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Melatonin at pharmacological concentrations suppresses osteoclastogenesis via the attenuation of intracellular ROS

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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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Author notes

  1. Long Zhou and Xi Chen contributed equally to this work.

Authors and Affiliations

  1. Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Soochow University, No. 188 Shizi Street, Suzhou, Jiangsu, 215006, China

    L. Zhou, X. Chen, J. Yan, M. Li, T. Liu, H. Yang & F. He

  2. Orthopaedic Institute, Medical College, Soochow University, No.708 Renmin Road, Suzhou, Jiangsu, 215007, China

    L. Zhou, X. Chen, J. Yan, M. Li, C. Zhu, G. Pan, Q. Guo, H. Yang & F. He

  3. Department of Orthopaedics, Suzhou Science & Technology Town Hospital, Suzhou, 215153, China

    L. Zhou

  4. School of Biology and Basic Medical Sciences, Medical College, Soochow University, Suzhou, 215123, China

    X. Chen

  5. Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics and Division of Exercise Physiology, West Virginia University, Morgantown, WV, 26505, USA

    M. Pei

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  1. L. Zhou
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Correspondence to H. Yang or F. He.

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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

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