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

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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