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.     

Expression level of c-FLIP versus Fas determines susceptibility to Fas ligand-induced cell death in murine thymoma EL-4 cells

The caspase-8 inhibitor c-FLIP blocks death receptor-mediated cell death and plays an essential role in the regulation of lymphocyte homeostasis and the immune escape of tumors. The murine thymoma cell line EL-4 was resistant to Fas ligand (FasL)-induced apoptosis by constitutive expression of FLIP (L). Cycloheximide downregulated the expression of FLIP (L) and markedly sensitized EL-4 cells to FasL-induced apoptosis. In contrast, DNA-damaging agents sensitized EL-4 cells to FasL-induced cell death via an increase of cell-surface Fas without any influence on FLIP (L) expression. Enforced expression of transfected Fas rendered EL-4 cells highly susceptible to FasL-induced cell death. These findings demonstrate that susceptibility to FasL-induced cell death mainly depends on the expression level of c-FLIP versus cell-surface Fas.     

The Fas/Fas Ligand Death Receptor Pathway Contributes to Phenylalanine-Induced Apoptosis in Cortical Neurons

Phenylketonuria (PKU), an autosomal recessive disorder of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene, leads to childhood mental retardation by exposing neurons to cytotoxic levels of phenylalanine (Phe). A recent study showed that the mitochondria-mediated (intrinsic) apoptotic pathway is involved in Phe-induced apoptosis in cultured cortical neurons, but it is not known if the death receptor (extrinsic) apoptotic pathway and endoplasmic reticulum (ER) stress-associated apoptosis also contribute to neurodegeneration in PKU. To answer this question, we used specific inhibitors to block each apoptotic pathway in cortical neurons under neurotoxic levels of Phe. The caspase-8 inhibitor Z-IETD-FMK strongly attenuated apoptosis in Phe-treated neurons (0.9 mM, 18 h), suggesting involvement of the Fas receptor (FasR)-mediated cell death receptor pathway in Phe toxicity. In addition, Phe significantly increased cell surface Fas expression and formation of the Fas/FasL complex. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited apoptosis caused by Phe. In contrast, blocking the ER stress-induced cell death pathway with salubrinal had no effect on apoptosis in Phe-treated cortical neurons. These experiments demonstrate that the Fas death receptor pathway contributes to Phe-induced apoptosis and suggest that inhibition of the death receptor pathway may be a novel target for neuroprotection in PKU patients.     

Inhibition of metalloproteinase cleavage enhances the cytotoxicity of Fas ligand

The Fas ligand (FasL)/Fas receptor (CD95) pathway is an important mediator of apoptosis in the immune system and can also mediate cancer cell death. Soluble FasL (sFasL), shed from the membrane-bound form of the molecule by a putative metalloproteinase (MP), may function to locally regulate the activity of membrane-bound FasL. Using a replication-defective recombinant adenovirus-expressing FasL (RAdFasL), we identified a variable ability of different carcinoma cells to respond to FasL-induced cytotoxicity and to shed sFasL. Blockade of FasL cleavage with an MP inhibitor significantly enhanced RAdFasL-induced apoptosis suggesting that sFasL may antagonize the effect of membrane-bound FasL. In support of this concept, a recombinant adenovirus expressing a noncleavable form of FasL (RAdD4) was found to be a potent inducer of apoptosis even at very low virus doses. Our results highlight the therapeutic potential of noncleavable FasL as an antitumor agent and emphasize the important role of MP via the production of sFasL in regulating the response of the Fas pathway. Moreover, these findings have general implications for the therapeutic exploitation of TNF family ligands and for the possible impact of MP-based therapies on the normal physiology of Fas/TNF pathways.     

A novel role of microtubular cytoskeleton in the dynamics of caspase-dependent Fas/CD95 death receptor complexes during apoptosis

The activation of cysteine-aspartic proteases or caspases and the dynamic arrangement of cytoskeletal components are crucial during apoptosis. Here we describe the fate of Fas downstream of the FasL-induced internalization step, including formation of caspase-dependent SDS-stable Fas complexes, which is mediated by cytoskeleton integrity. We show, in particular, that following FasL treatment, the Fas lower aggregate complex can be co-immunoprecipitated with tubulin and an active form of caspase-8 and that this interaction contributes to the propagation of FasL-induced cell death. The importance of cytoskeletal components during FasL-induced apoptosis is highlighted by our detection of a pool of microtubule-associated Fas complexes.     

Fas ligand induces cell-autonomous NF-kappaB activation and interleukin-8 production by a mechanism distinct from that of tumor necrosis factor-alpha

Fas ligand (FasL) has been well characterized as a death factor. However, recent studies revealed that FasL possesses inflammatory activity. Here we found that FasL induces production of the inflammatory chemokine IL-8 without inducing apoptosis in HEK293 cells. Reporter gene assays involving wild-type and mutated IL-8 promoters and NF-kappaB- and AP-1 reporter constructs indicated that an FasL-induced NF-kappaB and AP-1 activity are required for maximal promoter activity. FasL induced NF-kappaB activation with slower kinetics than did TNF-alpha, yet this response was cell autonomous and not mediated by secondary paracrine factors. The death domain of Fas, FADD, and caspase-8 were required for NF-kappaB activation by FasL. A dominant-negative mutant of IKKgamma inhibited the FasL-induced NF-kappaB activation. However, TRADD and RIP, which are essential for the TNF-alpha-induced NF-kappaB activation, were not involved in the FasL-induced NF-kappaB activation. Moreover, CLARP/FLIP inhibited the FasL- but not the TNF-alpha-induced NF-kappaB activation. These results show that FasL induces NF-kappaB activation and IL-8 production by a novel mechanism, distinct from that of TNF-alpha. In addition, we found that mouse FADD had a dominant-negative effect on the FasL-induced NF-kappaB activation in HEK293 cells, which may indicate a species difference between human and mouse in the FasL-induced NF-kappaB activation.     

Withdrawal of Survival Factors Results in Activation of the JNK Pathway in Neuronal Cells Leading to Fas Ligand Induction and Cell Death

The JNK pathway modulates AP-1 activity. While in some cells it may have proliferative and protective roles, in neuronal cells it is involved in apoptosis in response to stress or withdrawal of survival signals. To understand how JNK activation leads to apoptosis, we used PC12 cells and primary neuronal cultures. In PC12 cells, deliberate JNK activation is followed by induction of Fas ligand (FasL) expression and apoptosis. JNK activation detected by c-Jun phosphorylation and FasL induction are also observed after removal of either nerve growth factor from differentiated PC12 cells or KCl from primary cerebellar granule neurons (CGCs). Sequestation of FasL by incubation with a Fas-Fc decoy inhibits apoptosis in all three cases. CGCs derived from gld mice (defective in FasL) are less sensitive to apoptosis caused by KCl removal than wild-type neurons. In PC12 cells, protection is also conferred by a c-Jun mutant lacking JNK phosphoacceptor sites and a small molecule inhibitor of p38 mitogen-activated protein kinase and JNK, which inhibits FasL induction. Hence, the JNK-to-c-Jun-to-FasL pathway is an important mediator of stress-induced neuronal apoptosis.     

Nitric oxide negatively regulates Fas CD95-induced apoptosis through inhibition of ubiquitin-proteasome-mediated degradation of FLICE inhibitory protein

Stimulation of cell surface Fas (CD95) results in recruitment of cytoplasmic proteins and activation of caspase-8, which in turn activates downstream effector caspases leading to programmed cell death. Nitric oxide (NO) plays a key role in the regulation of apoptosis, but its role in Fas-induced cell death and the underlying mechanism are largely unknown. Here we show that stimulation of the Fas receptor by its ligand (FasL) results in rapid generation of NO and concomitant decrease in cellular FLICE inhibitory protein (FLIP) expression without significant effect on Fas and Fas-associated death domain (FADD) adapter protein levels. FLIP down-regulation as well as caspase-8 activation and apoptosis induced by FasL were all inhibited by the NO-liberating agent sodium nitroprusside and dipropylenetriamine NONOate, whereas the NO synthase inhibitor aminoguanidine and NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) had opposite effects, indicating an anti-apoptotic role of NO in the Fas signaling process. FasL-induced down-regulation of FLIP is mediated by a ubiquitin-proteasome pathway that is negatively regulated by NO. S-nitrosylation of FLIP is an important mechanism rendering FLIP resistant to ubiquitination and proteasomal degradation by FasL. Deletion analysis shows that the caspase-like domain of FLIP is a key target for S-nitrosylation by NO, and mutations of its cysteine 254 and cysteine 259 residues completely inhibit S-nitrosylation, leading to increased ubiquitination and proteasomal degradation of FLIP. These findings indicate a novel pathway for NO regulation of FLIP that provides a key mechanism for apoptosis regulation and a potential new target for intervention in death receptor-associated diseases.     

Programmed cell death of embryonic motoneurons triggered through the Fas death receptor

About 50% of spinal motoneurons undergo programmed cell death (PCD) after target contact, but little is known about how this process is initiated. Embryonic motoneurons coexpress the death receptor Fas and its ligand FasL at the stage at which PCD is about to begin. In the absence of trophic factors, many motoneurons die in culture within 2 d. Most (75%) of these were saved by Fas-Fc receptor body, which blocks interactions between Fas and FasL, or by the caspase-8 inhibitor tetrapeptide IETD. Therefore, activation of Fas by endogenous FasL underlies cell death induced by trophic deprivation. In the presence of neurotrophic factors, exogenous Fas activators such as soluble FasL or anti-Fas antibodies triggered PCD of 40-50% of purified motoneurons over the following 3-5 d; this treatment led to activation of caspase-3, and was blocked by IETD. Sensitivity to Fas activation is regulated: motoneurons cultured for 3 d with neurotrophic factors became completely resistant. Levels of Fas expressed by motoneurons varied little, but FasL was upregulated in the absence of neurotrophic factors. Motoneurons resistant to Fas activation expressed high levels of FLICE-inhibitory protein (FLIP), an endogenous inhibitor of caspase-8 activation. Our results suggest that Fas can act as a driving force for motoneuron PCD, and raise the possibility that active triggering of PCD may contribute to motoneuron loss during normal development and/or in pathological situations.     

The extracellular domains of FasL and Fas are sufficient for the formation of supramolecular FasL-Fas clusters of high stability

Using fluorescent variants of Fas and FasL, we show that membrane FasL and Fas form supramolecular clusters that are of flexible shape, but nevertheless stable and persistent. Membrane FasL-induced Fas clusters were formed in caspase-8- or FADD-deficient cells or when a cytoplasmic deletion mutant of Fas was used suggesting that cluster formation is independent of the assembly of the cytoplasmic Fas signaling complex and downstream activated signaling pathways. In contrast, cross-linked soluble FasL failed to aggregate the cytoplasmic deletion mutant of Fas, but still induced aggregation of signaling competent full-length Fas. Moreover, membrane FasL-induced Fas cluster formation occurred in the presence of the lipid raft destabilizing component methyl-beta-cyclodextrin, whereas Fas aggregation by soluble FasL was blocked. Together, these data suggest that the extracellular domains of Fas and FasL alone are sufficient to drive membrane FasL-induced formation of supramolecular Fas-FasL complexes, whereas soluble FasL-induced Fas aggregation is dependent on lipid rafts and mechanisms associated with the intracellular domain of Fas.     

Differential T cell response in central and peripheral nerve injury: connection with immune privilege.

The central nervous system (CNS), unlike the peripheral nervous system (PNS), is an immune-privileged site in which local immune responses are restricted. Whereas immune privilege in the intact CNS has been studied intensively, little is known about its effects after trauma. In this study, we examined the influence of CNS immune privilege on T cell response to central nerve injury. Immunocytochemistry revealed a significantly greater accumulation of endogenous T cells in the injured rat sciatic nerve than in the injured rat optic nerve (representing PNS and CNS white matter trauma, respectively). Use of the in situ terminal deoxytransferase-catalyzed DNA nick end labeling (TUNEL) procedure revealed extensive death of accumulating T cells in injured CNS nerves as well as in CNS nerves of rats with acute experimental autoimmune encephalomyelitis, but not in injured PNS nerves. Although Fas ligand (FasL) protein was expressed in white matter tissue of both systems, it was more pronounced in the CNS. Expression of major histocompatibility complex (MHC) class II antigens was found to be constitutive in the PNS, but in the CNS was induced only after injury. Our findings suggest that the T cell response to central nerve injury is restricted by the reduced expression of MHC class II antigens, the pronounced FasL expression, and the elimination of infiltrating lymphocytes through cell death.     

The anti-apoptotic protein lifeguard is expressed in breast cancer cells and tissues

Lifeguard (LFG) is an anti-apoptotic protein that inhibits Fas-mediated death in tumour cells. However, the molecular function of human LFG in the carcinogenesis of human breast cells is uncertain. We studied the expression and function of endogenous LFG in four breast cancer cell lines (MCF-7, MDA-MB-231, T-47D and HS 578T), a human breast epithelial cell line (HS 578Bst), and in healthy and cancerous breast tissues. Molecular (Western blot and RT-PCR) and immunohistochemical techniques were used to investigate the LFG expression. To investigate the breast cancer cell proliferation in the presence of Fas, we performed fluorescent cell viability assays. The possible association of Fas with LFG was analyzed by immunofluorescence microscopy. In this paper, we provide convincing evidence that LFG is overexpressed in several human breast cancer cell lines. More importantly, we found that the LFG expression correlates with high tumour grades in primary breast tumours. Finally, we demonstrated that Fas sensitivity is reduced in breast cancer cell lines expressing LFG. Our results indicated that LFG is strongly expressed in breast cancer epithelial cells. Moreover, the overexpression of LFG correlated with tumour grade and reduced Fas sensitivity. Our findings support the idea that LFG may have a role in the downregulation of apoptosis in breast cancer cells.     

The mycotoxin penicillic acid inhibits Fas ligand-induced apoptosis by blocking self-processing of caspase-8 in death-inducing signaling complex

Upon engagement with Fas ligand (FasL), Fas rapidly induces recruitment and self-processing of caspase-8 via the adaptor protein Fas-associated death domain (FADD), and activated caspase-8 cleaves downstream substrates such as caspase-3. We have found that penicillic acid (PCA) inhibits FasL-induced apoptosis and concomitant loss of cell viability in Burkitt's lymphoma Raji cells. PCA prevented activation of caspase-8 and caspase-3 upon treatment with FasL. However, PCA did not affect active caspase-3 in FasL-treated cells, suggesting that PCA primarily blocks early signaling events upstream of caspase-8 activation. FasL-induced processing of caspase-8 was severely impaired in the death-inducing signaling complex, although FasL-induced recruitment of FADD and caspase-8 occurred normally in PCA-treated cells. Although PCA inhibited the enzymatic activities of active recombinant caspase-3, caspase-8, and caspase-9 at similar concentrations, PCA exerted weak inhibitory effects on activation of caspase-9 and caspase-3 in staurosporine-treated cells but strongly inhibited caspase-8 activation in FasL-treated cells. Glutathione and cysteine neutralized an inhibitory effect of PCA on caspase-8, and PCA bound directly to the active center cysteine in the large subunit of caspase-8. Thus, our present results demonstrate that PCA inhibits FasL-induced apoptosis by targeting self-processing of caspase-8.     

Tissue inhibitor of metalloproteinases-3 facilitates Fas-mediated neuronal cell death following mild ischemia

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a natural inhibitor of metalloproteinases involved in matrix degradation and ectodomain shedding of many cell-surface proteins, including death receptors and/or their ligands. In the present study, we examined the role of TIMP-3 in Fas-mediated neuronal cell death following cerebral ischemia, using both gene deletion and pharmacological approaches. In culture, exposure of primary cortical neurons to 2 h of oxygen-glucose deprivation (OGD) resulted in delayed neuronal cell death that was dependent on activation of the death receptor, Fas. Cortical cultures derived from timp-3(-/-) mice displayed partial resistance against OGD-induced neuronal cell death and also displayed increased shedding of Fas ligand (FasL) into the culture media, compared to wild-type control cultures. Both the increased neuroprotection and increased FasL shedding in timp-3(-/-) cultures were reversed by addition of exogenous metalloproteinase inhibitors, recombinant TIMP-3 or GM6001. In vivo, timp-3(-/-) mice showed marked resistance to a brief (30 min) middle cerebral artery occlusion (MCAO), but were not protected against more severe lesions induced by 90 min of MCAO. These studies demonstrate that TIMP-3 facilitates Fas-mediated neuronal cell death following OGD and plays a pro-apoptotic role in mild cerebral ischemia.     

Reactive oxygen species are involved in FasL-induced caspase-independent cell death and inflammatory responses

Fas-mediated caspase-dependent cell apoptosis has been well investigated. However, recent studies have shown that Fas can induce nonapoptotic caspase-independent cell death (CICD) when caspase activity is inhibited. Currently, the molecular mechanism of this alternative cell death mediated by Fas remains unclear. In this study, we investigated the signaling pathway of Fas-induced CICD in mouse embryonic fibroblasts (MEFs) whose caspase function was disrupted by the pan-caspase inhibitor Z-VAD-FMK and its coupling to inflammatory responses. Our results revealed that receptor-interacting protein 1 and tumor necrosis factor receptor-associated factor 2 play important roles in FasL-induced CICD. This death is associated with intracellular reactive oxygen species (ROS) production from mitochondria, as a ROS scavenger (BHA), antioxidants (trolox, NAC), and a mitochondrial respiratory chain uncoupler (rotenone) could prevent this event. Furthermore, delayed and sustained JNK activation, mitochondrial membrane potential breakdown, and loss of intracellular GSH were observed. In addition to CICD, FasL also induces cyclooxygenase-2 and MIP-2 gene upregulation, and both responses are attributed to ROS-dependent JNK activation. Taken together, these results demonstrate alternative signaling pathways of Fas upon caspase inhibition in MEFs that are unrelated to the classical apoptotic pathway, but steer cells toward necrosis and an inflammatory response through ROS production.     

Fas activates lipolysis in a Ca2+-CaMKII-dependent manner in 3T3-L1 adipocytes

Fas (CD95) is a member of the tumor necrosis factor (TNF) receptor superfamily and plays a crucial role in the induction of apoptosis. However, like TNF, Fas can induce nonapoptotic signaling pathways. We previously demonstrated that mice lacking Fas specifically in adipocytes are partly protected from diet-induced insulin resistance, potentially via decreased delivery of FAs to the liver, as manifested by lower total liver ceramide content. In the present study, we aimed to delineate the signaling pathway involved in Fas-mediated adipocyte lipid mobilization. Treatment of differentiated 3T3-L1 adipocytes with membrane-bound Fas ligand (FasL) significantly increased lipolysis after 12 h without inducing apoptosis. In parallel, Fas activation increased phosphorylation of ERK1/2, and FasL-induced lipolysis was blunted in the presence of the ERK-inhibitor U0126 or in ERK1/2-depleted adipocytes. Furthermore, Fas activation increased phosphorylation of the Ca²⁺/calmodulin-dependent protein kinases II (CaMKII), and blocking of the CaMKII-pathway (either by the Ca²⁺ chelator BAPTA or by the CaMKII inhibitor KN62) blunted FasL-induced ERK1/2 phosphorylation and glycerol release. In conclusion, we propose a novel role for CaMKII in promoting lipolysis in adipocytes.