Ano5 Deficiency Leads to Abnormal Bone Formation via miR-34c-5p/KLF4/β-Catenin in Gnathodiaphyseal Dysplasia

Gnathodiaphyseal dysplasia (GDD) is a rare autosomal dominant genetic disease, mainly characterized by enlargement of the mandible, osteosclerosis, and frequent fracture of tubular bone. GDD is caused by heterozygous mutations in <i>Anoctamin 5</i> (<i>ANO5</i>). We have previously generated an <i>Ano5</i> knockout (KO) mice model and validated the phenotypes consistent with GDD patients, including enhanced bone formation and alkaline phosphatase (ALP) activity. Experiments have identified that <i>Ano5</i> deficiency elevated the osteogenesis of calvaria-derived osteoblasts (mCOBs). In this study, we found that <i>Ano5</i> deficiency notably inhibited miR-34c-5p expression. Krüppel-Like Factor 4 (<i>Klf4</i>), a target gene of miR-34c-5p confirmed by dual luciferase reporter assay, was up-regulated in <i>Ano5^-/-</i> mCOBs, accompanied by activated downstream canonical Wnt/β-catenin signaling and increased expression of β-catenin. Overexpression of miR-34c-5p in <i>Ano5^-/-</i> mCOBs inhibited osteogenic capacity by suppressing proliferative capacity, osteoblast-related factor levels, ALP activity, and matrix calcification through regulating KLF4/β-catenin signaling axis. Furthermore, miR-34c-5p adeno-associated virus (AAV) treatment in vivo rescued the abnormally thickened cortical bone and enhanced biomechanical properties in <i>Ano5</i>^-/- mice. Importantly, the serum level of P1NP, a marker of bone formation, was also significantly declined. We conclude that dysregulation of miR-34c-5p contributes to the enhanced osteogenesis in GDD by excessive activation of KLF4/β-catenin signaling axis under Ano5-deficient conditions. This study elucidates the pathogenesis of GDD and provides novel insights into the therapeutic strategies.