CLUSTERING OF SPECIFIC MOLECULES IN SHED VESICLES.

In several tumor cell lines serum addition causes release of vesicles that bud from the cell surface and can be purified from cell conditionated media. These vesicles are known to be involved in cell migration and tumor progression. We recently demonstrated that FGF-2, a growth factor devoid of the classical signaling sequence, is secreted as a component of these vesicles. In order to analyze how molecules are clustered in shed vesicles we followed their intracellular movements by immunofluorescence techniques. The role of cytoskeletal components was analyzed using molecules such as paclitaxol, nocodazole, colchicin and cytochalasin which destabilize their organization. In the absence of serum, no clear localization of FGF-2 was observed. After serum addition, FGF-2 was localized partially in the nucleus and nucleolus, and partially in granules near the plasma membrane. Nocodazole and paclitaxol, which interfere with microtubular organization, inhibit FGF-2 nuclear localization but do not appear to modify FGF-2 movements toward the plasma membrane. Cytocalasine, which interferes with actin polymerization, decreases FGF-2 clustering in granules localized near the cell membrane. In summary, microtubular organization seems to be required for FGF-2 nuclear localization while actin filaments appear to be needed for FGF-2 translocation toward the plasma membrane. In a different set of experiments, we analyzed localization of neutral ceramidase (ncDase) and of Sphingosine Kinase (SphK). Ceramidase catalyzes ceramide hydrolysis giving rise to sphingosine, which in turn can be phosphorilated to S1P by SphK. Sp1P is an important signaling molecule involved in induction of cell migration and apoptosis. SphK-1 was known to be shed into the extracellular medium by an unconventional mechanism, we hypothesized that shed vesicles could vehicle its release. We therefore analyzed the localization of membrane-bound isoforms of ceramidase (ncDase) and of SphK (SphK-1 and SphK-2) by western blotting and Immunofluorescence techniques. Immunolocalization showed that ncDase is located into the plasma membrane and in cellular extensions. The concentration of ncDase was found to be higher in extracts of shed vesicles than in cell extracts. SphK-1 was found to be localized in plasma membrane and in vesicles, which appear to be enriched in this enzyme. SphK-2 was preferentially located in the nucleus and it was not detected in vesicles. In conclusion, ncDase and SphK were found to be clustered in shed vesicles. In order to analyze the role of SphK-1, either in the shedding phenomenon or in vesicle functions, we used transiently transfected SK-Hep1 cells, which overexpress SphK or express a non-functional mutant of this enzyme

Shed vesicles are involved in the release of some leaderless proteins.

Most proteins destined for secretion in the extracellular matrix are characterized by the presence of N-terminal signal peptides which direct their translocation into the endoplasmic reticulum, they are subsequently transferred to the Golgi apparatus and then secreted in the extracellular space. A growing number of secreted proteins, are being identified which, however, lack signal peptides allowing their entrance into the endoplasmic reticulum. They include the inflammatory cytokine interleukin 1b, galactins, macrophage migration inhibitory factor (MIF), acid and basic fibroblast growth factors (FGF-1, FGF-2) and Sphingosine kinase1(SphK-1). These proteins are secreted from the cell by unconventional processes which are the subject of numerous studies. Several types of normal and tumor cells can release in the extracellular medium microvesicles, called esovesicles, which result from budding of their plasma membranes. The vesicle diameter ranges between 100nm and 1000nm, the vesicle composition and function depends on the kind of the cell from which they have been produced. We already reported that FGF-2, a secreted lectin that transmits proangiogenic signals, and which is recognized as a potential oncoprotein able to modulate tumour growth and malignancy (Sorensen et al 2006), is released from SkHep1 cells, and from transfected NIH 3T3 cells through vesicle shedding (Taverna et al.2003). Now we are trying to elucidate the intracellular route followed by the growth factor from the site of synthesis to vesicles budding from the cell membrane. Actin filaments appear to be a binary for this intracellular trafficking. After 6h of treatment with cytocalasine, a drug that interferes with actin polymerization, the amount of vesicles was in fact decreased and FGF-2 clustering in granules localized near the cell surface was avoided. On the contrary no effects were observed when cells were treated with drugs which interfere with microtubule polymerization or de-polymerization. We also observed that FGF-2 granules are not included in lipid-coated vesicles. We are also analyzing the possibility that esovesicles are involved in the secretion of another leader-less signalling protein: Sphingosine kinase1 (SphK1). SphK1 has been shown to regulate a wide variety of cellular processes, including promotion of cell proliferation, survival and motility (Spiegel et al. 2003). SphK1 is primarily localized in the cytosol; when a signal induces the phosphorylation of Ser 225 of SphK1 through the activation of MAPK and ERK1/2, the molecule is translocated in plasma membranes and the involvement of actin filaments in its targeting has been reported (Pitson et. al. 2003). Three SphK1 isoforms having a different number of amino acids (384, 398 and 470) have been identified, we found that extracellular vesicles are enriched in the 47kDa isoform. SphK assays with TLC confirm that the enzyme is present in shed vesicles and that it has enzymatic activity. The substrate Sphingosine is also present in esovesicles therefore shed vesicles are likely to be a site of Sphingosine 1 Phosphate production

An Active Form of Sphingosine Kinase-1 Is Released in the Extracellular Medium as Component of Membrane Vesicles Shed by Two Human Tumor Cell Lines

Expression of sphingosine kinase-1 (SphK-1) correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P), a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis

Urokinase Plasminogen Activator and Gelatinases Are Associated with Membrane Vesicles Shed by Human HT1080 Fibrosarcoma Cells

Membrane vesicles are shed by tumor cells both in vivo and in vitro. Although their functions are not well understood, it has been proposed that they may play multiple roles in tumor progression. We characterized membrane vesicles from human HT1080 fibrosarcoma cell cultures for the presence of proteinases involved in tumor invasion. By gelatin zymography and Western blotting, these vesicles showed major bands corresponding to the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2) and to the MMP-9. tissue inhibitor of metalloproteinase 1 complex. Both gelatinases appeared to be associated with the vesicle membrane. HT1080 cell vesicles also showed a strong, plasminogen-dependent fibrinolytic activity in 125I fibrin assays; this activity was associated with urokinase plasminogen activator, as shown by casein zymography and Western blotting. Urokinase was bound to its high affinity receptor on the vesicle membrane. Addition of plasminogen resulted in activation of the progelatinases associated with the vesicles, indicating a role of the urokinase-plasmin system in MMP-2 and MMP-9 activation. We propose that vesicles shed by tumor cells may provide a large membrane surface for the activation of membrane-associated proteinases involved in extracellular matrix degradation and tissue invasion

Membrane vesicles shed by oligodendroglioma cells induce neuronal apoptosis

In order to investigate the mechanism by which oligodendrogliomas cause neuronal damage, media conditioned by G26/24 oligodendroglioma cells, were fractionated into shed vesicles and vesicle-free supernatants, and added to primary cultures of rat fetal cortical neurons. After one night treatment with vesicles, a reproducible, dose-dependent, inhibitory effect on neurite outgrowth was already induced and, after 48-72 h of incubation, neuronal apoptosis was evident. Vesicle-free supernatants and vesicles shed by NIH-3T3 cells had no inhibitory effects on neurons. Western blot analyses showed that treated neurons expressed a decreased amount of neurofilament (NF), growth-associated protein (GAP-43) and microtubule-associated protein (MAP-2). Moreover procaspase-3 and -8 were activated while Bcl-2 expression was reduced. Vesicles were found positive for the proapoptotic molecule, Fas-ligand (Fas-L), and for the B isoform of Nogo protein, a myelin component with inhibitory effects on neurons. Nogo B involvement in the vesicle effects was analyzed both by testing the neutralizing capability of anti-Nogo antibodies and by removing the Nogo receptor from neurons by phospholipase C digestion. These treatments did not revert the vesicle effects. To test the role of Fas-L, vesicles were treated with functional anti-Fas-L monoclonals. Vesicle inhibitory and proapoptotic effects were reduced. Vesicles shed by ovarian carcinoma cells (OvCa), which are known to vehicle biologically active Fas-L, had similar effects on neurons to those of oligodendroglioma vesicles, and their inhibitory effects were also reduced by anti Fas-L antibodies. We therefore conclude that vesicles shed by G26/24 cells induce neuronal apoptosis at least partially by a Fas-L mediated mechanism