Most proteins destined for secretion in the extracellular matrix are characterized by the presence ofN-terminal signal peptides which direct their translocation into the endoplasmic reticulum, they aresubsequently 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 peptidesallowing their entrance into the endoplasmic reticulum. They include the inflammatory cytokineinterleukin 1b, galactins, macrophage migration inhibitory factor (MIF), acid and basic fibroblastgrowth factors (FGF-1, FGF-2) and Sphingosine kinase1(SphK-1). These proteins are secreted fromthe 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 diameterranges between 100nm and 1000nm, the vesicle composition and function depends on the kind ofthe cell from which they have been produced. We already reported that FGF-2, a secreted lectin thattransmits proangiogenic signals, and which is recognized as a potential oncoprotein able tomodulate tumour growth and malignancy (Sorensen et al 2006), is released from SkHep1 cells, andfrom 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 ofsynthesis to vesicles budding from the cell membrane. Actin filaments appear to be a binary for thisintracellular trafficking. After 6h of treatment with cytocalasine, a drug that interferes with actinpolymerization, the amount of vesicles was in fact decreased and FGF-2 clustering in granuleslocalized near the cell surface was avoided. On the contrary no effects were observed when cellswere treated with drugs which interfere with microtubule polymerization or de-polymerization. Wealso 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 anotherleader-less signalling protein: Sphingosine kinase1 (SphK1). SphK1 has been shown to regulate awide 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 thephosphorylation of Ser 225 of SphK1 through the activation of MAPK and ERK1/2, the molecule istranslocated in plasma membranes and the involvement of actin filaments in its targeting has beenreported (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 the47kDa isoform. SphK assays with TLC confirm that the enzyme is present in shed vesicles and thatit has enzymatic activity. The substrate Sphingosine is also present in esovesicles therefore shedvesicles are likely to be a site of Sphingosine 1 Phosphate production.
|Publication status||Published - 2006|