Targeting AHNAK ameliorates osteonecrosis of the femoral head and affects adipogenic pathways involving Smad/PPARγ

Aims: This study aimed to investigate the role of AHNAK in patients with osteonecrosis of the femoral head (ONFH) and its association with bone adipogenesis. Non-traumatic ONFH, primarily driven by steroid use and alcohol consumption, results in an imbalance between osteogenesis and adipogenesis. Understanding the function of AHNAK under ischaemic stress may help identify novel therapeutic targets for ONFH. Methods: Bone tissues from 19 ONFH patients and 15 control patients were analyzed using proteomics, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF). A steroid-induced ONFH mouse model and an ischaemia-mimicking in vitro model using MC3T3-E1 preosteoblasts were employed to examine AHNAK expression and function. AHNAK knockdown was performed using short hairpin RNA (shRNA) to assess its regulatory effects on adipogenic markers and Smad/PPARγ/C/EBPα signalling. Results: AHNAK expression was significantly upregulated in bone tissues of ONFH patients and in femoral heads of steroid-induced ONFH mice. This was accompanied by reduced osteocyte numbers and increased adipogenesis, as indicated by elevated perilipin 1 (PLIN1) expression. In vitro, ischaemic stress induced AHNAK and PLIN1 expression, whereas AHNAK knockdown reversed these effects and suppressed Smad1/5 phosphorylation and PPARγ activation. C/EBPα expression remained unchanged, suggesting a selective role of AHNAK in modulating the Smad/PPARγ axis during adipogenesis. Conclusion: AHNAK is a critical mediator of ischaemia-induced adipogenesis in ONFH through activation of the Smad/PPARγ signalling pathway, promoting the differentiation of preosteoblasts into adipocytes. Targeting AHNAK may offer a novel therapeutic strategy to reduce bone loss and marrow fat accumulation in ONFH, particularly in cases associated with steroids or metabolic dysfunction. Further studies are warranted to validate AHNAK as a clinical target.