Iron overload-induced osteocyte apoptosis stimulates osteoclast differentiation through increasing osteocytic RANKL production in vitro

Yang, Jiancheng; Dong, Dandan; Luo, Xinle; Zhou, Jianhua; Shang, Peng; Zhang, Hao

Affiliations

¹Department of Spine Surgery, People’s Hospital of Longhua, Affiliated Hospital of Southern Medical University, Shenzhen, China

²Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, China

³The Third School of Clinical Medicine, Southern Medical University, Shenzhen, China

⁴Research & Development Institute of Northwestern Polytechnical University in Shenzhen, Shenzhen, China

Abstract and keywords

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.

Keywords: Iron overload; Osteocytes; Cell apoptosis; Osteoclastogenesis; Bone resorption; Osteoporosis

Links

DOI: 10.1007/s00223-020-00735-x

PubMed: 32995951

WoS: 000573778800002

History

Received: 2020-05-13

Accepted: 2020-07-21

Online: 2020-09-29

Cited Works (9)

Year	Entry
2002	Zhao S, Kato Y, Zhang Y, Harris S, Ahuja SS, Bonewald LF. MLO‐Y4 osteocyte‐like cells support osteoclast formation and activation. J Bone Miner Res. November 2002;17(11):2068-2079.
2016	Cabahug-Zuckerman P, Frikha-Benayed D, Majeska RJ, Tuthill A, Yakar S, Judex S, Schaffler MB. Osteocyte apoptosis caused by hindlimb unloading is required to trigger osteocyte RANKL production and subsequent resorption of cortical and trabecular bone in mice femurs. J Bone Miner Res. July 2016;31(7):1356-1365.
2012	Kennedy OD, Herman BC, Laudier DM, Majeska RJ, Sun HB, Schaffler MB. Activation of resorption in fatigue-loaded bone involves both apoptosis and active pro-osteoclastogenic signaling by distinct osteocyte populations. Bone. May 2012;50(5):1115-1122.
2006	Aguirre JI, Plotkin LI, Stewart SA, Weinstein RS, Parfitt AM, Manolagas SC, Bellido T. Osteocyte apoptosis is induced by weightlessness in mice and precedes osteoclast recruitment and bone loss. J Bone Miner Res. April 2006;21(4):605-615.
2020	Robling AG, Bonewald LF. The osteocyte: new insights. Annu Rev Physiol. 2020;82:485-506.
2011	Nakashima T, Hayashi M, Fukunaga T, Kurata K, Oh-hora M, Feng JQ, Bonewald LF, Kodama T, Wutz A, Wagner EF, Penninger JM, Takayanagi H. Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nat Med. October 2011;17(10):1231-1234.
2014	Kennedy OD, Laudier DM, Majeska RJ, Sun HB, Schaffler MB. Osteocyte apoptosis is required for production of osteoclastogenic signals following bone fatigue in vivo. Bone. July 2014;64:132-137.
2011	Xiong J, Onal M, Jilka RL, Weinstein RS, Manolagas SC, O'Brien CA. Matrix-embedded cells control osteoclast formation. Nat Med. November 2011;17(10):1235-1242.
2020	McCutcheon S, Majeska RJ, Spray DC, Schaffler MB, Vazquez M. Apoptotic osteocytes induce RANKL production in bystanders via purinergic signaling and activation of pannexin channels. J Bone Miner Res. May 2020;35(5):966-977.

Cited By (1)

Year	Entry
2022	Ma J, Wang A, Zhang H, Liu B, Geng Y, Xu Y, Zuo G, Jia P. Iron overload induced osteocytes apoptosis and led to bone loss in Hepcidin^−/− mice through increasing sclerostin and RANKL/OPG. Bone. November 2022;164:116511.