Regulation of CD95 (Fas/APO-1)-induced apoptosis in human chondrocytes

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Abstract
Objective To examine the role of nuclear factor κB (NF‐κB) and caspases 3, 8, and 9 in CD95‐mediated apoptosis of normal chondrocytes. Methods First‐passage chondrocytes from normal human knee cartilage were stimulated with CD95 antibody, and cell death was determined by annexin V binding and by an enzyme‐linked immunosorbent assay. Activation of caspases 3, 8, and 9 was measured by Western blotting, and their role in death signaling was evaluated using caspase‐specific small peptide inhibitors. The influence of NF‐κB was determined by electrophoretic mobility shift assay (EMSA) and proteasome inhibition–dependent blocking of the degradation of inhibitor of NF‐κB. Results Low levels of NF‐κB activity were detected by EMSA in unstimulated chondrocytes. NF‐κB activity was increased in response to agonistic CD95 antibody. CD95 antibody–induced apoptosis was potentiated by the proteasome inhibitors MG‐132 and PS1, and this was associated with a reduced nuclear translocation of NF‐κB. Proteasome inhibitors also caused the induction of DNA fragmentation by tumor necrosis factor α. Procaspase 3 processing was enhanced by the proteasome inhibitor MG‐132. Procaspase 8 was undetectable by immunoblotting in whole cell lysates of chondrocytes, but caspase 8 messenger RNA was detected by reverse transcription–polymerase chain reaction. Furthermore, apoptosis induced by CD95 stimulation and proteasome inhibitors was blocked by the caspase 8–specific inhibitor Ac‐IETD‐CHO. Processing of procaspase 9 was not observed, and inhibition of CD95‐dependent cell death by the caspase 9 inhibitor Ac‐LEHD‐CHO was not significant. Conclusion These results suggest that CD95‐dependent cell death is enhanced by NF‐κB inhibition at and/or downstream of caspase 8 activation and that caspase 9 activation is not involved in CD95‐mediated apoptosis in chondrocytes.