title: Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-alpha, lipopolysaccharide, or peptidoglycan.
abstract: Focusing on the final step of osteoclastogenesis, we studied cell fusion from tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells into multinuclear cells. TRAP-positive mononuclear cells before generation of multinuclear cells by cell fusion were differentiated from RAW264.7 cells by treatment with receptor activator of nuclear factor kappa B ligand (RANKL), and then the cells were treated with lipopolysaccharide (LPS), followed by culturing for further 12 h. LPS-induced cell fusion even in the absence of RANKL. Similarly, tumor necrosis factor (TNF)-alpha and peptidoglycan (PGN) induced cell fusion, but M-CSF did not. The cell fusion induced by RANKL, TNF-alpha, and LPS was specifically blocked by osteoprotegerin (OPG), anti-TNF-alpha antibody, and polymyxin B, respectively. LPS- and PGN-induced cell fusion was partly inhibited by anti-TNF-alpha antibody but not by OPG. When TRAP-positive mononuclear cells fused to yield multinuclear cells, phosphorylation of Akt, Src, extracellular signal-regulated kinase (ERK), p38MAPK (p38), and c-Jun NH2-terminal kinase (JNK) was observed. The specific chemical inhibitors LY294002 (PI3K), PP2 (Src), U0126 (MAPK-ERK kinase (MEK)/ERK), and SP600125 (JNK) effectively suppressed cell fusion, although SB203580 (p38) did not. mRNA of nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP) during the cell fusion was quantified, however, there was no obvious difference among the TRAP-positive mononuclear cells treated with or without M-CSF, RANKL, TNF-alpha, LPS, or PGN. Collectively, RANKL, TNF-alpha, LPS, and PGN induced cell fusion of osteoclasts through their own receptors. Subsequent activation of signaling pathways involving PI3K, Src, ERK, and JNK molecules was required for the cell fusion. Although DC-STAMP is considered to be a requisite for cell fusion of osteoclasts, cell fusion-inducing factors other than DC-STAMP might be necessary for the cell fusion.
authors.affiliation: Department of Developmental and Reconstructive Medicine, Division of Orthodontics and Biomedical Engineering, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan. hotoke@nagasaki-u.ac.jp
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Hotokezaka
authors.forename: Eiko
authors.initials: E
authors.lastname: Sakai
authors.forename: Naoya
authors.initials: N
authors.lastname: Ohara
authors.forename: Yuka
authors.initials: Y
authors.lastname: Hotokezaka
authors.forename: Carmen
authors.initials: C
authors.lastname: Gonzales
authors.forename: Ken-ichiro
authors.initials: K
authors.lastname: Matsuo
authors.forename: Yuji
authors.initials: Y
authors.lastname: Fujimura
authors.forename: Noriaki
authors.initials: N
authors.lastname: Yoshida
authors.forename: Koji
authors.initials: K
authors.lastname: Nakayama
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002459:Cell Fusion; D002460:Cell Line; D007527:Isoenzymes / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008561:Membrane Fusion / Q000187:drug effects; D051379:Mice; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects*; D010457:Peptidoglycan / Q000494:pharmacology*; D053245:RANK Ligand / Q000378:metabolism*; D000071681:Tartrate-Resistant Acid Phosphatase; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D007527:Isoenzymes; D008070:Lipopolysaccharides; D010457:Peptidoglycan; D053245:RANK Ligand; D014409:Tumor Necrosis Factor-alpha; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase