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

尿激酶型纤溶酶原激活物(UPA)及其快速抑制物纤溶酶原激活物抑制物-1(PAI-1)在细胞迁移和血管生成中起重要作用。在本研究中，我们重点分析了参与细胞表面纤溶的这些因素的分子相互作用，以利用实时成像设备来调节细胞的迁移。取得的结果如下。PAI-1通过α_Vβ_5整合素抑制人纤维肉瘤细胞(HT1080)与玻璃连结蛋白(VN)的黏附，并刺激细胞从VN向IV型胶原(COL)迁移。PAI-1对VN和Col的黏附作用强于纤维连接蛋白(Fn)，而PAI-1仅干扰细胞对VN的黏附，对Col和Fn均无影响。从FN向COL迁移的细胞数量明显多于从VN或COL迁移的细胞，细胞似乎从亲和力较低的细胞外基质(ECM)迁移到较高亲和力的ECM。此外，整合素拮抗剂RGD肽和抗α_Vβ_5整合素抗体类似地抑制细胞与VN的附着，刺激细胞从VN向COL迁移，从而降低细胞对细胞外基质的亲和力，似乎刺激细胞向亲和力较高的细胞迁移。另一方面，uPA并不直接改变细胞黏附，而是逆转了PAI-1对VN细胞黏附的抑制作用和对细胞从VN向COL迁移的刺激作用，因此uPA和PAI-1可能通过α_Vβ_5整合素改变细胞对VN的黏附，从而改变肿瘤细胞的迁移。PAI-1对细胞迁移的促进作用可能与其促肿瘤作用有关。我们还成功地将绿色荧光蛋白(GFP)标记的uPAR转化HT1080细胞。我们开始分析uPAR、uPA和PAI-1在VN上迁移细胞表面的相互作用。

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.