Osteoporosis International

, Volume 28, Issue 12, pp 3325–3337 | Cite as

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

Original Article

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.

Keywords

Bone marrow monocytes Melatonin Osteoclasts Osteoporosis ROS SIRT1 

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

Supplementary material

198_2017_4127_MOESM1_ESM.docx (795 kb)
ESM 1(DOCX 794 kb)

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Copyright information

© International Osteoporosis Foundation and National Osteoporosis Foundation 2017

Authors and Affiliations

  • L. Zhou
    • 1
    • 2
    • 3
  • X. Chen
    • 1
    • 2
    • 4
  • J. Yan
    • 1
    • 2
  • M. Li
    • 1
    • 2
  • T. Liu
    • 1
  • C. Zhu
    • 2
  • G. Pan
    • 2
  • Q. Guo
    • 2
  • H. Yang
    • 1
    • 2
  • M. Pei
    • 5
  • F. He
    • 1
    • 2
  1. 1.Department of OrthopaedicsThe First Affiliated Hospital of Soochow University, Soochow UniversitySuzhouChina
  2. 2.Orthopaedic Institute, Medical CollegeSoochow UniversitySuzhouChina
  3. 3.Department of OrthopaedicsSuzhou Science & Technology Town HospitalSuzhouChina
  4. 4.School of Biology and Basic Medical Sciences, Medical CollegeSoochow UniversitySuzhouChina
  5. 5.Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics and Division of Exercise PhysiologyWest Virginia UniversityMorgantownUSA

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