Abstract
BACKGROUND: Bone morphogenetic protein 9 (BMP9) has been demonstrated to robustly induce osteogenic differentiation of mesenchymal stem cells (MSCs), offering substantial potential for advancements in bone tissue engineering. The purinergic receptor P2X7 has emerged as a crucial modulator of bone formation and bone metabolism. However, the precise role of P2X7 in BMP9-induced osteogenic differentiation of MSCs and the associated molecular mechanisms remain partially understood. This study aims to ascertain the exact function of P2X7 in BMP9-induced osteogenic differentiation of MSCs, and to unravel the relevant molecular mechanism. METHODS: Transcriptome sequencing, quantitative real-time polymerase chain reaction, Western blot, and chromatin immunoprecipitation assays were initially performed to validate the up-regulation of P2X7 by BMP9. Subsequently, the influence of P2X7 on BMP9-induced osteogenic differentiation of MSCs was assessed through ALP determination, calcium deposition analysis, Western blotting, ectopic bone formation model, and skull defect model. Finally, the mechanism through which P2X7 modulated BMP9-induced osteogenic differentiation of MSCs was investigated using intracellular Ca(2+) imaging, Western blot, molecular docking, immunoprecipitation and immunofluorescence staining. RESULTS: BMP9 was confirmed to upregulate P2X7 expression via Smad signaling. Activation of P2X7 significantly potentiated BMP9-induced osteogenic differentiation of MSCs and enhanced BMP9-promoted ectopic bone formation, then enhanced the repair of bone defect by BMP9. Conversely, inhibition of P2X7 elicited a contrary effect. Mechanistically, P2X7 activation promoted calcium influx, subsequently leading to the activation of CaMKII by phosphorylation. The activated CaMKII then interacted with GSK-3β, facilitating the inhibitory phosphorylation of GSK-3β at Serine 9 (Ser 9) residue. This may stabilize β-catenin and increase its nuclear translocation, thus finally mediating the osteogenic differentiation of MSCs induced by BMP9. CONCLUSION: This study clarifies that P2X7 may mediate BMP9-induced osteogenic differentiation of MSCs through the CaMKII/GSK-3β/β-catenin axis, providing novel insights into the molecular mechanism of BMP9-induced osteogenesis and a potential target for bone defect treatment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-026-02747-w.