PURPOSE: Bone morphogenetic protein 9 (BMP9) is a cytokine with potent osteoinductive activity, although its precise regulatory mechanisms remain incompletely elucidated. NOTUM, a secreted palmitoleoyl-protein carboxylesterase, has an unclear role in osteogenic differentiation. This study aims to explore the role and mechanism of NOTUM in BMP9-induced osteogenic differentiation of mouse embryonic fibroblasts (MEFs).
METHODS TO METHODS: In the BMP9-induced osteogenic differentiation model, Notum was either overexpressed or silenced, and the expression of RUNX2 and OPN as well as osteogenic signaling pathways were assessed by q-PCR and Western blot. ALP staining and Alizarin Red staining were subsequently performed to determine ALP activity and matrix mineralization. Furthermore, the osteogenic effect of Notum was evaluated using an in vivo subcutaneous ectopic osteogenesis model, supplemented by histochemical staining and micro-CT analysis.
RESULTS: BMP9 significantly downregulates NOTUM protein expression in MEF cells. NOTUM attenuated BMP9-induced expression of osteogenic markers RUNX2 and OPN, as well as ALP activity and matrix mineralization. In vivo ectopic osteogenesis assays yielded results consistent with in vitro observations. Mechanistically, NOTUM overexpression reduced β-catenin protein levels, while NOTUM knockdown elevated β-catenin accumulation during BMP9-mediated osteogenic differentiation. NOTUM overexpression up-regulated GSK-3β protein expression while suppressing its phosphorylation status during BMP9-induced osteogenesis, whereas NOTUM depletion produced the converse effects. Pharmacological activation of the Wnt/β-catenin signaling pathway rescued osteogenic marker expression, effectively counteracting NOTUM-mediated suppression of BMP9's osteogenic potential.
CONCLUSIONS: NOTUM serves as a novel regulator of BMP9-Induced osteogenesis via modulation of the Wnt/β-Catenin Axis, Suggesting its potential as a therapeutic target for bone regeneration.
