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