Role of phospholipase D in agonist-stimulated lysophosphatidic acid synthesis by ovarian cancer cells

Lysophosphatidic acid (LPA) is a receptor-active lipid mediator with a broad range of biological effects. Ovarian cancer cells synthesize LPA, which promotes their motility, growth, and survival. We show that a murine homolog of a human protein previously reported to hydrolyze LPA is a highly selective detergent-stimulated LPA phosphatase that can be used to detect and quantitate LPA. Use of this protein in novel enzymatic assay demonstrates that SK-OV-3 ovarian cancer cells release physiologically relevant levels of biologically active LPA into the extracellular space. LPA release is markedly increased by nucleotide agonists acting through a P2Y4 purinergic receptor. Promotion of LPA formation by nucleotides is accompanied by stimulation of phospholipase D (PLD) activity. Overexpression of both PLD1 and PLD2 in SK-OV-3 cells produces active enzymes, but only overexpression of PLD2 results in significant amplification of both nucleotide-stimulated PLD activity and LPA production. SK-OV-3 cells express and secrete a phospholipase A2 activity that can generate LPA from the lipid product of PLD, phosphatidic acid.Our results identify a novel role for nucleotides in the regulation of ovarian cancer cells and suggest an indirect but critical function for PLD2 in agonist-stimulated LPA production. Lysophosphatidic acid (LPA) is a receptor-active lipid mediator with a broad range of biological effects. Ovarian cancer cells synthesize LPA, which promotes their motility, growth, and survival. We show that a murine homolog of a human protein previously reported to hydrolyze LPA is a highly selective detergent-stimulated LPA phosphatase that can be used to detect and quantitate LPA. Use of this protein in novel enzymatic assay demonstrates that SK-OV-3 ovarian cancer cells release physiologically relevant levels of biologically active LPA into the extracellular space. LPA release is markedly increased by nucleotide agonists acting through a P2Y4 purinergic receptor. Promotion of LPA formation by nucleotides is accompanied by stimulation of phospholipase D (PLD) activity. Overexpression of both PLD1 and PLD2 in SK-OV-3 cells produces active enzymes, but only overexpression of PLD2 results in significant amplification of both nucleotide-stimulated PLD activity and LPA production. SK-OV-3 cells express and secrete a phospholipase A2 activity that can generate LPA from the lipid product of PLD, phosphatidic acid. Our results identify a novel role for nucleotides in the regulation of ovarian cancer cells and suggest an indirect but critical function for PLD2 in agonist-stimulated LPA production. Lysophosphatidic acid (LPA) is a naturally occurring lysophospholipid with diverse actions on a broad range of cell types. Responses to LPA include stimulation of cell growth, differentiation, survival, and alterations in cell morphology and motility. LPA binds to cell surface G-protein-coupled receptors termed LPA1-3. These receptors are widely expressed and signal through actions of members of both the Gq and Gi families of heterotrimeric G-proteins. Many, but possibly not all, of the effects of LPA on cells are mediated by these receptors (1Hla T. Lee M.J. Ancellin N. Paik J.H. Kluk M.J. Lysophospholipids—receptor revelations.Science. 2001; 294: 1875-1878Google Scholar, 2Tigyi G. Physiological responses to lysophosphatidic acid and related glycero-phospholipids.Prostaglandins. 2001; 64: 47-62Google Scholar). While the signaling actions of LPA have been well characterized, much less is known about the pathways and enzymes involved in the synthesis and inactivation of this mediator. LPA is a normal constituent of serum, where it is produced and released by activated platelets (3Gerrard J.M. Robinson P. Identification of the molecular species of lysophosphatidic acid produced when platelets are stimulated by thrombin.Biochim. Biophys. Acta. 1989; 1001: 282-285Google Scholar, 4Fourcade O. Simon M.F. Viode C. Rugani N. Leballe F. Ragab A. Fournie B. Sarda L. Chap H. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells.Cell. 1995; 80: 919-927Google Scholar, 5Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. The bioactive phospholipid lysophosphatidic acid is released from activated platelets.Biochem. J. 1993; 291: 677-680Google Scholar). LPA production has also been demonstrated for a small number of other cell types (6Pages G. Girard A. Jeanneton O. Barbe P. Wolf C. Lafontan M. Valet P. Saulnier-Blache J.S. LPA as a paracrine mediator of adipocyte growth and function.Ann. N. Y. Acad. Sci. 2000; 905: 159-164Google Scholar, 7Shen Z. Belinson J. Morton R.E. Xu Y. Xu Y. Phorbol 12-myristate 13-acetate stimulates lysophosphatidic acid secretion from ovarian and cervical cancer cells but not from breast or leukemia cells.Gynecol. Oncol. 1998; 71: 364-368Google Scholar). LPA can be synthesized both de novo and through pathways that are initiated by phospholipase-catalyzed degradation of precursor glycerophospholipids (8Gaits F. Fourcade O. le Balle F. Gueguen G. Gaige B. Gassama-Diagne A. Fauvel J. Salles J.P. Mauco G. Simon M.F. Chap H. Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis.FEBS Lett. 1997; 410: 54-58Google Scholar). Hydrolysis of lysophospholipids could generate LPA directly, and enzymes that catalyze this reaction with selectivity for lysophosphatidylcholine (lysoPC) have been reported (9Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma.Biochim. Biophys. Acta. 1986; 875: 31-38Google Scholar, 10Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production.J. Cell Biol. 2002; 158: 227-233Google Scholar). LPA can also be generated by phospholipase A (PLA)-catalyzed hydrolysis of phosphatidic acid (PA), which can be formed in stimulated cells through actions of inositol lipid-specific phospholipase C (PLC), diacylglycerol kinase, or phosphatidylcholine-specific phospholipase D (PLD) (11Snitko Y. Yoon E.T. Cho W. High specificity of human secretory class II phospholipase A2 for phosphatidic acid.Biochem. J. 1997; 321: 737-741Google Scholar). How LPA that is formed from cellular lipids accumulates in the extracellular space is not clear. Studies with platelets implicate membrane microvesicles released in response to agonist activation as key intermediates in LPA production, but the relevance of this pathway to the process of LPA production by other cell types is unknown (4Fourcade O. Simon M.F. Viode C. Rugani N. Leballe F. Ragab A. Fournie B. Sarda L. Chap H. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells.Cell. 1995; 80: 919-927Google Scholar). LPA plays a central role in the growth invasiveness and resistance to chemotherapeutics of ovarian cancer cells (12Xu Y. Fang X.J. Casey G. Mills G.B. Lysophospholipids activate ovarian and breast cancer cells.Biochem. J. 1995; 309: 933-940Google Scholar, 13Pustilnik T.B. Estrella V. Wiener J.R. Mao M. Eder A. Watt M.A. Bast Jr., R.C. Mills G.B. Lysophosphatidic acid induces urokinase secretion by ovarian cancer cells.Clin. Cancer Res. 1999; 5: 3704-3710Google Scholar, 14Mills G.B. Eder A. Fang X. Hasegawa Y. Mao M. Lu Y. Tanyi J. Tabassam F.H. Wiener J. Lapushin R. Yu S. Parrott J.A. Compton T. Tribley W. Fishman D. Stack M.S. Gaudette D. Jaffe R. Furui T. Aoki J. Erickson J.R. Critical role of lysophospholipids in the pathophysiology, diagnosis, and management of ovarian cancer.Cancer Treat. Res. 2002; 107: 259-283Google Scholar). Ovarian surface epithelial cells, from which these cancers are commonly derived, are resistant to many of these effects of LPA, suggesting that acquisition of enhanced LPA responsiveness is associated with transformation and that targeting LPA synthesis or signaling might provide a novel treatment strategy for the disease (14Mills G.B. Eder A. Fang X. Hasegawa Y. Mao M. Lu Y. Tanyi J. Tabassam F.H. Wiener J. Lapushin R. Yu S. Parrott J.A. Compton T. Tribley W. Fishman D. Stack M.S. Gaudette D. Jaffe R. Furui T. Aoki J. Erickson J.R. Critical role of lysophospholipids in the pathophysiology, diagnosis, and management of ovarian cancer.Cancer Treat. Res. 2002; 107: 259-283Google Scholar). Ovarian tumors grow in the peritoneal cavity, where patients accumulate ascites fluid. Ovarian cancer ascites contain physiologically significant levels of LPA, and it is likely that ovarian cancer cells themselves are the source of this LPA (14Mills G.B. Eder A. Fang X. Hasegawa Y. Mao M. Lu Y. Tanyi J. Tabassam F.H. Wiener J. Lapushin R. Yu S. Parrott J.A. Compton T. Tribley W. Fishman D. Stack M.S. Gaudette D. Jaffe R. Furui T. Aoki J. Erickson J.R. Critical role of lysophospholipids in the pathophysiology, diagnosis, and management of ovarian cancer.Cancer Treat. Res. 2002; 107: 259-283Google Scholar, 15Xu Y. Gaudette D.C. Boynton J.D. Frankel A. Fang X.J. Sharma A. Hurteau J. Casey G. Goodbody A. Mellors A. Characterization of an ovarian cancer activating factor in ascites from ovarian cancer patients.Clin. Cancer Res. 1995; 1: 1223-1232Google Scholar). Certain ovarian cancer cell lines have been shown to release LPA constitutively and in response to pharmacological agents, including Ca2+ ionophore, phorbol esters, and LPA itself. The use of primary alcohols that divert PA produced by PLD to biologically inactive phosphatidylalcohols implicates PLD in the pathway of LPA synthesis by ovarian cancer cells, but the mechanism involved is unclear (16Eder A.M. Sasagawa T. Mao M. Aoki J. Mills G.B. Constitutive and lysophosphatidic acid (LPA)-induced LPA production: role of phospholipase D and phospholipase A2.Clin. Cancer Res. 2000; 6: 2482-2491Google Scholar). Sensitive measurement of LPA is presently a complicated process that generally involves either radiolabeling approaches or combined liquid chromatography mass spectrometry. Here we show that a murine homolog of a human enzyme previously reported to hydrolyze LPA is a highly specific LPA phosphatase that can be used to detect and quantitate LPA. We have used this validated assay to identify a novel role for nucleotides and a specific PLD enzyme, PLD2, in the control of LPA synthesis by SK-OV-3 ovarian cancer cells. McCoy's tissue culture medium, penicillin, streptomycin, and fetal bovine serum were obtained from the tissue culture facility, UNC Lineberger Comprehensive Cancer Center, Chapel Hill, NC. Nucleotide agonists, ATP, ADP, uridine 5′ triphosphate (UTP), UDP, 2-methyl thio-adenosine triphosphate (2MeSATP), diadenosine tetraphosphate (Ap4A), and suramin were obtained from Sigma Chemical Co. (St. Louis, MO). Phospholipids, sphingosine 1-phosphate (S1P), and ceramide 1-phosphate (C1P) were from Avanti Polar Lipids (Alabaster, AL, or BIOMOL, Inc., Plymouth Meeting, PA), and phosphoinositides were purified from brain lipid extracts as described previously. Other reagents were from previously identified sources (17Sciorra V.A. Morris A.J. Sequential actions of phospholipase D and phosphatidic acid phosphohydrolase 2b generate diglyceride in mammalian cells.Mol. Biol. Cell. 1999; 10: 3863-3876Google Scholar, 18Sciorra V.A. Rudge S.A. Prestwich G.D. Frohman M.A. Engebrecht J. Morris A.J. Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes.EMBO J. 1999; 18: 5911-5921Google Scholar, 19Sciorra V.A. Rudge S.A. Wang J. McLaughlin S. Engebrecht J. Morris A.J. Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes.J. Cell Biol. 2002; 159: 1039-1049Google Scholar). The ovarian cancer cell line SK-OV-3 was a generous gift from Dr. Gordon Mills (University of Texas, MD Anderson Cancer Center, Houston, TX). These cells were also obtained from the American Type Culture Collection (Manassas, VA). SK-OV-3 cells from both sources behaved identically in the assays reported in this paper. Cells were propagated in McCoy's medium supplemented with 10% fetal bovine serum, 1.5 mM l-glutamine, and 100 U/ml of penicillin/streptomycin. Cells were generally used at low passage number and resuscitated from frozen stocks frequently during this study. In vivo PLD activity was measured by transphosphatidylation as described previously (17Sciorra V.A. Morris A.J. Sequential actions of phospholipase D and phosphatidic acid phosphohydrolase 2b generate diglyceride in mammalian cells.Mol. Biol. Cell. 1999; 10: 3863-3876Google Scholar, 18Sciorra V.A. Rudge S.A. Prestwich G.D. Frohman M.A. Engebrecht J. Morris A.J. Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes.EMBO J. 1999; 18: 5911-5921Google Scholar, 19Sciorra V.A. Rudge S.A. Wang J. McLaughlin S. Engebrecht J. Morris A.J. Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes.J. Cell Biol. 2002; 159: 1039-1049Google Scholar). Cells were seeded in 12-well dishes (5 × 105 cells/well) and allowed to attach in the complete growth medium. The medium was replaced 24 h later with McCoy's media containing 10% FCS and 2 μCi/well [3H]palmitic acid. After 24 h, the medium was removed, and cells were incubated in McCoy's medium without serum for 1 h. Butan-1-ol was added to a final concentration of 0.3%. The cells were incubated at 37°C for a further 10 min, and then reactions were initiated by the direct addition of the appropriate nucleotides from concentrated stock solutions. For investigations into the antagonist action of ATP on UTP-stimulated PLD activity, cells were preincubated with varying concentrations of ATP for 10 min and were then incubated for 10 min with different concentrations of UTP in the continuing presence of ATP. Unless otherwise noted, assays were for 10 min, after which the incubations were terminated by removal of the culture medium and addition of ice-cold MeOH-0.1 M HCl (1:1; v/v). Lipids were extracted using acidified organic solvents before resolution by TLC in the organic phase of a solvent comprised of 2,2,4-trimethylpentane-ethyl acetate-acetic acid-water (5:11:2:10; v/v/v/v) using an unlined, unequilibrated chromatography tank. [3H]phosphatidylbutanol (PtdBut) bands, identified by unlabeled or [14C]PtdBut standards, were scraped and quantitated by liquid scintillation counting. PLD activity was determined in vitro using previously described methods (17Sciorra V.A. Morris A.J. Sequential actions of phospholipase D and phosphatidic acid phosphohydrolase 2b generate diglyceride in mammalian cells.Mol. Biol. Cell. 1999; 10: 3863-3876Google Scholar, 18Sciorra V.A. Rudge S.A. Prestwich G.D. Frohman M.A. Engebrecht J. Morris A.J. Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes.EMBO J. 1999; 18: 5911-5921Google Scholar, 19Sciorra V.A. Rudge S.A. Wang J. McLaughlin S. Engebrecht J. Morris A.J. Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes.J. Cell Biol. 2002; 159: 1039-1049Google Scholar). LPA release by SK-OV-3 cells was measured by both an enzymatic procedure and a bioassay. For sample preparation, cells were seeded in 100 mm-diameter dishes and allowed to attach in the basic growth medium. The cells were grown to ∼75% confluence, and the medium was replaced 24 h later with McCoy's medium without serum. After 2 h of incubation, the medium was then removed, cells were incubated in McCoy's medium containing 1 mg/ml BSA, and cells were treated with vehicle or nucleotide agonists. The medium was collected and centrifuged at 100 g for 10 min to remove any unattached cells. Lipids were then extracted from the medium using acidified organic solvents with back extraction using a synthetic lower phase followed by three washes with a synthetic upper phase. The lower phases, which were removed by evaporation, were collected. Samples were stored in a small volume of CHCl3 at −20°C. A full-length cDNA encoding of this enzyme, identified by blast searches of the IMAGE Consortium Expressed Sequence Tag database, was obtained from Research Genetics, Inc. (Birmingham, AL) and sequenced. The sequence has been deposited in GenBank with accession number AF216223. Murine lysophosphatidic acid phosphatase (mLPAP) was expressed in insect cells using a baculovirus vector and purified using an N-terminally appended His6 epitope tag. In general, purifications were from a 225 cm2 flask of sf 9 cells that generated sufficient enzyme for 100 to 200 assays. Cells were lysed in 5 ml ice-cold 5 mM Tris (pH 7.4) containing protease inhibitors by brief sonication, and the lysate was cleared by centrifugation. The purification was conducted at 4°C. Proteins were bound to 1 ml of Talon Superflow resin (Clontech, Inc.) in a capped, 10 ml disposable column for 1 h, and then the caps were removed and the column was drained and washed with 3 × 10 ml of extraction buffer. For the final stages of the purification, the column was washed with 10 ml of 0.1 M Tris (pH 7.5), and then bound proteins were eluted with sequential 1 ml applications of 0.1 M Tris (pH 7.5) containing 150 mM imidazole. LPAP activity was determined using assay buffer containing 0.1 M Tris (pH 7.5), 100 μM lipid substrate, and 0.3 mM Triton X-100 (22Hiroyama M. Takenawa T. Purification and characterization of a lysophosphatidic acid-specific phosphatase.Biochem. J. 1998; 336: 483-489Google Scholar, 23Hiroyama M. Takenawa T. Isolation of a cDNA encoding human lysophosphatidic acid phosphatase that is involved in the regulation of mitochondrial lipid biosynthesis.J. Biol. Chem. 1999; 274: 29172-29180Google Scholar). In some experiments, the Triton X-100 concentration was varied. Recombinant LPAP was expressed and purified as described above. Lipids extracted from SK-OV-3 cell culture medium were dried and resuspended in 50 μl of 0.1 M Tris (pH 7.5) containing 0.6 mM Triton X-100 by vortexing followed by brief bath sonication, and then an equal volume of purified LPAP (∼1 μg) was added to a final volume of 100 μl. Standard amounts of LPA (0–100 pmol) were run in parallel to unknown samples. The phosphatase reactions were run to completion (generally 30–60 min) as determined by a separate assay with [32P]LPA. Released phosphate was quantitated by a highly sensitive cycling reaction in which maltose phosphorylase generates glucose substrate for glucose oxidase, and peroxide formed by this latter reaction reacts with Amplex Red to generate resorufin, which is quantitated spectrophotometrically. The enzymes and reagents required for the phosphate detection step of the assay were obtained from Molecular Probes, Inc. (Eugene, OR), and the assays were performed exactly as described by the manufacturer. For the quantitation step of the assay, 50 μl of each dephosphorylation reaction was transferred directly to individual wells of a 96-well microtiter plate. Fifty microliters of 0.1 M Tris (pH 7.5) containing Amplex Red, maltose, glucose oxidase, and horseradish peroxidase was added to each well. The reactions were incubated at room temperature in a plate-reading spectrophotometer (BioTek, Inc.), which shook the plate and took an absorbance reading at 563 nM every 30 min for 12 h. Progress curves for the reactions with known quantities of LPA generated a series of standard curves for the assay, which in turn were used to determine LPA mass in the unknown samples. We used cultured insect cells expressing the LPA2 (Edg4) receptor by means of a recombinant baculovirus constructed with a cDNA encoding human LPA2 vector, generously provided by Kevin Lynch (University of Virginia), to detect LPA activity. In these cells, stimulation of the LPA2 receptor by agonist binding produces rapid increases in intracellular Ca2+, which can be readily detected using fluorescent indicators (20Bandoh K. Aoki J. Hosono H. Kobayashi S. Kobayashi T. Murakami-Murofushi K. Tsujimoto M. Arai H. Inoue K. Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid.J. Biol. Chem. 1999; 274: 27776-27785Google Scholar). In brief, serum-free adapted sf 9 cells were cultured in suspension in SF900 II serum-free medium. Monolayer cultures of these cells were infected with baculoviruses at a multiplicity of 10 and cultured for 48 h. Cells were dislodged by gentle shaking, recovered by centrifugation, and resuspended at ∼106 cells/ml in Grace's medium without serum. The cells were loaded by incubation with 10 mM Fura-2 AM (Molecular Probes, Inc.) on ice for 1 h, during which time the cells were gently mixed periodically. The cells were then washed once in Grace's medium to remove unincorporated indicator followed by resuspension in SF900 II medium. After a further 30 min incubation at room temperature to allow deesterification of the trapped indicator, the cells were recovered by centrifugation and resuspended at 105 cells/ml in SF900 II medium. One milliliter of the cell suspension was added to a stirred cuvette, and fluorescence measurements were made using a SPEX Fluromax-2 fluorimeter. Excitation was at 340 nm and 380 nm, and the fluorescence signal was detected at 510 nm. The fluorescence ratio was used to monitor intracellular Ca2+. For control experiments, a 1 μM solution of oleyl LPA was prepared by sonication of a dried lipid film in serum-free Grace's medium containing 1 mg/ml BSA. Dried lipid samples prepared from cell culture media as described above were resuspended in the same manner and added directly to the cuvette. In some cases, extracted lipids were resuspended in 0.3 mM Triton X-100 and incubated with purified LPAP or heat-inactivated LPAP using the assay conditions described above. Enzyme-treated samples were reextracted as described for samples obtained from cell culture medium, dried, and resuspended in serum-free Grace's medium containing 1 mg/ml BSA before addition to the cuvette. After washing with PBS, SK-OV-3 cells were incubated at 37°C in phosphate-free DMEM containing 100 μCi/ml [32P]H3PO4. After 90 min, cells were washed three times with phosphate-free DMEM to remove unincorporated [32P]H3PO4, and they were then incubated at 37°C with nucleotide agonists and other pharmacological agents for 10 min in phosphate-free DMEM containing 1% BSA. In experiments using adenovirus vectors (see below), cells were infected 48 h before the experiment. The culture medium was collected and treated as described above to extract lipids, and the lower phases were dried under N2 and then dissolved in chloroform-methanol (1:1; v/v). Lipids were then separated by two-dimensional TLC using chloroform-methanol-28% ammonia (65:25:5; v/v/v) for the first dimension and chloroform-methanol-acetic acid-water (45:20:5:0.5; v/v/v/v) for the second dimension. Labeled lipids were detected and quantified using a PhosphorImager (Image Quant software, Molecular Dynamics, Sunnyvale, CA) and quantitated as arbitrary units. The identity of each 32P-labeled phospholipid was achieved by comigration with unlabeled internal standards visualized with iodine vapor. Total cellular RNA was extracted using Trizol reagent (Life Technologies, Grant Island, NY) according to the manufacturer's instructions. Confluent SK-OV-3 cells were directly lysed by adding Trizol in a 75 cm2 flask. The resulting RNA pellet was finally washed with 70% ice-cold ethanol, air dried, and redissolved in 100 μl of diethyl pyrocarbonate-treated water. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using the Titan-One Tube RT-PCR Kit (Roche Diagnostics Corp., Indianapolis, IN). Specific primers for the human PLD1 and PLD2, and P2X1, P2Y1, P2Y2, P2Y4, and P2Y6 receptors were constructed based on cDNA sequences previously determined by us or obtained from GenBank: PLD1 forward (5′-CGCATCCCCATTCCCACTAG-3′) and PLD1 reverse (5′-CACAGCAATTCAAGCCTGGT-3′) (313 bp); PLD2 forward (5′-CCGTTTCTGGCCATCTATGA-3′) and PLD2 reverse (5′-TGGCTGCATGTCTGGTGGAG-3′) (358 bp); P2X1 forward (5′-TCTCCGAGAGGCCGAGAACT-3′) and P2X1 reverse (5′-GGTAGTTGGTCCCGTTCTCC-3′) (380 bp); P2Y1 forward (5′-TACTACCTGCCGGCTGTCTA-3′) and P2Y1 reverse (5′-CTGAGTAGAAGAGGATGGGG-3′) (380 bp); P2Y2 forward (5′-GGCCCCTGGAATGACACCAT-3′) and P2Y2 reverse (5′-GCGCTGGTGGTGACAAAGTA-3′) (512 bp); P2Y4 forward (5′-GTTTGCTATGGACTCATGGC-3′) and P2Y4 reverse (5′-CACTAGTGCCAGGGAAGAGG-3′) (363 bp); P2Y6 forward (5′-GCACGGCCGTGTACACCCTAAA-3′) and P2Y6 reverse (5′-TACACACACTAGCCAGGCAGCC-3′) (269 bp). First-strand cDNA synthesis was carried out in a 50 μl volume at 50°C for 30 min. Amplification was performed using the following profile: 2 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 50–70°C gradient, 1 min at 68°C, and a final extension step of 7 min at 68°C. The products were separated in a 1.5% agarose gel containing 1 mg/ml ethidium bromide. To prepare lysates and subcellular fractions for in vitro enzyme assays, SK-OV-3 cells were washed in PBS and harvested by scraping in ice-cold lysis buffer containing 20 mM Tris (pH 7.5), 1 mM EGTA, 0.1 mM benzamidine, and 0.1 mM PMSF. Nuclei and broken cells were removed by centrifugation at 500 g for 10 min. The supernatant obtained was separated into a total membrane and cytosolic fraction by centrifugation at 35,000 g for 30 min. The membrane fraction was resuspended in lysis buffer. Adenovirus vectors for expression of wild-type and catalytically inactive mutants of PLD1 and PLD2 were generated using the AdEasy system (Stratagene, Inc., La Jolla, CA). In brief, PLD1 and PLD2 cDNAs were subcloned into pShuttle-cytomegalovirus (CMV), and the PLD cDNAs and CMV promoter were transferred into the adenovirus genome by homologous recombination in an adenovirus-packing cell line according to the manufacturer's instructions. The adenoviruses were propagated in HEK293 cells and high titer purified preparations generated. Subconfluent monolayers of SK-OV-3 cells in 6-well plates were infected with vector control, wild-type PLD1, or wild-type PLD2 adenoviruses at a multiplicity of ∼10. Viral infection was allowed to proceed for 6 h, and then the medium was changed and the cells incubated in McCoy's medium containing 10% serum for a further 48 h. For determinations of intact cell PLD activity, cells were labeled with [3H]palmitic acid for the final 24 h as described above. Vectors for expression of PLD1 and PLD2 with N-terminally appended enhanced green fluorescent protein (EGFP) tags (EGFP-PLD1 and EGFP-PLD2) have been described previously (18Sciorra V.A. Rudge S.A. Prestwich G.D. Frohman M.A. Engebrecht J. Morris A.J. Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes.EMBO J. 1999; 18: 5911-5921Google Scholar). SK-OV-3 cells were cultured in McCoy's medium supplemented with 10% of FBS. Thirty-five millimeter-diameter dishes of 50% confluent cells were transfected with 1 μg pCGN-hPLD1 or pCGN-hPLD2 using lipofectamine in Opti-MEM (Life Technologies, Inc.). The transfection medium was removed after 24 h and replaced with complete McCoy's medium. The cells were harvested 24 h later by washing in PBS followed by scraping into ice-cold lysis buffer containing 20 mM Tris (pH 7.5), 5 mM EGTA, 0.1 mM benzamidine, and 0.1 mM PMSF. The lysate was disrupted by sonication on ice with a probe-type sonicator, and the material was used in assays within 24 h. PLA activity was measured by monitoring conversion of [32P]PA or 1- or 2-[3H]palmitoyl PA into [32P]LPA or [3H]LPA using minor adaptations of previously described methods (21Higgs H.N. Glomset J.A. Identification of a phosphatidic acid-preferring phospholipase A1 from bovine brain and testis.Proc. Natl. Acad. Sci. USA. 1994; 91: 9574-9578Google Scholar). Briefly, the substrate preparation used contained final concentrations of 100 μM radiolabeled PA (0.2 μCi/assay) and 1 mM 1-palmitoyl,2-oleyl-PC in 400 mM Triton X-100 mixed micelles in 100 mM HEPES (pH 7.5), 2 mM DTT, 5 mM EDTA, and 1 mM ATP. The assay volume was 100 μl and reactions were carried out at 37°C for 30 min. At the end of the incubation, lipids were extracted using acidified organic solvents. Lipid extracts were analyzed by TLC with a solvent system of chloroform-methanol-20% NH4OH (60:35:5; v/v/v). Radiolabeled LPA and PA were localized by reference to unlabeled standards run in parallel. The appropriate regions were scraped from the plate and associated radioactivity quantitated by liquid scintillation counting. SDS-PAGE, Western blotting, and protein determination were performed as described previously (18Sciorra V.A. Rudge S.A. Prestwich G.D. Frohman M.A. Engebrecht J. Morris A.J. Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes.EMBO J. 1999; 18: 5911-5921Google Scholar). PLD1 and PLD2 were expressed in sf 9 cells using baculovirus vectors and purified as described previously (18Sciorra V.A. Rudge S.A. Prestwich G.D. Frohman M.A. Engebrecht J. Morris A.J. Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes.EMBO J. 1999; 18: 5911-5921Google Scholar). An LPA-selective phosphatase was previously purified from rat liver. Expression of a cDNA encoding as a likely human homolog of this enzyme in COS-7 cells resulted in increased rates of hydrolysis of LPA in cell extracts; however, the recombinant protein was not purified, and its selectivity for a range of phospholipid monoester substrates was not evaluated (22Hiroyama M. Takenawa T. Purification and characterization of a lysophosphati