Oxygen‑sensing histone demethylase KDM6A modulates chondrocyte‑to‑osteoblast transdifferentiation by activating the Wnt/β‑catenin pathway

Fracture healing is a complex biological process involving chondrocyte (CH) differentiation and endochondral ossification. A subset of CHs may transdifferentiate into osteoblasts, enhancing bone regeneration. The oxygen‑sensing histone demethylase lysine demethylase 6A (KDM6A) and local oxygen microenvironment are hypothesized to serve pivotal roles in modulating this transition; however, the precise regulatory mechanisms remain unclear. To assess the role of KDM6A, an oxygen‑sensitive histone demethylase, in endochondral ossification, an inducible cartilage‑specific Kdm6a‑knockout mouse model was generated. Single‑cell RNA sequencing (scRNA‑seq) analysis was performed in a mouse tibial fracture model to characterize CH subpopulations and their fate transitions during bone repair. scRNA‑seq identified distinct CH subpopulations, including chondrocyte‑derived osteoprogenitors (CDOPs), which acted as osteoblast precursors during endochondral ossification. Pseudotime trajectory analysis revealed a bifurcated differentiation pathway, with CDOPs exhibiting rapid osteoblast conversion. Functional enrichment analyses implicated the Wnt/β‑catenin pathway in this transition. In vitro, CHs isolated from bone callus of KDM6A‑knockout and control mice were induced to undergo transdifferentiation into osteoblasts under varying oxygen tensions. The expression levels of chondrogenic markers, osteogenic differentiation‑related indicators and canonical Wnt signaling molecules, as well as the levels of histone dimethylation of H3K27 (H3K27me2) and trimethylation of H3K27 (H3K27me3) at their promoter regions, were assessed. In vivo, the molecular and functional consequences of KDM6A deficiency were characterized through histopathological evaluation and bone microarchitecture analysis. In vitro, CHs cultured under normoxic conditions exhibited greater osteogenic differentiation than those cultured under hypoxic conditions. Conversely, loss of KDM6A impaired the pro‑osteogenic effect of normoxia on CH‑to‑osteoblast transdifferentiation, indicating the importance of KDM6A in oxygen‑mediated CH‑to‑osteoblast transdifferentiation. Mechanistically, chromatin immunoprecipitation analysis revealed that under normoxic conditions, KDM6A‑knockout CHs exhibited higher levels of the repressive histone marks H3K27me2 and H3K27me3 at the Wnt3a promoter region, as well as increased H3K27me3 levels at the Runt‑related transcription factor 2 (RUNX2) promoter region, compared with control cells. These findings indicated that KDM6A catalyzed the removal of H3K27 methylation at the promoters of Wnt3a and RUNX2, thereby relieving their transcriptional repression. In vivo, KDM6A‑knockout mice exhibited osteogenic defects and delayed fracture healing compared with control mice. KDM6A serves as a pivotal oxygen sensor that drives CH‑to‑osteoblast transdifferentiation and enhances fracture healing through Wnt/β‑catenin pathway activation. The KDM6A‑mediated oxygen response mechanism is a potential target for enhancing bone regeneration during fracture repair.