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.

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APA

Kühn, K., & Lotz, M. (2001). Regulation of CD95 (Fas/APO-1) - Induced apoptosis in human chondrocytes. Arthritis and Rheumatism, 44(7), 1644–1653. https://doi.org/10.1002/1529-0131(200107)44:7<1644::AID-ART287>3.0.CO;2-S

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