Proteome analysis of phosphorylated proteins associated with osteoclast formation using established osteoclast precursor cell line

使用已建立的破骨细胞前体细胞系对与破骨细胞形成相关的磷酸化蛋白质进行蛋白质组分析

基本信息

批准号：
16591835
负责人：
AMANO Shigeru
金额：
$ 2.3万
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：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

Osteoclasts are multinucleated giant cells with the capacity to resorb mineralized tissues. It is well known that osteoclasts are derived from monocyte/macrophage lineage. It is also well known that M-CSF and RANKL are dispensable factors for osteoclastogenesis. Binding of RANKL to its receptor, RANK, activates transcription factors including c-Fos, Mitf, PU.1, and NFATc 1, which are known to be important for osteoclastogenesis. However, a systematic analysis of qualitative and quantitative changes in nuclear proteins for osteoclastogenesis has not been performed. Recently, we established osteoclast precursor cell line 4B12 cells from the Mac-l^-c-Fms^+RANK^+ cell population in 14-day-old mouse embryonic calvarial bone cells. Therefore, we compared the proteomic changes between nuclear proteins prepared from 4B12 cells treated with M-CSF alone and nuclear proteins prepared from 4B12 cells treated with M-CSF and sRANKL using two dimensional gel electrophoresis (2D-PAGE). The 2D maps of … More nuclear proteins prepared from 4B12 cells treated with M-CSF alone displayed 327 protein spots, while the 2D maps of nuclear proteins prepared from 4B12 cells treated with M-CSF and sRANKL displayed 220 protein spots. Subtraction image analysis of both groups revealed that 101 spots newly appeared, 208 spots disappeared, 32 spots upregulated, 37 spots decreased in the SYPRO Ruby-stained nuclear proteins prepared from 4B12 cells treated with M-CSF and sRANK in comparison with M-CSF alone. Major 11 spots among the 101 spots, one spot (Mr 50kDa pI 5.5), 8 spots (Mr 50kDa pI 6-7), and 2 spots (Mr 60kDa pI 5.5-6.5) were stained by Pro-Q Diamond, suggesting that these proteins were phosphorylated. These spots were disappeared or reduced by treatment of p38 MAP kinase inhibitor SB202190(10 μM). These results suggested that the phosphorylated nuclear proteins may participate in osteoclastogenesis. Identification of these new proteins may lead to make discovery novel factors associated with the osteoclastogenesis, and provide to new clues to elucidate the mechanisms of control osteoclast specific differentiation. Less

破骨细胞是具有吸收矿化组织能力的多核巨细胞，众所周知，破骨细胞源自单核细胞/巨噬细胞谱系。还众所周知，M-CSF 和 RANKL 是破骨细胞生成中不可或缺的因子。 , RANK，激活转录因子，包括 c-Fos、Mitf、PU.1 和 NFATc 1，已知这些转录因子是然而，尚未对破骨细胞生成的核蛋白的定性和定量变化进行系统分析，最近，我们从 Mac-l^-c-Fms^+RANK^+ 细胞中建立了破骨细胞前体细胞系 4B12 细胞。因此，我们比较了仅用 M-CSF 处理的 4B12 细胞制备的核蛋白与 4B12 制备的核蛋白之间的蛋白质组变化。使用二维凝胶电泳 (2D-PAGE) 处理经 M-CSF 和 sRANKL 处理的细胞，从仅经 M-CSF 处理的 4B12 细胞制备的核蛋白二维图谱显示 327 个蛋白点，而制备的核蛋白二维图谱。用M-CSF和sRANKL处理的4B12细胞显示220个蛋白点，两组的减影图像分析显示新出现101个点，208个点。与仅使用 M-CSF 处理的 4B12 细胞制备的 SYPRO Ruby 染色核蛋白相比，101 个斑点中，1 个斑点消失，32 个斑点上调，37 个斑点减少（Mr 50kDa pI）。 5.5)、8 个点 (Mr 50kDa pI 6-7) 和 2 个点 (Mr 60kDa pI) 5.5-6.5)经Pro-Q Diamond染色，表明这些蛋白在p38 MAP激酶抑制剂SB202190(10μM)处理后被磷酸化，这些结果表明磷酸化的核蛋白可能参与破骨细胞形成。这些新蛋白的鉴定可能会导致发现与破骨细胞生成相关的新因子，并为阐明控制破骨细胞的机制提供新线索特异性分化较少