Enolase 2-mediated lactylation-dependent disruption of the GNL3-MDM2-p53 axis in age-related osteoarthritis.

Age-related osteoarthritis (OA) involves metabolic dysregulation and chondrocyte senescence. This study examined the nonmetabolic role of enolase 2 (ENO2) in OA pathogenesis and its therapeutic potential. Human aged and OA cartilage (n = 3 per group) underwent 18F-FDG positron emission tomography (PET)-computed tomography (CT) imaging, proteomic profiling, and immunohistochemistry. In vitro chondrocyte senescence models were generated by inducing doxorubicin-induced stress and serial passaging. Protein-protein interactions (ENO2-GNL3-MDM2) were validated by co-immunoprecipitation (IP), GST pull-down, and site-directed mutagenesis (E4A-ENO2 and K5R-GNL3 mutants). Lactylation was assessed using lactylomics and immunoprecipitation. The therapeutic effect of the ENO2-specific inhibitor POMHEX was evaluated in C57BL/6 J mice (n = 6 per group) via intra-articular injection for 16 weeks. Outcomes included histology, micro-CT, pain behavior, and gait analysis. Proteomics revealed ENO2 upregulation in aged human cartilage. In vitro, ENO2 overexpression promoted extracellular matrix catabolism, senescence, and glycolysis, whereas ENO2 knockdown attenuated these processes. Mediated by its Glu-4 residue, nuclear ENO2 bound GNL3 lactylated at Lys-5. This interaction displaced MDM2 from GNL3, resulting in MDM2 destabilization, impaired ubiquitination, p53 accumulation, and persistent senescence. Moreover, p53 transcriptionally activated ENO2, establishing a pathological positive feedback loop. Pharmacological inhibition of ENO2 with POMHEX disrupted ENO2-GNL3 binding, restored p53 degradation, reduced senescence markers in vitro, and mitigated cartilage degradation, subchondral bone sclerosis, and pain in aged mice. ENO2 promotes OA progression through a lactate-dependent, lactylation-mediated disruption of the GNL3-MDM2-p53 axis, leading to a senescent feedback loop. Targeting ENO2 may represent a novel disease-modifying therapeutic approach for age-related OA.