[Show abstract][Hide abstract] ABSTRACT: Osteoclasts are generated from monocyte/macrophage-lineage precursors in response to colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). CSF-1-mutated CSF-1(op/op) mice as well as RANKL(-/-) mice exhibit osteopetrosis (OP) caused by osteoclast deficiency. We previously identified RANKL receptor (RANK)/CSF-1 receptor (CSF-1R) double-positive cells as osteoclast precursors (OCPs), which existed in bone in RANKL(-/-) mice. Here we show that OCPs do not exist in bone but in spleen in CSF-1(op/op) mice, and spleen acts as their reservoir. IL-34, a newly discovered CSF-1R ligand, was highly expressed in vascular endothelial cells in spleen in CSF-1(op/op) mice. Vascular endothelial cells in bone also expressed IL-34, but its expression level was much lower than in spleen, suggesting a role of IL-34 in the splenic generation of OCPs. Splenectomy (SPX) blocked CSF-1-induced osteoclastogenesis in CSF-1(op/op) mice. Osteoclasts appeared in aged CSF-1(op/op) mice with up-regulation of IL-34 expression in spleen and bone. Splenectomy blocked the age-associated appearance of osteoclasts. The injection of 2-methylene-19-nor-(20S)-1α,25(OH)(2)D(3) (2MD), a potent analog of 1α,25-dihidroxyvitamin D(3), into CSF-1(op/op) mice induced both hypercalcemia and osteoclastogenesis. Administration of 2MD enhanced IL-34 expression not only in spleen but also in bone through a vitamin D receptor-mediated mechanism. Either splenectomy or siRNA-mediated knockdown of IL-34 suppressed 2MD-induced osteoclastogenesis. These results suggest that IL-34 plays a pivotal role in maintaining the splenic reservoir of OCPs, which are transferred to bone in response to diverse stimuli, in CSF-1(op/op) mice. The present study also suggests that the IL-34 gene in vascular endothelial cells is a unique target of vitamin D.
Proceedings of the National Academy of Sciences 06/2012; 109(25):10006-11. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fos plays essential roles in the osteoclastic differentiation of precursor cells generated by colony-stimulating factor 1 (CSF-1) and receptor activator of NF-κB ligand (RANKL; also known as tumor necrosis factor ligand superfamily member 11, Tnsf11). RANKL-deficient (RANKL(-/-)) mice and Fos(-/-) mice exhibit osteopetrosis due to an osteoclast deficiency. We previously reported that RANK-positive osteoclast precursors are present in bone of RANKL(-/-) mice but not Fos(-/-) mice. Here we report the role of Fos in RANK expression in osteoclast precursors. Medullary thymic epithelial cells and intestinal antigen-sampling microfold cells have been shown to express RANK. High expression of RANK was observed in some epithelial cells in the thymic medulla and intestine but not in osteoclast precursors in Fos(-/-) mice. RANK mRNA and protein levels in bone were lower in Fos(-/-) mice than RANKL(-/-) mice, suggesting that Fos-regulated RANK expression is tissue specific. When wild-type bone marrow cells were inoculated into Fos(-/-) mice, RANK-positive cells appeared along bones. RANK expression in wild-type macrophages was upregulated by coculturing with RANKL(-/-) osteoblasts as well as wild-type osteoblasts, suggesting that cytokines other than RANKL expressed by osteoblasts upregulate RANK expression in osteoclast precursors. CSF-1 receptor-positive cells were detected near CSF-1-expressing osteoblastic cells in bone in Fos(-/-) mice. CSF-1 upregulated RANK expression in wild-type macrophages but not Fos(-/-) macrophages. Overexpression of Fos in Fos(-/-) macrophages resulted in the upregulation of RANK expression. Overexpression of RANK in Fos(-/-) macrophages caused RANKL-induced signals, but failed to recover the RANKL-induced osteoclastogenesis. These results suggest that Fos plays essential roles in the upregulation of RANK expression in osteoclast precursors within the bone environment.
[Show abstract][Hide abstract] ABSTRACT: Osteoclasts are derived from the monocyte/macrophage lineage, but little is known about osteoclast precursors in circulation. We previously showed that cell cycle-arrested quiescent osteoclast precursors (QOPs) were detected along bone surfaces as direct osteoclast precursors. Here we show that receptor activator of NF-κB (RANK)-positive cells isolated from bone marrow and peripheral blood possess characteristics of QOPs in mice. RANK-positive cells expressed c-Fms (receptors of macrophage colony-stimulating factor) at various levels, but scarcely expressed other monocyte/granulocyte markers. RANK-positive cells failed to exert phagocytic and proliferating activities, and differentiated into osteoclasts but not into dendritic cells. To identify circulating QOPs, collagen disks containing bone morphogenetic protein-2 (BMP disks) were implanted into mice, which were administered bromodeoxyuridine daily. Most nuclei of osteoclasts detected in BMP-2-induced ectopic bone were bromodeoxyuridine-negative. RANK-positive cells in peripheral blood proliferated more slowly and had a much longer lifespan than F4/80 (a macrophage marker)-positive macrophages. When BMP disks and control disks were implanted in RANK ligand-deficient mice, RANK-positive cells were observed in the BMP disks but not in the controls. F4/80-positive cells were distributed in both disks. Administration of FYT720, a sphingosine 1-phosphate agonist, promoted the egress of RANK-positive cells from hematopoietic tissues into bloodstream. These results suggest that lineage-determined QOPs circulate in the blood and settle in the bone.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 08/2011; 26(12):2978-90. · 6.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: How are sites suitable for osteoclastogenesis determined? We addressed this issue using in vivo and in vitro experimental systems. We first examined the formation of osteoclasts in ectopic bone induced by BMP-2. When collagen disks which contained BMP-2 (BMP-2-disks) or vehicle (control-disks) were implanted into wild-type mice, osteoclasts and osteoblasts appeared in the BMP-2-disks, but not in the control disks. RANKL-deficient (RANKL(-/-)) mice exhibited osteopetrosis, with an absence of osteoclasts. BMP-2 and control disks were implanted into RANKL(-/-) mice, which were intraperitoneally injected with RANKL. Osteoclasts formed in the BMP-2-disks, but not in the control disks. In the BMP-2-disks, osteoclasts were observed in the vicinity of osteoblasts. Cell cycle-arrested quiescent osteoclast precursors (QOP) were identified as the committed osteoclast precursors in vitro. Experiments in vivo showed that QOPs survived for several weeks, and differentiated into osteoclasts in response to M-CSF and RANKL. QOPs were identified as RANK and c-Fms double-positive cells, and detected along bone surfaces in the vicinity of osteoblasts in RANKL(-/-) mice. QOPs were also observed in the ectopic bone induced by BMP-2 implanted into RANKL(-/-) mice, suggesting that QOPs were circulating. These results imply that osteoblasts support the homing of QOPs to bone tissues. In response to bone-resorbing stimuli, QOPs promptly differentiate into osteoclasts. Therefore, the distribution of QOPs appears to determine the correct site of osteoclastic development.
Advances in Experimental Medicine and Biology 01/2010; 658:21-30. · 2.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteoclasts are multinucleated cells that resorb bone. Although osteoclasts originate from the monocyte/macrophage lineage, osteoclast precursors are not well characterized in vivo. The relationship between proliferation and differentiation of osteoclast precursors is examined in this study using murine macrophage cultures treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB (RANK) ligand (RANKL). Cell cycle-arrested quiescent osteoclast precursors (QuOPs) were identified as the committed osteoclast precursors in vitro. In vivo experiments show that QuOPs survive for several weeks and differentiate into osteoclasts in response to M-CSF and RANKL. Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D(3) analogue given to those mice. Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice. These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs.
The Journal of Cell Biology 03/2009; 184(4):541-54. · 9.69 Impact Factor