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Iron Overload-Induced Osteocyte Apoptosis Stimulates Osteoclast Differentiation Through Increasing Osteocytic RANKL Production In Vitro

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Abstract

Iron overload is closely associated with osteoporosis, the potential cellular mechanism involved in decreased osteoblast differentiation and increased osteoclast formation. However, the effect of iron overload on the biological behavior in osteocytes has not been reported. This study aims to investigate the changes of osteocytic activity, apoptosis, and its regulation on osteoclastogenesis in response to iron overload. MLO-Y4 osteocyte-like cells and primary osteocytes from mice were processed with ferric ammonium citrate (FAC) and deferoxamine (DFO), the conditioned medium (CM) was harvested and co-cultured with Raw264.7 cells and bone marrow-derived macrophages (BMDMs) to induce them to differentiate into osteoclasts. Osteocyte apoptosis, osteoclast differentiation, osteocytic gene expression and protein secretion of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) was examined. Excessive iron has a toxic effect on MLO-Y4 osteocyte-like cells. Increased cell apoptosis in MLO-Y4 cells and primary osteocytes was induced by iron overload. The osteoclastic formation, differentiation-related gene expression, and osteoclastic bone‐resorption capability were significantly increased after treated with the CM from iron overload‐exposed osteocytes. Excessive iron exposure significantly promoted the gene expression and protein secretion of the RANKL in MLO‐Y4 cells. Addition of RANKL-blocking antibody completely abolished the increase of osteoclast formation and bone resorption capacity induced by the CM from osteocytes exposed to excessive iron. Moreover, the pan-caspase apoptosis inhibitor, QVD (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methylketone) was used to inhibit osteocyte apoptosis. The results showed osteocyte apoptosis induced by iron overload was reduced by QVD and accompanied by the decrease of soluble RANKL (sRANKL) in supernatant. The increase of osteoclast formation and bone resorption capacity induced by the CM from osteocytes exposed to excessive iron was significantly decreased by QVD. These results indicated that iron overload-induced osteocyte apoptosis is required to regulate osteoclast differentiation by increasing osteocytic RANKL production. This study, for the first time, reveals the indirect effect of iron overload on osteoclast differentiation through regulating osteocytes.

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Acknowledgement

We would like to thank Yi Lyu in the Key Laboratory for Space Bioscience and Biotechnology for the technical assistance.

Funding

This work sponsored by the Shenzhen Municipal Research Program of Health and Family Planning System (SZXJ2017060), the National Natural Science Foundation of China (51777171), and the Innovation Fund Program for Science and Technology in Longhua District of Shenzhen (2017008).

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Authors and Affiliations

  1. Department of Spine Surgery, People’s Hospital of Longhua, Affiliated Hospital of Southern Medical University, No. 38, Jinglong Construction Road, Shenzhen, 518109, Guangdong, China

    Jiancheng Yang, Xinle Luo, Jianhua Zhou & Hao Zhang

  2. Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 West Youyi Road, Beilin District, Xi’an, 710072, Shaanxi, China

    Jiancheng Yang, Dandan Dong & Peng Shang

  3. Research & Development Institute of Northwestern Polytechnical University in Shenzhen, Shenzhen, China

    Peng Shang

  4. The Third School of Clinical Medicine, Southern Medical University, Shenzhen, China

    Xinle Luo & Hao Zhang

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  1. Jiancheng Yang
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  3. Xinle Luo
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  4. Jianhua Zhou
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  5. Peng Shang
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by JY, DD, XL, and JZ. The first draft of the manuscript was written by JY and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Peng Shang or Hao Zhang.

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Jiancheng Yang, Dandan Dong, Xinle Luo, Jianhua Zhou, Peng Shang, and Hao Zhang declare that they have no conflict of interest.

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Animal operation was conducted in accordance with the ethical guidelines for animals of the Medical and Experimental Animal Ethics Committee of Northwestern Polytechnic University.

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Yang, J., Dong, D., Luo, X. et al. Iron Overload-Induced Osteocyte Apoptosis Stimulates Osteoclast Differentiation Through Increasing Osteocytic RANKL Production In Vitro. Calcif Tissue Int 107, 499–509 (2020). https://doi.org/10.1007/s00223-020-00735-x

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