CROSS-TALK BETWEEN THE PROTEOLYTIC AND NON-PROTEOLYTIC FUNCTIONS OF THE UROKINASE RECEPTOR

Urokinase (uPA) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homeostasis. Moreover the uPA-system plays an important role in many pathological events, such as tumour cell migration and dissemination. On the one hand, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. On the other hand, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin (VN). Although the existence of feedback loops between the functions of uPAR in extracellular proteolysis, cell adhesion and signalling has been described, some aspects of this cross-talk are still poorly understood and not characterized experimentally. We here report that cell surface plasminogen activation induces a potent negative feedback on cell adhesion to VN. The feedback is predominantly caused by proteolytic cleavage of the RGD-motif in VN catalyzed by both uPA and plasmin. In this process the cell-adhesive properties of VN are impaired by disruption of the integrin binding site and release of the somatomedin B (SMB) domain responsible for binding of uPAR. Cleavage of VN by uPA displays a remarkable receptor-dependence and requires concomitant binding of both uPA and VN to uPAR suggesting that the hydrolysis is accelerated by a mechanism of substrate presentation. VN represents the first described uPAR-dependent substrate of uPA and our findings therefore identify a potential novel function of uPAR in focusing the proteolytic activity of the plasminogen activation system onto extracellular matrix-associated VN. Additionally, SMB-containing N-terminal VN fragments are released by several cancer cell lines in vitro and are detectable in human urines samples. We have thus developed a clinical grade immunoassay for the detection and quantification of such fragments in urine samples with the aim of using the levels of urinary VN fragments as a novel cancer biomarker. Our working hypothesis is that this biomarker may be used as an indirect functional measurement of the uPA-system activity in the tumour tissue. Finally, we show that the specific urokinase inhibitor, plasminogen activator inhibitor 1 (PAI-1), blocks the negative feedback mediated by uPA and behaves as a potent uPA-dependent agonist of the interaction between uPAR and VN. Indeed, we report for the first time that the covalent complex between uPA and PAI-1 is endowed with higher agonistic activity compared to uPA. Taken together, these data might represent a molecular explanation of the poor clinical outcome observed in cancer patients with high levels of uPA and PAI-1.