Study on signal transduction in osteoclastogenesis for the development of anti-osteoporosis drugs.

破骨细胞发生的信号转导抑制被认为为开发抗骨质疏松药物提供了重要信息。我们已经成功地克隆了破骨细胞生成的重要因子RANKL，它在成骨细胞中作为膜相关因子表达，以响应许多骨吸收因子。利用破骨细胞形成的培养系统，我们研究了破骨细胞发生的信号转导，包括RANK-RANKL信号转导。研究结果如下：(1)在M-CSF存在下，TNFα刺激骨髓源性巨噬细胞的破骨细胞生成。(2)转染人PTH/PTHrP受体cDNA建立的人骨肉瘤细胞系SaOS-4/3亚克隆，在小鼠骨髓细胞共培养中支持破骨细胞形成以响应PTH。SaOS-4/3细胞RANKL和M-CSF mrna的表达在PTH的作用下上调。这两个因子均表达为膜相关因子。(3)碘霉素和A23187刺激小鼠骨髓细胞和成骨细胞共培养的破骨细胞形成。我们还发现，蛋白激酶C的激活剂PMA通过上调成骨细胞中RANKL的表达，在共培养中刺激破骨细胞形成。(4) BMP-2显著增强RANKL和M-CSF诱导的破骨细胞分化。在培养物中加入可溶形式的BMP- ia型受体不仅抑制RANKL和BMP-2诱导的破骨细胞形成，而且抑制RANKL单独支持的基础破骨细胞形成。骨髓巨噬细胞和成熟破骨细胞均表达BMP-2和BMP受体IA型mrna。这些发现为开发抗骨质疏松药物提供了重要信息。

Inhibition of signal transduction for osteoclastogenesis is thought to provide important information for development of anti-osteoporosis drugs. We have succeeded in molecular cloning of an essential factor for osteoclastogenesis, RANKL, which is expressed as a membrane associated factor in osteoblasts in response to many bone-resorbing factors. Using culture systems for osteoclast formation, we have studied signal transduction for osteoclastogenesis including RANK-RANKL signaling. The findings obtained from the study are as follows. (1) TNFα stimulated osteoclastogenesis in bone marrow-derived macrophage cultures in the presence of M-CSF.(2) SaOS-4/3, a subclone of the human osteosarcoma cell line SaOS-2, established by transfecting the human PTH/PTHrP receptor cDNA, supported osteoclast formation in response to PTH in co-culture with mouse bone marrow cells. Expression of mRNAs for RANKL and M-CSF by SaOS-4/3 cells was up-regulated in response to PTH.Both factors were expressed as membrane-associated factors. (3) Ionomycin and A23187 stimulated osteoclast formation in mouse co-cultures of bone marrow cells and osteoblasts. We also found that PMA, an activator of protein kinase C, stimulated osteoclast formation in the co-culture though up-regulation of RANKL expression in osteoblasts. (4) BMP-2 markedly enhanced osteoclast differentiation induced by RANKL and M-CSF.Addition of a soluble form of BMP receptor type-IA to the culture inhibited not only osteoclast formation induced by RANKL and BMP-2, but also the basal osteoclast formation supported by RANKL alone. Both bone marrow macrophages and mature osteoclasts expressed BMP-2 and BMP receptor type IA mRNAs. These findings provide important information for the development of anti-osteoporosis drugs.