Effects of urokinase receptor occupancy on plasmin generation and proteolysis of basement membrane by human tumor cells.
The goal of the present study was to assess the relative importance of receptor-bound and secreted plasminogen activator urokinase (u-PA) in generating cell-surface plasmin and fostering destruction of normal tissue by tumor cells. We first showed that active site-inhibited u-PA could displace endogenous u-PA from the surface of the human colon adenocarcinoma cell line HCT 116. We then prepared expression vectors for u-PA and for a mutant molecule in which the codon for the active site serine residue was changed to encode alanine. Expression of non-functional mutant u-PA decreased the level of cell-bound active u-PA by more than 95% via a mechanism that involved competition for receptor sites. Decreased cell-surface u-PA activity was associated with a decrease in cell-bound plasmin activity to undetectable levels, suggesting that receptor-bound u-PA plays an important role in the generation of plasmin on the cell surface. Transfectants that secreted eightfold to 20-fold elevated levels of active wild-type u-PA showed approximately 50% increases in cell-associated u-PA and only twofold to fourfold increases in cell-associated plasmin, suggesting that the role of secreted u-PA in generating cell-surface plasmin activity was relatively minor. In parent cells and both types of transfectants there was a good correlation between the amount of plasmin bound to the tumor cell surface and the extent to which a basement membrane substrate was degraded. These studies show that receptor-bound u-PA provides an efficient mechanism for plasmin generation on the surface of tumor cells, which, in turn, contributes significantly to their degradative potential.
Cohen
R L
RL
Cancer Research Institute, University of California-San Francisco 94143.
Xi
X P
XP
Crowley
C W
CW
Lucas
B K
BK
Levinson
A D
AD
Shuman
M A
MA
D000230:Adenocarcinoma; D001483:Base Sequence; D001485:Basement Membrane / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D003110:Colonic Neoplasms; D005341:Fibrinolysin / Q000096:biosynthesis*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010960:Plasminogen Activators / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D011956:Receptors, Cell Surface / Q000502:physiology*; D055293:Receptors, Urokinase Plasminogen Activator; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
D015345:Oligonucleotide Probes; C524898:PLAUR protein, human; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D010960:Plasminogen Activators; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator