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.

Every successful pregnancy requires proper embryo implantation. Low implantation rate is a major problem during infertility treatments using assisted reproductive technologies (ART) 1 . Here we report a new molecular influence on implantation through the lysophosphatidic acid (LPA) receptor LPA 3 2-4 . Targeted deletion of LPA 3 in mice resulted in significantly reduced litter size, which could be attributed to delayed implantation and altered embryo spacing. These two events led to delayed embryonic development, hypertrophic placentas shared by multiple embryos, and embryonic death. An enzyme demonstrated to influence implantation, cyclooxygenase-2 (COX-2) 5 , was downregulated in LPA 3 -deficient uteri during preimplantation. Down regulation of COX-2 led to reduced levels of prostaglandins that are critical for implantation 1 . Exogenous administration of the prostaglandins PGE 2 and cPGI into LPA 3 -deficient females rescued delayed implantation but did not rescue defects in embryo spacing. These data identify LPA 3 receptor-mediated signalling as a new influence on implantation and further indicate linkage between LPA signalling and prostaglandin biosynthesis.Multiple factors can adversely affect successful pregnancy. Two of these factors are failed synchronization between embryonic and endometrial development during implantation and occurrence of multiple gestations (especially monochorionic gestation), which can result in fetal demise 1,6-9 . These factors are particularly important for the clinical success and efficacy of ART. One molecular factor that has been previously implicated in female reproduction is the small, bioactive phospholipid LPA 10 . LPA has a range of influences that are mediated by at least four G protein-coupled receptors, LPA 1-4 2 . Deletion of LPA 1 and LPA 2 in mice revealed roles for these receptors in neural development, craniofacial formation, neuropathic pain, and altered cellular signalling, but without obvious effects on female reproduction 11-Correspondence and requests for materials should be addressed to J. Chun (e-mail:jchun@scripps.edu).. 6 These authors contributed equally to the work. Functional deletion of LPA 3 was achieved by replacing a fragment covering the untranslated region and the start codon in exon 2 with a neomycin-resistance gene in reverse orientation in R1 embryonic stem cells (supplementary Fig. S1). The LPA 3 -deficient mice were born with normal Mendelian frequency without sexual bias (supplementary Table S1), and appeared grossly normal (data not shown). However, LPA 3 -deficient females produced litter sizes of less than 50% compared to that from wild-type (WT) and LPA 3 heterozygote (Het) controls (supplementary Table S2), and showed a statistically significant prolongation of pregnancy (20.9±0.5 days, vs. 19.4±0.7 days in WT/Het controls, P<0.05). These phenotypes were independent of stud genotype, indicating defects in female reproduction. Supplementary InformationTowards determining whether LPA 3 deletion might directly affect the female...

CorrectionsArai, and Glenn D. Prestwich, which appeared in number 1, January 7, 2003, of Proc. Natl. Acad. Sci. USA (100, 131-136; First Published December 26, 2002; 10.1073͞pnas.0135855100), Fig. 4 should have appeared in color. The correct figure and its legend appear below. Fig. 4.LPA stimulates lipid accumulation, CD36 expression, and oxidized LDL uptake through a PPAR-responsive element. (a) LPA stimulates monocyte uptake of oxidized LDL. Freshly elutriated human monocytes were allowed to interact with an anti-ICAM3-coated well, which leads to rapid PPAR␥ expression (13), and then stimulated, or not (negative, oxLDL), with oleoyl LPA. Some cells were then briefly exposed to oxidized LDL before intracellular lipid stores were visualized with oil red O stain. (b) LPA increases the expression of CD36 on the surface of primary human monocytes. Monocytes engaging anti-ICAM3 were treated or not with LPA, and then recovered by gentle scraping and washing by centrifugation before their surface CD36 was assessed by flow cytometry. (c) LPA and the LPA analogs XY4 and XY8 stimulate CD36 promoter function only when the PPRE is present. RAW264.7 cells were transfected with the human CD36 promoter containing the PPRE (CD36 Ϫ273 ) or a reporter that lacks only this element (CD36 Ϫ261 ) and then stimulated with oleoyl LPA, azPC, XY4, or XY8. Expression of luciferase normalized to ␤-galactosidase was determined as above. (d) Anti-CD36 blocks LPA-stimulated accumulation of cholesterol from oxidized LDL. Freshly isolated human monocytes were treated as in a, but after being preincubated with a blocking anti-CD36 antibody before exposure to oxidized LDL. L ysophosphatidic acid (LPA) is a pluripotent lipid mediator controlling growth, motility, and differentiation (1, 2). It is the ovarian cancer-activating factor that is elevated in the serum of ovarian cancer patients (3), and it controls adipogenesis (4). LPA also is generated during platelet activation (5) to become a major growth factor of serum. LPA stimulates three G protein-linked, plasma membrane-associated receptors [LPA 1 , LPA 2 , and LPA 3 , formerly edg2, edg4, and edg7 (6)] that recognize extracellular LPA (7). However, control of complex processes including growth and differentiation is difficult to reconcile with these receptors (8), suggesting that undiscovered receptors for LPA may exist. LPA is a central component of cellular phospholipid metabolism and, because a role for intracellular LPA beyond this is unknown, the plasma membrane separates signaling LPA from metabolic LPA.The transcription factor peroxisome proliferator-activated receptor ␥ (PPAR␥) regulates genes that in general control energy metabolism (9). PPAR␥, like other members of its extended nuclear hormone receptor superfamily, is activated by binding an appropriate lipid ligand (10). Synthetic compounds, including the widely prescribed drug rosiglitazone, target PPAR␥ and activate transcription with high affinity. Anionic fatty acids and their oxidized derivatives also bind and activate PPAR...

Autotaxin (ATX, also known as Enpp2) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine to generate lysophosphatidic acid (LPA), a lipid mediator that activates G protein-coupled receptors to evoke various cellular responses. Here, we report the crystal structures of mouse ATX alone and in complex with LPAs with different acyl-chain lengths and saturations. These structures reveal that the multidomain architecture helps to maintain the structural rigidity of the lipid-binding pocket, which accommodates the respective LPA molecules in distinct conformations. They indicate that a loop region in the catalytic domain is a major determinant for the substrate specificity of the Enpp family enzymes. Furthermore, along with biochemical and biological data, these structures suggest that the produced LPAs are delivered from the active site to cognate G protein-coupled receptors through a hydrophobic channel.

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities that accounts for many biological properties of serum. LPA is thought to be produced during serum formation based on the fact that the LPA level is much higher in serum than in plasma. In this study, to better understand the pathways of LPA synthesis in serum, we evaluated the roles of platelets, plasma, and phospholipases by measuring LPA using a novel enzyme-linked fluorometric assay. First, examination of platelet-depleted rats showed that half of the LPA in serum is produced via a platelet-dependent pathway. However, the amount of LPA released from isolated platelets after they are activated by thrombin or calcium ionophore accounted for only a small part of serum LPA. Most of the platelet-derived LPA was produced in a two-step process: lysophospholipids such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine, and lysophosphatidylserine, were released from activated rat platelets by the actions of two phospholipases,

Autotaxin (ATX) is a tumor cell motility-stimulating factor originally isolated from melanoma cell supernatant that has been implicated in regulation of invasive and metastatic properties of cancer cells. Recently, we showed that ATX is identical to lysophospholipase D, which converts lysophosphatidylcholine to a potent bioactive phospholipid mediator, lysophosphatidic acid (LPA), raising the possibility that autocrine or paracrine production of LPA by ATX contributes to tumor cell motility. Here we demonstrate that LPA and ATX mediate cell motility-stimulating activity through the LPA receptor, LPA 1 . In fibroblasts isolated from lpa 1 ؊/؊ mice, but not from wild-type or lpa 2 ؊/؊ , cell motility stimulated with LPA and ATX was completely absent. In the lpa 1 ؊/؊ cells, LPA-stimulated lamellipodia formation was markedly diminished with a concomitant decrease in Rac1 activation. LPA stimulated the motility of multiple human cancer cell lines expressing LPA 1 , and the motility was attenuated by an LPA 1 -selective antagonist, Ki16425. The present study suggests that ATX and LPA 1 represent potential targets for cancer therapy.

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, LPA 1 , an LPA receptor responsible for LPAdriven 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 LPA 1 -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 LPA 1 , ATX, and LPC-producing enzymes are potential targets for cancer therapy, including GBM.

Autotaxin (ATX) is an ecto-enzyme responsible for lysophosphatidic acid (LPA) production in blood. ATX is present in various biological fluids such as cerebrospinal and seminal fluids and accounts for bulk LPA production in these fluids. ATX is a member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family and was originally isolated from conditioned medium of melanoma cells as an autocrine motility stimulating factor. LPA, a second-generation lipid mediator, binds to its cognate G protein-coupled receptors through which it exerts a number of biological functions including influencing cell motility and proliferation stimulating activity. Some of the biological roles of LPA can be mediated by ATX. However, there are other LPA-producing pathways independent of ATX. The accumulating evidences for physiological and pathological functions of ATX strongly support that ATX is an important therapeutic target. This review summarizes the historical aspects, structural basis, pathophysiological functions identified in mice studies and clinical relevance discovered by measuring the blood ATX level in human. The general features and functions of each NPP family member will be also briefly reviewed. The presence of the ATX gene in other model organisms and recently developed ATX inhibitors, both of which will be definitely useful for further functional analysis of ATX, will also be mentioned.