Study of roles of receptor tyrosine kinases in regulating osteoclast function.

本课题旨在从分子水平阐明破骨细胞功能调控的机制。为了实现这一目标，我们首先需要一个纯的功能性破骨细胞群，并成功地开发了一种分离成熟破骨细胞的新方法。利用分离的破骨细胞，我们着重阐明受体酪氨酸激酶在调节破骨细胞功能中的作用。在这项研究中，我们克隆了8个受体酪氨酸激酶，包括成纤维细胞生长因子受体-1（FGFR-1），酪氨酸3，血管上皮生长因子受体（Fit-1和Flk-1），胰岛素受体，c-fms和c-met，表达在成熟的破骨细胞。除这些受体酪氨酸激酶外，我们还检测了破骨细胞中的一些受体丝氨酸/苏氨酸激酶，如前列腺素（PG）E2受体亚型、EP 4和骨形态发生受体（BMPR-IA和BMPR-II）。我们发现通过FGFR-1、Tyro 3和VEGF受体的信号传导刺激骨细胞骨吸收和/或破骨细胞存活。这些受体与c-src、FAK、P13 K和42/44 p MAPK结合表达刺激作用。另一方面，PGE 2通过与GS-蛋白偶联的EP 4受体抑制骨吸收活性，而BMP-2通过其受体/Smads刺激骨吸收活性。因此，成熟的破骨细胞在其质膜上表达多种受体酪氨酸激酶和丝氨酸/苏氨酸激酶受体来调节其功能，这些结果为我们了解破骨细胞的调控机制提供了很多线索。

This project was undertaken to elucidate the mechanism for regulation of osteoclast function at molecular level. To address the aim, we first needed a pure population of functional osteoclasts, and succeeded in developing a new method for isolation of mature osteoclasts. Using the isolated osteoclasts, we focused on elucidating the roles of receptor tyrosine kinases in the regulation of osteoclastic function. In this study, we cloned eight receptor tyrosine kinases, including fibroblast growth factor receptor-1(FGFR-1),Tyro3, vascular epithelial growth factor receptors(Fit-1 and Flk-1), insulin receptor, c-fms, and c-met, expressed on mature osteoclasts. Besides these receptor tyrosine kinases, we also detected some receptor serine/threonine kinases such as a subtype of prostaglandin (PG) E2 receptor, EP4 and Bone morphogenetic receptors (BMPR-IA and BMPR-II) in osteoclasts. We found the signaling through FGFR-1,Tyro3, and VEGF receptors stimulated the osteoclastic bone resorption and/or the osteoclast survival. These receptors associated with c-src, FAK, P13K, and 42/44p MAPKs to express the stimulatory effects. On the other hand, PGE2 inhibited the osteoclastic bone-resorbing activity through the EP4 receptor coupled with Gs-protein, whereas BMP-2 stimulated the activity through its receptors/Smads. Thus, mature osteoclasts express many receptor tyrosine kinases as well as serine/threonine kinase receptors on their plasma membranes to regulate their functions, and these results provide us a lot of insights to understand the regulatory mechanism for osteoclast.