Transduction of signals initiating motility by extracellular matrix (ECM) molecules differed depending on the type of matrix molecule and whether the ligand was in solution or bound to a substratum. Laminin, fibronectin, and type IV collagen stimulated both chemotaxis and haptotaxis of the A2058 human melanoma cell line. Peak chemotactic responses were reached at 50-200 nM for laminin, 50-100 nM for fibronectin, and 200-370 nM for type IV collagen. Checkerboard analysis of each attractant in solution demonstrated a predominantly directional (chemotactic) response, with a minor chemokinetic component. The cells also migrated in a concentration-dependent manner to insoluble step gradients of substratum-bound attractant (haptotaxis). The haptotactic responses reached maximal levels at coating concentrations of 20 nM for laminin and type IV collagen, and from 30 to 45 nM for fibronectin. Pretreatment of cells with the protein synthesis inhibitor, cycloheximide (5 micrograms/ml), resulted in a 5-30% inhibition of both chemotactic and haptotactic responses to each matrix protein, indicating that de novo protein synthesis was not required for a significant motility response. Pretreatment of cells with 50-500 micrograms/ml of synthetic peptides containing the fibronectin cell- recognition sequence GRGDS resulted in a concentration-dependent inhibition of fibronectin-mediated chemotaxis and haptotaxis (70-80% inhibition compared to control motility); negative control peptide GRGES had only a minimal effect. Neither GRGDS nor GRGES significantly inhibited motility to laminin or type IV collagen. Therefore, these results support a role for the RGD-directed integrin receptor in both types of motility response to fibronectin. After pretreatment with pertussis toxin (PT), chemotactic responses to laminin, fibronectin, and type IV collagen were distinctly different. Chemotaxis to laminin was intermediate in sensitivity; chemotaxis to fibronectin was completely insensitive; and chemotaxis to type IV collagen was profoundly inhibited by PT. In marked contrast to the inhibition of chemotaxis, the hepatotactic responses to all three ligands were unaffected by any of the tested concentrations of PT. High concentrations of cholera toxin (CT; 10 micrograms/ml) or the cAMP analogue, 8-Br-cAMP (0.5 mM), did not significantly affect chemotactic or haptotactic motility to any of the attractant proteins, ruling out the involvement of cAMP in the biochemical pathway initiating motility in these cells. The sensitivity of chemotaxis induced by laminin and type IV collagen, but not fibronectin, to PT indicates the involvement of a PT-sensitive G protein in transduction of the signals initiating motility to soluble laminin and type IV collagen.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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...