Facilitation of fas mediated apoptosis of human chondrocytes by the proteasome inhibitor and actinomycin D.
OBJECTIVE
We investigated the susceptibility of chondrocytes to apoptosis induced by anti-Fas and various potentiators, and the relevant signaling pathway.


METHODS
Chondrocytes were cultured from cartilages obtained at the time of joint replacement surgery for knee osteoarthritis (OA) or femur neck fracture. Fas receptor ligation was performed with agonistic anti-Fas antibody (clone CH-11) at concentrations ranging from 0.5 to 1.0 micro g/ml. Mitogen activated protein kinase inhibitors SB203580 and PD98059, cycloheximide, bisindolylmaleimide, actinomycin D, or MG132 were added with anti-Fas to facilitate cell death. Chondrocyte surface expression of Fas was analyzed by FACS, and the expression of apoptosis related proteins analyzed by Western blot.


RESULTS
Cell death increased upon coculture with 0.5 micro g/ml of anti-Fas and 0.2 micro g/ml of actinomycin D or 20 micro M MG132. Apoptosis potentiated by actinomycin D or MG132 was effectively inhibited by caspase inhibitors, implicating the involvement of the caspase cascade in chondrocyte apoptosis. Compared with untreated cells or actinomycin D treated cells, cells treated with MG132 showed distinct shifts in the distribution of surface Fas fluorescence. Although concentrations of Bcl-2, Bax, FLICE inhibitory protein (FLIP), and Fas ligand were unaffected by MG132 or actinomycin D, both treatments led to a significant increase of p53. The expression of the p53 response proteins, MDM2 and p21, was elevated in MG132 treated chondrocytes.


CONCLUSIONS
Our results suggest that chondrocytes can be rendered sensitive to anti-Fas mediated apoptosis by the proteasome inhibitor MG132 and the transcription inhibitor actinomycin D. MG132 and actinomycin D show different characteristics in terms of apoptosis signaling.
Kim
Hyun A
HA
Department of Internal Medicine, Hallym University College of Medicine, Seoul, Korea.
Kim
Yong H
YH
Song
Yeong W
YW
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002358:Cartilage, Articular / Q000166:cytology; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008301:Maleimides / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D046988:Proteasome Endopeptidase Complex; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology; D019014:fas Receptor / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007211:Indoles; D007976:Leupeptins; D008301:Maleimides; D009097:Multienzyme Complexes; D011500:Protein Synthesis Inhibitors; D011725:Pyridines; D019014:fas Receptor; D003609:Dactinomycin; D003513:Cycloheximide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C088060:bisindolylmaleimide; C093642:SB 203580; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde