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

Salvatrice Rigogliuso, Simona Taverna, Maria Letizia Vittorelli, Monica Salamone

Risultato della ricerca: Other contribution


Sphingolipid metabolism is a dynamic process resulting in the formation of a number of bioactive metabolitesincluding 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 aninducer of cell growth arrest, differentiation and apoptosis. (Hannun et. al 1996; Science: 274:1855-1859). Ceramidasecatalyzes 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 J.Biol.Chem. 2000;275: 19513-19520) and recently, alternatively spliced variants of human SphK1 and SphK2, differing in their aminoterminal portions, have also been described (Billich et. al J.Biol.Chem. 2003; 278; 47408-47415). SphK1 and SphK2differ in their relative tissue distribution, sub cellular localization and biochemical activities, consistent with distinctbiologic functions for these two enzymes (Saba et al. Circ. Res. 2004; 94:724-734). SphK2 presents a nuclearlocalization 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 theactivation of MAPK and ERK1/2. Phosphorylation of SphK1results in its plasma membrane localization and in itsactivation. (Pitson et al. Embo J. 2003; 22: 5491-550). SphK1 is a cell surface-active kinase and an extracellularprotein.As several secreted proteins, like for instance FGF-1 and FGF-2, SphK1 molecule lacks a conventional leadersecretion signal sequence. The mechanism of its release from the cell occurs via a non classical pathway independent ofthe 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 extracellularmedium, was shown to regulate a wide variety of cellular processes, including promotion of cell proliferation, survivaland motility (Olivera et al. J.Biol.Chem. 2003; 278: 46452-46460). S1P is an important proangiogenic factor and itsability to promote capillary morphogenesis in endothelial cell is significantly enhanced when S1P is associated withFGF-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 itexerts a synergic role with vesicle associated FGF-2, in the induction of endothelial cell differentiation. We alsoconsidered 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 lipidicsubstrates.· 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 andSphK1 are simultaneously released in shed vesicles.· Shed vesicles exert chemiotactic effects on endothelial cells and have the ability to promote theirmorphogenesis in capillary-like structures.· Si
Lingua originaleEnglish
Stato di pubblicazionePublished - 2007

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