USP25 stabilises GLI1 through K48‐linked deubiquitination within a reciprocal regulatory circuit in osteoarthritis models

Abstract Background Osteoarthritis (OA) is characterised by progressive articular cartilage degeneration, yet effective disease‐modifying targets remain limited. The post‐translational mechanisms sustaining Hedgehog/ GLI1 signalling during OA progression remain incompletely understood. Methods Bioinformatic analyses of GEO datasets were performed to evaluate GLI1 expression across OA‐related tissue compartments. Independent cartilage datasets, paired human cartilage specimens, and primary human chondrocytes exposed to graded IL‐1β stimulation provided tissue‐ and cell‐relevant evidence. An unbiased deubiquitinase library screen combined with GLI1 ‐responsive luciferase assays was used to identify GLI1 ‐regulating deubiquitinases. The molecular function of USP25, its reciprocal regulation with GLI1 , and the preclinical chondroprotective activity and target dependence of AZ1 were investigated in vitro and in vivo. Results GLI1 dysregulation in OA cartilage and primary human chondrocytes supported subsequent screening, which prioritised USP25 for mechanistic study. In paired cartilage from 24 OA patients, USP25 protein abundance was higher in grossly degenerated than in relatively preserved regions and showed a modest positive correlation with GLI1 . USP25 directly interacted with GLI1 , removed K48‐linked polyubiquitin chains, and limited its proteasomal degradation. Reciprocal gain‐ and loss‐of‐function and rescue experiments indicated that GLI1 substantially, but not exclusively, mediated USP25‐associated inflammatory and cartilage‐catabolic phenotypes. GLI1 , in turn, bound the USP25 promoter and enhanced USP25 transcription, establishing a reciprocal regulatory circuit. AZ1 showed preclinical chondroprotective activity in primary human chondrocytes and DMM mice. Residual AZ1 activity after USP25 silencing indicated partial USP25 dependence. Conclusions These findings define a GLI1 ‐centred mechanistic branch of USP25 biology in OA‐relevant models and provide tissue‐level support for coordinated USP25‐ GLI1 expression in human OA cartilage. The reciprocal USP25‐ GLI1 circuit contributes to cartilage‐catabolic phenotypes, while AZ1 provides preclinical proof‐of‐concept as a pharmacological probe. Further studies are warranted to define target selectivity, long‐term safety, and clinical relevance.