Production of LPA by autotaxin and toxic phospholipases D : biochemical characterization and physiological implications

Abstract

Lysophosphatidic acid (LPA) is a lipid mediator with a wide variety of biological actions, particularly as an inducer of cell proliferation, migration and survival. LPA binds to at least five distinct G protein-coupled receptors and thereby activates multiple signal transduction pathways, including those of the small GTPases Ras, Rho, and Rac. LPA signaling has been implicated in such diverse processes as wound healing, brain development, vascular remodeling and tumor progression. LPA is produced by autotaxin (ATX), a member of the ecto-nucleotide pyrophosphatase/phosphodiesterase (NPP) family. ATX was isolated in the early 1990s as an autocrine motility factor from melanoma cells. At the time, however, it was unknown how ATX exerted this effect. It has now become clear that all biological effects of ATX, at least in vitro, can be explained by its activity as a lysophosolipase D that hydrolyses lysophosphatidylcholine (LPC) into LPA with subsequent activation of LPA receptors. Interestingly, ATX expression is upregulated in several human cancers and was found to enhance the angiogenic and metastatic potential of tumor cells. The studies presented in this thesis have focused on ATX, its regulation and role in development, as well as on exogenous, non-mammalian phospholipases D (PLDs) that similarly produce bioactive LPA from LPC. Chapter 1 presents a general overview on LPA, its signaling properties and how it is produced, with special focus on ATX In Chapter 2 we report that spider and bacterial PLDs (or sphingomyelinases D) hydrolyze albumin-bound LPC leading to LPA production. The Km values for LPC are well below the normal LPC levels in blood. Furthermore, introduction of the LPA1 receptor into LPA receptor-negative cells renders non-susceptible cells susceptible to PLD/SMase D, but only in LPC-containing media. We conclude that these toxic PLDs have intrinsic lysophospholipase D activity toward LPC. We speculate that the degradation of circulating LPC to LPA with consequent activation of LPA receptors may play a role in the pathophysiology of these exogenous SMases D. Chapter 3 describes regulation of ATXactivity by its bioactive products, LPA and S1P, as measured by a newly developed fluorescent probe that reports ATXactivity with high sensitivity. ATX follows the classical secretory pathway and its N-terminal putative transmembrane domain is cleaved off in this pathway before reaching the plasma membrane and being secreted as a soluble enzyme. Subsequently the catalytic activity of soluble ATX was examined. To this end, we used a newly invented fluorescence resonance energy transfer (FRET)-based phosphodiesterase sensor (termed CPF4) that reports ATX activity in conditioned media with superior sensitivity. CPF4 is a bis-para-nitrotrophenyl-phosphate-derived probe, in which both phenyl moieties are linked to coumarin and fluorescein, respectively, resulting in FRET with high efficiency. Cleavage of the phosphodiester group by ATX causes loss of FRET, providing a convenient ratiometric readout of enzyme activity. Using this probe, we demonstrate that ATX is potently and specifically inhibited by LPA and S1P. Kinetic analysis revealed that LPA and S1P act as mixed-type inhibitors with an inhibition constant (Ki) of ~0.1 _M. The inhibition of ATX by LPA and S1P is specific in that short-chain LPA (6:0) and other lipids have no effect, and the closely related ecto-phosphodiesterase NPP1, which lacks lysoPLD activity, is insensitive to LPA and S1P. Because ATX primarily functions as an LPA-generating lysoPLD, our findings imply that LPA is capable of controlling its own biosynthesis in the cellular environment. Product inhibition of ATX will prevent excessive accumulation of bioactive LPA in the extracellular milieu and may explain why steady-state plasma levels of LPA are very low despite the abundance of its precursor LPC. Our findings point to a novel role for S1P as an inhibitor of ATX, in addition to its well documented role as a receptor agonist. The important implication is that changes in extracellular S1P levels may greatly influence LPA production and signaling. Identification and characterization of the LPA/S1P binding site(s) is a major challenge for future studies and will likely facilitate the development of selective ATX inhibitors that could be of value in anticancer therapy. Finally, Chapter 4 reports the generation and analysis of ATX-deficient mice. These animals die at embryonic day 9.5 (E9.5) with profound vascular defects in yolk sac and embryo. Furthermore, at E8.5, ATX-deficient embryos showed allantois malformation, neural tube defects, and asymmetric headfolds. The onset of these abnormalities coincided with increased expression of ATX and LPA receptors in normal embryos. ATX heterozygous mice appear healthy but show half-normal ATX activity and plasma LPA levels. Our results reveal a critical role for ATX in vascular development, indicate that ATX is the major LPA-producing enzyme in vivo Since the observed phenotype is strongly reminiscent of the G_13 knockout mouse, we suggest that the vascular defects in ATX-deficient embryos may be explained by loss of LPA signaling through G_13. In conclusion, our studies show that the production of LPA has important roles in both physiological and pathophysiological conditions. LPA can be produced by spider and bacterial PLS SMases D which may play a role in the pathophysiology of secreted SMases D. Furthermore, the LPA-producing enzyme ATX was shown to be subject to inhibition by LPA and S1P at physiological relevant concentrations. Finally, ATX was shown to be essential for embryonic vascular development. Our studies provide a basis for future studies on the regulation of ATX and its role in physiological and pathophysiological processes, in particular tumor progression.