O-GlcNAcylation: A Morphologic Regulator of Osteoblast Differentiation

Published: 7 October 2024| Version 1 | DOI: 10.17632/5ybkzhyp8y.1
Contributor:
Ziyi Wang

Description

To explore the potential mechanisms which O-linked-Nacetylglucosaminylation (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.

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In this study, we re-analyzed a publicly available RNA-seq dataset (GSE138783) from the Gene Expression Omnibus (GEO) database, complemented by literature mining. Our approach identified mitochondria and the cytoskeleton as the primary targets of O-GlcNAcylation in osteoblasts. By cross-referencing multiple databases (O-GlcNAcAtlas, TFLink, and Ensembl Variation), we constructed a gene regulatory network to identify candidate transcription factors (TFs) involved in O-GlcNAcylation-regulated osteogenesis. Guided by our bioinformatics findings, we assessed morphological changes in mitochondria and the cytoskeleton following Ogt knockout using the CRISPR/Cas9 system in MC3T3-E1 osteoblast-like cells. We evaluated changes in mitochondrial and cytoskeletal function by applying an artificial intelligence (AI) model to time-lapse images of living cells. Finally, based on our bioinformatics analysis, Ezh2, Opa1, and Hspa9 were selected and preliminarily validated as key genes in O-GlcNAcylation-mediated functions of the cytoskeleton and mitochondria in osteoblasts.

Institutions

Okayama Daigaku

Categories

Bioinformatics, Western Blot, Real-Time Polymerase Chain Reaction, Immunofluorescence

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