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Elucidation of role of beta ig-3 derived from osteoclasts in bone resorption and formation

阐明破骨细胞衍生的 β ig-3 在骨吸收和形成中的作用

基本信息

批准号：
15591946
负责人：
HAKEDA Yoshiyuki
金额：
$ 2.24万
依托单位：
Meikai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Background : Osteoclastogenesis is promoted by a trigger molecule, receptor activator of NF-κB(RANKL). However, the molecular mechanism for RANKL-induced osteoclast formation and how the osteoclast progenitors are committed to differentiate into osteoclasts have remained to be investigated. Through our project to seek for master genes for osteoclast generation dependent of RANKL-signals, we identified beta ig-h3, which is induced to be expressed in osteoclast progenitors in response to RANKL. In this study, we attempted to elucidate the role of osteoclast progenitor-derived beta ig-h3 in bone resorption by osteoclasts and bone formation by osteoblasts.Methods : We employed in vitro culture system of M-CSF dependent bone marrow cells-derived osteoclast progenitors for osteoclast formation. We searched for genes expressed in osteoclast progenitors that are dependent on RANKL-stimuli by fluorescent differential display.Results and Discussion : In osteoclast progenitors, gene expression of … More beta ig-h3 was induced within early times after exposure to RANKL. The gene expression was also induced by TGF-β and further amplified by the simultaneous addition of RANKL and TGF-β. The induction occurred earlier than the expressions of osteoclast differentiation-related molecules such as tartrate-resistant acid phosphatase (TRAP), cathepsin K, and calcitonin receptors. Since other bone cells such as osteoblasts did not express beta ig-h3, the expression is likely to be specific for osteoclast lineage. Beta ig-h3 contains RGD sequence, and is considered to be an adherent protein that associates with some integrins. Among such adherent proteins, osteopontin is also well known to be expressed in osteoclasts. However, the osteopontin is also expressed in osteoblasts, and is not specific for osteoclasts. In addition, the osteopontin expression did not depended on RANKL. We cloned full-length gene of beta ig-h3, and confirmed from compelling expression in HEK293 cells that the adherent molecule was a secretion protein. Since beta ig-h3 interacts with some integrins, we examined the expression of integrins in osteoclast progenitors. Among these integrins, expression of a V and β3 integrins was linked to osteoclast formation as well as beta ig-h3. Neutralizing antibody against β3 blocked the osteoclast generation in a culture of osteoclast progenitors with M-CSF, TGF-β, and RANKL. Since the culture of osteoclast progenitors does not contain other cells, the molecule associated with β3 integrin is the progenitor-derived one. Taken together, beta ig-h3 would play a possible role in Osteoclastogenesis as a positive regulator. Less

背景：破骨细胞的形成是由一种触发分子，NF-κB受体激活因子（RANKL）促进的。然而，rankl诱导的破骨细胞形成的分子机制以及破骨细胞祖细胞如何分化为破骨细胞仍有待研究。通过我们寻找依赖RANKL信号的破骨细胞生成主控基因的项目，我们确定了β - ig-h3，该基因在RANKL的诱导下在破骨细胞祖细胞中表达。在这项研究中，我们试图阐明破骨细胞祖细胞衍生的β - ig-h3在破骨细胞骨吸收和成骨细胞骨形成中的作用。方法：采用M-CSF依赖性骨髓细胞源性破骨细胞祖细胞体外培养系统进行破骨细胞的形成。我们通过荧光差异显示寻找依赖rankl刺激的破骨细胞祖细胞中表达的基因。结果与讨论：在破骨细胞祖细胞中，RANKL暴露后早期诱导β - ig-h3基因表达增加。TGF-β诱导基因表达，同时加入RANKL和TGF-β进一步扩增基因表达。诱导发生的时间早于破骨细胞分化相关分子的表达，如抗酒石酸酸性磷酸酶（TRAP）、组织蛋白酶K和降钙素受体。由于其他骨细胞如成骨细胞不表达β - ig-h3，因此这种表达可能是破骨细胞谱系所特有的。β - ig-h3含有RGD序列，被认为是一种与某些整合素相关的粘附蛋白。在这些粘附蛋白中，骨桥蛋白也被认为在破骨细胞中表达。然而，骨桥蛋白也在成骨细胞中表达，而不是针对破骨细胞。骨桥蛋白的表达不依赖于RANKL。我们克隆了β ig-h3的全长基因，并通过在HEK293细胞中的显著表达证实了其为分泌蛋白。由于β - ig-h3与一些整合素相互作用，我们检测了整合素在破骨细胞祖细胞中的表达。在这些整合素中，a - V和β3整合素的表达与破骨细胞的形成以及β ig-h3有关。在M-CSF、TGF-β和RANKL的破骨细胞祖细胞培养中，抗β3中和抗体阻断了破骨细胞的生成。由于培养的破骨细胞祖细胞不含其他细胞，与β3整合素相关的分子是祖细胞衍生的分子。综上所述，β - ig-h3可能在破骨细胞形成中发挥积极调节作用。少