The stressosome,a caspase-8-activating signalling complex assembled in response to cell stress in an ATG5-mediated manner

Stress-induced apoptosis is mediated primarily through the intrinsic pathway that involves caspase-9. We previously reported that in caspase-9-deficient cells, a protein complex containing ATG5 and Fas-associated death domain (FADD) facilitated caspase-8 activation and cell death in response to endoplasmic reticulum (ER) stress. Here, we investigated whether this complex could be activated by other forms of cell stress. We show that diverse stress stimuli, including etoposide, brefeldin A and paclitaxel, as well as heat stress and gamma-irradiation, caused formation of a complex containing ATG5-ATG12, FADD and caspase-8 leading to activation of downstream caspases in caspase-9-deficient cells. We termed this complex the ‘stressosome’. However, in these cells, only ER stress and heat shock led to stressosome-dependent cell death. Using in silico molecular modelling, we propose the structure of the stressosome complex, with FADD acting as an adaptor protein, interacting with pro-caspase-8 through their respective death effector domains (DEDs) and interacting with ATG5-ATG12 through its death domain (DD). This suggests that the complex could be regulated by cellular FADD-like interleukin-1β-converting enzyme–inhibitory protein (cFLIP_L), which was confirmed experimentally. This study provides strong evidence for an alternative mechanism of caspase-8 activation involving the stressosome complex.     

A cross talk between cellular signalling and cellular redox state during heat-induced apoptosis in a rat histiocytoma

Increasing evidence provides support for oxidative stress to be closely linked to apoptosis. Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. Though heat shock is a universal phenomenon, BC-8, a macrophage-like cell line failed to mount a typical heat shock response. In the absence of heat shock proteins and functional p53, BC-8 cells undergo apoptosis through CD95 signaling. In the present study, we have investigated the role of ROS in the regulation of apoptosis in these cells. We show that cells transfected with hsp70 and functional p53 are resistant to heat-induced apoptosis through inhibition of CD95 expression and ROS induction. Furthermore, apoptosis in BC-8 cells resulted in two bursts of ROS generation, one correlated with heat stress and intracellular depletion of GSH and the other with Bax overexpression and cytochrome c release. Antioxidants could not protect these cells from heat-induced apoptosis and the death pathway seems to be dependent on initial signaling cascade subsequently altering the intracellular redox. Hence, our data suggest that ROS generation in BC-8 cells upon heat shock is facultative but not obligatory for apoptosis.     

The small heat shock protein alpha B-crystallin negatively regulates cytochrome c- and caspase-8-dependent activation of caspase-3 by inhibiting its autoproteolytic maturation

Caspases are universal effectors of apoptosis. The mitochondrial and death receptor pathways activate distinct apical caspases (caspase-9 and -8, respectively) that converge on the proteolytic activation of the downstream executioner caspase-3. Caspase-9 and -8 cleave procaspase-3 to produce a p24 processing intermediate (composed of its prodomain and large subunit), which then undergoes autoproteolytic cleavage to remove the prodomain from the active protease. Recently, several heat shock proteins have been shown to selectively inhibit the mitochondrial apoptotic pathway by disrupting the activation of caspase-9 downstream of cytochrome c release. We report here that the small heat shock protein alphaB-crystallin inhibits both the mitochondrial and death receptor pathways. In S-100 cytosolic extracts treated with cytochrome c/dATP or caspase-8, alphaB-crystallin inhibits the autoproteolytic maturation of the p24 partially processed caspase-3 intermediate. In contrast, neither the closely related small heat shock protein family member Hsp27 nor Hsp70 inhibited the maturation of the p24 intermediate. We also demonstrate that alphaB-crystallin co-immunoprecipitates with the p24 partially processed caspase-3 in vivo. Taken together, our results demonstrate that alphaB-crystallin is a novel negative regulator of apoptosis that acts distally in the conserved cell death machinery by inhibiting the autocatalytic maturation of caspase-3.     

Heat shock induces apoptosis independently of any known initiator caspase-activating complex

Adaptive responses to mild heat shock are among the most widely conserved and studied in nature. More intense heat shock, however, induces apoptosis through mechanisms that remain largely unknown. Herein, we present evidence that heat shock activates an apical protease that stimulates mitochondrial outer membrane permeabilization and processing of the effector caspase-3 in a benzyloxycarbonyl-VAD-fluoromethyl ketone (polycaspase inhibitor)- and Bcl-2-inhibitable manner. Surprisingly, however, neither FADD.caspase-8 nor RAIDD.caspase-2 PIDDosome (p53-induced protein with a death domain) complexes were detected in dying cells, and neither of these initiator caspases nor the endoplasmic reticulum stress-activated caspases-4/12 were required for mitochondrial outer membrane permeabilization. Similarly, although cytochrome c was released from mitochondria following heat shock, functional Apaf-1.caspase-9 apoptosome complexes were not formed, and caspase-9 was not essential for the activation of caspase-3 or the induction of apoptosis. Thus, heat shock does not require any of the known initiator caspases or their activating complexes to promote apoptotic cell death but instead relies upon the activation of an apparently novel apical protease with caspase-like activity.     

Caspases activation in hyperthermia-induced stimulation of TRAIL apoptosis

In leukemia cells, hyperthermia enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. The phenomenon is caspase-dependent and results in membrane changes leading to an increased recognition of TRAIL death receptors by TRAIL. Because either caspase-2 or an apical proteolytic event has been recently proposed to act as an initiator of the cell death mechanism induced by heat shock, we have investigated the hierarchy of caspase activation in cells exposed to the combined heat shock plus TRAIL treatment. We report here that caspases-2, -3, and -8 were the first caspases to be activated. As expected, caspase-8 is required and indispensable during the initiation of this death signaling. Caspase-2 may also participate in the phenomenon but, in contrast to caspase-8, its presence appears dispensable because its depletion by small interfering RNA is devoid of effects. Our observations also suggest a role of caspase-3 and of a particular cleaved form of this caspase during the early signals of heat shock plus TRAIL-induced apoptosis.     

Fever-like hyperthermia controls T Lymphocyte persistence by inducing degradation of cellular FLIPshort

Fever has a major impact on immune responses by modulating survival, proliferation, and endurance of lymphocytes. Lymphocyte persistence in turn is determined by the equilibrium between death and survival-promoting factors that regulate death receptor signaling in these cells. A potential integrator of death receptor signaling is the caspase-8 inhibitor c-FLIP, the expression of which is dynamically regulated, either rapidly induced or down-regulated. In this study, we show in activated primary human T lymphocytes that hyperthermia corresponding to fever triggered down-regulation of both c-FLIP-splicing variants, c-FLIPshort (c-FLIP(S)) and c-FLIPlong, with consequent sensitization to apoptosis mediated by CD95 (Fas/APO-1). The c-FLIP down-regulation and subsequent sensitization was specific for hyperthermic stress. Additionally, we show that the hyperthermia-mediated down-regulation was due to increased ubiquitination and proteasomal degradation of c-FLIP(S), the stability of which we have shown to be regulated by its C-terminal splicing tail. Furthermore, the induced sensitivity to CD95 ligation was independent of heat shock protein 70, as thermotolerant cells, expressing substantially elevated levels of heat shock protein 70, were not rescued from the effect of hyperthermia-mediated c-FLIP down-regulation. Our findings indicate that fever significantly influences the rate of lymphocyte elimination through depletion of c-FLIP(S). Such a general regulatory mechanism for lymphocyte removal has broad ramifications for fever-mediated regulation of immune responses.     

In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock-induced apoptosis

Activation of 'initiator' (or 'apical') caspases-2, -8 or -9 (refs 1-3) is crucial for induction of apoptosis. These caspases function to activate executioner caspapses that, in turn, orchestrate apoptotic cell death. Here, we show that a cell-permeable, biotinylated pan-caspase inhibitor (bVAD-fmk) both inhibited and 'trapped' the apical caspase activated when apoptosis was triggered. As expected, only caspase-8 was trapped in response to ligation of death receptors, whereas only caspase-9 was trapped in response to a variety of other apoptosis-inducing agents. Caspase-2 was exclusively activated in heat shock-induced apoptosis. This activation of caspase-2 was also observed in cells protected from heat-shock-induced apoptosis by Bcl-2 or Bcl-xL. Reduced sensitivity to heat-shock-induced death was observed in caspase-2(-/-) cells. Furthermore, cells lacking the adapter molecule RAIDD failed to activate caspase-2 after heat shock treatment and showed resistance to apoptosis in this setting. This approach unambiguously identifies the apical caspase activated in response to apoptotic stimuli, and establishes caspase-2 as a proximal mediator of heat shock-induced apoptosis.     

Caspase-10 sensitizes breast carcinoma cells to TRAIL-induced but not tumor necrosis factor-induced apoptosis in a caspase-3-dependent manner

Although signaling by death receptors involves the recruitment of common components into their death-inducing signaling complexes (DISCs), apoptosis susceptibility of various tumor cells to each individual receptor differs quite dramatically. Recently it was shown that, besides caspase-8, caspase-10 is also recruited to the DISCs, but its function in death receptor signaling remains unknown. Here we show that expression of caspase-10 sensitizes MCF-7 breast carcinoma cells to TRAIL- but not tumor necrosis factor (TNF)-induced apoptosis. This sensitization is most obvious at low TRAIL concentrations or when apoptosis is assessed at early time points. Caspase-10-mediated sensitization for TRAIL-induced apoptosis appears to be dependent on caspase-3, as expression of caspase-10 in MCF-7/casp-3 cells but not in caspase-3-deficient MCF-7 cells overcomes TRAIL resistance. Interestingly, neutralization of TRAIL receptor 2 (TRAIL-R2), but not TRAIL-R1, impaired apoptosis in a caspase-10-dependent manner, indicating that caspase-10 enhances TRAIL-R2-induced cell death. Furthermore, whereas processing of caspase-10 was delayed in TNF-treated cells, TRAIL triggered a very rapid activation of caspase-10 and -3. Therefore, we propose a model in which caspase-10 is a crucial component during TRAIL-mediated apoptosis that in addition actively requires caspase-3. This might be especially important in systems where only low TRAIL concentrations are supplied that are not sufficient for the fast recruitment of caspase-8 to the DISC.     

Activation of the heat shock response in a primary cellular model of motoneuron neurodegeneration-evidence for neuroprotective and neurotoxic effects

Pharmacological up-regulation of heat shock proteins (hsps) rescues motoneurons from cell death in a mouse model of amyotrophic lateral sclerosis. However, the relationship between increased hsp expression and neuronal survival is not straightforward. Here we examined the effects of two pharmacological agents that induce the heat shock response via activation of HSF-1, on stressed primary motoneurons in culture. Although both arimoclomol and celastrol induced the expression of Hsp70, their effects on primary motoneurons in culture were significantly different. Whereas arimoclomol had survival-promoting effects, rescuing motoneurons from staurosporin and H₂O₂ induced apoptosis, celastrol not only failed to protect stressed motoneurons from apoptosis under same experimental conditions, but was neurotoxic and induced neuronal death. Immunostaining of celastrol-treated cultures for hsp70 and activated caspase-3 revealed that celastrol treatment activates both the heat shock response and the apoptotic cell death cascade. These results indicate that not all agents that activate the heat shock response will necessarily be neuroprotective.     

Nuclear clusterin accumulation during heat shock response: implications for cell survival and thermo-tolerance induction in immortalized and prostate cancer cells

Clusterin (CLU), whose role is still debated, is differentially regulated in several patho-physiological processes and invariably induced during apoptosis. In heat shock response, CLU is considered a stress-inducible, pro-survival/cyto-protective factor via an HSE element present in his promoter. In both human prostate PNT1A and PC-3 epithelial cells we found that apoptotic stimuli induced nuclear localization of CLU (nCLU), and that overexpression of nCLU is pro-apoptotic. We show here that CLU time-course accumulation kinetic is different from that of HSP70 in these cells, thus other factor(s) might mediate HSF-1 activation and CLU expression. Sub-lethal heat shock inhibited the secretion of CLU (sCLU), leading to increased cytoplasm accumulation of CLU (cCLU) in association to cell survival. At difference, lethal heat stress caused massive accumulation of pro-apoptotic nCLU in cells dying by caspase-3-dependent apoptosis. Double heat stress (sub-lethal heat shock followed by recovery and lethal stress) induced HSP70 and thermo-tolerance in PNT1A cells, but not in PC-3 cells. In PNT1A cells, CLU secretion was inhibited and cCLU was accumulated, suggesting that cCLU might be pro-survival, while in PC-3 cells accumulation of nCLU was concomitant to caspase-3 induction and PARP activation instead. Thus, CLU expression/sub-cellular localization is strictly related to cell fate. In particular, nCLU and physiological levels of HSP70 affected cell survival in an antagonistic fashion. Prevalence of heat-induced nCLU, not allowing PC-3 cells to cope with heat shock, could be the rational explaining why malignant cells are more sensitive to heat when delivered by minimally invasive procedures for ablation of localized prostate cancer.     

Hsp27 inhibits 6-hydroxydopamine-induced cytochrome c release and apoptosis in PC12 cells

Cellular stress may stimulate cell survival pathways or cell death depending on its severity. 6-Hydroxydopamine (6-OHDA) is a neurotoxin that targets dopaminergic neurons that is often used to induce neuronal cell death in models of Parkinson's disease. Here we present evidence that 6-OHDA induces apoptosis in rat PC12 cells that involves release of cytochrome c and Smac/Diablo from mitochondria, caspase-3 activation, cleavage of PARP, and nuclear condensation. 6-OHDA also induced the heat shock response, leading to increased levels of Hsp25 and Hsp70. Increased Hsp25 expression was associated with cell survival. Prior heat shock or overexpression of Hsp27 (human homologue of Hsp25) delayed cytochrome c release, caspase activation, and reduced the level of apoptosis caused by 6-OHDA. We conclude that 6-OHDA induces a variety of responses in cultured PC12 cells ranging from cell survival to apoptosis, and that induction of stress proteins such as Hsp25 may protect cells from undergoing 6-OHDA-induced apoptosis.     

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.     

Heat induced expression of CD95 and its correlation with the activation of apoptosis upon heat shock in rat histiocytic tumor cells

The heat shock response is a universal phenomenon and is among the most highly conserved cellular responses. However, BC-8, a rat histiocytoma, fails to mount a heat shock response unlike all other eukaryotic cells. In the absence of induction of heat shock proteins, apoptotic cell death is activated in BC-8 tumor cells upon heat shock. We demonstrate here that stable transformants of BC-8 tumor cells transfected with hsp70 cDNA constitutively express hsp70 protein and are transiently protected from heat induced apoptosis for 6-8 h. In addition heat stress induces CD95 gene expression in these tumor cells. There is a delay in CD95 expression in hsp70 transfected cells suggesting a correlation between the cell surface expression of CD95 and the time of induction of apoptosis in this tumor cell line. Also expression of CD95 antigen appears to inhibit the interaction between heat shock factors and heat shock elements in these cells resulting in the lack of heat shock response.     

Herpes simplex virus 1 blocks caspase-3-independent and caspase-dependent pathways to cell death

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type HSV blocks the execution of the cell death program triggered by viral gene products, by the effectors of the immune system such as the Fas and tumor necrosis factor pathways, or by nonspecific stress agents such as either osmotic shock induced by sorbitol or thermal shock. A report from this laboratory showed that caspase inhibitors do not block DNA fragmentation induced by infection with the HSV-1 d120 mutant. To identify the events in programmed cell death induced and blocked by HSV-1, we examined cells infected with wild-type virus or the d120 mutant or cells infected and exposed to sorbitol. We report that: (i) the HSV-1 d120 mutant induced apoptosis by a caspase-3-independent pathway inasmuch as caspase 3 was not activated and DNA fragmentation was not blocked by caspase inhibitors even though the virus caused cytochrome c release and depolarization of the inner mitochondrial membrane. (ii) Cells infected with wild-type HSV-1 exhibited none of the manifestations associated with programmed cell death assayed in these studies. (iii) Uninfected cells exposed to osmotic shock succumbed to caspase-dependent apoptosis inasmuch as cytochrome c was released, the inner mitochondrial potential was lost, caspase-3 was activated, and chromosomal DNA was fragmented. (iv) Although caspase-3 was activated in cells infected with wild-type HSV-1 and exposed to sorbitol, cytochrome c outflow, depolarization of the inner mitochondrial membrane, and DNA fragmentation were blocked. We conclude that although d120 induces apoptosis by a caspase-3-independent pathway, the wild-type virus blocks apoptosis induced by this pathway and also blocks the caspase-dependent pathway induced by osmotic shock. The block in the caspase-dependent pathway may occur downstream of caspase-3 activation.     

The c-FLIPL Cleavage Product p43FLIP Promotes Activation of Extracellular Signal-regulated Kinase (ERK), Nuclear Factor κB (NF-κB), and Caspase-8 and T Cell Survival

Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIP_L can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIP_L. This triggers cleavage of c-FLIP_L at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.     

Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation

Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-overexpressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitro with purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation.     

IL-15 maintains T-cell survival via S-nitrosylation-mediated inhibition of caspase-3

Caspase activity is critical for both T-cell survival and death. However, little is known regarding what determines caspase activity in cycling T cells. Interleukin (IL)-2 and IL-15 confer very different susceptibilities to T-cell death. We therefore considered that IL-2 and IL-15 differentially regulate caspase activity to influence T-cell survival. We observed that IL-2-cultured primary murine effector T cells manifested elevated levels of caspase-3 activity compared with IL-15-cultured T cells. T cell receptor (TCR) restimulation further increased caspase activity and induced considerable cell death in IL-2-cultured T cells, but provoked only a minimal increase of caspase activity and cell death in IL-15-cultured T cells. IL-2 sensitization to cell death was caspase-3 mediated. Interestingly, increased active caspase-3 levels with IL-2 were independent of active initiator caspase-8 and caspase-9 that were similar with IL-2 and IL-15. Rather, caspase-3 activity was inhibited by posttranslational S-nitrosylation in IL-15-cultured T cells, but not in the presence of IL-2. This paralleled increased reactive nitrogen and oxygen species with IL-15 and reduced glycolysis. Taken together, these data suggest that the metabolic state conferred by IL-15 inhibits T-cell apoptosis in part by maintaining low levels of active caspase-3 via S-nitrosylation.