Caspase-10-Dependent Cell Death in Fas/CD95 Signalling Is Not Abrogated by Caspase Inhibitor zVAD-fmk Elodie Lafont1,2,3, Delphine Milhas1,2, Justin Teissie´ 4, Nicole Therville1,2, Nathalie Andrieu-Abadie1,2, Thierry Levade1,2, Herve´ Benoist1,2,3, Bruno Se´ gui1,2,3* 1 U858 INSERM (Institut National de la Sante´ et de la Recherche Me´dicale), De´partement Cancer, Equipe 14, Toulouse, France, 2 Institut Fe´de´ratif de Recherche 150, Institut de Me´decine Mole´culaire de Rangueil, Toulouse, France, 3 Universite´ Paul Sabatier (Toulouse III), Faculte´ des Sciences Pharmaceutiques, Toulouse, France, 4 IPBS (Institut de Pharmacologie et de Biologie Structurale) Unite´ Mixte de Recherche 5089 CNRS (Centre National de la Recherche Scientifique), Toulouse, France Abstract Background: Upon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non- apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase- 10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated. Methodology and Principal Findings: The present study shows that FasL-induced cell death was completely impaired in caspase-8- and caspase-10-doubly deficient (I9-2e) Jurkat leukaemia T-cell lines. Over-expressing of either caspase-8 or caspase-10 in I9-2e cells triggered cell death and restored sensitivity to FasL, further arguing for a role of both initiator caspases in Fas apoptotic signalling. In the presence of zVAD-fmk, FasL triggered an alternative form of cell death similarly in wild-type (A3) and in caspase-8-deficient Jurkat cells expressing endogenous caspase-10 (clone I9-2d). Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death. Noteworthy, ectopic expression of caspase-10 in I9-2e and HeLa cells restored the ability of FasL to trigger cell death in the presence of zVAD-fmk. As a matter of fact, FasL-triggered caspase-10 processing still occurred in the presence of zVAD-fmk. Conclusions and Significance: Altogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling. Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling. Citation: Lafont E, Milhas D, Teissie´ J, Therville N, Andrieu-Abadie N, et al. (2010) Caspase-10-Dependent Cell Death in Fas/CD95 Signalling Is Not Abrogated by Caspase Inhibitor zVAD-fmk. PLoS ONE 5(10): e13638. doi:10.1371/journal.pone.0013638 Editor: Frederic Rieux-Laucat, INSERM U768, Pavillon Kirmisson, France Received February 15, 2010; Accepted October 6, 2010; Published October 26, 2010 Copyright: 2010 Lafont et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by INSERM, Paul Sabatier University, and grant 3417 from the Association pour la Recherche sur le Cancer (ARC) (to BS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bruno.segui@inserm.fr Introduction Fas (CD95 or Apo-1) is a member of the TNF (tumour necrosis factor) receptor superfamily. Fas plays a crucial function in the regulation of T-cell homeostasis as illustrated by the development of an autoimmune lymphoproliferative syndrome (ALPS) in patients carrying gene mutations affecting Fas signalling [1,2,3]. Upon FasL (CD95L or CD178) challenge, the adaptor protein FADD (Fas-associated protein with death domain) is recruited to the Fas death domain [4]. FADD next interacts with caspase-8 [5] and -10 [6] to form the death-inducing signalling complex (DISC). Oligomerization of caspase-8 and -10 at the DISC level is responsible for the activation of the caspase cascade leading to apoptosis [5,6]. Caspase-8 and -10 cleave and activate effector caspase-3 and -7 [7,8,9], which in turn specifically cleave and inactivate a variety of substrates essential for survival leading to apoptosis [10]. Initiator caspases can trigger an alternative route of cell death involving mitochondria. This pathway requires the cleavage of Bid (Bcl-2 interacting domain), a pro-apoptotic member of the Bcl-2 superfamily [11,12,13,14]. FasL has also been reported to activate a caspase-independent cell death pathway, leading to necrosis rather than apoptosis [15,16]. This alternative pathway involves FADD and the kinase activity of RIP (receptor-interacting protein), which is recruited to the Fas receptor [15,16]. Caspase-8 and -10 can display non-apoptotic functions in cell signalling [17]. Moreover, initiator caspase-8 and -10 have been previously reported to activate signalling pathways independently of their catalytic activities. For instance, over-expression of the N- terminal part of caspase-8 containing the two death effector domains (DED) but lacking the catalytic site, triggered death- effector filament formation, leading to endogenous caspase activation and apoptosis in HeLa cells [18]. The DED of caspase-8 and -10 can activate NF-kB in a RIP-dependent PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13638