cFLIPL inhibits tumor necrosis factor-related apoptosis-inducing ligand-mediated NF-kappaB activation at the death-inducing signaling complex in human keratinocytes.
Human keratinocytes undergo apoptosis following treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via surface-expressed TRAIL receptors 1 and 2. In addition, TRAIL triggers nonapoptotic signaling pathways including activation of the transcription factor NF-kappaB, in particular when TRAIL-induced apoptosis is blocked. The intracellular protein cFLIP(L) interferes with TRAIL-induced apoptosis at the death-inducing signaling complex (DISC) in many cell types. To study the role of cFLIP(L) in TRAIL signaling, we established stable HaCaT keratinocyte cell lines expressing varying levels of cFLIP(L). Functional analysis revealed that relative cFLIP(L) levels correlated with apoptosis resistance to TRAIL. Surprisingly, cFLIP(L) specifically blocked TRAIL-induced NF-kappaB activation and TRAIL-dependent induction of the proinflammatory target gene interleukin-8. Biochemical characterization of the signaling pathways involved showed that apoptosis signaling was inhibited at the DISC in cFLIP(L)-overexpressing keratinocytes, although cFLIP(L) did not significantly impair enzymatic activity of the receptor complex. In contrast, recruitment and modification of receptor-interacting protein was blocked in cFLIP(L)-overexpressing cells. Taken together, our data demonstrate that cFLIP(L) is not only a central antiapoptotic modulator of TRAIL-mediated apoptosis but also an inhibitor of TRAIL-induced NF-kappaB activation and subsequent proinflammatory target gene expression. Hence, cFLIP(L) modulation in keratinocytes may not only influence apoptosis sensitivity but may also lead to altered death receptor-dependent skin inflammation.
University of Würzburg Medical School, Department of Dermatology, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany.
Tina
T
Wachter
Martin
M
Sprick
Dominikus
D
Hausmann
Andreas
A
Kerstan
Kirsty
K
McPherson
Giorgio
G
Stassi
Eva-B
EB
Bröcker
Henning
H
Walczak
Martin
M
Leverkus
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007249:Inflammation; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000502:physiology*; D015603:Keratinocytes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D011419:Propidium / Q000494:pharmacology; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D012260:Ribonucleases / Q000378:metabolism; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D012867:Skin / Q000473:pathology; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D011419:Propidium; D012313:RNA; D012260:Ribonucleases; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases