Inhibition of death receptor-mediated gene induction by a cycloheximide-sensitive factor occurs at the level of or upstream of Fas-associated death domain protein (FADD).
In HeLa cells, induction of apoptosis and nuclear factor kappaB (NF-kappaB) activation initiated by TRAIL/Apo2L or the agonistic Apo1/Fas-specific monoclonal antibody anti-APO-1 require the presence of cycloheximide (CHX). Inhibition of caspases prevented TRAIL/anti-APO-1-induced apoptosis, but not NF-kappaB activation, indicating that both pathways bifurcate upstream of the receptor-proximal caspase-8. Under these conditions, TRAIL and anti-APO-1 up-regulated the expression of the known NF-kappaB targets interleukin-6, cellular inhibitor of apoptosis 2 (cIAP2), and TRAF1 (TRAF, tumor necrosis factor receptor-associate factor). In the presence of CHX, the stable overexpression of a deletion mutant of the Fas-associated death domain molecule FADD comprising solely the death domain of the molecule but lacking its death effector domain (FADD-(80-208)) led to the same response pattern as TRAIL or anti-APO-1 treatment. Moreover, the ability of death receptors to induce NF-kappaB activation was drastically reduced in a FADD-deficient Jurkat cell line. TRAIL-, anti-APO-1-, and FADD-(80-208)-initiated gene induction was blocked by a dominant-negative mutant of TRAF2 or the p38 kinase inhibitor SB203580, similar to tumor necrosis factor receptor-1-induced NF-kappaB activation. CHX treatment rapidly down-regulated endogenous cFLIP protein levels, and overexpression of cellular FLICE inhibitory protein (cFLIP) inhibited death receptor-induced NF-kappaB activation. Thus, a novel functional role of cFLIP as a negative regulator of gene induction by death receptors became apparent.
Wajant
H
H
Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. harald.wajant@po.unistuttgart.de
Haas
E
E
Schwenzer
R
R
Muhlenbeck
F
F
Kreuz
S
S
Schubert
G
G
Grell
M
M
Smith
C
C
Scheurich
P
P
D048868:Adaptor Proteins, Signal Transducing; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D016328:NF-kappa B / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D047990:TNF Receptor-Associated Factor 1; D015533:Transcriptional Activation; D019014:fas Receptor / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D000590:Amino Acid Chloromethyl Ketones; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D016328:NF-kappa B; D011506:Proteins; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D047990:TNF Receptor-Associated Factor 1; C105190:TNFRSF10A protein, human; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D003513:Cycloheximide; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases