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

• Published in: BioFactors (Oxford) 2025-01, Vol.51 (1), p.e2131
• Main Authors: Weng, Yao, Wang, Ziyi, Sitosari, Heriati, Ono, Mitsuaki, Okamura, Hirohiko, Oohashi, Toshitaka
• Format: Article
• Language: English
• Subjects: Animals; Cell Differentiation; Cell Line; Cell Proliferation; Cytoskeleton - genetics; Cytoskeleton - metabolism; Endoplasmic Reticulum - genetics; Endoplasmic Reticulum - metabolism; Enhancer of Zeste Homolog 2 Protein - genetics; Enhancer of Zeste Homolog 2 Protein - metabolism; Glycosylation; Mice; Mitochondria - genetics; Mitochondria - metabolism; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - metabolism; Osteoblasts - cytology; Osteoblasts - metabolism; Osteogenesis - genetics; Osteogenesis - physiology
• ISSN: 0951-6433; 1872-8081; 1872-8081
• DOI: 10.1002/biof.2131

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.