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

t-PA受体基因表达调控生物学功能分析

基本信息

批准号：
17590193
负责人：
MATSUO Osamu
金额：
$ 2.18万
依托单位：
Kinki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590193/
关键词：
biotechnology angiogenesis proteolysis activity gene expression regulation t-PA t-PAR system 蛋白分解活性

项目摘要

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.

我们以前已经证明，组织型纤溶酶原激活剂受体（t-PAR）纯化的人脐静脉内皮细胞特异性相互作用的t-PA。根据这些数据，我们已经产生了在大肠杆菌中表达的重组t-PAR。t-PAR与t-PA特异性结合，经配体印迹分析证实。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表达质粒稳定转染内皮细胞（EC）。半定量PCR和Western blotting检测t-PAR mRNA和t-PAR/myc/His融合蛋白的表达，IAsys检测t-PA与t-PAR过表达的内皮细胞的结合能力。与对照组（LacZ转染组）相比，组成型表达t-PAR的内皮细胞与t-PA的结合信号增强。GFP灭活的t-PA对t-PAR的相互作用无影响。结果表明，t-PA与细胞表面t-PAR的结合不依赖于t-PA的催化活性。显色法检测纤溶活性，t-PAR过表达的内皮细胞在加入t-PA后显示出明显的纤溶酶原激活物（PA）活性。t-PAR诱导的PA活性可被抗t-PA抗体所抑制，提示t-PAR可使t-PA在细胞表面聚集，增强细胞周围的纤溶活性。此外，我们已经证明，t-PAR促进EC增殖后，加入t-PA。因此，t-PAR可能通过信号转导途径介导内皮细胞的增殖。