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Autotaxin (ATX), an exo-nucleotide pyrophosphatase and phosphodiesterase, was originally isolated as a potent stimulator of tumor cell motility. In order to study whether ATX expression a ects motility-dependent processes such as invasion and metastasis, we stably transfected full-length ATX cDNA into two nonexpressing cell lines, parental and ras-transformed NIH3T3 (clone7) cells. The e ect of ATX secretion on in vitro cell motility was variable. The ras-transformed, ATX-secreting subclones had enhanced motility to ATX as chemoattractant, but there was little di erence in the motility responses of NIH3T3 cells transfected with atx, an inactive mutant gene, or empty vector. In Matrigel TM invasion assays, all subclones, which secreted enzymatically active ATX, demonstrated greater spontaneous and ATX-stimulated invasion than appropriate controls. This di erence in invasiveness was not caused by di erences in gelatinase production, which was constant within each group of transfectants. In vivo studies with athymic nude mice demonstrated that injection of atx-transfected NIH3T3 cells resulted in a weak tumorigenic capacity with few experimental metastases. Combination of ATX expression with ras transformation produced cells with greatly ampli®ed tumorigenesis and metastatic potential compared to ras-transformed controls. Thus, ATX appears to augment cellular characteristics necessary for tumor aggressiveness.

Recent advances in understanding the complex biology of the microenvironment that underlies tumor invasion and migration have revealed novel and promising therapeutic targets. Pharmacological blockade of intra- and extracellular signaling events that regulate migration and survival of multiple cell types may disrupt the host-tumor conspiracy that allows escape from normal developmental regulation.

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Autotaxin (ATX)1 is a 125-kDa glycoprotein secreted by the human melanoma cell line A2058. ATX stimulates both random and directed motility in its producer cells (1), and its recent cloning and sequencing (2) has revealed homology with the active site of bovine intestinal 5Ј-nucleotide PDE (EC 3.1.4.1) (4) and extensive homology with the ectoprotein PC-1 (5), the brain-type PDE I-nucleotide pyrophosphatase gene 2 (6), and the rat neural differentiation antigen gp130 RB13-6 (7). ATX contains two tandem somatomedin B regions, the loop region of an EF-hand and a type I PDE catalytic site, and possesses 5Ј-nucleotide PDE activity (2) .Early studies on digestive enzymes responsible for RNA degradation identified a class of enzymes characterized by their reaction product, a 5Ј-monophosphate nucleotide, and their activity toward p-nitrophenyl-thymidine monophosphate (⌽-TMP) (8). This type I PDE activity has also been detected in a variety of mammalian tissues, their plasma membranes, and cell surfaces (9 -11). The unifying features of these activities, in addition to the reaction product, are the broad specificity for substrates and competitive inhibitors, the alkaline pH optimum, and the ability to hydrolyze the phosphodiester bond between the ␣-and ␤-phosphates in nucleoside polyphosphates. ATX possesses type I PDE activity and also induces a known biological response, the potent stimulation of cellular locomotion; thus it is possible to investigate the role of this enzyme reaction center in extracellular signal transduction.The reaction mechanism for type I PDE has been described as involving formation of nucleotidylated threonine as a covalently bound reaction intermediate (4), and PC-1 can be autophosphorylated on this threonine at the PDE catalytic center using [␥-32 P]ATP (12). Previous studies from this laboratory on ATX with point mutations at the PDE active site showed that the corresponding threonine in ATX (Thr 210 ) is required for its chemotactic, 5Ј-nucleotide PDE and threonine phosphorylation activities, and that phosphorylation-deficient, 5Ј-nucleotide PDE-competent ATX (K209L) is fully active in the stimulation of cellular motility (3). These findings suggested that the dephosphorylated state of ATX is a biologically active form and prompted us to investigate the relationship between the phosphorylation state and the catalytic properties of ATX. These earlier studies had also shown that phospho-ATX contains the ␥-and not the ␣-phosphate from ATP but addressed neither the stability of this construct nor the fate of the ␤-phosphate. In addition, unanswered questions remained concerning the nucleotide reaction products, the ability of ATX to use substrates other than ATP, and the possibility that the phosphorylation of ATX was due to the presence of a co-purifying protein kinase. We have resolved these issues by characterizing the enzymatic activities of ATX using homogeneously pure recombinant ATX (rATX) derived from the human teratocarcinoma cell line N-tera2D1 (13) and partially purified ATX (A2058 AT...

Diadenosine polyphosphates (Ap n As) 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 Ap n-1 from Ap n A. 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¢¢¢-P 1 ,P 3 -triphosphate (Ap 3 A), diadenosine 5¢,5¢¢¢-P 1 ,P 4 -tetraphosphate (Ap 4 A), and diadenosine 5¢,5¢¢¢-P 1 ,P 5 -pentaphosphate, (Ap 5 A), and the diguanosine polyphosphate, diguanosine 5¢,5¢¢¢-P 1 ,P 4 -tetraphosphate (Gp 4 G). Each of the three enzymes hydrolyzed Ap 3 A, Ap 4 A, and Ap 5 A at comparable rates. Gp 4 G was hydrolyzed by NPP1 and NPP2 at rates similar to Ap 4 A, but only at half this rate by NPP3. Hydrolysis was asymmetric, involving the a,b-pyrophosphate bond. Ap n A hydrolysis had a very alkaline pH optimum and was inhibited by EDTA. Michaelis constant (K m ) values for Ap 3 A were 5.1 lM, 8.0 lM, and 49.5 lM 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.Keywords: diadenosine polyphosphate; diguanosine polyphosphate; ectonucleotidase; nucleotide pyrophosphatase; nucleotide phosphodiesterase.Diadenosine polyphosphates [adenosine-(5¢)-oligophospho-(5¢)-adenosines, Ap n As] comprise two adenosine residues linked together by a polyphosphate chain through phosphoester bonds at their ribose 5¢ carbons. Ap n As are present intracellularly in prokaryotic and eukaryotic cells [1]. Recently, this group of nucleotides has attracted considerable interest because its members act as extracellular signaling molecules in a broad variety of tissues [2,3]. They are involved, for example, in the modulation of synaptic transmission and sensory nerve function [2-4], inhibition of platelet aggregation [5], or in the control of vascular tone [6][7][8][9]. Vasoactive effects were also observed with adenosine polyphosphoguanosines (Ap n Gs) and diguanosine polyphosphates (Gp n Gs) [10].The diadenosine polyphosphates diadenosine 5¢,5¢¢¢-P 1 ,P 3 -triphosphate (Ap 3 A), diadenosine 5¢,5¢¢¢-P 1 ,P 4 -tetraphosphate (Ap 4 A), and diadenosine 5¢,5¢¢¢-P 1 ,P 5 -pentaphosphate (Ap 5 A) are stored in chromaffin granules at millimolar concentrations together with noradrenaline and other nucleotides such as ATP and ADP [11,12]. In cholinergic synaptic vesicles, Ap 4 A and Ap 5 A were found to be co-stored with acetylcholine [13]. They can be released from secretory cells in a stimulus-dependent manner [2]. Besides the adrenal medulla, platelets are thought to represent the main source of Ap n As in blood. Stimulated platelets release, from their storage granules, a mixture of Ap n As (up to Ap 7 A), as well as Ap n Gs and Gp n Gs, together with ATP, ADP and serotonin [14,15]. Ap n ...

Lysophosphatidic acid (LPA), via interaction with its G-protein coupled receptors, is involved in various pathological conditions. Extracellular LPA is mainly produced by the enzyme autotaxin (ATX). Using fibroblast-like synoviocytes (FLS) isolated from synovial tissues of patients with rheumatoid arthritis (RA), we studied the expression profile of LPA receptors, LPAinduced cell migration, and interleukin (IL)-8 and IL-6 production. We report that FLS express LPA receptors LPA 1-3 . Moreover, exogenously applied LPA induces FLS migration and secretion of IL-8/IL-6, whereas the LPA 3 agonist L-sn-1-Ooleoyl-2-methyl-glyceryl-3-phosphothionate (2S-OMPT) stimulates cytokine synthesis but not cell motility. The LPA-induced FLS motility and cytokine production are suppressed by LPA 1/3 receptor antagonists diacylglycerol pyrophosphate and (S)-phosphoric acid mono-(2-octadec-9-enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl]-propyl) ester (VPC32183). Signal transduction through p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and Rho kinase is involved in LPA-mediated cytokine secretion, whereas LPA-induced cell motility requires p38 MAPK and Rho kinase but not p42/44 MAPK. Treatment of FLS with tumor necrosis factor-␣ (TNF-␣) increases LPA 3 mRNA expression and correlates with enhanced LPA-or OMPT-induced cytokine production. LPA-mediated superproduction of cytokines by TNF-␣-primed FLS is abolished by LPA 1/3 receptor antagonists. We also report the presence of ATX in synovial fluid of patients with RA. LPA 1/3 receptor antagonists and ATX inhibitors reduce the synovial fluid-induced cell motility. Together the data suggest that LPA 1 and LPA 3 may contribute to the pathogenesis of RA through the modulation of FLS migration and cytokine production. The above results provide novel insights into the relevance of LPA receptors in FLS biology and as potential therapeutic targets for the treatment of RA.

Autotaxin, a potent human tumor cell motility-stimulating exophosphodiesterase, was isolated and cloned from the human teratocarcinoma cell line NTera2D1. The deduced amino acid sequence for the teratocarcinoma autotaxin has 94% identity to the melanoma-derived protein, 90% identity to rat brain phosphodiesterase I/nucleotide pyrophosphatase (PD-I alpha), and 44% identity to the plasma cell membrane marker PC-I. Utilizing polymerase chain reaction screening of the CEPH YAC library, we localized the autotaxin gene to human chromosome 8q23-24. Northern blot analysis of relative mRNA from multiple human tissues revealed that autotaxin mRNA steady state expression is most abundant in brain, placenta, ovary, and small intestine.