Analysis of biological function by t-PA receptor gene expression regulation

我们先前已经证明，从人脐静脉内皮细胞中纯化的组织型纤溶酶原激活剂受体(t-PAR)与t-PA具有特异性的相互作用。根据这些数据，我们获得了在大肠杆菌中表达的重组t-PAR。T-PAR与t-PA特异性结合，通过配基印迹分析鉴定。在本研究中，为了研究t-PAR在细胞表面的生理作用，我们用myc/His标记的t-PAR表达载体稳定地转染内皮细胞。用半定量聚合酶链式反应和免疫印迹法检测了t-PA和t-PAR/myc/His融合蛋白的表达，并用IAsys检测了t-PA与过表达的内皮细胞的结合能力。与对照(LacZ)相比，t-PAR的结构性表达增加了内皮细胞与t-PA的结合信号。DFP灭活的t-PA对t-PAR的相互作用没有任何影响。提示t-PA与细胞表面t-PAR的结合不依赖于t-PA的催化活性。发色实验显示，t-PAR高表达的内皮细胞在加入t-PA后具有明显的纤溶酶原激活物活性。T-PAR诱导的PA活性可被t-PA抗体抑制，提示t-PAR可能使t-PA聚集在细胞表面，增强细胞周围的纤溶活性。此外，我们还证明了t-PAR在加入t-PA后促进了EC的增殖。因此，t-PAR可能通过信号转导途径介导内皮细胞的增殖。

We have previously demonstrated that tissue-type plasminogen activator receptor (t-PAR) purified from human umbilical vein endothelial cells specifically interacted with t-PA. Following these data, we have produced recombinant t-PAR expressed in E.coli. t-PAR bound to t-PA specifically, which was identified by ligand blot analysis. In the present study, to investigate the physiological role of t-PAR on cell surface, we have stably transfected endothelial cells (ECs) with a myc/His-tagged t-PAR expression plasmid. Expression of both the mRNA and t-PAR/myc/His fusion protein was confirmed by semi-quantitative PCR and western blotting.The binding ability of t-PA to t-PAR-overexpressed ECs was studied by using IAsys. Constitutive expression of t-PAR increased binding signal of ECs to t-PA compared with control (LacZ-transfected) ECs. The DFP-inactivated t-PA did not give any effect on the interaction to t-PAR. It was suggested that the binding of t-PA to t-PAR on the surface of cells was independent on catalytic activity of t-PA. On the fibrinolytic activity using chromogenic assay, t-PAR-overexpressed ECs showed marked plasminogen activator (PA) activity after the addition of t-PA. This t-PAR-induced PA activity could be inhibited by the addition of antibodies against t-PA, suggesting that t-PAR might concentrate t-PA on the surface of cell and enhanced the fibrinolytic properties around cells. Furthermore, we have demonstrated that t-PAR promoted proliferation of EC after the addition of t-PA. Thus, t-PAR could mediate proliferation of ECs via signal transduction pathway.