Real time imaging analysis of molecular interactions of factors involved in fibrinolysis on cell surface to modify cell migration

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Both urokinase plasminogen activator (uPA) and its fast inhibitor, I.e. plasminogen activator inhibitor-1 (PAI-1) play important role in cell migration and angiogenesis. In the present study we focused to analyze molecular interaction of these factors involved in fibrinolysis on cell surface to modify cell migration employing real-time imaging apparatus. Results obtained are follows. PAI-1 inhibited the adhesion of the human fibrosarcoma cell (HT-1080) to vitronectin (Vn) via α_Vβ_5 integrin, and stimulates cell migration from Vn toward collagen type IV (Col). The cell attached more strongly to Vn and Col than fibronectin (Fn), while PAI-1 interfered with cell attachment to Vn only, affecting neither Col nor Fn. The number of cells that migrated from Fn toward Col was significantly higher than that from Vn or Col. The cell appeared to migrate from the extracellular matrix (ECM) of lower affinity toward ECM of higher one. Moreover, an integrin antagonist, RGD peptide, and anti- α_Vβ_5 integrin antibody, which similarly inhibited cell attachment to Vn, stimulated cell migration from Vn toward Col. Thus decreasing cell affinity for ECM seems to stimulate cell migration toward the one with higher affinity. On the other hand, uPA did not modify cell attachment directly, but reversed PAI-1 mediated inhibitory effect on cell adhesion to Vn, and its stimulatory effect on cell migration from Vn toward Col. Thus tumor cell migration appeared to be modified by uPA and PAI-1 altering cell adhesion to Vn via α_Vβ_5 integrin. Such enhancement of cell migration by PAI-1 may be related to its tumor promoting effect. We also succeeded to transform HT1080 cells with Green Fluorescent Protein (GFP) labeled uPAR. We started to analyze the interaction between uPAR, uPA and PAI-1 on migrating cell surface on Vn.