The combination of mitogenic stimulation and DNA damage induces chondrocyte senescence

Abstract

Cellular senescence is a phenotypic state characterized by stable cell-cycle arrest, enhanced lysosomal activity, and the secretion of inflammatory molecules and matrix degrading enzymes. Senescence has been implicated in osteoarthritis (OA) pathophysiology; however, the mechanisms that drive senescence induction in cartilage and other joint tissues are unknown. While numerous physiological signals are capable of initiating senescence, one emerging theme is that damaged cells convert to senescence in response to sustained mitogenic stimulation. The goal of this study was to develop an in vitro articular cartilage explant model to investigate the mechanisms of senescence induction. This study utilized healthy cartilage derived from cadaveric equine stifles and human ankles. Explants were irradiated to initiate DNA damage, and mitogenic stimulation was provided through serum-containing medium and treatment with transforming growth factor β1 and basic fibroblastic growth factor. Readouts of senescence were a quantitative flow cytometry assay to detect senescence-associated β galactosidase activity (SA-β-gal), immunofluorescence for p16 and γH2AX, and qPCR for the expression of inflammatory genes. Human cartilage explants required both irradiation and mitogenic stimulation to induce senescence as compared to baseline control conditions (7.16% vs 2.34% SA-β-gal high, p=0.0007). These conditions also resulted in chondrocyte clusters within explants, a persistent DNA damage response, increased p16, and gene expression changes. Treatment of cartilage explants with mitogenic stimuli in the context of cellular damage reliably induces high levels of SA-β-gal activity and other senescence markers, which provides a physiologically relevant model system to investigate the mechanisms of senescence induction.