Inserm U858 Team 10, Dept of Biochemistry and Molecular Biology, Faculty of Medicine-Rangueil, University Paul Sabatier Toulouse-3, IFR-150, Toulouse, France.
Cell Signal. 2009 Dec;21(12):1925-34. doi: 10.1016/j.cellsig.2009.08.010. Epub 2009 Sep 6.
Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin alpha(v)beta(3), evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the alpha(v)beta(3) blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin alpha(v)beta(3) interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin alpha(v)beta(3), uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin alpha(v)beta(3) and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.
纤溶酶原激活物参与多种疾病的发病机制，如炎症性疾病和癌症。除了丝氨酸蛋白酶活性外，这些药物还触发参与细胞迁移、黏附和增殖的信号通路。我们之前报道过鞘脂代谢途径在纤溶酶原激活物有丝分裂效应中的作用，但中性鞘磷脂酶-2（NSMase-2）激活（鞘脂代谢途径的第一步）所涉及的信号机制知之甚少。本研究旨在探讨尿激酶型纤溶酶原激活物（uPA）如何激活 NSMase-2。我们报告说，uPA 及其催化失活的 N-氨基片段 ATF 触发 MMP-2、NSMase-2 和 ERK1/2 的顺序激活，这是 uPA 诱导的 ECV304 增殖所必需的，如 Marimastat（一种 MMP 抑制剂）、MMP-2 特异性 siRNA、MMP-2 缺陷和 NSMase 特异性 siRNA 的抑制作用所评估的那样。此外，在 uPA 刺激下，uPAR、MT1-MMP、MMP-2 和 NSMase-2 与整合素α（v）β（3）相互作用，这一点通过共免疫沉淀和免疫细胞化学实验得到证明。此外，α（v）β（3）阻断抗体抑制了 uPA 触发的 MMPs/uPAR/整合素α（v）β（3）相互作用、NSMase-2 激活、Ki67 表达和 ECV304 中的 DNA 合成。总之，uPA 触发整合素α（v）β（3）、uPAR 和 MMPs 之间的相互作用，导致 NSMase-2 和 ERK1/2 的激活和细胞增殖。这些发现强调了 uPA 的一个新的信号机制，并表明在 uPA 刺激下，uPAR、MMPs、整合素α（v）β（3）和 NSMase-2 形成一个信号复合物，参与 ECV304 细胞中的有丝分裂信号。
