Original Full Length Article Vascular expression of the chemokine CX3CL1 promotes osteoclast recruitment and exacerbates bone resorption in an irradiated murine model ☆ Ki Hoon Han a, ⁎, Jae Won Ryu a , Kyung-Eun Lim b , Soo-Han Lee c , Yuna Kim c , Chang Sun Hwang d , Je-Yong Choi b , Ki Ok Han d,e, ⁎⁎ a Department of Cardiology, School of Medicine, University of Ulsan, Asan Medical Center, Seoul 138-736, Republic of Korea b Department of Biochemistry and Cell Biology, School of Medicine, WCU Program, Skeletal Diseases Genome Research Center, Kyungpook National University, Daegu 700-422, Republic of Korea c Department of Cardiology and Pharmacology, School of Medicine, University of Ulsan, Asan Medical Center, Seoul 138-736, Republic of Korea d Department of Endocrinology and Metabolism, School of Medicine, Kwandong University, Seoul 100-380, Republic of Korea e Department of Endocrinology and Metabolism, G-SAM Medical Center, Gunpo-si 435-010, Republic of Korea abstract article info Article history: Received 28 August 2013 Revised 28 November 2013 Accepted 27 December 2013 Available online 5 January 2014 Edited by: Shu Takeda Keywords: Osteoclast Recruitment CX3CL1 Chemokine Radiation Bone loss Circulating osteoclast precursor cells highly express CX3C chemokine receptor 1 (CX3CR1), which is the only receptor for the unique CX3C membrane-anchored chemokine, fractalkine (CX3CL1). An irradiated murine model was used to evaluate the role of the CX3CL1–CX3CR1 axis in osteoclast recruitment and osteoclastogene- sis. Ionizing radiation (IR) promoted the migration of circulating CD11b+ cells to irradiated bones and dose- dependently increased the number of differentiated osteoclasts in irradiated bones. Notably, CX3CL1 was dra- matically upregulated in the vascular endothelium after IR. IR-induced production of CX3CL1 by skeletal vascular endothelium promoted chemoattraction of circulating CX3CR1+/CD11b+ cells and triggered homing of these osteoclast precursor cells toward the bone remodeling surface, a specific site for osteoclast differentiation. CX3CL1 also increased the endothelium-derived expression of other chemokines including stromal cell- derived factor-1 (CXCL12) and macrophage inflammatory protein-2 (CXCL2) by activating the hypoxia- inducible factor-1 α pathway. These effects may further enhance osteoclastogenesis. A series of in vivo experi- ments confirmed that knockout of CX3CR1 in bone marrow-derived cells and functional inhibition of CX3CL1 using a specific neutralizing antibody significantly ameliorated osteoclastogenesis and prevented bone loss after IR. These results demonstrate that the de novo CX3CL1–CX3CR1 axis plays a pivotal role in osteoclast recruit- ment and subsequent bone resorption, and verify its therapeutic potential as a new target for anti-resorptive treatment. © 2014 Elsevier Inc. All rights reserved. Bone 61 (2014) 91–101 Abbreviations: μCT, micro-computed tomography; BM, bone marrow; BMCs, bone marrow cells; BMD, bone mineral density; BMMs, bone marrow macrophages; BMT, bone marrow transplantation; BMU, basic multicellular unit; CX3CR1, CX3C chemokine receptor 1; ECs, endothelial cells; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; HAECs, human aortic endothelial cells; HIF, hypoxia-inducible factor; IFN, interferon; IL, interleukin; IR, ionizing radiation; KO, knockout; mAb, monoclonal antibody; M-CSF, macrophage colony stimulating factor; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; OPG, osteoprotegerin; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; Pre-osteoclasts, osteoclast precursor cells; RANKL, receptor acti- vator of nuclear factor κB ligand; Real-time RT-PCR, real-time reverse transcription polymerase chain reaction PCR; sry, sex determining region Y; TNF, tumor necrosis factor; TRAP, tartrate-resistant acid phosphatase; WT, wild type. ☆ Grants: This work was supported by grants from the Korea Healthcare Technology Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (A080016, A050020) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0072141). ⁎ Correspondence to: K.H. Han, Department of Cardiology, School of Medicine, University of Ulsan, Asan Medical Center, 388-1 Pungnap-2 dong Songpa-gu, Seoul 138-736, Republic of Korea. Fax: +82 2 486 5918. ⁎⁎ Correspondence to: K.O. Han, Department of Endocrinology and Metabolism, G-SAM Medical Center, 730 Dang-dong, Gunpo-si, Gyeonggi-do 430-733, Republic of Korea. Fax: +82 31 389 3787. E-mail addresses: steadyhan@amc.seoul.kr (K.H. Han), hankiok@medigate.net (K.O. Han). 8756-3282/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bone.2013.12.032 Contents lists available at ScienceDirect Bone journal homepage: www.elsevier.com/locate/bone