U0126 and PD98059, specific inhibitors of MEK, accelerate differentiation of RAW264.7 cells into osteoclast-like cells.
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.
Hotokezaka
Hitoshi
H
Division of Orthodontics and Biomedical Engineering, Department of Developmental and Reconstructive Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki-city, Japan. hotoke@net.nagasaki-u.ac.jp
Sakai
Eiko
E
Kanaoka
Kazuhiro
K
Saito
Kan
K
Matsuo
Ken-ichiro
K
Kitaura
Hideki
H
Yoshida
Noriaki
N
Nakayama
Koji
K
D000818:Animals; D002070:Butadienes / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D015151:Immunoblotting; D048728:MAP Kinase Kinase Kinase 1; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D008954:Models, Biological; D009000:Monocytes / Q000166:cytology; D009570:Nitriles / Q000494:pharmacology*; D010010:Osteoclasts / Q000166:cytology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011725:Pyridines / Q000494:pharmacology; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D015398:Signal Transduction; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases
D002070:Butadienes; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D008562:Membrane Glycoproteins; D009570:Nitriles; D011725:Pyridines; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; C113580:U 0126; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482578:Map3k1 protein, mouse; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one