Ano5 deficiency inhibits osteoclastogenesis and reduces ovariectomy-induced bone loss by regulating the ACSL4-dependent ferroptosis pathway

Ano5 loss alleviates osteoporosis by inhibiting osteoclastogenesis in OVX mice. • Ano5 deficiency triggers osteoclast ferroptosis by upregulating ACSL4 protein. • The Ano5 -ACSL4-ferroptosis axis is a therapeutic target for osteoporosis. Osteoporosis is driven by a pathological imbalance in bone remodeling, with osteoclast hyperactivation being a primary driver of bone loss. However, the intrinsic signaling nodes that govern osteoclast survival and function remain poorly understood. While loss-of-function mutations in Anoctamin 5 ( Ano5 ) are associated with genetic skeletal disorders, its precise involvement in metabolic osteoporosis remains elusive. In this study, we utilized an Ano5 -deficient ovariectomized (OVX) mouse model to investigate the role of Ano5 in bone homeostasis. We found that genetic depletion of Ano5 significantly attenuated the osteoporotic phenotype in OVX mice, evidenced by preserved bone microarchitecture, enhanced biomechanical properties, and suppressed bone resorptive activity. Mechanistically, Ano5 -deficient osteoclasts exhibited classical hallmarks of ferroptosis, including the accumulation of the lipid peroxidation end-product malondialdehyde (MDA), elevated reactive oxygen species (ROS), and a notable upregulation of the key lipid metabolic enzyme ACSL4 at the protein level. Importantly, the impaired osteoclastogenesis observed in Ano5 -deficient cells was effectively rescued by pharmacological inhibition of either ferroptosis (via Ferrostatin-1) or ACSL4 activity (via Rosiglitazone), which restored TRAP-positive osteoclast numbers, F-actin ring formation, and the expression of resorptive markers such as CTSK. Our findings identify Ano5 as a critical regulator of bone remodeling that modulates osteoclastogenesis through the ACSL4-dependent ferroptosis pathway. This study not only uncovers a previously unrecognized molecular axis in bone cell biology but also highlights the Ano5 -ACSL4-ferroptosis axis as a potential therapeutic target for the treatment of osteoporosis.