A novel therapeutic target for osteoarthritis: control of cellular plasticity and senescence using connexin43

Introduction: Osteoarthritis (OA) is a degenerative musculoskeletal disease, which affects approximately the 13% of western population. Nowadays, there is no effective treatment for OA to avoid disease progression or to promote cartilage regeneration. Connexin43 (Cx43) is a transmembrane protein increased in cartilage and synovium from OA patients. Cx43 forms membrane channels that allow the exchange of molecules and ions between two adjacent cells through gap junctions (GJs), or between a cell and its environment through hemichannels. In this study we investigated the involvement of Cx43 and GJ intercellular communication in the degradation of articular cartilage in chondrocytes from patients with OA. Material and methods: Primary chondrocytes were obtained from cartilage from OA and healthy donors. Protein levels were evaluated by western-blot, immunofluorescence and flow cytometry. RNA expression was evaluated by RT-qPCR. A scrape loading/dye transfer assay was used to evaluate cell communication. Cell senescence was analysed by flow cytometry or by light microscopy using beta-galactosidase assay. Results: Cx43 and GJs overactivities were correlated with the progression of OA, by promoting chronic cell dedifferentiation and senescence in vitro assays. We found that Cx43 overexpression activates factors involved in epithelial-to-mesenchymal transition, such as Twist-1. Increased levels of dedifferentiated cells, with high rates of cell proliferation, led to cell senescence via p53/p16(INK4a), activating the senescence-associated secretory phenotype (SASP) and promoting the synthesis and liberation of inflammatory factors, including the interleukin-6 (IL-6). Cx43 downregulation by using small molecules, such as oleuropein, or by genetic edition with CRISPR technology, led to the chondrocyte redifferentiation and an improved phenotype, with increased synthesis of extracellular matrix proteins such as Col2A1 and down-regulating the synthesis of MMPs, inflammation and senescence. Conclusions: Downregulation of Cx43 in OA chondrocytes restores regeneration by activating chondrocyte re-differentiation and decreasing cellular senescence. These results corroborate the use of Cx43 as an effective therapeutic target in order to restore cartilage regeneration and avoid OA progression.