Key points are not available for this paper at this time.
Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATX-null embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways. Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATX-null embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways. Autotaxin (ATX) 2The abbreviations used are: ATX, autotaxin; LPA, lysophosphatidic acid; S1P, sphingosine 1-phosphate; PECAM, platelet-endothelial cell adhesion molecule; E, embryonic day; lysoPLD, lysophospholipase D; RT-PCR, reverse transcription PCR. is a motogen-like phosphodiesterase originally isolated from conditioned medium of human melanoma cells (1Stracke M.L. Krutzsch H.C. Unsworth E.J. Arestad A. Cioce V. Schiffmann E. Liotta L.A. J. Biol. Chem. 1992; 267: 2524-2529Abstract Full Text PDF PubMed Google Scholar). Enforced expression of ATX in Ras-transformed NIH3T3 cells greatly enhances their invasive, tumorigenic, and metastatic potentials (2Nam S.W. Clair T. Campo C.K. Lee H.Y. Liotta L.A. Stracke M.L. Oncogene. 2000; 19: 241-247Crossref PubMed Scopus (161) Google Scholar). In addition, enhanced expression of ATX has been demonstrated in various malignant tumor tissues (3Mills G.B. Moolenaar W.H. Nat. Rev. Cancer. 2003; 3: 582-591Crossref PubMed Scopus (942) Google Scholar). Thus, ATX is implicated in tumorigenic and metastatic potentials of cancer cells. ATX is also expressed in various tissues and is present at high concentration in various biological fluids including plasma, serum, and seminal plasma (4Aoki J. Semin. Cell Dev. Biol. 2004; 15: 477-489Crossref PubMed Scopus (248) Google Scholar), implying specific roles of ATX in circulation. Recently, ATX was shown to have lysophospholipase D (lysoPLD) activity, which converts lysophosphatidylcholine to a bioactive lysophospholipid, lysophosphatidic acid (LPA) (5Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Crossref PubMed Scopus (797) Google Scholar, 6Tokumura A. Majima E. Kariya Y. Tominaga K. Kogure K. Yasuda K. Fukuzawa K. J. Biol. Chem. 2002; 277: 39436-39442Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar). ATX also converts sphingosylphosphorylcholine into another bioactive lysophospholipid, sphingosine 1-phosphate (S1P) in vitro (7Clair T. Aoki J. Koh E. Bandle R.W. Nam S.W. Ptaszynska M.M. Mills G.B. Schiffmann E. Liotta L.A. Stracke M.L. Cancer Res. 2003; 63: 5446-5453PubMed Google Scholar). Because LPA and S1P are regulators of cell motility and proliferation in various cell systems, they might be the effectors of the motogenic actions of ATX. LPA and S1P have been shown to have diverse roles in many biological processes that are mediated by G protein-coupled receptors (GPCRs) specific to LPA or S1P; there are five GPCRs for LPA (LPA1-5) and five for S1P (S1P1-5) with a number of putative GPCRs (8Ishii I. Fukushima N. Ye X. Chun J. Annu. Rev. Biochem. 2004; 73: 321-354Crossref PubMed Scopus (649) Google Scholar). Thus, ATX may exert its functions through these receptors. Indeed, ATX stimulates cell motility of tumor cells through one of the LPA receptors, LPA1 (9Hama K. Aoki J. Fukaya M. Kishi Y. Sakai T. Suzuki R. Ohta H. Yamori T. Watanabe M. Chun J. Arai H. J. Biol. Chem. 2004; 279: 17634-17639Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), and ATX positively or negatively modulates cell motility depending on S1P receptor subtypes (7Clair T. Aoki J. Koh E. Bandle R.W. Nam S.W. Ptaszynska M.M. Mills G.B. Schiffmann E. Liotta L.A. Stracke M.L. Cancer Res. 2003; 63: 5446-5453PubMed Google Scholar, 10Takuwa Y. Biochim. Biophys. Acta. 2002; 1582: 112-120Crossref PubMed Scopus (153) Google Scholar). To uncover the physiological role of ATX and to identify the endogenous product of ATX, we investigated ATX knock-out mice. In this study we show that ATX produces LPA, but not S1P, in circulating blood and that it contributes to blood vessel stability through novel LPA signaling pathways. ATX Knock-out Mice—ATX knock-out mice (atx-/-) in the genetic background 129/SvEvBrd were produced by and obtained from Lexicon Genetics (The Woodlands, TX). The ATX gene was targeted with an ATX gene-targeting vector, which was designed to replace the initiation codon and first 45 amino acids encoded by exons 1 and 2 with a lacZ-neo cassette in the vector pKOS (Fig. 1A). The ATX knock-out mice were backcrossed with C57BL/6j mice at least six times before being used. Mice were genotyped by both Southern blotting and PCR of genomic DNA. The PCR primers for detection of wild-type alleles are 5′-ccgaatctctccgatcactac-3′ and 5′-tccaacattaaagcgataacc-3′, and those for detection of mutant alleles are 5′-gcagcgcatcgccttctatc-3′ and 5′-tccaacattaaagcgataacc-3′. Flk-1-laxZ knock-in mice (11Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3361) Google Scholar) were kindly donated by Dr. Janet Rossant (The Hospital for Sick Children, Toronto, Canada). All mice used in this study were bred and maintained at the Animal Care Facility in the Graduate School of Pharmaceutical Sciences, the University of Tokyo, under specific pathogen-free conditions in accordance with institutional guidelines. Recombinant ATX—Two recombinant mouse ATX proteins (wild-type ATX and catalytically inactive T209A mutant ATX) with a His tag at the N terminus were expressed and purified using a baculovirus system and nickel column chromatography (HisTrap HP; GE Healthcare), respectively. Preparation of ATX-depleted Serum—To establish anti-mouse ATX monoclonal antibody, the recombinant mouse ATX protein (50 μg) was used to immunize rats (WKY/Izm strain) with Freund's complete adjuvant into the hind footpads. Cells from the enlarged medial iliac and inguinal lymph nodes were fused with mouse myeloma (PAI) cells. The antibody-secreting hybridoma cells were selected by screening with an enzyme-linked immunosorbent assay and immunoprecipitation. One clone (5E5 (rat IgG1)) was found to have activity to immunoprecipitate ATX in mouse serum. 5E5 reacted with mouse, human, and bovine ATX but does not react with rat ATX. The monoclonal antibody was purified from culture supernatant of hybridoma cells and was coupled to Sepharose 4B beads (2 mg/ml) (GE Healthcare). To deplete ATX from mouse serum, mouse serum (1 ml) was incubated with the 5E5-Sepahrose 4B (40 μl)for 2 h at 4 °C,andthe resulting supernatant was used for measuring lysoPLD activity and LPA production. ATX bound to the 5E5-Sepahrose 4B was eluted with 100 mm glycine, pH 2.5. This gave rise to a single 100-kDa band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, showing that 5E5 is specific to ATX. Immunohistochemistry, Whole-mount Immunostaining, and Whole-mount LacZ Staining—Tissue sections (5 μm) were dehydrated, embedded in paraffin, incubated in 3% (v/v) H2O2 for 20 min, incubated overnight at 4 °C with first antibodies (EPOS anti-a-SMA/HRP; DAKO), stained with diaminobenzidine according to the manufacturer's protocol, and counter-stained with hematoxylin. For whole-mount embryo immuno-staining, embryos were fixed in 4% paraformaldehyde, dehydrated, incubated with 5% hydrogen peroxide in methanol, rehydrated through a methanol series to phosphate-buffered saline, incubated in phosphate-buffered saline containing 4% bovine serum albumin and 0.1% Triton X-100, incubated with rat anti-PECAM monoclonal antibody (BD Biosciences), incubated with peroxidase-conjugated goat anti-rat IgG (American Qualex), and stained with diaminobenzidine as a peroxidase substrate. LacZ staining was performed as described (11Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3361) Google Scholar). Allantois Culture—Allantoides were dissected from embryos at E8.5 and cultured in Dulbecco's modified Eagle's medium containing glutamine and antibiotics (18Argraves W.S. Larue A.C. Fleming P.A. Drake C.J. Dev. Dyn. 2002; 225: 298-304Crossref PubMed Scopus (33) Google Scholar). To perform the vessel formation, the allantois explants were cultured for 24 h (37 °C; 5% CO2) in the presence or absence of the factors tested. In some cases, to examine the effect of factors on stabilization of preformed vessels the allantoides were first cultured for 24 h in the presence of amniotic fluids (isolated from E12.5 embryos) to allow formation of stable vascular networks and then cultured for an additional 18 h in the presence or absence of the factors tested. Cells were fixed with 4% paraformaldehyde and then immunostained with anti-PECAM monoclonal antibody. Determination of LPA and S1P Concentration, lysoPLD Activity, and Western Blotting—LPA and S1P concentrations in plasma and serum were determined as described previously (12Kishimoto T. Matsuoka T. Imamura K. Acta. 2003; PubMed Scopus Google Scholar, Y. F. Ohta J. Y. Biochem. 1995; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). lysoPLD activity was determined as described using lysophosphatidylcholine as (5Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Crossref PubMed Scopus (797) Google Scholar). Western blotting of ATX was performed as described using monoclonal antibody M. Kishi Y. Y. Y. Aoki J. Arai H. 2004; PubMed Scopus Google Scholar). In of ATX and LPA1 was in or sections by in using for ATX and LPA1 as previously described T. K. PubMed Google Scholar). were with at 2 in phosphate-buffered saline for and then in 4% saline for 20 min, by was with embryos and at was isolated using and reverse using the system for used to ATX, LPA receptors, and were described previously Y. M. Hama K. J. Yamori T. Aoki J. T. Arai H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was performed as described (9Hama K. Aoki J. Fukaya M. Kishi Y. Sakai T. Suzuki R. Ohta H. Yamori T. Watanabe M. Chun J. Arai H. J. Biol. Chem. 2004; 279: 17634-17639Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar) using detection system was to the number of or in ATX-null the physiological role of ATX, we investigated knock-out mice. The ATX gene was targeted with an ATX gene-targeting vector that was designed to replace the initiation codon and first 45 amino acids encoded by exons 1 and 2 with a lacZ-neo cassette in the vector pKOS (Fig. 1A). The of mutant mice was by Southern blotting and of genomic (Fig. of embryos the knock-out that ATX was not produced from the (Fig. The plasma lysoPLD activity and plasma ATX protein level in mice were about half the found in mice (Fig. and showing that ATX is responsible for of the lysoPLD activity in The plasma LPA concentrations in mice were about half of those in mice (Fig. In addition, in ATX-depleted mouse serum, which is by the mouse serum with Sepharose 4B beads coupled with monoclonal antibody both lysoPLD activity and LPA production at °C were (Fig. and that ATX is a that produces LPA in serum. the S1P in plasma (Fig. and serum from mice were the LPA heterozygous mice appeared and were to those in wild-type is responsible for bulk LPA production in LPA in plasma from and mice determined by a LPA were by about one half in mice. lysoPLD activity of ATX-depleted mouse serum. lysoPLD activity was determined by of from lysophosphatidylcholine using as a substrate. production of LPA in ATX-depleted mouse serum at ATX-depleted serum, and serum. S1P level in plasma from and mice. S1P were not was analyzed using are by in and of heterozygous produced that ATX are embryonic To embryos were embryos were isolated at various of embryos at least to embryonic day embryos were and at of them were in were in the embryo and number of and (Fig. The but were and In addition, were in (atx-/-) The of embryos vessels at and wild-type vessels (Fig. also a in and vessels were in with the and (Fig. and were more in with that were with blood cells (Fig. the vascular embryonic were present (Fig. that blood cells To the in the and we heterozygous mice, in which expression of is by the of the gene and is to cells (11Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3361) Google Scholar). and embryos with To this we first mice with mice and and embryos were by mice. but to (Fig. In at were found to have severe vascular they blood vessels (Fig. with this both and vessels were in at (Fig. at E8.5 vascular in the and but not in the (Fig. with antibodies PECAM, another the vascular of the in embryos vascular was also in the embryo Whole-mount anti-PECAM staining revealed and in embryos (Fig. In the were and in embryos (Fig. the both embryonic and blood vessels by the of their the blood vessels (Fig. and were both in and embryos (Fig. However, it was that allantois the vessel in the allantois (Fig. of the and embryo at with a specific of vascular that cells were present and the cells in both the (Fig. and embryo This that cell is not the of vascular in that blood vessel formation in embryos but the blood vessels to develop into mature vessels in the absence of of of at of of of number of embryos of embryo or in a in vascular in in the from and embryos at and are stained with and E. the blood vessels in and their absence in that are present in whole-mount LacZ staining of and embryos and and both and vessels are in vessels are in whole-mount of and embryos at of are shown at sections of and at were stained with and blood embryonic blood vessels. E, of and In embryos, the allantois a vessel to at allantois is and the vessel in the expression of cells in and at are in expression of ATX in both the and embryo (Fig. In expression in cells that the at E8.5 (Fig. ATX protein expression was in amniotic fluids in embryonic and its level was to the plasma level as by both Western blotting and lysoPLD activity (Fig. and As was in plasma, the lysoPLD activity and ATX protein in amniotic fluids from embryos at and were half the found in embryos (Fig. and Western also that ATX expression was not in or embryo at ATX protein was expressed in amniotic fluids (Fig. D and that of the ATX proteins produced in embryos are and in amniotic ATX and LPA of in the role of ATX and to its activity in vascular we the allantois culture system (18Argraves W.S. Larue A.C. Fleming P.A. Drake C.J. Dev. Dyn. 2002; 225: 298-304Crossref PubMed Scopus (33) Google Scholar). As reported by (18Argraves W.S. Larue A.C. Fleming P.A. Drake C.J. Dev. Dyn. 2002; 225: 298-304Crossref PubMed Scopus (33) Google Scholar), E8.5 allantois explants were cultured in the presence of serum, a of vessels was 24 h of culture (Fig. The amniotic fluids isolated from E12.5 wild-type embryos were found to have a but an more effect on vessel formation than serum (Fig. Allantois explants from embryos also a of vessels they were cultured in the presence of amniotic fluids isolated from E12.5 wild-type embryos (Fig. or serum LPA or recombinant ATX to this system effect on vessel formation (Fig. showing that ATX and LPA are not angiogenic. In the presence of amniotic the vessels that the first 24 h of culture their the 18 h of culture (Fig. However, in the absence of amniotic the preformed vessels were and (Fig. The recombinant ATX, but not catalytically inactive ATX, the disassembly (Fig. LPA also the disassembly of preformed S1P effect (Fig. that ATX in the amniotic fluids has a role in stabilizing vessels. To the of LPA blood we the expression of LPA receptors. and in that the LPA receptors LPA1 is expressed in the and embryo at and (Fig. it is expressed by cells in the than cells (Fig. of and were revealed to be Thus, we the effect of an LPA receptor Ki16425, on the effect of ATX and The of are for for and for H. K. N. A. E. J. T. M. Y. Watanabe T. M. M. Suzuki R. H. Sakai T. T. H. K. H. F. 2003; PubMed Scopus Google Scholar). not show activity H. K. N. A. E. J. T. M. Y. Watanabe T. M. M. Suzuki R. H. Sakai T. T. H. K. H. F. 2003; PubMed Scopus Google Scholar). at in the allantois culture system not the of ATX and LPA (Fig. Thus, it is that and are in the effect of ATX and ATX was originally as a cell for cancer cells (1Stracke M.L. Krutzsch H.C. Unsworth E.J. Arestad A. Cioce V. Schiffmann E. Liotta L.A. J. Biol. Chem. 1992; 267: 2524-2529Abstract Full Text PDF PubMed Google Scholar). ATX was found to have activity to a bioactive LPA (5Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Crossref PubMed Scopus (797) Google Scholar, 6Tokumura A. Majima E. Kariya Y. Tominaga K. Kogure K. Yasuda K. Fukuzawa K. J. Biol. Chem. 2002; 277: 39436-39442Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar, J. Taira A. Y. Kishi Y. Hama K. T. K. K. R. Arai H. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). LPA has been as a with biological including of cell motility (3Mills G.B. Moolenaar W.H. Nat. Rev. Cancer. 2003; 3: 582-591Crossref PubMed Scopus (942) Google Scholar). In this study to the physiological role of ATX we analyzed ATX knock-out mice. To ATX knock-out mice were embryonic showing that ATX is for embryonic vessel formation was in ATX-null embryos, novel roles of ATX and LPA in the formation of blood vessels in the embryonic In addition, the present study that ATX is responsible for bulk LPA production in but not for S1P production. The phenotype of ATX-null embryos was from those of LPA receptors reported so Fukushima N. Chun J. A. 2000; PubMed Scopus Google Scholar, I. Fukushima N. Ye X. Chun J. Biol. 2002; PubMed Scopus Google Scholar, X. Hama K. Inoue A. Suzuki H. T. Arai H. Aoki J. Chun J. Nature. PubMed Scopus Google Scholar). a that ATX and LPA to embryonic blood vessel formation through as LPA receptors. Nam S.W. Clair T. A. Schiffmann E. Liotta L.A. Stracke M.L. Cancer Res. Google Scholar) reported that of with purified ATX into mice in blood vessel formation the and that ATX human cells on to form these they that ATX was an present that ATX is not but it preformed vessels through an Because factors that vessel formation, ATX may to the vessel formation in with The formation of by and is not for embryonic but also for the of J. Nat. 1995; PubMed Scopus Google Scholar). ATX stimulates both cell proliferation and cell motility of cancer cells through LPA production (5Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Crossref PubMed Scopus (797) Google Scholar). In addition, of ATX is with malignant as small cell cancer Y. N. J. Cell Biol. PubMed Scopus Google Scholar), cell cancer M. J. T. J. M. J. Cancer. PubMed Scopus Google Scholar), Google Scholar, X. J. Google Scholar), cancer N. M. V. H. M. Res. 2002; Google Scholar, Lee J. H.C. Lee Lee H.Y. 2002; 19: PubMed Scopus Google Scholar), T. Kishi Y. Arai H. M. Aoki J. PubMed Scopus Google Scholar), cancer A. N. A. T. H. J. J. Cancer. 2004; PubMed Scopus Google Scholar), and Y. M. Hama K. J. Yamori T. Aoki J. T. Arai H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, ATX has been implicated in the of malignant In this study we that ATX has an additional role in blood vessel formation, by stabilizing preformed blood vessels. The present study the that ATX, in to proliferation and motility of tumor cells (5Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Crossref PubMed Scopus (797) Google Scholar), contributes to the of by stabilizing blood vessels in the of ATX is of S1P ATX also a to to S1P (7Clair T. Aoki J. Koh E. Bandle R.W. Nam S.W. Ptaszynska M.M. Mills G.B. Schiffmann E. Liotta L.A. Stracke M.L. Cancer Res. 2003; 63: 5446-5453PubMed Google Scholar). in this study that ATX is a for LPA, but not for S1P, in blood. is in that S1P is produced from sphingosine through mediated by of sphingosine 1 and sphingosine M.L. T. H. A. Y. R. M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Lee Y. R. K. W.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). vascular were also in sphingosine and S1P mutant embryos K. T. A. Biol. PubMed Scopus Google Scholar) and mutant embryos M. Y. Y. T. M.L. Wu T. J. Biol. 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PubMed Scopus Google Scholar), which the expression in and knock-out mice have not been reported so far. is that ATX its role in blood vessel stabilization through these LPA receptors signaling of LPA receptors and H. Moolenaar and for
Tanaka et al. (Sat,) studied this question.