Cytosolic Phospholipase A2 Is Required for Cytokine-induced Expression of Type IIA Secretory Phospholipase A2 That Mediates Optimal Cyclooxygenase-2-dependent Delayed Prostaglandin E2 Generation in Rat 3Y1 Fibroblasts*

Hiroshi Kuwata, Yoshihito Nakatani, Makoto Murakami, 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
Procedures
Results
Discussion
References

Activation of rat fibroblastic 3Y1 cells with interleukin-1beta (IL-1beta ) and tumor necrosis factor alpha  (TNFalpha ) induced delayed prostaglandin (PG) E2 generation over 6-48 h, which occurred in parallel with de novo induction of type IIA secretory phospholipase A2 (sPLA2) and cyclooxygenase (COX)-2, without accompanied by changes in the constitutive expression of type IV cytosolic PLA2 (cPLA2) and COX-1. Types V and IIC sPLA2s were barely detectable in these cells. Studies using an anti-type IIA sPLA2 antibody, sPLA2 inhibitors, and a type IIA sPLA2-specific antisense oligonucleotide revealed that IL-1beta /TNFalpha -induced delayed PGE2 generation by these cells was largely dependent on inducible type IIA sPLA2, which was functionally linked to inducible COX-2. Delayed PGE2 generation was also suppressed markedly by the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3), which attenuated induction of type IIA sPLA2, but not COX-2, expression. AACOCF3 inhibited the initial phase of cytokine-stimulated arachidonic acid release, and supplementing AACOCF3-treated cells with exogenous arachidonic acid partially restored type IIA sPLA2 expression. These results suggest that certain metabolites produced by the cPLA2-dependent pathway are crucial for the subsequent induction of type IIA sPLA2 expression and attendant delayed PGE2 generation. Some lipoxygenase-derived products might be involved in this event, since IL-1beta /TNFalpha -induced type IIA sPLA2 induction and PGE2 generation were reduced markedly by lipoxygenase, but not COX, inhibitors. In contrast, Ca2+ ionophore-stimulated immediate PGE2 generation was regulated predominantly by the constitutive enzymes cPLA2 and COX-1, even when type IIA sPLA2 and COX-2 were maximally induced after IL-1beta /TNFalpha treatment, revealing functional segregation of the constitutive and inducible PG biosynthetic enzymes.

    INTRODUCTION
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Abstract
Introduction
Procedures
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References

Two kinetically different prostaglandin (PG)1-generating pathways from endogenous arachidonic acid, the immediate and delayed phases, imply the recruitment of different sets of biosynthetic enzymes, expression and activation of which are tightly regulated by distinct transmembrane signalings (1). The immediate phase of PG biosynthesis, occurring within several minutes of stimulation, is elicited by agonists that mobilize intracellular Ca2+ and characterized by a burst release of arachidonic acid initiated by phospholipase A2 (PLA2) and subsequent conversion to bioactive PGs by the sequential actions of cyclooxygenase (COX) and terminal PG synthases. The delayed phase of PG biosynthesis is accompanied by the continuous supply of arachidonic acid and its conversion to PGs, often PGE2, over long culture periods following growth or proinflammatory stimuli.

Segregated utilization of the two COX isoforms in these two distinct phases has been demonstrated in several systems (2-4). COX-1 is constitutively expressed in most cells and tissues and is generally thought to serve certain physiologic housekeeping functions, whereas COX-2 is dramatically induced in response to a wide variety of stimuli, and is thought to contribute to the generation of PGs at certain stages of cell proliferation and differentiation and at sites of inflammation (5). The absolute requirement for COX-2 in the delayed PG generation, irrespective of the constitutive presence of COX-1, has been shown by studies using COX-2-selective inhibitors, antisense oligonucleotides, and knockout mice (2-4, 6). COX-1 is involved in immediate PG generation, such as thromboxane A2 generation by activated platelets (7) and the IgE-dependent immediate phase of PGD2 generation by mast cells (2, 4). In contrast, interleukin (IL)-1alpha -primed, platelet-derived growth factor-initiated immediate PGE2 generation by mouse calvaria cells depends on IL-1alpha -induced COX-2 in preference to constitutive COX-1 (8). Several explanations of the functional segregation of the two COX isoforms have been proposed, such as their different subcellular localizations (9) and their different substrate concentration requirements (10). In fact, the finding that the utilization of arachidonic acid by COX-2 is favored over COX-1 at low peroxide concentrations (10) appears consistent with the involvement of COX-1 in the burst of events that follow immediate activation and of COX-2 in the delayed phase, in which only a limited amount of arachidonic acid would be supplied continuously. Moreover, preferential coupling between particular PLA2s, COXs, and terminal PG synthases have been suggested by several recent studies (11-13).

The enzymatic properties of type IV 85-kDa cytosolic PLA2 (cPLA2), i.e. arachidonate selectivity, Ca2+-dependent translocation, and phosphorylation-dependent activation via kinases belonging to the mitogen-activated protein kinase family (14, 15), are in agreement with its role in immediate PG biosynthesis, which is usually accompanied by rapid and transient cytoplasmic Ca2+ mobilization. Under such conditions, cPLA2 translocates to the perinuclear envelope and endoplasmic reticular membranes (16, 17), where downstream COXs, 5-lipoxygenase (LOX), 5-LOX-activating protein, and some terminal PG and leukotriene synthases colocalize (9, 18-20). The functional association of cPLA2 with constitutive COX-1 resulting in immediate PG biosynthesis has been demonstrated in several studies (21, 22). cPLA2 has been also implicated in the inducible COX-2-dependent delayed PG generation lasting for hours (12, 23, 24) and COX-2-dependent immediate PGE2 generation (8) that are initiated and primed, respectively, by inflammatory stimuli.

Secretory PLA2 (sPLA2) enzymes comprise a growing family of distinct enzymes with similar molecular masses of about 14 kDa. To date, five mammalian sPLA2s, designated types I, IIA, IIC, V, and X, have been identified (1, 25-27). Type I sPLA2 is abundantly expressed in the pancreas, where it functions as a digestive enzyme for dietary phospholipids. It is also expressed in several nondigestive organs, where it may act as a regulator of cellular functions via specific sPLA2 receptor (28, 29). Type IIA sPLA2 was originally isolated from inflamed sites and inflammatory cells, is induced by proinflammatory stimuli, and is thought to play some roles in cellular inflammatory responses (1, 30). Type IIA sPLA2 has been shown to augment the delayed phase of PG generation by several cells in response to proinflammatory cytokines (31-34), and further support for these biological actions has been supplied by several animal studies (35-37). Recently, two groups have demonstrated that type V sPLA2, the transcript of which was originally found to be expressed in the heart and lung (38), is crucial for stimulus-initiated PG biosynthesis by mouse macrophages (39) and mast cells (40). Type IIC sPLA2 is abundantly expressed in rodent testes, whereas it is a nonfunctional pseudogene in human (41). The overall structures of types IIA, IIC, and V sPLA2s, the genes of which are tightly linked on human chromosome 1, are more closely related to one another than to that of type I sPLA2, the gene of which maps to human chromosome 12 (26). More recently, a novel group X sPLA2 has been cloned; it is distributed in immune tissues, exhibits some features characteristic of both types I and IIA sPLA2s, and maps to human chromosome 16 (27). Thus, the sPLA2 family is growing rapidly; therefore, it has been proposed that some of the previously described functions of sPLA2s, particularly type IIA sPLA2, need to be reevaluated, since studies based upon enzyme activities and using inhibitors or antibodies against type IIA sPLA2 may not discriminate these sPLA2s (26).

Here, we report the regulation of PGE2 biosynthesis in rat fibroblastic 3Y1 cells, which have the capacity to express high levels of type IIA sPLA2, cPLA2 and the two COXs and to exhibit both immediate and delayed PGE2 generation in response to appropriate stimuli. Pharmacologic, immunochemical, and genetic studies have provided evidence that both cPLA2 and type IIA sPLA2 are required for COX-2-dependent delayed, whereas cPLA2, but not type IIA sPLA2, is utilized for COX-1-dependent immediate, PGE2 biosynthesis by 3Y1 cells. Of particular interest is that functional cPLA2 may be crucial for the subsequent type IIA sPLA2 induction and attendant COX-2-dependent delayed PGE2 generation. Thus, this study (i) reconfirmed the involvement of type IIA sPLA2 in biological responses, (ii) demonstrated significant cross-talk between the two Ca2+-dependent PLA2s where one enzyme is required for the induction of the other, and (iii) revealed segregated coupling of discrete PLA2 and COX enzymes in the different phases of PG biosynthesis.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Mouse IL-1beta was purchased from Genzyme. Human TNFalpha was provided by Dr. H. Ishimaru (Asahi Chemical Industry). Rabbit antiserum to human cPLA2 was provided by J. D. Clark (Genetics Institute), mouse cPLA2 cDNA by M. Tsujimoto (RIKEN Institute), rabbit antiserum to mouse COX-1 by W. L. Smith (Michigan State University), mouse COX-2 cDNA and the COX-2 inhibitor NS-398 (42) by J. Trzaskos (Merck DuPont), rat type V sPLA2 cDNA by J. A. Tischfield (Indiana University School of Medicine), and mouse beta -actin cDNA by J. P. Arm (Harvard Medical School). Mouse COX-1 cDNA, rabbit antiserum to COX-2, arachidonic acid, and the PGE2 enzyme immunoassay kit were purchased from Cayman Chemical. The LOX inhibitors, including nordihydroguaiaretic acid (NDGA; general LOX inhibitor), AA-861 (5-LOX inhibitor), cinnamyl-3,4-dihydroxy-alpha -cyanocinnamate (12-LOX inhibitor) and 5,8,11,14-eicosatetraynoic acid (15-LOX inhibitor) (43), were purchased from BIOMOL Research Laboratories. Rat type IIA sPLA2 cDNA (44) and rabbit polyclonal antibody against rat type IIA sPLA2 (45) were prepared as described previously. The cDNA probes for rat cPLA2 and rat type IIC sPLA2 were obtained by the reverse transcriptase-polymerase chain reaction (RT-PCR) using RNA extracted from 3Y1 cells (34) and rat testis (41), respectively, as described previously. The antisense and sense oligonucleotides and RT-PCR primers were obtained from Greiner Japan. The type IIA sPLA2 inhibitor thielocin A1 (35, 36) was donated by Dr. T. Yoshida (Shionogi Pharmaceutical). A23187 and the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3) (47) were purchased from Calbiochem. Aspirin, dexamethasone, and heparin were purchased from Sigma. CellFectin reagent, Opti-MEM medium, and TRIzol reagent were from Life Technologies, Inc. The IL-3-dependent mouse bone marrow-derived mast cell line MC-MKM, which originated from BALB/cJ mice (48), was cultured in enriched medium supplemented with 50% (v/v) WEHI-3B-conditioned medium as a source of IL-3 and 100 ng/ml recombinant mouse c-kit ligand, which had been expressed using a baculovirus system (2).

Activation of 3Y1 Cells-- The 3Y1 cells were a gift from Dr. Y. Uehara (National Institute of Infectious Disease, Tokyo, Japan) and maintained in culture medium composed of Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical) supplemented with 10% (v/v) fetal calf serum (FCS), penicillin/streptomycin (100 units/ml and 100 µg/ml, respectively) (Flow Laboratories) and 2 mM glutamine (Life Technologies, Inc.). The media of 3Y1 cells that had attained 60-80% confluence in six-well plates (Iwaki) were replaced with 2 ml of DMEM supplemented with 2% FCS. After culture for 24 h, 1 ng/ml IL-1beta , 100 units/ml human TNFalpha , or both were added to the cultures to assess the delayed response. Replicated cells were activated with 1 µM A23187 for 10 min in DMEM supplemented with 2% FCS to assess the immediate response. The supernatants were taken for PGE2 enzyme immunoassay. For immunoblot analysis and the PLA2 assay (see below), the cells were trypsinized, washed once with 10 mM phosphate buffer, pH 7.4, containing 150 mM NaCl (phosphate-buffered saline), resuspended in 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA and 250 mM sucrose at 1 × 107 cells/ml, and disrupted by 1-min pulse sonication (50% work cycle, setting 4) with a Branson Sonifier (Branson Sonic Power Co.). As for the RNA blot analysis, TRIzol was directly added to the cell monolayer.

RNA Blotting-- All the procedures were performed as described elsewhere (34). Briefly, equal amounts (10 µg) of total RNA, purified using TRIzol reagent, 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 sequentially probed with cPLA2, type IIA sPLA2, COX-2, and beta -actin cDNA probes that had been labeled with [32P]dCTP (Amersham Life Science) by random priming (Takara Biomedicals). All hybridizations were carried out at 42 °C overnight 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, 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. The blots were visualized by autoradiography with Kodak X-Omat AR films and double intensifying screens at -80 °C.

The relative amount of each transcript was estimated by quantitating the associated radioactivity using a BAS-III bioimaging analyzer (Fuji Film). The -fold increase in steady-state mRNA was calculated as the ratio of radioactivity associated with a specific transcript in treated cells compared with that in untreated cells and was corrected for changes in steady-state levels of beta -actin transcript to adjust for differences in loading between lanes.

RT-PCR-- Specific primers for the PCR, based on the sequences of sPLA2s reported previously (38, 41, 45), were synthesized. The type IIA sPLA2 primers used were 5'-ATG AAG GTC CTC CTC CTG CTA G-3' and 5'-TCA GCA TTT GGG CTT CTT CC-3' (45), type IIC sPLA2 primers were 5'-ATG GAC CTC CTG GTC TCC TCA GG-3' and 5'-CTA GCA ATG AGT TTG TCC CTG C-3' (41), and type V sPLA2 primers were 5'-CAG GGG GCT TGC TAG AAC TCA A-3' and 5'-AAG AGG GTT GTA AGT CCA GAG G-3' (38). The RT-PCR was carried out using a RNA PCR kit (avian myeloblastosis virus) version-2 (Takara Biomedicals), according to the manufacturer's instructions, using 1 µg of total RNA from IL-1beta /TNFalpha -treated 3Y1 or mast cells as a template. Equal amounts of each RT product were amplified by PCR with ex Taq polymerase (Takara Biomedicals) for 30 cycles consisting of 30 s each at 94 °C, 55 °C and 72 °C. The amplified cDNA fragments were resolved electrophoretically on 1.5% (w/v) agarose gels and visualized by ethidium bromide. The fragments were then transferred onto Immobilon-N membranes and probed with [32P]dCTP-labeled cDNAs for types IIA, IIC, and V sPLA2s. Hybridization and subsequent washing were carried out as described above for RNA blot analysis.

Immunoblotting-- All the procedures were performed as described previously (34). Briefly, cell lysates were subjected to SDS-polyacrylamide gel electrophoresis under nonreducing (for type IIA sPLA2) and reducing (for cPLA2, COX-1, and COX-2) 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 washed once with 10 mM Tris-HCl (pH 7.2) containing 150 mM NaCl and 0.1% (v/v) Tween 20 (TBS-T) and then blocked for 1 h in TBS-T containing 3% (w/v) skim milk. After washing the membranes with TBS-T, antibodies against cPLA2, type IIA sPLA2, COX-1, and COX-2 were added at a dilution of 1:5,000 in TBS-T and incubated for 2 h. Following three washes with TBS-T, the membranes were treated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories Inc.; diluted 1:7,000) in TBS-T. After six washes with TBS-T, the protein bands were visualized using an ECL Western blot analysis system (Amersham Life Science).

Measurement of PLA2 Activity-- The PLA2 activities in the resulting lysates were measured after incubation with 100 mM Tris-HCl, pH 9.0, 4 mM CaCl2, and 2 µM 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoethanolamine (Amersham Life Science) or 1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphoethanolamine (NEN Life Science Products), as the substrates, for 30 min at 37 °C (34).

Transfection of Antisense Oligonucleotide into 3Y1 Cells-- The antisense (5'-TAG CAA CAG GAG GAC CTT CAT-3') and sense (5'-ATG AAG GTC CTC CTG TTG CTA-3') oligonucleotides for rat type IIA sPLA2, corresponding to the translation initiation site, 100 nM each, were incubated individually with CellFectin reagent (Life Technologies, Inc.) in 200 µl of Opti-MEM medium (Life Technologies, Inc.) for 15 min at room temperature and then added to cells that had attained 60-80% confluence in 6-well plates and been supplemented with 800 µl of Opti-MEM. After incubation for 6 h at 37 °C, the medium was replaced with 2 ml of DMEM supplemented with 2% FCS in the continued presence of 100 nM oligonucleotide. After culture for 24 h, 1 ng/ml IL-1beta and 100 units/ml TNFalpha were added to the culture to activate the cells.

Arachidonic Acid Release-- 3Y1 cells grown in 24-well plates were preincubated overnight with [3H]arachidonic acid (100 Ci/nmol; Amersham Life Science) that was diluted 1:500 with fresh culture medium. After three washes with fresh medium, 250 µl of fresh culture medium with or without IL-1beta and TNFalpha was added to each well and the amount of free [3H]arachidonic acid released into each supernatant during culture was measured. The amount of arachidonic acid released was calculated as a percentage, using the formula [S/(S + P)] × 100, where S and P are the radioactivities of equal portions of supernatant and cell pellet, respectively.

    RESULTS
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Procedures
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References

Cytokine-induced COX-2-dependent Delayed PGE2 Generation-- When rat fibroblastic 3Y1 cells were cultured with 1 ng/ml IL-1beta and 100 units/ml TNFalpha , PGE2 generation started to increase after culture for 6 h, increased markedly by 12 h, and reached its maximum to a plateau level after 24-48 h (Fig. 1A). The concentrations of the cytokines used shown in Fig. 1 and in subsequent experiments were based upon the results of dose-response experiments, in which the maximal response was observed when >1 ng/ml IL-1beta and >100 units/ml TNFalpha were combined (data not shown). Each cytokine alone elicited a similar, but weaker, effect than the combination of the two (data not shown).


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Fig. 1.   Time courses of PGE2 generation by (A) and PLA2 activity associated with (B) 3Y1 cells. 3Y1 cells were cultured for the indicated periods in the presence (solid circles) or absence (open circles) of 1 ng/ml IL-1beta and 100 units/ml TNFalpha . The values are means ± S.D. of three independent experiments.

Expression of the two COX enzymes, COX-1 and COX-2, was assessed by immunoblotting and RNA blotting (Fig. 2A). Expression of the 72-kDa COX-1 protein was constant during culture with IL-1beta /TNFalpha for 48 h, whereas that of the 72-kDa COX-2 protein was minimal during the initial 3 h, then increased markedly to reach its maximum to a plateau level after culture for 12-24 h and declined thereafter, and was preceded by marked induction of COX-2 mRNA expression, which was barely detectable before stimulation, increased after cytokine stimulation to reach a peak at 12 h (approximately 60-fold increase over the control level), and then declined gradually (Fig. 2A). Thus, IL-1beta /TNFalpha -induced COX-2 protein expression almost paralleled PGE2 generation (Fig. 1A). The COX-2-selective inhibitor NS-398 (1 ng/ml) (42) blocked IL-1beta /TNFalpha -induced PGE2 generation almost completely (Fig. 3A). Therefore, IL-1beta /TNFalpha -induced delayed PGE2 generation depends entirely on de novo induction of COX-2, irrespective of the continued presence of COX-1.


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Fig. 2.   Expression of prostanoid biosynthetic enzymes in 3Y1 cells. A, 3Y1 cells were stimulated with 1 ng/ml IL-1beta and 100 units/ml TNFalpha for the indicated periods and the expression of proteins (left) and transcripts (right) for cPLA2, type IIA sPLA2, COX-2, and COX-1 was assessed by RNA blotting and immunoblotting, respectively. B, expression of types IIA, IIC, and V sPLA2s in 3Y1 cells stimulated for 24 h with IL-1beta /TNFalpha and in BALB/cJ mouse bone marrow-derived mast cells maintained in c-kit ligand plus IL-3, assessed by RT-PCR. Representative results of four (A) and two (B) independent experiments are shown.


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Fig. 3.   Effects of various agents on delayed PGE2 generation by 3Y1 cells. A, 3Y1 cells were stimulated for 48 h with IL-1beta /TNFalpha in the absence (column 1) or presence of 10 µg/ml anti-type IIA sPLA2 antibody (column 2), 1 µg/ml thielocin A1 (column 3) or 1 ng/ml NS-398 (column 4). Values are expressed as relative PGE2 generation (%), calculated using the formula: (PGE2 produced in the presence of each inhibitor)/(PGE2 produced in the absence of inhibitor) × 100. B, 3Y1 cells were stimulated with IL-1beta /TNFalpha in the presence or absence of 1 mg/ml heparin for 48 h, and the PLA2 activity (left) and amount of PGE2 (right) released into the supernatants were assessed. C, 3Y1 cells were stimulated with IL-1beta /TNFalpha in the presence or absence of 1 µM dexamethasone (Dex) for 48 h, and the cell-associated PLA2 activity, PGE2 generation, and expression of cPLA2, type IIA sPLA2, COX-2, and COX-1 proteins and transcripts were assessed. Representative results of four (A), two (B), and three (C) independent experiments are shown.

Expression of PLA2-- Expression of the 85-kDa cPLA2 protein and mRNA was constitutive and did not alter significantly during culture with IL-1beta /TNFalpha for 48 h (Fig. 2A). Expression of the 14-kDa type IIA sPLA2 protein was minimal before stimulation and increased dramatically after culture with IL-1beta /TNFalpha for 6-12 h, reaching its maximum and plateau level after 12-48 h (Fig. 2A). Increased expression of type IIA sPLA2 protein was accompanied by that of its 0.9-kilobase mRNA, which was barely detectable during the initial 3 h, then increased to reach a peak at 24 h (approximately 60-fold increase over the control level), and tended to decline gradually thereafter (Fig. 2A). PLA2 activity associated with 3Y1 cells increased markedly over 6-24 h of culture with IL-1beta /TNFalpha (Fig. 1B), in parallel with type IIA sPLA2 protein expression (Fig. 2A). The increased PLA2 activity was sensitive to dithiothreitol, stable after acid extraction, and neutralized by an antibody raised against type IIA sPLA2, whereas the basal PLA2 activity was resistant to dithiothreitol, acid-labile, did not react with the anti-type IIA sPLA2 antibody, and was inhibited by the cPLA2 inhibitor AACOCF3 (data not shown).

To establish whether other sPLA2 family members were expressed in 3Y1 cells, the RT-PCR was carried out using primers specific for types IIC and V sPLA2s, as well as those for type IIA sPLA2. After 30 cycles of amplification, a strong signal for type IIA sPLA2 was obtained, whereas the signals for types IIC and V sPLA2s were barely detectable, in IL-1beta /TNFalpha -stimulated 3Y1 cells (Fig. 2B). Under the same PCR conditions, expression of types IIA, IIC, and V sPLA2s was detected clearly in BALB/cJ-derived bone marrow-derived mast cells (Fig. 2B), consistent with our previous findings (49), but in contrast to those of Reddy et al. (40), who detected only type V, but not types IIA and IIC, sPLA2 in the mouse mast cells they used. These different sPLA2 expression profiles may reflect mast cell heterogeneity under different culture conditions. Nevertheless, our RT-PCR studies indicate that, of the three closely related sPLA2 enzymes mapped to the same chromosome locus (26), type IIA sPLA2 is the predominant sPLA2 isozyme expressed in 3Y1 cells.

Involvement of Type IIA sPLA2 in Delayed PGE2 Generation-- To clarify the involvement of type IIA sPLA2 in IL-1beta /TNFalpha -induced PGE2 biosynthesis, we carried out several approaches using agents that inhibit sPLA2 activity, prevent type IIA sPLA2 association with plasma membranes, or reduce type IIA sPLA2 expression specifically. First, we examined the effects of the neutralizing anti-rat type IIA sPLA2 antibody (45) and the sPLA2-specific inhibitor thielocin A1 (46) on PGE2 generation. The addition of 10 µg/ml anti-rat type IIA sPLA2 antibody to 3Y1 cells stimulated for 48 h with IL-1beta /TNFalpha attenuated PGE2 generation almost to the basal level (Fig. 3A). Similar reduction of PGE2 generation was observed when the cells were stimulated with IL-1beta /TNFalpha in the presence of 10 µg/ml thielocin A1 (Fig. 3A). The induced sPLA2 activity was virtually abrogated, whereas the cPLA2 activity was not affected appreciably, by these agents at the concentrations we used (data not shown). Furthermore, neither the anti-type IIA sPLA2 antibody nor thielocin A1 inhibited recombinant types IIC and V sPLA2s, revealing their strict specificity to type IIA sPLA2.2

The association of type IIA sPLA2 with heparan sulfate proteoglycans on plasma membranes has been shown to be critical for its cellular functions (32-34). The addition of heparin to IL-1beta /TNFalpha -stimulated 3Y1 cells resulted in a marked increase in the sPLA2 activity in the supernatant (Fig. 3B) with a concomitant reduction in the cell-associated sPLA2 activity (data not shown). Solubilization of the cell surface-associated sPLA2 by heparin was accompanied by a marked reduction in PGE2 generation (Fig. 3B).

The glucocorticoid dexamethasone has been reported to inhibit induced expression of type IIA sPLA2 (50, 51), cPLA2 (52, 53), and COX-2 (54-56), although its effects differ according to cell type. Stimulation of 3Y1 cells for 48 h with IL-1beta /TNFalpha in the presence of 10-6 M dexamethasone reduced cell-associated PLA2 activity and PGE2 generation by ~80% (Fig. 3C). Dexamethasone reduced the expression of type IIA sPLA2 protein and mRNA markedly, without affecting the constitutive expression of cPLA2 and COX-1 or the induced expression of COX-2 (Fig. 3C). The failure of dexamethasone to down-regulate COX-2 expression is unusual (54-56), even though the COX-2 gene lacks a consensus glucocorticoid response element and dexamethasone did not affect the induction of reporter constructs containing the regulatory region of COX-2 (55).

In an attempt to obtain more conclusive evidence for the participation of type IIA sPLA2 in the delayed PGE2 generation, we used antisense technology to reduce type IIA sPLA2 protein expression specifically. As shown in Fig. 4, transfection of the antisense, but not the sense, oligonucleotide, directed to the translation initiation site of rat type IIA sPLA2, into 3Y1 cells markedly reduced IL-1beta /TNFalpha -induced delayed PGE2 generation, with a concomitant reduction in IL-1beta /TNFalpha -induced type IIA sPLA2 protein expression. The antisense oligonucleotide affected neither induced expression of COX-2 (Fig. 4) nor constitutive expression of cPLA2 (data not shown), implying that the selective suppression of type IIA sPLA2 expression was responsible for the reduced PGE2 generation.


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Fig. 4.   Effect of antisense oligonucleotide for type IIA sPLA2 on delayed PGE2 generation. 3Y1 cells treated with or without antisense or sense oligonucleotide for type IIA sPLA2 were stimulated with IL-1beta /TNFalpha for 48 h as described under "Experimental Procedures," and PGE2 generation (upper panel) and expression of type IIA sPLA2 and COX-2 proteins (bottom panel) were assessed. A representative result of three independent experiments is shown.

Role of cPLA2 in Type IIA sPLA2 Expression and Delayed PGE2 Generation-- Culture of 3Y1 cells with IL-1beta /TNFalpha for 48 h in the presence of AACOCF3 reduced PGE2 generation considerably almost to the basal level (Fig. 5A). This observation suggests that the AACOCF3-sensitive cPLA2 (47) is also involved in delayed PGE2 generation by 3Y1 cells, consistent with previous studies demonstrating the involvement of cPLA2 in the delayed responses of some cells (12, 23, 24). Interestingly, we found that AACOCF3 markedly suppressed IL-1beta /TNFalpha -induced expression of type IIA sPLA2 without affecting that of COX-2 (Fig. 5, B and C). The inhibitory effect of AACOCF3 on type IIA sPLA2 expression was evident at the mRNA level (Fig. 5C). The addition of excess exogenous arachidonic acid to AACOCF3-treated, IL-1beta /TNFalpha -stimulated cells restored type IIA sPLA2 expression to some extent (Fig. 5, B and C), although it did not exhibit this effect when added to unstimulated cells (data not shown). Restoration of type IIA sPLA2 expression was only minimal when exogenous arachidonic acid was supplied to AACOCF3-treated cells 24 h after starting the culture (Fig. 5C). As shown in Fig. 5D, IL-1beta /TNFalpha stimulation evoked rapid [3H]arachidonic acid release by [3H]arachidonic acid-prelabeled cells, and this release was abrogated by AACOCF3. These results suggest that activation of cPLA2 occurring during the early phase of cell activation following cytokine treatment is crucial for the subsequent induction of type IIA sPLA2 expression in 3Y1 cells.


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Fig. 5.   Effects of cPLA2 and COX inhibitors on IL-1beta /TNFalpha -induced type IIA sPLA2 expression. A, effect of 10 µM AACOCF3 on PGE2 generation by 3Y1 cells stimulated for 48 h with IL-1beta /TNFalpha . B, effects of 10 µM AACOCF3, 1 ng/ml NS-398, and 10 µg/ml aspirin on type IIA sPLA2 and COX-2 protein expression in cells stimulated for 48 h with IL-1beta /TNFalpha . C, effects of AACOCF3 and exogenous arachidonic acid on IL-1beta /TNFalpha -induced expression of type IIA sPLA2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. Lane 1, no stimulation; lane 2, IL-1beta /TNFalpha stimulation for 48 h; lane 3, IL-1beta /TNFalpha stimulation for 48 h in the presence of 10 µM AACOCF3; lane 4, IL-1beta /TNFalpha stimulation for 48 h in the presence of 10 µM AACOCF3 supplemented with 50 µM arachidonic acid from the start of the culture period; lane 5, IL-1beta /TNFalpha stimulation for 48 h in the presence of 10 µM AACOCF3 with 50 µM arachidonic acid supplemented 24 h after starting the culture. D, arachidonic acid release from 3Y1 cells. Cells prelabeled with [3H]arachidonic acid were treated for the indicated periods with (solid symbols) or without (open circles) IL-1beta /TNFalpha in the presence (triangles) or absence (circles) of 10 µM AACOCF3. In these studies, each inhibitor was added 2 h before stimulation with cytokines.

Neither aspirin, which inactivates both COX-1 and COX-2, nor NS-398, a COX-2-selective inhibitor, affected IL-1beta /TNFalpha -induced type IIA sPLA2 or COX-2 expression (Fig. 5B), suggesting that the induction of type IIA sPLA2 expression is independent of any endogenous PG. In contrast, treating the cells with the general LOX inhibitor NDGA suppressed IL-1beta /TNFalpha -induced type IIA sPLA2 expression and PGE2 generation in parallel, without affecting induction of COX-2 expression (Fig. 6A). Unlike AACOCF3-treated cells (Fig. 5, B and C), type IIA sPLA2 expression was not restored when exogenous arachidonic acid was added to NDGA-treated cells (Fig. 6B). These observations are in line with the hypothesis that exogenous arachidonic acid is metabolized to certain LOX products that facilitate type IIA sPLA2 induction in AACOCF3-treated (cPLA2-defective), but not NDGA-treated (LOX-defective), cells. Application of more specific LOX inhibitors, including AA-861 (5-LOX inhibitor), 5,8,11,14-eicosatetraynoic acid (ETYA; 15-LOX inhibitor), and cinnamyl-3,4-dihydroxy-alpha -cyanocinnamate (CDC; 12-LOX inhibitor), revealed that the inhibition of 12- and 15-LOXs led to dose-dependent reduction of sPLA2-IIA expression, whereas the 5-LOX inhibitor was without effect (Fig. 6C).


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Fig. 6.   Effects of LOX inhibitors on IL-1beta /TNFalpha -induced type IIA sPLA2 expression. A, effects of NDGA on PGE2 generation (upper panel) and type IIA sPLA2 and COX-2 protein expression (bottom panel) in 3Y1 cells stimulated for 48 h with IL-1beta /TNFalpha . B, effect of NDGA on type IIA sPLA2 mRNA expression after culture with IL-1beta /TNFalpha for 48 h in the presence or absence of exogenous arachidonic acid. Lane 1, IL-1beta /TNFalpha -stimulated cells; lane 2, cells stimulated with IL-1beta /TNFalpha in the presence of 10 µM NDGA; lane 3, cells stimulated with IL-1beta /TNFalpha in the presence of 10 µM NDGA and 50 µM arachidonic acid. C, effect of various inhibitors directed to 5-, 12-, and 15-LOXs on type IIA sPLA2 mRNA induction after 48-h stimulation with IL-1beta /TNFalpha . The indicated concentrations of each inhibitor were added 5 h before activation with cytokines. Representative results of two to four independent experiments are shown.

Involvement of cPLA2 and COX-1 in Ionophore-stimulated Immediate PGE2 Generation-- When 3Y1 cells were stimulated with 1 µM A23187, immediate PGE2 generation was elicited within 10 min, with PGE2 concentrations in the supernatants reaching approximately 20 ng/ml (Table I). A23187-initiated immediate PGE2 generation by cells pretreated for 12-48 h with IL-1beta /TNFalpha , in which both type IIA sPLA2 and COX-2 proteins had been maximally induced (Fig. 2A), did not differ significantly from that by control cells (Table I). Immediate PGE2 generation was inhibited by AACOCF3 almost completely, but not by NS-398 (Table I). We conclude, therefore, that A23187-induced immediate PGE2 generation by 3Y1 cells is mediated by cPLA2 and COX-1, even when enough type IIA sPLA2 and COX-2 to mediate IL-1beta /TNFalpha -induced delayed PGE2 generation are present in these cells.

                              
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Table I
A23187-induced immediate PGE2 generation by 3Y1 cells
3Y1 cells were cultured for 24 h with or without IL-1beta and TNFalpha , washed, and then activated with 1 µM A23187 for 10 min. NS-398 (1 ng/ml) was added to the cells during culture with cytokines and subsequent activation with A23187, and AACOCF3 (10 µM) was added 2 h before washing the cells and during activation with A23187. The resulting supernatants were taken for PGE2 assay. Under this experimental condition, induction of type IIA sPLA2 and COX-2 expression in cytokine-stimulated cells cultured in the presence of these inhibitors was comparable with that in replicate cells cultured in their absence. Similar results were obtained with cells pretreated for 12 and 48 h with IL-1beta and TNFalpha . Without A23187 stimulation, only <100 pg/well PGE2 was produced by these cells.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Several recent studies have shown that cPLA2 appears to be one of the most important PLA2 isozymes involved in regulating immediate eicosanoid generation, which is triggered by an increase in the cytoplasmic Ca2+ concentration (21, 22, 57-60). cPLA2 has also been implicated in delayed PG generation lasting for hours (12, 23, 24), although the mechanisms that activate cPLA2 under such Ca2+-less conditions remain to be elucidated. Several pieces of evidence have shown that sPLA2 enzymes also regulate certain phases of eicosanoid biosynthesis. Delayed PG generation initiated by proinflammatory stimuli is often accompanied by marked induction of type IIA sPLA2 expression and secretion, in which a relationship between type IIA sPLA2 function and cytokine-stimulated delayed PG generation has been suggested using type IIA sPLA2 inhibitors or anti-type IIA sPLA2 antibodies (31-33, 50, 51). We recently showed that cell surface-associated type IIA sPLA2 introduced forcibly into cells augmented the cytokine-induced, COX-2-dependent delayed PGE2 generation and proposed that cPLA2 and type IIA sPLA2 initiate and enhance delayed eicosanoid biosynthesis, respectively (24, 34). More recently, several investigators demonstrated that type V sPLA2, rather than type IIA sPLA2, plays an important role in relatively rapid eicosanoid biosynthesis in some murine cells expressing type V, but not IIA, sPLA2 (39, 40). These findings, together with the identification of types IIC (41) and X (27) sPLA2s, have provided new insights into the redundant functions of sPLA2 family members, which may explain why some inbred mouse strains in which the type IIA sPLA2 gene is naturally disrupted (61, 62) remain relatively normal throughout their lives.

In the present study, we examined the distinct roles of PLA2 enzymes and their functional coupling with downstream COX enzymes during particular phases of PG biosynthesis by the rat fibroblastic cell line 3Y1. Several lines of evidence suggest that sPLA2 type IIA plays an important role in COX-2-dependent delayed PGE2 generation. First, IL-1beta /TNFalpha -elicited delayed PGE2 generation was accompanied by concordant induction of type IIA sPLA2 and COX-2, with little change in the constitutive expression of cPLA2 and COX-1 and no detectable level of types IIC and V sPLA2 expression. Second, PGE2 generation, which was abolished by the COX-2 inhibitor NS-398, was suppressed markedly by a neutralizing antibody against type IIA sPLA2, by the sPLA2 inhibitor thielocin A1, by heparin, which inhibits type IIA sPLA2-mediated PG biosynthesis by preventing it from associating with cell surfaces (32-34), and by dexamethasone, which suppressed type IIA sPLA2 expression. Third, the antisense oligonucleotide directed to the translation initiation site for type IIA sPLA2 suppressed type IIA sPLA2 expression and reduced delayed PGE2 generation concomitantly. Finally, as mentioned below, cPLA2 or 12-/15-LOX inhibitors blocked type IIA sPLA2 expression, leading to reduced delayed PGE2 generation.

Intriguingly, the cPLA2 inhibitor AACOCF3, which suppressed IL-1beta /TNFalpha -induced PGE2 generation by 3Y1 cells at concentrations comparable to those generally used to assess cPLA2 function in cells (21, 57-59), markedly reduced IL-1beta /TNFalpha -induced type IIA sPLA2 expression without affecting COX-2 expression. The need for deacylation of arachidonate substrate was overridden by evaluating AACOCF3 in the presence of exogenous arachidonic acid, which partially prevented inhibition of type IIA sPLA2 expression by AACOCF3. Moreover, AACOCF3 blocked the [3H]arachidonic acid release from IL-1beta /TNFalpha -stimulated cells that occurred immediately after adding these cytokines. Similar inhibition of [3H]arachidonic acid release was observed with another cPLA2 inhibitor, methylarachidonyl fluorophosphate (12, 63) (data not shown). These results suggest that arachidonic acid released by cPLA2 at the early stage of cytokine stimulation is crucial for the subsequent type IIA sPLA2 induction and that the inhibitory effect of the cPLA2 inhibitor on delayed PGE2 generation may be, at least in part, due to suppression of type IIA sPLA2 expression in 3Y1 cells. Our results are not only in line with several reports that inhibitors or antisense oligonucleotides for cPLA2 suppressed the delayed phase of PG biosynthesis in several cell types (12, 23, 24), but also consistent with the recently proposed hypothesis that prior activation of cPLA2 is necessary for sPLA2 to act (24, 40, 63). That the counteracting effect of exogenous arachidonic acid on inhibition of type IIA sPLA2 expression by AACOCF3 was only partial, even when present in excess, predicts the need for some additional PLA2 metabolites, such as lysophospholipids or their derivatives, in the regulation of type IIA sPLA2 induction. Indeed, we recently found that lysophosphatidylcholine exhibited significant type IIA sPLA2-inducing activity.3 It should be noted that this kind of cross-talk between cPLA2 and type IIA sPLA2 may not be always applicable to other systems, since type IIA sPLA2-independent delayed PGE2 generation does occur in several cells, including those derived from type IIA sPLA2-deficient mice (11, 24, 40). In these cases, some other sPLA2s, such as type V sPLA2 (39, 40), may compensate for type IIA sPLA2, or cPLA2 alone is sufficient to remote delayed PG generation (24).

IL-1beta /TNFalpha -induced type IIA sPLA2 expression and PGE2 generation were also suppressed by inhibitors for 12- or 15-LOXs, but not by those for COX or 5-LOX, suggesting that certain arachidonate metabolites produced via the cPLA2 and 12- or 15-LOX pathway are required for type IIA sPLA2 induction. Although in our preliminary experiments the addition of 12-hydroxyeicosatetraenoic acid to 3Y1 cells restored sPLA2-IIA expression to some extent,3 further studies should be awaited to determine the precise roles of the particular 12-/15-LOX metabolites in type IIA sPLA2 expression. The importance of some LOX pathway products in the homeostasis of mammalian cells has been suggested by several studies. For instance, Honn and co-workers (64) recently reported that 12-/15-LOX metabolites were essential regulators of cell survival and apoptosis. Rao et al. (43) have shown that 12- and 15-hydroperoxyeicosatetraenoic acids induced the expression of c-fos and c-jun that form the transcription factor AP-1 in vascular smooth muscle cells. Some growth factors require arachidonic acid release and metabolism through the LOX pathway for induction of growth in certain cell types (65, 66). The proliferator-activated receptors, which were originally identified as a group of transcription factors that regulate gene expression of enzymes associated with lipid homeostasis (67), have been shown to be the nuclear receptors for particular arachidonate metabolites, such as J-series PGs (68) and leukotriene B4 (69).

In contrast to cytokine-induced delayed PGE2 generation, which requires both type IIA sPLA2 and cPLA2 that are linked cooperatively to COX-2 but not to COX-1, ionophore-stimulated immediate PGE2 generation is regulated predominantly by constitutive cPLA2 and COX-1, even when type IIA sPLA2 and COX-2 are maximally induced. Dissociation of endogenous type IIA sPLA2 from the immediate response is consistent with several previous observations (34, 70, 71), although there are some exceptions, particularly when excess exogenous type IIA enzyme is added to agonist-primed cells, in which it elicits the immediate response (72-74). In this regard, the role of type IIA sPLA2 in 3Y1 cells appears to differ from that of type V sPLA2 in certain mast cells, in which type V enzyme is reported to be crucial for immediate, rather than delayed, PGD2 generation (40). The failure of COX-2 to mediate the immediate response in 3Y1 cells is in line with the observation that IgE/antigen-mediated immediate PGD2 generation by mouse bone marrow-derived mast cells was entirely dependent upon COX-1, even when COX-2 is present in these cells following cytokine priming (2, 75). In contrast, we recently found that IL-1alpha -primed mouse calvarial osteoblasts (8) and lipopolysaccharide-primed rat peritoneal macrophages2 elicited immediate PGE2 generation in response to secondary Ca2+-mobilizing stimuli that was dependent upon COX-2 rather than COX-1. Therefore, it is likely that utilization of COX isoforms in the immediate response, relative to the absolute dependence of the delayed response on COX-2 (2-4, 6), appears to be cell type- or stimulus-specific and may reflect the presence of some particular machineries, such as different intracellular localizations (9), separate intracellular transport of arachidonic acid, the presence of presumptive COX isoform-selective co-regulatory factors (76), and selective coupling with the downstream terminal PG synthases (13).

In summary, we have reconfirmed that inducible type IIA sPLA2 is essential for optimal delayed PGE2 biosynthetic pathway, in which it is functionally linked to inducible COX-2, in 3Y1 cells. Furthermore, our results suggest that cPLA2 contributes to delayed PGE2 generation at least partly through the induction of type IIA sPLA2 via the 12-/15-LOX pathway. These results are in line with our recent findings that cPLA2 and COX-2 are functionally linked in cells derived from type IIA sPLA2-deficient mice and forced introduction of type IIA sPLA2 into these cells augmented the delayed PGE2 biosynthetic response (24). In contrast, immediate PGE2 generation is mediated predominantly by the constitutive enzymes cPLA2 and COX-1. These observations reveal the particular routes for functional cross-talk between and segregation of the constitutive and inducible enzymes in the different phases of PG biosynthetic pathway.

    ACKNOWLEDGEMENTS

We thank S. Shimbara, K. Tada, and T. Ando for their technical assistance; Drs. J. D. Clark, W. L. Smith, J. Trzaskos, J. A. Tischfield, J. P. Arm, and T. Yoshida for providing cDNAs, antibodies, and inhibitors; and Drs. Y. Uehara and H. Ishimaru for providing 3Y1 cells and recombinant TNFalpha , respectively.

    FOOTNOTES

* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and by 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.

1 The abbreviations used are: PG, prostaglandin; PLA2, phospholipase A2; sPLA2, secretory PLA2; cPLA2, cytosolic PLA2; COX, cyclooxygenase; LOX, lipoxygenase; IL, interleukin; TNFalpha , tumor necrosis factor alpha ; TBS-T, Tris-buffered saline containing 0.05% Tween 20; FCS, fetal calf serum; DMEM, Dulbecco's modified Eagle medium, RT-PCR, reverse transcriptase-polymerase chain reaction; PCR, polymerase chain reaction; AACOCF3, arachidonoyl trifluoromethyl ketone; NDGA, nordihydroguaiaretic acid.

2 H. Naraba, M. Murakami, H. Matsumoto, S. Shimbara, I. Kudo, A. Ueno, and S. Oh-ishi, submitted for publication.

3 H. Kuwata, M. Murakami, Y. Nakatani, and I. Kudo, unpublished observation.

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Discussion
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