Hypergravity reduces F-actin accumulation in osteoclasts, with attenuated bone resorption

Bone loss occurs in astronauts during prolonged spaceflight, thus indicating the sensitivity of skeletal homeostasis to altered gravitational environments. Previous studies have shown that microgravity affects osteoclast differentiation and bone resorption, which suggests that osteoclasts possess mechanisms to sense and respond to gravity-generated mechanical forces. For testing of the related mechanisms, hypergravity can be experimentally reproduced with use of a centrifuge. In the present study, osteoclasts derived from mouse bone marrow were subjected to hypergravity under three conditions: 30G exposure using a non-CO2 centrifuge system, and short- or long-term exposure to 3G or 5G using an incubator-compatible centrifuge system. Cytoskeletal organization and resorptive function were assessed using TRAP (tartrate-resistant acid phosphatase) staining, F-actin visualization, and dentin pit assays. In addition, phosphoproteomic analysis was performed after short-term exposure to 5G hypergravity. Hypergravity exposure for as brief as 30 minutes compromised F-actin ring integrity, reduced fluorescence intensity, and promoted nuclear repositioning toward actin rings, whereas tubulin and vinculin localization remained unchanged, and the structural alterations corresponded to attenuated resorption pit formation. Quantitative phosphoproteomic profiling revealed coordinated hypergravity-dependent changes in phosphorylation across multiple cellular modules, including cytoskeletal organization, membrane trafficking, intracellular signaling, and nuclear regulatory pathways. Together, these results indicate that osteoclasts are sensitive to gravity-generated mechanical loading, with hypergravity rapidly modifying F-actin-associated cytoskeleton properties and reprogramming phosphorylation-dependent signaling networks, ultimately attenuating bone-resorptive activity. These findings provide mechanistic insight into how osteoclasts respond to altered gravitational loading conditions and have implications for skeletal adaptation during spaceflight and under altered mechanical loading conditions on Earth.