The serine protease urokinase-type plasminogen activator (uPA) promotes matrix degradation
by many cell types, including the invasive extravillous trophoblast (EVT) of the human
placenta. The noncatalytic amino-terminal end of uPA binds to uPA receptors (uPARs)
expressed by these cells. A highly polarized expression of uPAR-bound uPA at the migration
front of EVT cells in situ suggests a functional role of uPA:uPAR interaction in EVT
cell migration. The present study examined whether uPA stimulates EVT cell migration,
independent of proteolytic function, and investigated some of the signaling pathways
involved. Using in vitro-propagated EVT cells in Transwell migration assays, both
uPA and its noncatalytic amino-terminal fragment (ATF) were shown to stimulate migration
through multiporous polycarbonate (pore size 8 microm) membranes. A uPAR-blocking
antibody inhibited basal and ATF-stimulated migration. Migration was found to be stimulated
by hypoxic conditions, which upregulates uPAR expression; this stimulation was abrogated
with the uPAR-blocking antibody, indicating the role of endogenous uPA in EVT cell
migration. Spectrofluorometric measurement of cytosolic calcium in cells treated with
uPA and ATF demonstrated a rapid rise in [Ca2+](i), which was prevented by pretreatment
of cells with thapsigargin, indicating a release from intracellular stores. Both basal
and ATF-mediated migratory responses were suppressed in the presence of selective
pharmacological inhibitors LY294002, U73122, and U0126, implicating the respective
roles of phosphatidinylinositol 3-kinase (PI 3-K), phospholipase C (PLC), and MEK1/2
in basal and ATF-stimulated migratory capacity. Taken together, these results demonstrate
that uPA:uPAR interaction stimulates EVT cell migration, independent of uPA enzymatic
activity, using the mitogen-activated protein kinase pathway and calcium signaling
events including the participation of PI 3-K and PLC. These findings are relevant
to clinical conditions of aberrant trophoblast migration, including spontaneous abortion,
preeclampsia, and choriocarcinoma.