Abstract
Summary
Osteoporosis is linked to age-related decline of melatonin production; however, the direct effects of melatonin on osteoclastogenesis remain unknown. Our study demonstrates that melatonin at pharmacological concentrations, rather than at physiological concentrations, significantly inhibits osteoclastogenesis. Melatonin-mediated anti-osteoclastogenesis involves a reactive oxygen species (ROS)-mediated but not a silent information regulator type 1 (SIRT1)-independent pathway.
Introduction
Osteoporosis is a bone disorder linked to impaired bone formation and excessive bone resorption. Melatonin has been suggested to treat osteoporosis due to its beneficial actions on osteoblast differentiation. However, the direct effects of melatonin on osteoclastogenesis in bone marrow monocytes (BMMs) remain unknown. This study was to investigate whether melatonin at either physiological or pharmacological concentrations could affect osteoclast differentiation.
Methods
Primary BMMs were isolated from the femurs and tibias of C57BL/6 mice and were induced toward multinucleated osteoclasts, in the presence of melatonin at either physiological (0.01 to 10 nM) or pharmacological (1 to 100 μM) concentrations. Tartrate-resistant acid phosphatase (TRAP) staining was used to label multinucleated osteoclasts and the levels of osteoclast-specific genes were evaluated. To further explore the underlying mechanisms, the roles of silent information regulator type 1 (SIRT1) and reactive oxygen species (ROS) were evaluated.
Results
We found that melatonin at pharmacological concentrations, rather than at physiological concentrations, significantly inhibited osteoclast formation in a dose-dependent manner. The number of TRAP-positive cells and the gene expression of osteoclast-specific markers were significantly downregulated in melatonin-treated BMMs. The melatonin-mediated repression of osteoclast differentiation involved the inhibition of the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. The treatment with SIRT1 inhibitors did not affect osteoclast differentiation but, when supplemented with exogenous hydrogen peroxide, a partial rescue of melatonin-suppressed osteoclastogenesis was observed.
Conclusion
Melatonin at pharmacological doses directly inhibited osteoclastogenesis of BMMs by a ROS-mediated but not a SIRT1-independent pathway.
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Abbreviations
- BMMs:
-
Bone marrow monocytes
- CCK-8:
-
Cell counting kit-8
- CTRL:
-
Control cells
- CTSK:
-
Cathepsin K
- DCFH2-DA:
-
2′,7′-Dichlorofluorescein diacetate
- GAPDH:
-
Glyceraldehyde-3-phosphate dehydrogenase
- H2O2 :
-
Hydrogen peroxide
- M-CSF:
-
Macrophage-colony stimulating factor
- MLT:
-
Melatonin
- MSCs:
-
Mesenchymal stem cells
- NAM:
-
Nicotinamide
- NF-κB:
-
Nuclear factor κ-light-chain-enhancer of activated B cells
- OC:
-
Osteoclasts
- OCG:
-
Osteoclastogenesis
- OSCAR:
-
Osteoclast-associated receptor
- RANKL:
-
Receptor activator of nuclear factor-κB ligand
- ROS:
-
Reactive oxygen species
- SIRT1:
-
Silent information regulator type 1
- TRAP:
-
Tartrate-resistant acid phosphatase
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Acknowledgments
The authors are grateful to Suzanne Danley (West Virginia University, USA) and Paula Sahyoun (University of Waterloo, Canada) for carefully reviewing and editing the manuscript. This work was supported by the National Natural Science Foundation of China (31570978, 21574091, 31400826), the Natural Science Foundation of Jiangsu Province (BK20140323), the National Institutes of Health (NIH) (AR062763-01A1, AR067747-01A1) and an Established Investigator Grant from Musculoskeletal Transplant Foundation (MTF) to M.P., and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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Zhou, L., Chen, X., Yan, J. et al. Melatonin at pharmacological concentrations suppresses osteoclastogenesis via the attenuation of intracellular ROS. Osteoporos Int 28, 3325–3337 (2017). https://doi.org/10.1007/s00198-017-4127-8
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DOI: https://doi.org/10.1007/s00198-017-4127-8