NRAGE,a p75 neurotrophin receptor-interacting protein,induces caspase activation and cell death through a JNK-dependent mitochondrial pathway

The p75 neurotrophin receptor (p75NTR) mediates signaling events leading to activation of the JNK pathway and cell death in a variety of cell types. We recently identified NRAGE, a protein that directly interacts with the p75NTR cytosolic region and facilitates p75NTR-mediated cell death. For the present study, we developed an inducible recombinant NRAGE adenovirus to dissect the mechanism of NRAGE-mediated apoptosis. Induced NRAGE expression resulted in robust activation of the JNK pathway that was not inhibited by the pharmacological mixed lineage kinase (MLK) inhibitor CEP1347. NRAGE induced cytosolic accumulation of cytochrome c, activation of Caspases-3, -9 and -7, and caspase-dependent cell death. Blocking JNK and c-Jun action by overexpression of the JNK-binding domain of JIP1 or dominant-negative c-Jun ablated NRAGE-mediated caspase activation and NRAGE-induced cell death. These findings identify NRAGE as a p75NTR interactor capable of inducing caspase activation and cell death through a JNK-dependent mitochondrial apoptotic pathway.     

Ceramide increases mitochondrial free calcium levels via caspase 8 and Bid: role in initiation of cell death

We investigated how the mitochondrial phase of ceramide-mediated cell death is initiated in nerve growth factor (NGF)-differentiated PC12 cells. We distinguished three independent effects of ceramide: free radical production; a transient increase in cytosolic free calcium; and a long-lasting increase in mitochondrial free calcium. Only the latter led to cell death, which could be prevented by buffering of mitochondrial calcium with the calcium binding protein calbindin D-28K ectopically expressed in mitochondria. We showed that mitochondrial calcium did not increase as a result of the increase in cytosolic free calcium levels. Rather, it appears to derive from the endoplasmic reticulum (ER) since dantrolene, which inhibits release of calcium from ER into cytosol through ryanodine receptors, prevented the increase in cytosolic free calcium but potentiated the increase in mitochondrial free calcium. This suggests that a transfer of calcium occurs directly, or very locally, between the two organelles. This transfer implicated activation of caspase 8 and cleavage of its substrate Bid, a previously unknown function of these cell death intermediaries. The increase in mitochondrial free calcium was also responsible for the release of cytochrome c into the cytosol, underlining the critical role it plays in ceramide-mediated cell death.     

Essential roles of receptor-interacting protein and TRAF2 in oxidative stress-induced cell death

Oxidative stress and reactive oxygen species (ROS) can elicit and modulate various physiological and pathological processes, including cell death. However, the mechanisms controlling ROS-induced cell death are largely unknown. Data from this study suggest that receptor-interacting protein (RIP) and tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2), two key effector molecules of TNF signaling, are essential for ROS-induced cell death. We found that RIP(-/-) or TRAF2(-/-) mouse embryonic fibroblasts (MEF) are resistant to ROS-induced cell death when compared to wild-type cells, and reconstitution of RIP and TRAF2 gene expression in their respective deficient MEF cells restored their sensitivity to H(2)O(2)-induced cell death. We also found that RIP and TRAF2 form a complex upon H(2)O(2) exposure, but without the participation of TNFR1. The colocalization of RIP with a membrane lipid raft marker revealed a possible role of lipid rafts in the transduction of cell death signal initiated by H(2)O(2). Finally, our results demonstrate that activation of c-Jun NH(2)-terminal kinase 1 is a critical event downstream of RIP and TRAF2 in mediating ROS-induced cell death. Therefore, our study uncovers a novel signaling pathway regulating oxidative stress-induced cell death.     

Developmental differences in HO-induced oligodendrocyte cell death: role of glutathione, mitogen-activated protein kinases and caspase 3

The molecular mechanisms underlying H(2)O(2)-induced toxicity were characterized in rat oligodendrocyte cultures. While progenitor cells were more sensitive than mature oligodendrocytes to H(2)O(2), the antioxidant, N-acetyl-L-cysteine, blocked toxicity at both stages of development. Differentiated oligodendrocytes contained more glutathione than did progenitors and were less susceptible to decreases in glutathione concentration induced by H(2)O(2) stress. As free radicals have been considered to serve as second messengers, we examined the effect of H(2)O(2) on activation of the mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinases (ERK) 1/2 and p38. H(2)O(2) caused a time- and concentration-dependent increase in MAPK phosphorylation, an effect that was totally blocked by N-acetyl-L-cysteine. Further exploration of potential mechanisms involved in oligodendrocyte cell death showed that H(2)O(2) treatment caused DNA condensation and fragmentation at both stages of development, whereas caspase 3 activation and poly (ADP-ribose) polymerase cleavage were significantly increased only in oligodendrocyte progenitors. The pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone, blocked DNA fragmentation in progenitors and produced a small but significant level of protection from H(2)O(2) toxicity in progenitors and mature oligodendrocytes. In contrast, inhibitors of both p38 and MEK reduced H(2)O(2)-induced death most significantly in oligodendrocytes. The poly (ADP-ribose) polymerase inhibitor, PJ34, reduced H(2)O(2)-induced toxicity on its own but was most effective when combined with benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone or PD169316. The finding that molecular mechanisms conferring resistance to reactive oxygen species toxicity are regulated during oligodendrocyte differentiation may be of importance in designing therapies for certain neurological diseases affecting white matter.     

The role of zinc in caspase activation and apoptotic cell death

In addition to its diverse role in many physiological systems, zinc (Zn) has now been shown to be an important regulator of apoptosis. The purpose of this review is to integrate previously published knowledge on Zn and apoptosis with current attempts to elucidate the mechanisms of action of this biometal. This paper begins with an introduction to apoptosis and then briefly reviews the evidence relating Zn to apoptosis. The major focus of this review is the mechanistic actions of Zn and its candidate intracellular targets. In particular, we examine the cytoprotective functions of Zn which suppress major pathways leading to apoptosis, as well as the more direct influence of Zn on the apoptotic regulators, especially the caspase family of enzymes. These two mechanisms are closely related since a decline in intracellular Zn below a critical threshold level may not only trigger pathways leading to caspase activation but may also facilitate the process by which the caspases are activated. Studies by our laboratory in airway epithelial cells show that Zn is co-localized with the precursor form of caspase-3, mitochondria and microtubules, suggesting this Zn is critically placed to control apoptosis. Further understanding the different pools of Zn and how they interact with apoptotic pathways should have importance in human disease.     

Calcium binding of ARC mediates regulation of caspase 8 and cell death

Apoptosis repressor with CARD (ARC) possesses the ability not only to block activation of caspase 8 but to modulate caspase-independent mitochondrial events associated with cell death. However, it is not known how ARC modulates both caspase-dependent and caspase-independent cell death. Here, we report that ARC is a Ca(2+)-dependent regulator of caspase 8 and cell death. We found that in Ca(2+) overlay and Stains-all assays, ARC protein bound to Ca(2+) through the C-terminal proline/glutamate-rich (P/E-rich) domain. ARC expression reduced not only cytosolic Ca(2+) transients but also cytotoxic effects of thapsigargin, A23187, and ionomycin, for which the Ca(2+)-binding domain of ARC was indispensable. Conversely, direct interference of endogenous ARC synthesis by targeting ARC enhanced such Ca(2+)-mediated cell death. In addition, binding and immunoprecipitation analyses revealed that the protein-protein interaction between ARC and caspase 8 was decreased by the increase of Ca(2+) concentration in vitro and by the treatment of HEK293 cells with thapsigargin in vivo. Caspase 8 activation was also required for the thapsigargin-induced cell death and suppressed by the ectopic expression of ARC. These results suggest that calcium binding mediates regulation of caspase 8 and cell death by ARC.     

Anandamide-induced cell death in primary neuronal cultures: role of calpain and caspase pathways

Anandamide (arachidonoylethanolamide or AEA) is an endocannabinoid that acts at vanilloid (VR1) as well as at cannabinoid (CB1/CB2) and NMDA receptors. Here, we show that AEA, in a dose-dependent manner, causes cell death in cultured rat cortical neurons and cerebellar granule cells. Inhibition of CB1, CB2, VR1 or NMDA receptors by selective antagonists did not reduce AEA neurotoxicity. Anandamide-induced neuronal cell loss was associated with increased intracellular Ca(2+), nuclear condensation and fragmentation, decreases in mitochondrial membrane potential, translocation of cytochrome c, and upregulation of caspase-3-like activity. However, caspase-3, caspase-8 or caspase-9 inhibitors, or blockade of protein synthesis by cycloheximide did not alter anandamide-related cell death. Moreover, AEA caused cell death in caspase-3-deficient MCF-7 cell line and showed similar cytotoxic effects in caspase-9 dominant-negative, caspase-8 dominant-negative or mock-transfected SH-SY5Y neuroblastoma cells. Anandamide upregulated calpain activity in cortical neurons, as revealed by alpha-spectrin cleavage, which was attenuated by the calpain inhibitor calpastatin. Calpain inhibition significantly limited anandamide-induced neuronal loss and associated cytochrome c release. These data indicate that AEA neurotoxicity appears not to be mediated by CB1, CB2, VR1 or NMDA receptors and suggest that calpain activation, rather than intrinsic or extrinsic caspase pathways, may play a critical role in anandamide-induced cell death.     

TRAF6 regulates cell fate decisions by inducing caspase 8-dependent apoptosis and the activation of NF-kappaB

Tumor necrosis factor receptor-associated factor 6 (TRAF6) functions as an adaptor, positively regulating the NF-kappaB pathway. Here we report a new function of human TRAF6, the direct stimulation of apoptosis. The mechanism of apoptosis induction results from the capacity of human TRAF6 to interact and activate caspase 8. Both the C-terminal TRAF domain of human TRAF6, which directly interacts with the death effector domain of pro-caspase 8, and the N-terminal RING domain, which is required for activation of caspase 8, are necessary for the induction of apoptosis. The role of endogenous TRAF6 in regulating apoptosis was confirmed by extinguishing TRAF6 expression with specific small-hairpin RNA that resulted in diminished spontaneous apoptosis and resistance to induced apoptosis. In contrast to the human molecule, murine TRAF6 displayed less ability to induce apoptosis and a greater capacity to stimulate NF-kappaB activity. Human and murine TRAF6 are similar except in the region between zinc finger 5 and the TRAF domains. Reciprocal transfer of this connecting region completely exchanged the ability of human and murine TRAF6 to induce apoptosis and activate NF-kappaB. Unique regions of TRAF6 therefore play an important role in determining cell fate.     

Prolonged activation of ERK1,2 induces FADD-independent caspase 8 activation and cell death

Prolonged ERK/MAPK activation has been implicated in neuronal cell death in vitro and in vivo. We found that HEK293 cells, recently reported to express neuronal markers, are exquisitely sensitive to long term ERK stimulation. Activation of an inducible form of Raf-1 (Raf-1:ER) in HEK293 cells induced massive apoptosis characterized by DNA degradation, loss of plasma membrane integrity and PARP cleavage. Cell death required MEK activity and protein synthesis and occurred via the death receptor pathway independently of the mitochondrial pathway. Accordingly, prolonged ERK stimulation activated caspase 8 and strongly potentiated Fas signaling. The death receptor adaptator FADD was found to be rapidly induced upon ERK activation. However using RNA interference and ectopic expression, we demonstrated that neither FADD nor Fas were necessary for caspase 8 activation and cell death. These findings reveal that prolonged ERK/MAPK stimulation results in caspase 8 activation and cell death. This work was supported by grant from Association pour la Recherche sur le Cancer (CNRS6543/ARC). S. Cagnol is supported by a fellowship from the Ligue Nationale contre le Cancer.     

Serum amyloid A protects murine macrophages from lethal toxin-mediated death

Lethal toxin, a key virulence factor produced by Bacillus anthracis, induces cell death, in part by disrupting numerous signaling pathways, in mouse macrophages. However, exposure to sublethal doses of lethal toxin allows some cells to survive. Because these pro-survival signaling events occur within a few hours after exposure to sublethal doses, we hypothesized that acute phase proteins might influence macrophage survival. Our data show that serum amyloid A (SAA) is produced in response to lethal toxin treatment. Moreover, pre-treatment of macrophages with exogenous SAA protected macrophages from lethal toxin-mediated death. Exogenous SAA activated the p38 mitogen activated protein kinase (MAP) kinase pathway, while lethal toxin mutants incapable of p38 activation were incapable of causing cell death. Chemical inhibition of the p38 activation pathway abrogated the protective effects of SAA. These data show that SAA affords protection against lethal toxin in mouse macrophages and link this response to the p38 pathway.     

It cuts both ways: reconciling the dual roles of caspase 8 in cell death and survival

Caspase 8 can initiate apoptosis, but it also has non-apoptotic roles; for example, it is required for embryonic development and immune cell proliferation. Recent work has indicated that the requirement for caspase 8 in development and immune cell proliferation is defined by suppression of receptor-interacting protein kinase 3 (RIPK3), a kinase that triggers an alternative form of cell death called programmed necrosis. Interestingly, these recent findings can be reconciled with earlier work on the non-apoptotic roles of caspase 8.     

Ras protects Rb family null fibroblasts from cell death: a role for AP-1

The retinoblastoma protein (Rb) controls cell proliferation, differentiation, and senescence and provides an essential tumor suppressive function that cells must eliminate to attain unlimited proliferative potential. Elimination of the Rb pathway also results in apoptosis, however, thereby providing an efficient surveillance mechanism to sense the loss of Rb. To become tumorigenic cells must thus overcome not only Rb function but also the apoptotic response caused by the loss of Rb function. We show that oncogenic Ras (RasV12) potently blocks cell death in Rb family member knockout mouse embryo fibroblasts (TKO cells). Activation of phosphatidylinositol 3-kinase and Raf by oncogenic Ras mediated this protection, implying that multiple Ras effector pathways are required, in concert, for this pro-survival signal. Although activation of Raf by selective Ras mutants protected TKO cells from cell death, pharmacologic inhibition of MEK had little effect on RasV12 protection, suggesting that a Raf-dependent, MEK-independent pathway was important for this effect. We show that this Raf-dependent protection occurred through activation of c-Jun and thus AP-1 activation. These observations could account for the dependence of Ras transformation on c-Jun activity and for the roles of AP-1 in oncogenesis. Our results support the concept of two oncogenic events cooperating to achieve a balance between immortalization and survival.     

Apoptosis repressor with caspase recruitment domain protects against cell death by interfering with Bax activation

Myocardial ischemia/reperfusion (I/R) is associated with an extensive loss of myocardial cells. The apoptosis repressor with caspase recruitment domain (ARC) is a protein that is highly expressed in heart and skeletal muscle and has been demonstrated to protect the heart against I/R injury (Gustafsson, A. B., Sayen, M. R., Williams, S. D., Crow, M. T., and Gottlieb, R. A. (2002) Circulation 106, 735-739). In this study, we have shown that transduction of TAT-ARCL31F, a mutant of ARC in the caspase recruitment domain, did not reduce creatine kinase release and infarct size after I/R. TAT-ARCL31F also failed to protect against hydrogen peroxide-mediated cell death in H9c2 cells, suggesting that the caspase recruitment domain is important in mediating ARC's protective effects. In addition, we report that ARC co-immunoprecipitated with the pro-apoptotic protein Bax, which causes cytochrome c release when activated. TAT-ARC, but not TAT-ARCL31F, prevented Bax activation and cytochrome c release in hydrogen peroxide-treated H9c2 cells. TAT-ARC was also effective in blocking cytochrome c release after ischemia and reperfusion, whereas TAT-ARCL31F had no effect on cytochrome c release. In addition, recombinant ARC protein abrogated Bax-induced cytochrome c release from isolated mitochondria. This suggests that ARC can protect against cell death by interfering with activation of the mitochondrial death pathway through the interaction with Bax, preventing mitochondrial dysfunction and release of pro-apoptotic factors.     

Bacterial GroEL-like heat shock protein 60 protects epithelial cells from stress-induced death through activation of ERK and inhibition of caspase 3

Bacterial heat shock proteins (hsps) can have various effects on human cells. We investigated whether bacterial hsp60s can protect epithelial cells from cell death by affecting the mitogen-activated protein kinase (MAPK) signal pathways. Cell protection was studied by adding bacterial hsp60s to skin keratinocyte cultures (HaCaT cell line) before UV radiation. The results show that hsp60 significantly protected against UV radiation-induced cell death. Effects of UV radiation and exogenous hsp60 on phosphorylation of MAPKs and on activation of caspase 3 were examined by Western blot analysis. UV radiation strongly induced phosphorylation of p38 MAPK and formation of active caspase 3. A p38 inhibitor, SB 203580, totally blocked UV radiation-mediated activation of caspase 3. Preincubation with hsp60 strongly induced phosphorylation of ERK1/2 and inhibited UV radiation-mediated activation of caspase 3. PD 98059, a specific inhibitor of the ERK1/2 pathway, blocked this inhibitory effect of exogenous hsp60. Studies on the association between activity of MAPKs or caspase 3 and cell death showed that the ERK1/2 pathway inhibitor reversed protective effect of hsp60 while specific inhibition of p38 and caspase 3 reduced cell death. These results indicate that in HaCaT cells UV radiation mediates cell death through activation of p38 followed by caspase 3 activation. Exogenous hsp60 partially protects against UV radiation-mediated epithelial cell death through activation of ERK1/2, which inhibits caspase 3 activation.     

The essential role of the death domain kinase receptor-interacting protein in insulin growth factor-I-induced c-Jun N-terminal kinase activation

Insulin-like growth factor I (IGF-I) plays an important role in cell survival, proliferation, and differentiation. Diverse kinases, including AKT/protein kinase B, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), can be activated by IGF-I. Here, we show that the receptor-interacting protein (RIP), a key mediator of tumor necrosis factor-induced NF-kappaB and JNK activation, plays a key role in IGF-I receptor signaling. IGF-I induced a robust JNK activation in wild type but not RIP null (RIP-/-) mouse embryonic fibroblast cells. Reconstitution of RIP expression in the RIP-/- cells restored the induction of JNK by IGF-I, suggesting that RIP is essential in IGF-I-induced JNK activation. Reconstitution experiments with different RIP mutants further revealed that the death domain and the kinase activity of RIP are not required for IGF-I-induced JNK activation. Interestingly, the AKT and ERK activation by IGF-I was normal in RIP-/- cells. The phosphatidylinositol 3-kinase inhibitor, wortmannin, did not affect IGF-I-induced JNK activation. These results agree with previous studies showing that the IGF-I-induced JNK activation pathway is distinct from that of ERK and AKT activation. Additionally, physical interaction of ectopically expressed RIP and IGF-IRbeta was detected by co-immunoprecipitation assays. More importantly, RIP was recruited to the IGF-I receptor complex during IGF-I-induced signaling. Furthermore, we found that IGF-I-induced cell proliferation was impaired in RIP-/- cells. Taken together, our results indicate that RIP, a key factor in tumor necrosis factor signaling, also plays a pivotal role in IGF-I-induced JNK activation and cell proliferation.     

Necdin protects embryonic motoneurons from programmed cell death

NECDIN belongs to the type II Melanoma Associated Antigen Gene Expression gene family and is located in the Prader-Willi Syndrome (PWS) critical region. Necdin-deficient mice develop symptoms of PWS, including a sensory and motor deficit. However, the mechanisms underlying the motor deficit remain elusive. Here, we show that the genetic ablation of Necdin, whose expression is restricted to post-mitotic neurons in the spinal cord during development, leads to a loss of 31% of specified motoneurons. The increased neuronal loss occurs during the period of naturally-occurring cell death and is not confined to specific pools of motoneurons. To better understand the role of Necdin during the period of programmed cell death of motoneurons we used embryonic spinal cord explants and primary motoneuron cultures from Necdin-deficient mice. Interestingly, while Necdin-deficient motoneurons present the same survival response to neurotrophic factors, we demonstrate that deletion of Necdin leads to an increased susceptibility of motoneurons to neurotrophic factor deprivation. We show that by neutralizing TNFα this increased susceptibility of Necdin-deficient motoneurons to trophic factor deprivation can be reduced to the normal level. We propose that Necdin is implicated through the TNF-receptor 1 pathway in the developmental death of motoneurons.     

Vanadyl bisacetylacetonate protects β cells from palmitate-induced cell death through the unfolded protein response pathway

Endoplasmic reticulum (ER) stress induced by free fatty acids (FFA) is important to β-cell loss during the development of type 2 diabetes. To test whether vanadium compounds could influence ER stress and the responses in their mechanism of antidiabetic effects, we investigated the effects and the mechanism of vanadyl bisacetylacetonate [VO(acac)₂] on β cells upon treatment with palmitate, a typical saturated FFA. The experimental results showed that VO(acac)₂ could enhance FFA-induced signaling pathways of unfolded protein responses by upregulating the prosurvival chaperone immunoglobulin heavy-chain binding protein/78-kDa glucose-regulated protein and downregulating the expression of apoptotic C/EBP homologous protein, and consequently the reduction of insulin synthesis. VO(acac)₂ also ameliorated FFA-disturbed Ca²⁺ homeostasis in β cells. Overall, VO(acac)₂ enhanced stress adaption, thus protecting β cells from palmitate-induced apoptosis. This study provides some new insights into the mechanisms of antidiabetic vanadium compounds.