Thapsigargin modulates osteoclastogenesis through the regulation of RANKL-induced signaling pathways and reactive oxygen species production.
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-kappaB and AP-1 activities.


BACKGROUND
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.


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-kappaB 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-kappaB activity, but suppressed RANKL-induced AP-1 activity and had little effect on ERK phosphorylation.


CONCLUSIONS
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.
Yip
Kirk H M
KH
Molecular Orthopaedic Laboratory, School of Surgery and Pathology, and Western Australian Institute for Medical Research, Nedlands, WA 6009, Australia.
Zheng
Ming H
MH
Steer
James H
JH
Giardina
Tindaro M
TM
Han
Renzhi
R
Lo
Susan Z
SZ
Bakker
Anthony J
AJ
Cassady
A Ian
AI
Joyce
David A
DA
Xu
Jiake
J
D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002352:Carrier Proteins / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D010010:Osteoclasts / Q000187:drug effects* / Q000378:metabolism; D010012:Osteogenesis; D053245:RANK Ligand; D017382:Reactive Oxygen Species / Q000378:metabolism*; D053246:Receptor Activator of Nuclear Factor-kappa B; D019284:Thapsigargin / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D053245:RANK Ligand; D017382:Reactive Oxygen Species; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D018808:Transcription Factor AP-1; D019284:Thapsigargin; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D002118:Calcium