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

Abstract

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-α, 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-α on a wide range of cell types has been documented; however, the direct effect of TNF-α 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-α. A co-culture system of osteocytes and TNF receptors I and II deficient osteoclast precursors treated with TNF-α show a significant increase in TRAP-positive cells while cultures without TNF-α failed to show TRAP-positive cells. Additionally, in vivo experiments of TNF-α 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-α show up-regulation of MAPKs phosphorylation measured by western blot, and adding MAPKs inhibitors to osteocytes cultured with TNF-α significantly decreases RANKL mRNA expression compared to osteocytes cultured with TNF-α alone. We also found that TNF-α activates the NF-κB pathway in osteocytes measured as a function of p65 subunit nuclear translocation. TNF-α directly affects osteocyte RANKL expression and increases osteoclastogenesis; our results demonstrate that osteocytes guard an important role in inflammatory bone resorption mediated by TNF-α.

Osteocyte-Related Cytokines Regulate Osteoclast Formation and Bone Resorption

“…Activated AKT also acts as an anti-apoptotic signal, and TNF-α stimulates the AKT pathway in a cell-type-specific manner [104]. We found a trend of activated AKT phosphorylation to levels that did not reach statistical significance [16]. The results of the study supported the role of osteocytes in their potential contribution to bone destruction by increasing RANKL expression via the direct action of TNF-α.…”

“…Inhibition of ERK1/2, p38, and JNK MAPKs via their respective inhibitors (U0126, SB203580, and SP600125) in osteocytes cultured with TNF-α suppressed RANKL mRNA expression to baseline levels compared with untreated cells. We also found that TNF-α activates the NF-κB pathway in osteocytes measured by p65 subunit nuclear translocation [16]. Activated AKT also acts as an anti-apoptotic signal, and TNF-α stimulates the AKT pathway in a cell-type-specific manner [104].…”

“…In our recent study, we evaluated the direct effects of TNF-α on osteocytes. In the study, we used highly purified primary osteocytes that were isolated by cell sorting from neonatal calvariae of DMP1-Topaz mice expressing green fluorescent protein under the control of the dentin matrix protein 1 promoter [16]. Primary osteocytes cultured with TNF-α showed significantly higher RANKL mRNA expression.…”

“…These results suggest that osteocyte RANKL affects bone resorption in both healthy and diseased individuals. In our recent study, we found that tumor necrosis factor-α (TNF-α) directly enhances osteocyte RANKL expression and promotes osteoclast formation [16] and that mechanical force induces TNF-α, which leads to sclerostin expression in osteocytes and sclerostin subsequently enhances osteoclast formation [17]. These results suggest that osteocyte-related cytokines affect osteoclast formation and bone resorption.…”

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“…Early in vitro studies demonstrated that TNF-α could induce both cartilage degradation [62] and bone resorption [63]. Recently, TNF-α was also shown to enhance RANK-L secretion by osteocytes which further promotes osteoclastogenesis [64]. Interestingly, some studies pointed out that TNF-α can also directly induce the differentiation of monocyte/macrophage lineage cells into osteoclasts by a RANK-L-independent mechanism [65][66][67].…”