Materials and methods br Results

Materials and methods
Results
Discussion ATX plays a significant role in initiating and sustaining tumor metastasis [43]. LPA stimulates cell proliferation, migration and survival by acting on its cognate G-protein-coupled receptors. Aberrant LPA production, receptor VX-809 and signaling probably contribute to cancer initiation, progression and metastasis. LPA production could prove to be an attractive target for therapy either directly (production) or indirectly (binding to its receptor(s)) [44]. Furthermore, as shown previously [1], [5], [6], [7], [13] adipocytes secrete large amounts of ATX massively, at least in culture. Therefore, the role of ATX might also be linked to the development of adipose tissue, through an as yet unknown mechanism. Indeed, it is probable that, despite a common catalytic site, proximate regions to which different substrates bind (cAMP and LPC, respectively) present slightly different features that may modify the overall activity. Furthermore, catalytic activity is the result of different physico-chemical parameters dependent not only on the substrates themselves, but also of the transmission of the chemical modification catalysed by the enzyme. Indeed, the naturally occurring enzyme has similar specificity toward both substrates (LPC and pNppp) than the cloned one. We report herein on the characteristics of ATX. According to the available literature, the data we gathered on the kinetics of the enzyme are in good accordance with the known data, for specific activity, Km for both substrates and Vmax. Furthermore, we attempted to determine the capacity of various published phosphodiesterase inhibitors (a set of 80 compounds coming from a commercial library) to inhibit autotaxin activity. Among these compounds, damnacanthal, a p56 kinase inhibitor [45] was identified as an interesting inhibitor. As compared with already known inhibitors of this enzyme activity namely, LPA, the product of the reaction and its derivatives [16], [17], [21], [46], the compounds described herein seemed to be as potent and maybe more specific. Of interest, the observation that the sensitivity of the two catalytic activities were different to some of the inhibitors (e.g. calmidazolium or quizanon), i.e. these compounds inhibited the two catalytic activities of ATX differently. Because both activities are reported to be catalysed at the same site [21], the only possible explanation for these discrepancies would be the different binding zones for the respective substrates, near the essential threonine 210 (human sequence). This suggests that compounds might be found that could fine-tune the lyso-PLD activity while leaving the PDE activity untouched, or vice-versa. Obviously, pharmacological agents as specific as possible of ATX are required to validate the potential role of this enzyme, and of the LPA pathway in several pathologies, e.g. metastasis [47], [48] and adiposity [7], [13].
Acknowledgement
Introduction and overview Lysophosphatidate (LPA) is the simplest phospholipid comprising of a glycerol backbone, one fatty acid and a phosphate head group. Its modest structure belies the multitude of physiological and pathophysiological processes it mediates. Enzymatic generation of LPA in plasma by lysophospholipase D activity was first described in 1986 by Akira et al. [1]. Autotaxin (ATX) is a 125-kDa secreted glycoprotein that was first isolated in 1992 from A2058 melanoma cells and described as an “autocrine motility factor” [2]. However, it would take another ten years before the plasma lysoPLD activity was isolated and found to be identical to ATX [3], [4], [5]. ATX, which is also known as ecto-nucleotide pyrophosphatase/phosphodiesterase family member 2 (ENPP2), is one of seven members of a pyrophosphatase/phosphodiesterase family. This family of secreted enzymes has diverse functions, but was originally described as being capable of degrading nucleotide phosphates. However, ATX/ENPP2 is unique among the ENPPs because of its lysoPLD activity. It generates LPA from lysophosphatidylcholine (LPC), the most abundant phospholipid in plasma with a plasma concentration of >200μM in human beings [6], [7] (Fig. 1A). Physiological plasma LPA levels are normally ⩽1μM in healthy subjects [8], with the predominant LPA species being C20:4 (39%), C18:2 (30%), and C18:1 (9%) [9]. The dynamic regulation of LPA concentrations in the blood is established by the balance of LPA production by ATX [10] versus the degradation of LPA by the ecto-activities of the lipid phosphate phosphatases (LPPs), particularly LPP1 [11], [12]. There may be some regulation of LPA concentrations caused by the competitive inhibition of LPA and sphingosine-1-phosphate (S1P) on ATX activity, but much of this work was performed using low μM concentrations of fluorescent LPC analogs for ATX [10], [13], [14], [15]. This would amplify the effects of the inhibition compared to the use of physiological concentrations of LPC at >200μM.