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Journal of Lipid Research, Vol. 46, 2488-2496, November 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

PGE₂ release is independent of upregulation of Group V phospholipase A₂ during long-term stimulation of P388D₁ cells with LPS¹

Ursula A. Kessen, Ralph H. Schaloske, Daren L. Stephens, Karin Killermann Lucas and Edward A. Dennis²

Departments of Chemistry and Biochemistry and Pharmacology, University of California–San Diego, La Jolla, CA 92093

¹ Trudy Forte served as Guest Editor for this manuscript.

Published, JLR Papers in Press, September 8, 2005. DOI 10.1194/jlr.M500325-JLR200

² To whom correspondence should be addressed. e-mail: edennis{at}ucsd.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
P388D₁ cells release arachidonic acid (AA) and produce prostaglandin E₂ (PGE₂) upon long-term stimulation with lipopolysaccharide (LPS). The cytosolic Group IVA (GIVA) phospholipase A₂ (PLA₂) has been implicated in this pathway. LPS stimulation also results in increased expression and secretion of a secretory PLA₂, specifically GV PLA₂. To test whether GV PLA₂ contributes to PGE₂ production and whether GIVA PLA₂ activation increases the expression of GV PLA₂, we utilized the specific GIVA PLA₂ inhibitor pyrrophenone and second generation antisense oligonucleotides (AS-ONs) designed to specifically inhibit expression and activity of GV PLA₂. Treatment of P388D₁ cells with antisense caused a marked decrease in basal GV PLA₂ mRNA and prevented the LPS-induced increase in GV PLA₂ mRNA. LPS-stimulated cells release active GV PLA₂ into the medium, which is inhibited to background levels by antisense treatment. However, LPS-induced PGE₂ release by antisense-treated cells and by control cells are not significantly different.

Collectively, the results suggest that the upregulation of GV PLA₂ during long-term LPS stimulation is not required for PGE₂ production by P388D₁ cells. Experiments employing pyrrophenone suggested that GIVA PLA₂ is the dominant player involved in AA release, but it appears not to be involved in the regulation of LPS-induced expression of GV PLA₂ or cyclooxygenase-2.

Abbreviations: AA, arachidonic acid; AS-ON, antisense oligonucleotide; GIVA, Group IVA; LPS, lipopolysaccharide; MAFP, methyl arachidonyl fluorophosphonate; PGE₂, prostaglandin E₂; PLA₂, phospholipase A₂; S-ON, sense oligonucleotide; sPLA₂, secreted phospholipase A₂

Supplementary key words antisense inhibitor • macrophage • lipopolysaccharide • prostaglandin E₂ • arachidonic acid • eicosanoid

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The phospholipase A₂ (PLA₂) superfamily encompasses a series of enzymes that catalyze the hydrolysis of the fatty acid esterified at the sn-2 position of glycerophospholipids, producing free fatty acid and lysophospholipids (1). Any free arachidonic acid (AA) liberated by these enzymes can subsequently be converted into prostaglandins (PGs) through the action of cyclooxygenases (COXs) and PG synthases.

Prominent members of the PLA₂ superfamily are the secreted phospholipase A₂s (sPLA₂s), the cytosolic PLA₂s (cPLA₂s), and the calcium-independent PLA₂s (iPLA₂s). Common features of the sPLA₂s are their relatively low molecular mass (about 14 kDa), a high abundance of disulfide bonds, and their requirement for micromolar Ca²⁺ for activity. cPLA₂s are characterized by a higher molecular mass (about 85 kDa) and their preference for AA-containing phospholipids (2–4). The iPLA₂s are distinguished from the other PLA₂s in that they do not require Ca²⁺ for activity.

Previous work has demonstrated that the murine macrophage-like P388D₁ cells release prostaglandin E₂ (PGE₂) upon long-term stimulation with bacterial lipopolysaccharide (LPS). Shinohara et al. (5) and Balsinde et al. (6) have suggested possible roles for the c- and sPLA₂s in the release of AA, resulting in PGE₂ production. In a hypothetical model, activation of the Group IV (GIV) cPLA₂ results in an increased expression of Group V (GV) sPLA₂, which, in turn, is largely responsible for the release of the AA that is subsequently converted into PGE₂, and GV PLA₂ gives rise to the upregulation of COX-2 expression. The model is based on experiments utilizing the chemical inhibitors methyl arachidonyl fluorophosphonate (MAFP), affecting Group IVA (GIVA) PLA₂; 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy) propane sulfonic acid (LY311727), affecting sPLA₂; and first-generation antisense oligonucleotides (AS-ONs). All three of these inhibitors have drawbacks. MAFP is not specific, inasmuch as it inhibits a number of enzymes in addition to GIVA PLA₂, namely GroupVI PLA₂ (7, 8), anandamide amidase (9, 10), and platelet-activating factor-acetylhydrolase (11). It also binds to the CB1 cannabinoid receptor (12, 13). The sPLA₂ inhibitor LY311727 is structurally related to Me-Indoxam. Mounier et al. (14) described recently that Me-Indoxam does not pass through the plasma membrane of mammalian cells and that it does not inhibit AA release from human embryonic kidney cells that have been transfected with GIIA or GX PLA₂s. The authors concluded that the sPLA₂s act inside the cell prior to their secretion. It is probable that LY311727 is not able to act inside the cells. Recent experiments from our laboratory employing LY311727 in P388D₁ cells support this hypothesis. The first-generation AS-ONs suffer from their low affinity toward target RNA molecules and their toxic side effects (15–18).

To test the hypothetical model described above, we employed second-generation AS-ONs consisting of a 5-10-5 2'-O-methoxy-ethyl RNA gapmer with a phosphorothioate backbone. These modifications increase the binding of the oligonucleotide to its target, stabilize the ON by preventing nuclease degradation, and support RNase H-mediated cleavage of the targeted mRNA (15–18). We used second-generation AS-ONs specifically designed to inhibit expression of GV PLA₂, and thus its activity, in order to examine specifically the role of GV PLA₂ in PGE₂ production. We also utilized the novel GIVA PLA₂ inhibitor pyrrophenone, which is more specific and potent than MAFP (19, 20).

We now show that treatment of P388D₁ cells with AS-ONs resulted in a marked decrease of basal GV PLA₂ mRNA, prevented the LPS-induced increase in GV PLA₂ mRNA as measured with real-time quantitative PCR (Q-PCR), and also caused a significant decrease in basal and stimulated GV PLA₂ protein and activity levels. Basal and stimulated PGE₂ levels were not affected by AS-ON treatment. Treatment of the cells with the novel GIVA PLA₂ inhibitor pyrrophenone resulted in a 60–70% inhibition of PGE₂ production but did not affect COX-2 protein levels. We show that the upregulation of GV PLA₂ is i) not mandatory for PGE₂ production by P388D₁ cells during long-term stimulation with LPS and ii) not dependent on GIVA PLA₂ activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials
Iscove's modified Dulbecco's medium (IMDM), OPTI-MEM, and penicillin/streptomycin were obtained from Gibco (Grand Island, NY). Fetal bovine serum was from VWR International (Bristol, CT). LPS (Escherichia coli 0111:B4) was obtained from Sigma Chemical Co. (St. Louis, MO). Nonradioactive 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS) were purchased from Avanti%20Polar%20Lipids">Avanti Polar Lipids (Alabaster, AL). 1-palmitoyl-2-[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine (radioactive DPPC) was purchased from Amersham Biosciences (Piscataway, NJ). [5,6,8,9,11,12,14,15-³H]AA (specific activity 100 Ci/mmol) was obtained from NEN Life Science Products (Boston, MA). The Complete Mini Protease Inhibitor Cocktail was from Roche Applied Science (Indianapolis, IN). Triton X-100 was purchased from Calbiochem (La Jolla, CA). The GV PLA₂ AS-ONs and control oligonucleotides were kindly provided by Dr. Frank Bennett of ISIS Pharmaceuticals (Carlsbad, CA). LY 311727 was the generous gift of Dr. Jerome Fleisch (Lilly Research Laboratories). Pyrrophenone was the generous gift of Dr. Takaski Ono (Shionogi Research Laboratories). MAFP and the polyclonal antibody directed against murine GV PLA₂ and the polyclonal antibody directed against COX-2 were purchased from Cayman Chemicals (Ann Arbor, MI). The monoclonal antibody against GAPDH was obtained from HyTest (Turku, Finland). Cytofectin was from Gene Therapy Systems (San Diego, CA). SYBR-Green PCR Master Mix was from Applied Biosystems (Foster City, CA). Primers used for the real-time Q-PCR were ordered from Proligo (Boulder, CO). M-MLV reverse transcriptase, RNase H, SeeBlue2 PreStained protein standard and the 12% Bis-Tris gels were from Invitrogen (Carlsbad, CA).

Cell culture
P388D₁ cells (MAB clone) (5), were maintained at 37°C in a humidified atmosphere at 90% air and 10% CO₂ in IMDM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete IMDM). For transfection experiments, the cells were seeded at a density of 10⁶ cells/2 ml/well in 6-well plates and allowed to adhere overnight. For experiments using chemical inhibitors, cells were seeded either at a density of 10⁶ cells/ml/well in 12-well plates or at a density of 3 x 10⁶ cells/2 ml/well in 6-well plates and allowed to adhere overnight. For experiments assaying AA-derived radioactivity release, cells were seeded in 12-well plates in the presence of 0.5 µCi ³H-labeled AA.

Stimulation of cells
Cells transfected with oligonucleotides were stimulated 5 h after transfection by adding 150 ng/ml LPS to the cells for 24 h. In experiments using chemical inhibitors assaying AA-derived radioactivity release or PGE₂ release, cells were washed three times in IMDM, incubated in the same medium for 1 h, and then stimulated by adding 100 ng/ml LPS for 18 h. Inhibitors were added during the starvation period 30 min prior to the addition of LPS.

Transient transfection of AS-ONs
The following ONs were used for transfection: GV sense oligonucleotide (S-ON) ISIS# 357 886 (5'-TTC CGG AGG AAG GGT CTA GG-3') and GV AS-ON ISIS# 314 510 (5'-CCT AGA CCC TTC CTC CGG AA-3'). The nucleotides shown in bold and plain text represent 2'-O-methylribonucleotides and 2'-deoxyribonucleotides, respectively, and are linked through thioester bonds. All transfection solutions were prepared in polystyrol 6- or 12-well plates. The oligonucleotides were transfected into the cells by using the transfection reagent Cytofectin. Solution A contained 4 µg of Cytofectin diluted in 200 µl of OPTI-MEM, and solution B contained 3 µl of oligonucleotide (100 µM stock) diluted in 200 µl OPTI-MEM. Both solutions were incubated for 10 min at room temperature. Subsequently, solution B was added drop-wise to solution A, and this transfection mix was incubated for another 15 min at room temperature. The cell medium was replaced by 600 µl fresh complete IMDM, and the transfection mix was then added to the cells. The cells were incubated for 5 h before stimulation with LPS.

Preparation of RNA and cDNA
Total cellular RNA was isolated from cells with the RNeasy Mini Kit from Qiagen (Valencia, CA) according to the manufacturer's instructions. The remaining DNA was digested by using the RNase-Free DNase Set from Qiagen. cDNA was synthesized by using M-MLV reverse transcriptase and oligo dT primer following the manufacturer's protocol. The remaining RNA was digested by incubating the samples with 20 units of RNase H at 37°C for 20 min.

Design of primers used for real-time Q-PCR
All the primers used are listed in reference (21) and were selected using the Primer 3 software (www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). They encompassed at least one intron of the gene. To determine which mRNAs are expressed in P388D₁ cells, we performed PCR using cDNA from either nontreated cells or cells treated with LPS for 18 h. The reactions contained 100 ng of cDNA, 1 µM of gene-specific primers (described in Ref. 21) and the SYBR Green PCR Master Mix in a total volume of 25 µl. PCR was performed on a BioRad iCycler Thermal Cycler. PCR cycles were identical to those used for real-time Q-PCR as described below. Three microliters of the PCR product was applied to 4% agarose gels (E-gels, Invitrogen). In cases in which two different primer pairs for a given gene did not produce a detectable band, we assumed that the mRNA was not present. Occasionally, there was a faint band visible at the correct size of the GIIF amplicon. However, even when using 500 ng of cDNA template for the real-time Q-PCR, we could not quantitate the cDNA encoding GIIF PLA₂ (data not shown). Similarly, cDNA encoding GIII PLA₂ could only be detected occasionally. This suggests that the amounts of GIIF and GIII messages are close to the detection limit. We confirmed the functionality of the primers by using cDNAs that were synthesized from commercially available total murine tissue RNA (Ambion, Austin, TX).

Real-time Q-PCR
The real-time Q-PCR for all samples was performed on the ABI 7700 Sequence Detection System from Applied Biosystems using SYBR green detection. All primers were shown to yield a single product by performing a dissociation curve after each real-time Q-PCR run. The real-time Q-PCR consisted of an initial hold at 95°C for 10 min, and then 40 cycles of 95°C for 15 s and 61°C for 1 min. The amount of template cDNA and the primer concentration that was used are described in reference (21). Gene expression was normalized to the house-keeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR runs were performed at the Rebecca and John Moores UCSD Cancer Center, Molecular Pathology, Quantitative Real-Time PCR Shared Resource. The amplicons were cloned into the pCR2.1-TOPO vector (Invitrogen) and the sequences verified by sequencing. DNA sequencing was performed by the DNA Sequencing Shared Resource, Rebecca and John Moores UCSD Cancer Center.

Preparation of cell extracts and cell media for immunoblotting and PLA₂ assays
The cell media were cleared of detached cells by centrifuging for 4 min at 4,000 rpm in an Eppendorf centrifuge. The centrifuged supernatants were transferred into a fresh tube, stored at –80°C, and thawed right before the assay. The adherent cells on the plate were frozen at –80°C for 15–30 min and subsequently thawed. Lysis buffer (0.5% Triton X-100 and 1 x "Complete Mini" in PBS) was added, and lysates were transferred into Eppendorf reaction tubes and sonicated three times for 15 s at intervals of 15 s.

Immunoblotting
Cell media (22.5 µl from an original volume of 1 ml) were mixed with 7.5 µl 4x NuPAGE LDS sample buffer (Invitrogen) and 2% ß-mercaptoethanol, boiled for 3 min, and subsequently run on 12% Bis-Tris SDS-polyacrylamide gels using MES buffer (Nupage, Invitrogen). Proteins were transferred onto nitrocellulose membranes. For detection of GV PLA₂, the membrane was blocked by incubating the membranes with 3% BSA and 1% goat serum in TBS buffer containing 0.05% Tween 20 (TBS-Tween) for 1 h before probing with a GV PLA₂-specific antibody (1:250 dilution in TBS-Tween-3% BSA) for 1 h. The membrane was washed three times in TBS-Tween and then incubated with secondary antibody (Biorad) for 1 h (goat anti-rabbit IgG-HRP conjugate in a 1:2,500 dilution in TBS-Tween-3% BSA). For detection of COX-2 and GAPDH protein, the membrane was blocked by incubating the membranes with 3% BSA in TBS-Tween for 1 h before probing with a COX-2-specific antibody (1:400 dilution in TBS-Tween-3% BSA) or a GAPDH-specific antibody (1:2,000 in TBS-Tween-3% BSA) for 1 h. The membrane was washed three times in TBS-Tween and then incubated with Protein A-HRP conjugate (Amersham) for 1 h in a 1:3,000 dilution in TBS-Tween-3% BSA.

For GV PLA₂, GAPDH, and COX-2 blots, the membrane was then washed three times with TBS-Tween and two times with 10 mM Tris-HCl, pH 7.4, and then developed using the Western Lightning ECL kit (Amersham Pharmacia Biotech).

PGE₂ assay
Cells were stimulated as described above. The media were removed and cleared by centrifugation, and the PGE₂ concentration was quantitated using a monoclonal PGE₂ EIA kit (Cayman Chemical, Ann Arbor, MI). The assays were conducted according to the manufacturer's protocol.

Lipid preparation
Lipid stock solutions were made by dispensing the volume of phospholipid solution in chloroform necessary for a total of 50 nmol per assay tube. The lipid was dried under nitrogen and lyophilized for at least 1 h to remove all traces of chloroform. Lipid was then resuspended in 3 ml 100 mM HEPES, pH 7.5, and small unilamellar vesicles (SUVs) were created by repeated sonication until the solution cleared. A small sample of the SUV preparation was then centrifuged at 15,000 g for 5 min to test for the precipitation of large lipid structures. The supernatant from the centrifuged sample was counted by liquid scintillation and compared with the counts derived from a noncentrifuged sample of equal volume. If the counts compared varied more than 10%, the sample was subjected to further sonication and the centrifugation test was repeated until the counts were in agreement. The SUV suspension (3 ml) was further diluted to 200 µl per assay tube by the addition of 100 mM HEPES, pH 7.5.

PLA₂ assay
In the GV PLA₂-specific assay (22) the lipids used contained 100 µM DPPC-POPS (3:1) doped with 100,000 cpm ¹⁴C-labeled DPPC. The assay buffer contained 100 mM HEPES, pH 7.5, 4 mM CaCl₂, 1 mg/ml BSA, and 4 µM MAFP. The total volume for each assay was 500 µl (200 µl lipid, 250 µl assay buffer, and 50 µl cell lysate or cell media). The amount of calcium added was adjusted to account for the addition of EDTA in the lysis buffer to give the final concentrations listed above. For inhibitor studies, 1 µM LY311727 was added to the substrate preparation and mixed 5 min prior to the addition of samples. Samples were incubated with substrate for 1 h at 40°C in a shaking water bath. The assay was then terminated by the addition of 2.5 ml Dole Reagent (isopropyl alcohol-heptane-0.5 M sulfuric acid; 400:100:20; v/v/v). Silica gel (0.1–0.2 g) was added to each tube, followed by 1.5 ml heptane and 1.5 ml deionized water. Each tube was vortexed for 15 s. One milliliter of the organic phase was removed and passed through a glass wool-plugged Pasteur pipet containing silica gel (0.1–0.2 g). Radioactivity was then eluted with 1 ml diethyl ether. Five milliliters of scintillation cocktail (Biosafe II, RPI, Mount Prospect, IL) was then added to the eluate, and the radioactivity was determined by scintillation counting.

Data presentation
All assays and real-time Q-PCR reactions were carried out in triplicate. Each set of experiments was performed at least three times with similar results. Unless otherwise indicated, the data presented are from representative experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Abundance of mRNAs from sPLA₂s identified in P388D₁ cells stimulated with LPS
Using PCR, we amplified portions of genes encoding five different sPLA₂s in the P388D₁ cells: GIB, GIIE, GIID, GV, and GXII. Using real-time Q-PCR, we measured the relative abundance of these mRNA species after long-term LPS stimulation. Among these sPLA₂s, only the GV PLA₂ mRNA was significantly upregulated, resulting in a 5-fold increase in GV message after 13 h of LPS treatment (Fig. 1). We did not detect mRNA for GIIA, GIIC, and GX (data not shown). mRNAs encoding GIIF and GIII were close to the detection limit (see Experimental Procedures section).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Abundance of mRNAs from secreted phospholipase A2s (PLA2s) identified in P388D1 cells. Cells (3 x 106) were plated in 6-well plates and allowed to adhere overnight. Cells were subsequently stimulated with 100 ng/ml lipopolysaccharide (LPS) for 18 h. Cells were lysed, RNA extracted, and reverse transcribed into cDNA. cDNA of the respective genes was quantified by real-time PCR. The abundance of PLA2 mRNA was normalized to GAPDH mRNA. mRNA for GIIA, GIIC, and GX was not detected, and GIIF and GIII were close to the detection limit. Data represent one out of three experiments, each measured in triplicate. Error bars indicate SD.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Effect of antisense oligonucleotides (AS-ONs) on GV PLA2 mRNA levels in P388D1 cells. Cells (106) were plated in 6-well plates and allowed to adhere overnight. Cells were transfected with 300 nM oligonucleotide using Cytofectin as transfection reagent in a total volume of 1 ml, incubated for 5 h, and subsequently stimulated with 150 ng/ml LPS for 24 h. A: Cells were subsequently lysed, RNA extracted, and reverse transcribed into cDNA. cDNA was quantified by real-time PCR. The mRNA abundance of GV mRNA was normalized to GAPDH mRNA. Control: no addition; Mock: only transfection reagent; GV S: GV sense oligonucleotide (S-ON) + transfection reagent; GV AS: GV AS-ON + transfection reagent. Data represent one out of three experiments, each measured in triplicate. Error bars indicate SD. B: Media (22.5 µl) were loaded on a 14% polyacrylamide gel and transferred onto nitrocellulose, and the membrane was immunostained with anti-mouse GV antibody (Cayman). Std: 1.5 ng purified rat GV protein; control: media of untreated cells; Mock: media of cells treated with transfection reagent only; GV S: media of cells treated with GV S-ON + transfection reagent; GV AS: media of cells treated with GV AS-ON + transfection reagent. The GV PLA2 band is indicated by the arrowhead. Data represent one out of three experiments.

GV PLA₂ activity in P388D₁ cell lysates and cell media
To be sure that the reduction of GV PLA₂ mRNA results also in a reduction of GV PLA₂ enzyme as measured by activity, we measured GV activity in cell lysates as well as in the media of cells that were either untreated or stimulated with LPS in the presence of either cytofectin alone, the S-ON, or the AS-ON (Fig. 3A). In lysates of cells that were either untreated, treated with Cytofectin, or transfected with S-ON or AS-ON, we detected only minimal GV activity. In lysates of cells stimulated with LPS, we measured a 3-fold elevation in GV PLA₂ activity. Pretreatment of the cells with Cytofectin alone or Cytofectin plus S-ON did not alter this increase. However, lysates of cells stimulated with LPS in the presence of AS-ON showed a decrease in activity to a level comparable to that of nonstimulated cells.

View larger version (23K): [in this window] [in a new window]   Fig. 3. GV PLA2 activity measured in the media and lysate of GV antisense-treated P388D1 cells. A: Cells (106) were plated in 6-well plates and allowed to adhere overnight. Cells were transfected with 300 nM oligonucleotide using Cytofectin as transfection reagent in a total volume of 1 ml, incubated for 5 h, and subsequently stimulated with 150 ng/ml LPS for 24 h. Cell lysates and the media were assayed for PLA2 activity under conditions specific for the GV enzyme using the Dole assay. B: Effect of LY311727 on GV PLA2 activity measured in the media of LPS-stimulated cells. Cells (2 x 106) cells were plated in 6-well plates and allowed to adhere overnight. Cells were then stimulated with 100 ng/ml LPS for 24 h. The media were assayed for GV PLA2 activity in the presence (+) or absence (–) of the indicated amounts of LY311727. Data represent one out of three experiments, each measured in triplicate, and show the extrapolated total activity in the lysate and in the media. Error bars in A and B indicate SD.

Effect of second-generation AS-ONs on PGE₂ levels in P388D₁ cells
LPS induced a 30-fold increase in PGE₂ levels in the media of the cells. The transfection reagent alone and the S-ON had no effect on PGE₂ levels in stimulated or nonstimulated cells. Strikingly, the addition of the GV PLA₂ AS-ON did not reduce LPS-stimulated PGE₂ release in P388D₁ macrophages (Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 4. Effect of AS-ONs against GV PLA2 mRNA on prostaglandin E2 (PGE2) release from P388D1 cells. Cells (106) were plated in 6-well plates and allowed to adhere overnight. Cells were transfected with 300 nM oligonucleotide using Cytofectin as transfection reagent in a total volume of 1 ml, incubated for 5 h, and subsequently stimulated with 150 ng/ml LPS for 24 h. The media were assayed for PGE2 by enzyme immunoassay. Control: no addition; Mock: only transfection reagent; GV S: GV S-ON + transfection reagent; GV AS: GV AS-ON + transfection reagent. Data represent the mean of three independent experiments, each measured in triplicate. Error bars indicate SD.

View larger version (30K): [in this window] [in a new window]   Fig. 5. Effect of LY311727 on AA and PGE2 release. Cells (106) were plated in 12-well plates and allowed to adhere overnight in the presence of 0.5 µCi 3H-labeled AA if necessary. Cells were then stimulated with 100 ng/ml LPS for 18 h in the presence (+) or absence (–) of LY311727. A: Determination of AA release by radioactivity detection of all arachidonic acid-derived products. Data represent one out of three experiments, each measured in triplicate. B: Media were assayed for PGE2 by enzyme immunoassay. Data represent one out of three experiments, each measured in triplicate. Error bars in A and B indicate SD.

It has now been shown that MAFP inhibits a number of enzymes (7–13). To test whether the reported effect of MAFP on GV PLA₂ levels was indeed due to GIVA PLA₂ inhibition, we employed the newer, very potent and specific GIV PLA₂ inhibitor pyrrophenone (19, 20). The inhibitory effect of pyrrophenone on long-term AA release is shown in Fig. 6A. The maximal effect on [³H]AA-derived radioactivity release was observed at a pyrrophenone concentration of 100 nM. Figure 6B shows the effects of 100 nM and 300 nM pyrrophenone on PGE₂ release. LPS stimulation of the cells led to an approximately 5-fold increase in AA-derived radioactivity release (Fig. 6A) and to a 12-fold increase in PGE₂ release (Fig. 6B). Both 100 nM and 300 nM pyrrophenone decreased the LPS-stimulated AA release by 80–90% and the LPS-stimulated PGE₂ release by 70%, indicating that GIVA PLA₂ plays a dominant role in the release of AA. Furthermore, long-term stimulation of cells with LPS induced the synthesis and secretion of catalytically active GV PLA₂ (Figs. 3, 6C) as well as the upregulation of COX-2 (Fig. 6D). However, neither 100 nM nor 300 nM pyrrophenone resulted in a pronounced decrease in the activity of GV PLA₂ (Fig. 6C), in the amount of secreted protein (data not shown), or in the upregulation of COX-2 (Fig. 6D).

View larger version (31K): [in this window] [in a new window]   Fig. 6. Effect of pyrrophenone on AA and PGE2 release, GV PLA2 activity, and COX-2 expression. Cells (106) were plated in 12-well plates and allowed to adhere overnight in the presence of 0.5 µCi 3H-labeled AA if necessary. Cells were then stimulated with 100 ng/ml LPS for 18 h. Pyrrophenone was added 30 min prior to LPS stimulation. A: Determination of AA release by scintillation counting of all radiolabeled AA-derived products. Data represent one out of two experiments, each measured in triplicate. Error bars indicate SD. B: Media were assayed for PGE2 by enzyme immunoassay. Data represent one out of three experiments for 100 nM pyrrophenone (error bars indicate SD) and one out of two experiments for 300 nM pyrrophenone (error bars indicate SD), each measured in triplicate. C: Cells (3 x 106) were plated in 6-well plates and allowed to adhere overnight. Cells were then stimulated with 100 ng/ml LPS for 24 h in the presence (+) or absence (–) of the indicated amounts of pyrrophenone. Media were assayed for GV PLA2 activity. Data represent the mean of three experiments for 100 nM pyrrophenone (error bars indicate SD) and the mean of two experiments for 300 nM pyrrophenone (error bars indicate SE), each measured in triplicate, and show the extrapolated total activity in the media. D: Protein (50 µg) from cell lysate was loaded on a 4–12% polyacrylamide gel, transferred onto nitrocellulose, and probed with anti-COX-2 and GAPDH antibody. Lane 1: control; lane 2: 100 nM pyrrophenone; lane 3: 300 nM pyrrophenone; Std: 20 ng purified ovine COX-2 protein (Cayman). The COX-2 and GAPDH bands are indicated by arrowheads. Data represent one out of three experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have explored the role of the GV PLA₂ in the production of PGE₂ upon long-term stimulation of P388D₁ cells with LPS. Specific second-generation AS-ON directed against GV PLA₂ reduced the expression of the enzyme. We succeeded in decreasing the basal and LPS-induced levels of mRNA encoding GV PLA₂ as well as in reducing the amount of GV protein and activity measured in the cell lysate and cell media under both basal and LPS-stimulated conditions.

Although GV PLA₂ activity was reduced almost to background levels, there was no significant effect on the amount of PGE₂ produced. These results suggest that the upregulation of GV PLA₂ does not play a prominent role in the production of PGE₂. This appears to be in contrast to the findings by Shinohara et al. (5), where first-generation AS-ONs were used. However, this type of ON has been shown to have toxic effects (for review, see Ref. 23) and to bind nonspecifically to proteins (24), which might explain this discrepancy.

For comparison, we also tested the sPLA₂ inhibitor LY311727 and found that i) 1 µM LY311727 inhibits GV activity in the media and ii) PGE₂ release was not affected by treatment of the cells with up to 50 µM of the compound. At 25 µM, no significant reduction of LPS-induced AA-derived radioactivity release was detectable, but at 50 µM, a moderate inhibition of AA release was observed. At this high concentration, we cannot rule out nonspecific effects on the cells. Shinohara et al. (5) also utilized this inhibitor and found a greater inhibition of AA release at a lower concentration. We do not have a good explanation for these differences. We did try several different batches of the inhibitor with essentially the same results. It is important to note that the LY311727 used in this study was efficient in in vitro experiments (Fig. 3B).

Mounier et al. (14) have recently shown that Me-Indoxam, a cell-impermeable sPLA₂ inhibitor structurally related to LY311727, does not have an effect on AA release from human embryonic kidney cells transfected with sPLA₂s GIIA or GX. The authors concluded that these sPLA₂s are active predominantly during the secretion process and not outside the cell. It is conceivable that LY311727 also is unable to cross the cell membrane and therefore cannot act inside the cell. From our results, we conclude that GV PLA₂ does not act on the cell surface of P388D₁ cells to release significant amounts of AA. We cannot exclude an intracellular role for GV PLA₂ in PGE₂ production that might occur prior to its secretion, similar to the mechanism suggested by Mounier et al. (14). However, the total amount of GV PLA₂ activity in lysates and media from cells stimulated with LPS was inhibited virtually to nonstimulated levels with GV AS-ON treatment. This result renders the aforementioned possibility unlikely, inasmuch as we would expect a decrease in PGE₂ production even if GV PLA₂ acted inside the cell. Low levels of GV PLA₂, however, might still contribute to some AA release in cells stimulated with LPS.

It is important to note that LPS-induced AA release is almost entirely blocked by the GIVA-specific inhibitor pyrrophenone. This suggests that GIVA PLA₂ is the dominant signaling component responsible for AA release and confirms previous results by our group utilizing the relatively nonspecific inhibitor MAFP (5).

We cannot rule out that by an unknown mechanism, LPS-induced activation of GIVA PLA₂ results in the activation of low amounts of GV PLA₂ enzyme present in the cell, even after treatment with AS-ON, which might be responsible for some AA release intracellularly. Recently, Arm and coworkers (25) have shown that peritoneal macrophages from GV PLA₂ knock-out mice displayed a 50% reduction in the amount of eicosanoids released upon stimulation with zymosan, indicating a secondary role for the enzyme in this pathway. Previous studies by Leslie and coworkers (26) had shown that zymosan-induced AA release and eicosanoid production were abolished in peritoneal macrophages from GIVA PLA₂ knock-out mice. This suggests that GV PLA₂ is subordinate to GIVA PLA₂ and plays a rather augmentative role in this pathway in peritoneal macrophages. To test whether basal levels of GV PLA₂ are sufficient for AA release in a similar augmentative manner in the P388D₁ cell line would require a complete knock-out of the gene. Unfortunately, to date, utilizing several approaches, we have not succeeded in disrupting the GV PLA₂ gene in P388D₁ cells.

We have shown that considerable amounts of active GV PLA₂ are secreted into the extracellular fluid upon LPS stimulation. The secreted enzyme is inhibited in vitro by 1 µM LY311727. However, as mentioned earlier, even 25 µM LY311727 was not effective in inhibiting the LPS-induced release of AA-derived radioactivity, and 50 µM resulted in only a moderate inhibition.

Why does the secreted active enzyme not act on the plasma membrane of P388D₁ cells? Recently, it has been shown that extracellular addition of the human GV enzyme to P388D₁ cells leads to AA release (6). However, this AA release might be due to detached cells, considering the results from Bezzine et al. (27), which indicate that GV PLA₂ is virtually inactive on adherent HEK293 or CHO-K1 cells. However, when cells were dislodged from the surface, fatty acid release was readily detected (27). At high concentrations of murine GV PLA₂ (1 µg/ml), release of arachidonate from HEK293 cells but not from RBL-2H3 cells was detectable (28). We cannot offer an easy explanation as to why the endogenous secreted enzyme does not act on P388D₁ cells in our system. It is conceivable that the presentation of phospholipid substrate on the cell surface is not correct.

If the GV PLA₂ is only active inside the cell, as discussed earlier, is there a function for the protein once it is released into the extracellular space? There are several conceivable roles: i) sPLA₂ might act in a paracrine manner, as previously suggested (29, 30); ii) GV PLA₂ could bind with high affinity to the M-type sPLA₂ receptor (31) and might thus exert functions through a cytokine-like action rather than through its catalytic activity; iii) recently, a role in LDL hydrolysis and macrophage foam cell formation was attributed to GV PLA₂ (32); iv) GV PLA₂ could serve to kill Gram-positive bacteria (33); and v) secretion of the enzyme might simply be a means to clear the cells from its intracellular activity and direct it into the degradative pathway. A degradative pathway for GV PLA₂ in neutrophils has previously been suggested by Kim et al. (34).

The signal transduction model developed by Shinohara et al. (5) and Balsinde et al. (6) proposes that GIVA PLA₂ regulates expression of GV PLA₂, which, in turn, upregulates COX-2. These results were based mainly on the chemical inhibitors MAFP, LY311727, and first-generation AS-ON directed against GV PLA₂. Here we used pyrrophenone, which is more specific than MAFP in inhibiting the GIVA enzyme. Treatment of the cells with pyrrophenone did not result in an inhibition of the LPS-induced increase in GV PLA₂ activity or COX-2 protein level. We attempted to confirm the results found with pyrrophenone by utilizing second-generation AS-ON to inhibit expression of GIVA PLA₂. However, these experiments were not conclusive, inasmuch as GIVA PLA₂ protein levels and GIVA PLA₂ activity were not affected by treatment with AS-ON. The antisense technology in general has practical limitations, because reduction of protein levels depends on the half-life of the protein (15).

In cells treated with GV AS-ON we did not detect a decrease in COX-2 protein in three independent experiments. In one experiment, we detected only a slight decrease (data not shown). If GV PLA₂ were significantly involved in the regulation of COX-2 expression, we would expect a prominent decrease in the amount of COX-2 protein produced in the presence of GV AS-ON.

Altogether, our results suggest that the GIVA PLA₂ is indispensable for AA release. However, it does not appear to play a role in the regulation of the expression of GV PLA₂ or COX-2. The role of the GV PLA₂ in P388D₁ cells is elusive. However, from our results, it is clear that the upregulation of the enzyme upon stimulation with LPS is not required for PGE₂ production.

	ACKNOWLEDGMENTS

 
The authors thank Raymond Deems for the many helpful discussions and for critically reading the manuscript. The authors thank Iveta Kalcheva for running the quantitative PCR shared resource. The authors are also grateful to Dr. Frank Bennett (ISIS Pharmaceuticals) for providing us with the second-generation antisense oligonucleotides. The authors thank Dr. Henk van den Bosch for providing rat GV PLA₂. The authors utilized the Rebecca and John Moores UCSD Cancer Center DNA Sequencing Shared Resource supported in part by NCI Cancer Center Support Grant #2 P30 CA23100-18. This work was supported by Biostar grant 01-10138 from UC BioStar/ISIS Pharmaceuticals, Inc. Research described in this article was supported in part by Philip Morris, Inc. (postdoctoral fellowship granted to U.A.K.).

Manuscript received July 27, 2005 and in revised form August 23, 2005.

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