Hotokezaka Hitoshi, Sakai Eiko, Kanaoka Kazuhiro, Saito Kan, Matsuo Ken-ichiro, Kitaura Hideki, Yoshida Noriaki, Nakayama Koji
Division of Orthodontics and Biomedical Engineering, Department of Developmental and Reconstructive Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki-city, Japan.
J Biol Chem. 2002 Dec 6;277(49):47366-72. doi: 10.1074/jbc.M208284200. Epub 2002 Sep 16.
Osteoclasts are multinucleated cells that differentiate from hematopoietic cells and possess characteristics responsible for bone resorption. To study the involvement of mitogen-activated protein kinases (MAPKs) in osteoclastogenesis of the murine monocytic cell line RAW264.7, which can differentiate into osteoclast-like cells in the presence of the receptor activator of nuclear factor kappa B ligand (RANKL), we treated the cells with specific inhibitors of p38 MAPK, PD169316 and SB203580, and specific inhibitors of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK), U0126 and PD98059. Each inhibitor blocked differentiation into osteoclast-like cells when the cells were plated at the standard cell density (2000-4000 cells per well (96-well)). However, the effect of MEK inhibitors on osteoclastogenesis varied according to the initial cell density during culture, because cell growth was clearly inhibited by them. When the cells were plated at more than 8000 cells per well, marked enhancement and acceleration of the differentiation were observed. In addition, immunoblot analysis revealed that phosphorylation of ERK was increased by treatment with the p38 inhibitors, whereas the MEK inhibitors increased phosphorylation of p38, which implies a seesaw-like balance between ERK and p38 phosphorylation. We suggest that osteoclastogenesis is regulated under a balance between ERK and p38 pathways and that the MEK/ERK pathway negatively regulates osteoclastogenesis while the p38 pathway does so positively. This is the first report that an inhibitor of signal transduction enhanced osteoclastogenesis.
破骨细胞是由造血细胞分化而来的多核细胞，具有负责骨吸收的特性。为了研究丝裂原活化蛋白激酶（MAPK）在小鼠单核细胞系RAW264.7破骨细胞生成中的作用（该细胞系在核因子κB受体活化剂配体（RANKL）存在下可分化为破骨细胞样细胞），我们用p38 MAPK的特异性抑制剂PD169316和SB203580，以及MAPK细胞外信号调节激酶（ERK）激酶（MEK）的特异性抑制剂U0126和PD98059处理细胞。当细胞以标准细胞密度（每孔（96孔板）2000 - 4000个细胞）接种时，每种抑制剂都阻断了向破骨细胞样细胞的分化。然而，MEK抑制剂对破骨细胞生成的影响因培养期间的初始细胞密度而异，因为它们明显抑制了细胞生长。当细胞以每孔超过8000个细胞接种时，观察到分化明显增强和加速。此外，免疫印迹分析显示，用p38抑制剂处理可增加ERK的磷酸化，而MEK抑制剂可增加p38的磷酸化，这意味着ERK和p38磷酸化之间存在跷跷板样的平衡。我们认为破骨细胞生成是在ERK和p38途径之间的平衡下进行调节的，MEK/ERK途径对破骨细胞生成起负调节作用，而p38途径起正调节作用。这是首次报道信号转导抑制剂增强破骨细胞生成的研究。
