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

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 (Ano5 KI/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.

Akt activation by SC79 stimulation can obviously rescue abnormal increased osteogenesis and decreased osteoclastogenesis in Ano5 KI/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.

Bone marrow-derived macrophages (BMMs) and mouse calvarial osteoblasts (mCOBs) were isolated from homozygous Ano5 KI/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.

Akt activation promoted the generation of TRAP-positive multinucleated osteoclasts and the formation of actin rings in Ano5 KI/KI BMMs cultures, accompanied by increased expression of Nfatc1, Trap, Dc-stamp, Mmp9, Ctsk, and Atp6v0d2. Additionally, Ano5 Cys360Tyr mutation down-regulated the Akt phosphorylation level in osteoblast. ALP activity and matrix mineralization capacity in Ano5 KI/KI osteoblast cultures were inhibited after SC79 stimulation, with reduced expression of Runx2, Opn, Col1a1, and Ocn.