Fas receptor clustering and involvement of the death receptor pathway in rituximab-mediated apoptosis with concomitant sensitization of lymphoma B cells to fas-induced apoptosis

Ab binding to CD20 has been shown to induce apoptosis in B cells. In this study, we demonstrate that rituximab sensitizes lymphoma B cells to Fas-induced apoptosis in a caspase-8-dependent manner. To elucidate the mechanism by which Rituximab affects Fas-mediated cell death, we investigated rituximab-induced signaling and apoptosis pathways. Rituximab-induced apoptosis involved the death receptor pathway and proceeded in a caspase-8-dependent manner. Ectopic overexpression of FLIP (the physiological inhibitor of the death receptor pathway) or application of zIETD-fmk (specific inhibitor of caspase-8, the initiator-caspase of the death receptor pathway) both specifically reduced rituximab-induced apoptosis in Ramos B cells. Blocking the death receptor ligands Fas ligand or TRAIL, using neutralizing Abs, did not inhibit apoptosis, implying that a direct death receptor/ligand interaction is not involved in CD20-mediated cell death. Instead, we hypothesized that rituximab-induced apoptosis involves membrane clustering of Fas molecules that leads to formation of the death-inducing signaling complex (DISC) and downstream activation of the death receptor pathway. Indeed, Fas coimmune precipitation experiments showed that, upon CD20-cross-linking, Fas-associated death domain protein (FADD) and caspase-8 were recruited into the DISC. Additionally, rituximab induced CD20 and Fas translocation to raft-like domains on the cell surface. Further analysis revealed that, upon stimulation with rituximab, Fas, caspase-8, and FADD were found in sucrose-gradient raft fractions together with CD20. In conclusion, in this study, we present evidence for the involvement of the death receptor pathway in rituximab-induced apoptosis of Ramos B cells with concomitant sensitization of these cells to Fas-mediated apoptosis via Fas multimerization and recruitment of caspase-8 and FADD to the DISC.     

The TRAF3-binding site of human molluscipox virus FLIP molecule MC159 is critical for its capacity to inhibit Fas-induced apoptosis

Members of the viral Flice/caspase-8 inhibitory protein (v-FLIP) family prevent induction of apoptosis by death receptors through inhibition of the processing and activation of procaspase-8 and -10 at the level of the receptor-associated death-inducing signaling complex (DISC). Here, we have addressed the molecular function of the v-FLIP member MC159 of the human molluscum contagiosum virus. MC159 FLIP powerfully inhibited both caspase-dependent and caspase-independent cell death induced by Fas. The C-terminal region of MC159 bound TNF receptor-associated factor (TRAF)3, was necessary for optimal TRAF2 binding, and mediated the recruitment of both TRAFs into the Fas DISC. TRAF-binding-deficient mutants of MC159 showed impaired inhibition of FasL-induced caspase-8 processing and Fas internalization, and had reduced antiapoptotic activity. Our findings provide evidence that a MC159/TRAF2/TRAF3 complex regulates a new aspect of Fas signaling, and identify MC159 FLIP as a molecule that targets multiple features of Fas-induced cell death.     

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.     

Helicobacter pylori sensitizes TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric epithelial cells through regulation of FLIP

Helicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulcer and gastric cancer. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Here we show that human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death receptor signaling. Human gastric epithelial cells are intrinsically resistant to TRAIL-mediated apoptosis. The induction of TRAIL sensitivity by H. pylori is dependent on the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex (DISC) through downregulation of cellular FLICE-inhibitory protein (FLIP). Overexpression of FLIP abolished the H. pylori-induced TRAIL sensitivity in human gastric epithelial cells. Our study thus demonstrates that H. pylori induces sensitivity to TRAIL apoptosis by regulation of FLIP and assembly of DISC, which initiates caspase activation, resulting in the breakdown of resistance to apoptosis, and provides insight into the pathogenesis of gastric damage in Helicobacter infection. Modulation of host apoptosis signaling by bacterial interaction adds a new dimension to the pathogenesis of Helicobacter.     

E1A sensitizes cells to tumor necrosis factor alpha by downregulating c-FLIP S

Tumor necrosis factor alpha (TNF-alpha) activates both apoptosis and NF-kappaB-dependent survival pathways, the former of which requires inhibition of gene expression to be manifested. c-FLIP is a TNF-alpha-induced gene that inhibits caspase-8 activation during TNF-alpha signaling. Adenovirus infection and E1A expression sensitize cells to TNF-alpha by allowing apoptosis in the absence of inhibitors of gene expression, suggesting that it may be disabling a survival signaling pathway. E1A promoted TNF-alpha-mediated activation of caspase-8, suggesting that sensitivity was occurring at the level of the death-inducing signaling complex. Furthermore, E1A expression downregulated c-FLIP(S) expression and prevented its induction by TNF-alpha. c-FLIP(S) and viral FLIP expression rescued E1A-mediated sensitization to TNF-alpha by restoring the resistance of caspase-8 to activation, thereby preventing cell death. E1A inhibited TNF-alpha-dependent induction of c-FLIP(S) mRNA and stimulated ubiquitination- and proteasome-dependent degradation of c-FLIP(S) protein. Since elevated c-FLIP levels confer resistance to apoptosis and promote tumorigenicity, interference with its induction by NF-kappaB and stimulation of its destruction in the proteasome may provide novel therapeutic approaches for facilitating the elimination of apoptosis-refractory tumor 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.     

Regulation of the Fas death pathway by FLICE-inhibitory protein in primary human B cells

The Fas/Fas ligand (L) system plays an important role in the maintenance of peripheral B cell tolerance and the prevention of misguided T cell help. CD40-derived signals are required to induce Fas expression on virgin B cells and to promote their susceptibility to Fas-mediated apoptosis. In the current study, we have analyzed the early biochemical events occurring upon Fas ligation in CD40L-activated primary human tonsillar B cells with respect to Fas-associated death domain protein (FADD), caspase-8/FADD-like IL-1beta-converting enzyme (FLICE), and c-FLICE inhibitory protein (FLIP). We report here that Fas-induced apoptosis in B cells does not require integrity of the mitochondrial Apaf-1 pathway and that caspase-8 is activated by association with the death-inducing signaling complex (DISC), i.e., upstream of the mitochondria. We show that both FADD and the zymogen form of caspase-8 are constitutively expressed at high levels in virgin B cells, whereas c-FLIP expression is marginal. In contrast, c-FLIP, but neither FADD nor procaspase-8, is strongly up-regulated upon ligation of CD40 or the B cell receptor on virgin B cells. Finally, we have found that c-FLIP is also recruited and cleaved at the level of the DISC in CD40L-activated virgin B cells. We propose that c-FLIP expression delays the onset of apoptosis in Fas-sensitive B cells. The transient protection afforded by c-FLIP could offer an ultimate safeguard mechanism against inappropriate cell death or allow recruitment of phagocytes to ensure efficient removal of apoptotic cells.     

Downregulation of FLIP by cycloheximide sensitizes human fat cells to CD95-induced apoptosis

Adipocyte apoptosis is an important regulator of adipocyte number in fat depots. We have previously shown that an inhibition of protein synthesis sensitizes human adipocytes for apoptosis. In vivo, dramatic changes in the fat cell's protein expression should be anticipated under special conditions such as calorie restriction. Here, we studied the underlying mechanism by which human preadipocytes and adipocytes are sensitized for death receptor induced apoptosis in vitro.The protein synthesis blocker cycloheximide (CHX) sensitized human fat cells for CD95-induced apoptosis in a caspase-dependent manner. Treatment with CHX differentially changed expression of pro- and anti-apoptotic proteins. Most noticeably, FLICE-like inhibitory protein (FLIP) expression rapidly decreased during CHX treatment. Reduction of FLIP levels resulted in undetectable amounts of FLIP at the CD95 death-inducing signaling complex (DISC) upon CD95 stimulation, thereby enhancing recruitment and activation at caspase-8. Down-regulation of FLIP by shRNA sensitized preadipocytes for CD95-induced apoptosis. In mice, adipose tissue mRNA levels of Flip were down-regulated upon fasting.In conclusion, we identify FLIP as an important regulator of apoptosis sensitivity in fat cells. Modulating adipocyte homeostasis by apoptosis might provide a new therapeutic concept to get rid of excess adipose tissue, and FLIP might be a possible target molecule.     

FLIP inhibits endothelial cell apoptosis during hyperoxia by suppressing Bax

High oxygen tension (hyperoxia) causes pulmonary cell death, involving apoptosis, necrosis, or mixed death phenotypes, though the underlying mechanisms remain unclear. In mouse lung endothelial cells (MLEC) hyperoxia activates both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) apoptotic pathways. We examined the hypothesis that FLIP, an inhibitor of caspase-8, can protect endothelial cells against the lethal effects of hyperoxia. Hyperoxia caused the time-dependent downregulation of FLIP in MLEC. Overexpression of FLIP attenuated intracellular reactive oxygen species generation during hyperoxia exposure, by downregulating extracellular-regulated kinase-1/2 activation and p47(phox) expression. FLIP prevented hyperoxia-induced trafficking of the death-inducing signal complex from the Golgi apparatus to the plasma membrane. Furthermore, FLIP blocked the activations of caspase-8/Bid, caspases -3/-9, and inhibited the mitochondrial translocation and activation of Bax, resulting in protection against hyperoxia-induced cell death. Under normoxic conditions, FLIP expression increased the phosphorylation of p38 mitogen-activated protein kinase leading to increased phosphorylation of Bax during hyperoxic stress. Furthermore, FLIP expression markedly inhibited protein kinase C activation and expression of distinct protein kinase C isoforms (alpha, eta, and zeta), and stabilized an interaction of PKC with Bax. In conclusion, FLIP exerted novel inhibitory effects on extrinsic and intrinsic apoptotic pathways, which significantly protected endothelial cells from the lethal effects of hyperoxia.     

Regulation of death complexes formation in tumor necrosis factor receptor signaling

TNFα stimulation triggers both cell death and survival programs. Since dysregulated apoptosis or cell growth can cause inflammatory diseases, cancer, or autoimmune disorders, it is important to understand the molecular mechanism of controlling cell death and survival by TNFR downstream signaling molecules. In this study, we used normal diploid cells, mouse embryonic fibroblasts (MEFs), to mimic the general TNFα-resistant phenomenon seen under physiological conditions. We elucidated the TNFα-induced death signaling complexes in TNF α-resistant WT MEFs and TNFα-sensitive MEFs that were cFLIP-, RelA-, TRAF2- or RIP1-deficient. Consistent with TNFα-mediated killing, we detected TNFα-induced high molecular weight complexes containing caspase-8 and FADD by gel filtration in the deficient MEFs, especially in those devoid of cFLIP. In addition to the presence of caspase-8-FADD in the TNFα-induced-death complex in the deficient MEFs, we also detected an intermediate protein complex containing RIP1, TRAF2 and caspase-8. Moreover, we demonstrated a correlation between TNFα-sensitivity and death-inducing complex ability in two transformed cell lines, E1A- and Ras- transformed MEFs and PDGF-B-transformed NIH-3T3 cells with PDGF-B signaling inhibited by the tyrosine kinase inhibitor STI571. Taken together, our results suggest the involvement of cFLIP-, RelA-, RIP1-, or TRAF2-related mechanisms for preventing FADD-caspase-8 interaction in wild-type MEFs.     

A caspase-8-independent component in TRAIL/Apo-2L-induced cell death in human rhabdomyosarcoma cells

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) belongs to the Tumor necrosis factor (TNF) family of death-inducing ligands, and signaling downstream of TRAIL ligation to its receptor(s) remains to be fully elucidated. Components of the death-inducing signaling complex (DISC) and TRAIL signaling downstream of receptor activation were examined in TRAIL - sensitive and -resistant models of human rhabdomyosarcoma (RMS). TRAIL ligation induced DISC formation in TRAIL-sensitive (RD, Rh18, Rh30) and TRAIL-resistant RMS (Rh28, Rh36, Rh41), with recruitment of FADD and procaspase-8. In RD cells, overexpression of dominant-negative FADD (DNFADD) completely abolished TRAIL-induced cell death in contrast to dominant-negative caspase- 8 (DNC8), which only partially inhibited TRAIL-induced apoptosis, growth inhibition, or loss in clonogenic survival. DNC8 did not inhibit the cleavage of Bid or the activation of Bax. Overexpression of Bcl-2 or Bcl-xL inhibited TRAIL-induced apoptosis, growth inhibition, and loss in clonogenic survival. Bcl-2 and Bcl-xL, but not DNC8, inhibited TRAIL-induced Bax activation. Bcl-xL did not inhibit the early activation of caspase-8 (<4 h) but inhibited cleavage of Bid, suggesting that Bid is cleaved downstream of the mitochondria, independent of caspase-8. Exogenous addition of sphingosine also induced activation of Bax via a caspase-8-and Bid-independent mechanism. Further, inhibition of sphingosine kinase completely protected cells from TRAIL-induced apoptosis. Data demonstrate that in RMS cells, the TRAIL signaling pathway circumvents caspase-8 activation of Bid upstream of the mitochondria and that TRAIL acts at the level of the mitochondria via a mechanism that may involve components of the sphingomyelin cycle.     

Crystal structure of a viral FLIP: insights into FLIP-mediated inhibition of death receptor signaling

Death receptor signaling is initiated by the assembly of the death-inducing signaling complex, which culminates in the activation of the initiator caspase, either caspase-8 or caspase-10. A family of viral and cellular proteins, known as FLIP, plays an essential role in the regulation of death receptor signaling. Viral FLIP (v-FLIP) and short cellular FLIP (c-FLIPS) inhibit apoptosis by interfering with death receptor signaling. The structure and mechanisms of v-FLIP and c-FLIPS remain largely unknown. Here we report a high resolution crystal structure of MC159, a v-FLIP derived from the molluscum contagiosum virus, which is a member of the human poxvirus family. Unexpectedly, the two tandem death effector domains (DEDs) of MC159 rigidly associate with each other through a hydrophobic interface. Structure-based sequence analysis suggests that this interface is conserved in the tandem DEDs from other v-FLIP, c-FLIPS, and caspase-8 and -10. Strikingly, the overall packing arrangement between the two DEDs of MC159 resembles that between the caspase recruitment domains of Apaf-1 and caspase-9. In addition, each DED of MC159 contains a highly conserved binding motif on the surface, to which loss-of-function mutations in MC159 map. These observations, in conjunction with published evidence, reveal significant insights into the function of v-FLIP and suggest a mechanism by which v-FLIP and c-FLIPS inhibit death receptor signaling.     

An essential role for membrane rafts in the initiation of Fas/CD95-triggered cell death in mouse thymocytes

Fas, a member of the tumor necrosis factor receptor family, can upon ligation by its ligand or agonistic antibodies trigger signaling cascades leading to cell death in lymphocytes and other cell types. Such signaling cascades are initiated through the formation of a membrane death-inducing signaling complex (DISC) that includes Fas, the Fas-associated death domain protein (FADD) and caspase-8. We report here that a considerable fraction of Fas is constitutively partitioned into sphingolipid- and cholesterol-rich membrane rafts in mouse thymocytes as well as the L12.10-Fas T cells, and Fas ligation promotes a rapid and specific recruitment of FADD and caspase-8 to the rafts. Raft disruption by cholesterol depletion abolishes Fas-triggered recruitment of FADD and caspase-8 to the membrane, DISC formation and cell death. Taken together, our results provide the first demonstration for an essential role of membrane rafts in the initiation of Fas-mediated cell death signaling.     

Homotypic FADD interactions through a conserved RXDLL motif are required for death receptor-induced apoptosis

Death receptors in the TNF receptor superfamily signal for apoptosis via the ordered recruitment of FADD and caspase-8 to a death-inducing signaling complex (DISC). However, the nature of the protein-protein interactions in the signaling complex is not well defined. Here we show that FADD self-associates through a conserved RXDLL motif in the death effector domain (DED). Despite exhibiting similar binding to both Fas and caspase-8 and preserved overall secondary structure, FADD RDXLL motif mutants cannot reconstitute FasL- or TRAIL-induced apoptosis and fail to recruit caspase-8 into the DISC of reconstituted FADD-deficient cells. Abolishing self-association can transform FADD into a dominant-negative mutant that interferes with Fas-induced apoptosis and formation of microscopically visible receptor oligomers. These findings suggest that lateral interactions among adapter molecules are required for death receptor apoptosis signaling and implicate self-association into oligomeric assemblies as a key function of death receptor adapter proteins in initiating apoptosis.     

Epoxycyclohexenone inhibits Fas-mediated apoptosis by blocking activation of pro-caspase-8 in the death-inducing signaling complex

Death receptors belong to the tumor necrosis factor receptor family. They can induce apoptosis following engagement with specific ligands and are known to play an important role in the regulation of the immune system. Here we report that epoxycyclohexenone (ECH) inhibits apoptosis induced by anti-Fas antibody, Fas ligand (FasL), or tumor necrosis factor-alpha but not by staurosporine, MG-132, C2-ceramide, or UV irradiation. These results suggest that ECH specifically blocks death receptor-mediated apoptosis. Neither the surface expression of Fas nor the Fas-FasL interaction was influenced by ECH. However, ECH did block the activation of pro-caspase-8 in the death-inducing signaling complex, although recruitment of Fas-associating death domain (FADD) and pro-caspase-8 was not affected. ECH inhibited the enzymatic activity of recombinant active caspase-8 at slightly lower concentrations than it did for active caspase-3 and active caspase-9 in vitro. However, in FasL-treated cells, ECH was only able to inhibit the activation of pro-caspase-8, and it had no effect on the already activated caspase-8 at a concentration that is effective at inhibiting Fas-induced apoptosis. ECH directly bound the large subunit of active caspase-8 that contains the active center cysteine and had a relatively higher affinity to pro-caspase-8. Moreover, compared with pro-caspase-3 and pro-caspase-9, pro-caspase-8 was predominantly depleted by biotinylated ECH with avidin beads in the cell lysates, suggesting that ECH preferentially affects pro-caspase-8. Thus, our results suggest that ECH blocks the self-activation of pro-caspase-8 in the death-inducing signaling complex and thus selectively inhibits death receptor-mediated apoptosis.     

Proliferative versus apoptotic functions of caspase-8 Hetero or homo: the caspase-8 dimer controls cell fate

Caspase-8, the initiator of extrinsically-triggered apoptosis, also has important functions in cellular activation and differentiation downstream of a variety of cell surface receptors. It has become increasingly clear that the heterodimer of caspase-8 with the long isoform of cellular FLIP (FLIP(L)) fulfills these pro-survival functions of caspase-8. FLIP(L), a catalytically defective caspase-8 paralog, can interact with caspase-8 to activate its catalytic function. The caspase-8/FLIP(L) heterodimer has a restricted substrate repertoire and does not induce apoptosis. In essence, caspase-8 heterodimerized with FLIP(L) prevents the receptor interacting kinases RIPK1 and -3 from executing the form of cell death known as necroptosis. This review discusses the latest insights in caspase-8 homo- versus heterodimerization and the implication this has for cellular death or survival. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Tocotrienol-induced caspase-8 activation is unrelated to death receptor apoptotic signaling in neoplastic mammary epithelial cells

Tocotrienols, a subclass in the vitamin E family of compounds, have been shown to induce apoptosis by activating caspase-8 and caspase-3 in neoplastic mammary epithelial cells. Since caspase-8 activation is associated with death receptor apoptotic signaling, studies were conducted to determine the exact death receptor/ligand involved in tocotrienol-induced apoptosis. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained in serum-free media. Treatment with 20 microM gamma-tocotrienol decreased+SA cell viability by inducing apoptosis, as determined by positive terminal dUTP nick end labeling (TUNEL) immunocytochemical staining. Western blot analysis showed that gamma-tocotrienol treatment increased the levels of cleaved (active) caspase-8 and caspase-3. Combined treatment with caspase inhibitors completely blocked tocotrienol-induced apoptosis. Additional studies showed that treatment with 100 ng/ml tumor necrosis factor-alpha (TNF-alpha), 100 ng/ml FasL, 100 ng/ml TNF-related apoptosis-inducing ligand (TRAIL), or 1 microg/ml apoptosis-inducing Fas antibody failed to induce death in +SA cells, indicating that this mammary tumor cell line is resistant to death receptor-induced apoptosis. Furthermore, treatment with 20 microM gamma-tocotrienol had no effect on total, membrane, or cytosolic levels of Fas, Fas ligand (FasL), or Fas-associated via death domain (FADD) and did not induce translocation of Fas, FasL, or FADD from the cytosolic to the membrane fraction, providing additional evidence that tocotrienol-induced caspase-8 activation is not associated with death receptor apoptotic signaling. Other studies showed that treatment with 20 microM gamma-tocotrienol induced a large decrease in the relative intracellular levels of phospho-phosphatidylinositol 3-kinase (PI3K)-dependent kinase 1 (phospho-PDK-1 active), phospho-Akt (active), and phospho-glycogen synthase kinase3, as well as decreasing intracellular levels of FLICE-inhibitory protein (FLIP), an antiapoptotic protein that inhibits caspase-8 activation, in these cells. Since stimulation of the PI3K/PDK/Akt mitogenic pathway is associated with increased FLIP expression, enhanced cellular proliferation, and survival, these results indicate that tocotrienol-induced caspase-8 activation and apoptosis in malignant +SA mammary epithelial cells is associated with a suppression in PI3K/PDK-1/Akt mitogenic signaling and subsequent reduction in intracellular FLIP levels.     

The adaptor protein FADD and the initiator caspase-8 mediate activation of NF-��B by TRAIL

Besides inducing apoptosis, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates NF-κB. The apoptosis signaling pathway of TRAIL is well characterized involving TRAIL receptors, Fas-associated protein with death domain (FADD) and caspase-8. In contrast, the molecular mechanism of TRAIL signaling to NF-κB remains controversial. Here, we characterized the receptor–proximal mediators of NF-κB activation by TRAIL. Deletion of the DD of TRAIL receptors 1 and 2 revealed that it is essential in NF-κB signaling. Because FADD interacts with the TRAIL receptor DD, FADD was tested. RNAi-mediated knockdown of FADD or FADD deficiency in JURKAT T-cell leukemia cells decreased or disabled NF-κB signaling by TRAIL. In contrast, TRAIL-induced activation of NF-κB was maintained upon loss of receptor interacting protein 1 (RIP1) or knockdown of FLICE-like inhibitory protein (FLIP). Exogenous expression of FADD rescued TRAIL-induced NF-κB signaling. Loss-of-function mutations of FADD within the RHDLL motif of the death effector domain, which is required for TRAIL-induced apoptosis, abrogated FADD''s ability to recruit caspase-8 and mediate NF-κB activation. Accordingly, deficiency of caspase-8 inhibited TRAIL-induced activation of NF-κB, which was rescued by wild-type caspase-8, but not by a catalytically inactive caspase-8 mutant. These data establish the mechanism of TRAIL-induced NF-κB activation involving the TRAIL receptor DD, FADD and caspase-8, but not RIP1 or FLIP. Our results show that signaling of TRAIL-induced apoptosis and NF-κB bifurcates downstream of caspase-8.     

Selective up-regulation of phosphatidylinositol 3'-kinase activity in Th2 cells inhibits caspase-8 cleavage at the death-inducing complex: a mechanism for Th2 resistance from Fas-mediated apoptosis.

In this study the mechanism of differential sensitivity of CD3-activated Th1- and Th2-type cells to Fas-mediated apoptosis was explored. We show that the Fas-associated death domain protein (FADD)/caspase-8 pathway is differentially regulated by CD3 activation in the two subsets. The apoptosis resistance of activated Th2-type cells is due to an incomplete processing of caspase-8 at the death-inducing signaling complex (DISC) whereas recruitment of caspase-8 to the DISC of Th1- and Th2-like cells is comparable. Activation of phosphatidylinositol 3'-kinase upon ligation of CD3 in Th2-type cells blocked caspase-8 cleavage to its active fragments at the DISC, thereby preventing induction of apoptosis. This study offers a new pathway for phosphatidylinositol 3'-kinase in mediating protection from Fas-induced apoptosis.