Vitronectin Concentrates Proteolytic Activity on the Cell Surface and Extracellular Matrix by Trapping Soluble Urokinase Receptor-Urokinase Complexes

Abstract

Urokinase-type-plasminogen activator (uPA) and its receptor are localized in the vessel wall where they are involved in cellular activation and remodelling processes. Besides the cell surface glycolipid (GPI)-anchored urokinase receptor (uPAR), which binds uPA with high affinity, recent evidence points to the existence of soluble uPAR (suPAR), as well. In the present study, the origin, binding mechanism, and cellular effects of suPAR were examined. Under basal conditions human vascular smooth muscle cells (HVSMC), human umbilical vein endothelial cells (HUVEC), and monocytic cells released 0.1 to 2 ng/mL suPAR, which was increased twofold to fivefold after phorbol ester (PMA) stimulation, as measured by a function-dependent enzyme-linked immunosorbent assay (ELISA). suPAR alone did not bind to HVSMC or HUVEC, but reduced cellular uPA binding by 50% to 70%. However, after removal of GPI-uPAR with phosphatidylinositol-specific phospholipase C, suPAR dose-dependently increased uPA binding by fourfold to fivefold. This increase in binding was completely inhibited by vitronectin (VN) and by a monoclonal antibody against VN, but not by other matrix proteins or antibodies. Thus, VN-mediated uPA binding to cells was regulated by the ratio of soluble to surface-associated uPAR. In a uPAR-deficient cell line (LM-TK−), suPAR increased uPA binding up to 10-fold, whereas the truncated receptor lacking the amino-terminal uPA-binding domain was ineffective. The formation of a ternary uPA/suPAR/VN-complex on the cell surface and the free extracellular matrix could be inhibited by a monoclonal antibody against VN, as well as by plasminogen activator inhibitor-1 (PAI-1). Moreover, VN-mediated binding of the uPA/suPAR-complex led to a fivefold increase in plasminogen activator activity. Through this novel pathway, VN concentrates the uPA/suPAR-complex to cell surfaces and extracellular matrix sites, leading to the accumulation of plasminogen activator activity required for cell migration and tissue remodelling processes.

Activation of hepatocyte growth factor by urokinase-type plasminogen activator is ionic strength-dependent

“…Although scHGF is genetically similar to plasminogen, the effects of ionic strength on scHGF cleavage are not specific for chloride anions, as previously demonstrated for plasminogen [19]. Our studies also demonstrate that soluble MET (HGF receptor) and Su-PAR (soluble u-PA receptor) inhibit scHGF cleavage, a potentially important result given by recent evidence demonstrating that naturally occurring forms of Su-PAR are present in the plasma and extracellular spaces [20,21].…”

Enhanced release of soluble urokinase receptor by endothelial cells in contact with peripheral blood cells

“…in endothelial cells, smooth muscle cells and keratinocytes [5]. The U937 monocytic cell line has recently been shown to produce suPAR in culture [23,27], but to our knowledge, the present report is the first demonstration that blood cells produce suPAR in the plasma and that in normal cell culture conditions mononuclear cells and endothelial cells release suPAR to their growth medium without stimulation.…”

Expression of urokinase plasminogen activator and its receptor during acute renal allograft rejection

“…Endothelial cells also synthesize and release uPAR upon inflammation. Indeed, after stimulation with phorbol 12-myristate 13acetate (PMA), endothelial cells produce appreciable quantities of soluble uPAR in vitro [37] and we found a distinct synthesis and expression of uPAR by endothelial cells in the kidney during vascular rejection.…”