Abstract
Toll-like receptor 3 (TLR3) is a pattern-recognition receptor known to initiate an innate immune response when stimulated by double-stranded RNA (dsRNA). Components of TLR3 signaling, including TIR domain-containing adapter inducing IFN-α (TRIF), have been demonstrated to contribute to dsRNA-induced cell death through caspase-8 and receptor interacting protein (RIP)1 in various human cancer cells. We provide here a detailed analysis of the caspase-8 activating machinery triggered in response to Poly(I:C) dsRNA. Engagement of TLR3 by dsRNA in both type I and type II lung cancer cells induces the formation of an atypical caspase-8-containing complex that is devoid of classical death receptors of the TNFR superfamily, but instead is physically associated to TLR3. The recruitment of caspase-8 to TLR3 requires RIP1, and is negatively modulated by cellular inhibitor of apoptosis protein (cIAP)2–TNF receptor-associated factor (TRAF)2–TNFR-associated death domain (TRADD) ubiquitin ligase complex, which regulates RIP1 ubiquitination. Intriguingly, unlike Fas- or TRAILR-dependent death signaling, caspase-8 recruitment and activation within the TLR3 death-signaling complex appears not to be stringently dependent on Fas-associated with death domain (FADD). Our findings uncover a novel aspect of the molecular mechanisms involved during apoptosis induced by the innate immune receptor TLR3 in cancer cells.
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Abbreviations
- DD:
-
death domain
- DISC:
-
death-inducing signaling complex
- DR:
-
death receptor
- dsRNA:
-
double-stranded RNA
- FADD:
-
Fas-associated with death domain
- FLIPL:
-
FLICE-like inhibitory protein
- ISRE:
-
interferon-stimulated response element
- MDA5:
-
melanoma differentiation-associated protein 5
- NSCLC:
-
non small cell lung cancer
- PI:
-
propidium iodide
- PKR:
-
protein kinase RNA-activated
- RHIM:
-
RIP homotypic interaction motif
- RIG-I:
-
retinoic acid-inducible gene I protein
- RIP:
-
receptor interacting protein
- Ter:
-
terminal
- TIR:
-
Toll-IL-1-receptor
- TLR:
-
Toll-like receptor
- TRADD:
-
TNFR-associated death domain
- TRAF:
-
TNF receptor-associated factor
- TRIF:
-
TIR domain-containing adapter inducing IFN-α
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Acknowledgements
We thank our colleagues who provided reagents, especially Patrick Mehlen for the Bcl-2-expressing plasmid, Wayne Fairbrother for the gift of Smac mimetic BV6, Isabelle Grosjean for her assistance with cultures, Stéphanie Brunet-Manquat and Isabelle Durand for their support with flow cytometry, Jean-Paul Pais De Barros for assistance with size-exclusion chromatography, and our colleagues in the laboratory for their restless support and encouragement. The Cancéropole Lyon Auvergne Rhone-Alpes (CLARA) funded the work and SL was supported by a grant from the Hospices Civils de Lyon. OM was supported by grants from the ANR (Agence Nationale de la Recherche 07-PCV-0031) and the European Community (ApopTrain Marie Curie RTN). GJ and NL were supported by fellowships from the Ligue Nationale contre le Cancer and a joint INSERM and Conseil Régional de Bourgogne fellowship, respectively. We gratefully acknowledge the help and advice of Nicolas Foray.
Author Contributions
YE, FT, FV, OM, and SL conceived and designed the experiments. YE, FT, FV, GJ, MB, AP, BV, NL, and PM-G performed the experiments and data analysis. TR, BS, and YP contributed to data analysis, discussions, and intellectual input. YE, FT, FV, OM, and SL wrote the paper with all authors providing detailed comments and suggestions. SL directed the project.
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Estornes, Y., Toscano, F., Virard, F. et al. dsRNA induces apoptosis through an atypical death complex associating TLR3 to caspase-8. Cell Death Differ 19, 1482–1494 (2012). https://doi.org/10.1038/cdd.2012.22
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DOI: https://doi.org/10.1038/cdd.2012.22
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