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

• Published in: Arthritis and rheumatism 2004-02, Vol.50 (2), p.498-506
• Main Authors: Grassi, Francesco, Piacentini, Anna, Cristino, Sandra, Toneguzzi, Stefania, Facchini, Andrea, Lisignoli, Gina
• Format: Article
• Language: English
• Subjects: Apoptosis - drug effects; Apoptosis - physiology; Biological and medical sciences; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cartilage, Articular - drug effects; Cartilage, Articular - metabolism; Cartilage, Articular - pathology; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 3; Caspase 8; Caspase Inhibitors; Caspases - genetics; Caspases - metabolism; Cells, Cultured; Chondrocytes - drug effects; Chondrocytes - metabolism; Chondrocytes - pathology; Diseases of the osteoarticular system; Dose-Response Relationship, Drug; Fas Ligand Protein; fas Receptor - metabolism; Flow Cytometry; Humans; Interferon-gamma - metabolism; Interferon-gamma - pharmacology; Intracellular Signaling Peptides and Proteins; Medical sciences; Membrane Glycoproteins - metabolism; Miscellaneous. Osteoarticular involvement in other diseases; Osteoarthritis; Osteoarthritis, Knee - metabolism; Osteoarthritis, Knee - pathology; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Signal Transduction
• ISSN: 0004-3591; 1529-0131
• DOI: 10.1002/art.20008

Objective 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‐γ (IFNγ) 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. Methods CD95‐induced apoptotic death of human OA chondrocytes was analyzed in the presence or absence of IFNγ 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. Results CD95‐induced apoptotic death of human OA chondrocytes was strongly counteracted by IFNγ 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 IFNγ treatment. Moreover, the FLIP:caspase 8 mRNA ratio increased significantly. FLIP up‐regulation by IFNγ 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 IFNγ. Conclusion These findings suggest that IFNγ 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.