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Address correspondence to Junken Aoki, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 Japan. Tel.: 81-3-5841-4723. Fax: 81-3-3818-3173. E-mail: jaoki{at}mol.f.u-tokyo.ac.jp
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Abstract |
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Key Words: lysoPLD; EDG receptor; lysophosphatidylcholine; chemotaxis; cell proliferation
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Introduction |
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In contrast to the extensive analysis of mechanisms underlying LPA signaling mediated by the LPA receptor family, the enzymes regulating LPA production and degradation have not been characterized fully. LPA can be produced by a variety of cells including platelets, fibroblasts, adipocytes, and ovarian cancer cells (Gerrard and Robinson, 1989; Eichholtz et al., 1993; Shen et al., 1998). LPA is also produced by the action of extracellular lysophospholipase D (lysoPLD) on lysophosphatidylcholine (LPC; Tokumura et al., 1986), which is present at high micromolar levels in plasma (Okita et al., 1997; Tokumura et al., 1999; Croset et al., 2000). LPA can be detected in various biological fluids such as serum, plasma, ascites, and saliva (Tokumura et al., 1986, 1999; Tigyi and Miledi, 1992), and its levels are elevated in diverse physiological and pathological conditions such as pregnancy, high cholesterol diet, and ovarian cancer (Xu et al., 1995; Tokumura et al., 2000, 2002). Plasma lysoPLD appears to mediate the production of LPA in plasma (Tokumura et al., 1986), potentially contributing to the aberrant LPA levels in pathophysiological states. To characterize the as yet uncloned lysoPLD and to further elucidate the biological function of LPA, we purified lysoPLD from biological fluids.
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Results and discussion |
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ATX/lysoPLD stimulates cell motility particularly in the presence of LPC
The cell motilitystimulating activity of ATX on tumor cells requires an intact catalytic domain (Lee et al., 1996), however, the ATX substrate essential for the ability of ATX to induce cell motility has yet to be identified. In the following experiments, we investigated whether the cell motilitystimulating activity of ATX (Stracke et al., 1992; Fig. 2 A) could be explained by its lysoPLD activity because of the observation that LPA, the product of lysoPLD, is an effective inducer of chemotaxis in multiple cell lineages (Imamura et al., 1993; Stam et al., 1998; Sturm et al., 1999; Manning et al., 2000). Consistent with previous observations (Stracke et al., 1992), recombinant ATX produced in Sf9 cells stimulated a dose-dependent chemotaxis of the human melanoma A-2058 cell line using a Boyden chamber assay (Fig. 2 A). Interestingly, the ability of recombinant ATX to induce motility was dramatically increased by the addition of LPC, a substrate for lysoPLD (Fig. 2 A). LPC alone was insufficient to alter cell motility (Fig. 2 A). The addition of nucleotide substrates of ATX, including ATP, ADP, and adenosine (up to 100 µM) failed to alter the effects of ATX on cell motility (unpublished data). In contrast, exogenous LPA was sufficient to stimulate motility of A-2058 melanoma cells (Fig. 2 B). ATX in the absence of exogenous LPC was sufficient to modestly increase cellular motility (Fig. 2 A). If the effects of ATX on cellular motility are due to the hydrolysis of LPC and the subsequent action of LPA on cells, LPC must be present in the cells or cell supernatants. One possible explanation is that cancer cells can release LPC into culture media, which may in turn serve as substrate for ATX, resulting in the production of LPA which, in turn, induces cellular motility (see Fig. 4 A and next paragraph).
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Here, we have demonstrated that lysoPLD is identical to ATX and provided evidence that LPA, the product of ATX/lysoPLD, mediates chemotaxis and proliferation of cancer cells. LPA is known to stimulate both chemotaxis (Imamura et al., 1993; Stam et al., 1998; Sturm et al., 1999; Manning et al., 2000) and proliferation (van Corven et al., 1989, 1992) of multiple cell lineages. The LPA receptors, in particular the EDG2 receptor analyzed here, can couple with pertussis toxinsensitive Gi (Hecht et al., 1996), which is consistent with the effect of pertussis toxin on cell motility induced by ATX. As assessed by quantitative PCR, all of the cancer cell lines (with the exception of RH7777) used here express EDG2 (unpublished data). Some tumor cells appear to be capable of secreting factors that stimulate their own motility, survival, and growth. Although autocrine secretion of motility factors might play a role in the initiation of tumor cell invasion, autocrine secretion of growth factors by tumor cells might contribute to the proliferation and survival of metastatic colonies. Both chemoattractant and proliferative activities can be stimulated by LPA, which can be produced by the action of ATX/lysoPLD on LPC. LPC is present at high levels in plasma, providing a potential source for paracrine or endocrine action of ATX. In the microenvironment of the tumor, LPC secreted by tumor cells may play an important role in ATX-mediated autocrine motility and proliferation of tumor cells.
LPC was detected in the culture media of various cell types (Fig. 4 A). Indeed, LPC levels in culture supernatants of RH7777 cells were 1 µM. The release of LPC (Fig. 4 A) from cells was dependent on the presence of BSA in the media (unpublished data), which is capable of removing LPC from the outer membrane leaflet. LPC is also present at significant levels in intact cells (unpublished data). Thus, LPC released from cells or in the extracellular leaflet of the cell membrane could be a source of substrate for lysoPLD. A similar mechanism for LPA production was proposed by van Dijk et al. (1998) using bacterial phospholipase D. Indeed, when cell supernatants of RH7777 cells, which release high levels of LPC, were incubated with lysoPLD, LPA was readily detectable (unpublished data). ATX/lysoPLD was also present in the supernatant of a number of cancer cell lines. Thus, there is the potential for an autocrine loop where both ATX/lysoPLD and LPC are produced by the same cells, leading to LPA production. Alternatively, paracrine loops may occur where cells produce one (but not both) of ATX/lysoPLD or LPC.
ATX/lysoPLD (also called NPP-2) shares significant homology (45% in amino acid level) with members of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, which includes PC-1/NPP-1 and gp130RB136/NPP-3. PC-1/NPP-1 and gp130RB136/NPP-3 have been implicated in multiple processes, including bone mineralization (Okawa et al., 1998; Nakamura et al., 1999) and signaling by insulin and by nucleotides (Bollen et al., 2000). As with ATX/lysoPLD, the biological effects of other NPP family members require a functional catalytic site. However, the physiological substrates for these two enzymes still remain to be identified. Indeed, it is possible that similar to ATX/lysoPLD, their substrates may be lipidlike molecules and potentially lysophospholipids.
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Materials and methods |
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Purification of lysoPLD
510% polyethleneglycol precipitate of FBS was loaded onto blue Sepharose 6 Fast Flow (Amersham Biosciences), and eluted with a linear gradient of NaCl (02 M). The active fractions were loaded onto Con A Sepharose (Amersham Biosciences) and eluted with 5 mM -methylmannopyranoside at room temperature. The active fractions from the eluate were sequentially loaded onto a BioAssist Q ion exchange column (TOSOH) and a HiTrapTM heparin column (Amersham Biosciences), and were eluted with a linear gradient of NaCl (00.5 M). Active fractions were loaded onto a RESOURCE® PHE hydrophobic column (Amersham Biosciences). Active fractions (flow-through fractions) were loaded onto an Econo-Pac CHT-II hydroxyapatite column (Bio-Rad Laboratories), and eluted with a linear gradient of Na2HPO4 (00.15 M). All column chromatography was performed at a neutral pH (7.5). The latter four-column chromatography steps were performed using ÄKTATM (Amersham Biosciences). Amino acid sequence analysis of purified lysoPLD was performed as described previously (Takeda et al., 2001).
LysoPLD assay
150-µl samples were incubated with 1 mM LPC (from egg) in the presence of 100 mM Tris-HCl, pH 9.0, 500 mM NaCl, 5 mM MgCl2, and 0.05% Triton X-100 for 1 h at 37°C. The liberated choline was detected by an enzymatic photometric method using choline oxidase (Asahi Chemical), horseradish peroxidase (Toyobo), and TOOS reagent (N-ethyl-N-(2-hydoroxy-3-sulfoproryl)-3-methylaniline; Dojindo Molecular Technologies, Inc.) as a hydrogen donor (Imamura and Horiuti, 1978; Tamaoku et al., 1982).
Plasmids and recombinant enzyme
Rat cDNA for ATX (corresponding to human autotaxin-T) was amplified by RT-PCR using a rat liver cDNA library as template DNA based on the sequence information in the database (Rattus norvegicus ectonucleotide pyrophosphatase/phosphodiesterase 2; GenBank/EMBL/DDBS accession no. NM_057104). A myc-tag was added at the COOH terminus. Transient transfection into CHO-K1 cells was performed using LipofectAMINETM (Invitrogen). The rat cDNA for ATX was also introduced into the baculovirus transfer vector pFASTBac-1 (Invitrogen), and recombinant baculovirus was prepared according to the manufacturer's protocol. Purification of recombinant ATX/lysoPLD protein was performed as described above from 1 liter of culture supernatant of Sf9 insect cells infected with ATX/lysoPLD recombinant baculovirus.
Chemotaxis assay
A-2058 cells were maintained in RPMI 1640 with 5% heat-inactivated FBS at 37°C and 5% CO2. Polycarbonate filters with 8-µm pores (Neuro Probe, Inc.) were coated with 13.3 mg/ml fibronectin (Sigma-Aldrich) in PBS for 60 min. A dry coated filter was placed on a 96-blind well chamber (Neuro Probe, Inc.) containing the indicated amounts of LPA (18:1; Avanti Polar Lipids, Inc.) or ATX/lysoPLD recombinant protein both in the presence or absence of 1 or 10 µM LPC (1-oleoyl; Avanti Polar Lipids, Inc.), and cells (200 µl, 8 x 104 per well) were added to the top wells. The ligand solution and cell suspension were prepared in the same buffer (serum-free RPMI 1640 medium containing 0.1% BSA). After incubation at 37°C in 5% CO2 for 4 h, the filter was disassembled. The cells on the filter were fixed with methanol and stained with a Diff-Quick staining kit (International Reagents Corp.). The top side of the filter was scraped free of cells. The number of cells that migrated to the bottom side was determined by measuring optical densities at 595 nm using a 96-well microplate reader (model 3550; Bio-Rad Laboratories). When LPC or LPA was added to the cells, it was suspended in serum-free media containing 0.1% BSA.
Proliferation assay
Human EDG2 cDNA (in pcDNA3 expression vector) was cloned as described previously (Bandoh et al., 1999). Rat hepatoma RH7777 cells stably expressing human LPA1/EDG2 were established as described previously (Fukushima et al., 1998). Cancer cell lines were obtained from American Type Culture Collection. MDA-MB-231 cells were maintained in RPMI 1640 with 5% heat-inactivated FBS at 37°C and 5% CO2. CHO-K1 and RH7777 cells were maintained in Ham's F12 and DME, respectively, with 10% heat-inactivated FBS at 37°C and 5% CO2. Cells were seeded in 96-well plates and cultured for 24 h. Cells were starved for 48 h by replacing the media with serum-free media (DME for RH7777 cells, Ham's F12 for CHO-K1 cells, and RPMI 1640 for other cancer cells) containing 0.1% BSA, followed by addition of the indicated amount of LPA or recombinant ATX/lysoPLD in the presence or absence of LPC (10 µM). The cells were further cultured for 48 h. Cell proliferation was evaluated by MTT hydrolysis using Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.). When LPC or LPA was added to the cells, it was suspended in serum-free media containing 0.1% BSA.
Lipid analysis
Phospholipids in reaction mixture, cells, or culture media were extracted by the method of Bligh and Dyer (1959) under acidic conditions by adjusting the pH to 3.0 with 1 N HCl to recover LPA efficiently. Lipids in the aqueous phase were reextracted and pooled with the previous organic phase. The extracted lipids were dried, dissolved in chloroform/methanol (1:1), and used for thin TLC analysis using chloroform/methanol/formic acid/H2O (60:30:7:3, vol/vol) for 1-D TLC, and chloroform/methanol/formic acid/H2O (60:30:7:3, vol/vol) and chloroform/methanol/28% ammonia/H2O (50:40:8:2, vol/vol) for 2-D TLC as organic solvents. Phospholipids were detected using iodine vapor. For detection of LPC in cultured cells, cells were cultured for 12 h in the presence of [32P]orthophosphate (50 µCi/ml) and serum-free media containing 0.1% BSA in 12-well plates before the lipid extraction. The radioactivities were detected using an image analyzer (model BAS 2000; Fuji Film). The recovery of lipids was monitored by the addition of trace amounts of 1-[3H]oleoylLPC or 1-[3H]oleoylLPA to the samples. Under the described conditions, recoveries of 1-[3H]oleoylLPC and 1-[3H]oleoylLPA were always >95%. Concentration of LPC was determined by an enzyme-linked fluorometric method. After the lipids in the samples were extracted and concentrated, LPC concentration was determined by fluorometry of H2O2 using 3-(4-hydroxyphenyl) propionic acid (7.5 mM) as a peroxidase donor (Tamaoku et al., 1982). H2O2 was generated by reaction of LPA with 500 U/ml monoglyceride lipase (Asahi Chemical Industry Co. Ltd.), 10 U/ml phosphocholine phosphodiesterase (GPCP; Asahi Chemical Industry Co. Ltd.), and 500 U/ml glycero-3-phosphate oxidase (Asahi Chemical Industry Co. Ltd.) in a buffer containing 50 mM Hepes, 2 mM CaCl2, 0.2% Triton X-100, and 100 U/ml peroxidase (Toyobo), pH 7.5, in a total volume of 1,500 µl. Fluorescence intensity at excitation at 320 nm/emission at 404 nm was measured by a fluorometer (Hitachi) 5 min after mixing the samples. LPC concentrations as low as 0.1 nmol could be readily detected. The assay was linear up to 10 nmol.
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Footnotes |
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K. Inoue's present address is Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui, Kanagawa 199-0195, Japan.
* Abbreviations used in this paper: ATX, autotaxin; EDG, endothelial differentiation gene; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; lysoPLD, lysophospholipase D; pNP-TMP, p-nitrophenyl-tri-monophosphate.
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Acknowledgments |
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This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology, and by the Human Frontier Special Program.
Submitted: 5 April 2002
Revised: 31 May 2002
Accepted: 31 May 2002
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