PPARγ-Induced SUMOylation Drives Fbxo6-Mediated Cx43 Ubiquitination to Alleviate Osteoarthritis

Previous studies from our group confirmed the chondroprotective effects of the PPARγ agonist pioglitazone. In the present study, we aimed to elucidate the mechanism by which PPARγ activation protects against osteoarthritis (OA), with a particular focus on the regulation of Connexin 43 (Cx43). OA models were established in mice and rats through exercise and intra-articular AGE-BSA injections. Cx43-knockout mice were used for genetic validation. Primary human and rat chondrocytes were treated with AGEs in vitro. Mechanistic investigations were performed using RNA-sequencing, co-immunoprecipitation, ubiquitination assays, cycloheximide chase assays, and functional tests. Cx43 expression was markedly upregulated in OA chondrocytes and inversely correlated with PPARγ levels. The PPARγ agonist pioglitazone alleviated OA and was accompanied by elevated PPARγ and reduced Cx43 protein levels without altering Cx43 mRNA levels. Genetic ablation of Cx43 attenuated OA severity, and under pioglitazone treatment, Cx43-KO mice did not exhibit additional protection compared with WT mice, whereas Cx43 overexpression reversed the drug’s protection. Mechanistically, PPARγ activation promoted Cx43 degradation via a post-translational pathway. Integrated RNA-sequencing and IP-MS analyses identified SUMO2 and the E3 ligase adaptor Fbxo6 as key mediators of this process. Specifically, PPARγ upregulated SUMO2, which mediated Cx43 SUMOylation, facilitating its recognition by Fbxo6. This led to Cx43 ubiquitination and proteasomal degradation. Mutation of either Cx43 SUMOylation site (K109 or K133) or the disruption of the Fbxo6 SUMO-interacting motif (SIM) abolished this regulation. Functionally, activation of the SUMO2/Fbxo6 axis protected chondrocytes from AGEs-induced dysfunction. PPARγ activation triggers SUMO2-mediated Cx43 SUMOylation, thereby promoting Fbxo6-dependent ubiquitination and degradation of Cx43. This novel PPARγ-SUMO2-Fbxo6 axis is essential for chondroprotection, and provides new mechanistic insight and potential therapeutic targets for OA treatment. Clinical trial number: not applicable.