Osteoclast-lineage Indian hedgehog sustains RANKL-induced osteoclastogenesis and bone resorption via the JNK/c-Fos/NFATc1 axis during postnatal bone remodeling.

Bone remodeling depends on the coordinated balance between osteoclast-mediated resorption and osteoblast-mediated formation. Although Indian hedgehog (Ihh) is known to regulate skeletal development and bone homeostasis, its direct involvement in osteoclastogenesis remains unclear. We generated a novel mouse model, Ctsk-CreERT2; Ihhfl/fl, to investigate the role of Ihh in osteoclast lineage cells. Skeletal phenotypes were assessed by radiography, micro-CT, histology, dynamic histomorphometry, and three-point bending tests. Bone marrow macrophages were isolated to assess osteoclast precursor viability, RANKL-induced osteoclast differentiation, F-actin ring formation, and bone resorptive activity using CCK-8 assays, TRAP staining, phalloidin staining, and resorption pit assays. Osteoclastogenic gene expression and signaling pathway activation were examined by RT-qPCR and Western blotting. IhhCKO mice exhibited reduced body weight and an osteosclerotic phenotype, characterized by increased metaphyseal trabecular bone mass, improved femoral peak load and stiffness, and reduced cortical bone parameters. Histological analyses revealed fewer TRAP-positive osteoclasts on trabecular surfaces, increased osteoblast-related parameters, and decreased cortical bone formation indices. In vitro, Ihh deficiency reduced osteoclast precursor viability, impaired RANKL-induced osteoclast formation, decreased F-actin ring number/size and nuclei per osteoclast, and markedly reduced resorption pit formation. Mechanistically, Ihh loss suppressed osteoclast marker and fusion-related genes and diminished RANKL-induced JNK phosphorylation, c-Fos, NFATc1, and CTSK expression. Osteoclast-lineage-derived Ihh is required for efficient RANKL-induced osteoclastogenesis and bone resorption, at least in part by sustaining the JNK/c-Fos/NFATc1 axis. These findings identify Ihh as an osteoclast-intrinsic regulator of postnatal bone remodeling.