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Constant hypoxia inhibits osteoclast differentiation and bone resorption by regulating phosphorylation of JNK and IκBα

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Abstract

Background

Osteoclasts are responsible for the bone loss in rheumatoid arthritis (RA). Hypoxia has been suggested to play key roles in pathological bone loss. However, the current understanding of the effects of hypoxia on osteoclastogenesis is controversial. Effects of hypoxia on both the formation and function of osteoclasts requires examination. In the current study, we aimed to explore the effect of hypoxia on osteoclast differentiation and the underlying mechanisms.

Methods

RAW264.7 cells and murine bone-marrow-derived monocytes were used to induce osteoclastogenesis in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL). Hypoxic conditions were maintained in a hypoxic chamber at 5% CO2 and 1% O2, balanced with N2. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. A bone resorption assay was carried out in vitro using bone slices. RT-PCR was conducted to detect osteoclast markers and transcription factors. The phosphorylation of nuclear factor-κBα (IκBα), c-Jun N-terminal kinase (JNK), extracellular regulated protein kinase (ERK), and p38 was detected by western blotting. Mann–Whitney U test or Student’s t test was used to compare differences between the two groups.

Results

TRAP staining and the bone resorption assay revealed that hypoxia-restrained osteoclast differentiation and bone resorption. Expression of osteoclast markers including cathepsin K, RANK, and TRAP decreased during osteoclast differentiation under hypoxic conditions (all P < 0.05). Hypoxia at 1% O2 did not affect cell viability, whereas it dramatically abated RANKL-dependent phosphorylation of the JNK-mitogen-activated protein kinases (MAPK) and IκBα pathways. Moreover, the expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) was inhibited under hypoxic conditions (all P < 0.05).

Conclusions

These results suggest that constant hypoxia at 1% O2 significantly restrains osteoclast formation and resorbing function without affecting cell viability. Constant hypoxia might inhibit RANKL-induced osteoclastogenesis by regulating NFATc1 expression via interfering the phosphorylation of JNK and IκBα.

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Acknowledgements

We gratefully thank the Medical Research Center of Peking University Third Hospital for kindly providing the RAW264.7 cells. We thank the Medical Research Center of Peking University Third Hospital for providing experimental equipment and technical support.

Funding

This work was supported by the National Natural Science Foundation of China (No. 81471599) and the National Young Scholars Foundation of China (No. 81501387), the Prairie Fire Program (LYJH-92).

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Author notes

  1. Zhenzhen Ma and Ruohan Yu contributed equally to this study.

Authors and Affiliations

  1. Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing, 100191, China

    Zhenzhen Ma, Ruohan Yu, Jinxia Zhao, Lin Sun, Leilei Jian, Changhong Li & Xiangyuan Liu

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Ma, Z., Yu, R., Zhao, J. et al. Constant hypoxia inhibits osteoclast differentiation and bone resorption by regulating phosphorylation of JNK and IκBα. Inflamm. Res. 68, 157–166 (2019). https://doi.org/10.1007/s00011-018-1209-9

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