TNF-α Directly Enhances Osteocyte RANKL Expression and Promotes Osteoclast Formation

Osteoimmunology peeks into the interaction of bone and the immune system, which has largely proved to be a multiplex reaction. Osteocytes have been shown to regulate bone resorption through the expression of RANKL in physiologic and pathologic conditions. TNF-alpha, a product of the immune system, is an important cytokine regulating bone resorption in inflammatory conditions either directly or by increasing RANKL and M-CSF expressions by osteoblasts and stromal cells. The effect of TNF-alpha on a wide range of cell types has been documented; however, the direct effect of TNF-alpha on osteocytes has not been established yet. In this study, primary osteocytes were isolated by cell sorting from neonatal calvaria of Dmp1-Topaz mice, which express the green fluorescent protein under the influence of dentin matrix protein 1 promoter. The results show that osteocytes have a significantly higher RANKL mRNA expression when cultured with TNF-alpha. A co-culture system of osteocytes and TNF receptors I and II deficient osteoclast precursors treated with TNF-alpha show a significant increase in TRAP-positive cells while cultures without TNF-alpha failed to show TRAP-positive cells. Additionally, in vivo experiments of TNF-alpha injected to mouse calvaria show an increase in TRAP-positive cell number in the suture mesenchyme and an increase in the percentage of RANKL-positive osteocytes compared to PBS-injected calvaria. Osteocytes cultured with TNF-alpha show up-regulation of MAPKs phosphorylation measured by western blot, and adding MAPKs inhibitors to osteocytes cultured with TNF-alpha significantly decreases RANKL mRNA expression compared to osteocytes cultured with TNF-alpha alone. We also found that TNF-alpha activates the NF-kappa B pathway in osteocytes measured as a function of p65 subunit nuclear translocation. TNF-alpha directly affects osteocyte RANKL expression and increases osteoclastogenesis; our results demonstrate that osteocytes guard an important role in inflammatory bone resorption mediated by TNF-alpha.