Inhibition of CD95 apoptotic signaling by interferon‐γ in human osteoarthritic chondrocytes is associated with increased expression of FLICE inhibitory protein

Abstract

Cartilage homeostasis dysregulation during osteoarthritis (OA) has been linked to an increased rate of apoptosis of chondrocytes, the only cell type resident in the cartilage. In addition, the CD95-CD95 ligand (the Fas system) has emerged as one of the major pathways of cell death in the cartilage. We undertook the present study to investigate the role of interferon-gamma (IFNgamma) in the regulation of the Fas system by analyzing the modulation of intracellular signaling molecules (FLICE inhibitory protein [FLIP] and caspases 3 and 8) in primary cultures of human OA chondrocytes. CD95-induced apoptotic death of human OA chondrocytes was analyzed in the presence or absence of IFNgamma using cell death immunoassay for apoptosis, real-time polymerase chain reaction for FLIP and caspase 8 expression, Western blotting for FLIP, and proteolytic activity for caspases 3 and 8. CD95-induced apoptotic death of human OA chondrocytes was strongly counteracted by IFNgamma treatment, although the surface expression of CD95 was slightly up-regulated by this cytokine. The messenger RNA (mRNA) expression of FLIP and caspase 8, mediators involved in CD95 signaling, revealed that FLIP expression in human OA chondrocytes was significantly up-regulated (2-fold increase) by IFNgamma treatment. Moreover, the FLIP:caspase 8 mRNA ratio increased significantly. FLIP up-regulation by IFNgamma was confirmed at the protein level. Caspase 8 and caspase 3 proteolytic activities, both induced in these cells by stimulation with anti-CD95, were also significantly down-modulated by IFNgamma. These findings suggest that IFNgamma impairs CD95-mediated signaling and apoptotic death in human chondrocytes. Its mechanism of action involves down-regulation of caspase 8 and caspase 3 activities and increased expression of the antiapoptotic protein FLIP, suggesting an interesting mechanism for the inhibition of chondrocyte apoptosis.