Zoledronate Inhibits RANKL-induced Osteoclast Differentiation via Suppression of Expression of NFATc1 and Carbonic Anhydrase 2
Clinical reports describe surgical dental treatments in patients receiving BP treatment as being associated with osteonecrosis of the jaw (ONJ), however, the role of BPs in this mechanism is unknown until now. On the other hand, reports have been published of ONJ cases, including treatment with anti-RANKL agents, denosumab, suggesting that these agents appear to have a mode of action similar to that of BPs. Such evidence from previous reports suggests that both BPs and anti-RANKL agents can regulate RANKL-mediated osteoclast differentiation, and the identification of novel BP targets in RANKL-mediated osteoclastogenesis may nullify the mechanism of bone-remodeling agent-induced ONJ.
In this study, we hypothesized that BPs suppresses osteoclast differentiation by regulation of RANKL-mediated genes. To prove the hypothesis, we performed a genome-wide screening of RANKL-induced osteoclast differentiation among cells treated with or without zoledronate.
[Material and Methods]To assess the effect of zoledronate on RANKL-induced osteoclast differentiation, we cultured mouse osteoclast precursor cells (OCPCs) with or without RANKL (100 ng/ml) and/or zoledronate (10 μM) in the presence of M-CSF (100 ng/ml) and performed the TRAP assay. To investigate changes in the expression pattern of genes associated with RANKL-induced osteoclast differentiation, we performed genome-wide screening of mouse OCPCs in either the presence or absence of RANKL with or without zoledronate. The molecular targets of zoledronate in the RANKL signal pathway and additional factors associated with osteoclastogenesis were analyzed by genome-wide screening. To verify the results from microarray analyses, we evaluated the effect of zoledronate on the mRNA expression of candidate genes in mouse OCPCs using real-time quantitative RT-PCR and western blotting. We also examined whether the inhibition of RANKL-mediated gene expression and osteoclast differentiation was because of the changes in gene expression induced by zoledronate in RANKL-treated cells by western blotting and immunofluorescence cytochemistry.
[Results] TRAP-positive multinucleated cells (MNCs) were observed in the RANKL-treated cells in the absence of zoledronate but not in those treated with zoledronate. Microarray analysis identified that among 31 genes on 44 entities of RANKL-inducible genes, the mRNA expression level of two genes, i.e., nuclear factor of activated T-cells c1 (NFATc1) and carbonic anhydrase 2 (CAII), was decreased in zoledronate-treated cells. Subsequent analyses verified that these two genes were significantly silenced by zoledronate treatment and that their expression was restored following inhibition of zoledronate action by geranylgeraniol (GGOH). Furthermore, although RANKL-induced MNC formation was significantly suppressed by zoledronate, MNC formation was recovered by the addition of GGOH. Zoledronate inhibited RANKL-induced osteoclast differentiation by suppression of NFATc1 and CAIIgene expression.
[Conclusion] We demonstrate that inhibition of NFATc1 and CAII by zoledronate was identified among RANKL-induced genes, suggesting that N-BPs may have inhibitory action on RANKL-induced osteoclast differentiation via regulation of these two genes. Knowledge of the molecular mechanisms of these genes may provide a novel aspect in the control and prevention of ONJ induced by bone-remodeling agents such as zoledronate and denosumab.
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