Anoctamin-5 deficiency enhances ATG9A-dependent autophagy, inducing osteogenesis and gnathodiaphyseal dysplasia–like bone formation

Mutations in the anoctamin-5 (ANO5) gene can lead to musculoskeletal disorders, with monoallelic (autosomal dominant) mutations typically presenting as skeletal abnormalities known as gnathodiaphyseal dysplasia (GDD). Clinically, GDD is characterized by thickened cortices of long bones and mandibles, narrowed medullary cavities, and increased bone fragility. While autophagy is necessary in regulating bone formation, the specific relationship between ANO5 and autophagy remains poorly understood. In this study, we demonstrated that Ano5 deficiency activates autophagy in mouse cranial osteoblasts (mCOBs), leading to enhanced osteogenic capacity in Ano5-/- mCOBs. The application of 3-methyladenine (3-MA) and chloroquine (CQ) reversed the excessive osteogenesis observed in Ano5-/- mCOBs. Further analysis revealed that Ano5 deficiency upregulated the expression of ATG9A, and silencing ATG9A significantly reduced both autophagy and osteogenic activity in Ano5-/- mCOBs. Additionally, AMP-activated protein kinase (AMPK) was found to positively regulate ATG9A, and inhibiting AMPK reduced ATG9A expression, which in turn mitigated excessive osteogenesis of Ano5-/- mCOBs. Moreover, in vivo experiments confirmed that treatment with 3-MA alleviated the bone phenotype abnormalities in Ano5-/- mice. These findings suggest that Ano5 negatively regulates autophagy, contributing to illuminate pathogenesis of GDD. Meanwhile, this research highlights potential therapeutic strategies targeting autophagy to pave the way for the clinical manifestations of GDD.