Spleen serves as a reservoir of osteoclast precursors through vitamin D-induced IL-34 expression in osteopetrotic op/op mice

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-(20 S)-1α,25(OH) ₂D 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.