Caspase-10-Dependent Cell Death in Fas/CD95 Signalling Is Not Abrogated by Caspase Inhibitor zVAD-fmk

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.

[1]  T. Kataoka,et al.  Caspase-8 Mediates Mitochondrial Release of Pro-apoptotic Proteins in a Manner Independent of Its Proteolytic Activity in Apoptosis Induced by the Protein Synthesis Inhibitor Acetoxycycloheximide in Human Leukemia Jurkat Cells* , 2009, Journal of Biological Chemistry.

[2]  R. Beyaert,et al.  Non-apoptotic functions of caspase-8. , 2008, Biochemical pharmacology.

[3]  Jun Wu,et al.  Cloning and characterization of a novel caspase-10 isoform that activates NF-kappa B activity. , 2007, Biochimica et biophysica acta.

[4]  H. Bobby Gaspar,et al.  Autoimmune lymphoproliferative syndrome: molecular basis of disease and clinical phenotype , 2006, British journal of haematology.

[5]  S. Straus,et al.  Causes and consequences of the autoimmune lymphoproliferative syndrome , 2006, Hematology.

[6]  A. Schäffer,et al.  Genetic alterations in caspase-10 may be causative or protective in autoimmune lymphoproliferative syndrome , 2006, Human Genetics.

[7]  C. Stroh,et al.  Unique and overlapping substrate specificities of caspase-8 and caspase-10 , 2006, Oncogene.

[8]  F. Rieux-Laucat Inherited and acquired death receptor defects in human Autoimmune Lymphoproliferative Syndrome. , 2006, Current directions in autoimmunity.

[9]  O. Cuvillier,et al.  Caspase-10 Triggers Bid Cleavage and Caspase Cascade Activation in FasL-induced Apoptosis* , 2005, Journal of Biological Chemistry.

[10]  R. Bárcia,et al.  Caspase involvement in RIP-associated CD95-induced T cell apoptosis. , 2003, Cellular immunology.

[11]  J. Teissié,et al.  Retroactive pathway involving mitochondria in electroloaded cytochrome c-induced apoptosis. Protective properties of Bcl-2 and Bcl-XL. , 2003, Experimental cell research.

[12]  T. Miyashita,et al.  Caspase‐8 and caspase‐10 activate NF‐κB through RIP, NIK and IKKα kinases , 2003 .

[13]  A. Grosse-Wilde,et al.  Caspase‐10 is recruited to and activated at the native TRAIL and CD95 death‐inducing signalling complexes in a FADD‐dependent manner but can not functionally substitute caspase‐8 , 2002, The EMBO journal.

[14]  J. Blenis,et al.  Death Receptor Recruitment of Endogenous Caspase-10 and Apoptosis Initiation in the Absence of Caspase-8* , 2001, The Journal of Biological Chemistry.

[15]  H. Chun,et al.  Caspase-10 is an initiator caspase in death receptor signaling , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[16]  A. Thorburn,et al.  An apoptosis signaling pathway induced by the death domain of FADD selectively kills normal but not cancerous prostate epithelial cells , 2001, Cell Death and Differentiation.

[17]  P. Vandenabeele,et al.  Apoptotic and necrotic cell death induced by death domain receptors , 2001, Cellular and Molecular Life Sciences CMLS.

[18]  S. Nagata,et al.  Necrotic Death Pathway in FAS Receptor Signaling , 2000, The Journal of cell biology.

[19]  Brian Seed,et al.  Fas triggers an alternative, caspase-8–independent cell death pathway using the kinase RIP as effector molecule , 2000, Nature Immunology.

[20]  W. Fiers,et al.  Structure/Function Analysis of p55 Tumor Necrosis Factor Receptor and Fas-associated Death Domain , 2000, The Journal of Biological Chemistry.

[21]  S. Spiegel,et al.  Involvement of Sphingosine in Mitochondria-dependent Fas-induced Apoptosis of Type II Jurkat T Cells* , 2000, The Journal of Biological Chemistry.

[22]  A. Fontana,et al.  Induction of antitumor immunity with Fas/APO-1 ligand (CD95L)-transfected neuroblastoma neuro-2a cells. , 1999, Journal of immunology.

[23]  S. Nagata,et al.  Caspase-independent Cell Killing by Fas-associated Protein with Death Domain , 1998, The Journal of cell biology.

[24]  J. Blenis,et al.  Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade , 1998, Current Biology.

[25]  Xiaodong Wang,et al.  Bid, a Bcl2 Interacting Protein, Mediates Cytochrome c Release from Mitochondria in Response to Activation of Cell Surface Death Receptors , 1998, Cell.

[26]  Junying Yuan,et al.  Cleavage of BID by Caspase 8 Mediates the Mitochondrial Damage in the Fas Pathway of Apoptosis , 1998, Cell.

[27]  J. Bertin,et al.  Death-effector Filaments: Novel Cytoplasmic Structures that Recruit Caspases and Trigger Apoptosis , 1998, The Journal of cell biology.

[28]  G M Cohen,et al.  Caspases: the executioners of apoptosis. , 1997, The Biochemical journal.

[29]  V. Dixit,et al.  Fas-associated Death Domain Protein Interleukin-1β-converting Enzyme 2 (FLICE2), an ICE/Ced-3 Homologue, Is Proximally Involved in CD95- and p55-mediated Death Signaling* , 1997, The Journal of Biological Chemistry.

[30]  G. Salvesen,et al.  FLICE Induced Apoptosis in a Cell-free System , 1997, The Journal of Biological Chemistry.

[31]  S. Srinivasula,et al.  In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Matthias Mann,et al.  FLICE, A Novel FADD-Homologous ICE/CED-3–like Protease, Is Recruited to the CD95 (Fas/APO-1) Death-Inducing Signaling Complex , 1996, Cell.

[33]  Arul M. Chinnaiyan,et al.  FADD, a novel death domain-containing protein, interacts with the death domain of fas and initiates apoptosis , 1995, Cell.

[34]  M. Lenardo Molecular regulation of T lymphocyte homeostasis in the healthy and diseased immune system , 2003, Immunologic research.

[35]  A. Thorburn,et al.  Caspase- and serine protease-dependent apoptosis by the death domain of FADD in normal epithelial cells. , 2003, Molecular biology of the cell.

[36]  T. Miyashita,et al.  Caspase-8 and caspase-10 activate NF-kappaB through RIP, NIK and IKKalpha kinases. , 2003, European journal of immunology.