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，破骨细胞是源自单核细胞/巨噬细胞谱系的。众所周知，M-CSF和RANKL是破骨构成的可分配因素。 RANKL与其受体的结合，将激活C-FOS，MITF，PU.1和NFATC 1在内的转录因子，这对于破骨细胞生成很重要。然而，尚未对核蛋白的定性和定量变化进行整体分析破骨细胞生成。最近，我们从Mac-l^-C-FMS^+等级^+细胞种群中建立了破骨细胞生成细胞系4B12细胞中的14天大小鼠胚胎钙质钙质骨细胞。因此，我们比较了由单独使用M-CSF处理的4B12细胞制备的核蛋白与使用M-CSF和SRANKL处理的4B12细胞制备的核蛋白之间的核蛋白之间的变化，并使用二维凝胶电泳（2D-PAGE）制备了核蛋白。 ……更多由单独用M-CSF处理的4B12细胞制备的核蛋白的2D地图显示出327个蛋白质斑点，而由M-CSF和SRANKL处理的4B12细胞制备的2D核蛋白显示了220个蛋白质斑点。两组的减法图像分析表明，新出现的101个斑点，208个斑点消失，32个斑点更新，在Sypro红宝石染色的核蛋白中，由M-CSF和SRANK处理的4B12细胞制备的Sypro红宝石染色的核蛋白降低，与仅与M-CSF相比。 Pro-Q Diamond染色了101个斑点（MR 50KDA PI 5.5），1个斑点（MR 50KDA PI 5.5），8个斑点（MR 50KDA PI 6-7）和2个斑点（MR 50KDA PI 5.5-6.5）。通过处理p38 MAP激酶抑制剂SB202190（10μM），这些斑点消失或减少。这些结果表明，磷酸化的核蛋白可能参与破骨细胞生成。这些新蛋白质的鉴定可能导致发现与破骨细胞发生相关的新因素，并提供了新的线索，以阐明控制破骨细胞生成的机理。较少的