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

BACKGROUND: 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. METHODS: F-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. RESULTS: 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. CONCLUSIONS: 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.