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

Authors

  • Kirk Hm Yip,

    1. Molecular Orthopaedic Laboratory, School of Surgery and Pathology, and Western Australian Institute for Medical Research, Nedlands, Australia
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  • Ming H Zheng,

    1. Molecular Orthopaedic Laboratory, School of Surgery and Pathology, and Western Australian Institute for Medical Research, Nedlands, Australia
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  • James H Steer,

    1. Pharmacology Unit, School of Medicine and Pharmacology, University of Western Australia, Nedlands, Australia
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  • Tindaro M Giardina,

    1. Pharmacology Unit, School of Medicine and Pharmacology, University of Western Australia, Nedlands, Australia
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  • Renzhi Han,

    1. Molecular Orthopaedic Laboratory, School of Surgery and Pathology, and Western Australian Institute for Medical Research, Nedlands, Australia
    2. Present address: Howard Hughes Medical Institute University of Iowa, College of Medicine, 400 EMRB, Iowa City, Iowa, USA
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  • Susan Z Lo,

    1. Pharmacology Unit, School of Medicine and Pharmacology, University of Western Australia, Nedlands, Australia
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  • Anthony J Bakker,

    1. Physiology Unit, School of Biomedical and Chemical Sciences, University of Western Australia, Nedlands, Australia
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  • A Ian Cassady,

    1. Institute for Molecular Biosciences and School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland, Australia
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  • David A Joyce,

    1. Pharmacology Unit, School of Medicine and Pharmacology, University of Western Australia, Nedlands, Australia
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  • Jiake Xu MD, PhD

    Corresponding author
    1. Molecular Orthopaedic Laboratory, School of Surgery and Pathology, and Western Australian Institute for Medical Research, Nedlands, Australia
    • Molecular Orthopaedic Laboratory, School of Surgery and Pathology, University of Western Australia, QEII Medical Centre, 2nd Floor, M Block, Nedlands, WA 6009, Australia

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  • The authors have no conflict of interest.

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.

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