O-GlcNAcylation Mediates BMP2-Induced Osteogenesis/Cementogenesis via NMIIA

The precise cellular origin and regulatory mechanisms underlying cementum development remain poorly understood, hindering progress toward ideal cementum regeneration. Bone morphogenetic protein 2 (BMP2), approved by the US Food and Drug Administration for clinical use due to its potent osteoinductive capacity, is a key candidate for such regulation. Activin receptor-like kinase 3 (Alk3)-mediated BMP signaling plays a crucial role in the development and structural maintenance of mineralized tissues, including teeth. However, the precise mechanism by which BMP signaling regulates periodontal tissue development mediated by periodontal ligament stem cells (PDLSCs), especially Gli1⁺ cells, remains unknown. Emerging evidence has indicated that O-GlcNAc glycosylation (O-GlcNAcylation), a dynamic posttranslational modification, modulates critical biological processes, such as transcription, translation, and cell fate determination. In this study, we demonstrate how BMP2 signaling enhances O-GlcNAcylation in a SMAD-dependent manner. Notably, we demonstrate that the lack of <i>Alk3</i> in Gli1⁺ cells resulted in reduced O-GlcNAcylation levels in vivo. Nonetheless, O-GlcNAcylation is identified as indispensable for PDLSC-mediated osteogenesis and cementogenesis both in vivo and in vitro. Moreover, deleting O-β-N-acetylglucosaminyltransferase (<i>Ogt</i>) in Gli1⁺ cells suppresses BMP signaling, consequently impairing cellular cementum formation and delaying alveolar socket healing. Mechanistically, we further revealed that the cytoskeleton, especially MYH9 (nonmuscle myosin IIA, NMIIA), is O-GlcNAcylated and is essential for BMP2-induced osteogenic/cementogenic differentiation. These findings demonstrate that O-GlcNAcylation is essential for cellular cementum formation by modulating the BMP signaling pathway in PDLSC differentiation and Gli1⁺ periodontal progenitors, highlighting its critical role in both tooth root development and alveolar bone repair.