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

SHP-1是一种磷酸酶，其磷酸酶活性的降低导致破骨细胞骨吸收活性的显著增加，提示SHP-1是破骨细胞活性的负向调节因子。在这里，我们计划通过设计激动剂来开发破骨细胞活性抑制剂，这将增加SHP-1与其结合蛋白之间的相互作用。我们已经用MALDI-TOF系统在转染SHP-1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。然而，这些蛋白是SHP-1的片段或非特异性结合蛋白，因此我们无法进入下一步，如使用siRNA筛选功能蛋白和开发SHP-1激动剂。为了解决这个问题，我们也曾尝试在小鼠巨噬细胞系RAW 264.7中使用GST pull-down法寻找SHP-1结合蛋白，但我们无法鉴定出该蛋白。尽管有这些结果，但应该提到的是，测量破骨细胞活性的新系统已经通过使用测量三维表面形态的设备建立起来，该设备是从该资助中购买的，用于筛选影响破骨细胞活性的功能蛋白。以往对破骨细胞活性的评价是通过测量吸收坑的二维面积获得的，对破骨细胞活性的评价还不确定。在不久的将来，该评价系统将成为药物开发的有力工具。

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.