An IL-2-dependent switch between CD95 signaling pathways sensitizes primary human T cells toward CD95-mediated activation-induced cell death

The CD95 (APO-1/Fas) system plays a critical role in activation-induced cell death (AICD) of T cells. We previously described two distinct CD95 (APO-1/Fas) signaling pathways: 1) type I cells show strong death-inducing signaling complex (DISC) formation and mitochondria-independent apoptosis and 2) DISC formation is reduced in type II cells, leading to mitochondria-dependent apoptosis. To investigate the relevance of these pathways, we set up an in vitro model that mimics the initiation and the down phase of an immune response, respectively. Freshly activated human T cells (initiation) are resistant toward CD95-mediated AICD despite high expression of CD95. We previously reported that these T cells show reduced DISC formation. In this study, we show that freshly activated T cells are CD95-type II cells that show high expression levels of Bcl-x(L) and display a block in the mitochondrial apoptosis pathway. Furthermore, we show that, upon prolonged culture (down phase), human T cells undergo a switch from type II to type I cells that renders T cells sensitive to CD95-mediated AICD. Finally, we demonstrate that this switch is dependent on the presence of IL-2. Our observations reveal for the first time that the existence of coexisting CD95 signaling pathways is of physiological relevance.     

MAPK/ERK signaling in activated T cells inhibits CD95/Fas-mediated apoptosis downstream of DISC assembly

When T cells are activated, the expression of the CD95 ligand is elevated, with the purpose of inducing apoptosis in target cells and to later eliminate the activated T cells. We have shown previously that mitogen-activated protein kinase (MAPK or ERK) signaling suppresses CD95-mediated apoptosis in different cellular systems. In this study we examined whether MAPK signaling controls the persistence and CD95-mediated termination of an immune response in activated T cells. Our results show that activation of Jurkat T cells through the T cell receptor immediately suppresses CD95-mediated apoptosis, and that this suppression is mediated by MAPK activation. During the phase of elevated MAPK activity, the activation of caspase-8 and Bid is inhibited, whereas the assembly of a functional death-inducing signaling complex (DISC) is not affected. These results explain the resistance to CD95 responses observed during the early phase of T cell activation and suggest that MAPK-activation deflects DISC signaling from activating caspase-8 and Bid. The physiological relevance of the results was confirmed in activated primary peripheral T cells, in which inhibition of MAPK signaling markedly sensitized the cells to CD95-mediated apoptosis.     

Isolation and analysis of components of CD95 (APO-1/Fas) death-inducing signaling complex

CD95 (APO-1/Fas) is an apoptosis-inducing receptor belonging to the tumor necrosis factor receptor superfamily. Multimerization of CD95 leads to instant recruitment of the signaling molecules FADD and caspase-8 to the activated receptor forming the death-inducing signaling complex (DISC). DISC formation is the first essential step of CD95 signaling and results in activation of caspase-8 starting a signaling cascade that leads to apoptosis. Here we describe a method for analyzing the CD95 DISC. The method is based on coimmunoprecipitation of the signaling molecules with the activated CD95 receptor followed by Western blot detection of associated molecules. Therefore, this method can analyze the very first signaling events during CD95-mediated apoptosis.     

TCR-mediated up-regulation of c-FLIPshort correlates with resistance toward CD95-mediated apoptosis by blocking death-inducing signaling complex activity

To investigate apoptosis resistance upon restimulation in human peripheral blood T lymphocytes, we used the following in vitro model. This model represents the main features of T cell reactivity: freshly isolated PHA-activated T cells cultured in IL-2 for a prolonged period of time develop a CD95 (APO-1/Fas) apoptosis-sensitive phenotype. These T cells represent activation-induced cell death-sensitive T cells during the down phase of an immune response. A fraction of apoptosis-sensitive activated T cells becomes apoptosis resistant upon TCR/CD3 restimulation. CD95 apoptosis sensitivity requires formation of a functional receptor associated death-inducing signaling complex (DISC), i.e., a protein complex of CD95 receptors, the adaptor Fas-associated death domain protein (FADD)/MORT1 and caspase-8 (FADD-like IL-1ss-converting enzyme (FLICE), MACH, Mch5). We identified activation of procaspase-8 at the DISC as the main target for the protective activity of TCR/CD3 restimulation. We found that procaspase-8 cleavage is reduced in T cells after TCR/CD3 restimulation. In addition, we detected up-regulation of c-FLIP(S) (the short splice variant of the cellular FLICE inhibitory protein) and strongly enhanced recruitment of c-FLIP(S) into the DISC. These data suggest that the recruitment of c-FLIP(S) into the DISC results in reduced DISC and caspase-8 activity.     

Two CD95 (APO-1/Fas) signaling pathways.

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).     

The role of receptor internalization in CD95 signaling

Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death-inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand-mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF-kappaB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates.     

Synthetic glycosidated phospholipids induce apoptosis through activation of FADD,caspase-8 and the mitochondrial death pathway

Apoptosis is modulated by extrinsic and intrinsic signaling pathways through the formation of the death receptor-mediated death-inducing signaling complex (DISC) and the mitochondrial-derived apoptosome, respectively. Ino-C2-PAF, a novel synthetic phospholipid shows impressive antiproliferative and apoptosis-inducing activity. Little is known about the signaling pathway through which it stimulates apoptosis. Here, we show that this drug induces apoptosis through proteins of the death receptor pathway, which leads to an activation of the intrinsic apoptotic pathway. Apoptosis induced by Ino-C2-PAF and its glucosidated derivate, Glc-PAF, was dependent on the DISC components FADD and caspase-8. This can be inhibited in FADD−/− and caspase-8−/− cells, in which the breakdown of the mitochondrial membrane potential, release of cytochrome c and activation of caspase-9, -8 and -3 do not occur. In addition, the overexpression of crmA, c-Flip or dominant negative FADD as well as treatment with the caspase-8 inhibitor z-IETD-fmk protected against Ino-C2-PAF-induced apoptosis. Apoptosis proceeds in the absence of CD95/Fas-ligand expression and is independent of blockade of a putative death-ligand/receptor interaction. Furthermore, apoptosis cannot be inhibited in CD95/Fas−/− Jurkat cells. Expression of Bcl-2 in either the mitochondria or the endoplasmic reticulum (ER) strongly inhibited Ino-C2-PAF- and Glc-PAF-induced apoptosis. In conclusion, Ino-C2-PAF and Glc-PAF trigger a CD95/Fas ligand- and receptor-independent atypical DISC that relies on the intrinsic apoptotic pathway via the ER and the mitochondria.     

Glutathione dependence of caspase-8 activation at the death-inducing signaling complex.

Apoptosis triggered by the death receptor CD95 (APO-1 or Fas) is pivotal for the homeostasis of the immune system. We investigated differential effects of glutathione depletion on CD95-triggered apoptosis in T and B cell lines as well as the glutathione dependence of caspase-8 activation. In B lymphoblastoid SKW6.4 cells, CD95-mediated apoptosis was prevented upstream of caspase-8 activation and caspase-3-like activity after acute glutathione depletion by diethyl maleate or cis-chloro-dinitrobenzene. Immunoprecipitation of the death-inducing signaling complex (DISC) revealed that the DISC was still formed in the glutathione-depleted state. The first cleavage step of procaspase-8 activation at the DISC, however, was inhibited. Accordingly, under cell-free conditions, radiolabeled procaspase-8 was processed at the immunoprecipitated DISC only after the addition of exogenous dithiothreitol or reduced glutathione. We also observed suppression of CD95-mediated apoptosis in glutathione-depleted CEM and H9 cells. Notably, Jurkat cells still died upon CD95 engagement under this condition, displaying incomplete nuclear fragmentation and a partial switch to necrosis; this may be explained by reduced cytochrome c/dATP-mediated caspase activation observed in cytosol from glutathione-depleted Jurkat cytosol. Our data indicate that the activation of caspase-8 at the DISC and hence CD95-mediated apoptosis induction shows a cell-specific requirement for intracellular glutathione.     

Palmitoylation of CD95 facilitates formation of SDS-stable receptor aggregates that initiate apoptosis signaling

Apoptosis signaling through CD95 (Fas/APO-1) involves aggregation and clustering of the receptor followed by its actin-dependent internalization. Internalization is required for efficient formation of the death-inducing signaling complex (DISC) with maximal recruitment of FADD, caspase-8/10 and c-FLIP occurring when the receptor has reached an endosomal compartment. The first detectable event during CD95 signaling is the formation of SDS-stable aggregates likely reflecting intense oligomerization of the receptor. We now demonstrate that these SDS-stable forms of CD95 correspond to very high molecular weight DISC complexes (hiDISC) and are the sites of caspase-8 activation. hiDISCs are found both inside and outside of detergent-resistant membranes. The formation of SDS-stable CD95 aggregates involves palmitoylation of the membrane proximal cysteine 199 in CD95. Cysteine 199 mutants no longer form SDS-stable aggregates, and inhibition of palmitoylation reduces internalization of CD95 and activation of caspase-8. Our data demonstrate that SDS-stable forms of CD95 are the sites of apoptosis initiation and represent an important early step in apoptosis signaling through CD95 before activation of caspases.     

Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation.

The death receptor CD95 (APO-1/Fas), the anticancer drug etoposide, and gamma-radiation induce apoptosis in the human T cell line Jurkat. Variant clones selected for resistance to CD95-induced apoptosis proved cross-resistant to etoposide- and radiation-induced apoptosis, suggesting that the apoptosis pathways induced by these distinct stimuli have critical component(s) in common. The pathways do not converge at the level of CD95 ligation or caspase-8 signaling. Whereas caspase-8 function was required for CD95-mediated cytochrome c release, effector caspase activation, and apoptosis, these responses were unaffected in etoposide-treated and irradiated cells when caspase-8 was inhibited by FLIPL. Both effector caspase processing and cytochrome c release were inhibited in the resistant variant cells as well as in Bcl-2 transfectants, suggesting that, in Jurkat cells, the apoptosis signaling pathways activated by CD95, etoposide, and gamma-radiation are under common mitochondrial control. All three stimuli induced ceramide production in wild-type cells, but not in resistant variant cells. Exogenous ceramide bypassed apoptosis resistance in the variant cells, but not in Bcl-2-transfected cells, suggesting that apoptosis signaling induced by CD95, etoposide, and gamma-radiation is subject to common regulation at a level different from that targeted by Bcl-2.     

Resistance of short term activated T cells to CD95-mediated apoptosis correlates with de novo protein synthesis of c-FLIPshort

In the early phase of an immune response, T cells are activated and acquire effector functions. Whereas these short term activated T cells are resistant to CD95-mediated apoptosis, activated T cells in prolonged culture are readily sensitive, leading to activation-induced cell death and termination of the immune response. The translation inhibitor, cycloheximide, partially overcomes the apoptosis resistance of short term activated primary human T cells. Using this model we show in this study that sensitization of T cells to apoptosis occurs upstream of mitochondria. Neither death-inducing signaling complex formation nor expression of Bcl-2 proteins is altered in sensitized T cells. Although the caspase-8 inhibitor c-FLIP(long) was only slightly down-regulated in sensitized T cells, c-FLIP(short) became almost undetectable. This correlated with caspase-8 activation and apoptosis. These data suggest that c-FLIP(short), rather than c-FLIP(long), confers resistance of T cells to CD95-mediated apoptosis in the context of immune responses.     

Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model

The CD95 (Fas/APO-1) death-inducing signaling complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at?the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the?stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC.     

Receptor-mediated endocytosis is not required for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.     

CD95 (APO-1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway

CD95 (APO-1/Fas) is a member of the tumor necrosis factor receptor family, which can trigger apoptosis in a variety of cell types. However, little is known of the mechanisms underlying cell susceptibility to CD95-mediated apoptosis. Here we show that human T cells that are susceptible to CD95-mediated apoptosis, exhibit a constitutive polarized morphology, and that CD95 colocalizes with ezrin at the site of cellular polarization. In fact, CD95 co-immunoprecipitates with ezrin exclusively in lymphoblastoid CD4(+) T cells and primary long-term activated T lymphocytes, which are prone to CD95-mediated apoptosis, but not in short-term activated T lymphocytes, which are refractory to the same stimuli, even expressing equal levels of CD95 on the cell membrane. Pre-treatment with ezrin antisense oligonucleotides specifically protected from the CD95-mediated apoptosis. Moreover, we show that the actin cytoskeleton integrity is essential for this function. These findings strongly suggest that the CD95 cell membrane polarization, through an ezrin-mediated association with the actin cytoskeleton, is a key intracellular mechanism in rendering human T lymphocytes susceptible to the CD95-mediated apoptosis.     

Loss of caspase-9 provides genetic evidence for the type I/II concept of CD95-mediated apoptosis

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.     

Regulation of CD95/Fas signaling at the DISC

CD95 (APO-1/Fas) is a member of the death receptor (DR) family. Stimulation of CD95 leads to induction of apoptotic and non-apoptotic signaling pathways. The formation of the CD95 death-inducing signaling complex (DISC) is the initial step of CD95 signaling. Activation of procaspase-8 at the DISC leads to the induction of DR-mediated apoptosis. The activation of procaspase-8 is blocked by cellular FLICE-inhibitory proteins (c-FLIP). This review is focused on the role in the CD95-mediated signaling of the death effector domain-containing proteins procaspase-8 and c-FLIP. We discuss how dynamic cross-talk between procaspase-8 and c-FLIP at the DISC regulates life/death decisions at CD95.     

GDP-mannose-4,6-dehydratase (GMDS) deficiency renders colon cancer cells resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor- and CD95-mediated apoptosis by inhibiting complex II formation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through binding to TRAIL receptors, death receptor 4 (DR4), and DR5. TRAIL has potential therapeutic value against cancer because of its selective cytotoxic effects on several transformed cell types. Fucosylation of proteins and lipids on the cell surface is a very important posttranslational modification that is involved in many cellular events. Recently, we found that a deficiency in GDP-mannose-4,6-dehydratase (GMDS) rendered colon cancer cells resistant to TRAIL-induced apoptosis, resulting in tumor development and metastasis by escape from tumor immune surveillance. GMDS is an indispensable regulator of cellular fucosylation. In this study, we investigated the molecular mechanism of inhibition of TRAIL signaling by GMDS deficiency. DR4, but not DR5, was found to be fucosylated; however, GMDS deficiency inhibited both DR4- and DR5-mediated apoptosis despite the absence of fucosylation on DR5. In addition, GMDS deficiency also inhibited CD95-mediated apoptosis but not the intrinsic apoptosis pathway induced by anti-cancer drugs. Binding of TRAIL and CD95 ligand to their cognate receptors primarily leads to formation of a complex comprising the receptor, FADD, and caspase-8, referred to as the death-inducing signaling complex (DISC). GMDS deficiency did not affect formation of the primary DISC or recruitment to and activation of caspase-8 on the DISC. However, formation of secondary FADD-dependent complex II, comprising caspase-8 and cFLIP, was significantly inhibited by GMDS deficiency. These results indicate that GMDS regulates the formation of secondary complex II from the primary DISC independent of direct fucosylation of death receptors.     

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

The involvement of the death adaptor protein FADD and the apoptosis-initiating caspase-8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death-inducing signalling complex (DISC) that forms upon ligand-induced receptor cross-linking. However, the role of caspase-10, the other death-effector-domain-containing caspase besides caspase-8, in death receptor signalling has been controversial. Here we show that caspase-10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC, and that FADD is necessary for its recruitment to and activation at these two protein complexes. With respect to the function of caspase-10, we show that it is not required for apoptosis induction. In addition, caspase-10 can not substitute for caspase-8, as the defect in apoptosis induction observed in caspase-8-deficient cells could not be rescued by overexpression of caspase-10. Finally, we demonstrate that caspase-10 is cleaved during CD95-induced apoptosis of activated T cells. These results show that caspase-10 activation occurs in primary cells, but that its function differs from that of caspase-8.