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, Downloaded from and Edward A. Dennis2 Departments of Chemistry and Biochemistry and Pharmacology, University of California–San Diego, La Jolla, CA 92093 www.jlr.org Abstract P388D1 cells release arachidonic acid (AA) and pro- Any free arachidonic acid (AA) liberated by these enzymes duce prostaglandin E2 (PGE2) upon long-term stimulation with can subsequently be converted into prostaglandins (PGs) lipopolysaccharide (LPS). The cytosolic Group IVA (GIVA) through the action of cyclooxygenases (COXs) and PG syn- phospholipase A2 (PLA2) has been implicated in this path- thases. at Univ of California - San Diego Serials/Biomed Lib 0699, on December 24, 2020 way. LPS stimulation also results in increased expression Prominent members of the PLA2 superfamily are the se- and secretion of a secretory PLA2, specifically GV PLA2. To creted phospholipase A2s (sPLA2s), the cytosolic PLA2s test whether GV PLA2 contributes to PGE2 production and (cPLA s), and the calcium-independent PLA s (iPLA s). whether GIVA PLA2 activation increases the expression of 2 2 2 GV PLA2, we utilized the specific GIVA PLA2 inhibitor pyr- Common features of the sPLA2s are their relatively low rophenone and second generation antisense oligonucleotides molecular mass (about 14 kDa), a high abundance of di- (AS-ONs) designed to specifically inhibit expression and ac- sulfide bonds, and their requirement for micromolar tivity of GV PLA . Treatment of P388D cells with antisense 2ϩ 2 1 Ca for activity. cPLA2s are characterized by a higher mo- caused a marked decrease in basal GV PLA2 mRNA and pre- lecular mass (about 85 kDa) and their preference for AA- vented the LPS-induced increase in GV PLA2 mRNA. LPS- containing phospholipids (2–4). The iPLA2s are distin- stimulated cells release active GV PLA2 into the medium, which is inhibited to background levels by antisense treatment. guished from the other PLA2s in that they do not require Ca2ϩ for activity. However, LPS-induced PGE2 release by antisense-treated cells and by control cells are not significantly different. Previous work has demonstrated that the murine mac- Collectively, the results suggest that the upregulation of GV rophage-like P388D1 cells release prostaglandin E2 (PGE2) PLA2 during long-term LPS stimulation is not required for upon long-term stimulation with bacterial lipopolysaccha- PGE2 production by P388D1 cells. Experiments employing ride (LPS). Shinohara et al. (5) and Balsinde et al. (6) pyrrophenone suggested that GIVA PLA is the dominant 2 have suggested possible roles for the c- and sPLA2s in the player involved in AA release, but it appears not to be involved release of AA, resulting in PGE production. In a hypo- in the regulation of LPS-induced expression of GV PLA or 2 2 thetical model, activation of the Group IV (GIV) cPLA re- cyclooxygenase-2.—Kessen, U. A., R. H. Schaloske, D. L. 2 sults in an increased expression of Group V (GV) sPLA2, Stephens, K. Killermann Lucas, and E. A. Dennis. PGE2 re- lease is independent of upregulation of Group V phospholi- which, in turn, is largely responsible for the release of the pase A2 during long-term stimulation of P388D1 cells with AA that is subsequently converted into PGE2, and GV LPS. J. Lipid Res. 2005. 46: 2488–2496. PLA2 gives rise to the upregulation of COX-2 expression. The model is based on experiments utilizing the chemical Supplementary key words antisense inhibitor • macrophage • lipo- inhibitors methyl arachidonyl fluorophosphonate (MAFP), polysaccharide • prostaglandin E • arachidonic acid • eicosanoid 2 affecting Group IVA (GIVA) PLA2; 3-(3-acetamide-1-benzyl- 2-ethylindolyl-5-oxy) propane sulfonic acid (LY311727), affecting sPLA2; and first-generation antisense oligonucle- 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 glycerophospholip- ids, producing free fatty acid and lysophospholipids (1). Abbreviations: AA, arachidonic acid; AS-ON, antisense oligonucle- otide; GIVA, Group IVA; LPS, lipopolysaccharide; MAFP, methyl arachi- donyl fluorophosphonate; PGE2, prostaglandin E2; PLA2, phospholipase A2; S-ON, sense oligonucleotide; sPLA2, secreted phospholipase A2. Manuscript received 27 July 2005 and in revised form 23 August 2005. 1 Trudy Forte served as Guest Editor for this manuscript. Published, JLR Papers in Press, September 8, 2005. 2 To whom correspondence should be addressed. DOI 10.1194/jlr.M500325-JLR200 e-mail: [email protected] Copyright © 2005 by the American Society for Biochemistry and Molecular Biology, Inc. 2488 Journal of Lipid Research Volume 46, 2005 This article is available online at http://www.jlr.org otides (AS-ONs). All three of these inhibitors have draw- (specific activity 100 Ci/mmol) was obtained from NEN Life Sci- backs. MAFP is not specific, inasmuch as it inhibits a num- ence Products (Boston, MA). The Complete Mini Protease In- hibitor Cocktail was from Roche Applied Science (Indianapolis, ber of enzymes in addition to GIVA PLA2, namely GroupVI PLA (7, 8), anandamide amidase (9, 10), and platelet- IN). Triton X-100 was purchased from Calbiochem (La Jolla, CA). 2 The GV PLA AS-ONs and control oligonucleotides were kindly activating factor-acetylhydrolase (11). It also binds to the 2 provided by Dr. Frank Bennett of ISIS Pharmaceuticals (Carls- CB1 cannabinoid receptor (12, 13). The sPLA2 inhibitor bad, CA). LY 311727 was the generous gift of Dr. Jerome Fleisch LY311727 is structurally related to Me-Indoxam. Mounier (Lilly Research Laboratories). Pyrrophenone was the generous et al. (14) described recently that Me-Indoxam does not gift of Dr. Takaski Ono (Shionogi Research Laboratories). MAFP pass through the plasma membrane of mammalian cells and the polyclonal antibody directed against murine GV PLA2 and that it does not inhibit AA release from human em- and the polyclonal antibody directed against COX-2 were pur- bryonic kidney cells that have been transfected with GIIA chased from Cayman Chemicals (Ann Arbor, MI). The monoclo- or GX PLA s. The authors concluded that the sPLA s act nal antibody against GAPDH was obtained from HyTest (Turku, 2 2 Finland). Cytofectin was from Gene Therapy Systems (San Di- inside the cell prior to their secretion. It is probable that ego, CA). SYBR-Green PCR Master Mix was from Applied Biosys- LY311727 is not able to act inside the cells. Recent experi- tems (Foster City, CA). Primers used for the real-time Q-PCR Downloaded from ments from our laboratory employing LY311727 in P388D1 were ordered from Proligo (Boulder, CO). M-MLV reverse tran- cells support this hypothesis. The first-generation AS-ONs scriptase, RNase H, SeeBlue2 PreStained protein standard and suffer from their low affinity toward target RNA molecules the 12% Bis-Tris gels were from Invitrogen (Carlsbad, CA). and their toxic side effects (15–18). To test the hypothetical model described above, we em- Cell culture Њ ployed second-generation AS-ONs consisting of a 5-10-5 2Ј- P388D1 cells (MAB clone) (5), were maintained at 37 C in a www.jlr.org O-methoxy-ethyl RNA gapmer with a phosphorothioate humidified atmosphere at 90% air and 10% CO2 in IMDM sup- plemented with 10% fetal bovine serum, 100 U/ml penicillin, backbone. These modifications increase the binding of and 100 ␮g/ml streptomycin (complete IMDM). For transfection the oligonucleotide to its target, stabilize the ON by pre- experiments, the cells were seeded at a density of 106 cells/2 ml/ at Univ of California - San Diego Serials/Biomed Lib 0699, on December 24, 2020 venting nuclease degradation, and support RNase H-medi- well in 6-well plates and allowed to adhere overnight. For experi- ated cleavage of the targeted mRNA (15–18). We used sec- ments using chemical inhibitors, cells were seeded either at a den- ond-generation AS-ONs specifically designed to inhibit sity of 106 cells/ml/well in 12-well plates or at a density of 3 ϫ 106 expression of GV PLA2, and thus its activity, in order to ex- cells/2 ml/well in 6-well plates and allowed to adhere overnight. For experiments assaying AA-derived radioactivity release, cells were amine specifically the role of GV PLA2 in PGE2 produc- seeded in 12-well plates in the presence of 0.5 ␮Ci 3H-labeled AA. tion. We also utilized the novel GIVA PLA2 inhibitor pyr- rophenone, which is more specific and potent than MAFP Stimulation of cells (19, 20). Cells transfected with oligonucleotides were stimulated 5 h af- We now show that treatment of P388D1 cells with AS-ONs ter transfection by adding 150 ng/ml LPS to the cells for 24 h. In resulted in a marked decrease of basal GV PLA2 mRNA, experiments using chemical inhibitors assaying AA-derived ra- prevented the LPS-induced increase in GV PLA2 mRNA as dioactivity release or PGE2 release, cells were washed three times measured with real-time quantitative PCR (Q-PCR), and in IMDM, incubated in the same medium for 1 h, and then stim- also caused a significant decrease in basal and stimulated ulated by adding 100 ng/ml LPS for 18 h. Inhibitors were added during the starvation period 30 min prior to the addition of LPS. GV PLA2 protein and activity levels. Basal and stimulated PGE2 levels were not affected by AS-ON treatment. Treat- Transient transfection of AS-ONs ment of the cells with the novel GIVA PLA2 inhibitor pyr- The following ONs were used for transfection: GV sense oligo- rophenone resulted in a 60–70% inhibition of PGE2 pro- nucleotide (S-ON) ISIS# 357 886 (5Ј-TTC CGG AGG AAG GGT duction but did not affect COX-2 protein levels.