2005 Fiscal Year Final Research Report Summary
Cloning and analysis of PEDF receptor as an anti-angiogenic factor.
| Project/Area Number |
16390537
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pathobiological dentistry/Dental radiology
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
NAKAHAMA Ken-ichi Tokyo Medical and Dental Univ., Cellular Physiological Chem., Associate Prof., 大学院・医歯学総合研究科, 助教授 (60281515)
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| Co-Investigator(Kenkyū-buntansha) |
MORITA Ikuo Tokyo Medical and Dental Univ., Cellular Physiological Chem., Professor, 大学院・医歯学総合研究科, 教授 (60100129)
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| Project Period (FY) |
2004 – 2005
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| Keywords | PEDF / cancer / promoter / cloning / receptor |
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| Research Abstract |
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 mutants
We 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 mutants
To 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 PEDF
We 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.
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