Spider and bacterial sphingomyelinases D target cellular lysophosphatidic acid receptors by hydrolyzing lysophosphatidylcholine

Bites by Loxosceles spiders can produce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, hemolysis, and persistent inflammation. The causative factor is a sphingomyelinase D (SMaseD) that cleaves sphingomyelin into choline and ceramide 1-phosphate. A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor. However, the molecular basis for SMaseD toxicity is not well understood, which hampers effective therapy. Here we show that the spider and bacterial SMases D hydrolyze albumin-bound lysophosphatidylcholine (LPC), but not sphingosylphosphorylcholine, with K(m) values ( approximately 20-40 microm) well below the normal LPC levels in blood. Thus, toxic SMases D have intrinsic lysophospholipase D activity toward LPC. LPC hydrolysis yields the lipid mediator lysophosphatidic acid (LPA), a known inducer of platelet aggregation, endothelial hyperpermeability, and pro-inflammatory responses. Introduction of LPA(1) receptor cDNA into LPA receptor-negative cells renders non-susceptible cells susceptible to SmaseD, but only in LPC-containing media. Degradation of circulating LPC to LPA with consequent activation of LPA receptors may have a previously unappreciated role in the pathophysiology of secreted SMases D.     

Autotaxin的结构与功能

Autotaxin(ATX)是一个分泌型糖蛋白,具有磷酸二酯酶(PDE)活性,是胞外焦磷酸酶/磷酸二酯酶(ENPP)家族的一员.ATX还具有溶血磷脂酶D(lysoPLD)活性,能够以溶血磷脂酰胆碱(lysophosphatidylcholjne,LPC)为底物催化生成溶血磷脂酸(lysophosphatidic acid,LPA).ATX在很多肿瘤细胞中都有高表达,在肿瘤的发生、发展过程中有着重要作用,被认为是肿瘤治疗中一个可能的靶位.此外,ATX在神经系统、免疫系统中也发挥重要作用.目前已经建立了一系列快速检测ATX活性的方法,并在此基础上研发了相关疾病的诊断技术.基于ATX的多功能性,对其表达调控机理的研究和抑制剂的开发成为当前的研究热点.     

自分泌运动因子-溶血磷脂酸轴的生物学功能与调控

自分泌运动因子(autotaxin,ATX)也称作磷酸二酯酶Iα,是核苷酸焦磷酸酶/磷酸二酯酶家族(nucleotide pyrophosphatases,NPPs)中的一员,因而也称作NPP2.ATX是NPPs中唯一具有溶血磷脂酶D(lysophospholipase D,lysoPLD)活性的成员,它可以将溶血磷脂...     

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 approximately 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.     

Quantitation of glycerophosphorylcholine by flow injection analysis using immobilized enzymes

A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically.Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute.The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic- spectrophotometric procedure.An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described.Abbreviations GPC sn-glycerol-3-phosphorylcholine - GPE sn-glycerol-3-phosphorylethanolamine - GPS sn-glycerol-3-phosphorylserine - GPA sn-glycerol-3-phosphoric acid - PDE glycerophosphorylcholine-phosphodiesterase - GPA-Ox glycerophosphate oxidase - Cho-Ox choline oxidase     

Ecto-lysophospholipase C controls lysophospholipid uptake and metabolism in porcine kidney epithelial cell line LLC-PK1

Lysophosphatidylcholine (LPC) has diverse biological activities through different mechanisms including its conversion into other types of lipid mediators such as lysophosphatidic acid and 2-arachidonoylglycerol. Previously, we found that a large portion of the fluorescent analog of alkyl type LPC (Bodipy-lysoPAF) on porcine kidney epithelial cells (LLC-PK1) was degraded to monoalkylglycerol by lysophospholipase C-like activity and then quickly internalized into the cells. In this study, we investigated whether exogenous fluorescently labeled LPC (NBD-LPC) itself was also metabolized and internalized by a similar mechanism. LLC-PK1 cells converted NBD-LPC to either NBD-MG, possibly due to lysophospholipase C-like activity of ecto-nucleotide pyrophosphatase/phosphodiesterase-6, or to free fatty acid (FA), due to lysophospholipase activity in the culture medium at both sites. The resultant NBD-MG was further degraded to NBD-FA by lipase activity before or after its uptake into the cells, and a portion of NBD-FA was finally released into the culture medium on the opposite side.     

Human blood platelet 3': 5'-cyclic nucleotide phosphodiesterase. Isolation of low-Km and high-Km phosphodiesterase.

Human blood platelet contained at least three kinetically distinct forms of 3': 5'-cyclic nucleotide phosphodiesterase (3': 5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) (F I, F II, and F III) which were clearly separated by DEAE-cellulose column chromatography. Although a few properties of the platelet phosphodiesterases such as their substrate affinities and DEAE-cellulose profile resembled somewhat those of the three 3': 5'-cyclic nucleotide phosphodiesterase in rat liver reported by Russell et al. [10], there were pronounced differences in some properties between the platelet and the liver enzymes: (1) the platelet enzymes hydrolyzed both cyclic nucleotides and lacked a highly specific cyclic guanosine 3': 5'-monophosphate (cyclic GMP) phosphodiesterase and (2) kinetic data of the platelet enzymes indicated that cyclic adenosine 3': 5'-monophosphate (cyclic AMP) and cyclic GMP interact with a single catalytic site on the enzyme. F I was a cyclic nucleotide phosphodiesterase with a high Km for cyclic AMP and a negatively cooperative low Km for cyclic GMP. F II hydrolyzed cyclic AMP and cyclic GMP about equally with a high Km for both substrates. F III was low Km phosphodiesterase which hydrolyzed cyclic AMP faster than cyclic GMP. Each cyclic nucleotide acted as a competitive inhibitor of the hydrolysis of the other nucleotide by these three fractions with Ki values similar to the Km values for each nucleotide suggesting that the hydrolysis of both cyclic AMP and cyclic GMP was catalyzed by a single catalytic site on the enzyme. However, cyclic GMP at low concentration (below 10 muM) was an activator of cyclic AMP hydrolysis by F I. Papaverine and EG 626 acted as competitive inhibitors of each fraction with virtually the same Ki value in both assays using either cyclic AMP or cyclic GMP as the substrate. The ratio of cyclic AMP hydrolysis to cyclic GMP hydrolysis by each fraction did not vary significantly after freezing/thawing or heat treatment. These facts also suggest that both nucleotides were hydrolyzed by the same catalytic site on the enzyme. The differences in apparent Ki values for inhibitors such as cyclic nucleotides, papaverine and EG 626 would indicate that three enzymes were different from each other. Centrifugation in a continuous sucrose gradient revealed sedimentation coefficients F I and II had 8.9 S and F III 4.6 S. The molecular weight of these forms, determined by gel filtration on a Sepharose 6B column, were approx. 240 000 (F I and II) and 180 000 (F III). F III was purified extensively (70-fold) from homogenate, with a recovery of approximately 7%.     

Identification of human plasma lysophospholipase D,a lysophosphatidic acid-producing enzyme,as autotaxin,a multifunctional phosphodiesterase

We purified human plasma lysophospholipase D that produces physiologically active lysophosphatidic acid and showed that it is a soluble form of autotaxin, an ecto-nucleotide pyrophosphatase/phosphodiesterase, originally found as a tumor cell motility-stimulating factor. Its lower K(m) value for a lysophosphatidylcholine than that for a synthetic substrate of nucleotide suggests that lysophosphatidylcholine is a more likely physiological substrate for autotaxin and that its predicted physiological and pathophysiological functions could be mediated by its activity to produce lysophosphate acid, an intercellular mediator. Recombinant autotaxin was found to have lysophospholipase D activity; its substrate specificity and metal ion requirement were the same as those of the purified plasma enzyme. The activity of lysophospholipase D for exogenous lysophosphatidylcholine in human serum was found to increase in normal pregnant women at the third trimester of pregnancy and to a higher extent in patients in threatened preterm delivery, suggesting its roles in induction of parturition.     

Alkaline phosphodiesterase I release from eucaryotic plasma membranes by phosphatidylinositol-specific phospholipase C. I. The release from rat organs

From various rat organs, alkaline phosphodiesterase I was liberated by the action of phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis. Especially, a large amount of alkaline phosphodiesterase I was released from slices of small intestine, testis, lung, and kidney, but not from pancreas and liver. The release of the enzyme induced by phospholipase C was dependent on, or proportional to, the reaction time and the concentrations of the phospholipase C and the weight of the slices of small intestine or testis. Furthermore, little enzyme was released from the homogenate of pancreas. These results suggest an important role of phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membranes of rat small intestine and pancreas. The alkaline phosphodiesterase I released from slices of rat small intestine and testis had a molecular weight of about 240,000, and was activated by Mg2+ and Ca2+ but inhibited by EDTA. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9, having the Km values of 0.36 mM (small intestine) and 0.25 mM (testis). The intestinal enzyme differed from the testis enzyme in pI values, thermostability, and Arrhenius plot having a single breakpoint.     

Release of alkaline phosphodiesterase I from rat kidney plasma membrane produced by the phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis

The release of plasma-membrane-bound enzymes by phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis was investigated. Among the ectoenzymes of plasma membrane tested, alkaline phosphodiesterase I was released markedly from rat kidney cortex slices, in addition to alkaline phosphatase and 5'-nucleotidase. Other membrane-bound enzymes; alanine aminopeptidase, leucine aminopeptidase, dipeptidyl peptidase, leucine aminopeptidase, dipeptidyl peptidase IV, esterase and gamma-glutamyl transpeptidase could not be liberated from the treated slices. Alkaline phosphodiesterase I was released linearly from rat kidney slices with the concentration of phosphatidylinositol-specific phospholipase C, but little enzyme was released from rat liver slices. Alkaline phosphodiesterase I separated from kidney tissue with n-butanol still retained phosphatidylinositol and was transformed into a lower molecular weight form by phosphatidylinositol-specific phospholipase C. This suggests an important function for phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membrane of rat kidney cells. The alkaline phosphodiesterase I released from rat kidney had a molecular weight of about 240,000 and an isoelectric point (pI) of 5.4. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9 and had a Km value of 0.3 mM. The enzyme was activated by Mg2+ and Ca2+, but was inhibited by EDTA. Strong inhibition took place on the addition of adenosine 5'-phosphosulfate or the nucleotide pyrophosphates, i.e., UDP-galactose and alpha, beta-methylene ATP.     

The hydrolysis of lysophospholipids and nucleotides by autotaxin (NPP2) involves a single catalytic site

Autotaxin (NPP2) is a tumor cell motility-stimulating factor that displays both a nucleotide pyrophosphatase/phosphodiesterase activity and a recently described lysophospholipase D activity. The hydrolysis of nucleotides is a metal-assisted reaction that occurs via a nucleotidylated threonine in the catalytic site. We show here that the catalytic site threonine and the metal-coordinating residues are also essential for the hydrolysis of lysophospholipids. In comparing the substrate specificity of NPP2 and the closely related NPP1 and NPP3, we found that only NPP2 displayed a lysophospholipase D activity, whereas NPP1 and NPP3 had a much higher nucleotide pyrophosphatase activity.     

Extracellular metabolism-dependent uptake of lysolipids through cultured monolayer of differentiated Caco-2 cells

Glycerophospholipids are known to be hydrolyzed in the intestinal lumen into free fatty acids and lysophospholipids that are then absorbed by the intestinal epithelial cells. A monolayer of enterocyte-differentiated Caco-2 cell is often used to assess the intestinal bioavailability of nutrients. In this study, we examined how differentiated Caco-2 cells process lysoglycerolipids such as lysophosphatidylcholine (LPC). Our findings were twofold. (1) Caco-2 cells secreted both a lysophospholipase A-like enzyme and a glycerophosphocholine-phosphodiesterase enzyme into the apical, but not basolateral, lumen, suggesting that food-derived LPC is converted to a free fatty acid, sn-glycerol-3-phosphate, and choline through two sequential enzymatic reactions in humans. The release of the latter enzyme was differentiation-dependent. (2) Fatty acid-releasing activities toward exogenous fluorescent LPC, lysophosphatidic acid and monoacylglycerol were shown to be higher on the apical membranes of Caco-2 cells than on the basolateral membranes. These results suggest that human intestinal epithelial cells metabolize lysoglycerolipids by two distinct mechanisms involving secreted or apical-selective expression of metabolic enzymes.     

Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production

Autotaxin (ATX) is a tumor cell motility-stimulating factor, originally isolated from melanoma cell supernatants. ATX had been proposed to mediate its effects through 5'-nucleotide pyrophosphatase and phosphodiesterase activities. However, the ATX substrate mediating the increase in cellular motility remains to be identified. Here, we demonstrated that lysophospholipase D (lysoPLD) purified from fetal bovine serum, which catalyzes the production of the bioactive phospholipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC), is identical to ATX. The Km value of ATX for LPC was 25-fold lower than that for the synthetic nucleoside substrate, p-nitrophenyl-tri-monophosphate. LPA mediates multiple biological functions including cytoskeletal reorganization, chemotaxis, and cell growth through activation of specific G protein-coupled receptors. Recombinant ATX, particularly in the presence of LPC, dramatically increased chemotaxis and proliferation of multiple different cell lines. Moreover, we demonstrate that several cancer cell lines release significant amounts of LPC, a substrate for ATX, into the culture medium. The demonstration that ATX and lysoPLD are identical suggests that autocrine or paracrine production of LPA contributes to tumor cell motility, survival, and proliferation. It also provides potential novel targets for therapy of pathophysiological states including cancer.     

Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases.

Diadenosine polyphosphates (ApnAs) act as extracellular signaling molecules in a broad variety of tissues. They were shown to be hydrolyzed by surface-located enzymes in an asymmetric manner, generating AMP and Apn-1 from ApnA. The molecular identity of the enzymes responsible remains unclear. We analyzed the potential of NPP1, NPP2, and NPP3, the three members of the ecto-nucleotide pyrophosphatase/phosphodiesterase family, to hydrolyze the diadenosine polyphosphates diadenosine 5',5"'-P1,P3-triphosphate (Ap3A), diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), and diadenosine 5',5"'-P1,P5-pentaphosphate, (Ap5A), and the diguanosine polyphosphate, diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). Each of the three enzymes hydrolyzed Ap3A, Ap4A, and Ap5A at comparable rates. Gp4G was hydrolyzed by NPP1 and NPP2 at rates similar to Ap4A, but only at half this rate by NPP3. Hydrolysis was asymmetric, involving the alpha,beta-pyrophosphate bond. ApnA hydrolysis had a very alkaline pH optimum and was inhibited by EDTA. Michaelis constant (Km) values for Ap3A were 5.1 micro m, 8.0 micro m, and 49.5 micro m for NPP1, NPP2, and NPP3, respectively. Our results suggest that NPP1, NPP2, and NPP3 are major enzyme candidates for the hydrolysis of extracellular diadenosine polyphosphates in vertebrate tissues.     

A novel phosphodiesterase from cultured tobacco cells.

A novel phosphodiesterase was purified from cultured tobacco cells to a state which appeared homogeneous on polyacrylamide gel electrophoresis. The enzyme hydrolyzed various phosphodiester and pyrophosphate bonds, including p-nitrophenyl thymidine 5'-phosphate, p-nitrophenyl thymidine 3'-phosphate, cyclic nucleotides, ATP, NAD+, inorganic pyrophosphate, dinucleotides, and poly(adenosine diphosphate ribose), which is a polymer synthesized from NAD+. However, it did not hydrolyze highly polymerized polynucleotides. The molecular weight of the native enzyme was estimated as 270 000 to 280 000 by gel filtration on Sephadex G-200 and Bio-Gel A-5m. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was composed of subunits with molecular weights calculated to be 75 000. The enzyme did not require divalent cations for activity being fully active in the presence of ethylenediaminetetraacetic acid. The pH optimum for the enzyme was approximately 6 with p-ni-trophenyl thymidine 5'-phosphate or adenosine cyclic 3',5'monophosphate, and 5.3 with NAD+. Double reciprocal plots of the initial velocity against the concentration of p-nitrophenyl thymidine 5'-phosphate gave two apparent Km values of 0.17 and 1.3 mM, suggesting the presence of at least two active sites.     

Adenosine-3':5'-monophosphate phosphodiesterase from Rhizobium

The phosphodiesterase (PDE) activity of adenosine-3':5'-monophosphate was detected in the cells of tubercular bacteria of Rhizobium lupini and Rhizobium japonicum. The specific activity of three Rhizobium forms, e.g. bacteroids from lupine root tubercles, free-nitrogen-fixing culture and vegetative cells grown on a mannitol--yeast agar, were compared. In the bacteroids PDE is represented both by soluble and membrane-bound forms. The optimal enzyme activity is revealed in an alkaline medium, whereas the curve of PDE activity dependence on pH has a broad maximum. PDE is inhibited by methylxanthines, the inhibiting effect being stronger than that of theophylline.     

Identification of human intestinal alkaline sphingomyelinase as a novel ecto-enzyme related to the nucleotide phosphodiesterase family

Alkaline sphingomyelinase (alk-SMase) hydrolyzes dietary sphingomyelin and generates sphingolipid messengers in the gut. In the present study, we purified the enzyme, identified a part of the amino acid sequence, and found a cDNA in the GenBank coding for the protein. The cDNA contains 1841 bp, and the open reading frame encodes 458 amino acids. Transient expression of the cDNA linked to a Myc tag in COS-7 cells increased alk-SMase activity in the cell extract by 689-fold and in the medium by 27-fold. High activity was also identified in the anti-Myc immunoprecipitated proteins and the proteins cross-reacted with anti-human alk-SMase. Northern blotting of human intestinal tissues found high levels of alk-SMase mRNA in the intestine and liver. The amino acid sequence shared no similarity with acid and neutral SMases but was related to the ecto-nucleotide phosphodiesterase (NPP) family with 30-36% identity to human NPPs. Alk-SMase has a predicted signal peptide domain at the N terminus and a signal anchor domain at the C terminus. The ion-binding sites and the catalytic residue of NPPs were conserved, but the substrate specificity domain was modified. Alk-SMase had no detectable nucleotidase activity, but its activity against sphingomyelin could be inhibited by orthovanadate, imidazole, and ATP. In contrast to NPPs, alk-SMase activity was not stimulated by divalent metal ions but inhibited by Zn2+. Differing from NPP2, the alk-SMase cleaved phosphocholine but not choline from lysophosphatidylcholine. Phylogenetic tree indicated that the enzyme is a new branch derived from the NPP family. Two cDNA sequences of mouse and rat that shared 83% identity to human alk-SMase were identified in the GenBank. In conclusion, we identified the amino acid and cDNA sequences of human intestinal alk-SMase, and found that it is a novel ecto-enzyme related to the NPP family with specific features essential for its SMase activity.     

A nucleotide phosphodiesterase with preference for 2',5'-phosphodiester bonds from Ehrlich ascites carcinoma

We have previously reported that many tumor cell lines express a 5'-nucleotide phosphodiesterase (phosphodiesterase I, EC 3.1.4.1) with properties clearly distinguishable from enzymes of normal tissues (Biochim. Biophys. Acta (1988) 966, 99-106). Such an enzyme with 5'-nucleotide phosphodiesterase activity was purified from Ehrlich ascites carcinoma by measuring the cleavage of thymidine 5'-monophosphate p-nitrophenyl ester (TMP-NP). The enzyme is a soluble protein, has a pH optimum of 7.5, and the molecular mass estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 67 kDa. The enzyme does not hydrolyze other chromogenic substrates for phosphodiesterases, nor pyrophosphate bond of various nucleotides which are cleaved by 5'-nucleotide phosphodiesterases of normal tissues. But, it hydrolyzes dinucleotides to form 5'-phosphates, and is more active on 2',5'- than on 3',5'-phosphodiester bonds. These results indicate that the TMP-NP splitting enzyme in Ehrlich ascites carcinoma cells is a 2',5'-phosphodiesterase.     

Regulation and biological activities of the autotaxin-LPA axis

Autotaxin (ATX), or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2), is an exo-enzyme originally identified as a tumor cell autocrine motility factor. ATX is unique among the NPPs in that it primarily functions as a lysophospholipase D, converting lysophosphatidylcholine into the lipid mediator lysophosphatidic acid (LPA). LPA acts on specific G protein-coupled receptors to elicit a wide range of cellular responses, ranging from cell proliferation and migration to neurite remodeling and cytokine production. While LPA signaling has been studied extensively over the last decade, we are only now beginning to explore the properties and biological importance of ATX as the major LPA-producing phospholipase. In this review, we highlight recent advances in our understanding of the ATX-LPA axis, giving first an update on LPA action and then focusing on ATX, in particular its regulation, its link to cancer and its vital role in vascular development.     

Autotaxin is overexpressed in glioblastoma multiforme and contributes to cell motility of glioblastoma by converting lysophosphatidylcholine to lysophosphatidic acid

Autotaxin (ATX) is a multifunctional phosphodiesterase originally isolated from melanoma cells as a potent cell motility-stimulating factor. ATX is identical to lysophospholipase D, which produces a bioactive phospholipid, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC). Although enhanced expression of ATX in various tumor tissues has been repeatedly demonstrated, and thus, ATX is implicated in progression of tumor, the precise role of ATX expressed by tumor cells was unclear. In this study, we found that ATX is highly expressed in glioblastoma multiforme (GBM), the most malignant glioma due to its high infiltration into the normal brain parenchyma, but not in tissues from other brain tumors. In addition, LPA1, an LPA receptor responsible for LPA-driven cell motility, is predominantly expressed in GBM. One of the glioblastomas that showed the highest ATX expression (SNB-78), as well as ATX-stable transfectants, showed LPA1-dependent cell migration in response to LPA in both Boyden chamber and wound healing assays. Interestingly these ATX-expressing cells also showed chemotactic response to LPC. In addition, knockdown of the ATX level using small interfering RNA technique in SNB-78 cells suppressed their migratory response to LPC. These results suggest that the autocrine production of LPA by cancer cell-derived ATX and exogenously supplied LPC contribute to the invasiveness of cancer cells and that LPA1, ATX, and LPC-producing enzymes are potential targets for cancer therapy, including GBM.