Antisense Inhibition of Group VI Ca2+-independent Phospholipase A2 Blocks Phospholipid Fatty Acid Remodeling in Murine P388D1 Macrophages
1997; Elsevier BV; Volume: 272; Issue: 46 Linguagem: Inglês
10.1074/jbc.272.46.29317
ISSN1083-351X
AutoresJesús Balsinde, Marı́a A. Balboa, Edward A. Dennis,
Tópico(s)Adipose Tissue and Metabolism
ResumoA major issue in lipid signaling relates to the role of particular phospholipase A2 isoforms in mediating receptor-triggered responses. This has been difficult to study because of the lack of isoform-specific inhibitors. Based on the use of the Group VI Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL), we previously suggested a role for the iPLA2 in mediating phospholipid fatty acid turnover (Balsinde, J., Bianco, I. D., Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92: 8527–8531). We have now further evaluated the role of the iPLA2 in phospholipid remodeling by using antisense RNA technology. We show herein that inhibition of iPLA2 expression by a specific antisense oligonucleotide decreases both the steady-state levels of lysophosphatidylcholine and the capacity of the cell to incorporate arachidonic acid into membrane phospholipids. These effects correlate with a decrease in both iPLA2 activity and protein in the antisense-treated cells. Collectively these data provide further evidence that the iPLA2 plays a major role in regulating phospholipid fatty acyl turnover in P388D1 macrophages. In stark contrast, experiments with activated cells confirmed that the iPLA2 does not play a significant role in receptor-coupled arachidonate mobilization in these cells, as manifested by the lack of an effect of the iPLA2antisense oligonucleotide on PAF-stimulated arachidonate release. A major issue in lipid signaling relates to the role of particular phospholipase A2 isoforms in mediating receptor-triggered responses. This has been difficult to study because of the lack of isoform-specific inhibitors. Based on the use of the Group VI Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL), we previously suggested a role for the iPLA2 in mediating phospholipid fatty acid turnover (Balsinde, J., Bianco, I. D., Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92: 8527–8531). We have now further evaluated the role of the iPLA2 in phospholipid remodeling by using antisense RNA technology. We show herein that inhibition of iPLA2 expression by a specific antisense oligonucleotide decreases both the steady-state levels of lysophosphatidylcholine and the capacity of the cell to incorporate arachidonic acid into membrane phospholipids. These effects correlate with a decrease in both iPLA2 activity and protein in the antisense-treated cells. Collectively these data provide further evidence that the iPLA2 plays a major role in regulating phospholipid fatty acyl turnover in P388D1 macrophages. In stark contrast, experiments with activated cells confirmed that the iPLA2 does not play a significant role in receptor-coupled arachidonate mobilization in these cells, as manifested by the lack of an effect of the iPLA2antisense oligonucleotide on PAF-stimulated arachidonate release. The phospholipase A2(PLA2) 1The abbreviations used are: PLA2, phospholipase A2: cPLA2, Group IV Ca2+-dependent cytosolic phospholipase A2; iPLA2, Group VI Ca2+-independent cytosolic phospholipase A2; sPLA2, Ca2+-dependent secretory phospholipase A2; AA, arachidonic acid; BEL, bromoenol lactone; LPS, bacterial lipopolysaccharide, PAF, platelet-activating factor; lyso-PC, lysophosphatidylcholine; lyso-PE, lysophosphatidylethanolamine.1The abbreviations used are: PLA2, phospholipase A2: cPLA2, Group IV Ca2+-dependent cytosolic phospholipase A2; iPLA2, Group VI Ca2+-independent cytosolic phospholipase A2; sPLA2, Ca2+-dependent secretory phospholipase A2; AA, arachidonic acid; BEL, bromoenol lactone; LPS, bacterial lipopolysaccharide, PAF, platelet-activating factor; lyso-PC, lysophosphatidylcholine; lyso-PE, lysophosphatidylethanolamine.superfamily of enzymes includes a heterogeneous collection of proteins with diverse roles in cell function (1Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (754) Google Scholar). The PLA2s catalyze the hydrolysis of the sn-2 fatty acyl moiety of phospholipids, generating a free fatty acid and a 2-lysophospholipid, both of which may serve significant biological roles. The latter reaction is particularly relevant when the free fatty acid generated is arachidonic acid (AA), as this is the common precursor of the biologically active eicosanoids, i.e. the prostaglandins, leukotrienes, thromboxane, and lipoxins (2Smith W.L. Am. J. Physiol. 1992; 263: F181-F191PubMed Google Scholar). Based on sequence data, nine different PLA2 Groups have been identified to date (1Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (754) Google Scholar). However, based on biochemical properties and structural features, the PLA2 superfamily can be subdivided into three main types, i.e. the Ca2+-dependent secretory enzymes (sPLA2), the Ca2+-dependent cytosolic enzymes (cPLA2) and the Ca2+-independent cytosolic enzymes (iPLA2). It is difficult to demonstrate specificity of function for a single PLA2 isoform in vivo because most of the PLA2 inhibitors currently available are not isoform-specific. However, bromoenol lactone (BEL) had been regarded as a specific iPLA2 inhibitor since it manifests greater than 1000-fold selectivity for the iPLA2 versus the sPLA2 and cPLA2(3Hazen S.L. Zupan L.A. Weiss R.H. Getman D.P. Gross R.W. J. Biol. Chem. 1991; 266: 7227-7232Abstract Full Text PDF PubMed Google Scholar, 4Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). We recently found that BEL inhibits AA esterification into P388D1 cell phospholipids in a dose-dependent and saturatable manner, with the decrease being directly related to inhibition of both cellular iPLA2 activity and steady-state lysophospholipid levels (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar). These data, along with the finding that the process takes place in a Ca2+-independent manner, led us to implicate the iPLA2 as the enzyme providing the lysophospholipid acceptors employed in the reaction (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar, 6Balsinde J. Dennis E.A. Eur. J. Biochem. 1996; 235: 480-485Crossref PubMed Scopus (29) Google Scholar). Recently however, BEL has been found to inhibit another cellular phospholipase, the Mg2+-dependent phosphatidate phosphohydrolase, with similar potency to that shown for the iPLA2 (7Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 31937-31941Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The nucleotide sequence of the macrophage iPLA2 has recently been elucidated (8Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). This has now allowed us to achieve the specific inhibition of the iPLA2 by using antisense RNA technology. In this manner, the inherent lack of specificity associated with the use of chemical inhibitors such as BEL is circumvented. We have previously taken advantage of this technique to unravel the very important role that sPLA2 plays in AA metabolism in P388D1 cells (9Barbour S.E. Dennis E.A. J. Biol. Chem. 1993; 268: 21875-21882Abstract Full Text PDF PubMed Google Scholar, 10Balsinde J. Barbour S.E. Bianco I.D. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11060-11064Crossref PubMed Scopus (127) Google Scholar, 11Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). We report herein our results on the antisense inhibition of the macrophage iPLA2, which provide further evidence for the involvement of this enzyme in regulating fatty acid remodeling reactions among membrane phospholipids. Mouse P388D1 macrophage-like cells were obtained from the American Type Culture Collection (Rockville, MD). Iscove's modified Dulbecco's medium (endotoxin <0.05 ng/ml) was from Whittaker Bioproducts (Walkersville, MD). Fetal bovine serum was from Hyclone Labs. (Logan, UT). Nonessential amino acids were from Irvine Scientific (Santa Ana, CA). [5,6,8,9,11,12,14,15-3H]Arachidonic acid (specific activity 100 Ci/mmol) and [methyl-3H]choline chloride (specific activity 79 Ci/mmol) were obtained from New England Nuclear (Boston, MA). 1-Palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine (specific activity 59 mCi/mmol) and [2-14C]ethanolamine (specific activity 57 mCi/mmol) were from Amersham Corp. BEL was from Biomol (Plymouth Meeting, PA). Bacterial lipopolysaccharide (LPS) and platelet-activating factor (PAF) were from Sigma. Group VI iPLA2 antiserum was generously provided by Dr. S. Jones (Genetics Institute, Cambridge, MA) (8Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 12Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S.S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) A 20-base-long antisense corresponding to nucleotides 59–78 in the murine Group VI iPLA2 sequence (8Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) was utilized (ASGVI-18; 5′-CUC CUU CAC CCG GAA UGG GU). As a control, the sense complement of ASGVI-18 was used (SGV-18; 5′-ACC CAU UCC GGG UGA AGG AG). Both ASGVI-18 and SGVI-18 contained phosphorothioate linkages to limit degradation. The transfection procedure was adapted from that described by Locati et al. (13Locati M. Lamorte G. Luini W. Introna M. Bernasconi S. Mantovani A. Sozzani S. J. Biol. Chem. 1996; 271: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) for inhibition of the cPLA2. Briefly, 2.5 × 105 cells were cultured in the presence of different oligonucleotide concentrations in Iscove's modified Dulbecco's medium for 4 h at 37 °C in a humidified atmosphere at 90% air and 10% CO2. A final concentration of 10% fetal bovine serum was then added, and the cells were kept in culture for an additional 48-h time period. The culture medium was supplemented with 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and nonessential amino acids. Oligonucleotide treatment and culture conditions were not toxic for the cells as assessed by the trypan blue dye exclusion assay and by quantitating adherent cell protein. After the treatment described above, the cells were placed in phosphate-buffered saline containing 1 mm EGTA for 60 min, washed, and then exposed to exogenous [3H]AA (5 nm, 0.5 μCi/ml). After 10 min, supernatants were removed, and the cell monolayers were gently washed with buffer containing 5 mg/ml albumin to remove the labeled AA that had not been incorporated into cellular lipids. The cell monolayers were scraped twice with 0.5% Triton X-100, and total lipids were extracted according to Bligh and Dyer (14Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (41814) Google Scholar). Phospholipids were separated from the rest of cellular lipids by thin-layer chromatography withn-hexane/ethyl ether/acetic acid (70:30:1). In this system, phospholipids remain at the origin of the plate. Radioactivity content in phospholipids was quantitated by liquid scintillation counting. When BEL was used (25 μm), it was added to the cells 30 min before addition of [3H]AA. The cells were labeled with 0.5 μCi/ml [3H]choline or 0.5 μCi/ml [14C]ethanolamine for 2 days. When oligonucleotides were used, they were added to the cells at the same time as the radioactive compounds. Cellular uptake of the radioactive compounds was not affected by the oligonucleotides. Afterward, the cell monolayers were scraped in 0.5 ml of 0.5% Triton X-100. For separation of lyso-PC and lyso-PE, the lipids were extracted with ice-cold n-butyl alcohol and separated by thin-layer chromatography with chloroform/methanol/acetic acid/water (50:40:6:0.6) as a solvent system. Spots corresponding to lyso-PC or lyso-PE were scraped into scintillation vials, and the amount of radioactivity was estimated by liquid scintillation counting. The cells were washed twice with serum-free medium and homogenized by 25 strokes in a Dounce homogenizer in a buffer consisting of 20 mmTris-HCl, 2 mm EDTA, 10 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 20 μmleupeptin, 20 μm aprotinin, 0.1% 2-mercaptoethanol, pH 7.5. The homogenates were centrifuged at 500 × g for 5 min at 4 °C to separate nuclei. Samples (50 μg) were separated by SDS-PAGE (10% acrylamide gel) and transferred to Immobilon-P (Millipore). Nonspecific binding was blocked by incubating the membranes with a buffer consisting of 5% nonfat milk, 10 mm Tris-HCl, pH 8.0, 1 mm EDTA, 150 mm NaCl, and 0.1% Triton X-100 for 60 min. Membranes were then incubated with anti-iPLA2 antiserum at a 1:200 dilution for 30 min and then treated with horseradish peroxidase-conjugated protein A (Amersham Life Science, Inc.). Bands were detected by enhanced chemiluminescence (ECL, Amersham Life Science, Inc.). Homogenates from P388D1cells were prepared by sonication as described previously (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar). iPLA2 activity in the homogenates was assayed in a buffer consisting of 100 mm Hepes, 400 μm Triton X-100, 100 μm phospholipid substrate (1-palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine; 300,000 cpm), 5 mm EDTA, and the indicated amounts of ATP in a final volume of 0.2 ml (pH 7.5) (15Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 16Ackermann E.J. Kempner E.S. Dennis E.A. J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar). The mixture was incubated at 40 °C for 1 h with shaking. The reaction was stopped by adding 0.75 ml of choloroform/methanol (1:2). The products were extracted according to Bligh and Dyer (14Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (41814) Google Scholar) and separated by thin-layer chromatography with n-hexane/ethyl ether/acetic acid (70:30:1) as a mobile phase. The standard procedure for activating the cells with LPS and PAF has been previously described (4Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Briefly, the cells (106 cells/ml), labeled for 20 h with 0.5 μCi/ml of [3H]AA, were placed in serum-free medium for 1 h before the addition of LPS (200 ng/ml) for 1 h. Subsequently, 10 μl of a 10 mg/ml albumin solution was added per well (final concentration of albumin in each well was 0.1 mg/ml), and after 5 min, PAF (100 nm) was added. After a 15-min incubation period, supernatants were removed, cleared of detached cells by centrifugation, and assayed for radioactivity by liquid scintillation counting. The iPLA2 of P388D1 cells, now designated as a Group VI PLA2(1Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (754) Google Scholar), is an 80–85 kDa protein that shows no specificity for the fatty acid present at the sn-2 position of the phospholipid (16Ackermann E.J. Kempner E.S. Dennis E.A. J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar). This enzyme appears to be implicated in regulating basal phospholipid remodeling in P388D1 cells (4Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar). Fig.1 A shows that treatment of the P388D1 cells with an iPLA2 antisense oligonucleotide (referred to as ASGVI-18) led to a marked decrease in their capacity to incorporate exogenous [3H]AA into membrane phospholipids. This inhibition was not due to loss of cell viability, as judged by trypan blue exclusion and by quantitation of adherent cell protein. Cell viability was further assessed by monitoring [3H]thymidine incorporation, which was the same in control or oligonucleotide-treated cells (not shown). AA incorporation experiments carried out in the presence of 1 mm CaCl2 in the incubation medium instead of 1 mm EGTA gave the same results, in agreement with previous data (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar). More importantly, ASGVI-18 reduced the iPLA2activity of cell homogenates by 75–80% (Fig. 1 B). A polyclonal antibody against the Group VI iPLA2 has recently become available (12Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S.S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). This antibody was generated to a glutathione S-transferase fusion of the C-terminal half of the hamster Group VI iPLA2 (12Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S.S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Using this antibody, we observed a 55–60% decrease of the immunoreactive band, detected by Western blot, in homogenates from ASGVI-18-treated cells compared with control or sense-treated cells (Fig. 1 C). The dose dependence and time course of the effect of the iPLA2 antisense oligonucleotide was investigated, and the results are shown in Fig. 2. Concentrations of ASGVI-18 below 1 μm exerted little effect; whereas, maximal effects were observed at an oligonucleotide concentration of 10 μm (Fig. 2 A). Oligonucleotide concentrations higher than 10 μm induced excessive detachment of the cells from the plastic wells and, therefore, could not be used. Consistent with an antisense-type inhibition, ASGVI-18 decreases cellular AA esterification at all times measured (Fig. 2 B). The above experiments determined incorporation of AA at short times in cells that were already deficient in iPLA2. Thus it was important to determine whether iPLA2 depletion changes the endogenous AA pools. To that end, experiments were conducted wherein the radioactive AA was added at the time the cells were exposed to the oligonucleotides. As AA incorporation into phospholipids in P388D1 takes place very rapidly (10Balsinde J. Barbour S.E. Bianco I.D. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11060-11064Crossref PubMed Scopus (127) Google Scholar), it will be completed long before the cellular iPLA2 levels begin to drop as a result of the antisense treatment. Thus, the relative distribution of AA among phospholipids in iPLA2-depleted cells can be determined and compared with that in control untreated cells. As shown in Table I, the relative distribution of AA among phospholipids did not change after ASGVI-18 treatment, indicating that ASGVI-18 treatment does not change the endogenous AA pools.Table IEffect of ASGVI-18 on the distribution of AA among phospholipidsPhospholipidControlSGVI-18ASGVI-18%PC33 ± 1028 ± 328 ± 5PE55 ± 560 ± 357 ± 5PI15 ± 314 ± 120 ± 6P388D1 cells were labeled with [3H]AA (0.5 μCi/ml) at the time they were treated with SGVI-18, ASGVI-18, or neither (control) as indicated. After 2 days, the amount of AA in the different lipid classes was determined after separation by thin-layer chromatography. Data are given as a percentage of the total radioactivity found in all phospholipid classes and are expressed as means ± S.E. of three independent determinations. Open table in a new tab P388D1 cells were labeled with [3H]AA (0.5 μCi/ml) at the time they were treated with SGVI-18, ASGVI-18, or neither (control) as indicated. After 2 days, the amount of AA in the different lipid classes was determined after separation by thin-layer chromatography. Data are given as a percentage of the total radioactivity found in all phospholipid classes and are expressed as means ± S.E. of three independent determinations. Fig. 3 shows that ASGVI-18 treatment of P388D1 cells resulted in a 50–55% decrease of the steady-state levels of lyso-PC, which corresponds well with the decrease in AA incorporation into phospholipids shown in Fig.1 A. Lyso-PC levels in SGVI-18-transfected cells were the same as those found in control untreated cells (Fig. 3). Within experimental error, no effect of ASGVI-18 on lyso-PE levels could be detected (data not shown). Collectively, the above data suggest that selective inhibition of iPLA2 expression by antisense RNA technology reduces AA incorporation due to a decrease in the amount of cellular lyso-PC acceptors available for the esterification reaction. These data are consistent with our previous results using BEL to inhibit the iPLA2 (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar). However, it is now known that BEL also inhibits the Mg2+-dependent phosphatidate phosphohydrolase, which is another key enzyme in phospholipid metabolism (7Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 31937-31941Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 17Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar). To verify the specificity of BEL in our previous studies (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar), it seemed important to study the effects of BEL on AA esterification in antisense-treated cells. The results from these experiments are summarized in Fig. 4. In agreement with our previous data (8Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), AA incorporation into phospholipids was blocked up to 60% by BEL in control and SGVI-18-treated cells. In ASGVI-18-treated cells, which already showed a 60% decrease in AA incorporation, BEL was ineffective in further increasing this inhibition (Fig. 4). We have previously found that BEL has no effect on the relative distribution of AA among the different phospholipids of P388D1 cells (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar). The above data suggest that the Group VI iPLA2 may function as a housekeeping enzyme involved in the regulation of basal deacylation/reacylation reactions among phospholipids. To further assess its role in cellular function, we sought to assess its role during receptor activation conditions. The effect of ASGVI-18 on LPS/PAF-stimulated AA release from P388D1 cells is shown in Fig.5. Stimulation of murine P388D1 macrophages with nanomolar amounts of the inflammatory mediator PAF results in negligible cellular responses unless the cells are first treated with LPS. LPS acts just as a primer,i.e. it does not stimulate the P388D1 cells by itself but enables the cells to optimally respond to PAF (23Asmis R. Dennis E.A. Ann. N. Y. Acad. Sci. 1994; 744: 1-10Crossref PubMed Scopus (5) Google Scholar). The sense and antisense oligonucleotides both slightly decreased the AA release response, which was detected both in resting and PAF-activated cells. However, the ratio of stimulated versus unstimulated release remained the same under all conditions (Fig. 5). This result indicates that the iPLA2 does not play a significant role in mediating agonist-induced AA mobilization in these cells. It is now well established that the availability of free AA limits the synthesis of the biologically active eicosanoids (2Smith W.L. Am. J. Physiol. 1992; 263: F181-F191PubMed Google Scholar). As AA is mainly found esterified at the sn-2 position of cellular phospholipids, PLA2 has emerged as a key enzyme responsible for controlling the levels of free fatty acid (18Dillon D.A. Chen X. Zeimetz G.M. Wu W.-I. Waggoner D.W. Dewald J. Brindley D.N. Carman G.M. J. Biol. Chem. 1997; 272: 10361-10366Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, the amount of free AA available for eicosanoid synthesis represents a balance between what is being liberated by the activated PLA2s, minus what is reincorporated back into phospholipids by the highly active acyltransferases (19Chilton F.H. Fonteh A.N. Surette M.E. Triggiani M. Winkler J.D. Biochim. Biophys. Acta. 1996; 1299: 1-15Crossref PubMed Scopus (208) Google Scholar). Thus, free fatty AA levels are also efficiently controlled by the AA reacylation pathway (19Chilton F.H. Fonteh A.N. Surette M.E. Triggiani M. Winkler J.D. Biochim. Biophys. Acta. 1996; 1299: 1-15Crossref PubMed Scopus (208) Google Scholar). Even under resting conditions, the capacity of certain cells such as macrophages to incorporate AA into phospholipids via reacylation reactions is exceedingly high (6Balsinde J. Dennis E.A. Eur. J. Biochem. 1996; 235: 480-485Crossref PubMed Scopus (29) Google Scholar, 20Kuwae T. Schmid P.C. Johnson S.B. Schmid H.H.O. J. Biol. Chem. 1990; 265: 5002-5007Abstract Full Text PDF PubMed Google Scholar, 21Kuwae T. Schmid P.C. Schmid H.H.O. Biochim. Biophys. Acta. 1997; 1344: 74-86Crossref PubMed Scopus (33) Google Scholar). Thus these cells should possess a basal PLA2 activity high enough to account for their high AA reacylation capacity. Interestingly, AA incorporation into phospholipids in macrophages is Ca2+-independent,i.e. it takes place normally at free Ca2+concentrations lower than 10 nm (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar). As under these conditions neither the cPLA2 nor the sPLA2 must be active, these results suggest that the enzyme providing lysophospholipid acceptors for the AA reacylation pathway is an iPLA2. Further evidence for such a role was obtained by the use of BEL (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar). Using this inhibitor, we found a direct correlation between endogenous iPLA2 activity, steady-state lysophospholipid levels, and AA incorporation capacity of the cells (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar). However, BEL, being selective for the iPLA2 over the other PLA2s (3Hazen S.L. Zupan L.A. Weiss R.H. Getman D.P. Gross R.W. J. Biol. Chem. 1991; 266: 7227-7232Abstract Full Text PDF PubMed Google Scholar, 4Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar), is not devoid of other effects. For example, we have recently found that BEL also potently blocks the Mg2+-dependent phosphatidate phosphohydrolase, a key enzyme in cellular phospholipid metabolism (7Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 31937-31941Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The inherent problems associated with the use of chemical inhibitors can be potentially circumvented by inhibiting expression of the iPLA2 with antisense RNA oligonucleotides. Using this strategy, we have succesfully achieved the specific inhibition of the macrophage Group VI PLA2 and confirmed our previous findings with BEL. Thus, the iPLA2 antisense oligonucleotide ASGVI-18 reduces the cellular iPLA2activity by 75–80% (Fig. 1 B) and iPLA2 protein by at least 50–60% (Fig. 1 C), which results in a decrease of the capacities of the cells to incorporate AA into phospholipids (Figs. 1 A and 2) as well as the steady-state lyso-PC levels (Fig. 3). These latter effects correspond precisely, and are specific, as parallel experiments using the sense control SGVI-18 did not reproduce any of the effects induced by ASGVI-18. Specificity of ASGVI-18 is also stressed by the fact that sPLA2 antisense oligonucleotides do not affect macrophage AA esterification (10Balsinde J. Barbour S.E. Bianco I.D. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11060-11064Crossref PubMed Scopus (127) Google Scholar). These results provide definitive evidence for the key role that the iPLA2 plays in regulating basal phospholipid remodeling reactions in macrophages. Strikingly, ASGVI-18 treatment was found to significantly decrease the steady state of lyso-PC levels but not of lyso-PE. It is likely that our inablity to detect any effect of ASGVI-18 on lyso-PE levels merely reflects some sort of experimental limitations. However, it could indicate as well that the Group VI iPLA2 preferentially attacks PC over PE in vivo even though the enzyme has been found to lack headgroup specificity in vitro (12Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S.S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 16Ackermann E.J. Kempner E.S. Dennis E.A. J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar). It is possible that PC pools within the cells are more accesible to iPLA2 attack than the PE pools. Interestingly, in regards to AA metabolism, it is lyso-PC, not lyso-PE, that is the major acceptor for esterification of free AA (19Chilton F.H. Fonteh A.N. Surette M.E. Triggiani M. Winkler J.D. Biochim. Biophys. Acta. 1996; 1299: 1-15Crossref PubMed Scopus (208) Google Scholar). Over time, the AA accumulates into PE as a consequence of direct transfer from PC by CoA-independent transacylase, not by direct acylation of lyso-PE with AA (19Chilton F.H. Fonteh A.N. Surette M.E. Triggiani M. Winkler J.D. Biochim. Biophys. Acta. 1996; 1299: 1-15Crossref PubMed Scopus (208) Google Scholar). Therefore, selective inhibition of lyso-PC but not of lyso-PE would result in decreased labeling of phospholipids with AA, and the ratio AA-PC to AA-PE remaining unchanged. This is exactly what was found in the experiments determining the effect of ASGVI-18 treatment on AA distribution among phospholipids (Table I). Thus, the current data give additional support to the notion that the different phospholipid classes and subclasses serve different roles for AA incorporation and redistribution within different pools (19Chilton F.H. Fonteh A.N. Surette M.E. Triggiani M. Winkler J.D. Biochim. Biophys. Acta. 1996; 1299: 1-15Crossref PubMed Scopus (208) Google Scholar). Under our experimental conditions, we were not able to achieve complete inhibition of the iPLA2 but did achieve a 60% or 80% disappearance at best, as judged by protein content or activity, respectively. This reduces AA incorporation into phospholipids by about 60% in antisense-treated cells. It could thus be argued that achieving 100% inhibition of Group VI iPLA2 expression would result in almost complete ablation of the capacity of the cell to incorporate AA into phospholipids. However, the fact that the decrease in iPLA2 protein detected by Western blot does not correspond with the decrease in cellular iPLA2 activity suggests the possibility that, in addition to the iPLA2, the antibody used may be recognizing another 85 kDa-protein in P388D1macrophages. If this were the case, then the remainder of the 85-kDa protein band that is not blocked by ASGVI-18 could correspond to an antigenically related protein. Moreover, it is also possible that the 20% of iPLA2 activity that is not eliminated by ASGVI-18, corresponds to another iPLA2 distinct from the Group VI enzyme. The possibility that the P388D1 cells contain more than one iPLA2 form has previously been considered (16Ackermann E.J. Kempner E.S. Dennis E.A. J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar). Finally, BEL, either alone (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar) or together with the iPLA2antisense (Fig. 4) fails to completely inhibit the response. Saturation of inhibition by BEL is reached at about 60–70% (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar). Altogether, these facts seem to suggest that the cell possesses other mechanisms for regulating AA esterification besides the one provided by the Group VI iPLA2. It should be remarked here that the current experiments were done in the absence of Ca2+. Therefore, if a second PLA2 distinct from the Group VI enzyme was involved in regulating AA esterification, it would have to be another Ca2+-independent isoform. As discussed elsewhere (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar, 6Balsinde J. Dennis E.A. Eur. J. Biochem. 1996; 235: 480-485Crossref PubMed Scopus (29) Google Scholar), the contribution of the de novo biosynthetic pathway to AA incorporation into phospholipids is minimal in P388D1 macrophages. This contrasts with results obtained with peritoneal macrophages (21Kuwae T. Schmid P.C. Schmid H.H.O. Biochim. Biophys. Acta. 1997; 1344: 74-86Crossref PubMed Scopus (33) Google Scholar) and neutrophils (22Chilton F.H. Murphy R.C. Biochem. Biophys. Res. Commun. 1987; 145: 1126-1133Crossref PubMed Scopus (41) Google Scholar), wherein the de novo pathway has been demonstrated to provide a minor but significant route for the generation of highly polyunsaturated phospholipid species such as 1,2-diarachidonoyl-sn-glycero-3-phosphocholine. However the phospholipids of peritoneal macrophages and neutrophils are already very enriched in AA (21Kuwae T. Schmid P.C. Schmid H.H.O. Biochim. Biophys. Acta. 1997; 1344: 74-86Crossref PubMed Scopus (33) Google Scholar, 22Chilton F.H. Murphy R.C. Biochem. Biophys. Res. Commun. 1987; 145: 1126-1133Crossref PubMed Scopus (41) Google Scholar). Such a circumstance likely explains why a significant portion of the free AA is shunted to the low-affinity biosynthetic route in these cells. In contrast, P388D1cells are very scarce in endogenous AA (23Asmis R. Dennis E.A. Ann. N. Y. Acad. Sci. 1994; 744: 1-10Crossref PubMed Scopus (5) Google Scholar). Hence, most of the AA incorporation in these cells occurs through the high-affinity remodeling pathway (5Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 8527-8531Crossref Scopus (255) Google Scholar, 6Balsinde J. Dennis E.A. Eur. J. Biochem. 1996; 235: 480-485Crossref PubMed Scopus (29) Google Scholar). Nonetheless, recent data have highlighted the fact that the sn-1 position of phospholipids is also extensively remodeled in macrophages (21Kuwae T. Schmid P.C. Schmid H.H.O. Biochim. Biophys. Acta. 1997; 1344: 74-86Crossref PubMed Scopus (33) Google Scholar), which means that a phospholipase A1 could also eventually participate in regulating AA esterification via fatty acid remodeling. Such a possibility, as well as the possible involvement of the recently described CoA-dependent transacylase reaction (24Sugiura T. Kudo N. Ojima T. Mabuchi-Itoh K. Yamashita A. Waku K. Biochim. Biophys. Acta. 1995; 1255: 167-176Crossref PubMed Scopus (29) Google Scholar, 25Blank M.L. Fitzgerald V. Smith Z.L. Snyder F. Biochem. Biophys. Res. Commun. 1995; 210: 1052-1058Crossref PubMed Scopus (7) Google Scholar), is currently under investigation in our laboratory. In summary, in the present study we have utilized antisense RNA technology to obtain independent conclusive confirmation that the macrophage Group VI iPLA2 does play an important role in modulating phospholipid fatty acid turnover by providing the 2-lysophospholipid acceptors required for the reaction. In addition, our results demonstrate that the Group VI iPLA2 does not appear to play a significant role in the stimulation of AA release mediated via the PAF surface receptor.
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