Oral malodorous compound induces osteoclast differentiation without receptor activator of nuclear factor κb ligand

Background: Hydrogen sulfide (H₂S), the main component of halitosis, is one of the etiologic factors for periodontitis. We recently reported that H₂S may induce pathologic changes in rat alveolar bone. The objective of this study is to determine the effect of H₂S on osteoclast differentiation. Methods: Murine macrophage cells RAW264 were cultured in medium lacking nuclear factor kB ligand (receptor activator of nuclear factor kB ligand) in 5% CO₂ with air at 37°C for 24 hours; then 0.05, 0.5, or 5 ng/ml H₂S was added to the CO ₂-air mix for 4 days. The controls received the CO₂-air mix with no H₂S. Cell differentiation was evaluated by counting the tartrate-resistant acid-phosphatase (TRAP)-positive cells. Extracellular signaling-regulated kinase1/2 (ERK1/2) and mitogen-activated protein kinase p38 phosphorylation were examined by Western blotting. The bone-resorption activity was determined with the resorption assay of calcium phosphate. Results: There were significantly more TRAP-positive cells at a concentration of 0.05 ng/ml H₂S than at the other concentrations (P<0.001). Cathepsin K protein, a specific marker for osteoclasts, was expressed in the H2S-induced multinuclear cells. Resorption of calcium phosphate significantly increased in the H₂S-induced TRAP-positive cells cultured on plates coated with calcium phosphate apatite (P<0.01). The phosphorylation of ERK1/2 and p38 were accelerated by H₂S, and increased with time. PD98059 and SB203580, specific inhibitors of ERK1/2 and p38, suppressed the activation of these enzymes and osteoclast differentiation by H₂S. Conclusion: Results demonstrate that H₂S at physiologic concentrations in mouth air induces osteoclasts from RAW264 cells.