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

本研究旨在从分子水平阐明破骨细胞功能的调控机制。为了达到这个目的，我们首先需要一个具有功能的破骨细胞的纯群体，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，我们重点阐明了受体酪氨酸激酶在调节破骨细胞功能中的作用。在本研究中，我们克隆了8个在成熟破骨细胞上表达的受体酪氨酸激酶，包括成纤维细胞生长因子受体-1(FGFR-1)、Tyro3、血管内皮生长因子受体(FIT-1和Flk-1)、胰岛素受体、c-fms和c-met。除了这些受体酪氨酸激酶，我们还在破骨细胞中检测到一些受体丝氨酸/苏氨酸激酶，如前列腺素(PG)E2受体的一个亚型，EP4和骨形态发生受体(BMPR-IA和BMPR-II)。我们发现通过FGFR-1、Tyro3和VEGF受体的信号刺激破骨细胞性骨吸收和/或破骨细胞存活。这些受体与c-src、FAK、P13K和42/44P MAPK一起表达刺激效应。另一方面，PGE2通过EP4受体与Gs蛋白偶联抑制破骨细胞性骨吸收活性，而BMP-2通过其受体/Smads激活破骨细胞性骨吸收活性。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，这些结果为我们理解破骨细胞的调控机制提供了许多启示。

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.