Iron Overload-Induced Osteocyte Apoptosis Stimulates Osteoclast Differentiation Through Increasing Osteocytic RANKL Prod
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ORIGINAL RESEARCH
Iron Overload‑Induced Osteocyte Apoptosis Stimulates Osteoclast Differentiation Through Increasing Osteocytic RANKL Production In Vitro Jiancheng Yang1,2 · Dandan Dong2 · Xinle Luo1,4 · Jianhua Zhou1 · Peng Shang2,3 · Hao Zhang1,4 Received: 13 May 2020 / Accepted: 21 July 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
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 RANKLblocking 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-Omethylaspartyl-[-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. Keywords Iron overload · Osteocytes · Cell apoptosis · Osteoclastogenesis · Bone resorption · Osteoporosis
* Peng Shang [email protected] * Hao Zhang [email protected] 1
Department of Spine Surgery, People’s Hospital of Longhua, Affiliated Hospital of Southern Medical University, No. 38, Jinglo
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