nCDase and SphK-1 localization in vesicles shed by tumour cells and their biological roles.

Sphingolipid metabolism is a dynamic process resulting in the formation of a number of bioactive metabolites including ceramide, ceramide-1-phosphate, sphingosine e sphingosine-1-phosphate (S1P). (Pyne and Pyne; Biochem. J. 2000; 349:385-402). Following sphingomyelinase activation, sphingomyelin is hydrolyzed to ceramide, which is considered to be an inducer of cell growth arrest, differentiation and apoptosis. (Hannun et. al 1996; Science: 274:1855-1859). Ceramidase catalyzes the deacylation of ceramide to produce a free fatty acid and sphingosine. The enzyme sphingosine kinase (SphK) catalyzes the formation of S1P from sphingosine and ATP (Olivera et al. J.Biol.Chem. 1998; 273:12576- 12583). Two distinct SphK isoforms, SphK1 and SphK2, have been cloned and characterized. (Liu et.al. J.Biol.Chem. 2000; 275: 19513-19520) and recently, alternatively spliced variants of human SphK1 and SphK2, differing in their amino terminal portions, have also been described (Billich et. al J.Biol.Chem. 2003; 278; 47408-47415). SphK1 and SphK2 differ in their relative tissue distribution, sub cellular localization and biochemical activities, consistent with distinct biologic functions for these two enzymes (Saba et al. Circ. Res. 2004; 94:724-734). SphK2 presents a nuclear localization signal sequence and is localized in the cell nucleus (Igarashi et. al. J.Biol.Chem 2003; 278: 46832-46839). SphK1 is primarily localized in the cytosol. PMA and TNFα induce the phosphorylation of SphK1 Ser 225, through the activation of MAPK and ERK1/2. Phosphorylation of SphK1results in its plasma membrane localization and in its activation. (Pitson et al. Embo J. 2003; 22: 5491-550). SphK1 is a cell surface-active kinase and an extracellular protein. As several secreted proteins, like for instance FGF-1 and FGF-2, SphK1 molecule lacks a conventional leader secretion signal sequence. The mechanism of its release from the cell occurs via a non classical pathway independent of the endoplasmic reticulum/Golgi system but requiring functional actin dynamics (Ancellin et al J.Biol.Chem. 2002; 277: 6667-6675). SphK1 activity, and therefore production of S1P at the cell periphery and/or in the extracellular medium, was shown to regulate a wide variety of cellular processes, including promotion of cell proliferation, survival and motility (Olivera et al. J.Biol.Chem. 2003; 278: 46452-46460). S1P is an important proangiogenic factor and its ability to promote capillary morphogenesis in endothelial cell is significantly enhanced when S1P is associated with FGF-2 (Harvey et al. J Lab. Clin. Med 2002; 140: 188-198). Since we already reported that FGF-2 release occurs by vesicle shedding (Taverna et. al. J.Biol.Chem. 2003; 278: 51911-51919), we hypothesized and tried to demonstrate the possibility that S1P is produced in shed vesicles and that it exerts a synergic role with vesicle associated FGF-2, in the induction of endothelial cell differentiation. We also considered the hypothesis that enzymes involved in sphingolipid degradation could play a role in vesicle shedding. Our experimental dates indicate: · nCDase and SphK1 are both present in shed vesicles in biologically active forms, together with their lipidic substrates. · The enzymes of sphingolipid metabolism are not involved in the process of vesicle shedding. · In SK Hep-1 hepatocarcinoma cells, which we used in most of our experiments, FGF-2 and both nCDase and SphK1 are simultaneously released in shed vesicles. · Shed vesicles exert chemiotactic effects on endothelial cells and have the ability to promote their morphogenesis in capillary-like structures. · Since these effects are typical of both FGF-2 and S1P, to neutralize effects of vesicle-associated FGF-2, we denatured the protein components of vesicles by a 10 minutes treatment at 100 C°. This treatment is known todenature FGF-2 (Vemuri et al. 1994; J Pharm Pharmacol. 46: 481-486) while S1P is reported to be stable at temperatures below 120 C° (Harvey et al. J Lab. Clin. Med 2002; 140: 188-198). Heat treatment substantially decreased the chemiotactic effect of vesicles but only had a small effect on their capability to induce formation of capillary-like structures. · Promotion of endothelial cell morphogenesis was increased in vesicles shed by cells overexpressing SphK1 and it was decreased in cells expressing a dominant negative SphK1 isoform. In summary our dates show that shed vesicles are vehicles for both FGF-2 and SphK1 secretion, and that FGF-2 and S1P, produced in the vesicle membrane by the joined catalytic action of nCDase and SphK1, exert a synergic action on endothelial cells inducing angiogenesis.