OGT mediated HDAC5 O-GlcNAcylation promotes osteogenesis by regulating the homeostasis of epigenetic modifications and proteolysis.

BACKGROUND: O-GlcNAc transferase (OGT) is responsible for attaching O-linked N-acetylglucosamine (O-GlcNAc) to proteins, regulating diverse cellular processes ranging from transcription and translation to signaling and metabolism. This study focuses on the role and mechanisms of OGT in osteogenesis. MATERIALS AND METHODS: We found that OGT is downregulated in osteoporosis by bioinformatics analysis, determined its role in osteogenic differentiation by using OGT inhibitors (or OGA inhibitors) as well as conditional knockout OGT mice in vitro and in vivo, and explored and specific mechanisms by quantitative proteomic analysis and RNA-seq, qRT-PCR, western blotting, immunofluorescence, H&E, ALP, ARS, Masson staining, IHC, micro CT, etc. RESULTS: we revealed that OGT positively influenced osteogenesis and osteoblast differentiation in vitro as well as ovariectomy (OVX) mice in vivo. Consistently, mice with conditionally depleted OGT exhibited a reduction in bone mass, while O-GlcNAcylation enhancer could partially recover bone mass in ovariectomy (OVX) mice. Mechanistically, quantitative proteomic analysis and high-throughput RNAseq further reveals that HDAC5 is one of the endogenous O-GlcNAcylation substrates, and O-GlcNAcylation of HDAC5 on Thr934 promotes its translocation to lysosomes and subsequent degradation, thus, elevating the O-GlcNAcylation level of HDAC5 leads to its cytoplasmic cleavage, consequently diminished its nuclear entry and enhanced DNA transcription. The OGT-mediated O-GlcNAcylation of HDAC5 modulates the balance between its cytoplasmic proteolysis and nuclear entry, thereby impacting the Notch signaling pathway and DNA epigenetic modifications then playing a role in osteogenesis. CONCLUSION: OGT is a regulator that promotes osteoblast differentiation and bone regeneration. Additionally, it highlights the critical function of HDAC5 O-GlcNAcylation in controlling epigenetics. This study offers fresh perspectives on osteogenesis and O-GlcNAcylation, proposing that the OGT-mediated O-GlcNAcylation of HDAC5 could be a promising target for osteoporosis treatment. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: On one side, OGT might potentially be used as a new biomarker for clinical diagnosis of osteoporosis (OP) in the future. On the other side, small molecule inhibitors of HDAC5, a glycosylation substrate of OGT, or OGT agonists such as silymarin, could all potentially serve as therapeutic targets for the prevention or treatment of OP in the future.