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The functional analysis of RANKL signaling in osteoclast for developing the agonist of SHP-1

破骨细胞中RANKL信号传导的功能分析用于开发SHP-1激动剂

基本信息

批准号：
16390530
负责人：
AOKI Kazuhiro
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

项目摘要

The SHP-1 is the phosphatase and the reduction of its phosphatase activity leads to the significant increase of bone resorption activity of osteoclasts, suggesting that SHP-1 works as a negative regulator of osteoclast activity. Here we have planned to develop the inhibitor of osteoclast activity by designing the agonist, which would increase the interaction between SHP-1 and its binding protein. We had already found SHP-1 binding proteins by using the MALDI-TOF system in SHP-1-transfected 293Tcells. The binding proteins were La binding protein, ribosome protein L4, protein tyrosine phosphatase 1c, KRT19. These proteins were, however, the fragments of SHP-1 or non-specific binding proteins, thereby we could not get in the next step such as screening for the functional proteins by using siRNA and develop the agonist of SHP-1. To solve this problem, we have also tried to find the SHP-1 binding protein by using the GST pull-down assay in murine macrophage cell line, RAW 264.7, but we could not identify the proteins. In spite of these results, it should be mentioned that the new system for measuring osteoclast activity has been established by using the device for measuring the 3-dimensional surface morphology which was purchased from this grant for screening the functional proteins which affects osteoclast activity. Previously the assessment of osteoclast activity was obtained by measuring 2-dimentinal area of the resorption pits, and the evaluation of the osteoclast activity has not been certain. In the near future, this evaluation system would be a powerful tool for the drug development.

SHP-1是磷酸酶，其磷酸酶活性的降低导致破骨细胞的骨吸收活性显着增加，表明SHP-1作为破骨细胞活性的负调节剂。在这里，我们计划通过设计激动剂来开发破骨细胞活性的抑制剂，这将增加SHP-1与其结合蛋白之间的相互作用。我们已经通过使用 MALDI-TOF 系统在 SHP-1 转染的 293T 细胞中发现了 SHP-1 结合蛋白。结合蛋白为La结合蛋白、核糖体蛋白L4、蛋白酪氨酸磷酸酶1c、KRT19。但这些蛋白都是SHP-1的片段或非特异性结合蛋白，因此无法进行下一步利用siRNA筛选功能蛋白以及开发SHP-1激动剂的步骤。为了解决这个问题，我们还尝试在小鼠巨噬细胞系RAW 264.7中使用GST Pull-down实验来寻找SHP-1结合蛋白，但我们无法识别这些蛋白。尽管有这些结果，应该提到的是，通过使用本次拨款购买的用于筛选影响破骨细胞活性的功能蛋白的用于测量3维表面形态的装置，建立了用于测量破骨细胞活性的新系统。以往对破骨细胞活性的评估是通过测量吸收坑的二维面积来获得的，对破骨细胞活性的评估尚不明确。在不久的将来，这个评价系统将成为药物开发的有力工具。