Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro.
Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatment with various stimuli, including camptothecin, tumor necrosis factor-alpha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase activities in the lysates were characterized and quantitated with peptide cleavage profiles. To confirm that the results were not related to the immortalized nature of the cell line, primary human chondrocytes also were shown to undergo apoptosis under similar conditions, which resulted in increased cytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, Z-VAD-FMK, and the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activity and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, had no effect. Human chondrocytes were stably and transiently transfected with a type-II collagen gene (COL2A1) regulatory sequence driving a luciferase reporter as a specific marker of chondrocyte gene expression. Treatment of the cells with camptothecin or tumor necrosis factor-alpha plus cycloheximide significantly inhibited COL2A1 transcriptional activity. Significantly, cotreatment with Z-VAD-FMK or Ac-DMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibition was sufficient to maintain cell functionality as assessed by the retention of type-II collagen promoter activity.
Department of Bone and Cartilage Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA. Mark_E_Nuttall@sbphrd.com
M E
ME
Nuttall
D P
DP
Nadeau
P W
PW
Fisher
F
F
Wang
P M
PM
Keller
W E
WE
DeWolf
M B
MB
Goldring
A M
AM
Badger
D
D
Lee
M A
MA
Levy
M
M
Gowen
M W
MW
Lark
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000502:physiology*; D002460:Cell Line; D019902:Chondrocytes / Q000502:physiology*; D003094:Collagen / Q000235:genetics; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D015854:Up-Regulation
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D009842:Oligopeptides; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C096429:L 709049; D003094:Collagen; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases