A novel stromal cell source of RANKL during sustained osteoclast activation in disuse osteoporosis

Prolonged physical inactivity causes mechanical unloading of the bone, leading to disuse osteoporosis. Although various cellular and molecular changes have been reported, the mechanisms underlying disuse osteoporosis remain incompletely understood. Using the spiral wire immobilization (SWI) model, we observed that hindlimb immobilization in mice caused trabecular bone loss within 14 days after SWI initiation. Time-course analysis revealed that trabecular bone loss progressed rapidly shortly after SWI initiation, with little progression at later time points. During the early phase of bone loss, enhancement of bone resorption was sustained following transient suppression of bone formation. Administration of an antibody against receptor activator of nuclear factor-κB ligand (RANKL), which inhibits osteoclastogenesis, alleviated bone loss, suggesting that osteoclast-mediated bone resorption contributes substantially to bone loss during hindlimb immobilization. Transcriptomic analysis of the osteocyte-enriched bone cell fraction revealed SWI-enhanced expression of bone development-related genes, whereas expression of the Tnfsf11 gene encoding RANKL remained unchanged. However, single-cell RNA sequencing of bone marrow cells from RANKL fate-mapping mice demonstrated that SWI increased RANKL expression specifically in adipogenic C-X-C motif chemokine ligand 12-abundant reticular (adipo-CAR) cells, but not in osteoblasts or osteocytes. These findings clarify the contribution of osteoclastic bone resorption and identify adipo-CAR cells as a novel RANKL source. This study offers new insights into the pathogenesis of disuse osteoporosis and informs the development of therapeutic and preventive strategies for physical inactivity-associated bone loss. KEY POINTS: Prolonged physical inactivity can lead to disuse osteoporosis, yet the cellular and molecular mechanisms remain poorly understood. To elucidate its pathogenesis, we utilized a spiral wire immobilization (SWI) mouse model to investigate bone loss at cellular and molecular levels. Rapid bone loss driven by sustained enhancement of osteoclastic bone resorption was effectively suppressed by early anti-receptor activator of nuclear factor-κB ligand (RANKL) antibody treatment targeting osteoclast activity. SWI induced RANKL expression specifically in adipogenic C-X-C motif chemokine ligand 12-abundant reticular (adipo-CAR) cells, identifying them as a previously unrecognized cellular source of osteoclastogenic signalling. This study provides molecular insight into the pathogenesis of disuse osteoporosis and underscores the importance of early intervention targeting osteoclast activation, thereby informing new therapeutic strategies.