Originally published In Press as doi:10.1074/jbc.M608458200 on February 11, 2007
J. Biol. Chem., Vol. 282, Issue 16, 11676-11686, April 20, 2007
Function, Activity, and Membrane Targeting of Cytosolic Phospholipase A2
in Mouse Lung Fibroblasts*
Moumita Ghosh
,
Robyn Loper,
Farideh Ghomashchi
,
Dawn E. Tucker,
Joseph V. Bonventre¶,
Michael H. Gelb, and
Christina C. Leslie||1
From the
Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, the Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195, the ¶Renal Division, Brigham and Women's Hospital, Boston, Massachusetts 02115, and the ||Departments of Pathology and Pharmacology, University of Colorado School of Medicine, Aurora, Colorado 80045
Received for publication, September 4, 2006
, and in revised form, January 19, 2007.
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ABSTRACT
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Group IVA cytosolic phospholipase A2 (cPLA2
) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA2–/– lung fibroblasts stimulated with A23187
[GenBank]
or serum. cPLA2+/+ fibroblasts preferentially released arachidonic acid, but A23187
[GenBank]
-stimulated cPLA2–/– fibroblasts nonspecifically released multiple fatty acids. Arachidonic acid release from cPLA2 –/– fibroblasts was inhibited by the cPLA2 inhibitors pyrrolidine-2 (IC50, 0.03 µM) and Wyeth-1 (IC50, 0.1 µM), implicating another C2 domain-containing group IV PLA2. cPLA2 –/– fibroblasts contain cPLA2
and cPLA2
but not cPLA2
or cPLA2
. Purified cPLA2 exhibited much higher lysophospholipase and PLA2 activity than cPLA2 and was potently inhibited by pyrrolidine-2 and Wyeth-1, which did not inhibit cPLA2. In contrast to cPLA2, cPLA2 expressed in Sf9 cells mediated A23187
[GenBank]
-induced arachidonic acid release, which was inhibited by pyrrolidine-2 and Wyeth-1. cPLA2 exhibits specific activity, inhibitor sensitivity, and low micromolar calcium dependence similar to cPLA2 and has been identified as the PLA2 responsible for calcium-induced fatty acid release and prostaglandin E2 production from cPLA2 –/– lung fibroblasts. In response to ionomycin, EGFP-cPLA2 translocated to ruffles and dynamic vesicular structures, whereas EGFP-cPLA2 translocated to the Golgi and endoplasmic reticulum, suggesting distinct mechanisms of regulation for the two enzymes.
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INTRODUCTION
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Mammals contain a number of phospholipases A2 (PLA2)2 that include secreted forms, intracellular group IV cytosolic PLA2s (GIV cPLA2), and group VI calcium-independent PLA2s (GVI iPLA2) (1, 2). The presence of diverse PLA2s provides cells with differentially regulated pathways for the hydrolysis of fatty acids from phospholipid. Intracellular PLA2s exhibit multiple enzymatic activities (PLA2, PLA1, lysophospholipase, transacylase) to varying degrees, which can potentially result in the formation of a diverse number of phospholipid breakdown products (3, 4). The direct products of PLA2 action, lysophospholipids and fatty acids, can themselves act as cellular mediators or serve as precursors for the formation of mediators such as platelet-activating factor and eicosanoids.
There are six enzymes classified as GIV PLA2s: cPLA2 (GIVA), cPLA2 (GIVB), cPLA2
(GIVC), cPLA2 (GIVD), cPLA2 (GIVE), and cPLA2 (GIVF) (4). These enzymes contain a conserved Ser/Asp active site dyad and an Arg residue, which are critical for catalytic activity. cPLA2 has been studied extensively because it is the only PLA2 that exhibits specificity for hydrolysis of sn-2 arachidonic acid from phospholipids (4–6). Arachidonic acid is the precursor of a large number of biologically active oxygenated metabolites including prostaglandins and leukotrienes. cPLA2 is a highly regulated enzyme, which is important in controlling the availability of free arachidonic acid in cells for the production of eicosanoids (7). cPLA2 is regulated by phosphorylation and an increase in intracellular calcium. Calcium binds to the calcium- and phospholipid-binding C2 domain on cPLA2, which promotes its translocation from the cytosol to the Golgi, endoplasmic reticulum, and nuclear envelope, where it can access substrate (4, 8–11). cPLA2 is phosphorylated on serine residues in the catalytic domain. Phosphorylation of Ser-505 by mitogen-activated protein kinases occurs in response to diverse agonists and is required for cPLA2-mediated release of arachidonic acid in stimulated cells (12, 13).
Much less is known about the regulation and physiological function of the other GIV PLA2s (cPLA2, -, -, -, -) (4). cPLA2 is the only GIV enzyme that does not contain a C2 domain (14, 15). It contains fatty acyl and C-terminal farnesyl groups, and is constitutively bound to membrane (16, 17). Human cPLA2 is expressed most abundantly in heart and skeletal muscle; however, its role in these tissues is unknown. In contrast, the mouse cPLA2 homologue is only 50% homologous to human cPLA2 and is expressed exclusively in oocytes (18). cPLA2, cPLA2, and cPLA2 form a gene cluster near cPLA2 in humans and mice and have more homology to cPLA2 than to cPLA2 (4, 19, 20). Human cPLA2 is associated with psoriatic lesions and is expressed in stratified squamous epithelium (19). Human cPLA2 is widely expressed and occurs as multiple splice variants (14, 21, 22). It contains a novel, N-terminal-truncated JmjC domain immediately upstream of the C2 domain. We have recently found that the principle form of cPLA2 translated in human cells is a novel splice variant (cPLA23) that contains an internal deletion in the catalytic domain (22). cPLA23 exhibits calcium-dependent PLA2 activity but is constitutively bound to mitochondria and early endosomes in cells, suggesting a mechanism of regulation and function distinct from cPLA2. cPLA2, cPLA2, and cPLA2 have been cloned from mouse tissues; however, only preliminary information is available about their biochemical properties, and nothing is known of their functional roles (20).
It is well documented that cPLA2 functions to release arachidonic acid for the production of eicosanoids. However, eicosanoid production is not completely ablated in the cPLA2 knock-out mouse indicating a role for other PLA2s in mediating arachidonic acid release (23, 24). We previously isolated mouse lung fibroblasts (MLF) from cPLA2 wild type and knock-out mice and demonstrated a primary role for cPLA2 in mediating arachidonic acid release and prostaglandin E2 (PGE2) production (25). However, we found that cPLA2–/– MLFs (MLF–/–) release lower, but significant, levels of arachidonic acid and produce PGE2 in response to calcium ionophore and serum (25). We have identified cPLA2 and cPLA2 in MLF–/– and provide evidence here that cPLA2 is the enzyme that mediates calcium-dependent arachidonic acid release.
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EXPERIMENTAL PROCEDURES
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Materials—[5,6,8,9,11,12,14,15-3H]Arachidonic acid (100 Ci/mmol), 1-palmitoyl-2-[14C]arachidonyl-phosphatidylcholine (PC) (48 mCi/mmol), 1-[14C]palmitoyl-2-lyso-PC (55 mCi/mmol), 1-palmitoyl-2-[14C]arachidonyl-phosphatidylethanolamine (PE) (48 mCi/mmol), and 1-palmitoyl-2-[14C]oleoyl-PC (55 mCi/mmol) were from PerkinElmer Life Sciences. 1-Palmitoyl-[14C]linoleoyl-PE (55 mCi/mmol) was from Amersham Biosciences. 1-Palmitoyl-2-arachidonyl-PE, 1-palmitoyl-2-linoleoyl-PE, and 1-hexadecyl-2-arachidonyl-PC were from Avanti%20Polar%20Lipids">Avanti Polar Lipids. 1-Palmitoyl-2-arachidonyl-PC, dioleoylglycerol, bovine serum albumin (BSA), fatty acid-free BSA, pluronic acid, and anti-His6 monoclonal antibody were from Sigma. 1-Arachidonyl-2-hexadecyl-PC was prepared as described (26). PLA2 inhibitors indoxam, pyrrolidine-2, and Wyeth-1 were synthesized as described previously (27–30). Dulbecco's modified Eagle's medium (DMEM) was from BioWhittaker. Penicillin-streptomycin-L-glutamine solution was from Invitrogen. Fetal bovine serum was from Irvine Scientific. Bromoenol lactone was purchased from Biomol. Silica gel LC-Si SPE columns were from Sigma-Supelco. Protease inhibitor mixture tablets were from Roche Applied Science. The Total RNA Isolation kit was purchased from Promega, and the Advantage reverse transcription-PCR kit, BD SMART RACE cDNA amplification kit, and EGFP vector were from Clontech. The TA cloning vector was from Invitrogen. The plasmid isolation kit, RNeasy mini kit, and nickel-nitrilotriacetic acid-agarose beads were from Qiagen. iScript cDNA synthesis kit was obtained from Bio-Rad.
Culture of MLF and Assays for Fatty Acid Release and PGE2 Production—Lung fibroblasts were isolated from wild type (MLF+/+) and cPLA2 knock-out (MLF–/–) mice, and SV40 immortalized MLF (IMLF) were generated as described previously (31). Fibroblasts were plated in 24-well tissue culture plates at a density of 2.5 x 104 cells/well in supplemented DMEM (10% fetal bovine serum, 0.1% nonessential amino acids, 1 mM sodium pyruvate, and 1% penicillin-streptomycin-L-glutamine solution) and incubated for 6 h at 37 °C with 5% CO2. Cells were washed twice with serum-free DMEM and incubated in serum-free DMEM containing 0.1% BSA and radiolabeled fatty acids (0.2 µCi of [3H]arachidonic acid, 0.5 µCi of [3H]palmitic acid, 0.25 µCi of [3H]oleic acid, and 0.5 µCi of [14C]linoleic acid/well). After incubation overnight, the cells were washed three times with DMEM containing 0.1% fatty acid-free BSA and stimulated with agonists in albumin-containing medium for the times indicated. For inhibitor experiments, cells were preincubated with inhibitors for 15 min prior to stimulation. The culture medium was removed and centrifuged at 15,000 rpm for 15 min. Cells were solubilized with 0.1% Triton-X-100. The level of radioactivity in the culture medium and in the cells was measured, and the amount released was calculated as a percentage of the total (released plus cellular) radioactivity.
To measure the effect of inhibitors on PGE2 production, MLF–/– were incubated overnight in serum-free medium containing transforming growth factor to up-regulate cyclooxygenase-2 as reported previously (25). The cells were incubated with and without pyrrolidine-2 and Wyeth-1 (both at 10 µM) for 15 min and then stimulated with A23187
[GenBank]
(2 µg/ml) for 45 min. PGE2 in the culture medium was quantified by enzyme-linked immunosorbent assay (Elisa Tech, Aurora, CO).
Quantitative Real-time PCR—The primers and probes used for real-time PCR of mouse cPLA2, cPLA2, cPLA2, and cPLA2 were obtained from Applied Biosystems (premade Taqman gene expression assays). Assay IDs for mouse cPLA2, cPLA2, cPLA2, and cPLA2 are Mm 01271073_g1, Mm 01338177_g1, Mm 01279782_m1, and Mm 00625711_m1, respectively. Total RNA was isolated from MLF and IMLF using a Qiagen RNeasy mini kit, and 1 µg of total RNA was used to make cDNA using iScript cDNA synthesis kit from Bio-Rad following the manufacturer's instructions. Each PCR reaction (25 µl) contained 500 ng of cDNA, PCR master mix, and premade Taqman gene expression assay components containing a 6-carboxyfluorescein reporter dye at the 5'-end of the Taqman probe and a nonfluorescent quencher at the 3'-end of the probe. Rodent glyceraldehyde-phosphate dehydrogenase was used as a control to verify the quality of cDNA template. Real-time PCR was performed and analyzed by the dual-labeled fluorogenic probe method using an ABI Prism 7000 sequence detector from Applied Biosystems.
Cloning of Mouse cPLA2 and cPLA2—To clone cPLA2 cDNA from IMLF–/–, cells were cultured in supplemented DMEM; total RNA was isolated, 1 µg of which was used to generate cDNA. PCR analysis was performed using 10 µl of cDNA for cPLA2 and 5 µl of cDNA for glyceraldehyde-phosphate dehydrogenase following the manufacturer's instructions (Clontech Advantage reverse transcription-PCR kit). Specific primers used for mouse cPLA2 were as follows: 5'-gtctacaagcttatgcaggcaaaggtg-3', 5'-gccaactttggcggtaccggcaagagc-3', 5'-gctcttgccggtaccgccaaagttggc-3', and 5'-cagctgggatcctcactccggcctaaac-3'. The primers were designed based on the mouse cPLA2 genomic sequence available from NCBI (gi:211429) to amplify the full-length cDNA in two fragments. The PCR products were cloned into the TA cloning vector, and the fragments were sequenced and then assembled into the full-length clone using the internal KpnI site present in the PCR products.
Mouse cPLA2 was cloned from IMLF–/– cells and from mouse thyroid using the following primer sets: 5'-ctgggacctgagctgctactgctgg-3', 5'-gaatactactcccgggaaaagagag-3', 5'-ctctcttttcccgggagtagtattc-3', and 5'-gtttaaagtcttccctctccctcag-3'. These were designed based on the mouse cPLA2 sequence (NCBI NM_001024145) to amplify the full-length cDNA in two fragments. PCR products were cloned into the TA cloning vector, and the fragments were sequenced and then assembled into the full-length cPLA2 clone using the internal SmaI site present in the PCR products. For immunofluorescence microscopy, cPLA2 and cPLA2 cDNAs were cloned into the EGFP vector in the XhoI/HindIII and XhoI/BamHI sites, respectively.
Production of Recombinant Baculoviruses and Expression in Sf9 Cells—Mouse cPLA2 cDNA was cloned into the baculovirus vector pAcHLT in the StyI/NotI sites and cPLA2 in the XhoI/SacI sites. Recombinant baculovirus was generated by co-transfection of Sf9 cells with cPLA2-containing constructs and linearized baculovirus DNA (Baculogold) following the manufacturer's instructions (BD Biosciences-Pharmingen), and amplified by standard protocols. To determine the expression of cPLA2s, Sf9 cells were plated in a 12-well tissue culture plate at a density of 0.5 x 106 cells/well and infected with recombinant viruses at different multiplicities of infection for 1 h. The virus-containing medium was replaced with fresh medium, and cells were incubated for 48 h. Expression of His6-cPLA2 and His6-cPLA2 was determined by Western blot analysis using anti-His6 monoclonal antibodies. His6-cPLA2 and His6-cPLA2 expressed in Sf9 cells were affinity-purified using nickel-agarose beads following the manufacturer's instructions (Qiagen). The concentration of the enzymes in eluted fractions was determined by comparing the intensity of Coomassie-stained bands on SDS-polyacrylamide gels with a standard curve made with BSA and also by the bicinchoninic acid method.
Western Blotting—Cells were washed with phosphate-buffered saline and then scraped into ice-cold lysis buffer (50 mM Hepes, pH 7.4, 150 mM sodium chloride, 1.5 mM magnesium chloride, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 200 µM sodium vanadate, 10 mM terasodium pyrophosphate, 100 mM sodium fluoride, 300 nM p-nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). After incubation on ice for 30 min, lysates were centrifuged at 15,000 rpm for 15 min, and protein concentration in the supernatant was determined. Lysates were boiled for 5 min in Laemmli electrophoresis sample buffer, and the proteins (15–25 µg total protein) were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membrane. After blocking with 5% milk for 1 h, membranes were incubated overnight at 4 °C with monoclonal anti-His6 antibody in 20 mM Tris, pH 7.6, 137 mM NaCl, and 0.05% Tween containing 5% milk and then incubated with anti-mouse IgG horseradish peroxidase antibody (1:5000 dilution) for 30 min at room temperature. The immunoreactive proteins were detected using the Amersham Biosciences ECL system.
Assay for Arachidonic Acid Release from Sf9 Cells—Sf9 cells were plated in 24-well plates (2.5 x 105 cells/well) in 500 µl of TNM-FH medium containing 10% fetal bovine serum (complete medium) and incubated at 27 °C for 15 min. The medium was removed and baculoviruses added in 150 µl of complete medium. After incubation for 1 h, complete medium (350 µl/well) was added and the cells incubated 25–30 h followed by incubation overnight in complete medium (500 µl/well) containing 0.2 µCi [3H]arachidonic acid. The cells were washed three times with serum-free TNM-FH medium containing 0.1% human serum albumin and stimulated for 45 min with A23187
[GenBank]
(2 µg/ml). The level of released [3H]arachidonic acid was determined as described above for fibroblasts. A portion of the Triton lysate was used for Western blotting to determine the relative level of expression of the cPLA2s.
Enzyme Assays—PLA2 activity was assayed using 1-palmitoyl-2-[14C]arachidonyl-PC, 1-palmitoyl-2-[14C]oleoyl-PC, 1-palmitoyl-2-[14C]arachidonyl-PE, and 1-palmitoyl-2-[14C]linoleoyl-PE as substrates. To prepare substrate, solvents were evaporated from the lipid mixture under a stream of nitrogen, 50 mM Hepes buffer, pH 7.4, was added and the lipid mixture sonicated at 4 °C for 10 s on ice using a microprobe (Braun Instruments) to form small unilamellar vesicles. The reaction mixture (50 µl final volume) contained 30 µM phospholipid substrate (100,000 dpm), 9 µM dioleoylglycerol (which was co-sonicated with the substrate), 150 mM sodium chloride, 1 mg/ml fatty acid free BSA, 1 mM EGTA, and 5 mM CaCl2. For assays with palmitoyl-arachidonyl-PE, dioleoylglycerol was not added. Reactions were started by the addition of affinity-purified enzyme (50 ng-1 µg) and incubated at 37 °C for the times indicated. Free fatty acids were extracted using Dole reagent (propan-2-ol:heptane:1 N H2SO4, 20:5:1) and separated by silicic acid chromatography as described previously using unlabeled oleic acid (25 µg) as carrier lipid (32).
The calcium dependence of the PLA2 activity of cPLA2 and cPLA2 was measured using 1-palmitoyl-2-[14C]arachidonyl-PC. Vesicles were made by extrusion through two 0.2-µm Nucleopore membranes as described previously (33) in buffer (10 mM MOPS, 100 mM KCl, 0.5 mM EGTA, pH 7.2) to give 4–4.5 mM total phospholipid at a final specific radioactivity of 2.7 Ci/mol. The phospholipid concentration in the stock solution after extrusion was calculated from the initial concentration and the yield of radioactivity. The same buffer containing various concentrations of free calcium from 0 to 20 µM was prepared by fluorimetric titration using fluo-3 and Calcium Green 5N as described previously (34). A small aliquot of extruded vesicle stock was added to give 200 µM total phospholipid in each assay. Reactions (80 µl) were carried out in various calcium buffers supplemented with 0.5 mg/ml fatty acid-free BSA for 2 min at 37 °C. Reactions were quenched and analyzed for radiolabeled free arachidonic acid as described earlier (35).
Reactions to study the PLA2 and PLA1 activities were carried out as follows. 1-Hexadecyl-2-arachidonyl-PC or 1-arachidonyl-2-hexadecyl-PC was sonicated at 60 µM in 50 mM Hepes, pH 7.4, to form a stock solution of small unilamellar vesicles as described (36). Reaction mixtures contained 30 µM phospholipid in 250 µl of 50 mM Hepes, pH 7.4, 150 mM NaCl, 1 mg/ml fatty acid-free BSA, 1 mM EGTA, 5 mM CaCl2, and either cPLA2 (10 ng), cPLA2 (7.5 µg), or cPLA2 (20 ng). After 20 min at 37 °C, reactions were processed for arachidonic acid analysis using gas chromatography/mass spectrometry as described with d8-arachidonic acid (Cayman Chemicals, Inc.) as an internal standard (37).
Lysophospholipase activity was measured using 1-[14C] palmitoyl-2-lyso-PC sonicated in 50 mM Hepes, pH 7.4, to make micelles as described previously (38). Assays contained 50 µM substrate (120,000 dpm), 1 mM EGTA, and 5 mM CaCl2 in a final volume of 50 µl. Reactions were started by adding affinity-purified enzyme and incubated at 37 °C for the times indicated. Free fatty acid product was extracted using Dole reagent. After vortexing, the upper heptane phase was removed and dried under a stream of nitrogen, and 0.5 ml of heptane added. Radiolabeled free fatty acids were measured by liquid scintillation spectrometry. For inhibitor experiments, enzymes were preincubated for 2 min at 37 °C with inhibitors, and reactions were started by addition of substrate.
Immunofluorescence Microscopy—IMLF–/– were transfected with 10 µg of EGFP-cPLA2 and EGFP-cPLA2 cDNA using nucleofection technology (Amaxa Biosystems), with solution T, following the manufacturer's instructions. Transfected fibroblasts were plated in 35-mm glass-bottom MatTek plates at a density of 0.5 x 106/cm2 and incubated for 48 h. Cells were then washed twice with serum-free DMEM and incubated in serum-free DMEM containing 0.1% BSA overnight. For live cell imaging, fibroblasts were washed with and incubated in Hanks' balanced salt solution buffered with 25 mM Hepes. Cells were stimulated with 0.5 µM ionomycin and imaged at 37 °C with an inverted Zeiss 200M microscope with a 175-watt xenon lamp using a x63 oil immersion objective and GFP filters. Images were acquired every 5 s for a total of 10 min with a charge-coupled device camera from Sensicam, and data were analyzed using Intelligent Imaging Innovations Inc. (3I) software.
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RESULTS
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Fatty Acid Release from IMLF+/+ and IMLF –/– and Effect of PLA2 Inhibitors—We previously reported that primary MLF–/– and immortalized MLF–/– release arachidonic acid and produce PGE2 in response to A23187
[GenBank]
and serum indicating the presence of a calcium-regulated PLA2 that can initiate lipid mediator production (25). To characterize the PLA2, we compared the types of fatty acids released from stimulated IMLF+/+ and IMLF–/–. The cells were incubated with radiolabeled fatty acids to determine the acyl chain specificity of the PLA2 (Fig. 1, A and B). IMLF+/+ stimulated with A23187
[GenBank]
or mouse serum release relatively greater amounts of arachidonic acid than other fatty acids. This is consistent with a primary role for cPLA2 in mediating arachidonic acid release (25). In contrast IMLF–/– stimulated with A23187
[GenBank]
do not preferentially release arachidonic acid but release similar levels of saturated, monounsaturated, and polyunsaturated fatty acids. As previously shown, the release of arachidonic acid from IMLF–/– stimulated with mouse serum is lower than with A23187
[GenBank]
(25); however, the results also show nonspecific release of multiple fatty acids (Fig. 1B). Thus the PLA2 in IMLF–/– does not exhibit acyl chain specificity. It is interesting to note that the release of arachidonic acid from IMLF–/– is about 65% lower than from IMLF+/+; however, the % release of the other fatty acids (16:0, 18:1, 18:2) is similar in IMLF–/– and IMLF+/+. This suggests that the release of these fatty acids from IMLF+/+ is not due to cPLA2 but is mediated by another PLA2 that is present in both IMLF–/– and IMLF+/+.
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FIGURE 1.
Comparison of fatty acids released from IMLF+/+ and IMLF–/–. IMLF+/+ and IMLF–/– were incubated overnight in medium containing various radiolabeled fatty acids and then either left unstimulated (US, white bar) or stimulated with A23187 (2 µg/ml) (black bar) for 45 min (A) or with 10% mouse serum (MS, black bar) for 30 min (B). The amount of fatty acids released into the medium is expressed as a percentage of the total cellular incorporated radioactivity. Results are the average ± S.E. of four independent experiments.
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FIGURE 2.
Effect of PLA2 inhibitors on arachidonic acid release from IMLF+/+ and IMLF–/–. A, [3H]arachidonic acid-labeled IMLF–/– were treated with PLA2 inhibitors (10 µM) or Me2SO (vehicle control) for 15 min and then left unstimulated (US, light gray bars) or incubated with A23187 (2 µg/ml) (dark gray bars) for 45 min. The amount of [3H]arachidonic acid released into the medium is expressed as a percentage of the total cellular incorporated radioactivity. IMLF+/+ and IMLF–/– were preincubated with the indicated amounts of pyrrolidine-2 (B) or Wyeth-1 (C) for 15 min and then stimulated with 2 µg/ml A23187. The amount of [3H]arachidonic acid released is expressed as a percentage of the total incorporated radioactivity after subtracting the % release from unstimulated cells treated with inhibitors. Data shown are the average ± S.E. of three independent experiments.
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To determine the type of PLA2 involved in release of arachidonic acid from IMLF–/–, the cells were treated with PLA2 inhibitors and stimulated with A23187.
[GenBank]
The iPLA2 inhibitor bromoenol lactone, the sPLA2 inhibitor indoxam, and the cPLA2 inhibitors pyrrolidine-2 and Wyeth-1 were tested (29, 30). Although IMLF–/– lack cPLA2, pyrrolidine-2 and Wyeth-1 were used because they may inhibit another GIV cPLA2 present in IMLF. Pyrrolidine-2 and Wyeth-1 are the only inhibitors that block A23187
[GenBank]
-stimulated arachidonic acid release from IMLF–/– (Fig. 2A). Pyrrolidine-2 and Wyeth-1 inhibit arachidonic acid release from IMLF+/+ with an IC50 of 
0.01 and 0.05 µM, respectively, and from IMLF–/– with an IC50 of 0.03 and 0.1 µM, respectively (Fig. 2, B and C).
We previously reported that MLF–/– produce PGE2 in response to A23187
[GenBank]
and serum (25), suggesting that arachidonic acid released by the PLA2 couples to cyclooxygenases for PGE2 production. This is supported by data showing that pyrrolidine-2 and Wyeth-1 block PGE2 production from A23187
[GenBank]
-stimulated MLF–/– by 91 and 50%, respectively (Table 1). MLF–/– were incubated overnight with transforming growth factor prior to stimulation to up-regulate cyclooxygenase-2. These experiments were carried out with MLF–/– rather than IMLF–/– because IMLF–/– produce very little PGE2 due to a defect in the expression of microsomal PGE synthase from inactivation of p53 by SV40 (25).
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TABLE 1
PGE2 production from A23187-stimulated MLF–/–
MLF–/– were cultured overnight with transforming growth factor (TGF) (2 ng/ml) to up-regulate cyclooxygenase 2 and then left unstimulated (US) or treated with A23187 (2 µg/ml) for 45 min. Fibroblasts were incubated with 10 µM pyrrolidine-2 (Pyr-2) or Wyeth-1 for 15 min prior to stimulation with A23187. PGE2 levels in the culture medium were measured by enzyme-linked immunosorbent assay. A representative experiment of triplicate samples is shown ± S.D.
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Identification of cPLA2 and cPLA2 in IMLF–/–—Because pyrrolidine-2 does not inhibit iPLA2 or sPLA2s (39) and the PLA2 in IMLF–/– is calcium-regulated, the results suggested another C2 domain-containing GIV PLA2 may be responsible for arachidonic acid release in these cells. Real-time PCR analysis revealed that IMLF–/– contain transcripts for cPLA2 (GIVB) and cPLA2 (GIVF) but not cPLA2 (GIVE) or cPLA2 (GIVD) (Fig. 3A). The level of expression of cPLA2 and cPLA2 is similar in IMLF+/+ and IMLF–/–, indicating that the ablation of cPLA2 does not result in their up-regulation. Similar results were obtained for primary MLF+/+ and MLF–/– (data not shown).
Cloning of cPLA2 cDNA from IMLF–/– demonstrated that it has a C2 domain as well as a catalytic domain that contains the active site residues previously identified in human cPLA21 (14, 21). An alignment of the amino acid sequence of mouse cPLA2 and human cPLA21 is shown in Fig. 3B. The C2 and catalytic domains of mouse cPLA2 have 82% amino acid identity with human cPLA21. We have recently identified three transcripts of human cPLA2 in BEAS-2B lung epithelial cells (22). cPLA21 is identical to the form originally cloned (14, 21), and cPLA22 and cPLA23 contain internal deletions in the catalytic domain (22). We found that only cPLA23 is translated in BEAS-2B cells. Only one transcript of mouse cPLA2 is found in IMLF–/–, and its catalytic domain is most similar to human cPLA21 because it does not contain the internal deletion. This was determined by 3'-RACE PCR using three gene-specific primers to different regions in the catalytic domain of mouse cPLA2 and the universal primer. One striking difference between mouse and human cPLA2 is that mouse cPLA2 does not contain the N-terminal extension with the truncated JmjC domain found in human cPLA2 (Fig. 3B) (14, 21). We confirmed the lack of a truncated JmjC domain in mouse cPLA2 by 5'-RACE analysis. This is consistent with the prediction in the NCBI Database that separate adjacent genes encode a complete JmjC domain (accession number BC016255
[GenBank]
) and cPLA2 (accession number BC098210
[GenBank]
) on mouse chromosome 2.
Mouse cPLA2 cloned from IMLF–/– is homologous to the sequence previously reported for mouse cPLA2 cloned from thyroid with the exception of a number of base differences leading to amino acid changes. We also cloned cPLA2 from mouse thyroid and confirmed that it is identical to cPLA2 in IMLF–/–. The sequences of cPLA2 (accession number DQ888308
[GenBank]
) and cPLA2 (accession number DQ904008
[GenBank]
) from IMLF–/– can be found in the NCBI Database.
Enzymatic Properties and Inhibitor Sensitivity of Purified cPLA2 and cPLA2—To determine whether cPLA2 and cPLA2 have characteristics of the PLA2 responsible for mediating arachidonic acid release from IMLF–/–, the enzymes were expressed as N-terminal His6-tagged proteins in Sf9 cells using baculoviruses, affinity-purified, and their enzymatic properties and inhibitor sensitivity characterized. cPLA2 has lysophospholipase activity against palmitoyl-lyso-PC and lower PLA2 activity against both palmitoyl-arachidonyl-PC and palmitoyl-arachidonyl-PE (Fig. 4, A and B). We tested the ability of pyrrolidine-2 and Wyeth-1 to inhibit the activity of cPLA2 using palmitoyl-lyso-PC as substrate (Fig. 4C). The results demonstrate that concentrations of pyrrolidine-2 and Wyeth-1 that are more than sufficient to inhibit arachidonic acid release from IMLF–/– have no effect on the activity of mouse cPLA2. In contrast, the activity of cPLA2 under the same assay conditions is inhibited by over 90% by pyrrolidine-2 and Wyeth-1 (Fig. 4C).
Affinity-purified His6-tagged cPLA2 exhibits greater lysophospholipase and PLA2 activity than cPLA2 (Fig. 5, A and B). cPLA2 does not exhibit sn-2 acyl chain specificity, because it hydrolyzes both sn-2 arachidonic acid and oleic acid from PC with similar activity (Fig. 5B). cPLA2 hydrolyzes arachidonic acid from PE with slightly lower activity than from PC (Fig. 5C). The PLA2 activity of cPLA2 is very sensitive to inhibition by pyrrolidine-2 and Wyeth-1 with IC50 values of 0.002 and 0.02 µM, respectively (Fig. 5D). The analog of pyrrolidine-2 in which the ketone carbonyl has been reduced to the tertiary alcohol did not inhibit cPLA2 or cPLA2 when tested in vitro at a concentration up to 1 µM (data not shown). This is consistent with a mechanism of inhibition of both enzymes in which the active site serine residue (Ser-228 in cPLA2) adds to the ketone carbonyl of pyrrolidine-2 to form a hemi-ketal. This also explains why removal of the 2-fluorine atoms from pyrrolidine-2 renders the compound inactive as an inhibitor (30).
A summary of the specific activities of cPLA2 and cPLA2 is shown in Table 2. Overall cPLA2 has 300-fold greater lysophospholipase and 400–800-fold greater PLA2 activity than cPLA2. The release of sn-2 fatty acids could occur through a PLA2 mechanism and/or by sequential deacylation of the sn-1 fatty acid (PLA1 reaction) followed by release of sn-2 fatty acid by lysophospholipase activity. We measured the specific activity of the various cPLA2s for hydrolysis of 1-hexadecyl-2-arachidonyl-PC (PLA2 activity) or 1-arachidonyl-2-hexadecyl-PC (PLA1 activity) using sonicated vesicles of the pure phospholipid. The use of ester/ether phospholipids ensures that only one round of lipolysis can occur, i.e. no lysophospholipase activity is possible because phospholipases do not cleave ether-linked aliphatic groups. Estimates of the specific activities came from a single time point assay (20 min). The results show that all three cPLA2 isoforms (, , ) have PLA2 and PLA1 activity, with the former being about 2-fold higher than the latter (Table 3). To be sure that the 1-arachidonyl-2-hexadecyl-PC was not contaminated by the positional isomer in which the arachidonyl group is in the sn-2 position, we submitted the 2-hexadecyl-PC synthetic precursor to combined reverse-phase high pressure liquid chromatography/electrospray ionization mass spectrometry using the previously reported method for lysophospholipid analysis (40). Based on the signal-to-noise ratio for the 2-hexadecyl-PC peak, we estimated that there was less than 2% of the positional isomer present. Based on this estimate and the data in Table 3, we conclude that cPLA2 and cPLA2 display significant PLA1 activity. The activity for cPLA2 was very low; however, the amount of arachidonic acid generated was 30- and 4-fold higher than that measured with no enzyme control in the reactions with 1-arachidonyl-2-hexadecyl-PC and 1-hexadecyl-2-arachidonyl-PC, respectively.
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TABLE 3
Specific activity of three cPLA2 isoforms
The PLA1 and PLA2 activity of affinity-purified cPLA2, cPLA2, and cPLA2 was investigated with different substrates as described under "Experimental Procedures."
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cPLA2 Expressed in Sf9 Cells Mediates A23187
[GenBank]
-induced Arachidonic Acid Release—Our results suggest that cPLA2 is the PLA2 in IMLF–/– responsible for fatty acid release in response to A23187.
[GenBank]
To compare the ability of cPLA2 and cPLA2 to release arachidonic acid in cells in response to A23187
[GenBank]
, the enzymes were expressed in Sf9 cells. We have used this cell model to study the properties and regulation of cPLA2, which releases arachidonic acid in response to A23187
[GenBank]
when expressed in Sf9 cells (41, 42). As shown in Fig. 6A, His6-cPLA2 has a much greater capacity than cPLA2 to release arachidonic acid in response to A23187
[GenBank]
when expressed in Sf9 cells. Sf9 cells were infected with two concentrations of baculovirus, resulting in similar levels of expression of cPLA2 and cPLA2 as shown by Western blot analysis using anti-His6 antibody. In Sf9 cells expressing His6-cPLA2, A23187
[GenBank]
-stimulated arachidonic acid release is 10-fold greater than vector control cells but is only slightly above control levels in cells expressing His6-cPLA2. As we observed for arachidonic acid release from IMLF–/–, the release of arachidonic acid from A23187
[GenBank]
-stimulated Sf9 cells expressing His6-cPLA2 is inhibited by pyrrolidine-2 (IC50 0.1 µM) and Wyeth-1 (IC50 1.0 µM) (Fig. 6B).
Calcium Sensitivity of cPLA2—Our results demonstrate that cPLA2 is activated in cells in response to increases in intracellular calcium. The calcium dependence of cPLA2 was investigated in more detail by measuring the effect of calcium on the activity of purified cPLA2 in vitro. The PLA2 activity of cPLA2 assayed using palmitoyl-arachidonyl-PE is stimulated by calcium 5-fold above the low level of activity observed in the absence of calcium (Fig. 7A). The effect of calcium concentration on PLA2 activity of cPLA2 and cPLA2 was compared directly using palmitoyl-arachidonyl-PC as substrate (Fig. 7B). The results of three experiments show that the calcium dependence of cPLA2 (KCa 0.82 ± 0.45, 1.5 ± 0.3, 0.87 ± 0.3 µM) and cPLA2 (KCa 1.5 ± 0.3, 0.95 ± 0.15, 1.7 ± 0.4 µM) is comparable. At saturating calcium levels the specific activity of cPLA2 is 2-fold higher than cPLA2 (Fig. 7B). The activity in the absence of calcium is 16 and 7% of maximal activity at saturating calcium for cPLA2 and cPLA2, respectively.
Calcium-induced Translocation of EGFP-cPLA2 and EGFP-cPLA2 in IMLF–/–—The ability of cPLA2 to release arachidonic acid in response to calcium ionophore and the stimulation of its activity by physiological levels of calcium suggests that it may translocate to membrane in response to increases in intracellular calcium as reported for cPLA2. This was investigated by comparing translocation of EGFP-cPLA2 and EGFP-cPLA2 in ionomycin-stimulated IMLF–/– by live cell imaging. EGFP-cPLA2 exhibits primarily diffuse cytosolic localization in unstimulated cells (Fig. 8A, a). In response to ionomycin, EGFP-cPLA2 rapidly translocates to membrane ruffles and to dynamic vesicular structures (Fig. 8A, b–f). An enlarged image of a portion of this cell (3.3 min after ionomycin) shows the presence of numerous fluorescent vesicles (Fig. 8A, g). A video of this cell shows the dynamic localization of cPLA2 to these structures (Movie 1 in supplemental data). Images of another cell depicting the translocation of cPLA2 to membrane ruffles in response to ionomycin are shown in Fig. 8A, h–j. In comparison, EGFP-cPLA2 expressed in IMLF–/– rapidly translocates to Golgi and endoplasmic reticulum in response to ionomycin as observed in other cell types (Fig. 8B, a–c) (11, 43).
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DISCUSSION
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cPLA2 plays an important role in production of eicosanoids through its ability to specifically release arachidonic acid from stimulated cells. However, there is ample evidence that other PLA2s participate in the release of arachidonic acid for production of eicosanoids, including certain secreted and GVI PLA2s (27, 29, 44, 45). Our study is the first to implicate another member of the GIV PLA2 family as mediating agonist-induced arachidonic acid release and eicosanoid production. We previously reported that lung fibroblasts from the cPLA2 knockout mouse release arachidonic acid and produce PGE2 in response to A23187
[GenBank]
and mouse serum (25). Our data indicates that this is due to the calcium-dependent activation of cPLA2. The cPLA2 transcript is highly expressed in mouse thyroid and at lower levels in stomach, large intestine, and prostate (20). The mRNA for cPLA2 was not detected in whole mouse lung; however, the lung comprises over 20 different cell types, and cell type-specific expression of cPLA2 is possible. Mouse lung fibroblasts contain both cPLA2 and cPLA2; however, the enzymatic properties of cPLA2, its ability to mediate calcium-dependent release of arachidonic acid, and its sensitivity to inhibition by pyrrolidine-2 and Wyeth-1 support its role in mediating arachidonic acid release and PGE2 production from cPLA2–/– mouse lung fibroblasts. The ability of two structurally different inhibitors, pyrrolidine-2 and Wyeth-1, to block arachidonic acid release from cells and the activity of purified cPLA2 with similar IC50 values is strong evidence supporting a role for cPLA2 in mediating arachidonic acid release from stimulated IMLF–/–.
Our results demonstrate that cPLA2 and cPLA2 share similar properties. They both have lysophospholipase, PLA1, and PLA2 activity, they have similar specific activities, and they are activated by low micromolar levels of calcium. The data showing that cPLA2 and cPLA2 are both potently inhibited by pyrrolidine-2 and Wyeth-1 complicate the use of these inhibitors to specifically implicate cPLA2 in mediating arachidonic acid release from cells. The similar inhibitor sensitivity of cPLA2 and cPLA2 is in contrast to the results with mouse cPLA2, which is not inhibited by pyrrolidine-2 and Wyeth-1. Human cPLA2 and human cPLA2 also are not inhibited by pyrrolidine-2 and pyrrolidine-1, respectively (22, 39). The basis for the differences in sensitivity of the GIV PLA2 family to pyrrolidine-2 and Wyeth-1 is not clear at this time.
One difference noted between cPLA2 and cPLA2 is that cPLA2 does not have specificity for arachidonic acid but releases multiple fatty acids, including palmitic, oleic, linoleic, and arachidonic acids, in response to A23187.
[GenBank]
The release of these fatty acids may be through its PLA1, PLA2, and lysophospholipase activities acting sequentially to deacylate diacylphospholipids. We confirmed that pyrrolidine-2 inhibits the release of oleic and palmitic acids from A23187
[GenBank]
-stimulated IMLF–/– (data not shown), which is consistent with a role for cPLA2 in mediating the nonspecific release of fatty acids. Despite the similarities in inhibitor sensitivities, cPLA2 and cPLA2 clearly have differences in the active site that influence acyl-chain specificity.
In contrast to cPLA2, mouse cPLA2 has very low enzymatic activity and only weakly releases arachidonic acid in response to A23187
[GenBank]
when expressed in Sf9 cells. We recently reported that human cPLA2 also has low enzymatic activity and that neither mouse nor human cPLA2 is inhibited by cPLA2 inhibitors (22). Analysis of the mouse cPLA2 transcript present in lung fibroblasts demonstrates that it does not contain the truncated JmjC domain found in the N terminus of human cPLA2. The truncated JmjC domain of human cPLA2 is created by skipping exons 7 and 8 of the cPLA2 gene (4). However, a complete JmjC domain (image clone AAH25390
[GenBank]
) can be transcribed from the first 8 exons of the human cPLA2 gene (4). This transcript stops prematurely after the first 8 exons of the human cPLA2 gene and thus lacks the C2 and catalytic domains of cPLA2. In contrast separate adjacent genes on mouse chromosome 2 encode a complete JmjC domain and cPLA2. Both mouse and human cPLA2 are similarly situated near the gene cluster encoding cPLA2, -, and - (4, 20). cPLA2 has very low enzymatic activity when assayed in vitro; however, it is possible that it has a unique substrate and/or cofactors in vivo. The physiological function and mechanisms of the regulation of cPLA2 remain to be elucidated.
The finding that lung fibroblasts contain three GIV PLA2s suggests that they play different physiological functions and are regulated by distinct mechanisms. This is emphasized by the observation that cPLA2 and cPLA2 translocate to different subcellular sites in response to ionomycin. Although we were unable to express mouse EGFP-cPLA2 in IMLF–/– for microscopy, we had previously observed that human cPLA2 does not translocate in response to calcium ionophore but is constitutively localized on mitochondria and early endosomes (22). In addition to differences in localization of cPLA2 and cPLA2, the phosphorylation sites found in cPLA2 are not conserved in cPLA2. Thus cPLA2 and cPLA2 provide differentially regulated pathways for fatty acid release, eicosanoid production, and the regulated turnover of lysophospholipids.
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FOOTNOTES
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* This work was supported by National Institutes of Health Grants HL61378 and HL34303 (to C. C. L.) and HL50040 (to M. H. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this
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ABSTRACT
INTRODUCTION
