Ano5Cys360Tyr mutation leads to bone dysfunction of gnathodiaphyseal dysplasia via disturbing Akt signaling

Abstract

Background: Gnathodiaphyseal dysplasia (GDD) is a rare autosomal dominant genetic disease characterized by osteosclerosis of the tubular bones and cemento-osseous lesions of the mandibles. Anoctamin 5 (ANO5) is the pathogenic gene, however, the specific molecular mechanism of GDD remains unclear. Herein, a knockin (Ano5KI/KI) mouse model expressing the human mutation p.Cys360Tyr was used to investigate the role of Akt signaling in enhanced osteogenesis and decreased osteoclastogenesis in GDD. Methods: Bone marrow-derived macrophages (BMMs) and mouse calvarial osteoblasts (mCOBs) were isolated from homozygous Ano5KI/KI mice and treated with SC79, a specific Akt activator. The differentiation and F-actin ring formation of osteoclasts were examined by TRAP and phalloidin staining, respectively. Osteoblast differentiation and mineralization were examined by ALP and alizarin red staining. The expression of bone remodeling-related factors was measured by qRT-PCR. Results: Akt activation promoted the generation of TRAP-positive multinucleated osteoclasts and the formation of actin rings in Ano5KI/KI BMMs cultures, accompanied by increased expression of Nfatc1, Trap, Dc-stamp, Mmp9, Ctsk, and Atp6v0d2. Additionally, Ano5Cys360Tyr mutation down-regulated the Akt phosphorylation level in osteoblast. ALP activity and matrix mineralization capacity in Ano5KI/KI osteoblast cultures were inhibited after SC79 stimulation, with reduced expression of Runx2, Opn, Col1a1, and Ocn. Conclusion: Akt activation by SC79 stimulation can obviously rescue abnormal increased osteogenesis and decreased osteoclastogenesis in Ano5KI/KI mouse model, which demonstrated that disturbed Akt signaling pathway may play a pivotal role in the pathogenesis of GDD, and an Akt activator is probable a therapeutic target for GDD.

Keywords

• Ano5
• GDD
• Akt
• Osteoblast
• Osteoclast