Summary
Megakaryocytopoiesis is governed in the bone marrow microenvironment by cellular interactions
that include various adhesion receptor systems and pericellular proteolysis for proper
regulation of cell motility and differentiation. In order to define the role of cell
surface molecules required for these processes, we searched for protease receptors
on these cells. In an in vitro system utilizing different cell lines of the megakaryoblastic
lineage (MEG-01, Dami), low level surface expression of the urokinase (uPA) receptor
was noted. Following stimulation with phorbolester (PMA), a 3-6 fold higher expression
of uPA receptor over a period of up to 5 days could be observed by fluorescent activated
cell-sorting as well as by direct ligand-binding of amino-terminal fragment of uPA
or vitronectin. Together with elevated expression of αIIbβ3-integrin (glycoprotein
IIb/IIIa complex), double immuno-fluorescence staining of stimulated cells confirmed
the increased cell surface localization of uPA receptor. Semi-quantitative RT-PCR,
ligand blot analysis and measurement of cell-bound proteolytic activity revealed a
differentiation-dependent upregulation of the uPA receptor expression in megakaryoblastic
cell lines as in monocytic cells. Due to its glycolipid anchorage, incubation with
phosphatidyl-inositol-specific phospholipase C reduced uPA receptor-mediated ligand
binding by about 60%. uPA receptor mRNA was expressed in cultured megakaryocytes derived
from bone marrow, whereas no uPA receptor mRNA was detectable in platelets. These
results indicate a differentiation-dependent increase in the expression of uPA receptor
in megakaryoblastic cells. The characteristics of surface expression and functionality
of the receptor on megakaryocytic cells may influence their maturation by regulating
cellular communication in the bone marrow micro-environment.
