Cellular Arachidonate-releasing Function of Novel Classes of Secretory Phospholipase A2s (Groups III and XII)*

Makoto MurakamiDagger §, Seiko MasudaDagger , Satoko ShimbaraDagger , Sofiane Bezzine||, Michael Lazdunski||, Gérald Lambeau||, Michael H. Gelb**, Satoshi Matsukura||, Fumio Kokubu||, Mitsuru Adachi||, and Ichiro KudoDagger

From the Dagger  Department of Health Chemistry, School of Pharmaceutical Sciences, and the  First Department of Internal Medicine, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan, the ** Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195-1700, and the || Institut de Pharmacologie Moleculaire et Cellulaire, CNRS-UPR 411, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France

Received for publication, November 6, 2002, and in revised form, December 19, 2002

    ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Here we report cellular arachidonate (AA) release and prostaglandin (PG) production by novel classes of secretory phospholipase A2s (sPLA2s), groups III and XII. Human group III sPLA2 promoted spontaneous AA release, which was augmented by interleukin-1, in HEK293 transfectants. The central sPLA2 domain alone was sufficient for its in vitro enzymatic activity and for cellular AA release at the plasma membrane, whereas either the unique N- or C-terminal domain was required for heparanoid-dependent action on cells to augment AA release, cyclooxygenase-2 induction, and PG production. Group III sPLA2 was constitutively expressed in two human cell lines, in which other sPLA2s exhibited different stimulus inducibility. Human group XII sPLA2 had a weak enzymatic activity in vitro and minimally affects cellular AA release and PG production. Cells transfected with group XII sPLA2 exhibited abnormal morphology, suggesting a unique functional aspect of this enzyme. Based on the present results as well as our current analyses on the group I/II/V/X sPLA2s, general properties of cellular actions of a full set of mammalian sPLA2s in regulating AA metabolism are discussed.

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INTRODUCTION
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Secretory phospholipase A2 (sPLA2)1 comprises a family of Ca2+-dependent lipolytic enzymes with a conserved Ca2+-binding loop and His-Asp dyad at the catalytic site (1, 2). To date, 10 sPLA2 enzymes (groups IB, IIA, IIC, IID, IIE, IIF, V, X, III, and XII) have been identified in mammals (1, 2). In general, sPLA2s exhibit tissue- and species-specific expression, which suggests that their cellular behaviors and functions differ.

The group I/II/V/X sPLA2s represent a class of enzymes with a molecular mass of 14-18 kDa and 6-8 conserved disulfides (1, 2). sPLA2-IB and -X, but not the other enzymes, have an N-terminal prepropeptide, and the proteolytic cleavage of this prepropeptide is a regulatory step for generation of an active enzyme (3, 4). sPLA2-IB is abundantly present in pancreatic juice, and its main function has been thought to be the digestion of dietary phospholipids, although recent data with sPLA2-IB knockout mice have demonstrated no appreciable defects in this process (5). sPLA2-IIA, a prototypic inflammatory PLA2, and other group II subfamily sPLA2s (IID, IIE, IIF, and V) are inducible in various tissues with inflammation or damage (6-13). On the bases of current biochemical and cell biological studies, sPLA2s in the I/II/V/X branch may participate in various biological events, including arachidonate (AA) release from cellular membranes (see below) (9-18), host defense against bacteria (19, 20), atherosclerosis (21, 22), blood coagulation (23), and cancer (24).

Beyond the essential role of cytosolic PLA2alpha (cPLA2alpha ) in the initiation of stimulus-coupled AA metabolism, the I/II/V/X class of sPLA2s also has the ability to augment AA metabolism by multiple mechanisms (1). sPLA2s that show high interfacial binding to zwitterionic phosphatidylcholine (PC), such as sPLA2-X and -V, are capable of releasing AA from the PC-rich outer leaflet of the plasma membrane of quiescent cells (the external plasma membrane pathway) (17, 25-27). Cationic, heparin-binding, group II subfamily sPLA2s, such as sPLA2-IIA, -IID, -IIE, and -V, show marked preference for anionic phospholipids over PC and utilize the heparan sulfate proteoglycan (HSPG)-shuttling pathway (14-18, 28). In this regulatory pathway, these enzymes are captured by HSPGs (typically glypican, a glycosylphosphatidylinositol-anchored HSPG) in caveolae or rafts on activated cells and then internalized into vesicular membrane compartments that are enriched in the perinuclear area, where downstream cyclooxygenases (COXs) are located (16, 17, 28). This spatiotemporal co-localization of sPLA2s and COXs in the perinuclear compartments may allow efficient supply of AA between these enzymes. Recent evidence implies that the clathrin-independent, caveolae/raft-mediated endocytosis, and associated vesicular traffic is directed toward a rapid cycling pathway via the Golgi and endoplasmic reticulum (29). Occurrence of the HSPG-shuttling pathway appears to be cell type- and stimulus-specific, and in certain cases HSPGs exhibit a negative regulatory effect on the heparin-binding sPLA2s by facilitating their internalization and subsequent lysosomal degradation (30, 31). sPLA2-IIF, an anionic group II subfamily sPLA2 with poor affinity for HSPG, may interact stably with the plasma membrane through its unique C-terminal extension and releases AA (11), although the possibility that this enzyme also functions after internalization cannot be ruled out. In addition, cellular actions of several sPLA2s can be mediated by sPLA2 receptors independent of their enzymatic activity (32). Targeted disruption of the M-type sPLA2 receptor gene results in reduced inflammatory response in mice (33). The M-type sPLA2 receptor can also act as a negative regulator for sPLA2s by inhibiting their enzymatic functions in serum and by promoting their internalization and subsequent degradation (34, 35).

Besides the I/II/V/X branch, two distinct classes of sPLA2, namely the group III and group XII branches, have been recently identified in mammals (36-38). Structurally, these two novel sPLA2s show homology with the I/II/V/X sPLA2s only within the Ca2+ loop and catalytic site His-Asp dyad. Human sPLA2-III is a 56-kDa protein containing a long N-terminal domain, a central sPLA2 domain that is homologous to bee venom group III sPLA2, and a long C-terminal domain (36). sPLA2-XII, which harbors an unusual Ca2+ loop, is distantly related to other classes of sPLA2s (37, 38). This enzyme is expressed in antigen-activated helper T cells in the mouse (38). However, cellular functions of these two novel sPLA2s have not yet been described. To expand our current understanding of the sPLA2 actions on cells, we studied the cellular AA-releasing and prostaglandin (PG)-biosynthetic properties of human sPLA2-III and -XII by expressing these enzymes in HEK293 cells, as we have previously done with the I/II/V/X sPLA2s (9-12, 14-18).

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Materials-- Human embryonic kidney 293 (HEK293) cells (Human Science Research Resources Bank), human lung epithelial BEAS-2B cells (American Type Cell Collection, Manassas, VA), and human colon adenocarcinoma HCA-7 cells (a generous gift from Dr. M. Tsujii (Osaka University) and Dr. R. DuBois (Vanderbilt University Medical Center and VA Medical Center)) were cultured in RPMI 1640 medium (Nissui Pharmaceutical Co.) containing 10% (v/v) fetal calf serum (FCS; Bioserum). The cDNAs for human sPLA2-III (36), human sPLA2-XII (37), human COX-1 and COX-2 (15), and rat glypican-1 (16) were described previously. HEK293 cells stably expressing human sPLA2-V, human sPLA2-IIF, and human COX-2 were described previously (11, 14, 15). The enzyme immunoassay kits for PGE2 and the COX-2 inhibitor NS-398 were purchased from Cayman Chemicals. The goat anti-human COX-1 and anti-human COX-2 antibodies were purchased from Santa Cruz Biosciences, Inc. (Santa Cruz, CA). A23187 was purchased from Calbiochem. Human interleukin (IL)-1beta , interferon (IFN)-gamma , and tumor necrosis factor alpha  (TNF-alpha ) were purchased from Genzyme. LipofectAMINE 2000 reagent, Opti-MEM medium, TRIzol reagent, geneticin, zeocin, and mammalian expression vectors (pCR3.1, pRc-CMV, and pcDNA3.1 series of vectors containing a neomycin- or zeocin-resistant gene) were obtained from Invitrogen. Fluorescein isothiocyanate-conjugated anti-mouse and anti-rabbit IgGs and horseradish peroxidase-conjugated anti-goat IgG were purchased from Zymed Laboratories Inc.. Mouse monoclonal anti-FLAG antibody, anti-FLAG antibody-conjugated agarose, and heparin were from Sigma. The lipoxygenase-inhibitory antioxidant nordihydroguaiaretic acid (NDGA) was purchased from BIOMOL. Heparin-Sepharose was purchased from Amersham Biosciences. Rabbit antiserum for human sPLA2-XII was prepared as described previously (39).

Establishment of Transfectants-- Establishment of HEK293 transformants was performed as described previously (12-16). Briefly, 1 µg of plasmid (sPLA2 cDNAs subcloned into the pRc-CMV or pCR3.1 vector) was mixed with 2 µl of LipofectAMINE 2000 in 100 µl of Opti-MEM medium for 30 min and then added to cells that had attained 40-60% confluence in 12-well plates (Iwaki Glass) containing 0.5 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 1 ml of fresh culture medium. After overnight culture, the medium was replaced with 1 ml of fresh medium, and culture was continued at 37 °C in an incubator flushed with 5% CO2 in humidified air. The cells were cloned by limiting dilution in 96-well plates in culture medium supplemented with 1 mg/ml geneticin. After culture for 3-4 weeks, wells containing a single colony were chosen, and the expression of each protein was assessed by RNA blotting. The established clones were expanded and used for the experiments as described below.

In order to establish double transformants expressing sPLA2 and glypican, cells expressing each sPLA2 were subjected to a second transfection with glypican cDNA subcloned into pCDNA3.1/Zeo(+) using LipofectAMINE 2000. Three days after the transfection, the cells were used for the experiments or seeded into 96-well plates and cloned by culturing in the presence of 50 µg/ml zeocin to establish stable transformants.

To assess functional coupling between sPLA2 and either of the two COX isozymes, cells stably expressing sPLA2 were transfected with COX-1 or COX-2 subcloned into pCDNA3.1 using LipofectAMINE 2000. Three days after the transfection, the cells were activated with A23187 to measure PGE2 generation and were subjected to immunoblotting to examine COX-1 or COX-2 expression (see below).

Measurement of sPLA2 Activity-- sPLA2 activity was assayed by measuring the amounts of radiolabeled fatty acids released from the substrate 1-palmitoyl-2-[14C]arachidonoyl-phosphatidylethanolamine (2-AA-PE), 1-palmitoyl-2-[14C]linoleoyl-PE (2-LA-PE), 2-AA-PC, or 2-LA-PC (Amersham Biosciences). Each substrate in ethanol was dried up under N2 stream and was dispersed in water by sonication. Each reaction mixture (total volume 250 µl) consisted of appropriate amounts of the required sample, 100 mM Tris-HCl (pH 7.4), 4 mM CaCl2, and 10 µM substrate. After incubation for 10-30 min at 37 °C, [14C]AA or [14C]LA was extracted, and radioactivity was quantified, as described previously (14-18).

Expression of Recombinant sPLA2s by the Baculovirus System-- Baculovirus expression of recombinant sPLA2 proteins was performed using the BAC-to-BAC baculovirus expression system (Invitrogen). Briefly, sPLA2 cDNAs were subcloned into the baculovirus expression vector pFASTBAC1 (Invitrogen) at appropriate restriction enzyme sites. Recombinant sPLA2 proteins were first expressed in Sf9 insect cells and then amplified in High Five insect cells (Invitrogen) according to the manufacturer's instructions. Culture supernatants and cell lysates (4-5 days after infection) were used for subsequent experiments. Sf9 cells and High Five cells were maintained in Grace's insect medium (Invitrogen) supplemented with 10% FCS and Express Five SFM serum-free medium (Invitrogen), respectively.

Heparin Binding-- Recombinant sPLA2s (culture supernatants from baculovirus-infected High Five cells) were incubated with various amounts of heparin-Sepharose beads in 10 mM Tris-HCl (pH 7.4) containing 150 mM NaCl (TBS) for 2 h at 4 °C, and PLA2 activities remaining in the supernatants were assayed.

RNA Blotting-- Approximately equal amounts (~5 µg) of total RNA obtained from the cells were applied to separate lanes of 1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore Corp.). The resulting blots were then probed with the respective cDNA probes that had been labeled with [32P]dCTP (Amersham Biosciences) by random priming (Takara Biomedicals). All hybridizations were carried out as described previously (14-18).

SDS-PAGE/Immunoblotting-- Lysates from 105 cells were subjected to SDS-PAGE using 7.5-12.5% gels under reducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schuell) using a semidry blotter (MilliBlot-SDE system; Millipore). After blocking with 3% (w/v) skim milk in TBS containing 0.05% Tween 20 (TBS-Tween), the membranes were probed with the respective antibodies (1:20,000 dilution for COX-1, 1:5,000 dilution for COX-2, and 1:20,000 for FLAG epitope in TBS-Tween) for 2 h, followed by incubation with horseradish peroxidase-conjugated anti-goat (for COXs) or anti-mouse (for FLAG) IgG (1:5,000 dilution in TBS-Tween) for 2 h and were visualized using the ECL Western blot system (PerkinElmer Life Sciences) (14-18).

Reverse Transcription (RT)-PCR-- Synthesis of cDNA was performed using 0.5 µg of total RNA from human cell lines and avian myeloblastosis virus reverse transcriptase, according to the manufacturer's instructions supplied with the RNA PCR kit (Takara Biomedical). Subsequent amplification of the cDNA fragments was performed using 1 µl of the reverse-transcribed mixture as a template with specific primers for each sPLA2. For amplification of sPLA2-IB, -IIA, -IID, -IIE, -IIF, -V, -X, and -XII, a set of 23-bp oligonucleotide primers corresponding to 5'- and 3'-nucleotide sequences of their open reading frames were used as primers (40-45). For amplification of sPLA2-III, primers directed for the sPLA2 domain and N-terminal domain were used (see below). The PCR condition was 94 °C for 30 s and then 35 cycles of amplification at 94 °C for 5 s and 68 °C for 4 min, using the Advantage cDNA polymerase mix (Clontech). The PCR products were analyzed by 1% agarose gel electrophoresis with ethidium bromide. The gels were further subjected to Southern blot hybridization using sPLA2 cDNAs as probe.

Construction of sPLA2-III Mutants-- sPLA2-III mutants were produced by PCR with the Advantage cDNA polymerase mix using sPLA2-III/pRc-CMV as a template. The condition of PCR was 25 cycles at 94, 55, and 72 °C for 30 s each. The primers used were as follows: III-5' primer (5'-ATGGGGGTTCAGGCAGGGCTG-3'), III-3' primer (TCACTGGCTCCAGGACTTCTG-3'), III-S-S primer (5'-GATGGACCATGCCTGGCACAC-3'), III-S-AS primer (TCAAGTTGGGGAGGTGGCCCG-3'), III-HQ-S primer (5'-TGCCGGGAACAAGACCGCTGC-3'), and III-HQ-AS primer (5'-GCAGCGGTCTTGTTCCCGGCA-3'). In order to obtain sPLA2-III wild type (WT) and the truncated mutants III-S, III-N+S, and III-S+C (see "Results"), the primer sets III-5' and III-3', III-S-S and III-S-AS, III-5' and III-S-AS, and III-S-S and III-3', respectively, were used. In order for the mutants III-S and III-S+C to be secreted when expressed in cells, the signal sequence for human group IIA sPLA2 (40) was linked to the 5'-end of the III-S-S primer and PCR-amplified. To attach the FLAG epitope at the C terminus, the FLAG antisense oligonucleotide 5'-TTACTTGTGATCGTCGTCCTTGTAGTC-3' were directly linked to the 5'-ends of the antisense primers. In order to construct the catalytically inactive mutant III-N+S-HQ (see "Results"), the first PCR was conducted with III-5' and III-HQ-AS primers or with III-HQ-S and III-S-AS primers using sPLA2-III-WT cDNA as a template. The resulting two primary PCR fragments were mixed, 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. The secondary PCR product with specific mutation was obtained after 25 additional PCR cycles with III-5' and III-S-AS primers. A similar strategy was used to prepare III-S-HQ. 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 Biomedicals) and an autofluorometric DNA sequencer 310 Genetic Analyzer (Applied Biosystems) to confirm the sequences.

Activation of HEK293 Cells-- HEK293 cells (5 × 104/ml) were seeded into each well of 48-well plates. To assess fatty acid release (14-18), [3H]AA or [3H]oleic acid (OA) (both from Amersham Biosciences) (0.1 µCi/ml) was added to the cells in each well on day 3, when they had nearly reached confluence, and culturing was continued for another day. After three washes with fresh medium, 100 µl of RPMI 1640 with or without 10 µM A23187 with 1% FCS or 1 ng/ml IL-1beta and/or 10% FCS was added to each well, and the amount of free [3H]AA or [3H]OA released into the supernatant was measured. The percentage release was calculated using the formula (S/(S + P)) × 100, where S and P represent the radioactivity measured in the supernatant and cell pellet, respectively. The supernatants from replicate cells were subjected to the PGE2 enzyme immunoassay.

To assess transcellular PGE2 biosynthesis (11, 15), two cell populations (2.5 × 104 cells/ml for each) were added to the same wells of 48-well plates (100 µl/well) and cultured for 4 days. Then the cells were stimulated with IL-1beta in medium containing 10% FCS for 4 h, and PGE2 released into the supernatants was quantified.

Exogenous sPLA2 Assay-- Subconfluent cells grown in 48-well plates were incubated with recombinant sPLA2s (culture supernatants from baculovirus-infected High Five cells) for 1 h, and PGE2 released into the supernatants was quantified.

Confocal Laser Microscopy-- Cells grown on collagen-coated cover glasses (Iwaki Glass) were fixed with 3% paraformaldehyde for 30 min in phosphate-buffered saline (PBS). After three washes with PBS, the fixed cells were sequentially treated with 1% (w/v) bovine serum albumin (for blocking) and 0.2% (v/v) Triton X-100 (for permeabilization) in PBS for 1 h, with anti-FLAG antibody (1:500 dilution) for 1 h in PBS containing 1% albumin and then with fluorescein isothiocyanate-goat anti-mouse IgG (1:500 dilution) for 1 h in PBS containing 1% albumin. After six washes with PBS, the cells were mounted on glass slides using Perma Fluor (Japan Tanner), and the sPLA2 signal was visualized using a laser-scanning confocal microscope (IX70; Olympus), as described previously (16, 18).

Statistical Analysis-- Data were analyzed by Student's t test. Results are expressed as the mean ± S.E., with p = 0.05 as the limit of significance.

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Human sPLA2-III

Enzymatic Properties-- Human sPLA2-III-WT and its truncated mutants, III-S, III-N+S, and III-S+C, (structures illustrated in Fig. 1A) were each transfected into HEK293 cells. Stable transfectants expressing WT and truncated enzymes, with or without C-terminal FLAG epitope, were screened by Northern blotting using a specific sPLA2-III cDNA probe (Fig. 1B) or immunoblotting using an anti-C-terminal FLAG tag antibody (Fig. 1C), and clones in which their expression levels were almost comparable with one another were used in subsequent studies. As shown in Fig. 1C, III-WT, III-N+S, III-S+C, and III-S were expressed as major immunoreactive proteins with predicted molecular masses of 56, 32, 42, and 17 kDa, respectively. Flanking the C terminus with the FLAG epitope did not significantly affect in vitro and cellular functions, as described below.


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Fig. 1.   Expression of the WT and truncated forms of sPLA2-III in HEK293 cells. A, structures of the WT and truncated forms (S, N+S, and S+C) of sPLA2-III. In the case of III-S and III-S+C, a signal peptide for human sPLA2-IIA was fused at their N termini. The C terminus of each protein was tagged with the FLAG epitope as required for the experiments. B and C, expression levels of the WT and truncated forms of sPLA2-III in their HEK293 transfectants were assessed by Northern blotting (5 µg of total RNA per lane) (B) and in the case of FLAG-tagged enzymes by SDS-PAGE/immunoblotting (105 cell equivalents/lane) (C). D, in vitro enzymatic activity of the WT and truncated forms of sPLA2-III expressed in HEK293 transfectants. Aliquots (5-20 µl) of the culture supernatants were taken for PLA2 assay using 2-AA-PE, 2-AA-PC, 2-LA-PE, and 2-LA-PC as substrates. Values (percentage of substrate hydrolysis equivalent to 107 cells) are means ± S.E. of five independent experiments.

Culture supernatants of these transfectants were assayed for PLA2 activity using PE and PC bearing AA or LA at their sn-2-position as substrates. Under our PLA2 assay condition, the WT and truncated enzymes exhibited comparable PLA2 activity with similar substrate specificity (Fig. 1D). Of the four substrates tested, 2-LA-PE was the best substrate, being hydrolyzed 2-3 and 6-8 times more efficiently than 2-LA-PC and 2-AA-PE, respectively. 2-AA-PC was hydrolyzed ~2-fold faster than 2-AA-PE, whereas 2-LA-PE was hydrolyzed ~2-fold faster than 2-LA-PC.

Cellular Functions-- To assess the fatty acid-releasing function of sPLA2-III in cells, sPLA2-III-WT-transfected and control HEK293 cells were preincubated overnight with [3H]AA or [3H]OA, washed, and then cultured for 4 h with or without 10% FCS and/or IL-1. As shown in Fig. 2A, in the presence of 10% FCS, the WT enzyme significantly increased the release of both [3H]AA and [3H]OA almost in parallel. The further addition of IL-1 resulted in an increase in [3H]AA, but not [3H]OA, release (Fig. 2A). Comparing these properties of sPLA2-III with other sPLA2s reported so far, FCS-dependent, fatty acid nonselective release is similar to that by the plasma membrane-acting enzymes, such as sPLA2-X and -V, and IL-1 augmentation of AA release is reminiscent of that by the HSPG-shuttled enzymes, such as sPLA2-IIA, -IID, -IIE, and -V (9, 10, 14-18).


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Fig. 2.   Cellular fatty acid release by sPLA2-III. A, control or sPLA2-III-WT-expressing cells, which were prelabeled with [3H]AA or [3H]OA, were incubated for 4 h with 1% (-) or 10% (+) FCS with (+) or without (-) 1 ng/ml IL-1beta to assess the release of these fatty acids. B and C, comparison of the [3H]AA-releasing property between the WT and truncated (S, N+S, and S+C) sPLA2-III. [3H]AA-prelabeled cells were cultured for 4 h in varied combinations of FCS and IL-1beta (B) or for the indicated periods in the presence of 10% FCS plus 1 ng/ml IL-1beta (IL-1/FCS) (C). D, [3H]AA release by III-N+S, III-S, and their point mutants (HQ), in which the catalytic center His was replaced with Gln, following 4-h incubation with IL-1/FCS. Values are mean ± S.E. of 3-5 independent experiments in A, B, and D, and a representative result of three independent experiments is shown in C.

In agreement with the in vitro enzymatic activity (Fig. 1), the release of [3H]AA (Fig. 2B) and [3H]OA (data not shown) by cells transfected with the three truncated enzymes (III-S, III-N+S, and III-S+C) was similar to that by cells transfected with the WT enzyme. As shown in Fig. 2C, [3H]AA release proceeded gradually over 8 h of culture. Thus, the sPLA2 domain alone is essential and sufficient for cellular fatty acid release. As a notable difference, [3H]AA release by cells expressing III-WT, III-N+S, or III-S+C was significantly augmented by IL-1, whereas this augmentation was not observed appreciably in cells expressing III-S (i.e. the sPLA2 domain alone) (Fig. 2B). When III-N+S-HQ and III-S-HQ, in which the putative catalytic center His in III-N+S and III-S was, respectively, replaced by Gln, were transfected into HEK293 cells, no in vitro PLA2 activity was detected (data not shown), and an increase in cellular [3H]AA release was not observed (Fig. 2D), despite their reasonable expression levels (data not shown). This result indicates that the catalytic activity is an absolute requirement for the enzymatic action of sPLA2-III on both phospholipid vesicles and cellular membranes.

The AAs released by III-WT, -N+S, and -S+C following IL-1 stimulation were each efficiently converted to PGE2 (Fig. 3A). This PGE2 production was ablated by the COX-2 inhibitor NS-398 (data not shown), revealing functional coupling between sPLA2-III and COX-2 in the IL-1-stimulated delayed response. Remarkably, PGE2 production by cells transfected with III-S was less than that by cells transfected with III-WT, -N+S, and -S+C (Fig. 3A), despite the fact that all four proteins produced similar amounts of AA (Fig. 2B). Kinetic experiments demonstrated that PGE2 production by III-WT increased linearly over 1-8 h, thus lagging behind AA release (Fig. 2C), whereas PGE2 production by III-S was increased only modestly over the whole culture period (Fig. 3B). As shown in the top panel of Fig. 3A, III-WT, III-N+S, and III-S+C augmented IL-1-induced expression of COX-2 markedly relative to replicate control cells, whereas COX-2 induction by III-S was less than that by III-WT, -N+S, and -S+C (although it was still higher than control cells). Time course experiments showed that COX-2 mRNA induction in III-WT-expressing cells peaked at 1 h and declined to a plateau level after 4-8 h, whereas that in control and III-S-expressing cells reached a peak by 1 h and disappeared thereafter (Fig. 3B, inset). These results suggest that sPLA2-III has the ability to enhance COX-2 expression, as in the case of several HSPG-binding group II subfamily sPLA2s (16-18), and that the poor PGE2-biosynthetic action of the sPLA2 domain alone is, at least in part, due to its poor ability to induce COX-2.


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Fig. 3.   Coupling between sPLA2-III and COXs. A, PGE2 generation by parent HEK293 cells and cells transfected with the WT or truncated (S, N+S, and S+C) sPLA2-III after 4 h of incubation with IL-1/FCS. Endogenous COX-2 mRNA expression in the presence (+) or absence (-) of IL-1beta was assessed by Northern blotting (top). B, time course of PGE2 production by parental cells (open circles) and cells transfected with III-WT (closed circles) or III-S (closed squares) after incubation with IL-1/FCS. COX-2 mRNA expression in these cells at each time point, assessed by Northern blotting, is shown in the inset. C, parental and III-WT-transfected cells were transfected with mock (-) and COX-1 or COX-2 (+) plasmids (1 µg). Three days after transfection, the cells were stimulated with 10 µM A23187 for 30 min to assess PGE2 release. D, parental cells and cells expressing III-WT or III-S were transfected with the indicated amounts of the COX-1 plasmid. Three days after transfection, A23187-stimulated PGE2 production was assessed. COX-1 protein expression, as assessed by immunoblotting, is shown in the top panel. E, [3H]AA release by parental cells (open circles) and cells expressing III-WT (closed circles) or III-S (closed squares) after stimulation with A23187 over 30 min. F, transcellular PGE2 production. Parental cells and cells transfected with III-WT, III-S, or human sPLA2-IIF were cocultured with COX-2-transfected (+) or parental (-) HEK293 cells, and PGE2 production after 4-h incubation with IL-1/FCS was assessed. Values are mean ± S.E. of three independent experiments in A, E, and F, and a representative result of 2-4 reproducible experiments is shown in B-D.

To elucidate functional coupling between sPLA2-III and COX enzymes more directly, we performed cotransfection experiments, in which HEK293 cells expressing sPLA2-III were subsequently transfected with either COX-1 or COX-2, and PGE2 production following A23187 stimulation was examined. Expression of COX-1 and COX-2 was verified by immunoblotting (data not shown). As shown in Fig. 3C, PGE2 production by either COX-1 or COX-2 was markedly augmented by coexpression of sPLA2-III-WT. When the PGE2-biosynthetic activities of III-WT and -S were compared in this cotransfection analysis, PGE2 production by III-WT via the overexpressed COX-1 (Fig. 3D) or COX-2 (data not shown) was significantly higher than that produced by III-S, although A23187-induced immediate [3H]AA releases by III-WT and -S were comparable over 30 min of incubation period (Fig. 3E). These results suggest that the sPLA2 domain alone is coupled with downstream COX enzymes less efficiently than is III-WT, even if COX enzymes are equivalently expressed in cells. III-N+S and III-S+C were coupled with the overexpressed COX-1 with the same potency as III-WT (data not shown), indicating that either the N- or C-terminal domain can confer onto the sPLA2 domain the ability to efficiently couple to COX enzymes.

We next performed the transcellular PGE2-biosynthetic assay (11, 15), in which sPLA2-III-expressing and COX-2-expressing HEK293 cells were cocultured. As was observed with sPLA2-IIF used as a positive control (11), coculture of sPLA2-III-WT-expressing cells with COX-2-expressing cells resulted in a marked increase in the production of PGE2 (Fig. 3F), indicating that the WT enzyme is capable of supplying AA to COX-2 in neighboring cells to propagate PGE2 production in a paracrine manner. Coculturing sPLA2-III-S-expressing cells with COX-2-expressing cells also increased PGE2 production significantly, yet the amount of PGE2 produced by III-S was again reproducibly lower than that produced by the WT enzyme (Fig. 3F).

Heparanoid Dependence-- When recombinant FLAG-tagged sPLA2-III-S, -N+S, and -S+C, which were expressed by the baculovirus/insect cell system (see below), were incubated with incremental amounts of heparin-conjugated beads, III-N+S and III-S+C were more efficiently absorbed than III-S to the beads (Fig. 4A, left) under the condition where they were equally precipitated by anti-FLAG antibody-conjugated beads (Fig. 4A, right), indicating that both N- and C-terminal domains facilitate the binding of sPLA2-III to heparanoids. To assess whether sPLA2-III action on cells depends on cell surface HSPG, as does the HSPG-shuttled group II subfamily sPLA2s (14-18), the effect of exogenous heparin, which solubilizes the HSPG-bound pool of sPLA2s and thereby suppresses their cellular functions (14-18), was examined. As shown in Fig. 4B, AA release by sPLA2-III-WT in the absence of IL-1 (i.e. FCS-dependent release) was insensitive to exogenous heparin, whereas IL-1-augmented AA release was reversed by heparin to a level comparable with FCS-dependent release (Fig. 4B, left). This heparin effect resembled its effect on sPLA2-V-mediated AA release, where IL-1-stimulated (reflection of the HSPG-shuttling route), but not FCS-dependent (reflection of the plasma membrane route), AA release was suppressed by heparin (Fig. 4B, right). AA release by III-S, which was largely unaffected by IL-1 as noted above (Fig. 2), was unaffected by exogenous heparin (Fig. 4B, middle). Moreover, IL-1-stimulated PGE2 production by WT-expressing cells was reduced to a level comparable with that by III-S-expressing cells following heparin treatment (Fig. 4C). In contrast, the inhibitory effect of heparin on PGE2 production by III-S-expressing cells was only minimal (Fig. 4C).


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Fig. 4.   Heparanoid dependence of sPLA2-III-mediated AA metabolism. A, absorption of insect cell-derived recombinant FLAG-tagged sPLA2-III (S, N+S, and S+C) by heparin- or anti-FLAG antibody-conjugated beads. The culture supernatants of baculovirus-infected High Five cells were diluted with TBS (5-fold for S+C and 50-fold for S and N+S to adjust their enzyme activities to be equivalent) and mixed with heparin- or anti-FLAG antibody-conjugated beads. After 2-h incubation, remaining PLA2 activities in the supernatants were measured. B and C, effect of exogenous heparin on cellular AA release. [3H]AA-prelabeled HEK293 cells expressing sPLA2-III-WT or sPLA2-III-S and human sPLA2-V were incubated for 5 h with 0.5 mg/ml heparin and then cultured for an additional 4 h in medium containing 10% FCS with (+) or without (-) IL-1 in the continued presence of heparin to assess [3H]AA release (B) and PGE2 production (C). D and E, effect of glypican coexpression. HEK293 cells expressing suboptimal levels of sPLA2-III-WT or III-S were subjected to second transfection with glypican. The expression of III-WT, III-S, and glypican was as- sessed by Northern blotting (D). These cells were prelabeled with [3H]AA and analyzed for IL-1/FCS-dependent [3H]AA release (top) and PGE2 production (bottom) after 4-h treatment with IL-1/FCS. F, effect of NDGA. HEK293 cells expressing III-WT or III-S were cultured for 4 h with IL-1/FCS in the presence (+) or absence (-) of 10 µM NDGA, and PGE2 released into the supernatants was quantified. Values are mean ± S.E. of 3-5 independent experiments.

Since the function of the HSPG-shuttled group II subfamily sPLA2s is augmented by overexpression of glypican, a glycosylphosphatidylinositol-anchored HSPG that acts as a functional adaptor for these sPLA2s (16, 18), we next assessed the effect of glypican coexpression on sPLA2-III-mediated AA metabolism. Expression of glypican in III-WT- or III-S-expressing cells was verified by Northern blotting (Fig. 4D). As shown in Fig. 4E, coexpression of glypican markedly enhanced AA release and PGE2 production by the WT enzyme. AA release and PGE2 production by III-S were increased by glypican to a much lesser extent (Fig. 4E). A small augmentation of III-S function by glypican overexpression may be due to its weak affinity for heparanoids (Fig. 4A, left) or to some other unknown mechanisms. Overexpression of glypican alone did not affect AA release, as previously reported (16).

The function of the HSPG-shuttled group II subfamily sPLA2s, but not that of the plasma membrane-acting sPLA2s, is markedly attenuated by 12/15-lipoxygenase-inhibitable antioxidants (such as NDGA), leading to the suggestion that stimulus-induced membrane modification involves lipid-oxidative events (11, 46). In agreement with this notion, IL-1-augmented AA release (data not shown) and PGE2 production (Fig. 4F) by sPLA2-III-WT were reduced partially, to the level of those by III-S, by treatment of the transfectants with NDGA, whereas NDGA failed to affect PGE2 production by III-S. This suggests that III-WT, as has been proposed for the other HSPG-shuttled sPLA2s (11, 46-48), is accessible to particular membrane compartments that undergo oxidative modification after cytokine signaling.

Effect of Exogenous sPLA2-III on AA Metabolism-- The ability of the sPLA2 domain of sPLA2-III to elicit spontaneous (FCS-dependent) AA release suggests that it can act on the external plasma membrane, as does sPLA2-X and -V (17, 25-27). To explore this possibility further, we aimed to examine the effects of exogenous sPLA2-III on AA metabolism in mammalian cells. To this end, we expressed FLAG-tagged recombinant sPLA2-III in High Five insect cells using the baculovirus system in order to obtain recombinant enzyme in abundance. As assessed by immunoblotting using the anti-FLAG antibody, recombinant III-S, III-N+S, and III-S+C were expressed abundantly in the insect High Five cells as the expected sizes (Fig. 5A). The expression levels of III-S and III-N+S were comparable, whereas that of III-S+C was approximately one-tenth that of III-S and III-N+S. III-WT was not expressed appreciably in High Five cells (possibly due to rapid proteolytic degradation). Similar results were obtained when the enzymes were expressed in Sf9 insect cells (data not shown). The in vitro PLA2 activity of III-S was comparable with that of III-N+S and was about 10 times higher than III-S+C (Fig. 5B), in agreement with their expression levels (Fig. 5A). Enzymatic properties (e.g. substrate specificity, pH dependence, and Ca2+ requirement) of these insect cell-derived truncated enzymes were similar to those of enzymes expressed in HEK293 cells (data not shown).


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Fig. 5.   Recombinant expression of truncated sPLA2-III mutants by the baculovirus system and their effects on PGE2 production by HEK293 cells. A, expression of the FLAG-tagged, truncated forms of sPLA2-III in High Five insect cells. High Five cells grown in 12-well plates were infected with baculovirus bearing the truncated sPLA2-III cDNAs for 5 days, harvested, and lysed in 1 ml of PBS, and 10-µl aliquots were subjected to SDS-PAGE/immunoblotting using anti-FLAG antibody. B, in vitro enzymatic activity of recombinant sPLA2-III expressed in High Five cells. The indicated amounts of the culture supernatants of the baculovirus-infected High Five cells were taken for PLA2 assay. A representative result of two reproducible experiments is shown. C, effects of recombinant III-S and III-N+S on PGE2 production by COX-2-expressing HEK293 cells. The cells were incubated for 1 h with the culture supernatants of control or sPLA2-expressing High Five cells that were diluted (1:10) with RPMI 1640 containing 10% FCS, and PGE2 released into the supernatants was measured. A representative result of three independent preparations is shown. Values are mean ± S.E. of three independent experiments.

We then investigated the effects of recombinant III-S and III-N+S, which were secreted from the baculovirus-infected High Five cells at comparable levels (Fig. 5, A and B), on PGE2 production by HEK293 cells. Thus, culture supernatants of control (i.e. no baculovirus infection) and of III-S- or III-N+S-expressing High Five cells were diluted 10 times with RPMI 1640 plus 10% FCS and added to COX-2-transfected HEK293 cells. As shown in Fig. 5C, both exogenous III-N+S and III-S markedly increased PGE2 production by COX-2-transfected HEK293 cells after a 1-h incubation.

Subcellular Localization-- We next performed indirect immunofluorescent confocal microscopy to assess subcellular distribution of sPLA2-III-WT and truncated mutants in HEK293 transfectants. As shown in Fig. 6, signals for III-WT, III-N+S, and III-S+C were detected in the cytoplasmic punctate regions that excluded the nucleus, whereas III-S was distributed mainly on the plasma membrane but not in the cytosol. In addition, intense staining was seen at the spindle edges of cell adhesion sites in cells transfected with III-WT or III-N+S and to a lesser extent with III-S+C (Fig. 6).


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Fig. 6.   Subcellular distribution of sPLA2-III. HEK293 cells transfected with the FLAG-tagged sPLA2-III-WT and truncated forms (III-S, III-N+S, and III-S+C) were subjected to immunofluorescent staining using anti-FLAG antibody and fluorescein isothiocyanate-conjugated anti-IgG. Detailed procedures are described under "Experimental Procedures."

Expression in Human Cell Lines-- We next looked for human cell lines that expressed sPLA2-III endogenously and found that BEAS-2B, a human lung epithelial cell line (Fig. 7, A and B), and HCA-7, a human adenocarcinoma cell line (Fig. 7C), expressed this enzyme in addition to several other sPLA2s. RT-PCR for sPLA2-III was carried out using two different sets of primers (III-5'/III-HQ-AS and III-5'/III-S-AS), each of which amplified a single band with a predicted size (Fig. 7A). Subsequent Southern hybridization using a sPLA2-III-specific cDNA probe confirmed that these bands indeed corresponded to the expected portions of the enzyme (data not shown). In BEAS-2B cells, sPLA2-III was constitutively expressed and decreased after stimulation with cytokines (TNF-alpha and IFN-gamma ) (Fig. 7B). In comparison, the expression of sPLA2-IIA and sPLA2-V was tightly controlled by cytokines in that sPLA2-IIA was induced by TNF-alpha and suppressed by IFN-gamma , and sPLA2-V expression required both TNF-alpha and IFN-gamma (Fig. 7B). sPLA2-X was constitutively expressed (Fig. 7B), and other sPLA2s (IB, IID, IIE, IIF, and XII) were undetectable (data not shown).


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Fig. 7.   Expression of various endogenous sPLA2s in human cell lines. A, detection of endogenous sPLA2-III in BEAS-2B cells by RT-PCR. Two different primer sets were used to amplify sPLA2-III cDNA fragments in lanes 1 (III-5' and III-HQ-AS) and 2 (III-5' and III-S-AS). Specific bands were visualized by ethidium bromide in agarose gels. B and C, expression of various sPLA2s in BEAS-2B (B) and HCA-7 (C) cells with or without stimulation with 10 ng/ml IL-1alpha , IFN-gamma , or TNF-alpha for 24 h. Specific bands were detected by RT-PCR followed by Southern hybridization. A-C, representative results of two reproducible experiments are shown. D, effects of recombinant III-S and III-N+S on PGE2 production by HCA-7 cells (n = 3). The procedure is the same as in Fig. 5C.

sPLA2-III was also constitutively expressed in HCA-7 cells, in which its expression was unaffected by IL-1 (Fig. 7C). In this cell line, sPLA2-IIA was absent in unstimulated cells and was strongly induced by IL-1; sPLA2-IID, -IIF, -V, and -X were constitutively expressed, among which only sPLA2-V was up-regulated by IL-1; and other sPLA2s (IB, IIE, and XII) were undetectable (Fig. 7C). When sPLA2-III-S and sPLA2-N+S, which were produced by the baculovirus system (Fig. 5), were exogenously added to HCA-7 cells, there was a substantial increase in PGE2 production (Fig. 7D).

Human sPLA2-XII

Enzymatic Properties-- Human sPLA2-XII cDNA was transfected into HEK293 cells, and the expression of the enzyme in stable transfectants obtained after drug selection was assessed by Northern blotting (Fig. 8A, inset) and Western blotting (see below). In our PLA2 assay using the four substrates (PE and PC bearing sn-2-AA or -LA), sPLA2-XII-expressing cells displayed no detectable PLA2 activity in both culture supernatants and cell lysates. The enzyme activity was still below the detection limit even when recombinant sPLA2-XII was overexpressed by the baculovirus/High Five cell system (data not shown). It is unlikely that sPLA2-XII was inappropriately expressed in our system, since it was detected by Northern and Western blottings as readily as other sPLA2s transfected in HEK293 or insect cells. Thus, sPLA2-XII does not exhibit detectable enzymatic activity toward PE and PC in our PLA2 assay, in line with a previous report that the activity of this enzyme is extremely low as compared with most other sPLA2s (38).


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Fig. 8.   Properties of HEK293 cells transfected with sPLA2-XII. A, cellular fatty acid release. Control and sPLA2-XII-transfected cells were prelabeled with [3H]AA or [3H]OA, and the release of these fatty acids after 4-h incubation with 1% (-) or 10% (+) FCS with (+) or without (-) 1 ng/ml IL-1beta was assessed. The expression of sPLA2-XII was assessed by Northern blotting (inset). B, PGE2 production. Control cells and cells stably transfected with sPLA2-XII, sPLA2-III, or sPLA2-IIF (positive control) were transfected with the indicated concentrations of the COX-1 plasmid. Three days after transfection, the cells were stimulated for 30 min with 10 µM A23187 to assess PGE2 release. COX-1 expression was assessed by immunoblotting (top). C, detection of sPLA2-XII protein in the sPLA2-XII-transfected (+) and parental (-) HEK293 cells by immunoblotting using anti-sPLA2-XII antibody. D, immunofluorescent staining on sPLA2-XII-transfected or control cells using anti-sPLA2-XII antibody. Two typical versions of sPLA2-XII staining, in which the cells exhibited abnormal morphologies, are shown (b and c). Control cells did not show positive signals (a).

Effects on AA Metabolism-- [3H]AA and [3H]OA release by sPLA2-XII-expressing cells in the presence of 10% FCS was increased minimally relative to that by control cells, even in the presence of IL-1 (Fig. 8A). There was no appreciable increase in A23187-stimulated immediate [3H]AA release in sPLA2-XII-expressing cells (data not shown). sPLA2-XII-expressing cells did not display increased PGE2 production relative to control cells even when cells were cotransfected with COX-1 (Fig. 8B) or COX-2 (data not shown), although sPLA2-III and -IIF showed marked functional COX-1 coupling under the same experimental condition. sPLA2-XII also failed to increase PGE2 production when HEK293 cells expressing this enzyme were cocultured with those expressing COX-2 in the transcellular assay (data not shown).

Microscopic Analyses-- Our antibody raised against human sPLA2-XII specifically recognized a single 18-kDa protein in HEK293 transfectants on Western blotting (Fig. 8C). Using this antibody, we performed immunostaining of sPLA2-XII expressed in HEK293 cells by confocal microscopy. We noted that a population of sPLA2-XII-expressing cells exhibited unusual morphology; some cells appeared thin and long (Fig. 8D, panel b), whereas other cells were giant and multinucleated (Fig. 8D, panel c). In both situations, immunoreactivity of sPLA2-XII was detected throughout the intracellular regions excluding the nucleus. These unique morphological features were not observed in cells transfected with other sPLA2s (9-11, 14-18).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

To complete our current understanding of the AA-releasing capacity of the full set of mammalian sPLA2s in transfected cells (HEK293), we have herein examined the AA-releasing and PGE2-biosynthetic functions of the two recently discovered sPLA2s, group III and XII. These two sPLA2s show homology with other sPLA2s only in the catalytic site and Ca2+-binding loop (36, 38) and thus appear to have diverged from the group I/II/V/X sPLA2s at early evolutional stages. Group III enzymes were originally identified in bee venom (49); subsequently in venom from scorpion (imperatoxin I and phospholipin) (50, 51), lizard (52), and jellyfish (53); and more recently in Drosophila (in which five distinct group III sPLA2-related genes have been found) by scanning of public databases (2). Bee venom group III sPLA2 has been shown to induce AA release when added exogenously to several cell types and elicits various biological effects in vivo (54-58). Imperatoxin I, a scorpion venom group III sPLA2, inhibits ryanodine binding to Ca2+ release channels probably dependent upon its catalytic activity (50). However, it has remained unknown whether its mammalian homolog exerts a similar biological effect and, if so, how its function is regulated. Group XII sPLA2s, first cloned from humans (37) and mice (38), have also been found in the genomic databases of various low vertebrate species, yet there has been no functional assessment of this group of enzymes.

sPLA2-III-- Human sPLA2-III is made up of a central group III sPLA2 domain flanked by N- and C-terminal regions (36). Although no data base entries with significant homology to the N- and C-terminal domains can be found, both domains are highly cationic and are predicted to fold separately from the sPLA2 domain (36). Assessment of in vitro enzymatic activities of sPLA2-III-WT and the truncated mutants (III-S, III-N+S, and III-S+C) expressed in HEK293 cells (Fig. 1) and High Five insect cells (Fig. 5) demonstrates that the central sPLA2 domain alone is sufficient for catalytic function and neither N- nor C-terminal domain profoundly modulates the catalytic function of the sPLA2 domain. This is in line with the observation that recombinant bee venom sPLA2 expressed as an N-terminal fusion protein exhibits the same catalytic activity as the recombinant protein after removal of the N-terminal fusion peptide (59) and implies that the presence of the N-terminal extension (and presumably the C-terminal region, which is also not part of the catalytic site (36, 60)) does not interfere with the catalytic activity of sPLA2-III. This contrasts with sPLA2-IB and -X, for which proteolytic removal of the prepropeptide is essential for full enzymatic activity (3, 4). This difference exists most likely because, unlike the I/II/V/X sPLA2s, which contain a hydrogen bond network linking the N terminus to catalytic residues (1, 2), the N terminus of bee venom group III enzyme (and probably human sPLA2-III) does not form part of the active site structure (60).

The present cellular study suggests that sPLA2-III-WT, as well as the mutants harboring either the N- or C-terminal domain, can act on cells through the HSPG pathway after IL-1 stimulation. The highly cationic nature of the N-terminal (pI 9.1) and C-terminal (pI 11.3) domains, in contrast to the central sPLA2 domain that is acidic (pI 5.4) (36), may allow their electrostatic interaction with anionic heparin (or other anionic components). Although the affinity of III-N+S and III-S+C for heparin is weaker than that of sPLA2-IIA, sPLA2-IID, and sPLA2-V (data not shown), it appears to be still sufficient to promote cellular function. Indeed, the IL-1-augmented components of AA release and PGE2 production by sPLA2-III-WT are sensitive to heparin treatment, and, conversely, overexpression of glypican, a glycosylphosphatidylinositol-anchored HSPG to which HSPG-shuttled sPLA2s bind (16, 18), results in marked increases in AA release and PGE2 production by III-WT (Fig. 4), providing strong support for a functional link between sPLA2-III-WT and cellular HSPG. Moreover, the immunocytostaining study suggests that either the N- or C-terminal domain is essential for intracellular localization of the enzyme (Fig. 6). This is reminiscent of the previous finding that the heparin-binding group II subfamily sPLA2s (IIA, IID, IIE, and V) can be internalized into cells in HSPG- and caveolae- or raft-dependent manners, followed by cytoplasmic vesicle formation and intracellular membrane hydrolysis (16, 18, 28).

Without cell stimulation, sPLA2-III-WT and all of the truncated mutants elicit spontaneous (FCS-dependent), nonselective fatty acid release (Fig. 2). This pattern is very similar to that of sPLA2-X, which acts on the PC-rich outer leaflet of the plasma membrane (4, 17, 25, 27), and suggests that sPLA2-III, via its sPLA2 domain, can act on cells through the external plasma membrane pathway. The facts that sPLA2-III (and its truncated mutants) has significant activity toward PC (Fig. 1D), that the cellular AA-releasing function of III-S is insensitive to exogenous heparin treatment or is poorly augmented by glypican overexpression (Fig. 4), that III-S is distributed on the plasma membrane (Fig. 6), and that exogenous enzyme is capable of increasing PGE2 production in two cell types (Figs. 5C and 7D) support this notion.

Collectively, we conclude that sPLA2-III can utilize the HSPG-shuttling pathway, where its unique N- and C-terminal domains play a role in entering the HSPG-shuttling route (and thereby affecting the targeting of the enzyme) and its core sPLA2 domain exerts cellular membrane hydrolysis (Fig. 9). Although the N- and C-terminal domains have no sequence homology, both of them contribute to heparanoid binding based on the presence of basic residues. By comparison, the group II subfamily sPLA2s have clusters of heparin-binding cationic residues on their core surfaces (61, 62). Strikingly, the N-terminal (and probably C-terminal) domain of sPLA2-III also facilitates the distribution of this enzyme into the spindle edges of cell adhesion sites (Fig. 6). So far, none of the other sPLA2s exhibit this unique localization (10, 11, 16-18). This suggests that some anionic components or binding molecules that preferentially associate with the N-terminal domain may exist in the spindle edges of HEK293 cells, although functional consequences of this localization remain to be elucidated. Note that the N- and C-terminal domains found in the five Drosophila group III enzymes have no sequence homology with those of human enzyme (2), suggesting that their roles are different.


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Fig. 9.   Diagram of domain-function relationship of human sPLA2-III. A central sPLA2 domain is essential for the enzyme function and the plasma membrane action of the enzyme. Either the N- or C-terminal domain mediates HSPG binding, intracellular distribution, COX-2 induction, and efficient COX coupling. These domains, the N-terminal domain in particular, facilitate unique localization of this enzyme to the spindle edges of cell adhesion sites.

Since group III enzyme purified from bee venom consists of only the sPLA2 core (49), it may be anticipated that human sPLA2-III also undergoes proteolytic processing and maturation in mammalian cells. The presence of a basic doublet KE at the end of the N-terminal domain and several basic residues including basic doublets in the C-terminal domain (36) suggests that their proteolytic removal by subtilisin-like protease in the Golgi (63). Indeed, besides the major bands with expected sizes, several shorter products are faintly detected in HEK293 cells transfected with sPLA2-III (e.g. 30-kDa band in III-WT and III-N+S) in our immunoblot analysis (Fig. 1C). In addition, sPLA2-III appears to be susceptible to endogenous protease(s) in High Five insect cells, in which III-S+C and III-N+S (and probably III-WT, which was almost undetectable) are rapidly degraded during storage (data not shown). At present, the maturation process of sPLA2-III in mammalian cells remains obscure. It would be important to determine which forms of the enzyme (III-WT, III-N+S, III-S+C, III-S, or other processed forms) are truly present and functioning in vivo.

Interestingly, coexpression experiments demonstrate that III-WT (as well as III-N+S and III-S+C) is more efficiently coupled with COX than III-S (Fig. 3, D-F). Although the precise reasons for this result are unclear, it reminds us of the central dogma in the eicosanoid field that subcellular location of the biosynthetic enzymes is a critical determinant for their optimal functional coupling. It is now obvious that the perinuclear co-localization of sequential biosynthetic enzymes, including cPLA2alpha , COXs, and terminal PG synthases in the COX pathway and cPLA2alpha , 5-lipoxygenase, 5-lipoxygenase-activating protein, and terminal leukotriene synthases in the lipoxygenase pathway, is crucial for their functional coupling in activated cells (64-68). Indeed, our preliminary experiments have shown that the native cPLA2alpha , which translocates to the perinuclear membrane, is more efficiently coupled with COX than the cPLA2alpha mutant, which moves to the plasma membrane.2 In human neutrophils, the AA released by cPLA2alpha at the perinuclear membrane, but not that released by sPLA2-V at the plasma membrane, can be metabolized to leukotriene by the perinuclear 5-lipoxygenase (69). Considering the scenario that the HSPG-shuttled sPLA2s can be internalized and cause membrane hydrolysis in the perinuclear region (16, 18, 28), it would be speculated that the AA released by the internalized sPLA2s can be more efficiently supplied to adjacent COX in the perinuclear membrane than the AA released from cell surface by the plasma membrane-acting sPLA2s (including III-S), although the latter AA can be accessible to the perinuclear COX (possibly by diffusion across the cytosol or with the aid of fatty acid transfer proteins), as has been observed with sPLA2-X (4, 17, 25, 27) and even III-S (Fig. 3, D and F). Although intracellular membrane hydrolysis by the HSPG-shuttled sPLA2s will need further study, our present data may shed light on the intracellular action of these sPLA2s and its importance in efficient coupling with downstream enzymes.

The sPLA2-III transcript is detected in the kidney, heart, liver, and skeletal muscle by Northern blotting (36). In two cell lines that endogenously express sPLA2-III, sPLA2-III expression is constitutive and not cytokine-inducible, unlike the group II subfamily sPLA2s, sPLA2-IIA and sPLA2-V, that are induced by diverse sets of cytokines (Fig. 7). This implies that the transcriptional regulations and possibly functions of these sPLA2s are distinct in these cells. Although exogenous sPLA2-III-S modestly increased PGE2 production by HCA-7 cells (Fig. 7D), it remains to be elucidated whether endogenous sPLA2-III also participates in PGE2 production or exhibits other functions. Expression of multiple sPLA2s in a colon adenocarcinoma cell line is intriguing to note, since the COX-2-derived PGE2 has been implicated in the exacerbation of colorectal cancer (70-72). Although targeted disruption of cPLA2alpha (70), COX-2 (71), or the PGE2 receptor EP2 (72) each reduces the incidence of colorectal cancer in Apc mutant mice, the phenotype of the cPLA2alpha knockout mice is milder than that of the COX-2 or EP2 knockout mice, suggesting that some other PLA2s can contribute to supplying AA to COX-2 in colorectal cancer. Possible involvement of sPLA2s in colon cancer development is now under investigation.

Bee venom group III enzyme has been shown to evoke several cellular and in vivo responses via the N-type sPLA2 receptor independently of its catalytic activity (2, 32). It is tempting to speculate that human sPLA2-III may represent an endogenous ligand for the N-type receptor. Although the molecular entity of the N-type sPLA2 receptor is still obscure, it has been recently shown that some neurotoxic sPLA2s, including bee venom sPLA2, bind to calmodulin with high affinity (73). Since calmodulin is a cytosolic protein, this finding supports the view that neurotoxic sPLA2s have to be internalized to exert their effect. Whether calmodulin indeed acts as a functional binding protein for sPLA2-III and whether another high affinity binding site(s) for sPLA2-III exists need to be addressed. Further elucidation of the N-type receptor may provide insights into the regulatory functions of sPLA2-III. Additionally, a recent finding that a peptide derived from bee venom sPLA2 inhibits replication of human immunodeficiency virus by blocking the virus entry into host cells (74) suggests a possible role of human counterpart in this process.

sPLA2-XII-- sPLA2-XII has an unusual structure among the sPLA2 family members in that only 3 of its 11 cysteines correspond to cysteines of other sPLA2s (37, 38). Here we show that sPLA2-XII fails to increase cellular fatty acid release and PGE2 production despite its expression in HEK293 cells (Fig. 8, A and B). It is thus likely that sPLA2-XII is incapable of mobilizing cellular AA due to its weak catalytic activity, although we cannot rule out the possibility that at higher expression levels it can influence AA metabolism. The putative sPLA2-XII in zebrafish represented in genomic databases contains a leucine in place of histidine in the catalytic center, strongly suggesting that the zebrafish sPLA2-XII has little or no catalytic activity (37). This supports the idea that the catalytic activity of sPLA2-XII may not be critical for its cellular function. The appearance of multinucleated giant cells in a population of sPLA2-XII-transfected cells (Fig. 8D) is noteworthy, which suggests a potential role of this enzyme in membrane fusion or cell division, although the molecular mechanisms are unclear. In these multinucleated cells, the main sPLA2-XII immunoreactivity appears to be enriched in endoplasmic reticulum. Similar intracellular localization of mouse sPLA2-XII has been reported in baby hamster kidney cells transfected with this enzyme (38). Immunohistochemistry using our anti-sPLA2-XII antibody to determine cell types that endogenously express this enzyme will help us to understand its physiological and pathological functions.

    CONCLUSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

Using the strategy of overexpression of AA-metabolic enzymes in HEK293 cells and several other cell lines, we have uncovered some of the functional properties of the full set of mammalian sPLA2s. The ability of sPLA2s to release AA from quiescent cells is highly dependent upon their interfacial binding to PC enriched in the outer leaflet of the plasma membrane (X > V > IIF ~ III > IB IIA; IIC, IID, IIE, and XII are almost inactive in this route). These sPLA2s induce stimulus-independent, nonselective fatty acid release. sPLA2s that poorly act on the PC-rich membrane can promote AA release from activated cells with support of HSPG as an adapter (IIA ~ V > IID > III > IIE; IB, IIC, X, and XII are nonfunctional in this route). These sPLA2s prefer anionic membranes, bind heparanoids, and promote stimulus-dependent, AA-selective release and COX-2 induction. These sPLA2s (an exception is sPLA2-IIF, whose function does not depend on HSPG (11)) may be sorted into caveolae/rafts and internalized into particular membrane compartments that are assumed to be rich in AA and anionic membrane surfaces. Certain lipid-hydrolyzing products (fatty acids, lysophospholipids, or their derivatives) spatiotemporally generated by these sPLA2s in these compartments may be linked to COX-2 induction. The occurrence of these distinct regulatory pathways depends on cell types and stimuli.

Although this series of studies has provided useful information about sPLA2 behaviors and functions, much work is still needed to determine the complete set of functions of each sPLA2 in vivo. Each sPLA2 displays different tissue distribution and stimulus inducibility, implying that the sPLA2 members exhibit nonredundant functions in each tissue. Some sPLA2s may act as ligands (like cytokines), rather than enzymes, to transduce signals via the distinct classes of sPLA2 receptors (32). Some sPLA2s may play roles in defense against bacterial infection (which appears to be true for sPLA2-IIA) (19, 20), lipoprotein metabolism (21, 22, 75, 76), and other biological events (23, 24, 77). Determining the precise localization of each sPLA2 in various physiological and pathological tissues, developing inhibitors specific for each sPLA2, and targeted disruption or transgenic expression of each sPLA2 will yield more informative and conclusive answers.

    ACKNOWLEDGEMENTS

We thank Drs. M. Tsujii and R. DuBois for providing HCA-7 cells.

    FOOTNOTES

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

§ To whom correspondence should be addressed. Tel.: 81-3-3784-8197; Fax: 81-3-3784-8245; E-mail: mako@pharm.showa-u.ac.jp.

Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M211325200

2 M. Murakami, W. Cho, and I. Kudo, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: sPLA2, secretory phospholipase A2; PLA2, phospholipase A2; cPLA2alpha , cytosolic PLA2alpha ; COX, cyclooxygenase; AA, arachidonic acid; OA, oleic acid; LA, linoelic acid; PG, prostaglandin; PGE2, prostaglandin E2; PC, phosphatidylcholine; PE, phosphatidylethanolamine; HSPG, heparan sulfate proteoglycan; FCS, fetal calf serum; NDGA, nordihydroguaiaretic acid; RT-PCR, reverse transcriptase-PCR; IL-1, interleukin-1; TNF-alpha , tumor necrosis factor alpha ; IFN-gamma , interferon-gamma ; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; WT, wild type; 2-LA-PE, 1-palmitoyl-2-[14C]linoleoyl-PE; 2-LA-PC, 1-palmitoyl-2-[14C]linoleoyl-PC; 2-AA-PC, 1-palmitoyl-2-[14C]arachidonoyl-PC; 2-AA-PE, 1-palmitoyl-2-[14C]archidonoyl-PE.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

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