CaMKIIγ inhibition attenuates osteoclastogenesis via the NF-κB-NFATc1 axis and protects against ovariectomy-induced osteoporosis.

Osteoporosis features excessive osteoclast-mediated bone resorption, and long-term safety concerns with current antiresorptives and rebound after discontinuation underscore the need for new therapeutic targets. Calcium/calmodulin-dependent kinases (CaMKs) have been linked to osteoclastogenesis, yet which CaMKII isoform is functionally essential and therapeutically actionable remains incompletely defined. Here, we investigated the role of calcium/calmodulin-dependent protein kinase II gamma (CaMKIIγ) in the osteoclast lineage, examined its downstream signaling mechanisms, and evaluated whether its pharmacological inhibition protects against ovariectomy-induced bone loss. CaMKIIγ expression increased during osteoclastogenesis induced by receptor activator of NF-κB ligand (RANKL), and shRNA-mediated Camk2g knockdown in RAW 264.7 cells markedly reduced multinucleated osteoclast formation. Functionally, Camk2g knockdown disrupted F-actin ring organization and reduced osteoclast resorptive pit formation. Berbamine (BBM), a reported CaMKIIγ inhibitor, phenocopied these inhibitory effects at non-cytotoxic concentrations during osteoclast differentiation, with maximal inhibition when applied during early differentiation. RNA sequencing and quantitative PCR revealed coordinated repression of osteoclastogenic gene programs upon CaMKIIγ inhibition. Pathway enrichment analyses implicated NF-κB signaling, and western blotting confirmed that Camk2g knockdown and BBM preferentially attenuated early RANKL-induced p65 phosphorylation, accompanied by reduced downstream nuclear factor of activated T cells 1 (NFATc1) induction. In ovariectomized mice, BBM administration at 25 or 50 mg/kg per day by intraperitoneal injection partially preserved trabecular microarchitecture and reduced osteoclast presence, as shown by micro-CT and histological staining. These findings identify CaMKIIγ as a regulator of osteoclastogenesis and support CaMKIIγ inhibition as a potential strategy to prevent postmenopausal bone loss.