Stimulation of Tumor Cell Motility Linked to Phosphodiesterase Catalytic Site of Autotaxin

Our group has recently demonstrated (Gesta, S., Simon, M., Rey, A., Sibrac, D., Girard, A., Lafontan, M., Valet, P., and Saulnier-Blache, J. S. (2002) J. Lipid Res. 43, 904 -910) the presence, in adipocyte conditioned-medium, of a soluble lysophospholipase D-activity (LPLDact) involved in synthesis of the bioactive phospholipid lysophosphatidic acid (LPA). In the present report, LPLDact was purified from 3T3F442A adipocyte-conditioned medium and identified as the type II ecto-nucleotide pyrophosphatase phosphodiesterase, autotaxin (ATX). A unique ATX cDNA was cloned from 3T3F442A adipocytes, and its recombinant expression in COS-7 cells led to extracellular release of LPLDact. ATX mRNA expression was highly up-regulated during adipocyte differentiation of 3T3F442A-preadipocytes. This up-regulation was paralleled by the ability of newly differentiated adipocytes to release LPLDact and LPA. Differentiation-dependent up-regulation of ATX expression was also observed in a primary culture of mouse preadipocytes. Treatment of 3T3F442A-preadipocytes with concentrated conditioned medium from ATX-expressing COS-7 cells led to an increase in cell number as compared with concentrated conditioned medium from ATX non-expressing COS-7 cells. The specific effect of ATX on preadipocyte proliferation was completely suppressed by co-treatment with a LPA-hydrolyzing phospholipase, phospholipase B. Finally, ATX expression was found in mature adipocytes isolated from mouse adipose tissue and was substantially increased in genetically obesediabetic db/db mice when compared with their lean siblings. In conclusion, the present work shows that ATX is responsible for the LPLDact released by adipocytes and exerts a paracrine control on preadipocyte growth via an LPA-dependent mechanism. Up-regulations of ATX expression with adipocyte differentiation and genetic obesity suggest a possible involvement of this released protein in the development of adipose tissue and obesity-associated pathologies.Because of its ability to store extra energy as triacylglycerol (lipogenesis) and to release fatty acids and glycerol (lipolysis), adipose tissue plays a crucial role in energy balance. In obesity, excessive accumulation of triacylglycerol in adipocytes (hypertrophy) results from an alteration in the balance between lipogenic and/or lipolytic activities of the adipocytes.It is now recognized that, beside their involvement in lipid homeostasis, adipocytes also produce and secrete numerous factors. Among them are endocrine peptides (leptin, adiponectin, angiotensinogen, etc.) which may play an important role in the development of morbid complications of obesity such as cardiovascular diseases, hypertension, diabetes, and cancer. Other adipocyte-secreted factors (tumor necrosis factor, fatty acids, eicosanoids, lysophosphatidic acid, etc.) are produced locally and may influence adipose tissue development and/or metabolism by exerting autocrine/paracrine effects on the different cells composing adipose tissue (adipocytes, preadipocytes, and endothelial...

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“…3). Similar results were obtained when transfecting COS-7 cells with a pcDNA vector expressing human ATX (30,31).…”

Section: Co-purification Of Adipocyte Lysophospholipase D Activitysupporting

confidence: 71%

Autotaxin Is Released from Adipocytes, Catalyzes Lysophosphatidic Acid Synthesis, and Activates Preadipocyte Proliferation

Ferry

Tellier

Try

et al. 2003

Journal of Biological Chemistry

215

201

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“…3A and 4) and were predicted to display a similar spatial arrangement relative to the residue that forms the catalytic intermediate, i.e. a Thr in NPPs (6,42,43) and a Ser in AP and iPGM (32,44,45). Arylsulfatases have a similar catalytic core, but differ from AP and iPGMs in that they have only a single metal-binding site and that the residue that forms the catalytic intermediate is an oxidated cysteine (46).…”

Section: Resultsmentioning

confidence: 99%

Structural and Catalytic Similarities between Nucleotide Pyrophosphatases/Phosphodiesterases and Alkaline Phosphatases

Gijsbers

Ceulemans

Stalmans

et al. 2001

Journal of Biological Chemistry

157

164

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Nucleotide pyrophosphatases/phosphodiesterases (NPPs) generate nucleoside 5-monophosphates from a variety of nucleotides and their derivatives. Here we show by data base analysis that these enzymes are conserved from eubacteria to higher eukaryotes. We also provide evidence for the existence of two additional members of the mammalian family of ecto-NPPs. Homology searches and alignment-assisted mutagenesis revealed that the catalytic core of NPPs assumes a fold similar to that of a superfamily of phospho-/sulfo-coordinating metalloenzymes comprising alkaline phosphatases, phosphoglycerate mutases, and arysulfatases. Mutation of mouse NPP1 in some of its predicted metalcoordinating residues (D358N or H362Q) or in the catalytic site threonine (T238S) resulted in an enzyme that could still form the nucleotidylated catalytic intermediate but was hampered in the second step of catalysis. We also obtained data indicating that the ability of some mammalian NPPs to auto(de)phosphorylate is due to an intrinsic phosphatase activity, whereby the enzyme phosphorylated on Thr-238 represents the covalent intermediate of the phosphatase reaction. The results of site-directed mutagenesis suggested that the nucleotide pyrophosphatase/phosphodiesterase and the phosphatase activities of NPPs are mediated by a single catalytic site.

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“…Invasion was assayed in Biocoat Matrigel TM invasion chambers (Mulvaney et al, 1998). The lower chambers contained either partially puri®ed rATX (Lee et al, 1996a) diluted in 0.1% bovine serum albumin-DMEM or this same medium alone. Chemotaxis assays were incubated for 4 h, invasion chambers for 20 ± 24 h at 378C.…”

Section: In Vitro Motility and Invasion Assaysmentioning

confidence: 99%

“…The media were partially puri®ed by lectin a nity chromatography with concanavalin A-agarose (Vector Labs), samples were separated by gel electrophoresis, and immunoblots were prepared as previously described (Lee et al, 1996a).…”

Section: Western Blot Analysismentioning

confidence: 99%

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Autotaxin (ATX), a potent tumor motogen, augments invasive and metastatic potential of ras-transformed cells

et al. 2000

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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.

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Stimulation of Tumor Cell Motility Linked to Phosphodiesterase Catalytic Site of Autotaxin

Abstract: A family of extracellular type I phosphodiesterases has recently been isolated by cDNA cloning, but a physiological function linked to the phosphodiesterase active site has remained unknown. We now present evidence that the phosphodiesterase catalytic site,

Cited by 84 publications

References 33 publications

Autotaxin Is Released from Adipocytes, Catalyzes Lysophosphatidic Acid Synthesis, and Activates Preadipocyte Proliferation

Autotaxin Is Released from Adipocytes, Catalyzes Lysophosphatidic Acid Synthesis, and Activates Preadipocyte Proliferation

Structural and Catalytic Similarities between Nucleotide Pyrophosphatases/Phosphodiesterases and Alkaline Phosphatases

Autotaxin (ATX), a potent tumor motogen, augments invasive and metastatic potential of ras-transformed cells

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