Inhibition of Autotaxin by Lysophosphatidic Acid and Sphingosine 1-Phosphate

Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and sphingosine 1-phosphate (S1P) in a mixed-type manner (Ki ∼ 10–7 m). The homologous ecto-phosphodiesterase NPP1, which lacks lysophospholipase D activity, is insensitive to LPA and S1P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S1P as an inhibitor of ATX, in addition to its well established role as a receptor ligand. Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and sphingosine 1-phosphate (S1P) in a mixed-type manner (Ki ∼ 10–7 m). The homologous ecto-phosphodiesterase NPP1, which lacks lysophospholipase D activity, is insensitive to LPA and S1P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S1P as an inhibitor of ATX, in addition to its well established role as a receptor ligand. Autotaxin (ATX) 1The abbreviations used are: ATX, autotaxin; BSA, bovine serum albumin; FRET, fluorescence resonance energy transfer; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; NPP, ectonucleotide pyrophosphatase/phosphodiesterase; PLD, phospholipase D; lyso-PLD, lysophospholipase D; SPC, sphingosylphosphorylcholine; S1P, sphingosine 1-phosphate; pNP, para-nitrophenolate; pNP-TMP, para-nitrophenyl thymidine-5′-monophosphate; bis-pNPP, bis(para-nitrotrophenyl) phosphate; GFP, green fluorescent protein; HA, hemagglutinin.1The abbreviations used are: ATX, autotaxin; BSA, bovine serum albumin; FRET, fluorescence resonance energy transfer; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; NPP, ectonucleotide pyrophosphatase/phosphodiesterase; PLD, phospholipase D; lyso-PLD, lysophospholipase D; SPC, sphingosylphosphorylcholine; S1P, sphingosine 1-phosphate; pNP, para-nitrophenolate; pNP-TMP, para-nitrophenyl thymidine-5′-monophosphate; bis-pNPP, bis(para-nitrotrophenyl) phosphate; GFP, green fluorescent protein; HA, hemagglutinin. is a member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family of ecto-enzymes that hydrolyze phosphodiester bonds in various nucleotides and nucleotide derivatives (1Bollen M. Gijsbers R. Ceulemans H. Stalmans W. Stefan C. Crit. Rev. Biochem. Mol. Biol. 2000; 35: 393-432Crossref PubMed Scopus (252) Google Scholar, 2Clair T. Lee H.Y. Liotta L.A. Stracke M.L. J. Biol. Chem. 1997; 272: 996-1001Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 3Goding J.W. Grobben B. Slegers H. Biochim. Biophys. Acta. 2003; 1638: 1-19Crossref PubMed Scopus (288) Google Scholar). ATX, also termed NPP2, was originally isolated as an autocrine motility factor for melanoma cells (4Stracke 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, 5Murata J. Lee H.Y. Clair T. Krutzsch H.C. Arestad A.A. Sobel M.E. Liotta L.A. Stracke M.L. J. Biol. Chem. 1994; 269: 30479-30484Abstract Full Text PDF PubMed Google Scholar) and later found to enhance the invasive and metastatic potential of Ras-transformed NIH3T3 cells in nude mice and to induce an angiogenic response in Matrigel plug assays (6Nam S.W. Clair T. Campo C.K. Lee H.Y. Liotta L.A. Stracke M.L. Oncogene. 2000; 19: 241-247Crossref PubMed Scopus (159) Google Scholar, 7Nam S.W. Clair T. Kim Y.S. McMarlin A. Schiffmann E. Liotta L.A. Stracke M.L. Cancer Res. 2001; 61: 6938-6944PubMed Google Scholar). ATX mRNA is overexpressed in various human cancers, adding support to a link between ATX and tumor progression (8Mills G.B. Moolenaar W.H. Nat. Rev. Cancer. 2003; 3: 582-591Crossref PubMed Scopus (923) Google Scholar). Expression analysis has further suggested a normal physiological role for ATX in neurogenesis, oligodendrocyte differentiation, and myelination (9Fuss B. Baba H. Phan T. Tuohy V.K. Macklin W.B. J. Neurosci. 1997; 17: 9095-9103Crossref PubMed Google Scholar, 10Bachner D. Ahrens M. Betat N. Schroder D. Gross G. Mech. Dev. 1999; 84: 121-125Crossref PubMed Scopus (69) Google Scholar). The mode of action of ATX/NPP2 has long been elusive because the biological effects of ATX could not be explained by nucleotide hydrolysis. The surprise came when it was discovered that ATX is identical to plasma lysophospholipase D (lyso-PLD) and acts by hydrolyzing lysophospatidylcholine (LPC) into lysophosphatidic acid (LPA) (11Umezu-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 (774) Google Scholar, 12Tokumura 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 (601) Google Scholar), a lipid mediator that signals cell proliferation, migration, and survival via specific G protein-coupled receptors (13Moolenaar W.H. van Meeteren L.A. Giepmans B.N. BioEssays. 2004; 26: 870-881Crossref PubMed Scopus (485) Google Scholar). It has now become clear that de novo production of LPA can fully account for the biological effects of ATX observed in cell culture. The lysophospholipid substrate range of ATX has recently been broadened by showing that the enzyme can also hydrolyze sphingosylphosphorylcholine (SPC) to yield sphingosine 1-phosphate (S1P) (14Clair 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), a bioactive lipid with signaling properties very similar to those of LPA while acting on distinct receptors (15Postma F.R. Jalink K. Hengeveld T. Moolenaar W.H. EMBO J. 1996; 15: 2388-2392Crossref PubMed Scopus (265) Google Scholar, 16Hla T. Lee M.J. Ancellin N. Paik J.H. Kluk M.J. Science. 2001; 294: 1875-1878Crossref PubMed Scopus (471) Google Scholar, 17Ishii I. Fukushima N. Ye X. Chun J. Annu. Rev. Biochem. 2004; 73: 321-354Crossref PubMed Scopus (632) Google Scholar). The physiological significance of the SPC-to-S1P conversion is debatable, however, because the reported Km of ATX for SPC (14Clair 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) is 3 orders of magnitude higher than the normal SPC levels in plasma and serum (18Liliom K. Sun G. Bunemann M. Virag T. Nusser N. Baker D.L. Wang D.A. Fabian M.J. Brandts B. Bender K. Eickel A. Malik K.U. Miller D.D. Desiderio D.M. Tigyi G. Pott L. Biochem. J. 2001; 355: 189-197Crossref PubMed Scopus (144) Google Scholar). Rather than through SPC hydrolysis, S1P is thought to originate largely from the phosphorylation of sphingosine by sphingosine kinases (19Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol. 2003; 4: 397-407Crossref PubMed Scopus (1724) Google Scholar). Mutational analysis has revealed that the lyso-PLD and nucleotide phosphodiesterase activities of ATX originate from the same catalytic site (20Koh E. Clair T. Woodhouse E.C. Schiffmann E. Liotta L. Stracke M. Cancer Res. 2003; 63: 2042-2045PubMed Google Scholar, 21Gijsbers R. Aoki J. Arai H. Bollen M. FEBS Lett. 2003; 538: 60-64Crossref PubMed Scopus (98) Google Scholar). Unexpectedly, the other two members of the NPP family (NPP1 and NPP3) lack intrinsic lyso-PLD activity despite their close homology to ATX (21Gijsbers R. Aoki J. Arai H. Bollen M. FEBS Lett. 2003; 538: 60-64Crossref PubMed Scopus (98) Google Scholar). Given the differences in substrate specificity, it is not surprising that the NPPs appear to have largely unrelated physiological functions. The founding member, NPP1, hydrolyzes ATP into pyrophosphate, an inhibitor of calcification, and thereby regulates bone mineralization, whereas the third member, NPP3, promotes differentiation and invasion of glial cells by an unknown mechanism (3Goding J.W. Grobben B. Slegers H. Biochim. Biophys. Acta. 2003; 1638: 1-19Crossref PubMed Scopus (288) Google Scholar). An unresolved question concerns the regulation of ATX activity. One puzzling observation is that LPA levels in plasma or freshly isolated blood are very low (22Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (572) Google Scholar, 23Baker D.L. Morrison P. Miller B. Riely C.A. Tolley B. Westermann A.M. Bonfrer J.M. Bais E. Moolenaar W.H. Tigyi G. J. Am. Med. Assoc. 2002; 287: 3081-3082Crossref PubMed Scopus (148) Google Scholar, 24Sano T. Baker D. Virag T. Wada A. Yatomi Y. Kobayashi T. Igarashi Y. Tigyi G. J. Biol. Chem. 2002; 277: 21197-21206Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), yet plasma ATX is constitutively active and its substrate LPC abundantly present (> 100 μm) (25Croset M. Brossard N. Polette A. Lagarde M. Biochem. J. 2000; 345: 61-67Crossref PubMed Scopus (211) Google Scholar). This suggests that ATX is negatively regulated in vivo, but physiological or pharmacological inhibitors of ATX have not been identified to date. In the present study we sought to examine how ATX activity is regulated in the extracellular milieu. To this end, we used a newly invented fluorescence resonance energy transfer (FRET)-based phosphodiesterase sensor (termed CPF4; see Ref. 26Takakusa H. Kikuchi K. Urano Y. Sakamoto S. Yamaguchi K. Nagano T. J. Am. Chem. Soc. 2002; 124: 1653-1657Crossref PubMed Scopus (166) Google Scholar) that, as we show here, reports ATX activity in conditioned media with superior sensitivity. Using this assay system, we demonstrate that ATX, secreted by the classical export route, is potently and specifically inhibited by LPA and S1P at biologically relevant concentrations. These results have important implications for lysophospholipid action and signaling in general and ATX targeting in particular. Cell Culture and Materials—HEK293T cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. All phospholipids were obtained from Avanti Polar Lipids Inc. (Alabaster, AL). Brefeldin A, monensin, BSA, fatty acid-free BSA (99%), para-nitrophenyl thymidine-5′-monophosphate (pNP-TMP), and bis(para-nitrotrophenyl) phosphate (bis-pNPP) were from Sigma. Highly purified PLD from Streptomyces chromofuscus, sphingomyelinase D from Loxosceles laeta, and sphingomyelinase D from Corynebacterium pseudotuberculosis were kindly provided by M. Roberts (Boston College), D. Tambourgi (Instituto Butantan, Sao Paulo, Brazil), and S. Billington (University of Arizona), respectively. cDNA Cloning—RNA extracted from human diploid foreskin fibroblasts was used to generate cDNA using Invitrogen reverse transcriptase. ATX cDNA was isolated using ATX-specific primers. The stop codon was removed and KpnI and NotI restriction sites were introduced at the 5′ and 3′ sites, respectively. After digestion, ATX was ligated in pcDNA3 vector with 3′ Myc tag (ATX-Myc), 5′ HA tag or 5′ HA tag and 3′ Myc tag and in that the ATX were identical to human ATX The H.Y. Clair T. Woodhouse E.C. S. Liotta L.A. Stracke M.L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) was using the and cells were with ATX using the phosphate cells were to Dulbecco's modified Eagle's medium for medium was for to cell was used further and for the of ATX by and activity was very in ATX activity between of conditioned were using and with and and were using the was used for ATX ATX cDNA to the at the 5′ was into the vector The was used for generating to which were grown in medium the of was After of medium containing secreted ATX was by low and a The was a and the were with a phosphate The were an and were with a of of was as by and The yield was 3 of from of secreted of human was by of its extracellular to the site of the from and into a vector by the After the was into and a was isolated by single cell The enzyme was purified from by of and on a was by and lyso-PLD lyso-PLD activity, LPC and LPC μm) were and the was in and and BSA was The was by the addition of conditioned Lipids were extracted with All of the were in acid and extracted with of as (22Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (572) Google Scholar). In and the was and the was extracted were with of and were by on in Lipids were by activity and was by of medium was to of and containing or in After for 3 at of was by the at in a was as H. Kikuchi K. Urano Y. Sakamoto S. Yamaguchi K. Nagano T. J. Am. Chem. Soc. 2002; 124: 1653-1657Crossref PubMed Scopus (166) Google Scholar) and as a in ATX in or conditioned Dulbecco's modified Eagle's medium with was with or the and was at a of 2 fluorescence was in a at at and was using with or Meeteren L.A. Giepmans B.N. Billington Tambourgi Moolenaar W.H. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) were with at ATX and were by using a ATX in is as a of of a very a single and a catalytic ATX to yield a enzyme M.L. Clair T. Liotta L.A. 1997; PubMed Scopus Google Scholar), yet is ATX biosynthesis and In it the of ATX is on the cell a for the present we ATX in cells using and and ATX in cell as well as in the in ATX is in cell whereas ATX in the medium lacks the HA with secreted ATX by of the The and ATX of the classical export by ATX is in but not at the plasma in receptors primarily to the cell the same experimental results we that ATX the classical and that of its the than at the plasma ATX and established that ATX is not as a plasma we to examine the catalytic activity of To this end, we used conditioned medium from cells further in ATX purified from cell The as a H.Y. Clair T. Woodhouse E.C. S. Liotta L.A. Stracke M.L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The lyso-PLD activity of ATX was by the conversion of to LPA using LPC hydrolysis by ATX at a for at The Km for LPC was at in with reported μm) (11Umezu-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 (774) Google Scholar, 12Tokumura 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 (601) Google Scholar, 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) and in the range of normal LPC levels in plasma (25Croset M. Brossard N. Polette A. Lagarde M. Biochem. J. 2000; 345: 61-67Crossref PubMed Scopus (211) Google Scholar). as potential of lyso-PLD activity, we observed that LPC hydrolysis was inhibited by LPA and S1P to phospholipids not show an 2 and results not This suggests that ATX is to by analysis of lyso-PLD is however, by with the LPC hydrolysis which of LPA in of its a of LPA the of the assay the of ATX by LPA to be than the To the of lipid and the of LPC hydrolysis we of the that ATX uses a mechanism for the hydrolysis of lipid and non-lipid (20Koh E. Clair T. Woodhouse E.C. Schiffmann E. Liotta L. Stracke M. Cancer Res. 2003; 63: 2042-2045PubMed Google Scholar, 21Gijsbers R. Aoki J. Arai H. Bollen M. FEBS Lett. 2003; 538: 60-64Crossref PubMed Scopus (98) Google Scholar). non-lipid we pNP-TMP, a NPP and the phosphodiester bis-pNPP, a substrate for and PubMed Scopus Google Scholar, Y. J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. Roberts J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The pNP, was in hydrolysis of with a Km of than the reported by 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 (601) Google Scholar). ATX was also of hydrolyzing in with a (21Gijsbers R. Aoki J. Arai H. Bollen M. FEBS Lett. 2003; 538: 60-64Crossref PubMed Scopus (98) Google Scholar). hydrolysis were at but not because of substrate at higher results not to is observed with LPC as a the hydrolysis of and by ATX was inhibited by LPA and S1P μm) and It that, LPA and S1P their of the of the of as a we observed that ATX activity was inhibited by normal serum at of of the in fetal calf In BSA when at high The observation that is the for LPA and S1P suggests that ATX by BSA is largely to LPA was to a we found that its to ATX was not can be from the for the to ATX was close to using a substrate of to the a of ATX the assay of we a newly phosphodiesterase sensor termed H. Kikuchi K. Urano Y. Sakamoto S. Yamaguchi K. Nagano T. J. Am. Chem. Soc. 2002; 124: 1653-1657Crossref PubMed Scopus (166) Google Scholar). is a in which both are to and in with high of the phosphodiester by a phosphodiesterase from of FRET, a of enzyme activity H. Kikuchi K. Urano Y. Sakamoto S. Yamaguchi K. Nagano T. J. Am. Chem. Soc. 2002; 124: 1653-1657Crossref PubMed Scopus (166) Google Scholar). the fluorescence is insensitive to in the physiological range H. Kikuchi K. Urano Y. Sakamoto S. Yamaguchi K. Nagano T. J. Am. Chem. Soc. 2002; 124: 1653-1657Crossref PubMed Scopus (166) Google Scholar). of is their high sensitivity, of very low of as a substrate for in a of fluorescence and a in of substrate hydrolysis, which can be in was with the of the hydrolysis revealed with an Km as low as μm) for in medium the same Km was found with purified ATX from cell and that conditioned medium not of inhibitors of ATX the It is that the of ATX for is orders of magnitude higher than that for and nucleotides is with an Km of P. Clair T. J.W. K. J. H. J. Biochem. 2003; PubMed Scopus Google with bis-pNPP, a hydrolysis are observed at as high as of the to bis-pNPP, which and into a substrate for ATX, that the into a in substrate the catalytic of hydrolysis was than that observed with the other the assay superior at a of ATX activity was The of the assay was 3 of that of the assay by at This the for ATX activity in conditioned media and it to be to the of the of in ATX activity in biological also is a substrate for secreted the PLD from S. and the and from C. pseudotuberculosis and Loxosceles Meeteren L.A. Giepmans B.N. Billington Tambourgi Moolenaar W.H. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the used for ATX, of was to not It that the secreted ATX can hydrolyze of ATX by LPA and of its superior and the assay was used in further analysis of ATX the of LPA and S1P for ATX activity using a substrate close to the those the for is very similar to the found with or LPC as substrate and results not S1P inhibited ATX activity with the same as observed for LPA sphingosine and fatty not ATX activity. has been reported to the NPP (1Bollen M. Gijsbers R. Ceulemans H. Stalmans W. Stefan C. Crit. Rev. Biochem. Mol. Biol. 2000; 35: 393-432Crossref PubMed Scopus (252) Google Scholar, 2Clair T. Lee H.Y. Liotta L.A. Stracke M.L. J. Biol. Chem. 1997; 272: 996-1001Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), we not of or ATP on ATX activity not the effects of LPA and S1P were specific for ATX in that the activity of the ecto-phosphodiesterase was insensitive to lipid of ATX by LPA was on the of the was by and whereas The of a long suggests that LPA and S1P with a on The effects of LPA and S1P were that both on the same that, in to the G protein-coupled receptors I. Fukushima N. Ye X. Chun J. Annu. Rev. Biochem. 2004; 73: 321-354Crossref PubMed Scopus (632) Google Scholar), ATX not between LPA and S1P as the mechanism of by LPA and S1P. revealed that LPA and S1P are mixed-type a in and an in Km In other of ATX by has both a and a from a of a and of the active site for its analysis an of for LPA and for S1P, well the biologically active range of LPA and S1P. that are than the reported Km for the LPC substrate μm) (11Umezu-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 (774) Google Scholar, 12Tokumura 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 (601) Google Scholar, 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), that ATX LPA and S1P than it its physiological have a novel and phosphodiesterase sensor to show that the catalytic activity of ATX, secreted by the classical export is potently and specifically inhibited by LPA and S1P. analysis revealed that LPA and S1P as mixed-type inhibitors with an of The of ATX by LPA and S1P is specific in that and other have and the ecto-phosphodiesterase NPP1, which lacks lyso-PLD activity, is insensitive to LPA and S1P. ATX primarily functions as an lyso-PLD, that LPA is of its own biosynthesis in the of ATX of bioactive LPA in the extracellular and plasma levels of LPA are very low despite the of its serum LPA levels T. Baker D. Virag T. Wada A. Yatomi Y. Kobayashi T. Igarashi Y. Tigyi G. J. Biol. Chem. 2002; 277: 21197-21206Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) suggests that plasma ATX also be regulated by as yet unknown an important that to be further Our to a novel role for S1P as an inhibitor of ATX, in addition to its well role as a receptor The important is that in extracellular S1P levels LPA production and the S1P in blood are T. Lee M.J. Ancellin N. Paik J.H. Kluk M.J. Science. 2001; 294: 1875-1878Crossref PubMed Scopus (471) Google Scholar, L. Yatomi Y. Y. K. Y. J. 1999; PubMed Scopus Google Scholar), close to the for ATX reported here ATX activity in plasma could be by S1P Our results further that ATX has a site for LPA and S1P, not present in a study has that, in addition to its catalytic ATX an that can be to ATP T. Krutzsch H.C. Liotta L.A. Stracke M.L. Biochem. Biophys. Res. 1997; PubMed Scopus Google Scholar). in with reveal this is in an site for LPA and S1P. and of the is a for and the of ATX inhibitors that could be of in Giepmans for with cDNA and and Billington for purified with