Cloning and analysis of PEDF receptor as an anti-angiogenic factor.

本课题的目的是鉴定PEDF的受体，并分析其功能。1、PEDF抗血管生成活性部位的鉴定。(1)细胞外基质结合域突变体我们寻找了PEDF发挥抗血管生成活性的关键区域。定点诱变用于制备PEDF突变体。这些突变体表达肝素结合缺陷型PEDF（R148 A）、葡萄糖基化缺陷型（N284 Q）和胶原结合缺陷型（D299 N）。将稳定表达这些突变体的HeLa细胞移植到裸鼠体内。3周后，定量肿瘤大小和肿瘤中的血管密度。PEDF野生型表达肿瘤小于模拟表达肿瘤。另一方面，表达D299 N突变体的肿瘤与表达模拟物的肿瘤大小相同。肿瘤内微血管密度与肿瘤大小相关。这些结果表明PEDF的胶原结合结构域是发挥抗血管生成作用所必需的。(2)C端缺失突变体为了寻找PEDF的最小必需序列，用一系列缺失突变体表达载体转染HEK 293细胞。不幸的是，这些截短的突变蛋白保留在细胞中。因此，我们决定使用全长PEDF作为PEDF受体的配体。我们成功地用镍柱纯化了His 6-PEDF-DsRed融合蛋白。接下来，我们将开始PEDF受体的表达克隆。2、PEDF的转录调控我们也发现了PEDF的转录调控。我们分离了PEDF的12 kb启动子区。我们研究了EPC-1/PEDF启动子活性是否受到细胞老化的影响，使用人二倍体肺成纤维细胞培养。我们发现EPC-1/PEDF基因的启动子/增强子区位于转录起始位点上游1760 bp处，受衰老和细胞周期的调控。

The aim of this project was to identify the receptor of PEDF, and analyze the function of this receptor.1, Identification of active site of PEDF in anti-angiogenic effect.(1) Extracellular matrix binding domain mutantsWe searched an essential region to exert anti-angiogenic activity of PEDF. Site direct mutagenesis was used to make PEDF mutants. These mutants express heparin binding deficient PEDF (R148A), glucosylation deficient (N284Q) and collagen binding deficient (D299N). HeLa cells expressing these mutants stably were grafted to nude mouse. After 3 weeks, tumor size and blood vessel density in the tumor were quantified. The PEDF wild type expressing tumor was smaller than mock expressing tumor. On the other hand, D299N mutant expressing tumor was the same size as the mock expressing tumor. The density of microvessel in tumor was correlated with the size of tumor. These results suggest the collagen binding domain of PEDF is essential to exert anti-angiogenic effect.(2) C-terminus deletion mutantsTo find the minimal essential sequence of PEDF, HEK293 cells were transfected with a series of deletion mutant expression vectors. Unfortunately, these truncated mutant proteins retained in the cell. Therefore, we decided to use the full-length PEDF as a ligand for PEDF receptor. Now we succeeded in the purification of His6-PEDF-DsRed fusion protein using nickel column. Next, we will begin the expression cloning of PEDF receptor.2, Transcriptional regulation of PEDFWe also find the transcriptional regulation of PEDF. We isolated the 12kb promoter region of PEDF. We examined whether or not EPC-1/PEDF promoter activity was affected by the cellular ageing using human diploid lung fibroblast cells in culture. We find the promoter/enhancer region of EPC-1/PEDF existed at more than 1760bp upstream from the transcriptional initiation site of the gene, and was regulated by both aging and cell cycle.