Identification of proteins cleaved downstream of caspase activation in monocytes undergoing macrophage differentiation.
We have shown previously that caspases were specifically involved in the differentiation of peripheral blood monocytes into macrophages while not required for monocyte differentiation into dendritic cells. To identify caspase targets in monocytes undergoing macrophagic differentiation, we used the human monocytic leukemic cell line U937, whose macrophagic differentiation induced by exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) can be prevented by expression of the baculovirus caspase-inhibitory protein p35. A comparative two-dimensional gel proteomic analysis of empty vector- and p35-transfected cells after 12 h of exposure to 20 nm TPA, followed by mass spectrometry analysis, identified 38 differentially expressed proteins. Those overexpressed in p35-expressing cells (n = 16) were all full-length, whereas half of those overexpressed in control cells (n = 22) were N- or C-terminal cleavage fragments. The cleavage or degradation of seven of these proteins was confirmed in peripheral blood monocytes undergoing macrophage colony-stimulating factor-induced macrophagic differentiation. In U937 cells exposed to TPA, these proteolytic events can be inhibited by expression of a caspase-8 dominant negative mutant or the cowpox virus CrmA caspase inhibitor. These cleavages provide new insights to analyze the role of caspases in this specific differentiation program.
Cathelin
Séverine
S
INSERM UMR 517, IFR 100, Faculty of Medicine, 7 Boulevard Jeanne d'Arc, F-21079 Dijon Cedex, France.
Rébé
Cédric
C
Haddaoui
Lamya
L
Simioni
Nicolas
N
Verdier
Frédérique
F
Fontenay
Michaëla
M
Launay
Sophie
S
Mayeux
Patrick
P
Solary
Eric
E
D002273:Carcinogens / Q000494:pharmacology; D053181:Caspase 8; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000276:immunology; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation / Q000276:immunology; D006801:Humans; D008264:Macrophages / Q000166:cytology* / Q000201:enzymology*; D009000:Monocytes / Q000166:cytology* / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D034741:RNA, Small Interfering; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D020298:U937 Cells; D014764:Viral Proteins / Q000235:genetics
D002273:Carcinogens; D061945:Caspase Inhibitors; D010446:Peptide Fragments; D020543:Proteome; D034741:RNA, Small Interfering; D014764:Viral Proteins; C492234:p35 protein, Baculovirus; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases; D013755:Tetradecanoylphorbol Acetate