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Anoctamin-5 deficiency enhances ATG9A-dependent autophagy, inducing osteogenesis and gnathodiaphyseal dysplasia–like bone formation
Shuai Zhang, … , Hongyu Li, Ying Hu
Shuai Zhang, … , Hongyu Li, Ying Hu
Published March 11, 2025
Citation Information: JCI Insight. 2025;10(8):e189817. https://doi.org/10.1172/jci.insight.189817.
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Research Article Bone biology Genetics

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

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Abstract

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.

Authors

Shuai Zhang, Shengnan Wang, Sirui Liu, Xiu Liu, Mingyue Zhang, Huichong Xu, Xiaoyu Wang, Hongyu Li, Ying Hu

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Figure 1

Autophagic flux is promoted by the absence of Ano5 in mCOBs.

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Autophagic flux is promoted by the absence of Ano5 in mCOBs.
(A and B) W...
(A and B) Western blot analysis of p62, OCN, LC3B-II, and COL1A1 protein levels in mCOBs from the Ano5+/+ and Ano5–/– groups during osteogenic differentiation. (C) Western blot showing the accumulation of LC3B-II in mCOBs from the Ano5+/+ and Ano5–/– groups after CQ treatment. (D) TEM images of mCOBs following 14 days of osteogenic induction, displaying autophagosomes (black arrowheads) and autolysosomes (red arrowheads). Scale bars: 2 μm (top) and 1 μm (bottom). (E) Quantitation of autophagosomes and autolysosomes observed in TEM images. (F and H) Confocal microscopy of mCOBs infected with mCherry-GFP-LC3B adenovirus, assessed on day 0 and day 14 of osteogenic induction. Representative images show autophagic activity. Scale bars: 25 μm (top rows) and 10 μm (bottom rows). The zoomed-in images in F are magnified ×800, and those in H are magnified ×1200. (G and I) Quantification of autophagosomes (mCherry+GFP+, yellow puncta) and autolysosomes (mCherry+GFP–, red puncta) in F and H, respectively. Data are represented as mean ± SD. *P < 0.05; NS, P > 0.05, as assessed by 1-way ANOVA followed by Tukey’s post hoc test.

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