OGT regulates histone demethylation and alleviates osteoarthritis progression by O-GlcNAcylation of KDM6B

• Using conditional knockout mice, we firstly clarified the critical role of OGT-mediated O-GlcNAcylation in osteoarthritis (OA) progression and cartilage development. • Overexpression of OGT attenuates OA progression, highlighting its therapeutic potential. • We demonstrated, for the first time, that OGT regulates histone modifications by O-GlcNAcylating KDM6B. • We revealed that O-GlcNAcylation stabilizes KDM6B expression by suppressing its ubiquitin-mediated degradation. • We identified and experimentally validated serine 215 as the major O-GlcNAcylation site of KDM6B Osteoarthritis (OA) is a common orthopedic disease characterized by cartilage degeneration, osteophyte formation, and synovial hyperplasia. Although dysregulated O-GlcNAcylation has been implicated in various musculoskeletal and inflammatory disorders, its specific role and molecular targets in OA pathogenesis remain largely unexplored. In this study, we aimed to conduct a multidimensional investigation to demonstrate and delineate the chondrocyte-specific functions and mechanisms of O-GlcNAc transferase (OGT) during OA progression. To explore the mechanisms of OA progression, we analyzed OGT expression using GEO data and clinical cartilage samples. Cartilage-specific OGT knockout mice ( Acan-CreER T2 ; Ogt flox/flox ) were generated and subjected to DMM surgery. To determine the effects of OGT in OA, histological and immunohistochemical studies were conducted. RNA-seq and CUT&Tag were were employed to elucidate the underlying molecular mechanisms. OGT expression is reduced in OA cartilage, and its overexpression delays cartilage degeneration, while cartilage-specific OGT deletion accelerates OA progression. Mechanistically, we found that OGT interacts with and O-GlcNAcylates KDM6B at serine 215, thereby inhibiting its ubiquitination, enhancing protein stability, and promoting extracellular matrix gene expression by demethylating H3K27me3 at Col2a1 and Col6a6 loci. OGT is a key regulator of OA that exerts its function by modulating the epigenetic state of chondrocytes. Loss of OGT in chondrocytes not only disrupts normal cartilage development but also accelerates OA progression under pathological conditions, thereby providing new experimental evidence for understanding OA pathogenesis and informing the development of potential therapeutic strategies.