Regulation of CD95 (Fas/APO-1)-induced apoptosis in human chondrocytes.
OBJECTIVE
To examine the role of nuclear factor kappaB (NF-kappaB) 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-kappaB was determined by electrophoretic mobility shift assay (EMSA) and proteasome inhibition-dependent blocking of the degradation of inhibitor of NF-kappaB.


RESULTS
Low levels of NF-kappaB activity were detected by EMSA in unstimulated chondrocytes. NF-kappaB 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-kappaB. Proteasome inhibitors also caused the induction of DNA fragmentation by tumor necrosis factor alpha. 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.


CONCLUSIONS
These results suggest that CD95-dependent cell death is enhanced by NF-kappaB inhibition at and/or downstream of caspase 8 activation and that caspase 9 activation is not involved in CD95-mediated apoptosis in chondrocytes.
Kühn
K
K
The Scripps Research Institute, La Jolla, California 92037, USA.
Lotz
M
M
D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D002358:Cartilage, Articular / Q000166:cytology; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000201:enzymology; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D019281:Dimerization; D004586:Electrophoresis; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D016328:NF-kappa B / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D019014:fas Receptor / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; D009097:Multienzyme Complexes; D016328:NF-kappa B; D009842:Oligopeptides; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D019014:fas Receptor; C096429:L 709049; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex