O-GlcNAc transferase controls osteoblast differentiation through modulation of extracellular vesicle composition and calcium homeostasis.

Osteoblast differentiation and bone matrix mineralization depend on tightly regulated calcium signaling and extracellular vesicle (EV)-mediated communication. O-GlcNAc transferase (OGT), a nutrient-sensing enzyme that adds O-linked N-acetylglucosamine to proteins, regulates diverse signaling pathways, but its role in osteoblast-derived EVs is still not well defined. OGT-knockout MC3T3-E1 cells (KO-Ogt) was generated to investigate how OGT controls EV calcium content and osteogenic signaling. EVs from differentiated control and KO-Ogt cells were isolated, characterized, and analyzed for calcium content. The osteoinductive capacity of EVs was evaluated by measuring osteogenic markers expression, alkaline phosphatase activity, and Alizarin Red staining in recipient MC3T3-E1 cells. Public RNA-seq data (GSE138783) from cells with altered global O-GlcNAc level were reanalyzed to identify vesicle and calcium-related pathways. Calcineurin protein levels in control and KO-Ogt cells, as well as NFAT1 localization on the EVs recipient cells were also examined. KO-Ogt cells showed a marked reduction in OGT and global O-GlcNAcylation level. While EV size distribution and morphology seemed to be unaltered, the EVs from KO-Ogt cells contained significantly less calcium. KO-Ogt EVs also failed to support osteoblast differentiation, as shown by decreased differentiation markers expression, and impaired mineral deposition. Transcriptomic reanalysis revealed OGT-dependent enrichment of vesicle trafficking and calcium homeostasis pathways, including upstream regulators of the calcineurin-NFAT axis. These findings identify OGT as a critical regulator of osteoblast differentiation by remodeling EV calcium cargo and modulating cellular calcium homeostasis.