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Originally published In Press as doi:10.1074/jbc.M609641200 on December 20, 2006

J. Biol. Chem., Vol. 282, Issue 9, 6532-6539, March 2, 2007
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A Single Enzyme Catalyzes Both Platelet-activating Factor Production and Membrane Biogenesis of Inflammatory Cells

CLONING AND CHARACTERIZATION OF ACETYL-CoA:LYSO-PAF ACETYLTRANSFERASE*Formula

Hideo Shindou{ddagger}1, Daisuke Hishikawa{ddagger}, Hiroki Nakanishi, Takeshi Harayama{ddagger}, Satoshi Ishii{ddagger}§1, Ryo Taguchi||, and Takao Shimizu{ddagger}12

From the {ddagger}Department of Biochemistry and Molecular Biology, and Department of Metabolome, Faculty of Medicine, University of Tokyo, and §Precursory Research for Embryonic Science and Technology (PRESTO) and ||Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency, Hongo 7-3-1, Tokyo 113-0033, Japan

Received for publication, October 13, 2006 , and in revised form, December 1, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Platelet-activating factor (PAF) is a potent proinflammatory lipid mediator eliciting a variety of cellular functions. Lipid mediators, including PAF are produced from membrane phospholipids by enzymatic cascades. Although a G protein-coupled PAF receptor and degradation enzymes have been cloned and characterized, the PAF biosynthetic enzyme, aceyl-CoA:lyso-PAF acetyltransferase, has not been identified. Here, we cloned lyso-PAF acetyltransferase, which is critical in stimulus-dependent formation of PAF. The enzyme is a 60-kDa microsomal protein with three putative membrane-spanning domains. The enzyme was induced by bacterial endotoxin (lipopolysaccharide), which was suppressed by dexamethasone treatment. Surprisingly, the enzyme catalyzed not only biosynthesis of PAF from lyso-PAF but also incorporation of arachidonoyl-CoA to produce PAF precursor membrane glycerophospholipids (lysophosphatidylcholine acyltransferase activity). Under resting conditions, the enzyme prefers arachidonoyl-CoA and contributes to membrane biogenesis. Upon acute inflammatory stimulation with lipopolysaccharide, the activated enzyme utilizes acetyl-CoA more efficiently and produces PAF. Thus, our findings provide a novel concept that a single enzyme catalyzes membrane biogenesis of inflammatory cells while producing a prophlogistic mediator in response to external stimuli.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Platelet-activating factor (PAF3;1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipid mediator that activates a G protein-coupled receptor (1–3) and results in pleiotropic and potent biological effects, including platelet activation, airway constriction, and hypotension (1). PAF is synthesized in various cells and tissues via two distinct pathways, the de novo and remodeling pathways (2, 4, 5), and the latter is regulated by extracellular signals and plays a critical role in stimulus-coupled PAF biosynthesis (2, 4–6). PAF synthesis induced by extracellular signals has been reported in murine peritoneal cells stimulated by calcium ionophore (7) or by PAF (8), in human eosinophils stimulated by fMet-Leu-Phe (9), in human neutrophils stimulated by acid stress (10), and in murine peritoneal macrophages stimulated by lipopolysaccharide (LPS) (11). In the remodeling pathway, the precursor of PAF, 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF), is synthesized from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (1-alkyl-phosphatidylcholine; PC) by the action of phospholipase A2 (2, 4, 12, 13). Subsequently, lyso-PAF is converted to PAF by acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAF acetyltransferase) (EC 2.3.1.67 [EC] ) (14). PAF is then rapidly degraded to lyso-PAF by PAF acetylhydrolases (15). Alternatively, lyso-PAF is again transformed into PC by the action of lysophosphatidylcholine (LPC) acyltransferase (2.3.1.23 [EC] ) (16).

A G protein-coupled PAF receptor was cloned in our laboratory (17), and PAF acetylhydrolases have been cloned and characterized by others (18, 19). Lyso-PAF acetyltransferase was initially demonstrated and partially characterized by Wykle et al. (14) in 1980. Since this first report, the enzyme activity has been detected in microsomes of rat spleen and lung as well as porcine leukocytes (14, 20). Although several groups have attempted to identify and characterize the enzyme from various sources (2, 4, 14, 21, 22), it has not yet been purified nor cDNA-cloned.

PC is a major component of cellular membranes and also plays an important role as a PAF precursor. PC is formed from diacylglycerol by a de novo pathway, originally described by Kennedy (23) in 1961 but is also generated from LPC by a remodeling pathway. Rapid turnover of the sn-2 acyl moiety of glycerophospholipids was described by Lands (Lands' cycle) (24–26) and is attributed to activation of phospholipases A2 and lysophospholipid acyltransferases. Recently, we (27) and Chen et al. (28) independently cloned one of LPC acyltransferases designated LPCAT1, which is highly expressed in alveolar type II cells. Since PC synthesis occurs in a variety of different tissues, additional LPC acyltransferases may be present for membrane biogenesis.

Using the previously reported LPCAT1 (27) and an extensive genomic data base search as well as 5'- and 3'-RACE, we have identified a lyso-PAF acetyltransferase gene. The enzyme is primarily expressed in inflammatory cells and is induced by LPS. Surprisingly, the enzyme also catalyzes incorporation of arachidonoyl-CoA to produce PAF precursor membrane glycerophospholipids (LPC acyltransferase activity). Thus, we designated this enzyme LysoPAFAT/LPCAT2. Although this enzyme possesses both acetyltransferase and acyltransferase activity, only the acetyltransferase activity was enhanced by acute inflammatory signals. To our knowledge, this is the first documentation of a cDNA for LysoPAFAT/LPCAT2, a critically important enzyme in the biogenesis of PAF and in membrane homeostasis of inflammatory cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Various lysophospholipids and acyl-CoAs were obtained from Avanti%20Polar%20Lipids">Avanti Polar Lipids (Alabaster, AL). LPS from Salmonella minnesota, 4-amidinophenylmethanesulfonyl fluoride, dexamethasone (DEX), estradiol-17beta, and linoleoyl-CoA (C18:2) were purchased from Sigma. [3H]Acetyl-CoA (185 GBq/mmol) and [3H]lyso-PAF (6.25 TBq/mmol) were obtained from Amersham Biosciences (Buckinghamshire, UK). [1-14C]Arachidonoyl-CoA (2.22 GBq/mmol) was purchased from Moravec Biochemicals (Mercury Lane, CA). ODN1826 and poly(I:C) were purchased from InvivoGen (San Diego, CA).

Cloning of LysoPAFAT/LPCAT2—The mLysoPAFAT/LPCAT2 gene was identified based upon sequence similarity to the LPCAT1 gene (27) and LPA acyltransferase {zeta} gene (29) through a comprehensive basic local alignment search tool (BLAST) search. A 1.6-kb cDNA clone encoding the full-length mLysoPAFAT/LPCAT2 (DDBJ accession number AB244716) was cloned by PCR amplification using the forward primer 5'-CTAGCTAGCCACCATGGATTACAAGGATGACGATGACAAGAACCGATGCGCCGAGGCGGCCGC-3', the reverse primer 5'-CCGCTCGAGTCAGTCCACCTTTTTGTCTGAGGTGCCCTC-3', and a mouse spleen cDNA library as a template. The FLAG epitope (DYKDDDDK) was attached to the N terminus of mLysoPAFAT/LPCAT2 by PCR using a forward primer. Amplified PCR products were cloned into the pCXN2.1 vector, a slightly modified version of pCXN2 (30) with multiple cloning sites, and sequenced. Similarly, hLysoPAFAT/LPCAT2 cDNA (DDBJ accession number AB244718) was amplified by PCR and inserted into the pCXN2.1 vector.

Quantitative Real Time RT-PCR—Mouse total RNAs were prepared using the Absolutely RNA RT-PCR Miniprep Kit (Stratagene), and first strand cDNA was subsequently synthesized using Superscript II (Invitrogen). The PCRs were performed using FastStart DNA Master SYBR Green I (Roche Applied Science). The primers for mLysoPAFAT/LPCAT2 were designed to amplify a 167-bp fragment: forward primer, 5'-GTCCAGCAGACTACGATCAGTG-3'; reverse primer, 5'-CTTATTGGATGGGTCAGCTTTTC-3'. The primers for hLysoPAFAT/LPCAT2 were designed to amplify a 176-bp fragment: forward primer, 5'-TTGCTTCCAATTCGTGTCTTATT-3'; reverse primer, 5'-ATCCCATTGAAAAGAACATAGCA-3'.

Expression of FLAG-mLysoPAFAT/LPCAT2 in CHO-K1 Cells—After 48 h of transfection with FLAG-tagged enzyme using Lipofectamine 2000 (Invitrogen), cells in 10-cm dishes were scraped into 1 ml of ice-cold buffer containing 20 mM Tris-HCl (pH 7.4), 300 mM sucrose, and a proteinase inhibitor mixture, Complete (Roche Applied Science), and then sonicated three times on ice for 30 s. After centrifugation for 10 min at 800 x g, the supernatant was collected and centrifuged at 100,000 x g for 1 h. The resulting pellet was resuspended in buffer containing 20 mM Tris-HCl (pH 7.4), 300 mM sucrose, and 1 mM EDTA. Protein concentration was measured by the method of Bradford (31), using a commercially prepared protein assay solution (Bio-Rad) and bovine serum albumin (fraction V, fatty acid-free; Sigma) as a standard.

Confocal Microscopy—CHO-K1 cells were seeded onto 6-cm dishes before transfection. Six µg each of pCXN2.1 vector or FLAG-mLysoPAFAT/LPCAT2 were transfected using Lipofectamine 2000. 48 h post-transfection, vector- or FLAG-tagged enzyme-transfected cells were incubated with 2.5 µg/ml 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) (Invitrogen) and 10 µg/ml M5 anti-FLAG mouse monoclonal antibody (Sigma) in 1/4x permeabilization buffer (Beckman Coulter, Marseille, France). After washing, cells were incubated with 10 µg/ml Alexa Fluor 546 goat anti-mouse IgG (Eugene, OR) for 30 min. Confocal microscopy was performed with an LSM510 laser-scanning microscope (Carl Zeiss) equipped with a x63 water immersion objective lens (numerical aperture = 1.2). FLAG-mLysoPAFAT/LPCAT2 was monitored by excitation at 543 nm with a helium/neon laser and by emission with a 585-nm long path filter. For the detection of DiOC6(3), the excitation was at 488 nm with an argon laser, and emissions were taken with a 505–550-nm band pass filter.

Assay of Lyso-PAF Acetyltransferase—The lyso-PAF acetyltransferase activity was determined according to the method of Kume et al. (20), except for washing resin one time before and eight times after application of the reaction mixture. Briefly, 100 µM [3H]acetyl-CoA (1.11 GBq/mmol) and protein were incubated at 37 °C for 10 min in the presence or absence of 20 µM lyso-PAF (Cayman). Subsequently, the product was bound to C8 resin (Millipore), washed, and eluted. The difference between the radioactivities obtained in the presence and absence of lyso-PAF corresponded to lyso-PAF acetyltransferase activity.

Assay of Lysophospholipid Acetyltransferase and Acyltransferase by TLC—The acyltransferase activity was measured in two ways: (i) conversion of [1-14C]lyso-PAF (293 MBq/mmol) to PC in the presence of acetyl- and acyl-CoA and (ii) the transfer of [3H]acetyl-CoA (1.11 GBq/mmol)- or [14C]arachidonoyl-CoA (1.11–2.035 GBq/mmol) to lysophospholipids to form phospholipids. The reaction mixture contained 20 mM Tris-HCl (pH 7.4), 2 mM CaCl2, 1 mg/ml PC, 5 mM 2-mercaptoethanol, 20 µM 4-amidinophenylmethanesulfonyl fluoride (Sigma), a proteinase inhibitor mixture, 10 or 100 µM acyl-CoA, 20 µM lysophospholipid, and enzyme in a total volume of 100 µl. After incubation at 37 °C for 10 min, the reaction was stopped by the addition of 0.3 ml of chloroform/methanol (1:2, v/v). Total lipids were extracted using the Bligh-Dyer method (32) and subsequently analyzed by TLC in chloroform/methanol/acetic acid/water (50:25:8:4, v/v/v/v). Bands at positions corresponding to the expected product were visualized by I2 vapor, cut from the plate, placed in Microscinti-O (Packard Bioscience), and analyzed in a liquid scintillation counter LS6500 (Beckman).

Radioligand Binding Assay—The method of PAF-PAF receptor (PAFR) binding assay was described previously (7, 33). Briefly, the membrane fraction containing 158 fmol of PAFR from hearts and skeletal muscles of PAFR transgenic mice (7, 34) were mixed with 25 nM [3H]WEB 2086 and the lipid extract in a 96-well plate. After incubation at 25 °C for 90 min, receptor-bound [3H]WEB 2086 was collected by filtration through a UniFilter-GF/C (PerkinElmer Life Sciences) using a MicroMate 196 simultaneous 96-well harvester (PerkinElmer Life Sciences), and the filter was washed and dried. Subsequently, the radioactivities were counted with a TopCount microplate scintillation counter (PerkinElmer Life Sciences).

Electrospray Ionization Mass Spectrometry Analysis of PAF— Extracted lipid from the acetyltransferase assay was identified by electrospray ionization mass spectrometry analysis. The analysis was performed using a 4000 Q-TRAP quadrupole-linear ion trap hybrid mass spectrometer (Applied Biosystems/MDS Sciex, Concord, Canada) with an Ultimate 3000 high pressure liquid chromatography system (DIONEX Co.) combined with an HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland). The extracted lipids were subjected to electrospray ionization mass spectrometry analysis by flow injection without liquid chromatography separation. The solvent was acetonitrile, methanol, 50 mM ammonium formate, pH 7.4 (v/v/v, 45/50/5), and the flow rate was 10 µl/min. The scan range and speed were set at m/z 500–600 and 1000 Da/s, respectively. The trap fill time was set at 5 ms, and the ion spray voltage was set at -4500 V in the negative ion mode. Nitrogen was used as curtain and collision gas. The declustering potential was set at 20 V to minimize in-source fragmentation. Both Q1 and Q3 resolution were set to unit mass. The collision energy used was varied according to the desired experiment. The method to identify phosphatidylcholine species was described previously (35).

siRNA Transfection—hLysoPAFAT/LPCAT2 siRNAs (siRNA ID numbers 140446, 140447, and 140448; Ambion) and control siRNA (silencer negative control 1; Ambion) were transfected using siPORT amine transfection agent according to the manufacturer's protocol. The siRNA transfection was performed for 2 days in HEK293 cells.

Short Term LPS Stimulation—After transfection of RAW264.7 cells with LysoPAFAT/LPCAT2 using Lipofectamine 2000, cells were pretreated with or without 20 µM SB 203580 for 1 h and subsequently stimulated with 100 ng/ml LPS for 30 min. For preparation of cell extracts, the cells were scraped into 600 µl of an ice-cold buffer containing 20 mM Tris-HCl, (pH 7.4), 50 mM beta-glycerophosphate, 1 mM sodium orthovanadate, 5 mM 2-mercaptoethanol, 20 µM 4-amidino-phenylmethanesulfonyl fluoride, and Complete, and the collected cells were sonicated twice on ice for 30 s. Intact cells, cellular debris, and mitochondria were removed by centrifugation at 9000 x g for 10 min at 4 °C. Enzyme activities were measured as described above.

Isolation and Stimulation of Mouse Peritoneal Cells—Mouse peritoneal macrophages induced by thioglycollate (Difco) was prepared as described in detail previously (11). The cells were treated with 100 ng/ml LPS, 0.8 µM ODN1826, or 1 µg/ml poly(I:C), in the presence or absence of 100 nM DEX or 100 nM estradiol-17beta for 16 h. After treatment, the cells were washed with an ice-cold buffer containing 20 mM Tris-HCl (pH 7.4) and 300 mM sucrose. Cell extracts were prepared by the same method as described for experiments using RAW264.7 cells, and the enzyme activity was measured.

After treatment with microbial components for 16 h, total RNA was collected using the Absolutely RNA RT-PCR miniprep kit. Likewise, at 4 h after intraperitoneal injection with 2 ml of 2% casein, peritoneal exudate neutrophils were harvested from the peritoneal cavity, and their total RNA was prepared.

Statistics—Data are presented as mean ± S.E. or S.D. p values less than 0.05 were considered statistically significant. All statistical calculations were performed using Prism 4 (GraphPad Software) and StatView-J, version 5.0 (Abacus Concepts, Berkeley, CA).

Mice—C57BL/6J mice were obtained from Clea Japan, Inc. (Tokyo, Japan). Mice were maintained in a light-dark cycle with light from 8:00 to 20:00 at 21 °C. Mice were fed with a standard laboratory diet and water ad libitum. All animal studies were conducted in accordance with the guidelines for Animal Research at The University of Tokyo and were approved by the University of Tokyo Ethics Committee for Animal Experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification and Cloning of LysoPAFAT/LPCAT2—The mouse LysoPAFAT/LPCAT2 (mLysoPAFAT/LPCAT2) gene was identified based upon sequence homology with the previously reported LPCAT1 (27). The putative open reading frame of mLysoPAFAT/LPCAT2 encoded a 544-amino acid protein of 60.3 kDa, containing three putative transmembrane domains (36) and several conserved motifs found in members of the lysophospholipid acyltransferase family (27, 37). mLysoPAFAT/LPCAT2 contained putative EF-hand-like motifs (aa 374–494, E-value = 3e*-10) predicted by a conserved domain data base (available on the World Wide Web at www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) (38) and showed 48.2% amino acid sequence homology to mouse LPCAT1. It exhibited 88.4% amino acid sequence homology to human LysoPAFAT/LP-CAT2 (hLysoPAFAT/LPCAT2) (Fig. 1). The presence of the C-terminal sequence motif KKXX suggests that the protein is localized to the endoplasmic reticulum (ER) (39).

Tissue Distribution and Subcellular Localization of mLyso-PAFAT/LPCAT2 mRNA—The tissue distribution of mLyso-PAFAT/LPCAT2 was analyzed by quantitative PCR. We found the highest level of mLysoPAFAT/LPCAT2 expression in resident macrophages, casein-induced neutrophils, followed by skin, colon, spleen, and thioglycollate-induced macrophages (Fig. 2A). To facilitate immunocytochemical analysis of mLysoPAFAT/LPCAT2, we constructed an expression vector encoding FLAG-tagged mLysoPAFAT/LPCAT2. It was transfected into CHO-K1, and the enzyme distribution was examined by confocal microscopy after 48 h. FLAG-mLyso-PAFAT/LPCAT2 exhibited significant enzyme activities (Fig. 3, A and B). Cells were stained for ER-Golgi using DiOC6(3). The subcellular distribution pattern of FLAG-mLysoPAFAT/LPCAT2 was similar to that of DiOC6(3), suggesting that the enzyme is present mainly in the ER and Golgi (Fig. 2B).


Figure 1
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  		FIGURE 1.
Amino acid sequence alignment of mouse and human LysoPAFAT/LPCAT2. The predicted conserved lysophospholipid acyltransferase motifs (motifs 1–3) are boxed. The putative transmembrane domains are underlined. Amino acids conserved in both species are marked with asterisks. The ER localization sequence KKXX is present at the C terminus. The transmembrane motifs were determined using HMMTOP (available on the World Wide Web at www.enzim.hu/hmmtop/index.html).

 
Substrate Selectivity of mLysoPAFAT/LPCAT2—We next examined the acetyltransferase activity of mLysoPAFAT/LPCAT2 using a variety of 20 µM lysophospholipid acceptors and 100 µM [3H]acetyl-CoA as a donor. mLysoPAFAT/LPCAT2 had detectable acetyltransferase activity toward LPC (Fig. 3A) and alkyl-LPC (Fig. 3B). The enzyme had a higher activity toward C16 lysophospholipids than C18 at the sn-1 moiety, which served as an acetyl acceptor (Fig. 3B). The acetyltransferase activity of mLysoPAFAT/LPCAT2 was linear for the first 40 min at 37 °C, and the enzyme exhibited calcium-dependent activity with a pH optimum around 7.4 (data not shown). To confirm whether the product of the acetyltransferase reaction is PAF, we first performed a competitive receptor binding assay using PAFR prepared from PAFR-transgenic mice (7, 34). Lipid extracts from the acetyltransferase reaction of mLy-soPAFAT/LPCAT2 with lyso-PAF competed for binding of [3H]WEB2086 to PAFR, indicating that the enzyme indeed produced PAF (supplemental Fig. 1). More definitively, the reaction product was identified by mass spectrometry (MS) using a 4000 Q-TRAP mass spectrometer (supplemental Fig. 2).

Human LysoPAFAT/LPCAT2 siRNA Decreases Lyso-PAF Acetyltransferase Activity—To investigate whether endogenous lyso-PAF acetyltransferase activity was decreased by transfection with an siRNA against hLysoPAFAT/LPCAT2, we transfected three hLysoPAFAT/LPCAT2 siRNAs (siRNA ID 140446, 140447, and 140448; Ambion) into HEK293 cells. We chose HEK293 cells, because the cells exhibit a high endogenous enzymic activity and high transfection efficiency (data not shown). All siRNAs decreased mRNA levels of hLysoPAFAT/LPCAT2 by 70–80% and lyso-PAF acetyltransferase activity by 50–60% (Fig. 3C). Control siRNA (silencer negative control 1; Ambion) had no apparent effect on either enzyme activity or mRNA expression. Thus, hLysoPAFAT/LPCAT2 appears to be the principal enzyme for PAF production in HEK293 cells.

LysoPAFAT/LPCAT2 Possesses LPC Acyltransferase Activity— Next, we examined the acyl-CoA selectivity of mLysoPAFAT/LPCAT2 using [3H]lyso-PAF (C18) as an acceptor. At a high concentrations of acyl-CoAs (>20 µM), mLysoPAFAT/LPCAT2 showed both acetyltransferase and arachidonoyltransferase (acyltransferase) activities (Fig. 4). At a low concentration (<10 µM), arachidonoyl-CoA was a better substrate for LysoPAFAT/LPCAT2 than acetyl-CoA (Fig. 4). Medium chained fatty acyl-CoAs were poor substrates at both high and low concentrations of acyl-CoA (data not shown). These results suggest that mLysoPAFAT/LPCAT2 exhibits both lyso-PAF acetyltransferase and LPC acyltransferase activities. The apparent Km values of the enzyme for acetyl-CoA and for arachidonoyl-CoA were 50.4 and 21.1 µM, respectively (Fig. 4).

Enzyme Activation by an Inflammatory Stimulus—Next, we investigated the activation of the enzyme by an inflammatory stimulus, such as LPS. To examine the response to LPS stimulation, mLysoPAFAT/LPCAT2 was transfected into the macrophage cell line, RAW264.7, which expresses the LPS receptor, Toll-like receptor 4 (TLR4), and cells were stimulated by LPS for 30 min in the presence or absence of the p38 mitogen-activated protein kinase inhibitor SB 203580 (Tocris Cookson). The acetyltransferase activity of mLysoPAFAT/LPCAT2 was increased by LPS stimulation, but the effect was decreased in the presence of SB 203580; the acyltransferase activity of the enzyme was unchanged (Fig. 5). The endogenous lyso-PAF acyltransferase in RAW264.7 cells was activated by LPS, and this activation was blocked in the presence of SB 203580 (data not shown).


Figure 2
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  		FIGURE 2.
Tissue distribution and subcellular localization of mLy-soPAFAT/LPCAT2. A, the expression levels of mLysoPAFAT/LPCAT2 and beta-actin mRNA in 20 mouse tissues were analyzed by quantitative RT-PCR, and the levels of mLysoPAFAT/LPCAT2 mRNA were normalized to those of beta-actin mRNA in each tissue. The highest level of mLysoPAFAT/LPCAT2 expression was observed in resident macrophages. B, CHO-K1 cells were transfected with FLAG-mLysoPAFAT/LPCAT2 and subjected to immunocytochemical analysis 48 h post-transfection. ER-Golgi and FLAG-mLysoPAFAT/LPCAT2 were visualized using DiOC6(3) (green) and the M5 anti-FLAG peptide antibody (red), respectively. The subcellular distribution pattern of FLAG-mLysoPAFAT/LPCAT2 was similar to that of DiOC6(3) (Merge), suggesting that the enzyme is present mainly in the ER and Golgi. DiOC6(3) is an ER-Golgi marker. The scale bars correspond to 20 µm. Results are representative of two independent experiments with similar results.

 
Induction of mLysoPAFAT/LPCAT2 mRNA—Next, we examined induction of mLysoPAFAT/LPCAT2 mRNA in response to long term treatment with Toll-like receptor agonists. Mouse thioglycollate-induced macrophages were treated with LPS (a TLR4 ligand), ODN1826 (a TLR9 ligand), or poly(I:C) (a TLR3 ligand) for 16 h in the presence or absence of DEX or estradiol-17beta. As shown in Fig. 6A, the endogenous lyso-PAF acetyltransferase activity was enhanced 2.4- and 2.2-fold by LPS and ODN1826 treatment, respectively. Moreover, augmentation of the enzyme activity by LPS treatment was suppressed in the presence of DEX but not estradiol-17beta. Similar results were obtained for the LPC acyltransferase activity (data not shown). The enzyme activation by ODN1826 also tended to be reduced by DEX. Poly(I:C) did not affect the lyso-PAF acetyltransferase activity in macrophages.


Figure 3
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  		FIGURE 3.
Substrate selectivity of mLysoPAFAT/LPCAT2 and siRNA transfection with hLysoPAFAT/LPCAT2 siRNAs. A, lysophospholipid acetyltransferase assays were performed by TLC with 20 µM lysophospholipid (1-acyl- and 1-alkyl-LPA, LPC, lysophosphatidylglycerol (LPG), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), or lysophosphatidylserine (LPS)) and 100 µM [3H]acetyl-CoA in the presence of 1 µg of the microsomal fractions from vector-transfected (open bars) or mLysoPAFAT/LPCAT2-transfected (closed bars) cells. 1-O-Alkenyl-LPC (heart), LPI (liver), and LPS (brain) were from bovine tissues. Other lysophospholipids contained a palmitoyl group at the sn-1 position. B, several LPC (1-acyl-LPC C16, C18, 1-alkyl-LPC C16, C18, and 1-alkenyl-LPC) acetyltransferase activities were measured. mLysoPAFAT/LPCAT2 possessed lysophospholipid acetyltransferase activities toward 1-O-alkyl-LPC, 1-acyl-LPC, and 1-O-alkenyl-LPC. ND, not detectable. C, three hLyso-PAFAT/LPCAT2 siRNAs and a control siRNA were transfected into HEK293 cells. After 48 h, mRNA levels (left) and lyso-PAF acetyltransferase activities (right) were measured, as described under "Experimental Procedures." Endogenous lyso-PAF acetyltransferase activity was reduced by each hLy-soPAFAT/LPCAT2 siRNA. The data represent the mean ± S.D. of triplicate measurements. Statistical analyses were performed by analysis of variance and Tukey's multiple comparison test (p < 0.01). Two independent experiments were performed with similar results.

 


Figure 4
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  		FIGURE 4.
Acetyltransferase and acyltransferase activities of mLysoPAFAT/LPCAT2. Lyso-PAF acetyltransferase and acyltransferase assays were performed by TLC with the indicated concentrations of acetyl-CoA (2:0-CoA) and arachidonoyl-CoA (20:4-CoA) using 20 µM lyso-PAF. The inset shows a Lineweaver-Burk plot to calculate Km values. The data represent the mean ± S.D. of triplicate measurements. The results are representative of two independent experiments with similar results.

 
The mLysoPAFAT/LPCAT2 mRNA levels were increased 7.3- and 4.8-fold by LPS and ODN1826 treatment, respectively. Furthermore, induction of mLysoPAFAT/LPCAT2 by LPS was repressed by DEX treatment (Fig. 6B). ODN1826 also enhanced mLysoPAFAT/LPCAT2 levels, which tended to be reduced by DEX treatment. The expression level of mLyso-PAFAT/LPCAT2 was not changed by poly(I:C) treatment, similar to the lack of effect on the enzyme activity (Fig. 6A). Induction of an IFN{gamma}-inducible gene (IP-10) used as a positive control was observed by PCR after poly(I:C) stimulation under these conditions (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first report of isolation of a cDNA for LysoPAFAT/LPCAT2, a critically important enzyme in the biosynthesis of PAF. After long term treatments with a TLR4 or TLR9 agonist, the expression level of mLysoPAFAT/LPCAT2 mRNA was up-regulated, but not with a TLR3 agonist. Surprisingly, this enzyme catalyzed not only PAF biosynthesis (lyso-PAF acetyltransferase) but also generation of membrane glycerophospholipids (LPC acyltransferase), which are major membrane constituents and precursors of PAF. In the resting conditions, the enzyme prefers arachidonoyl-CoA to produce membrane lipids. However, under acute inflammatory stimulation by activating the TLR4, only the acetyltransferase activity of the enzyme was enhanced, and PAF production was augmented (Fig. 7).


Figure 5
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  		FIGURE 5.
Enzyme activation by an inflammatory stimulus. RAW264.7 cells transfected with vector or mLysoPAFAT/LPCAT2 were stimulated with 100 ng/ml LPS in the presence or absence of SB 203580, and enzyme activity assays were performed subsequently. The difference between the activities obtained with vector and mLysoPAFAT/LPCAT2 corresponded to transfected enzyme activity. Endogenous acetyltransferase activities of nonstimulation, LPS stimulation, and LPS stimulation with SB203580 were 300.2, 469.0, and 259.4 pmol/min/mg protein, respectively. Endogenous acyltransferase activities were 604.0, 900.2, and 572.2 pmol/min/mg protein. Statistical analyses were performed by using analysis of variance with Fisher's projected least significant difference (PLSD) test (p < 0.05). The results are expressed as mean ± S.E. of three independent experiments, each performed in triplicate.

 
Characterization of Lyso-PAF Acetyltransferase—mLyso-PAFAT/LPCAT2 possessed lyso-PAF acetyltransferase activity but did not show 1-alkyl-LPA acetyltransferase activity, which catalyzes the first step of the de novo PAF biosynthesis pathway (Fig. 3, A and B) (2, 4). Using a heterologous overexpression system, we found that the enzyme was predominantly localized to the ER-Golgi complex (Fig. 2B). The exact localization of the endogenous protein in native cells remains to be determined. Additionally, LysoPAFAT/LPCAT2 possessed putative EF-hand-like motifs and showed calcium-dependent activity. These correlations remain to be further clarified. Using hLysoPAFAT/LPCAT2 siRNA transfection of HEK293 cells, both the mRNA and the enzyme activity of endogenous lyso-PAF acetyltransferase were significantly reduced (Fig. 3C), indicating that LysoPAFAT/LPCAT2 constitutes a major (50–80%) lyso-PAF acetyltransferase, in HEK293 cells. It is possible that other enzyme(s) are present also to catalyze PAF production in other tissues and cells.

LPC Acyltransferase Activity of LysoPAFAT/LPCAT2—mLyso-PAFAT/LPCAT2 exhibited not only significant lyso-PAF acetyltransferase activity but also LPC acyltransferase activity (Fig. 4) in vitro, indicating that mLysoPAFAT/LPCAT2 can produce both PAF and PC. These data agree well with the previous studies showing that the lyso-PAF acetyltransferase activity in neutrophils was competed by long chain acyl-CoAs (40, 41). Recently, we and another group identified an LPC remodeling enzyme, designated LPCAT1, which is highly expressed in lung (27, 28). In contrast, LysoPAFAT/LPCAT2 is predominantly expressed in inflammatory cells with modest expression in skin, brain, and colon. Because PC is biosynthesized in all cell types, a different class of LPCATs may exist in addition to LPCAT1 (27, 28) and LysoPAFAT/LPCAT2 (present study).


Figure 6
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  		FIGURE 6.
Induction of LysoPAFAT/LPCAT2 by microbial components. Thioglycollate-induced mouse macrophages were treated with 100 ng/ml LPS, 0.8 µM ODN1826, or 1 µg/ml poly(I:C) for 16 h in the presence or absence of 100 nM DEX or 100 nM estradiol-17beta. Lyso-PAF acetyltransferase activity (A) and expression of mLysoPAFAT/LPCAT2 mRNA (B) were analyzed. The open bars indicate beta-actin as a control. Statistical analyses were performed by analysis of variance and Tukey's multiple comparison test (p < 0.05). The results are expressed as mean ± S.E. of three independent experiments.

 
An Acute Inflammatory Response—Upon acute inflammatory stimulation by LPS, the acetyltransferase activity of mLysoPAFAT/LPCAT2 was enhanced (Fig. 5). Similar activation has been observed with endogenous lyso-PAF acetyltransferase in mouse peritoneal macrophages (11). Nixon et al. reported that lyso-PAF acetyltransferase in human neutrophils is directly activated by p38 mitogen-activated protein kinase (42). It is possible that LysoPAFAT/LPCAT2 is modified and activated by phosphorylation after LPS-stimulation. In contrast, the LPC acyltransferase activity of the enzyme was not changed by short term LPS stimulation (Fig. 5). The cellular signaling pathway mediating LysoPAFAT/LPCAT2 activation by LPS remains to be elucidated. In addition to putative conformational changes of the enzyme, both enzyme activities may be regulated by the ratio of two substrates (acetyl-CoA and arachidonoyl-CoA) in cells.


Figure 7
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  		FIGURE 7.
Regulation of LysoPAFAT/LPCAT2 in mouse macrophages by inflammatory signals. LysoPAFAT/LPCAT2 activity is increased by two distinct pathways in mouse macrophages. Although LysoPAFAT/LPCAT2 catalyzes both acetyltransferase and acyltransferase, only acetyltransferase activity was enhanced under acute inflammatory conditions. GR, glucocorticoid receptor; p38, p38 mitogen-activated protein kinase; 20:4-CoA, arachidonoyl-CoA. See the first paragraph under "Discussion" for details.

 
Induction of LysoPAFAT/LPCAT2 by Bacterial Components— Bacterial infections increase PAF production (1, 43, 44), and a TLR4 agonist (LPS) and a TLR9 agonist (ODN1826) induced the enzyme activity and mRNA levels of LysoPAFAT/LPCAT2. LPS activates both MyD88 (myeloid differentiation primary response gene 88) and TRIF (Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-beta) adaptor protein (45–47). These results suggest that LysoPAFAT/LPCAT2 expression is regulated in a MyD88-dependent manner in the innate immune system, because TLR9 and TLR3 mediate MyD88- and TRIF-dependent pathways, respectively (45, 48, 49). Induction of mLysoPAFAT/LPCAT2 was attenuated by DEX treatment (Fig. 6). Ogawa et al. (50) reported that glucocorticoid receptor signaling represses LPS-mediated up-regulation of a large set of related inflammatory response genes, such as cyclooxygenase-2 and interleukin-6. Furthermore, DEX inhibits LPS-induced plasma PAF release (51). It will be important to determine the transcriptional regulation and cis-elements of LysoPAFAT/LPCAT2 in future studies.

LPS-induced accumulation of LysoPAFAT/LPCAT2 increases acyltransferase activity in addition to the acetyltransferase activity. The biological significance of up-regulation of the acyltransferase activity may be related to the fact that under long term LPS stimulation, cytosolic and secretory phospholipase A2 are activated, leading to increased release of free fatty acids and lysophospholipids from PC (52, 53). Lysophospholipids are toxic to cells because of their detergent effects. Alternatively, active membrane remodeling is required during inflammatory responses, such as phagocytosis or chemotaxis. LysoPAFAT/LPCAT2 may play an important role in the regulation of lysophospholipid and PAF levels and in the storage of PC as PAF precursor membrane glycerophospholipids. Conclusion—We have isolated a new enzyme that catalyzes PAF production and membrane biogenesis (LysoPAFAT/LPCAT2). Further studies are needed to elucidate the roles of mLysoPAFAT/LPCAT2 in vivo and to determine its potential as a novel therapeutic target for various diseases involving PAF biosynthesis. It will be important to characterize both acetyltransferase and acyltransferase activities of the enzyme, including identification of binding sites for each substrate (acetyl-CoA and arachidonoyl-CoA) and differential regulation of individual enzyme activity. Molecular cloning and characterization of this first LysoPAFAT/LPCAT2 will enable us to better understand the biochemical mechanisms underlying PAF and phospholipid biosynthesis in inflammatory cells.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB244716 (mouse) and AB244718 (human).

* This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Culture, Sports, and Technology of Japan (to T. S. and S. I.). 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 fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2. Back

1 Supported by the Center for NanoBio Integration at the University of Tokyo. Back

2 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5802-2925; Fax: 81-3-3813-8732; E-mail: tshimizu{at}m.u-tokyo.ac.jp.

3 The abbreviations used are: PAF, platelet-activating factor; LPS, lipopolysaccharide; PC, phosphatidylcholine; lyso-PAF acetyltransferase, acetyl-CoA: lyso-PAF acetyltransferase; LPC, lysophosphatidylcholine; DEX, dexamethasone; ER, endoplasmic reticulum; PAFR, PAF receptor; mLysoPAFAT and hLysoPAFAT, mouse and human LysoPAFAT, respectively; siRNA, small interfering RNA; DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide. Back


   ACKNOWLEDGMENTS
 
We are grateful to Drs. A. Yamashita (Teikyo University), T. Yokomizo, (Kyushu University), K. Kume (Kumamoto University), M. Nakamura, T. Takahashi, Y. Kita, and Y. Iizuka (University of Tokyo) and all members of our laboratory for valuable suggestions and F. Hamano (University of Tokyo) for technical assistance. We also thank Dr. J. Miyazaki for supplying pCXN2.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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