Thapsigargin Modulates Osteoclastogenesis Through the Regulation of RANKL-Induced Signaling Pathways and Reactive Oxygen Species Production

Abstract

The mechanism by which TG modulates osteoclast formation and apoptosis is not clear. In this study, we showed a biphasic effect of TG on osteoclast formation and apoptosis through the regulation of ROS production, caspase-3 activity, cytosolic Ca²+, and RANKL-induced activation of NF-κB and AP-1 activities. Introduction: Apoptosis and differentiation are among the consequences of changes in intracellular Ca²+ levels. In this study, we investigated the effects of the endoplasmic reticular Ca²+-ATPase inhibitor, thapsigargin (TG), on osteoclast apoptosis and differentiation. Materials and Methods: Both RAW264.7 cells and primary spleen cells were used to examine the effect of TG on RANKL-induced osteoclastogenesis. To determine the action of TG on signaling pathways, we used reporter gene assays for NF-κB and activator protein-1 (AP-1) activity, Western blotting for phospho-extracellular signal-related kinase (ERK), and fluorescent probes to measure changes in levels of intracellular calcium and reactive oxygen species (ROS). To assess rates of apoptosis, we measured changes in annexin staining, caspase-3 activity, and chromatin and F-actin microfilament structure. Results: At concentrations that caused a rapid rise in intracellular Ca²+, TG increased caspase-3 activity and promoted apoptosis in osteoclast-like cells (OLCs). Low concentrations of TG, which were insufficient to measurably alter intracellular Ca²+, unexpectedly suppressed caspase-3 activity and enhanced RANKL-induced osteoclastogenesis. At these lower concentrations, TG potentiated ROS production and RANKL-induced NF-κB activity, but suppressed RANKL-induced AP-1 activity and had little effect on ERK phosphorylation. Conclusion: Our novel findings of a biphasic effect of TG are incompletely explained by our current understanding of TG action, but raise the possibility that low intensity or local changes in subcellular Ca²+ levels may regulate intracellular differentiation signaling. The extent of cross-talk between Ca²+ and RANKL-mediated intracellular signaling pathways might be important in determining whether cells undergo apoptosis or differentiate into OLCs.