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

破骨细胞是多核巨细胞，具有吸收矿化组织的能力。众所周知，破骨细胞来源于单核细胞/巨噬细胞谱系。众所周知，M-CSF和RANKL是破骨细胞形成不可或缺的因子。RANKL与其受体RANK结合，激活c-Fos、Mitf、PU.1和NFATc 1等转录因子，已知这些转录因子对破骨细胞的发生很重要。然而，对核蛋白在破骨细胞发生中的定性和定量变化的系统分析尚未进行。最近，我们从14日龄小鼠胚胎颅骨骨细胞的Mac-l^-c-Fms^+RANK^+细胞群中建立了破骨细胞前细胞系4B12细胞。因此，我们使用二维凝胶电泳（2D-PAGE）比较了M-CSF单独处理4B12细胞制备的核蛋白与M-CSF和sRANKL处理4B12细胞制备的核蛋白之间的蛋白质组学变化。M-CSF单独处理的4B12细胞的核蛋白2D图显示327个蛋白点，而M-CSF和sRANKL联合处理的4B12细胞的核蛋白2D图显示220个蛋白点。两组的减法图像分析显示，与M-CSF单独处理的4B12细胞相比，M-CSF和sRANK处理的SYPRO红宝石染色核蛋白新出现101个斑点，消失208个斑点，上调32个斑点，降低37个斑点。101个斑点中的11个斑点，1个斑点（Mr 50kDa pI 5.5）， 8个斑点（Mr 50kDa pI 6-7）和2个斑点（Mr 60kDa pI 5.5-6.5）被Pro-Q Diamond染色，表明这些蛋白被磷酸化。p38 MAP激酶抑制剂SB202190（10 μM）处理后，这些斑点消失或减少。这些结果提示磷酸化的核蛋白可能参与了破骨细胞的发生。这些新蛋白的鉴定可能有助于发现与破骨细胞发生相关的新因子，并为阐明破骨细胞特异性分化的调控机制提供新的线索。少

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