bottom), which correlated with the intensities of the sPLA2-IIA protein bands visualized by immunoblotting (Fig. 4A, top). Expression of sPLA2-IIA alone increased A23187-induced AA release that was not accompanied by PGE2 generation, whereas coexpression of sPLA2-IIA and COX-1 enhanced PGE2 generation markedly (Fig. 4B). Coexpression of sPLA2-IIA and COX-2 also resulted in significant increase in PGE2 generation, although, unlike cPLA2/COX-2 double transfectants (Fig. 2B), they did not synergize at the level of AA release (Fig. 4B). A23187-induced PGE2 generation by sPLA2-IIA/COX-1 or -2 double transformants reached a maximum within 10 min and plateaued thereafter (Fig. 4C). Furthermore, the transfectants stably expressing both sPLA2-IIA and BKR (Fig. 3D), in which sPLA2-IIA expression level was comparable to that in cells expressing sPLA2-IIA alone (Fig. 4A), released more AA than the control cells following 30 min of exposure to BK (Fig. 3E), and the AA released by sPLA2-IIA in response to BK stimulation was converted to PGE2 by COX-1 and COX-2 when each COX cDNA was transfected into the sPLA2-IIA/BKR double transfectants (Fig. 3F). Thus, sPLA2-IIA appeared to couple functionally with both COX-1 and COX-2 in the immediate response.


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Fig. 4.   Coexpression of sPLA2-IIA and COX-1 or COX-2. A, expression of sPLA2-IIA, COX-1 and COX-2 proteins, assessed by immunoblotting, in 293 cells transfected with each cDNA alone or in combination (top) and sPLA2 activity released into the supernatants (bottom). B, AA release (top) and PGE2 generation (bottom) by sPLA2-IIA, COX-1, and COX-2 single transfectants and cotransfectants after stimulation for 30 min with 10 µM A23187. C, time course of A23187-induced PGE2 generation in transfectants expressing sPLA2-IIA/COX-1 (solid squares) or sPLA2-IIA/COX-2 (solid circles) and control cells (open circles). D, AA release (top) and PGE2 generation (bottom) by the sPLA2-IIA, COX-1 and COX-2 single transfectants and cotransfectants after stimulation for 4 h with 1 ng/ml IL-1beta plus 10% FCS. E, time course of IL-1/FCS-induced PGE2 generation by control cells (open circles) and transfectants expressing sPLA2-IIA/COX-1 (solid triangles), sPLA2-IIA/COX-2 (solid circles), and COX-2 alone (solid squares). F, effects of COX inhibitors. Cells expressing sPLA2-IIA/COX-1 and sPLA2-IIA/COX-2 were pretreated for 2 h with 1 µg/ml valeryl salicylate (VS) or 5 ng/ml NS-398 (NS), washed, and then activated for 30 min with A23187 or for 4 h with IL-1/FCS to assess PGE2 generation. Means ± S.E. of six to seven independent experiments are shown in B and D, and representative results of three to four independent experiments are shown in A, C, E, and F.

Expression of sPLA2-IIA alone increased IL-1/FCS-induced delayed AA release, accompanied by a modest increase in PGE2 generation, which depended on endogenous COX-2 (Fig. 4D), as reported previously (32). Although delayed PGE2 generation by cells coexpressing sPLA2-IIA and COX-1 was comparable to that by cells expressing sPLA2-IIA alone, leading further support to the hypothesis that COX-1 is dissociated from the delayed response, it was augmented significantly when sPLA2-IIA and COX-2 were coexpressed (Fig. 4D). Kinetic experiments showed that delayed PGE2 generation by sPLA2-IIA/COX-2 double transformants proceeded throughout the culture period (Fig. 4E). Valeryl salicylate and NS-398 reduced sPLA2-IIA/COX-1- and sPLA2-IIA/COX-2-mediated A23187-induced immediate PGE2 generation, respectively (Fig. 4F, top), and sPLA2-IIA/COX-2-mediated delayed PGE2 generation was suppressed almost completely by NS-398 (Fig. 4F, bottom).

Our previous study (32), as well as those of others (26, 27), demonstrated that sPLA2-V compensates for sPLA2-IIA during stimulus-initiated AA release. The expression profiles of sPLA2-V, COX-1, and COX-2 in 293 cells transfected with their cDNAs alone or in combination are shown in Fig. 5A. The expression levels of sPLA2-V were verified by RNA blotting and enzyme assay (Fig. 5A, bottom). Expression of sPLA2-V alone increased A23187-induced immediate and IL-1/FCS-induced delayed AA release in a manner similar to sPLA2-IIA (data not shown) (32). Coexpression of sPLA2-V and COX-1 markedly increased immediate (Fig. 5B) but not delayed (Fig. 5C) PGE2 generation. Coexpression of sPLA2-V and COX-2 consistently enhanced immediate (Fig. 5B) and delayed (Fig. 5C) PGE2 generation to approximately 1.5- and 2.7-fold, respectively, that by cells expressing COX-2 alone.


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Fig. 5.   Coexpression of sPLA2-V and COX-1 or COX-2. A, expression of COX-1 and COX-2, assessed by immunoblotting, in 293 cell transfectants (top) and that of sPLA2-V, assessed by enzyme activity released into the supernatants (bottom) and RNA blotting (Inset). B, PGE2 generation by the sPLA2-V, COX-1 and COX-2 single transfectants and cotransfectants after stimulation for 30 min with 10 µM A23187. C, PGE2 generation by the sPLA2-V and/or COX-1 or -2 transfectants after stimulation for 4 h with 1 ng/ml IL-1beta plus 10% FCS. D, dominant negative effect of the catalytically inactive sPLA2-IIA mutant (G30S) on sPLA2-V-mediated AA release. The sPLA2-IIA mutant G30S was coexpressed in 293 cells stably expressing sPLA2-V, and the distributions of sPLA2-V activity in the supernatant (S) and cell surface-associated fraction (C) (left) and IL-1/FCS-stimulated AA release (right) were compared with those of replicate cells not subjected to G30S cotransfection. E, dominant negative effect of the catalytically inactive sPLA2-IIA mutants (G30S and H48E) on sPLA2-IIA-mediated AA release. The indicated amounts of plasmids containing cDNAs for the sPLA2-IIA mutants G30S, H48E, and KE4 were each cotransfected into 293 cells stably expressing sPLA2-IIA, and IL-1/FCS-stimulated delayed AA release was examined. Means ± S.E. of four independent experiments are shown in B and C, and representative results of three independent experiments are shown in A, D, and E.

In previous studies, we showed that sPLA2-IIA and sPLA2-V, but not sPLA2-IIC, each has a cluster of cationic residues in the C-terminal domain that is essential for its association with cell surface proteoglycan and for its AA-releasing functions (32, 41). In order to gain further insight into the similar actions of sPLA2-IIA and sPLA2-V, we transfected a sPLA2-IIA mutant, G30S, which is catalytically inactive but possesses normal cell binding capacity (32), into 293 cells expressing sPLA2-V (Fig. 5D). We reasoned that if sPLA2-IIA and sPLA2-V share the common binding site on the plasma membrane, sPLA2-IIA(G30S) would displace sPLA2-V from the binding site, thereby solubilizing the cell surface-bound sPLA2-V and eventually leading to a reduction in sPLA2-V-mediated AA release. As shown in Fig. 5D, about 65% of sPLA2-V, assessed by enzymatic activity, was present as the cell surface-bound form. Coexpression of sPLA2-IIA(G30S) altered the distribution of sPLA2-V between the supernatant and cell surface, where the cell surface-bound portion of sPLA2-V decreased to half that in the nonmutant replicate cells. Accordingly, this reduced sPLA2-V-mediated AA release in response to A23187 (data not shown) or IL-1/FCS (Fig. 5D) by approximately 50%. sPLA2-IIA(G30S) also reduced sPLA2-IIA-mediated AA release in a plasmid concentration-dependent manner (Fig. 5E), and this was accompanied by a decrease in the amount of cell-associated native sPLA2-IIA (data not shown). The dominant-negative effect of this particular sPLA2-IIA mutant was confirmed by the observations that another mutant, sPLA2-IIA(H48E), which also lacks catalytic activity but has intact cell binding ability (32), blocked sPLA2-IIA-mediated AA release in a manner similar to sPLA2-IIA(G30S), whereas sPLA2-IIA(KE4), which has normal PLA2 activity and impaired cell binding ability, failed to do so (Fig. 5E). Thus, this experiment confirmed that sPLA2-IIA and sPLA2-V exhibit their AA-releasing effects through binding to a common component (proteoglycan or alternative) on the cell surface.

PGE2 Generation by Transfectants Expressing iPLA2 and COX-- Accumulating evidence suggests that iPLA2 plays a crucial role in phospholipid remodeling (30, 31). In support of this, we showed recently that iPLA2 overexpression in 293 cells led to increased spontaneous fatty acid release, which showed virtually no link with PGE2 generation via endogenous COX-2 (32). To ascertain whether iPLA2 could participate in PG biosynthesis or not, we herein established 293 transfectants stably expressing both iPLA2 and either COX-1 or COX-2, the expression of which was assessed by RNA blotting and immunoblotting, respectively (Fig. 6A). Unexpectedly, we found that treatment of iPLA2-expressing cells with A23187 induced marked AA release (Fig. 6B, top). Furthermore, coexpression studies demonstrated that the AA released by iPLA2 was efficiently metabolized to PGE2 by COX-1 in marked preference to COX-2 (Fig. 6B, bottom). These results suggest that although iPLA2 is a Ca2+-independent enzyme when assayed in vitro, it is subject to regulation by Ca2+ in vivo, probably indirectly, thereby acting as a sort of signaling PLA2. In marked contrast, iPLA2-mediated AA release failed to link with COX-1 for delayed PGE2 biosynthesis (Fig. 6C). Moreover, delayed PGE2 generation by cells coexpressing iPLA2 and COX-2 was almost equal to that by cells expressing COX-2 alone (Fig. 6C), providing further support for our previous observation (32) that iPLA2 plays a minimal role in the delayed phase of COX-2-dependent PGE2 biosynthesis.


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Fig. 6.   Coexpression of iPLA2 and COX-1 or COX-2. A, expression of COX-1 and COX-2 in 293 cell transfectants, assessed by immunoblotting (top), and that of iPLA2, assessed by RNA blotting (bottom). B, AA release (top) and PGE2 generation (bottom) by iPLA2, COX-1, and COX-2 single transfectants and cotransfectants after stimulation for 30 min with 10 µM A23187. C, AA release (top) and PGE2 generation (bottom) by the iPLA2, COX-1, and COX-2 single transfectants and cotransfectants after stimulation for 4 h with 1 ng/ml IL-1beta plus 10% FCS. Means ± S.E. of five to eight independent experiments are shown in B and C, and representative results of three independent experiments are shown in A.

Transcellular PGE2 Generation by sPLA2----- The fact that sPLA2s represent the only group of PLA2 enzymes that are able to gain access to other cells in microenvironments prompted us to ask whether sPLA2s promote PG biosynthesis by neighboring cells, namely the transcellular PG-biosynthetic response. To explore this, 293 cells expressing each PLA2 and those expressing COX-2 were cocultured, and the PGE2 produced by the COX-2-expressing cells after stimulation with IL-1/FCS for 4 h was quantified. Whereas PGE2 generation increased only in an additive manner when COX-2-expressing cells and cPLA2-expressing (Fig. 7A) or iPLA2-expressing (Fig. 7B) cells were cocultured, it increased synergistically when COX-2-expressing cells and sPLA2-V-expressing (Fig. 7A) or sPLA2-IIA-expressing (Fig. 7B) cells were cocultured, even though the amounts of AA released by the individual PLA2s were comparable in each set of experiments. The failure of cPLA2 and iPLA2 to participate in transcellular PGE2 generation in this setting would appear to eliminate the possibility that AA released from PLA2-expressing cells was incorporated into COX-2-expressing cells to be converted to PGE2. Neither the sPLA2-IIA mutant G30S nor KE4 increased PGE2 generation by COX-2-expressing cells, even though the expression levels of the mutants and the native enzyme were similar (32), indicating that both catalytic activity and cell surface binding ability are essential for promotion of the transcellular PG-biosynthetic pathway by sPLA2-IIA (Fig. 7C).


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Fig. 7.   Transcellular PG biosynthesis by sPLA2s. COX-2-expressing 293 cells and 293 cells expressing cPLA2, sPLA2-V (A), sPLA2-IIA, and iPLA2 (B) were cocultured for 3 days and then stimulated with IL-1/FCS for 4 h to assess AA release and PGE2 generation. C, cells expressing COX-2 and those expressing native or mutated sPLA2-IIA in coculture were stimulated with IL-1/FCS. Representative results of four independent experiments are shown. D, schematic model of transcellular PG biosynthesis following IL-1 stimulation. cPLA2 couples only with COX-2 expressed in the same cell, whereas COX-2 may activate cPLA2 through an unknown mechanism. sPLA2-IIA and -V not only couple with COX-2 expressed in the same cell in a autocrine manner, but also act on another COX-2-expressing cell in a paracrine manner. Similarly, sPLA2/COX-1-dependent transcellular PGE2 generation occurs when cells are stimulated with A23187 (see text).

We also performed similar coculture experiments using cells expressing each PLA2 and COX-1. After stimulation with A23187 for 30 min, PGE2 generation by COX-1-expressing cells was potentiated more efficiently by sPLA2s than by cPLA2. For instance, in a representative assay, the amount of PGE2 generated by cocultured COX-1- and cPLA2-expressing cells reached no more than 2 ng/well, whereas that produced by COX-1- and sPLA2-V-expressing cells in coculture reached 7 ng/well.

    DISCUSSION

Although PLA2/COX coupling during the two phases of the PG biosynthetic pathway in various cell types has been studied by several investigators, including us, the rapidly increasing number of PLA2 isozymes, the limitations of the technologies used to distinguish them, and the occurrence of cell-specific events has led to rather confusing results (4-11). Therefore, we recently carried out reconstitution analysis of five distinct PLA2s (cPLA2, sPLA2s-IIA, -V, -IIC, and iPLA2) in 293 and CHO cells in an attempt to define their roles in stimulus-induced and constitutive AA release (32). In the present study, we introduced the two COX isoforms, COX-1 and COX-2, into this system, in order to obtain information about general aspects of the functional coupling and segregation of particular PLA2 isozymes and COXs. Our results provided evidence that the signaling PLA2s, namely cPLA2, sPLA2-IIA and sPLA2-V, can each link functionally with both COX-1 and COX-2 and predominantly with COX-2 in the immediate and delayed PG-biosynthetic responses, respectively, whereas iPLA2, a phospholipid remodeling PLA2 that dissociates from COX-2-dependent delayed PG generation, can promote Ca2+-dependent immediate AA release, which is preferentially linked with COX-1 (Fig. 8). Moreover, coculture experiments revealed a distinct role of sPLA2 as a regulator of transcellular PG biosynthesis, a role not played by the intracellular PLA2 enzymes (Fig. 7D).


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Fig. 8.   Diagram of PLA2/COX coupling. cPLA2, sPLA2-IIA, and sPLA2-V release AA rather specifically, and the AA is, in turn, metabolized to PGE2 via COX-1 and COX-2 during the immediate responses induced by A23187 and BK and via COX-2, but not COX-1, during the delayed response induced by IL-1 plus FCS. iPLA2 releases both AA and oleic acid (OA) in the presence of FCS and plays a role in phospholipid remodeling. Furthermore, iPLA2-derived AA is utilized for the immediate PG biosynthesis via COX-1. The function of sPLA2-IIC is unknown.

Several studies have shown that utilization of the constitutive isozyme COX-1 is often, but not always, associated with immediate PG biosynthesis (4-6, 10, 11, 14), and that the induction of COX-2 expression is accompanied by a concomitant increase in PG generation throughout the culture period (4-6, 9-11, 15) and by the priming for enhancement of the immediate response (8, 11, 42), even though both isozymes exhibited some compensatory functions under certain conditions (43). Nevertheless, the closer association of COX-2 than COX-1 with inflammation, development, and tumorigenesis, which has been recently verified by studies on mice subjected to targeted disruption of either COX gene (14, 15, 44, 45), appears to agree with the proposal that COX-2 plays a prominent role in the prolonged generation of PGs, which would modify the nuclear events associated with cell differentiation and replication.

Analyses of COX transfectants have provided some clues about the mechanisms responsible for the functional differences between the two isoforms. Our results confirmed that utilization of COX-1 is restricted to the immediate response, whereas COX-2 is quite active in both the immediate and delayed responses, when AA is supplied endogenously. Comparison of the conversion of exogenous AA to PGE2 by COX-1 and COX-2 transformants revealed that low concentrations of AA are utilized predominantly by COX-2, whereas high concentrations of AA are utilized preferentially by COX-1. The latter finding is reminiscent of that of Reddy and Herschman (12), who were the first to demonstrate that COX-1 and COX-2 utilize exogenous and endogenous AA, respectively. While our study was underway, Shitashige et al. (13) reported a similar result that only COX-2 was functional at low AA concentrations and further showed that intracellular peroxides may contribute to COX-2 activation under conditions of low AA concentrations. Our results also appear to be compatible with the recent subtle kinetic studies on purified enzymes in that COX-2 has a lower threshold for hydroperoxide activation than COX-1, thereby enabling COX-2 to oxygenate AA in the presence of low peroxide concentrations (46, 47), and that negative allosteric regulation of COX-1 by low AA concentrations has the overall effect of increasing the rate of COX-2-mediated PG formation severalfold (48).

The different sensitivities of the two COX isozymes to high and low concentrations of AA could account, at least in part, for their segregated utilization during immediate and delayed PG biosynthesis from endogenous AA. It seems reasonable to speculate that during the immediate response, when a burst of AA is released in a short time, the local concentration of AA reaches a level high enough to activate both COX-1 and COX-2, whereas limited amounts of AA may be supplied gradually during the delayed phase, under which conditions only COX-2 remains active. This hypothesis is further supported by the finding that COX-1-dependent immediate PGE2 generation by 293 cells transfected with COX-1 alone was only modest, but it was enhanced considerably when COX-1 and the signaling PLA2s, which markedly increased the free AA levels, were coexpressed. We presume that the failure of several earlier studies to demonstrate the COX-1-dependent immediate response in several cell types, irrespective of the presence of COX-1 (12, 13, 42), was due to limitation of the supply of AA by PLA2 under the experimental conditions used. Mouse mast cells (4) and rat 3Y1 fibroblasts (10) utilize COX-1 predominantly in the immediate PG-biosynthetic response even when both COX isozymes are copresent, probably because cPLA2, which is abundantly expressed in these cells, may liberate in an intracellular local area rather high amounts of AA that reach a level enough to elicit preferred COX-1-dependent PG biosynthesis. Although the functional difference between COX-1 and COX-2 was suggested to be due to their different subcellular locations (49), recent immunohistochemical studies by Smith and co-workers (16) clearly demonstrated that any specific connection between COX-2 and the generation of products that might function in the nucleus would result from differences in the expression of activities of COX-1 and COX-2, not from gross differences in their subcellular distributions. Our preliminary immunocytostaining study also showed that the subcellular distributions of COX-1 and COX-2 in 293 transfectants are almost identical.2

We observed that transfection of cells with COX-2 alone induced spontaneous PGE2 generation, with a concomitant increase in AA release. This AA release was suppressed by the cPLA2 inhibitor MAFP, but not by the other PLA2 or COX inhibitors tested, and cPLA2 is the only PLA2 isozyme detectable in the parental 293 cells examined so far. Moreover, coexpression of COX-2 and cPLA2, but no other PLA2s, led to a marked increase in AA release during both the immediate and delayed phases. These observations led us to formulate the hypothesis that COX-2 has the ability to activate cPLA2 through an as yet unknown, COX activity-independent, positive feedback mechanism. This may provide new insight into the mechanism responsible for cPLA2 activation in the delayed response, which is not accompanied by cytoplasmic Ca2+ signaling. A direct physical interaction between cPLA2 and COX-2 is unlikely, because cPLA2 is located in the cytosol (18), whereas COXs face the luminal sides of the perinuclear and endoplasmic reticular membranes (50). There may be an accessory protein that affects the functional cPLA2/COX-2 interaction. However, the possibility that MAFP inhibits an unidentified PLA2 isozyme, which is activated by and functionally linked with COX-2, cannot be ruled out at present.

In our previous study, we classified cPLA2, sPLA2-IIA, and sPLA2-V as signaling PLA2s, the AA-releasing effects of which were dependent upon the cellular activation state, and each of them, if properly expressed, could mediate both immediate and delayed AA release (32). We found here that AA released by these three signaling PLA2s was metabolized to PGE2 through COX-1 and COX-2 in the immediate response and COX-2 in the delayed response. Segregated utilization of cPLA2 and sPLA2 during these two PG-biosynthetic phases, which was observed in some previous studies (5, 6, 10, 11), may, therefore, depend on the expression levels or subcellular localizations of these PLA2s, which probably differ in different cell types, at the moment PG generation takes place.

A remarkable difference between cPLA2 and sPLA2 is the ability of the latter, but not the former, to mediate transcellular PG biosynthesis. In contrast to cPLA2, which contributes to PG biosynthesis only when COX coexists in the same cell, sPLA2-IIA and sPLA2-V are secreted extracellularly, bind to the cell surfaces of neighboring COX-expressing cells, and then contribute to remote stimulus-dependent PG biosynthesis. This property reflects a distinct role of the secretory group of PLA2s as an amplifier of a signal from a single cell to its surrounding microenviromnent. This paracrine route of sPLA2 action may be compatible with the role of sPLA2 seen in mast cell-fibroblast interactions (51, 52) and may also apply to other cell systems. Given that abundant sPLA2-IIA (and probably sPLA2-V in the mouse, in which sPLA2-V is distributed in a wide variety of tissues, whereas sPLA2-IIA expression is restricted to the intestine)3 accumulates in inflammatory exudates and exacerbates the process of inflammation (53), COX-2-expressing cells would appear to be the major targets of sPLA2 responsible for the propagation of sustained PG biosynthesis at inflamed sites.

The concentrations of sPLA2-IIA produced by 293 transfectants, estimated by the enzyme activities and the intensities of the immunoblot bands, reached as high as 10 to ~50 ng/ml, comparable to those produced by cytokine-stimulated cells, such as fibroblasts (10), macrophages (11) and liver cells (54), and sufficient to promote PG generation in these cells. In contrast, a single addition of purified or recombinant sPLA2-IIA to target cells generally requires concentrations of the order of µg/ml to exert its actions (9, 24, 25, 52, 55-60), as we also observed with our 293 transfectant system.2 The striking difference between the amounts of sPLA2-IIA required by these different systems, i.e. endogenously produced (ng/ml) versus exogenously added (µg/ml), implies that the continued supply of sPLA2-IIA, which occurs in the former situation, may be an important factor for its adequate actions during cellular (particularly prolonged) responses. As sPLA2-IIA is believed to be produced continuously at inflamed sites, it is likely that the former case is most relevant to pathophysiologic conditions in vivo. Moreover, the higher sensitivity of the cells to endogenously produced than exogenously added sPLA2-IIA predicts the presence of particular machinery, through which newly produced sPLA2-IIA is readily transported into the compartments where AA-rich phospholipid pools are present. Therefore, caution should be exercised in interpreting the results of studies involving single addition of excess sPLA2-IIA, which may not always reflect in vivo circumstances.

We previously observed that iPLA2 overexpression did not induce endogenous COX-2-dependent delayed PGE2 generation (32). We have now confirmed this observation by demonstrating that iPLA2 failed to mediate the delayed response even when COX-2 was overexpressed. To our surprise, A23187 elicited marked iPLA2-induced AA release, and this AA was metabolized to PGE2 via COX-1 in marked preference to COX-2. Gross and co-workers (28, 29) showed that iPLA2-dependent AA release was activated by A23187 in smooth muscle cells and suggested that calmodulin might be involved in this regulatory step, and our results of AA release appear to be in line with their observations. However, the apparently preferred coupling between iPLA2 and COX-1 in the immediate response, despite the fact that the amount of AA released induced by iPLA2 was comparable to that released by other PLA2s, cannot be simply explained by any of the theories we have proposed. We suppose that iPLA2 releases AA in closer proximity to COX-1 than COX-2 and/or iPLA2-derived AA is virtually inaccessible to COX-2. Because iPLA2 is known to form a multimeric complex (61) and even exists as multiple splice variants that affect enzyme activity (62) in cells, it may bind to a certain cofactor that facilitates iPLA2/COX-1 interaction following Ca2+ signaling. Nonetheless, these results reveal an unexplored bifunctional mode of iPLA2 actions in the regulation of phospholipid remodeling and signaling pathways.

Finally, although the approaches used have yielded valuable information, we have to be careful of the limitation of this study that the cell model may still not fully mimic other cells that produce COX products. Important components may be missing from the cell machinery that could alter the interactions under study. For instance, specific fatty acid-binding proteins might be expressed and play a role, undefined receptors might be involved, specific protein kinase cascades could vary, and subcellular disposition of AA stores might differ, according to cell types.

    ACKNOWLEDGEMENTS

We thank Drs. J. A. Tischfield, S. S. Jones, J. Trzaskos, W. L. Smith, and R. M. Kramer for providing cDNAs, antibodies, and inhibitors. We thank Drs. H. Naraba, Y. Kosugi, A. Ueno, and S. Oh-ishi for the cloning of rat BKR B2 cDNA.

    FOOTNOTES

* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan and Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency.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.: 81-3-3784-8196; Fax: 81-3-3784-8245; E-mail: kudo{at}pharm.showa-u.ac.jp.

The abbreviations used are: PLA2, phospholipase A2; sPLA2, secretory PLA2; cPLA2, cytosolic PLA2; iPLA2, Ca2+-independent PLA2; AA, arachidonic acid; PG, prostaglandin; COX, cyclooxygenase; IL-1, interleukin-1; FCS, fetal calf serum; MAFP, methyl arachidonylfluorophosphate; CHO, Chinese hamster ovary; BK, bradykinin; BKR, bradykinin receptor.

2 T. Kambe, M. Murakami, S. Yamamoto, H. Kuwata, R. Takamiya, Y. Wakabayashi, M. Suematsu, and I. Kudo, unpublished observations.

3 H. Sawada, M. Murakami, and I. Kudo, unpublished observations.

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