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

作为抗血管生成因子的 PEDF 受体的克隆和分析。

• 批准号: 16390537
• 负责人: NAKAHAMA Ken-ichi
• 金额: $ 9.28万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390537/
• 关键词: PEDF; cancer; promoter; cloning; receptor; 血管新生; 癌治療; 発現クローニング

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.

本课题的目的是鉴定PEDF的受体，并分析该受体的功能。1、PEDF抗血管生成活性位点的鉴定。(1)细胞外基质结合域突变体我们寻找PEDF发挥抗血管生成活性的重要区域。使用定点直接诱变来制备PEDF突变体。这些突变体表达肝素结合缺陷 PEDF (R148A)、糖基化缺陷 (N284Q) 和胶原结合缺陷 (D299N)。将稳定表达这些突变体的HeLa细胞移植到裸鼠上。 3周后，对肿瘤大小和肿瘤中的血管密度进行量化。 PEDF野生型表达肿瘤比模拟表达肿瘤小。另一方面，表达D299N突变体的肿瘤与模拟表达肿瘤的大小相同。肿瘤中微血管的密度与肿瘤的大小相关。这些结果表明PEDF的胶原结合结构域对于发挥抗血管生成作用至关重要。(2)C末端缺失突变体为了找到PEDF的最小必需序列,用一系列缺失突变体表达载体转染HEK293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。现在我们成功地利用镍柱纯化了His6-PEDF-DsRed融合蛋白。接下来我们将开始PEDF受体的表达克隆。2、PEDF的转录调控我们还发现了PEDF的转录调控。我们分离了 PEDF 的 12kb 启动子区域。我们使用培养的人二倍体肺成纤维细胞检查了 EPC-1/PEDF 启动子活性是否受到细胞老化的影响。我们发现EPC-1/PEDF的启动子/增强子区域存在于该基因转录起始位点上游1760bp以上，并且受到衰老和细胞周期的调节。

项目成果

The effects of amniotic membrane on retinal pigment epithelial cell differentiation.

• 发表时间: 2005-01
• 期刊: Molecular vision
• 影响因子: 2.2
• 作者: K. Ohno-Matsui;S. Ichinose;K. Nakahama;Takeshi Yoshida;A. Kojima;M. Mochizuki;I. Morita

Age- and cell cycle-dependent changes in EPC-1/PEDF promoter activity in human diploid fibroblast-like (HDF) cells

• DOI: 10.1007/s11010-006-2680-0
• 发表时间: 2006-12-01
• 期刊: MOLECULAR AND CELLULAR BIOCHEMISTRY
• 影响因子: 4.3
• 作者: Kojima, Toshihiko;Nakahama, Ken-ichi;Morita, Ikuo
• 通讯作者: Morita, Ikuo

Involvement of the collagen I-binding motif in the anti-angiogenic activity of pigment epithelium-derived factor

• DOI: 10.1016/j.bbrc.2005.07.140
• 发表时间: 2005-09-30
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Hosomichi, J;Yasui, N;Morita, I
• 通讯作者: Morita, I