Regulation of TNF-related apoptosis-inducing ligand-mediated death-signal pathway in human beta cells by Fas-associated death domain and nuclear factor kappaB.
Transfectants of human CM and NES2Y beta cell lines and primary islets transfected by FADD-DN (dominant-negative form of Fas-associated death domain), a mutant of FADD and/or a superrepressor of nuclear factor kappaB (NF-kappaB) (AdIkappaB(SA)2), were examined for their susceptibility to the TRAIL (TNF-related apoptosis-inducing ligand)-induced death signal pathway, compared with controls, wild-type cells, and vector transfectants in caspase fluorescence, Western blot, electrophoretic mobility shift, apoptosis, and cytotoxicity assays. FADD-DN inhibited caspase-8 activation induced by TRAIL in the transfectants of CM and NES2Y cells. TRAIL-induced apoptosis and cytotoxicity to the FADD-DN transfectants were decreased in comparison to those responses in controls (CM, p < 0.01 and p < 0.01; NES2Y, p < 0.05, and p < 0.02, respectively). When CM, NES2Y, and primary islet cells were transfected by AdIkappaB(SA)2, TRAIL-induced IkappaB degradation and nuclear translocation of NF-kappaB p50/p65 were blocked. TRAIL-induced apoptosis and cytotoxicity to AdIkappaB(SA)2 transfectants of these cells were also reduced (CM, p < 0.02 and p < 0.02; NES2Y, p < 0.01 and p < 0.01, respectively, and islet p < 0.01 for cytotoxicity). Finally, cytotoxicity induced by TRAIL in CM and NES2Y cells transfected with both FADD-DN and AdIkappaB(SA)2 was reduced, compared with that observed in these cells transfected with either FADD-DN alone or AdIkappaB(SA)2 alone, suggesting that FADD and NF-kappaB have synergistic proapoptotic regulatory effects on the susceptibility of beta cell lines and islet cells to TRAIL-induced destruction.
Ou
D
D
Department of Pediatrics, Faculty of Medicine, University of British Columbia, BC, Vancouver, Canada. daweiou@interchange.ubc.ca
Wang
X
X
Metzger
D L
DL
Robbins
M
M
Huang
J
J
Jobin
C
C
Chantler
J K
JK
James
R F L
RF
Pozzilli
P
P
Tingle
A J
AJ
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000502:physiology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000494:pharmacology*; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D024202:Electrophoretic Mobility Shift Assay; D053200:Fas-Associated Death Domain Protein; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D052002:NF-kappa B p50 Subunit / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D012460:Sulfasalazine / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand; D051996:Transcription Factor RelA / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D012460:Sulfasalazine; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases