O‐GlcNAcylation regulates osteoblast differentiation through the morphological changes in mitochondria, cytoskeleton, and endoplasmic reticulum

Abstract To explore the potential mechanisms which O ‐linked‐N‐acetylglucosaminylation ( O ‐GlcNAcylation) regulates osteogenesis, a publicly RNA‐seq dataset was re‐analyzed with literature‐mining and showed the primary targets of O ‐GlcNAcylation in osteoblasts are mitochondria/cytoskeleton. Although the O ‐GlcNAcylation‐regulated mitochondria/cytoskeleton has been extensively studied, its specific role during osteogenesis remains unclear. To address this, we knocked out Ogt (Ogt‐KO) in MC3T3‐E1 osteoblastic cells. Then, significantly reduced osteoblast differentiation, motility, proliferation, mitochondria–endoplasmic reticulum (Mito–ER) coupling, volume of ER, nuclear tubulins, and oxygen metabolism were observed in Ogt‐KO cells. Through artificial intelligence (AI)‐predicted cellular structures, the time‐lapse live cells imaging with reactive‐oxygen‐species/hypoxia staining showed that lower cell proliferation and altered oxygen metabolism in the Ogt‐KO cells were correlated with the Mito–ER coupling. Bioinformatics analysis, combined with correlated mRNA and protein expression, suggested that Ezh2 and its downstream targets ( Opa1 , Gsk3a , Wnt3a , Hif1a , and Hspa9 ) may be involved in O ‐GlcNAcylation‐regulated Mito–ER coupling, ultimately impacting osteoblast differentiation. In conclusion, our findings indicate that O‐GlcNAcylation‐regulated osteoblast differentiation is linked to morphological changes in mitochondria, cytoskeleton, and ER, with Ezh2 potentially playing a crucial role.