Identification of the Major Prostaglandin Glycerol Ester Hydrolase in Human Cancer Cells*

Background: Prostaglandin glycerol esters are rapidly hydrolyzed in biological systems. Results: Complementary approaches demonstrated that lysophospholipase A2 hydrolyzes prostaglandin glycerol esters. Conclusion: Lysophospholipase A2 is a major prostaglandin glycerol ester-specific hydrolase in human cancer cells. Significance: Perturbation of lysophospholipase A2 provides a means to understand prostaglandin glycerol ester function in vivo. Prostaglandin glycerol esters (PG-Gs) are produced as a result of the oxygenation of the endocannabinoid, 2-arachidonoylglycerol, by cyclooxygenase 2. Understanding the role that PG-Gs play in a biological setting has been difficult because of their sensitivity to enzymatic hydrolysis. By comparing PG-G hydrolysis across human cancer cell lines to serine hydrolase activities determined by activity-based protein profiling, we identified lysophospholipase A2 (LYPLA2) as a major enzyme responsible for PG-G hydrolysis. The principal role played by LYPLA2 in PGE2-G hydrolysis was confirmed by siRNA knockdown. Purified recombinant LYPLA2 hydrolyzed PG-Gs in the following order of activity: PGE2-G > PGF2α-G > PGD2-G; LYPLA2 hydrolyzed 1- but not 2-arachidonoylglycerol or arachidonoylethanolamide. Chemical inhibition of LYPLA2 in the mouse macrophage-like cell line, RAW264.7, elicited an increase in PG-G production. Our data indicate that LYPLA2 serves as a major PG-G hydrolase in human cells. Perturbation of this enzyme should enable selective modulation of PG-Gs without alterations in endocannabinoids, thereby providing a means to decipher the unique functions of PG-Gs in biology and disease.

We chose to investigate the hydrolase responsible for PG-G metabolism in human cancer cell lines because of the high PGE 2 -G hydrolytic activity detected in preliminary experiments, the ease of cell maintenance, and the potential for straightforward biochemical and genetic manipulation. The various enzymes described above are serine hydrolases, so we explored the possibility that the PGE 2 -G hydrolase(s) in human cancer cells is(are) a member of this superfamily. Serine hydrolases are a diverse class of enzymes that include lipases, proteases, and esterases (32,33), and many class members are involved in lipid biosynthesis and metabolism (9 -12). A unifying feature of the serine hydrolase family is a catalytic mechanism that involves the activation of a serine nucleophile for attack on substrates containing esters, amides, or thioester bonds (33). This conserved mechanism has enabled the development of irreversible fluorophosphonate probes that can covalently modify the active site serine and render the enzyme catalytically inactive (32). Nomura et al. (34,35) coupled fluorophosphonate probe binding with mass spectrometric proteomics techniques, known as activity-based protein profiling with multidimension protein identification technology, to determine the relative activity levels of serine hydrolases across different cancer cell lines. Utilizing these inventories and comparing the relative activities of individual serine hydrolases to PGE 2 -G hydrolase activities has allowed us to identify lysophospholipase A2 (LYPLA2) as a principal hydrolase responsible for PG-G metabolism in human cells.
Lysophospholipases compose an important class of serine hydrolases that metabolize lysophospholipids to form free fatty acid and the glycerol phosphate-containing head group (36). Thus, we have identified a novel function and substrate for LYPLA2. Specifically, we identify LYPLA2 as the serine hydrolase responsible for hydrolysis of PG-Gs across a number of different cancer cell lines. siRNA knockdown and cDNA overexpression validated the involvement of LYPLA2 in PG-G hydrolysis. Active enzyme was expressed and purified in Escherichia coli, which allowed for kinetic evaluation of an array of different substrates. In contrast to other PG-G-hydrolyzing enzymes, we found that LYPLA2 exerted no action on 2-AG or AEA but did hydrolyze 1-AG.
siRNA Knockdown of LYPLA1 and LYPLA2 in Multiple Cancer Cell Types-The siRNAs for LYPLA2 were as follows: siRNA, sense 5Ј-GCUCCGGACUGUUGUCACAtt and antisense 5Ј-UGUGACAACAGUCCGGAtt; and siRNA2, sense 5Ј-GGGUCCAGUUCAAGACAUAtt and antisense 5Ј-UAU-GUCUUGAACUGGACCCtt. The siRNAs for LYPLA1 were sense 5Ј-GGGUUUUCUCAGGGAGGAGtt and antisense 5Ј-CUCCUCCCUGAGAAAACCCtt. MDA-MB-231 cells were plated at 1.0 ϫ 10 6 cells per 100 mm 2 and allowed to incubate for 24 h to 40 -60% confluence. siRNA was introduced into the cells as a complex with DharmaFECT 1 according to the manufacturer's recommendations. Briefly, siRNA-DharmaFECT complexes were formed for 20 min in serum-free Opti-MEM I. RPMI 1640 medium from each plate was removed and replaced with 5 ml of serum-free Opti-MEM I medium followed by addition of 1 ml of transfection solution. Cells were cultured for 24 h at 37°C. The medium was subsequently replaced with RPMI 1640 medium containing 10% FBS and incubated at 37°C for an additional 48 h before harvesting.
PC3 cells were plated at 1.0 ϫ 10 6 cells per 100 mm 2 and allowed to incubate for 24 h to give 40 -60% confluence. Complex formation with siRNA and Lipofectamine 2000 was performed according to the manufacturer's recommendations. Briefly, siRNA-Lipofectamine complexes were formed for 20 min in serum-free Opti-MEM I. The medium in each plate was removed and replaced with serum-free Opti-MEM I medium followed by addition of 1 ml of transfection solution. The cells were cultured for 8 h at 37°C. The medium was subsequently replaced with DMEM/F-12 supplemented with 10% (v/v) FBS. Cells were incubated at 37°C for an additional 48 h before harvesting.
LNCaP cells were plated at 1.0 ϫ 10 6 cells per 100 mm 2 and allowed to incubate for 24 h to 40 -60% confluence. Complex formation with siRNA and Lipofectamine RNAiMAX was performed according to the manufacturer's recommendations. Briefly, siRNA-Lipofectamine complexes were formed for 5 min in serum-free Opti-MEM I. One ml of transfection solu-tion was added to each plate, and the cells were cultured for 72 h at 37°C before harvesting.
LYPLA1 and LYPLA2 cDNA Overexpression in HEK293 Cells-HEK293 cells were plated at a density of 3 ϫ 10 5 cells per well in a 6-well plate in RPMI 1640 medium supplemented with 10% FBS and incubated for 24 h at 37°C. LYPLA1 or LYPLA2 cDNA was prepared by adding 45 l of TurboFect to 250 l of Opti-MEM I followed by 15 l (1.5 g) of cDNA. The cells were incubated at room temperature for 30 min. The cDNA solution was added to each well dropwise, and cells were incubated at 37°C for an additional 48 h before harvesting.
Preparation of Cell Lysates-Cells were harvested by scraping, pelleted by centrifugation, and washed once with phosphate-buffered saline. The cells were resuspended in 500 l of ice-cold 25 mM Tris buffer (pH 7.5) containing 0.1 mM of both EDTA and DTT. Cells were lysed by sonication (Virsonic Cell Disrupter model 16-850, 10 ϫ 10-s pulses at relative output of 0.5, on ice), and the cytosolic fractions were separated by centrifugation (100,000 ϫ g for 1 h). Protein concentrations were determined using the BCA reagent kit according to the manufacturer's instruction (Pierce).
PG-G Hydrolase Assay-Hydrolytic activity was determined by adding 10 nmol of PGE 2 -G to 100 l of cell lysates (250 g/ml total protein) at 37°C. Reactions were quenched after 2 h by addition of 1 ml of ethyl acetate containing deuterated internal standard (PGE 2 -d 4 ). The organic layer was removed, evaporated to near dryness under nitrogen, and reconstituted with 50% methanol. Samples were analyzed by LC-MS/MS for the hydrolytic product, PGE 2 , which was quantified against the internal standard using stable isotope dilution.
Serine Hydrolase Inhibition-The general serine hydrolase probe, FP-TAMRA, was used to determine the involvement of serine hydrolases in the metabolism of PG-Gs. FP-TAMRA (100 nM) or DMSO was added to 100 l of MDA-MB-231 cytosol and allowed to incubate for 30 min at 37°C. PG-G hydrolase activity was determined as described above, except that samples were incubated from 0 to 60 min to obtain a full hydrolysis time course.
His-tagged LYPLA2 E. coli Purification-All purification was performed at 4°C. E. coli cell pellets were resuspended in 20 mM sodium phosphate buffer (pH 7.4) containing 500 mM NaCl, 20 mM imidazole, and 0.1 mM DTT. Cells were lysed by sonication (10 ϫ 10-s pulses at relative output of 0.5, on ice), and the cytosolic fraction was separated by centrifugation (100,000 ϫ g for 1 h). HIS-Select nickel affinity beads pre-equilibrated with Buffer A (20 mM sodium phosphate buffer (pH 7.4), 500 mM NaCl, 20 mM imidazole, and 0.1 mM DTT) were added to the cytosolic fraction, and protein was bound overnight. Beads were packed into a column and washed with 4 column volumes of Buffer A. His-tagged protein was eluted by linear gradient from 0 to 100% Buffer B (20 mM sodium phosphate buffer (pH 7.4), 500 mM NaCl, and 500 mM imidazole). Eluted protein was collected and concentrated using a Millipore 3000 molecular weight cutoff centrifugal filter. Protein was loaded onto a 120-ml HiPrep 16/60 Sephacryl S-200 HR size exclusion column pre-equilibrated with 2 column volumes of running buffer (Tris buffer (pH 7.5), 0.1 mM EDTA, 0.1 mM DTT). The column was eluted with 1 column volume of running buffer at a flow rate of 0.5 ml/min; protein was collected, and purity was validated by SDS-PAGE. The organic layer was evaporated to dryness under nitrogen, reconstituted in 50% methanol, and analyzed by LC-MS/MS for PGE 2 .
LYPLA2 Inhibition in RAW264.7 Murine Macrophage-like Cells-The effects of LYPLA2 inhibition on PG-G production were tested by exposure of cells to Compound 1. RAW264.7 cells were plated at 3 ϫ 10 6 cells per 100-mm 2 plate and incubated for 24 h in DMEM supplemented with 10% FBS. Medium was replaced with serum-free DMEM containing either 1 g/ml of lipopolysaccharide (LPS) (Sigma), 10 M Compound 1, or a combination of both, and allowed to incubate for 6 h. Cells treated with LPS were then treated with 5 M ionomycin (Calbiochem) for 45 min. Ionomycin stimulates the production of 2-AG and AA from endogenous sources (40). Lipids were extracted from the cell medium by addition of 2 volumes of ethyl acetate containing PGE 2 -d 4 , PGE 2 -G-d 5 , and AA-d 8 internal standards. The organic layer was dried under nitrogen, and the residue was reconstituted in 50% methanol for analysis of PGs, PG-Gs, and AA by LC-MS/MS.
LC-MS Analytical Procedures-Analysis of prostanoids was accomplished by reverse phase chromatography followed by mass spectrometric detection by selected reaction monitoring (SRM) using a Shimadzu LC-20 HPLC system coupled to an Applied Biosystems 3200 QTRAP mass spectrometer. Separation of PGs and lyso-PCs was achieved by gradient elution of a Phenomenex Luna C18 column (50 ϫ 2.0 mm, 3 m particle size). Solvent A was HPLC-grade water with 0.1% formic acid, and solvent B was acetonitrile (ACN) with 0.1% formic acid. Samples were injected onto the column with a starting condition of 80% solvent A and 20% solvent B at a flow rate of 0.5 ml/min. A linear gradient of increasing solvent B to 98% was run over 2 min and then held for 1. Separation of PGs and PG-Gs was accomplished with a gradient formed between solvent A, HPLC-grade water with 5 mM ammonium acetate (pH 3.6), and solvent B, ACN supplemented with 6% (v/v) of solvent A. Samples were injected onto the column with a starting condition of 70% solvent A and 30% solvent B at a flow rate of 0.6 ml/min. A linear gradient of increasing solvent B to 100% was run over 3. Separation of AA was achieved by gradient elution. Solvent A was HPLC-grade water with 80 M silver acetate, and solvent B was methanol with 118 M silver acetate. Samples were injected onto the column with a starting condition of 80% solvent A and 20% solvent B at a flow rate of 0.4 ml/min. A linear gradient of increasing solvent B to 100% was run over 1 min and then held for 2 min. SRM transition was as follows: AA m/z 518.9 3 411.1 and AA-d 8 m/z 526.9 3 419.1.
Resolution of AG isomers was achieved by gradient elution using an Agilent Eclipse XDB-C18 column (150 ϫ 2.1 mm, 3.5 m particle size). Solvent A was HPLC-grade water with 2 mM ammonium acetate, and solvent B was methanol with 10 mM ammonium acetate. Samples were injected onto the column with a starting condition of 15% solvent A and 85% solvent B at a flow rate of 0.325 ml/min. A linear gradient of increasing solvent B to 95% was run over 9.5 min, followed by a sharp increase of B to 100% over 0.5 min and then held at 100% for an additional 3 min. SRM transitions were as follows: 1-AG and 2-AG transition m/z 396. Separation of choline-containing lysophospholipids was achieved by gradient elution using a Thermo C4 reverse phase column. Solvent A was HPLC-grade water with 0.1% formic acid, and solvent B was ACN with 0.1% formic acid. Samples were injected onto the column with a starting condition of 30% solvent A and 70% solvent B at a flow rate of 0.5 ml/min. A linear gradient of increasing solvent B to 99% was run over 2 min and then held for 1 min. RSM transitions were as follows: Separation of all other lysophospholipids was achieved by gradient elution using a Thermo C4 reverse phase column. Solvent A was HPLC-grade water with 0.1% formic acid, and solvent B was ACN with 0.1% formic acid. Samples were injected onto the column with a starting condition of 50% solvent A and 50% solvent B at a flow rate of 0.5 ml/min. A linear gradient of increasing solvent B to 99% was run over 4 min and then held for 2. Resolution of plasmalogens was achieved by gradient elution using a Thermo C4 reverse phase column. Solvent A was HPLC-grade water with 0.1% formic acid, and solvent B was a 2:1 ratio of 2-propanol to ACN with 0.1% formic acid. Samples were injected onto the column with a starting condition of 50% solvent A and 50% solvent B at a flow rate of 0.5 ml/min. A linear gradient of increasing solvent B to 100% was run over 2 min and then held for 5 min. SRM transitions were as follows: 18

Characterization of PGE 2 -G Hydrolysis in Cancer Cells-
MDA-MB-231 breast cancer cells were initially tested for hydrolytic activity by quantitatively monitoring the formation of PGE 2 from exogenously provided PGE 2 -G by LC-MS/MS. Cytosolic fractions demonstrated PGE 2 -G hydrolase activity that was linear with time for up to 60 min ( Fig. 2A). Initial studies demonstrated that the majority of hydrolytic activity was present in the cytosol, with no activity detectable in the membrane fraction (data not shown). Importantly, serine hydrolase inventories have been identified and published for multiple cancer cell lines, including MDA-MB-231. This led us to explore whether a member of the serine hydrolase family was responsible for PGE 2 -G hydrolysis. MDA-MB-231 cytosol was preincubated with an irreversible serine hydrolase inhibitor, FP-TAMRA, and assessed for hydrolytic activity following incubation. As shown in Fig. 2A, fluorophosphonate treatment abolished hydrolysis of PGE 2 -G. This supported the hypothesis that a serine hydrolase is responsible for PGE 2 -G hydrolysis in MDA-MB-231 cells.
Nomura et al. (34,35) recently profiled serine hydrolases in a series of human cancer cell lines. The serine hydrolase proteome was enriched by covalently labeling the enzymes with fluorophosphonate molecules bound to biotin followed by avidin chromatography. The purified serine hydrolases were identified by multidimensional liquid chromatography mass spectrometry-based proteomic analysis of tryptic digests. Spectral counting revealed the relative levels of serine hydrolase activities across a number of aggressive and nonaggressive cancer cell types (34,35). Utilizing the cancer cell types investigated in these serine hydrolase inventories, we quantified PGE 2 -G hydrolysis by the cytosolic fractions of two breast cancer cell lines, MDA-MB-231 and MCF7, and two prostate cancer cell lines, PC3 and LNCaP.   DECEMBER 5, 2014 • VOLUME 289 • NUMBER 49 cytosolic serine hydrolases (supplemental Table 1) (34, 35), we were able to identify one enzyme, acyl-protein thioesterase 2 (APT2), also referred to lysophospholipase A 2 (LYPLA2), that correlated across all data sets.

Identification of Prostaglandin Glycerol Ester Hydrolase
LYPLA2 Involvement in PGE 2 -G Hydrolysis-To investigate the role LYPLA2 plays in PGE 2 -G hydrolysis, siRNA knockdown of enzyme expression was initially conducted in MDA-MB-231 cells using two distinct siRNAs. The level of LYPLA2 protein was markedly reduced (95 and 69%) in MDA-MB-231 cells upon transfection with siRNA and siRNA2, respectively, as determined by Western blot analysis (Fig. 3, C and D). Cytosolic fractions obtained from the siRNA-and siRNA2-treated MDA-MB-231 cells displayed a respective 60 and 50% reduction in hydrolysis of PGE 2 -G compared with control cells (Fig. 3A). PC3 and LNCaP cell lines transfected with the LYPLA2-directed siRNA also displayed significant decreases in the levels of LYPLA2 (90 and 86%, respectively) as compared with the cells transfected with the control siRNA (Fig. 3, B and E). Comparisons of PGE 2 -G hydrolytic activity from cytosol obtained from control and LYPLA2 knockdown cells (Fig. 3A) demonstrated a reduction in hydrolysis of 60% in PC3 cells and 25% in LNCaP cells. The minor decrease in LNCaP hydrolytic activity may be due to lower expression levels of LYPLA2 (34,35), which also correlate with a smaller total amount of PGE 2 -G hydrolytic activity in this cell line. In contrast, cells that demonstrated high levels of PGE 2 -G hydrolytic activity were the most affected by siRNA knockdown of LYPLA2, with decreases in activity ranging from 60 to 80%. Taken together, siRNA knockdown across multiple cell lines supported the hypothesis that LYPLA2 constitutes a major PG-G hydrolytic enzyme in human cancer cells.
To further validate LYPLA2 involvement in PGE 2 -G hydrolysis, the cDNA for human LYPLA2 was transfected into HEK293 cells. Untransfected HEK293 cells express no detectable levels of LYPLA2 by Western blot (Fig. 4A) and had low levels of PGE 2 -G hydrolase activity (Fig. 4B). In contrast, HEK293 cells transfected with LYPLA2 showed high expression of LYPLA2 by Western blotting (Fig. 4A) and a substantial increase in PGE 2 -G hydrolytic activity compared with fractions from untransfected cells (Fig. 4B). Combined with our siRNA knockdown data, these overexpression data confirm LYPLA2 as a major PGE 2 -G hydrolytic enzyme in human cells.
LYPLA1 Involvement in PGE 2 -G Hydrolysis-LYPLA2 is a member of the serine hydrolase family responsible for lysophospholipid metabolism. A second isoform, lysophospholipase A1 (LYPLA1), is also present in all tested cancer cell lines. Similarly to LYPLA2, LYPLA1 is responsible for hydrolysis of lysophospholipids, and the two enzymes share 60% sequence homology. To investigate the role LYPLA1 plays in PGE 2 -G hydrolysis, siRNA knockdown of LYPLA1 was conducted in MDA-MB-231 cells. The levels of LYPLA1 protein were markedly reduced (70%) in MDA-MB-231 cells upon transfection with siRNA as determined by Western blot analysis (Fig. 5A). Cytosolic fractions obtained from MDA-MB-231 cells following LYPLA1 knockdown displayed a small but significant reduction in hydrolysis of PGE 2 -G compared with that of control cells (Fig. 5B).
To further test LYPLA1 involvement in PGE 2 -G hydrolysis, cDNA for human LYPLA1 was transfected into HEK293 cells. The cytosolic fraction from the transfected cells demonstrated high expression of LYPLA1 compared with that of control cells as indicated by Western blot (Fig. 5C). Importantly, upon over- expression of LYPLA1, HEK293 cytosolic fractions showed no significant increase in PGE 2 -G hydrolytic activity when compared with those from untransfected cells (Fig. 5D). Thus, unlike the siRNA knockdown data, the overexpression data do not confirm LYPLA1 as a serine hydrolase responsible for PGE 2 -G hydrolysis in highly hydrolytically active cancer cell lines.
Expression and Activity of Recombinant Human LYPLA1 and LYPLA2-Recombinant LYPLA1 and LYPLA2 were successfully expressed in E. coli as determined by Coomassie staining (Fig. 6B). Expression of human LYPLA1 in E. coli caused no significant increase in PGE 2 -G hydrolysis, whereas expression of human LYPLA2 in E. coli was accompanied by a significant increase in PGE 2 -G hydrolytic activity (Fig. 6A). E. coli expres-sion data in combination with the siRNA knockdown and cDNA overexpression data in HEK cells all confirm that LYPLA2 is the major hydrolase responsible for PGE 2    6C). Consistent with the predicted molecular weight of LYPLA2, the His-tagged hLYPLA2 was purified as a single 25-kDa band (Fig. 6D). On the basis of Coomassie Blue staining, the expressed His-tagged hLYPLA2 was Ͼ95% pure.
LYPLA2 had lower hydrolytic efficiency toward PG-Gs relative to its activity with saturated lysophospholipids. LYPLA2 showed a greater catalytic efficiency for PGE 2 -G (K cat /K m ϭ 1.1 min Ϫ1 M Ϫ1 ) than for PGF 2␣ -G and PGD 2 -G (k cat /K m ϭ 0.44 and 0.14 min Ϫ1 M Ϫ1 , respectively).
Commercially available glyceryl prostaglandins are an equilibrium mixture of the 2-glyceryl ester (minor) and the 1(3)-glyceryl ester. To evaluate the regiochemistry of hydrolysis, we modified the HPLC conditions to enable separation of the 2-and the 1(3)-isomers of PGE 2 -G. HPLC analysis of the incubation mixtures of PGE 2 -G and LYPLA2 demonstrated that the 1(3)-glyceryl ester was selectively hydrolyzed; no hydrolysis of the 2-glyceryl ester was observed (data not shown).
Neither of the endocannabinoids, 2-AG or AEA, was hydrolyzed by LYPLA2. Because commercial 2-AG is nearly pure 2-glyceryl ester, we incubated 2-AG in PBS overnight to allow it to equilibrate with the 1(3)-AG isomer. Addition of LYPLA2 to this equilibrium mixture demonstrated hydrolysis of the 1(3)-AG but no hydrolysis of 2-AG. The k cat /K m value for hydrolysis of 1-AG was lower than that of PGE 2 -G ( Table 1). Consideration of the substrate specificity data summarized in Table 1 suggests that LYPLA2 only hydrolyzes 1(3)-glyceryl esters.
Bovine serum albumin (BSA) increases the rate of PGD 2 -G hydrolysis by human MAGL (23), so we determined the relative activity of LYPLA2 to a series of substrates in the presence of 0.5% BSA. The activity of LYPLA2 toward PGE 2 -G increased, whereas the activity toward 1-AG, 16:0-LPC, and 18:0-LPC decreased (Fig. 8). In fact, PGE 2 -G is the preferred substrate of LYPLA2 in the presence of BSA.
Inhibition of Recombinant LYPLA2 Results in Decreased Hydrolysis of PG-Gs-Selective inhibitors of LYPLA2 and the related enzyme LYPLA1 have recently been discovered (39). We assessed the effects of these inhibitors, named Compound 1 (LYPLA2-specific inhibitor) and Compound 21 (LYPLA1-specific inhibitor) (Fig. 9A), on the hydrolytic activity of LYPLA2. Compound 1 inhibited the PGE 2 -G hydrolysis activity of purified LYPLA2 with an IC 50 of 904 nM (Fig. 9B), comparable with previously reported values (510 nM) for the inhibition of hydrolysis of a fluorescent substrate, resorufin acetate (39). Compound 21, however, did not affect LYPLA2 activity. An amidated PGE 2 -G structural analog, PGE 2 -SA, was also assessed for LYPLA2 inhibition. PGE 2 -SA had no effect on the hydrolysis of PGE 2 -G, indicating the enzyme may have a very tight requirement for substrate binding. The MAGL inhibitor JZL-184 inhibited LYPLA2 at a concentration of 29 M, which is ϳ500-fold higher than the concentrations at which it inhibits MAGL ( Fig. 9) (42).
LYPLA2 Inhibition Increases PG-G Levels in RAW264.7 Murine Macrophage-like Cells-To assess the physiological relevance of PG-G hydrolysis by LYPLA2, murine RAW264.7 macrophage-like cells were stimulated to produce PGE 2 -G and PGD 2 -G by priming cells with LPS (1 g/ml) to induce expression of COX-2 ( Fig. 10) followed by treatment with the calcium ionophore, ionomycin (5 M), to promote release of 2-AG/AA. We determined whether inhibition of LYPLA2 by Compound 1 could affect the amount of PG-Gs produced in stimulated RAW264.7 cells. Upon simulation, RAW264.7 cells produce high levels of PGD 2 and PGE 2 and lower levels of PGD 2 -G and PGE 2 -G, all of which are secreted into the culture medium, as reported previously (Fig. 10) (20). Pretreatment of stimulated RAW264.7 cells with 10 M compound 1 prior to addition of ionomycin, increased the levels of PGD 2 /PGE 2 -G in the medium compared with those in the medium of uninhibited cells (40 versus 26 pmol). A concomitant decrease in PGD 2 /

DISCUSSION
Endocannabinoids are lipid mediators that elicit a variety of physiological effects, including analgesia and suppression of inflammation (6,7,43). COX-2 oxygenates 2-AG to form PG-Gs, which display effects that are frequently opposite those of endocannabinoids, e.g. hyperalgesia and neuroinflammation (13)(14)(15)(16)(17)(18)(19). However, complete elucidation of the effects of PG-Gs in vivo has been challenging due to their instability to enzymatic hydrolysis (20). These studies  have identified a serine hydrolase, LYPLA2, as a major enzyme responsible for PGE 2 -G hydrolysis in human cancer cells.
LYPLA2 was expressed in E. coli and purified to apparent homogeneity to determine its specific activity against a range of substrates. LYPLA2 exhibited higher catalytic efficiency against lysophospholipids than against PG-Gs when assays were conducted in the absence of albumin. However, the inclusion of albumin in the assay buffer led to a reversal of substrate specificity such that PGE 2 -G was hydrolyzed more rapidly than other substrates, including lysophospholipids. Among the PG-Gs tested, PGE 2 -G was the preferred substrate, followed by PGF 2␣ -G and PGD 2 -G. The PG-Gs assayed as substrates were an equilibrium mixture of the 1(3)-and 2-glycerol esters; the 1(3)-glycerol ester composes 85% of the mixture. Monitoring the isomeric composition in the presence of LYPLA2 under conditions in which hydrolysis was faster than isomerization revealed that LYPLA2 selectively hydrolyzed the 1(3)-but not the 2-isomer. LYPLA2 also displayed no activity against 2-AG or AEA but did hydrolyze 1(3)-AG at rates lower than PGE 2 -G. In fact, in the presence of albumin, PGE 2 -G was hydrolyzed nearly 10-fold faster than 1(3)-AG. Fig. 11 summarizes the lipid substrates for LYPLA2.
LYPLAs are widely distributed in multiple human tissues (36). Interestingly, LYPLA2 has a narrower range of lysophospholipid substrates than LYPLA1. The latter enzyme, which is 60% identical to LYPLA2 in sequence, hydrolyzes a range of lysophospholipids, including lyso-PC, lyso-, lyso-PS, lysophosphatidylglycerol, and lysophosphatidylinositol. In contrast, LYPLA2 hydrolyzes only lyso-PC, and lyso-PS. Yet, LYPLA2 hydrolyzes PGE 2 -G and other PG-Gs, whereas LYPLA1 does not. LYPLA1 and LYPLA2 also catalyze depalmitoylation of proteins that associate with membranes by virtue of the presence of palmitoyl-cysteine residues in the proteins. LYPLA1 removes the palmitate group from G protein ␣ subunits and H-Ras, whereas LYPLA2 acts on GAP-43 (45,46). Because of their acyl protein hydrolase activities, LYPLA1 and LYPLA2 are also known as APT1 and APT2, respectively.
Because multiple enzymes hydrolyze PG-Gs with different rates and under different conditions, selective inhibitors or genetic manipulations will be important in dissecting the contributions of individual hydrolases to PG-G hydrolysis in intact cells or tissues. Compound 1, which had been previously reported to inhibit LYPLA2 (39), exhibited 32-fold selectivity for inhibition of PGE 2 -G hydrolysis compared with the MAGL inhibitor JZL-184, and greater than 100-fold selectivity compared with the LYPLA1 inhibitor compound 21. JZL-184 is a potent MAGL inhibitor but is frequently used in vitro and in vivo at concentrations much higher than its stated IC 50 for MAGL of 50 nM (42). Compound 1 significantly reduced PGE 2 -G hydrolysis by LYPLA2 in vitro and increased endogenous levels of PGD 2 -G/PGE 2 -G in LPS-activated RAW macrophages. This is the first demonstration of the elevation of a lipid substrate by inhibition of LYPLA2 in intact cells.
The specificity of LYPLA2 for 1(3)-glycerol ester substrates suggests that isomerization of the initially formed 2-glycerol esters of PGE 2 -G/PGD 2 -G occurred following LPS treatment of the RAW cells. The half-life to spontaneous acyl migration of 2-glycerol esters to 1(3)-glycerol esters is ϳ10 min in serumfree medium but reduces to ϳ3 min in the presence of serum (47). Because the synthesis of PGE 2 -G/PGD 2 -G took place over 45 min following addition of ionomycin to trigger 2-AG release, there was ample time for isomerization to occur. The possibility also exists that isomerization of 2-AG to 1(3)-AG occurred during this time course and that addition of compound 1 inhibited 1(3)-AG hydrolysis by LYPLA2 leading to elevated levels of PGE 2 -G/PGD 2 -G following COX-2 oxygenation. This possibility seems less likely than compound 1 inhibition of LYPLA2 hydrolysis of PGE 2 -G/PGD 2 -G because 1(3)-AG is an inferior substrate for LYPLA2 compared with glyceryl prostaglandins and because 1(3)-AG is a poorer substrate than 2-AG for oxygenation by COX-2.
The biological effects of PG-Gs that have been documented in the literature have all been recorded with commercial material, which are primarily the 1(3)-glycerol esters. It is not known whether the 2-glycerol esters of PG-Gs have more potent or different effects in the same assays. Therefore, it seems likely that LYPLA2 plays an important role in controlling the biological effects of endocannabinoid-derived prostaglandin glycerol esters. This study demonstrates that it is a major and perhaps the major glyceryl prostaglandin hydrolase in human cancer FIGURE 11. Lipid substrates for hydrolysis by LYPLA2. DECEMBER 5, 2014 • VOLUME 289 • NUMBER 49 cells. However, the multiplicity of enzymes that hydrolyze these compounds, either the 2-or the 1(3)-isomers, suggests that different enzymes will play significant roles in controlling the activities of glyceryl prostaglandins depending on the cell type and physiological state. It will be exciting to dissect the contributions of individual enzymes to PG-G hydrolysis using a combination of chemical biological and genetic approaches.