Downregulation of inhibitor of apoptosis proteins in apoptotic human chondrocytes treated with tumor necrosis factor-alpha and actinomycin D

SummaryObjectiveApoptosis of chondrocytes plays a pivotal role in cartilage degeneration. Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine and has been assumed to cause the degradation of human cartilage. To investigate the mechanisms of TNF-α-mediated apoptosis of human chondrocytes from a point of view of the balance between the caspase-cascade and the expression of inhibitor of apoptosis proteins (IAPs), although both of them are induced with TNF-signals.MethodsThe expression of TNF-receptors (TNF-Rs) in normal human articular chondrocyte (NHAC-kn) was examined with immunocytochemistry. Subconfluent cultures of NHAC-kn were tested with TNF-α and/or actinomycin D (actD), and the induction of apoptosis was evaluated by the frequency of apoptotic cells visualized with nuclear staining using Hoechst 33342. The activation of caspases and the expression of IAPs were examined with Western blot analyses.ResultsNHAC-kn expressed TNF-R1 and -R2. When NHAC-kn was treated with TNF-α (10 ng/ml) and actD (0.2 μg/ml) for 24 h, the frequency of apoptotic cells increased to more than 25%. TNF-α alone, however, induced the apoptosis insufficiently (up to 8.3%), even when used at the concentration of 100 ng/ml for 48 h. In apoptotic human chondrocytes induced with TNF-α (10 ng/ml) and actD (0.2 μg/ml), the caspase-3, -8, and -9 were activated and the protein expression of XIAP and c-IAP1 decreased.ConclusionsIn apoptotic human chondrocytes induced with TNF-α and actD, the balance between caspase activation and IAPs' expression lay with the executioner caspase (caspase-3) and led to decreased expression of XIAP and c-IAP1.