From the Department of Health Chemistry, School of
Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai,
Shinagawa-ku, Tokyo 142, Japan and the § Department of
Medical and Molecular Genetics, Indiana University School of Medicine,
Indianapolis, Indiana 46202-5251
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ABSTRACT |
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We examined the relative contributions of five distinct mammalian phospholipase A2 (PLA2) enzymes (cytosolic PLA2 (cPLA2; type IV), secretory PLA2s (sPLA2s; types IIA, V, and IIC), and Ca2+-independent PLA2 (iPLA2; type VI)) to arachidonic acid (AA) metabolism by overexpressing them in human embryonic kidney 293 fibroblasts and Chinese hamster ovary cells. Analyses using these transfectants revealed that cPLA2 was a prerequisite for both the calcium ionophore-stimulated immediate and the interleukin (IL)-1- and serum-induced delayed phases of AA release. Type IIA sPLA2 (sPLA2-IIA) mediated delayed AA release and, when expressed in larger amounts, also participated in immediate AA release. sPLA2-V, but not sPLA2-IIC, behaved in a manner similar to sPLA2-IIA. Both sPLA2s-IIA and -V, but not sPLA2-IIC, were heparin-binding PLA2s that exhibited significant affinity for cell-surface proteoglycans, and site-directed mutations in residues responsible for their membrane association or catalytic activity markedly reduced their ability to release AA from activated cells. Pharmacological studies using selective inhibitors as well as co-expression experiments supported the proposal that cPLA2 is crucial for these sPLA2s to act properly. The AA-releasing effects of these sPLA2s were independent of the expression of the M-type sPLA2 receptor. Both cPLA2, sPLA2s-IIA, and -V were able to supply AA to downstream cyclooxygenase-2 for IL-1-induced prostaglandin E2 biosynthesis. iPLA2 increased the spontaneous release of fatty acids, and this was further augmented by serum but not by IL-1. Finally, iPLA2-derived AA was not metabolized to prostaglandin E2. These observations provide evidence for the functional cross-talk or segregation of distinct PLA2s in mammalian cells in regulating AA metabolism and phospholipid turnover.
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INTRODUCTION |
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Phospholipase A2 (PLA2)1 represents a growing family of enzymes that catalyze the hydrolysis of glycerophospholipids at the sn-2 position, liberating free fatty acids including arachidonic acid (AA), a precursor of bioactive eicosanoids, and lysophospholipids. PLA2 has been implicated in diverse cellular responses, such as signal transduction, host defense, blood coagulation, digestion, and membrane remodeling. According to an updated classification, PLA2s can be subdivided into several groups based upon their structures and enzymatic characteristics (1).
Secretory PLA2s (sPLA2s) are low molecular mass (~14 kDa) enzymes with a rigid tertiary structure configured by 6-8 disulfide bridges. They require a millimolar concentration of Ca2+ to exert their enzymatic action and have little fatty acid selectivity when assayed in vitro (2). In mammals, five sPLA2 enzymes have been identified so far. Type I sPLA2 (sPLA2-I) is abundantly expressed in the pancreas, where it functions as a digestive enzyme for dietary phospholipids, and is present in relatively small amounts in several non-digestive organs, where it may act as a regulator of cellular functions via the M-type sPLA2 receptor (3-5). Type IIA sPLA2 (sPLA2-IIA; often referred to as sPLA2 in the literature) was originally isolated from inflammatory fluids and cells (6, 7). Later, this isozyme was shown to be induced by proinflammatory stimuli in many if not all cells (8-10) and is therefore thought to play a role in inflammatory responses (11). Some mouse strains genetically lacking sPLA2-IIA are more susceptible to colon cancer, implying an anti-tumorigenic property (12). Type V sPLA2 (sPLA2-V) is expressed in the heart, and to a lesser extent in several other tissues (13), and is the primary sPLA2 expressed in several murine inflammatory cells (14, 15). Type IIC sPLA2 (sPLA2-IIC) is expressed in rodent testes but is a non-functional pseudogene in humans (16). Type X sPLA2 (sPLA2-X) is the most recently discovered sPLA2 isozyme that exhibits some features characteristic of both sPLA2s-I and -IIA, and is expressed mainly in immune tissues (17). A phylogenetic tree derived from alignment of these sPLA2s reveals that three related isozymes (sPLA2s-IIA, -V, and -IIC), the genes for which are tightly linked to human chromosome 1, would have emerged from recent gene duplication events, whereas sPLA2s-I and -X, the genes for which map to human chromosomes 12 and 16, respectively, are more distant offshoots (2). Despite many efforts to prove the involvement of sPLA2 isozymes, especially sPLA2-IIA, in AA metabolism, confirmation has not been possible until recently because of conflicting results from different experimental systems. Nevertheless, our recent studies have provided convincing evidence that sPLA2-IIA can promote AA metabolism under certain conditions, especially when "membrane rearrangement" is induced by particular stimuli such as the proinflammatory cytokines, interleukin (IL)-1, and tumor necrosis factor (18-21). In addition, sPLA2-V has recently been shown to act as an effector of AA metabolism in mouse macrophage-like P388D1 cells (14) and mast cells (15). However, it remains unclear how these sPLA2s exert their actions on cellular AA metabolism, whether they are functionally redundant or discriminative, and which prostanoid-biosynthetic responses (immediate or delayed) they preferentially affected.
Type IV cytosolic PLA2 (cPLA2) is a ubiquitously distributed 85-kDa enzyme, the activation of which has been shown to be tightly regulated by postreceptor transmembrane signaling (22). In vitro, cPLA2 requires a submicromolar Ca2+ concentration for effective hydrolysis of its substrate, AA-containing glycerophospholipids (23, 24). The N-terminal CALB domain is responsible for Ca2+-dependent translocation of cPLA2 from the cytosol to perinuclear and endoplasmic reticular membranes (25, 26), where several eicosanoid-generating enzymes, such as the two cyclooxygenase (COX) isozymes and 5-lipoxygenase, are co-localized (27, 28). cPLA2 has multiple phosphorylation sites, among which the mitogen-activated protein kinase-directed site (Ser505) is the most crucial for in vivo activation of cPLA2 (29). These enzymatic features of cPLA2 are consistent with its role in immediate eicosanoid biosynthesis occurring within minutes of stimulation, which is usually accompanied by rapid and transient cytoplasmic Ca2+ mobilization and mitogen-activated protein kinase activation. The AA thus liberated is supplied to constitutive COX-1 and 5-lipoxygenase to be converted into prostanoids and leukotrienes, respectively (30-33). cPLA2 has also been implicated in delayed, COX-2-dependent prostanoid generation lasting for hours despite the absence of Ca2+ signaling in this setting (15, 19, 34, 35), although in some experimental systems cPLA2 failed to supply AA to COX-2 during the delayed response unless the cells were exposed to a secondary Ca2+-mobilizing stimulus (36, 37). Increased expression of cPLA2 induced by proinflammatory stimuli has been reported to be linked to an ongoing delayed response (19, 34, 35, 38) or to priming for increased immediate response (21, 36, 37, 39, 40).
Another distinctive group of PLA2s is Ca2+-independent PLA2 (iPLA2). To date, three major classes of iPLA2 have been cloned, namely 26-kDa lysosomal PLA2 (41), intracellular types I (42, 43) and II (44), and extracellular (45) platelet-activating factor acetylhydrolases, which exhibit restricted substrate specificities toward platelet-activating factor and oxidized phospholipids, and type VI cytosolic iPLA2 (46). The latter is an 85-kDa protein that exists as a multimeric complex of ~300 kDa, exhibits no fatty acid selectivity, and contains eight ankyrin motifs (46). Ongoing studies by Dennis and co-workers (47, 48) have so far provided support for the notion that it is involved in phospholipid remodeling. On the other hand, conflicting results have demonstrated that type VI iPLA2 mediates stimulus-initiated AA release (49, 50).
In general, mammalian cells contain more than one PLA2, making understanding of the regulation of AA metabolism by individual PLA2 isozymes more complicated. Studies using chemical inhibitors, antibodies, or even antisense oligonucleotides are limited in their ability to identify the particular PLA2 isozyme involved in certain biological events, since these agents often cannot gain access to certain cellular compartments and they could also cause cross-inhibition or undesirable side effects, thereby leading to misinterpretations. To reconcile the proposed functions of PLA2 enzymes, we have taken advantage of transfection analyses of each PLA2 cDNA, alone or in combination, into mammalian cell lines so as to assess gain-of-function. This approach has enabled us to compare overlaps or differences in the functions of five distinct PLA2s, including cPLA2, sPLA2s-IIA, -V, -IIC, and iPLA2. Our results have confirmed that cPLA2, sPLA2-IIA, and sPLA2-V are involved in "signaling," whereas iPLA2 plays a role in "membrane remodeling," but not vice versa. sPLA2s-IIA and -V, which have the capacity to bind to cell-surface proteoglycans, are functionally redundant, and their action requires the activation of cPLA2. Moreover, cPLA2, sPLA2s-IIA, and -V each are efficiently coupled with COX-2-dependent delayed PGE2 generation, whereas iPLA2 is not linked with it.
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EXPERIMENTAL PROCEDURES |
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Materials--
Mouse cPLA2 cDNA was provided by
Dr. M. Tsujimoto (RIKEN Institute); human sPLA2-IIA
cDNA by Dr. H. Ishimaru (Asahi Chemical Industry); human
sPLA2-V cDNA by Dr. T. Kamimura (Teijin Co. Ltd.); hamster iPLA2 cDNA by Dr. S. S. Jones (Genetics
Institute); mouse -actin cDNA by Dr. J. P. Arm (Harvard
Medical School); mouse M-type sPLA2 receptor cDNA by
Drs. K. Hanasaki and H. Arita (Shionogi Pharmaceutical); and the COX-2
inhibitor NS-398 (51) by Dr. J. Trzaskos (Merck DuPont). Human COX-1
and COX-2 cDNAs, the cPLA2 inhibitor methyl
arachidonylfluorophosphate (MAFP) (52), and the PGE2 enzyme
immunoassay kit were purchased from Cayman Chemical. Mouse
sPLA2-IIA cDNA (18), rabbit polyclonal antibody against rat sPLA2-IIA (53), rat sPLA2-V cDNA (54),
and rat sPLA2-IIC cDNA (16) were prepared as described
previously. The sPLA2-IIA inhibitor LY311727 (55) was
donated by Dr. R. M. Kramer (Lilly). Human and mouse IL-1
s were
purchased from Genzyme. A23187 and the cPLA2 inhibitor
arachidonoyl trifluoromethyl ketone (AACOCF3) (32) were
purchased from Calbiochem. CellFectin reagent, Opti-MEM medium, and
TRIzol reagent were obtained from Life Technologies, Inc. Porcine
sPLA2-I was purchased from Boehringer Mannheim.
Transfection of Mammalian Cells with cDNAs for Each PLA2-- Mouse cPLA2 and human sPLA2-IIA cDNAs were subcloned into the mammalian expression vector pBK-CMV (Stratagene), mouse sPLA2-IIA cDNA into pCDNA3.1 (Invitrogen), rat sPLA2-IIC and hamster iPLA2 cDNAs into pCR3.1 (Invitrogen), and rat sPLA2-V into pCEP4 (13) or pCDNA3.1. Each cDNA was transfected into human embryonic kidney 293 cells and Chinese hamster ovary (CHO)-K1 cells (both obtained from Riken Cell Bank) using CellFectin reagent according to the manufacturer's instructions. Briefly, 1 µg of plasmid was mixed with 5 µl of CellFectin in 200 µl of Opti-MEM medium for 15 min and then added to cells that had attained 40-60% confluency in 6-well plates (Iwaki) in 1 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 2 ml of fresh culture medium comprising RPMI 1640 (Nissui Pharmaceutical) containing 10% 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 analyses, the cells were harvested 3 days after transfection and were 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, Inc.), except for rat sPLA2-V/pCEP4-transfected 293 cells, which were selected in the presence of 50 µg/ml hygromycin (Sigma). After culture for 2-4 weeks, wells containing a single colony were chosen, and the expression of each PLA2 was assessed by RNA blotting and, in the case of sPLA2s, by measuring sPLA2 activity released into the supernatants. The established clones were expanded and used for the experiments as described below.
For co-transfection experiments, a 293 transformant expressing cPLA2 was subjected to a second transfection with sPLA2-IIA or iPLA2 cDNA, which had been subcloned into pCDNA3.1/Zeo (+) (Invitrogen) using LipofectAMINE reagent (Life Technologies, Inc.). 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 PLA2s.RNA Blotting--
Approximately equal amounts (~10 µg) of
total RNA 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 PLA2, COX-1, COX-2, or -actin
cDNA probes that had been labeled with [32P]dCTP
(Amersham Pharmacia Biotech) by random priming (Takara Shuzo). All
hybridizations were carried out at 42 °C for 16 h in a solution
comprising 50% (v/v) formamide, 0.75 M NaCl, 75 mM sodium citrate, 0.1% (w/v) SDS, 1 mM EDTA,
10 mM sodium phosphate, pH 6.8, 5× Denhardt's solution
(Sigma), 10% (w/v) dextran sulfate (Sigma), and 100 µg/ml salmon
sperm DNA (Sigma). The membranes were washed three times at room
temperature with 150 mM NaCl, 15 mM sodium
citrate, 1 mM EDTA, 0.1% SDS, and 10 mM sodium
phosphate, pH 6.8, for 5 min each time, followed by two washes at
55 °C with 30 mM NaCl, 3 mM sodium citrate,
1 mM EDTA, 0.1% SDS, and 10 mM sodium
phosphate, pH 6.8, for 15 min each time. The blots were visualized by
autoradiography using Kodak X-OMAT AR films and double intensifying
screens at
80 °C
SDS-PAGE/Immunoblotting-- Cell lysates were applied to SDS-polyacrylamide gels (10% for cPLA2 and 15% for sPLA2-IIA) and electrophoresed under reducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schuell) using a semi-dry blotter (MilliBlot-SDE system; Millipore), according to the manufacturer's instructions. The membranes were probed with antibodies against the respective PLA2s and visualized using the ECL Western blot analysis system (Amersham Pharmacia Biotech) as described previously (18).
Measurement of the sPLA2 Activity Expressed by Cells-- Cells in 1 ml of culture medium were seeded into each well of 24-well plates at a density of 5 × 104 cells/ml. 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 various concentrations of NaCl or 1 mg/ml heparin (Sigma). This allowed cell surface-associated sPLA2-IIA to be recovered quantitatively in the medium without causing significant cell death, as described previously (18). The PLA2 activity was assayed by measuring the amounts of free fatty acid released from the substrates 1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphoethanolamine (NEN Life Science Products) or 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoethanolamine (Amersham Pharmacia Biotech). Each reaction mixture consisted of an aliquot of the required sample, 100 mM Tris-HCl, pH 7-9, 4 mM CaCl2, and 2 µM substrate. After incubation for 10-30 min at 37 °C, the 14C-fatty acids released were extracted by Dole's method (56), and the radioactivity was counted.
Heparin-Sepharose Affinity Chromatography-- Approximately 25 ml of culture supernatants from 293 cell stable transformants with sPLA2s-IIA, -V, or -IIC were applied to a heparin-Sepharose (Amersham Pharmacia Biotech) column (1 × 5 cm) pre-equilibrated with 10 mM Tris-HCl, pH 7.4, containing 150 mM NaCl at a flow rate of 20 ml/h. After extensive washing, the bound proteins were eluted using 10 mM Tris-HCl, pH 7.4, with a 0.15-1 M NaCl gradient. The PLA2 activities of each fraction were measured as described above.
Cell Activation--
Transfectants stably expressing each
PLA2 (5 × 104 cells in 1 ml of culture
medium) were seeded into 24-well plates. In order to assess AA release,
0.1 mCi/ml [3H]AA (Amersham Pharmacia Biotech) was added
to the cells 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 with or without 10 µM
A23187, 1 ng/ml IL-1, and/or 10% FCS was added to each well, and
the amount of free [3H]AA released into the supernatant
during culture (up to 30 min with A23187 and up to 4 h with
IL-1
) was measured. To assess oleic acid (OA) release, 0.5 µCi/ml
[3H]OA (NEN Life Science Products) was added to the cells
instead of [3H]AA. The percentage release of AA or OA 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 the addition of radiolabeled fatty acids were taken for
PGE2 enzyme immunoassay.
Site-directed Mutagenesis-- Site-specific mutations were introduced by mismatched primer polymerase chain reaction (PCR) with ex Taq polymerase (Takara Shuzo) as described previously (18), using mouse sPLA2-IIA or rat sPLA2-V cDNAs as a template. Primers I and II corresponded to the N-terminal sense and the C-terminal antisense oligonucleotides of each sPLA2 cDNA. Primer I sequences were 5'-ATG AAG GTC CTC CTC CTG C-3' and 5'-ATG AAG CGC CTC CTC ACG CTG-3', and primer II sequences were 5'-TCA GCA TTT GGG CTT CTT CCC TTT GC-3' and 5'-ATT AGC AGA GGA AGT TGG GGT A-3', for sPLA2s-IIA and -V, respectively. Two primary PCR fragments were produced using primer I plus the invert mutagenic primer and primer II plus the forward mutagenic primer for 20 cycles at 94, 55, and 72 °C for 30 s each. The two primary PCR fragments were mixed, then denatured at 94 °C for 5 min, annealed at 37 °C for 30 min and then 55 °C for 2 min, and extended at 72 °C for 4 min during each cycle. Secondary PCR products with specific mutations were obtained after 20 additional PCR cycles with primers I and II. The sequences of the forward mutated primers were 5'-TGC CAC TGT AGC CTG GGT GG-3' (G30S; sPLA2-IIA), 5'-TGT GTG ACT CAG GAC TGT TGT-3' (H48E; sPLA2-IIA), 5'-GCT TGT GAC GGG GAG CTG GTC-3' (R94G/K95E; sPLA2-V), and 5'-ACT GCC TGA GCA GCA ACC TCT GG-3' (R101S/R102S; sPLA2-V), in which corresponding residues were replaced at the underlined sites. Another sPLA2-IIA mutant, KE4, has been described previously (18).
Each PCR product was ligated into the pCR3.1 and was transfected into Top10F' supercompetent cells (Invitrogen). The plasmids were isolated and sequenced using a Taq cycle sequencing kit (Takara Shuzo) and an autofluorometric DNA sequencer DSQ-1000L (Shimadzu) to confirm the mutations. Plasmids containing each of the mutated cDNAs were then transfected into 293 cells as described above. ![]() |
RESULTS |
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A23187-induced Immediate AA Release by cPLA2 and sPLA2 Transformants-- cDNAs encoding mouse cPLA2, human and mouse sPLA2-IIAs, rat sPLA2-V, and rat sPLA2-IIC were each subcloned into mammalian expression vectors and transfected into 293 cells. After appropriate selections, several drug-resistant transformants stably expressing substantial levels of each PLA2 were isolated. Expression of cPLA2 and the three sPLA2s was barely detectable in parental 293 cells but was readily detected in the respective transformants (Fig. 1A, top panels). To assess immediate AA release, we stimulated these established PLA2 transformants with the calcium ionophore A23187. AA release reached a peak within 30 min after stimulation (data not shown). Whereas treatment of the control 293 cells with 10 µM A23187 for 30 min resulted in minimal AA release, all of the established cPLA2 transformants released severalfold more AA than the control cells (Fig. 1A). Of the established sPLA2-IIA transformants, three clones expressing relatively high sPLA2-IIA levels released more AA than the control cells in response to A23187, whereas AA release by the two clones with lower sPLA2-IIA expression did not differ significantly from that of the control cells (Fig. 1, A and B). A23187 stimulation of the sPLA2-V transformants also resulted in increased release of AA relative to the control cells, except in one clone, which expressed a lower level of sPLA2-V and did not show increased AA release (Fig. 1, A and B). In marked contrast, none of the sPLA2-IIC transformants exhibited an A23187-induced increase in AA release (Fig. 1A), despite the fact that sPLA2 activities secreted from the sPLA2-IIC transformants were higher than those of the sPLA2-V transformants (Fig. 1B).
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IL-1- and FCS-induced Delayed AA Release by cPLA2 and
sPLA2 Transformants--
When 293 cells were stimulated
with 1 ng/ml IL-1 in the presence of 10% FCS, delayed AA release,
extending over 1-4 h to reach a plateau by 10 h, occurred.
Delayed AA release was significantly higher in all transformants
expressing cPLA2, sPLA2-IIA, or
sPLA2-V than in the control cells (Fig.
2A). Modest enhancement was
consistently observed even in the clones expressing lower levels of
sPLA2s-IIA and -V (Fig. 2A) which failed to
augment A23187-induced immediate AA release (Fig. 1A). CHO
cells transfected with cPLA2, sPLA2-IIA, or
sPLA2-V also exhibited increased
cytokine-dependent delayed AA release, although there was
substantial background AA release in the control CHO cells, probably
due to constitutively high expression of endogenous cPLA2
(Fig. 2B). On the other hand, none of the transformants
expressing sPLA2-IIC released more AA than the control
cells during the delayed response (Fig. 2, A and
B). These observations confirm that cPLA2,
sPLA2-IIA, and sPLA2-V, but not
sPLA2-IIC, are capable of promoting both the
ionophore-induced immediate and the cytokine-initiated delayed phases
of AA release when appropriately expressed. It should be noted that the
absolute PLA2 activity of sPLA2-V is only about
a tenth of that of sPLA2-IIA (Fig. 1B), yet AA
release by activated cells expressing them was almost comparable (Figs.
1A and 2A). Thus, higher AA
release/PLA2 activity ratio of sPLA2-V than
that of sPLA2-IIA suggests that sPLA2-V may be
more effective at AA release than sPLA2-IIA.
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sPLA2-IIA and sPLA2-V Are Cell Surface-binding PLA2s-- To assess the molecular basis of the functional similarities and/or differences in the abilities of the three sPLA2s to regulate AA release by transfected cells, we compared their enzymatic characteristics using culture supernatants for each transformant. The results are summarized in Table I. Under our PLA2 assay conditions, sPLA2s-IIA, -IIC, and -V all exhibited maximal enzymatic activities in the presence of millimolar Ca2+ concentrations at neutral to alkaline pH values and hydrolyzed phosphatidylethanolamine several times as efficiently as phosphatidylcholine with no apparent AA selectivity (data not shown). These close similarities suggest that the failure of sPLA2-IIC to mediate AA release was not due to abnormal enzymatic properties. The sPLA2 inhibitor thielocin A1 and an antibody raised against rat sPLA2-IIA, which we had used in earlier studies (20, 21, 57), were quite specific for sPLA2-IIA (Table I) (21). LY311727, a structurally designed sPLA2 inhibitor (55), was also fairly selective for sPLA2-IIA, although at higher concentrations it inhibited sPLA2-V as well (data not shown).
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Functional Interaction between cPLA2 and sPLA2s-- When 293 cells expressing cPLA2 were treated with 10 µM MAFP (52), a cPLA2 inhibitor, A23187-induced immediate AA release was markedly reduced (Fig. 6A). This agent also suppressed the increased, but not constitutive, AA release induced by IL-1 and FCS in the cPLA2 transformants (Fig. 6A). Small but significant stimulus-initiated AA release from the control cells was also consistently suppressed by MAFP (Fig. 6A). Although the control 293 cells produced minimal amounts of PGE2 following stimulation with IL-1 and FCS, the cPLA2 transformants efficiently metabolized the released AA to PGE2 (Fig. 6B). This PGE2 generation was mediated by inducible COX-2, as evidenced by the observations that NS-398, a COX-2-selective inhibitor, almost completely blocked PGE2 generation (Fig. 6B) and that COX-2 is the only COX isozyme detectable in 293 cells after 2-5 h of stimulation with IL-1 and FCS (Fig. 6C). Although FCS alone elicited a small increase in AA release in the cPLA2 transformants (Fig. 6A), this AA was not metabolized to PGE2 (Fig. 6B), indicating that IL-1 signaling is required for the PGE2 biosynthetic steps downstream of the AA-releasing step to take place. Delayed PGE2 generation by the cPLA2 transformants was markedly suppressed by 10 µM AACOCF3, a cPLA2 inhibitor, but not by 10 µM LY311727, a sPLA2-IIA inhibitor (Fig. 6B). AA released by the cPLA2 transformants following A23187 stimulation was not metabolized to PGE2 (data not shown), likely because the expression of both COXs was below the detection level during the immediate response (Fig. 6C). Exogenous added AA was barely converted to PGE2 by 293 cells (data not shown), further supporting that the constitutive expression of COX-1 is extremely low in this cell line. We also failed to detect the expression of both COX proteins in unstimulated 293 cells, assessed by immunoblotting (data not shown).
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The M-type sPLA2 Receptor Is Not Involved in sPLA2s-IIA- or -V-mediated AA Release-- To determine whether the cloned M-type sPLA2 receptor was involved in sPLA2-mediated AA release by 293 cells, control and cPLA2-overexpressing 293 cells were treated with porcine pancreatic sPLA2-I, a known M-type sPLA2 receptor ligand (3-5), in the presence or absence of IL-1. There was, however, no increase in AA release on incubation with excess sPLA2-I (1 µg/ml), whether or not IL-1 was added to the culture (Fig. 8). Moreover, the transcript for the M-type sPLA2 receptor was undetectable in 293 cells (data not shown). These results indicate that the increased AA release observed in cells transfected with sPLA2-IIA or sPLA2-V may not have occurred through an sPLA2 receptor-dependent process. Supporting this idea, the sPLA2-I G30S mutant has been reported to bind to the M-type sPLA2 receptor and to activate cellular AA metabolism normally (59), whereas the sPLA2-IIA G30S mutant failed to stimulate AA release (Fig. 4).
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Fatty Acid Selectivity-- Consistent with the in vitro substrate specificity of cPLA2 (22-24), cPLA2-expressing 293 cells released AA in marked preference to OA after stimulation with A23187 (Fig. 9A) and with IL-1 and FCS (Fig. 9B). Surprisingly, sPLA2-IIA and sPLA2-V also displayed AA-selective release from A23187- (Fig. 9A) and IL-1/FCS-activated cells (Fig. 9B). OA release by both these PLA2 transformants was almost identical to that of the control cells and increased significantly after treatment with FCS but not with IL-1 (Fig. 9B). These results suggest that although sPLA2s show little fatty acid selectivity when phospholipid vesicles are used as the substrate in vitro (2, 11), they liberate AA rather specifically in vivo or that [3H]AA, but not [3H]OA, was incorporated into a particular phospholipid pool in the compartment to which sPLA2s are accessible. As described below, most of the OA released appeared to be derived from iPLA2 in this system.
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Effects of iPLA2 Overexpression-- Finally, hamster iPLA2 was stably overexpressed in 293 cells (Fig. 10A), and its role in fatty acid release was evaluated. As shown in Fig. 10B, basal AA release was significantly increased in cells expressing iPLA2 compared with the control cells. This AA release was further augmented by FCS (Fig. 10B) and continued to rise throughout the culture period (Fig. 10C). Unlike the cPLA2, sPLA2-IIA, and sPLA2-V transformants, the addition of IL-1 to FCS did not alter AA release appreciably in the iPLA2 transformants (Fig. 10B). OA release increased in parallel with AA in the iPLA2 transformants (Fig. 10B), indicating that, consistent with its in vitro activity (46), iPLA2 exhibits no fatty acid preference in vivo. In cells co-transfected with iPLA2 and cPLA2, AA release after IL-1 and FCS stimulation increased only in an additive manner (Fig. 10B), implying no functional cross-talk between these two enzymes. Most intriguingly, iPLA2-derived AA was not metabolized to PGE2, even though the total amounts of AA released from the cPLA2 and iPLA2 transformants were comparable (Fig. 10D). Thus, our results totally support the hypothesis by Dennis and colleagues (47, 48) that iPLA2 is the PLA2 isozyme specifically involved in the regulation of phospholipid turnover but is not crucial for signal transduction. Attempts to investigate the effect of iPLA2 on the immediate response were unsuccessful, since iPLA2 transformants tended to be dead and detach from the plates during exposure to A23187.
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DISCUSSION |
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Taking advantage of overexpression analyses, we examined the functions of cPLA2, various sPLA2s and iPLA2 in the regulation of AA metabolism. We showed that (i) cPLA2 is a key regulator of stimulus-initiated immediate and delayed AA release; (ii) sPLA2-IIA and sPLA2-V, which are heparin-binding PLA2s, each are capable of mediating cytokine-induced delayed AA release as well as ionophore-induced immediate AA release depending on their expression levels; (iii) both catalytic and cell surface-binding domains are essential for sPLA2s to function; (iv) cPLA2 is required for sPLA2s to act properly but not vice versa; (v) both cPLA2 and sPLA2s are linked to COX-2, which is responsible for PGE2 biosynthesis; (vi) iPLA2 is involved in the basal fatty acid release; and (vii) iPLA2 does not couple with COX-2-dependent PGE2 biosynthesis. Thus, our studies have provided unequivocal evidence for cross-talk between the signaling PLA2s cPLA2 and sPLA2s, the potentially redundant mode of action of the two closely related sPLA2-IIA and sPLA2-V isozymes, the functional segregation of the signaling PLA2s from iPLA2 working as the "remodeling" PLA2, and the functional coupling of signaling PLA2 isozymes with downstream COX-2.
As has been shown by a number of reports (11, 22-26, 29-33), there is no doubt that cPLA2 regulates the initiation of AA metabolism in response to stimuli that mobilize intracellular Ca2+. Consistently, all of the cPLA2 transformants established here exhibited increased AA release in response to A23187. The role of cPLA2 in the cytokine-induced delayed response has been also demonstrated by several studies using selective inhibitors (19-21) and antisense oligonucleotides (34, 35). Our present studies have led to confirmation that cPLA2 is certainly involved in IL-1-induced delayed AA release but only when FCS is also present. The requirement for both inflammatory (IL-1) and growth (FCS) stimuli for cPLA2 activation may explain why inflammatory cytokines failed to activate cPLA2 during the delayed response in some previous studies performed in the presence of low FCS concentrations (36, 37). Proinflammatory stimuli that induce delayed AA metabolism, such as IL-1, tumor necrosis factor, and lipopolysaccharide, strongly activate members of the mitogen-activated protein kinase family (60), which in turn phosphorylate cPLA2 at Ser505 to increase its intrinsic activity (29, 61), without accompanying Ca2+ signaling. Not only do growth stimuli stimulate mitogen-activated protein kinases, but they may also induce delayed and long-lasting intracellular Ca2+ oscillation that tends to be localized in specific subcellular areas (62, 63). We therefore assume that spatially and temporally synchronous coupling between local oscillatory Ca2+ elevations and phosphorylation may converge on the prolonged activation of cPLA2, leading to delayed AA release. The essential role of cPLA2 in the immediate and delayed eicosanoid biosynthetic pathways has recently been confirmed by studies using cPLA2 knock-out mice, in which peritoneal macrophages failed to generate PGE2, leukotrienes, and platelet-activating factor in response to ionophore and lipopolysaccharide, respectively (64).
Analyses using sPLA2 transformants have provided unambiguous data on unexplored aspects of the regulation of AA release by each sPLA2 isozyme. All of the sPLA2-IIA transformants augmented IL-1/FCS-induced delayed AA release. These data are consistent with those reported for several current studies, including ours, which demonstrated correlation between sPLA2-IIA induction and delayed PG generation (8-10, 18-21, 57). A23187-induced immediate AA release was also augmented by some, but not all, sPLA2-IIA transformants, in which relatively high levels (>~0.1 µg/ml) of sPLA2-IIA expression were evident. The latter finding is compatible with the previous observation that immediate eicosanoid biosynthesis is enhanced by exogenous sPLA2-IIA in the µg/ml range (10, 65, 66). No sPLA2-IIA-dependent AA release occurred without agonist stimulation. These results confirm that sPLA2-IIA acts on agonist-treated, but not untreated, cells, and suggest that IL-1, in conjunction with FCS, is a more effective inducer of membrane rearrangement, which renders cells susceptible to sPLA2-IIA, than the calcium ionophore A23187. This might explain why some earlier studies failed to detect appreciable effects of sPLA2-IIA on the immediate response despite its apparent participation in the delayed response (18, 20, 21). Proinflammatory stimuli may cause alterations in membrane lipid packing and asymmetry, which are thought to induce increased cellular sPLA2-IIA sensitivity. More recently, sphingomyelin turnover, one of the primary responses following ligation of the receptors for proinflammatory cytokines (67, 68), has been proposed to perturb the outer leaflet of the membrane, thereby modulating the activity of sPLA2s acting on biological membranes (69, 70).
We have also provided evidence that sPLA2-V functions in a manner similar to sPLA2-IIA. sPLA2-V mediated cytokine-induced delayed AA release, as well as A23187-induced immediate AA release when it was expressed in larger amounts, whereas it did not act on unstimulated cells. Like sPLA2-IIA, sPLA2-V is a heparin-binding sPLA2, existing in a cell-surface proteoglycan-associated form. Clustered cationic residues in the C-terminal region of sPLA2-V were crucial for its cell surface association, and mutations in these residues disrupted its AA-releasing activity in activated cells. In contrast, sPLA2-IIC, which does not contain the C-terminal basic amino acid cluster, neither associated with the cell surface nor mediated stimulus-initiated AA release. Therefore, we conclude that association with cell-surface proteoglycans is necessary for sPLA2s to act on activated cells. Proteoglycan anchoring might raise local concentrations of sPLA2s-IIA or -V in certain compartments, where they can effectively attack phospholipids in locally deranged membranes. An alternative possibility is that, as reported with fibroblast growth factors (71), proteoglycan-bound enzymes might be presented more readily to the second subunit of the sPLA2-recognizing protein which serves as a signal-transducing receptor. Nevertheless, the observation that catalytically inactive sPLA2-IIA mutants failed to mediate AA release irrespective of their normal binding to the cell surface indicates that enzymatic activity appears to be essential for sPLA2 functions.
As far as examined a number of established transformants stably expressing each sPLA2, sPLA2-IIA consistently showed activity severalfold greater than those of sPLA2-V when phosphatidylethanolamine was used as a substrate at pH 7.4. Similar results were obtained from transient expression experiments; when each sPLA2 was subcloned into the same vector (pCDNA3.1) and then transfected into 293 cells under the same condition, PLA2 activities released into the supernatants 3 days after the transfection were always sPLA2-IIA > IIC > V in order. Nevertheless, these differences in enzymatic characteristics will need more careful evaluation in a future study using purified enzymes under varying assay conditions. Indeed, in the presence of deoxycholate, sPLA2-V hydrolyzes phosphatidylcholine more efficiently than phosphatidylethanolamine (13). It is also formally possible that the post-transcriptional processing (e.g. protein synthesis, folding, and degradation) of sPLA2-V may differ from that of sPLA2-IIA, resulting in poorer sPLA2-V protein production, in 293 cells.
It is not surprising that sPLA2-IIA and sPLA2-V exhibit redundant functions, because a phylogenetic tree reveals that these two isozymes are most closely related to each other (2, 17). This functional similarity may explain why sPLA2-IIA-deficient mice exhibit no abnormalities during development or inflammation (12), since sPLA2-V may compensate for sPLA2-IIA in some cell types. Indeed, we have recently noted that sPLA2-V appears to be the primary sPLA2 isozyme expressed in the mouse, being distributed in a variety of tissues and cells,2 whereas sPLA2-IIA expression is restricted to the intestine, where it has been reported to be a possible modifier of a tumor suppressor gene (12). The predominant role of sPLA2-V in AA metabolism in mouse macrophage-like P388D1 cells (14) and mouse mast cells (15) therefore appears reasonable. sPLA2-IIA is the primary sPLA2 isozyme in the rat, in which it is distributed in many tissues and cells related to inflammatory responses, and its expression is dramatically increased upon exposure to proinflammatory stimuli (8-10, 20). The tissue and cellular distribution of sPLA2 isozymes in the human is similar to that in the rat, with sPLA2-IIA distributed in a wide variety of tissues (6, 7, 17), whereas sPLA2-V is expressed mainly in the heart (13). Our preliminary data suggest that, like sPLA2-IIA, sPLA2-V is an inducible enzyme in response to proinflammatory stimuli.2
The inability of sPLA2-IIC to mediate AA release from activated cells suggests that the function of this isozyme is apart from the signal transduction. sPLA2-IIC expression is rather restricted to the testes of the rat and mouse, whereas the human sPLA2-IIC gene lacks part of an exon and frequently contains a nonsense mutation (72). In mouse testis, sPLA2-IIC is expressed mainly in spermatocytes and spermatids undergoing meiosis (73). It would be of interest to test whether sPLA2-IIC has some function in the sperm acrosome reaction and the fusion of sperm and oocyte plasma membranes (74, 75), in which PLA2 is believed to play a key role. Cells undergoing apoptosis in such tissues are an attractive candidate for the sPLA2-IIC target, since we have recently shown that hydrolysis of apoptotic cell membranes by sPLA2-IIA does not require anchoring to proteoglycans on the plasma membranes (76).
Our results raised the fundamental question of how cPLA2 and sPLA2s-IIA or -V interact with each other. The studies using cPLA2 inhibitors, which suppressed AA release by cPLA2 and by sPLA2s-IIA and -V transformants to a similar extent, support the previously proposed hypothesis that endogenous cPLA2 is required for sPLA2s to exert their functions (15, 19, 20, 58). Conversely, the sPLA2-IIA inhibitor showed no inhibitory effect on cPLA2-mediated AA release, indicating that sPLA2-IIA is not needed for the action of cPLA2. The co-expression experiments revealed that cPLA2 and sPLA2 indeed act in synergy. The simplest explanation for these events is that sPLA2s-IIA and -V activate intracellular cPLA2, which eventually releases AA in a selective manner. Indeed, this route is compatible with the recent studies using rat mesangial cells (77), human neutrophils (78), human astrocytoma (79), and mouse osteoblasts (19), in which sPLA2-IIA caused translational (19) or post-translational (77-79) activation of cPLA2, probably through the production of lipid mediators (19, 77, 78) or through the putative sPLA2 receptor-dependent and catalysis-independent process (79). However, several lines of evidence argue against this speculation in our 293 cell system. Endogenous cPLA2 expression by itself was minimal and appeared to be insufficient to mobilize AA metabolism in parental 293 cells after A23187 or IL-1/FCS stimulation. Moreover, the M-type sPLA2 receptor, the only ligand so far known for which is sPLA2-I (3-5), appeared not to be involved in sPLA2-mediated AA release in 293 cells. In support of this, the M-type sPLA2 receptor of various species (except for the rabbit) has been reported to have no affinity or very low affinity for sPLA2-IIA (3-5), and in our preliminary experiments, co-expression of sPLA2s-IIA or -V with the M-type sPLA2 receptor in 293 cells did not significantly alter the level of AA release mediated by these sPLA2s alone.3 However, our results support an alternative mechanism proposed by Dennis and co-workers (58) that cPLA2 plays a role in membrane rearrangement by producing certain products that contribute to destabilization of the membrane, thereby influencing the subsequent activation of sPLA2s. This pathway has been further supported by recent studies using mouse mast cells (15) and rat fibroblasts (20). Considering all these points together, we propose that cPLA2 is required for sPLA2s-IIA or -V to liberate AA from rearranged cell membranes, although the possibility that sPLA2s activate cPLA2 through a novel unidentified sPLA2 receptor cannot be completely ruled out, as has been recently suggested by Nieto and colleagues (79). Nonetheless, bidirectional interaction between cPLA2 and sPLA2s may represent a general action leading to optimal AA release in mammalian cells, and the role of sPLA2s could be to amplify the primary responses initiated by intracellular cPLA2. It has been reported that sPLA2-IIA effectively hydrolyzes membrane vesicles shed from activated platelets (80) and the membranes of cells undergoing apoptosis (76). These observations imply diverse patterns of actions for sPLA2.
Dennis and colleagues (47, 48) have reported that iPLA2 contributes to remodeling of membrane phospholipids as a sort of housekeeping enzyme. In support of their proposal, iPLA2 overexpression led to an increase in basal fatty acid release, which was augmented by FCS but not by IL-1. Of particular interest was the finding that AA liberated by iPLA2 was not metabolized to PGE2. This is consistent with a recent study by Balsinde et al. (81), which showed that iPLA2 is not involved in stimulus-initiated AA release in P388D1 cells, by means of an antisense technique. This finding conflicts with several earlier reports describing iPLA2 involvement in agonist-induced AA release, most of which were based upon the inhibitory effects of the iPLA2 inhibitor bromoenol lactone (49, 50). However, evidence is accumulating that this agent also blocks certain other enzymes in the transmembrane signaling cascades (82), and therefore the involvement of iPLA2 in the signal transduction remains uncertain. It is also unlikely that iPLA2 affects stimulus-initiated AA release indirectly by altering AA susceptibility to the signaling PLA2s, at least in 293 cells, because co-transfection of cPLA2 and iPLA2 did not alter the efficiency of cPLA2-mediated AA release. Based upon these observations, it seems reasonable to conclude that iPLA2 may liberate AA from different phospholipid pools which are not accessible to COX-2.
A complex finding is that AA was released in marked preference to OA even in sPLA2-transfected cells. This was unexpected, since none of the sPLA2 isozymes discriminates between the fatty acids at the sn-2 position of glycerophospholipid substrates when assayed in vitro. This AA selectivity of sPLA2s is further manifested by the fact that iPLA2 released both AA and OA in a parallel fashion. Several previous studies provided some evidence that sPLA2 might act as an AA-preferential enzyme on mammalian cell membranes (83, 84), and our present results are in line with these observations. There might be a certain cellular co-factor that contributes to altering the substrate specificity of sPLA2s so that they act as AA-selective enzymes. An alternative explanation for these results is that sPLA2s may act on certain phospholipid pools in which [3H]AA is preferentially enriched, whereas iPLA2 may function in other intracellular compartments where both [3H]AA and [3H]OA are equally incorporated. The interpretation of these observations should need a special caution, since radiolabeled fatty acids added exogenously are often incorporated into separate phospholipid pools and do not always reflect the intracellular movement of endogenous fatty acids. The conclusion of fatty acid selectivity should await the measurement of the mass amounts of the fatty acids released.
Different PLA2s, acting on different cellular AA pools at different locations and being regulated by separate but interacting mechanisms, confer on the system great versatility in ensuring that both immediate and delayed AA-derived mediators are efficiently generated during cellular responses. Moreover, the different functions of the signaling and remodeling PLA2s predict the presence of specific machineries that transport AA toward eicosanoid-biosynthetic enzymes and CoA-dependent or -independent transacylases, respectively.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Tsujimoto, H. Ishimaru, T. Kamimura, S. S. Jones, J. P. Arm, J. Trzaskos, R. M. Kramer, K. Hanasaki, and H. Arita for providing cDNAs, antibodies, and inhibitors.
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FOOTNOTES |
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* This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science 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.
¶ Supported by National Institutes of Health Grant DK38185.
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.
1 The abbreviations used are: PLA2, phospholipase A2; sPLA2, secretory PLA2; cPLA2, cytosolic PLA2; iPLA2, Ca2+-independent PLA2; AA, arachidonic acid; OA, oleic acid; PG, prostaglandin; COX, cyclooxygenase; IL-1, interleukin-1; FCS, fetal calf serum; AACOCF3, arachidonoyl trifluoromethyl ketone; MAFP, methyl arachidonylfluorophosphate; PCR, polymerase chain reaction; CHO, Chinese hamster ovary.
2 M. Murakami, H. Sawada, and I. Kudo, unpublished observations.
3 M. Murakami, K. Hanasaki, H. Arita, and I. Kudo, unpublished observations.
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