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PTPC-dependent apoptotic signaling between ER and mitochondria in response to ER stress. Experimentally induced ER stress (A23187, TG, TN) leads to IP3R-mediated release of ER lumenal Ca 2+ , mitochondrial accumulation through VDAC and Ca 2+ -dependent opening of PTPC. The opening of PTPC then promotes PMM, release of apoptogenic factors and subsequent nuclear apoptosis. The proteins involved in the apoptotic pathway triggered by ER stress was determined by pharmacological studies using inhibitors of IP3R (2-APB, 2-AminoethoxydiPhenyl Borate), VDAC (DIDS, dihydro-4,4 ′ diisothiocyanostilbene- 2,2 ′ -disulphonic acid and NADH) and PTPC (CsA, cyclosporine A and the protonophore CCCP, carbonyl cyanide 3- chlorophenylhydrazon). The anti-apoptotic protein Bcl-2 was demonstrated to inhibit PTPC opening in response to ER stress, whereas this latter process was favored by the pro-apoptotic protein Bax.
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The ndoplasmic reticulum (ER) function is critical for multiple cellular activities. Hence, impairment of the physiological function of the ER, such as accumulation of unfolded proteins or disturbance of lumenal calcium homeostasis, leads to an evolutionarily conserved adaptive response known as the ER stress response. Activation of this self-prote...
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... quenched by the Co 2+ , a phenomenon that can be recorded by flow cytometry (Figure 2). We observed that ER stress agents, which generate a sustained accumulation of Ca 2+ into mitochondria, led to PTPC opening, MMP and subsequent nuclear apoptosis, whereas physiological stimuli such as the IP3-mobilizing agent histamine, which triggered only a transient increase in [Ca 2+ ] m , did not cause mitochondrial alterations nor apoptosis (150). Thus, PTPC appears to play a key role in the Ca 2+ -dependent apoptotic communication between ER and mitochondria during ER stress. By taking advantage of cell lines overexpressing Bcl-2 or vMIA (viral mitochondrial inhibitor of apoptosis, encoded by the Cytomegalovirus UL37 gene) or knock down for Bax or Bak, we demonstrated that (i) Bcl-2 and vMIA prevent ER stress- mediated PTPC opening and (ii) Bax invalidation decreased MPT, whereas Bak invalidation did not modify the response to ER stressors (150). Accordingly, a protection against ER stress-mediated MMP was observed in other cell systems after overproduction of Bcl-x L or vMIA (144). The protective effect of vMIA may rely on its ability to interact with the PTPC member ANT (but not VDAC) (159) or to sequester Bax in an inactive form at the mitochondria (160, 161). Members of Bcl-2 family are well-known regulators of MMP, which directly interact with mitochondrial membranes to control the release of IMS proteins (162). In the apoptotic signaling pathway triggers by ER-dependent Ca 2+ mobilization, Bcl-2 family proteins are thought to regulate cell death not by acting on mitochondria but through their localization at the ER. Indeed, Bcl-2 ER seems to protect cell against Ca 2+ -mediated MMP by reducing the amount of ER releasable Ca 2+ (163, 164) and/or by functionally interacting with IP3R (165, 166). Conversely, Bax ER could sensitize cells to ER stress by increasing the pool of Ca 2+ releasable from the ER (167, 168), probably by regulating the phosphorylation state of IP3R (169). A pharmacological approach allowed us to better define the molecular mechanisms of Ca 2+ transfer and the role of PTPC in the lethal dialogue between ER and mitochondria in whole cells. In addition, to study these processes free from all of the complex side reactions that occur in cells, we have developed an original cell-free system in which we can confronted gradient-purified mitochondria and ER vesicles purified by ultracentrifugation (150). These pure organelles can be isolated from mouse liver or brain or from human cancer cell lines. In both whole cells and cell-free system treated with ER stress agents (e.g. TG, TN, A23187), we demonstrated that BAPTA-AM (a Ca 2+ chelator) and 2- APB (2-AminoethoxydiPhenyl Borate (170)), an inhibitor of IP3R, led to partial inhibition of ER stress-mediated Ca 2+ entry in mitochondria and nuclear apoptosis, in accord with the well-known role of IP3R in Ca 2+ -dependent apoptosis (reviewed in (171)). Besides, 2-APB also delays PTPC opening, mitochondrial swelling and dissipation of ∆Ψ m , indicating the important role of IP3R in PTPC-dependent mitochondrial apoptosis triggers by Ca 2+ accumulation into mitochondria. To enter the mitochondrial matrix Ca 2+ moves from the cytosol to the IMS across the OM and then from the IMS to the matrix across the IM. Evidence has been presented that Ca 2+ delivery across IM occurs through the Ca 2+ uniporter, a possibly gated channel, whom molecular identity and nature remain unknown (172, 173). We have seen in the previous section that Ca 2+ can cross the OM through VDAC (111, 112, 174). In the presence of VDAC blockers, such as DIDS (4,4 ′ - Diisothiocyanatostilbene-2,2 ′ -disulfonic acid) and NADH (in the cell-free system), mitochondrial Ca 2+ uptake, PTPC opening, mitochondrial alterations and nuclear apoptosis are significantly reduced. Our results therefore demonstrate that VDAC can efficiently participate in IP3R-released Ca 2+ entry into mitochondria in response to ER stressors TG and TN, in cellulo as well as in cell-free system. By contrast, it appears that Ca 2+ accumulation in mitochondrial matrix does not involved other core proteins of PTPC since inhibitors of ANT (BA, Bongkrekic acid) and cyclophilin D (CsA) were unable to modify mitochondrial Ca 2+ uptake. On the other hand, BA and CsA, which prevent PTPC opening, also reduced mitochondrial swelling, loss of ∆Ψ m and hypoploidy induced by ER toxins, indicating that PTPC is required for ER-stress induced mitochondrial alterations and apoptosis. Along the same lines, knockdown of cyclophilin D by siRNA significantly inhibits Ca 2+ dependent MMP and cytochorme c release provoked by TG treatment of human leukemic cells, corroborating the important role of PTPC members in ER stress-induced apoptosis (175). In summary, when functions (TN) or Ca homeostasis (TG) of the ER is severely perturbed, a stress response is activated and a PTPC-dependent apoptotic dialogue is engaged between ER and mitochondria (Figure 3): Ca 2+ released from the ER through IP3R is taken up by mitochondria via VDAC and/or other OM channels, accumulates then in the matrix where it stimulates the opening of PTPC, which in turn promotes mitochondrial swelling, rupture of the OM and liberation of apoptogenic factors. In conclusion, the data discussed above settled Ca 2+ and PTPC as key components of the lethal crosstalk between ER and mitochondria. Nevertheless, one can not rule out that signaling molecules other than Ca 2+ ion could be released from ER to target PTPC and induce apoptosis. In agreement, we have observed that the ER stressor BFA provokes the opening of PTPC and subsequent mitochondrial apoptosis without inducing Ca 2+ release from ER (Figure 4). The nature of the molecule(s) involved in BFA-mediated lethal communication between ER and mitochondria is still unidentified and requires further investigation. In addition, renewed attention has been recently given to the gangliosides, as intracellular messengers of cell death and potential mediators of apoptosis in response to ER stress (176). For example, among the different gangliosides, GD3, found at the ER- mitochondria contacts sites (177), was demonstrated to promote opening of PTPC in a Ca -independent manner (178) and thus appears as an interesting signaling molecule of the PTPC-mediated ER-mitochondria lethal interplay. However, future research will be fundamental in better understanding the molecular mechanisms that connect PTPC to the apoptotic crosstalk between ER and mitochondria. Our work is supported in part by grants from the Association pour la Recherche contre le Cancer (ARC, #3902), the Institut National du Cancer (INCa) and PRES UniverSud Paris. The authors want to thank Dr Vito De Pinto for kindly providing the VDAC structural model previously published (179). 1. Lin J. H, P. Walter and T. S. Yen: Endoplasmic Reticulum Stress in Disease Pathogenesis. Annu Rev Pathol 3, 399-425 ...
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The function of the endoplasmic reticulum (ER) can be impaired by changes to the extra- and intracellular environment, such as disruption of calcium homeostasis, expression of mutated proteins, and oxidative stress. In response to disruptions to ER homeostasis, eukaryotic cells activate canonical branches of signal transduction cascades, collective...
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... On the other hand, it is known that severe ER stress induces apoptosis through the mitochondrial pathway to attenuate damage to cells [53]. Thus, we evaluated Moe's effect on DEHP-induced mitochondrial alterations. ...
Moringa oleifera (MO) is a medicinal plant that has been shown to possess antioxidant, anticarcinogenic and antibiotic activities. In a rat model, MO extract (MOe) has been shown to have a protective effect against brain damage and memory decline. As an extending study, here, we have examined the protective effect of MOe against oxidative stress and apoptosis caused in human neuroblastome (SH-SY5Y) cells by di-(2-ethylhexyl) phthalate (DEHP), a plasticizer known to induce neurotoxicity. Our data show that MOe prevents oxidative damage by lowering reactive oxygen species (ROS) formation, restoring mitochondrial respiratory chain complex activities, and, in addition, by modulating the expression of vitagenes, i.e., antioxidant proteins Nrf2 and HO-1. Moreover, MOe prevented neuronal damage by partly inhibiting endoplasmic reticulum (ER) stress response, as indicated by decreased expression of CCAAT-enhancer-binding protein homologous protein (CHOP) and Glucose-regulated protein 78 (GRP78) proteins. MOe also protected SH-SY5Y cells from DEHP-induced apoptosis, preserving mitochondrial membrane permeability and caspase-3 activation. Our findings provide insight into understanding of molecular mechanisms involved in neuroprotective effects by MOe against DEHP damage.
... Interestingly, cardiac myocytes also do not express plasmalemmal BK channels, and NS11021 shows therapeutic efficacy against IR injury in these cells and in ex vivo models of cardiac IR [18,[21][22]. Nevertheless, our studies did not rule out the possibility that NS11021 activates BK channels at other non-mitochondrial organelles considering that cross-talk between intracellular organelles is well recognized [69][70][71][72][73][74]. Indeed, BK channels have been discovered in many cellular organelles as is nicely reviewed by Sing et al. [75]. ...
Kidneys from deceased donors used for transplantation are placed in cold storage (CS) solution during the search for a matched recipient. However, CS causes mitochondrial injury, which may exacerbate renal graft dysfunction. Here, we explored whether adding NS11021, an activator of the mitochondrial big-conductance calcium-activated K+ (mitoBK) channel, to CS solution can mitigate CS-induced mitochondrial injury. We used normal rat kidney proximal tubular epithelial (NRK) cells as an in vitro model of renal cold storage (18 h) and rewarming (2 h) (CS + RW). Western blots detected the pore-forming α subunit of the BK channel in mitochondrial fractions from NRK cells. The fluorescent K+-binding probe, PBFI-AM, revealed that isolated mitochondria from NRK cells exhibited mitoBK-mediated K+ uptake, which was impaired ~70% in NRK cells subjected to CS + RW compared to control NRK cells maintained at 37 °C. Importantly, the addition of 1 M NS11021 to CS solution prevented CS + RW-induced impairment of mitoBK-mediated K+ uptake. The NS11021–treated NRK cells also exhibited less cell death and mitochondrial injury after CS + RW, including mitigated mitochondrial respiratory dysfunction, depolarization, and superoxide production. In summary, these new data show for the first time that mitoBK channels may represent a therapeutic target to prevent renal CS-induced injury.
... Furthermore, apoptosis is a programmed cell death that has two pathways (extrinsic and intrinsic), the intrinsic mitochondrial pathway is usually triggered by signals from other organs such as the endoplasmic reticulum (ER) (Sharaf El Dein et al., 2009). Unfolder protein response (UPR) is a mechanism induced by stressed ER, if normal conditions are not restored, ER stress would lead to mitochondrial apoptosis (Hetz, 2012). ...
... Cytosolic cytochrome c binds to apoptotic protease activation factor 1 (Apaf-1), which then initiates cleavage and activation of caspase-9, which subsequently activates the downstream executioner caspases-3, -6 and -7 (Li et al. 1997). The mitochondrial pathway of apoptosis is usually triggered by intracellular stimuli or signals emanating from other organelles such as the endoplasmic reticulum (ER) (Sharaf El Dein et al. 2009). Disruption of ER homeostasis induces ER stress, which subsequently leads to Unfolded Protein Response (UPR), a pro-survival process stimulated to restore normal ER functions. ...
The mycotoxin citrinin (CTN) is a natural contaminant of various human foods that may produce serious adverse health problems. Several studies demonstrated that citrinin exerts cytotoxic and genotoxic effects in both in vivo and in vitro systems. However, the precise mechanisms of action (MOA), particularly in intestinal cells remain unclear. The aim of the present study was to examine the precise MOA of citrinin in vitro. Data demonstrated that CTN significantly decreased the number of viable human intestinal HCT116 cells and induced apoptotic events including (1) decrease in ΔѰm indicative of mitochondrial membrane permeabilization, (2) activation of caspase 3, (3) elevated production of reactive oxygen species (ROS) and (4) relative persistence of plasma membrane integrity. Further, the genetic deficiency of the pro-apoptotic protein Bax protected cells against CTN-induced apoptosis, indicating that Bax is required for CTN-mediated toxicity. It was also found that CTN triggered endoplasmic reticulum (ER) stress and activated different arms of the unfolded protein response (UPR) as demonstrated by increase in expression of GRP78 (glucose-regulated protein-78), GRP94 (glucose-regulated protein-94), GADD34 (growth arrest and DNA damage-inducible protein-34), the protein disulfide isomerase associated 6 (PDIA6), CHOP (C/EBP-homologous protein) and the splicing of XBP1 (X-Box Binding Protein 1). Pretreatment of cells with the chemical chaperone 4-phenylbutyrate (PBA), known to alleviate ER stress, prevented significantly the apoptotic process triggered by CTN. Taken together, these results suggest that CTN exerts its cytotoxic effects in HCT116 cells by inducing apoptosis, at least in part, through induction of ER stress.
... It is well known that ER stress, such as the accumulation of unfolded or misfolded protein, triggers the mitochondrialmediated pathway, the caspase-dependent and the caspaseindependent death pathways [35,36]. Previous studies have shown that ZEA treatment increased the expression of the ER resident chaperones GRP78, ATF4, GADD34 and XBP1 [22,37]. ...
Zearalenone (ZEA) is a contaminant of human food and animal feedstuffs that causes health hazards. However, the signal pathways underlying ZEA toxicity remain elusive. The aims of this study were to determine which pathways are involved in ZEA-induced cell death and investigate the effect of apoptosis inducing factor (AIF) on cell death during ZEA treatment in the immortalized goat Leydig cell line hTERT-GLC. This study showed that ZEA-induced cell death in hTERT-GLCs works via endoplasmic reticulum (ER) stress, the caspase-dependent pathway, the caspase-independent pathway and autophagy. Recombinant lentiviral vectors were constructed to silence AIF expression in hTERT-GLCs. Flow cytometry results showed that knockdown of AIF diminished ZEA-induced cell apoptosis in hTERT-GLCs. Furthermore, we found AIF depletion down-regulated phosphoIRE1α, GRP78, CHOP and promoted the switch of LC3-I to LC3-II. Therefore, ZEA induces cytotoxicity in hTERT-GLCs via different pathways, while AIF-mediated signaling plays a critical role in ZEA-induced cell death in hTERT-GLCs.
... Severe ER stress is known to induce apoptosis through the mitochondrial pathway to eliminate damaged cells (Sharaf el dein et al. 2009). In addition, we have previously reported that α-ZOL and β-ZOL induce cell death by activating the mitochondrial pathway of apoptosis . ...
α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) are the major metabolites of Zearalenone (ZEN) and are known to induce many toxic effects. In the present study, we investigated the involvement of endoplasmic reticulum (ER) stress in α- and β-ZOL-mediated toxicity in human kidney cells (HEK293) and evaluated the effect of a common dietary compound Crocin (CRO), from saffron. We show that α- and β-ZOL treatment induces ER stress as evidenced by the upregulation of the 78 kDa glucose-regulated protein (GRP78) and the Growth arrest and DNA damage-inducible protein (GADD34). Activation of the ER stress response is associated with activation of the mitochondrial pathway of apoptosis. This apoptotic process is characterized by an increase in ROS generation and lipid peroxidation, a loss of mitochondrial transmembrane potential (ΔΨm) and activation of caspases. We also demonstrate that the antioxidant properties of CRO help to prevent ER stress and reduce α- and β-ZOL-induced apoptosis in HEK293 cells. Our results suggest that saffron consumption might be helpful to prevent α- and β-ZOL-induced ER stress and toxicity.
... Apoptosis is a highly regulated cell death program triggered through the extrinsic (or death receptors) pathway or the intrinsic (mitochondrial) pathway. The mitochondrial pathway of apoptosis is usually triggered by intracellular stimuli or signals emanating from other organelles such as the endoplasmic reticulum (ER) (Sharaf el dein et al., 2009). Disruption of ER homeostasis induces ER stress that, in turn, leads to the Unfolded Protein Response (UPR), a pro-survival process that is induced to restore normal ER function. ...
... Severe ER stress is known to induce apoptosis through the mitochondrial pathway to eliminate damaged cells (Sharaf el dein et al., 2009). In addition, we have previously reported that ZEN induces cell death by activating the mitochondrial pathway of apoptosis . ...
Mycotoxins are considered to be significant contaminants of food and animal feed. Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. ZEN has been shown to be cytotoxic, genotoxic, and mutagenic in different cell types. In the present study, we investigated the involvement of endoplasmic reticulum (ER) stress in ZEN-mediated toxicity in human intestine (HCT116) and kidney (HEK293) cells and evaluated the effects of the two common dietary compounds Quercetin (QUER) and Crocin (CRO). We show that ZEN treatment induces ER stress and activates the unfolded protein response (UPR) as evidenced by XBP1 mRNA splicing and upregulation of GRP78, ATF4, GADD34, PDIA6, and CHOP. Activation of the ER stress response is associated with activation of the mitochondrial pathway of apoptosis. This apoptotic process is characterized by an increase in ROS generation and lipid peroxidation, a loss of mitochondrial transmembrane potential (ΔΨm), and an activation of caspases and DNA damages. We also demonstrate that the antioxidant properties of QUER and CRO help to prevent ER stress and reduce ZEN-induced apoptosis in HCT116 and HEK293 cells. Our results suggest that antioxidant molecule might be helpful to prevent ZEN-induced ER stress and toxicity.
... Apoptosis is a highly regulated cell death program triggered through the extrinsic (or death receptors) pathway or the intrinsic (or mitochondrial) pathway. The mitochondrial pathway of apoptosis is usually triggered by intracellular stimuli or signals emanating from other organelles such as the endoplasmic reticulum (ER) (Sharaf El Dein et al., 2009). Disruption of ER homeostasis induces ER stress which in turn leads to the unfolded protein response (UPR), a prosurvival process induced to restore normal ER function. ...
... It is now well established that the mitochondrial pathway of apoptosis can be initiated by a large variety of intracellular stress signals including ER stress (Sharaf El Dein et al., 2009). Impairment of the physiological functions of the ER, such as accumulation of misfolded proteins and disruption of the luminal Ca 2þ homeostasis, causes the so-called ER stress. ...
Patulin (PAT) is a toxic metabolite produced by several filamentous fungi of the genera of Penicillium, Aspergillus and Byssochlamys. PAT is the most common mycotoxin found in apples and apple-based products including juice, compotes, cider and baby food. Exposure to this mycotoxin has been reported to induce intestinal and kidney injuries. This study investigated the mechanism of PAT-induced toxicity in human colon carcinoma (HCT116) and embryonic kidney cells (HEK293). We demonstrated that PAT activated endoplasmic reticulum (ER) and Unfolded Protein Response as evidenced by up-regulation of GRP78 and GADD34, splicing of XBP1 mRNA and expression of the pro-apoptotic factor CHOP. This ER stress response was accompanied by the induction of the mitochondrial apoptotic pathway. Apoptosis occurred with ROS production, drop in mitochondrial membrane potential and caspase activation. Further, we showed that deficiency of the pro-apoptotic protein Bax or Bak protected cells against PAT-induced apoptosis. The treatment of cells with the ROS scavenger N-acetylacysteine (NAC) inhibits the ER stress response and prevents mitochondrial apoptosis. Collectively, our data provide new mechanistic insights in the signaling pathways of the cell death induced by PAT and demonstrate that PAT induces cytotoxicity through a ROS-dependent mechanism involving ER stress and activation of mitochondrial apoptotic pathway in human intestinal and kidney cells.
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... En ce qui concerne l'effet intrinsèque de chaque protéine, il n'y a pas de mode d'action univoque à tout un groupe, mais on sait que directement ou indirectement les protéines des trois catégories agissent sur la perméabilisation d'une ou des membranes mitochondriales. Plusieurs de ces protéines, incluant Bax et Bcl-2, ont également été retrouvées dans la membranes du RE et participent à la régulation de la communication apoptotique entre cet organite et la mitochondrie en réponse au un stress RE (Sharaf El Dein,Gallerne et al. 2009). ...
Des altérations de fonctions physiologiques du réticulum endoplasmique (RE) induisent un processus appelé stress RE. Dans le domaine cardiovasculaire, plusieurs travaux ont montré que le stress RE contribue au développement de la majorité des pathologies cardiaques. En réponse au stress RE, la réponse UPR (Unfolded Protein Response) est activée afin de restaurer l’homéostasie du RE et de permettre la survie de la cellule. Néanmoins, dans le cas d’un stress RE excessif ou prolongé, les altérations ne pouvant plus être compensées, la cellule est éliminée par apoptose contribuant au développement de la pathologie cardiaque. Une thérapie prometteuse pour lutter contre ce type de pathologie consisterait donc à moduler la réponse au stress RE afin d’inhiber l’apoptose des cardiomyocytes. Au cours de ma thèse, je me suis intéressé aux modifications induites en réponse au stress RE dans le cœur et au rôle de la sirtuine 1 (SIRT1) dans la modulation de cette réponse. SIRT1 est une déacétylase activée par différents stress cardiaques et connue pour favoriser la survie cellulaire. D’une part, j’ai mis en évidence que le stress RE induit une modification importante de l’architecture des cardiomyocytes et en particulier une augmentation des contacts RE/mitochondries associée à une altération de la fonction mitochondriale. D’autre part, en utilisant une lignée cellulaire (H9c2), des cardiomyocytes de rat adulte et des souris invalidées pour SIRT1, j’ai démontré in vitro et in vivo (i) que SIRT1 est activée et joue un rôle cardioprotecteur en réponse au stress RE, (ii) que SIRT1 limite la réponse UPR en régulant spécifiquement la voie PERK, et (iii) que SIRT1 régule la voie PERK en déacétylant le facteur d’initiation de la traduction, eIF2 sur deux résidus lysine. Ces résultats montrent donc pour la première fois que SIRT1 est impliquée dans la régulation de la réponse apoptotique au stress RE des cardiomyocytes et suggèrent que cette déacétylase serait une cible thérapeutique intéressante pour prévenir l’apoptose dans les pathologies cardiaques liées au stress RE.
