Deficiency of αB crystallin augments ER stress-induced apoptosis by enhancing mitochondrial dysfunction

Endoplasmic reticulum (ER) stress is linked to several pathological conditions including age-related macular degeneration. Excessive ER stress initiates cell death cascades which are mediated, in part, through mitochondrial dysfunction. Here, we identify αB crystallin as an important regulator of ER stress-induced cell death. Retinal pigment epithelial (RPE) cells from αB crystallin (-/-) mice, and human RPE cells transfected with αB crystallin siRNA, are more vulnerable to ER stress induced by tunicamycin. ER stress-mediated cell death is associated with increased levels of reactive oxygen species, depletion of glutathione in mitochondria, decreased superoxide dismutase activity, increased release of cytochrome c, and activation of caspases 3 and 4. The ER stress signaling inhibitors, salubrinal and 4-(2-aminoethyl) benzenesulfonyl fluoride, decrease mitochondrial damage and reduce RPE apoptosis induced by ER stress. Prolonged ER stress decreases levels of αB crystallin, thus exacerbating mitochondrial dysfunction. Overexpression of αB crystallin protects RPE cells from ER stress-induced apoptosis by attenuating increases in Bax, CHOP, mitochondrial permeability transition, and cleaved caspase 3. Thus, these data collectively demonstrate that αB crystallin provides critical protection of mitochondrial function during ER stress-induced RPE apoptosis.     

BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress

Bax inhibitor-1 (BI-1) is an evolutionarily conserved endoplasmic reticulum (ER) protein that suppresses cell death in both animal and plant cells. We characterized mice in which the bi-1 gene was ablated. Cells from BI-1-deficient mice, including fibroblasts, hepatocytes, and neurons, display selective hypersensitivity to apoptosis induced by ER stress agents (thapsigargin, tunicamycin, brefeldin A), but not to stimulators of mitochondrial or TNF/Fas-death receptor apoptosis pathways. Conversely, BI-1 overexpression protects against apoptosis induced by ER stress. BI-1-mediated protection from apoptosis induced by ER stress correlated with inhibition of Bax activation and translocation to mitochondria, preservation of mitochondrial membrane potential, and suppression of caspase activation. BI-1 overexpression also reduces releasable Ca(2+) from the ER. In vivo, bi-1(-/-) mice exhibit increased sensitivity to tissue damage induced by stimuli that trigger ER stress, including stroke and tunicamycin injection. Thus, BI-1 regulates a cell death pathway important for cytopreservation during ER stress.     

Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER-mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders.     

Inhibition of PKR protects against tunicamycin-induced apoptosis in neuroblastoma cells

Endoplasmic reticulum (ER) dysfunction is thought to play a significant role in several neurological disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, cerebral ischemia, and the prion diseases. ER dysfunction can be mimicked by cellular stress signals such as disruption of calcium homeostasis, inhibition of protein glycosylation, and reduction of disulfide bonds, which results in accumulation of misfolded proteins in the ER and leads to cell death by apoptosis. Tunicamycin, which is an inhibitor of protein glycosylation, induces ER stress and apoptosis. In this study, we examined the involvement of double stranded (ds) RNA-activated protein kinase PKR in tunicamycin-induced apoptosis. We used overexpression of the trans-dominant negative, catalytically inactive mutant K296R to inhibit PKR activity in neuroblastoma cells. We demonstrate that inhibition of PKR activation in response to tunicamycin protects neuronal cells from undergoing apoptosis. Furthermore, K296R overexpressing cells show defective PKR activation, delayed eIF2α phosphorylation, dramatically delayed ATF4 expression. In addition, both caspase-3 activation and C/EBP homologous protein (CHOP, also known as GADD153) induction, which are markers of apoptotic cells, are absent from K296R overexpression cells in response to tunicamycin. These results establish that PKR activation plays a major regulatory role in induction of apoptosis in response to ER stress and indicates the potential of PKR as possible target for neuroprotective therapeutics.     

Tunicamycin-induced ER stress upregulates the expression of mitochondrial HtrA2 and promotes apoptosis through the cytosolic release of HtrA2

Recent studies provide some evidence that the HtrA2 protein is intimately associated with the pathogenesis of neurodegenerative disorders and that endoplasmic reticulum (ER) quality control and ER stress-associated cell death play critical roles in neuronal cell death. However, little is known about the intimate relationship between HtrA2 and ER stress-associated cellular responses. In the present study, we have demonstrated that the HtrA2 protein level was gradually and significantly increased by up to 10-fold in the mitochondria under tunicamycin (Tm)-induced ER stress, which eventually promoted cell death through the release of HtrA2 into the cytoplasm. Using an ecdysoneinducible mammalian expression system, we demonstrate that the extent of cell death in 293-HtrA2 cells was approximately 20 times higher under Tm-induced ER stress, indicating that the increase in the HtrA2 protein level in the mitochondria itself is necessary but not sufficient for the promotion of cell death. Taken together, these results suggest that HtrA2 may serve as a mediator of ER stress-induced apoptosis and ER-mitochondrial cross-talk in some cellular processes.     

Caspase-2 cleavage of BID is a critical apoptotic signal downstream of endoplasmic reticulum stress

The accumulation of misfolded proteins stresses the endoplasmic reticulum (ER) and triggers cell death through activation of the multidomain proapoptotic BCL-2 proteins BAX and BAK at the outer mitochondrial membrane. The signaling events that connect ER stress with the mitochondrial apoptotic machinery remain unclear, despite evidence that deregulation of this pathway contributes to cell loss in many human degenerative diseases. In order to "trap" and identify the apoptotic signals upstream of mitochondrial permeabilization, we challenged Bax-/- Bak-/- mouse embryonic fibroblasts with pharmacological inducers of ER stress. We found that ER stress induces proteolytic activation of the BH3-only protein BID as a critical apoptotic switch. Moreover, we identified caspase-2 as the premitochondrial protease that cleaves BID in response to ER stress and showed that resistance to ER stress-induced apoptosis can be conferred by inhibiting caspase-2 activity. Our work defines a novel signaling pathway that couples the ER and mitochondria and establishes a principal apoptotic effector downstream of ER stress.     

Inhibition of glycolysis attenuates 4-hydroxynonenal-dependent autophagy and exacerbates apoptosis in differentiated SH-SY5Y neuroblastoma cells

How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE.     

Proteome-wide study of endoplasmic reticulum stress induced by thapsigargin in N2a neuroblastoma cells

Disturbances in intraluminal endoplasmic reticulum (ER) Ca²⁺ concentration leads to the accumulation of unfolded proteins and perturbation of intracellular Ca²⁺ homeostasis, which has a huge impact on mitochondrial functioning under normal and stress conditions and can trigger cell death. Thapsigargin (TG) is widely used to model cellular ER stress as it is a selective and powerful inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPases. Here we provide a representative proteome-wide picture of ER stress induced by TG in N2a neuroblastoma cells. Our proteomics study revealed numerous significant protein expression changes in TG-treated N2a cell lysates analysed by two-dimensional electrophoresis followed by mass spectrometric protein identification. The proteomic signature supports the evidence of increased bioenergetic activity of mitochondria as several mitochondrial enzymes with roles in ATP-production, tricarboxylic acid cycle and other mitochondrial metabolic processes were upregulated. In addition, the upregulation of the main ER resident proteins confirmed the onset of ER stress during TG treatment. It has become widely accepted that metabolic activity of mitochondria is induced in the early phases in ER stress, which can trigger mitochondrial collapse and subsequent cell death. Further investigations of this cellular stress response in different neuronal model systems like N2a cells could help to elucidate several neurodegenerative disorders in which ER stress is implicated.     

Loss of mitofusin 2 promotes endoplasmic reticulum stress

The outer mitochondrial membrane GTPase mitofusin 2 (Mfn2) is known to regulate endoplasmic reticulum (ER) shape in addition to its mitochondrial fusion effects. However, its role in ER stress is unknown. We report here that induction of ER stress with either thapsigargin or tunicamycin in mouse embryonic fibroblasts leads to up-regulation of Mfn2 mRNA and protein levels with no change in the expression of the mitochondrial shaping factors Mfn1, Opa1, Drp1, and Fis1. Genetic deletion of Mfn2 but not Mfn1 in mouse embryonic fibroblasts or cardiac myocytes in mice led to an increase in the expression of the ER chaperone proteins. Genetic ablation of Mfn2 in mouse embryonic fibroblasts amplified ER stress and exacerbated ER stress-induced apoptosis. Deletion of Mfn2 delayed translational recovery through prolonged eIF2α phosphorylation associated with decreased GADD34 and p58(IPK) expression and elevated C/EBP homologous protein induction at late time points. These changes in the unfolded protein response were coupled to increased cell death reflected by augmented caspase 3/7 activity, lactate dehydrogenase release from cells, and an increase in propidium iodide-positive nuclei in response to thapsigargin or tunicamycin treatment. In contrast, genetic deletion of Mfn1 did not affect ER stress-mediated increase in ER chaperone synthesis or eIF2α phosphorylation. Additionally, ER stress-induced C/EBP homologous protein, GADD34, and p58(IPK) induction and cell death were not affected by loss of Mfn1. We conclude that Mfn2 but not Mfn1 is an ER stress-inducible protein that is required for the proper temporal sequence of the ER stress response.     

Calcium signaling at the endoplasmic reticulum: fine-tuning stress responses

Endoplasmic reticulum (ER) calcium signaling is implicated in a myriad of coordinated cellular processes. The ER calcium content is tightly regulated as it allows a favorable environment for protein folding, in addition to operate as a major reservoir for fast and specific release of calcium. Altered ER homeostasis impacts protein folding, activating the unfolded protein response (UPR) as a rescue mechanism to restore proteostasis. ER calcium release impacts mitochondrial metabolism and also fine-tunes the threshold to undergo apoptosis under chronic stress. The global coordination between UPR signaling and energetic demands takes place at mitochondrial associated membranes (MAMs), specialized subdomains mediating interorganelle communication. Here we discuss current models explaining the functional relationship between ER homeostasis and various cellular responses to coordinate proteostasis and metabolic maintenance.     

Endoplasmic reticulum stress-induced apoptosis requires bax for commitment and Apaf-1 for execution in primary neurons

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with various pathophysiological conditions including neurodegenerative diseases and ischemia. However, the mechanism by which ER stress induces neuronal apoptosis remains controversial. Here we identify the pathway of apoptosis carried out in sympathetic neurons triggered to die by ER stress-inducing agent tunicamycin. We find that ER stress induces a neuronal apoptotic pathway which upregulates BH3-only genes DP5 and Puma. Importantly, we show that ER stress commits neurons to die before cytochrome c release and this commitment requires Bax activation and c-jun N-terminal kinase signaling. Furthermore, ER stress engages the mitochondrial pathway of death as neurons release cytochrome c and Apaf-1 deficiency is sufficient to block apoptosis. Our findings identify a critical function of Bax in committing neurons to ER stress-induced apoptosis and clarify the importance of the apoptosome as the non-redundant caspase activation pathway to execute neuronal apoptosis in response to ER stress.     

Intracellular methylglyoxal induces oxidative damage to pancreatic beta cell line INS-1 cell through Ire1α-JNK and mitochondrial apoptotic pathway

An increased intracellular methylglyoxal (MGO) under hyperglycemia led to pancreatic beta cell death. However, its mechanism in which way with MGO induced beta cell death remains unknown. We investigated both high glucose and MGO treatment significantly inclined intracellular MGO concentration and inhibited cell viability in vitro. MGO treatment also triggered intracellular advanced glycation end products (AGEs) formation, declined mitochondrial membrane potential (MMP), increased oxidative stress and the expression of ER stress mediators Grp78/Bip and p-PERK; activated mitochondrial apoptotic pathway, which could mimic by Glo1 knockdown. Aminoguanidine (AG), a MGO scavenger, however, prevented AGEs formation and MGO-induced cell death by inhibiting oxidative stress and ER stress. Furthermore, both antioxidant N-acetylcysteine (NAC) and ER stress inhibitor 4-phenylbutyrate (4-PBA) could attenuate MGO-induced cell death through ameliorating ER stress. MGO treatment down-regulated Ire1α, a key ER stress mediator, increased JNK phosphorylation and activated mitochondrial apoptosis; down-regulated Bcl-2 expression which could be attenuated by the JNK inhibitor SP600125 and further inhibited cytochrome c leakage from mitochondria and blocked the conversion of pro caspase 3 into cleaved caspase 3, all these might contribute to the inhibition of INS-1 cell apoptosis. Ire1α down-regulation by Ire1α siRNAs mimicked MGO-induced cytotoxicity by activating the JNK phosphorylation and mitochondrial apoptotic pathway. In summary, we demonstrated that increased intracellular MGO induced cytotoxicity in INS-1 cells primarily by activating oxidative stress and further triggering mitochondrial apoptotic pathway, and ER stress-mediated Ire1α-JNK pathway. These findings may have implication on new mechanism of glucotoxicity-mediated pancreatic beta-cell dysfunction.     

Regulation of calnexin sub-cellular localization modulates endoplasmic reticulum stress-induced apoptosis in MCF-7 cells

The endoplasmic reticulum (ER) is the cellular compartment where proteins enter the secretory pathway, undergo post-translational modifications and acquire a correct conformation. If these functions are chronically altered, specific ER stress signals are triggered to promote cell death through the intrinsic apoptotic pathway. Here, we show that tunicamycin causes significant alteration of calnexin sub-cellular distribution in MCF-7 cells. Interestingly, this correlates with the absence of both tunicamycin-induced calnexin phosphorylation as well as tunicamycin-induced cell death. Under these conditions, calnexin-associated Bap31, an ER integral membrane protein, is subjected to a caspase-8 cleavage pattern within a specific sub-compartment of the ER. These results suggest that MCF-7 resistance to ER stress-induced apoptosis is partially mediated by the expression level of calnexin which in turn controls its sub-cellular localization, and its association with Bap31. These data may delineate a resistance mechanism to the ER stress-induced intrinsic apoptotic pathway.     

DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death

Damage to endoplasmic reticulum (ER) homeostasis that cannot be corrected by the unfolded protein response activates cell death. Here, we identified death-associated protein kinase (DAPk) as an important component in the ER stress-induced cell death pathway. DAPk-/- mice are protected from kidney damage caused by injection of the ER stress-inducer tunicamycin. Likewise, the cell death response to ER stress-inducers is reduced in DAPk-/- primary fibroblasts. Both caspase activation and autophagy induction, events that are activated by ER stress and precede cell death, are significantly attenuated in the DAPk null cells. Notably, in this cellular setting, autophagy serves as a second cell killing mechanism that acts in concert with apoptosis, as the depletion of Atg5 or Beclin1 from fibroblasts significantly protected from ER stress-induced death when combined with caspase-3 depletion. We further show that ER stress promotes the catalytic activity of DAPk by causing dephosphorylation of an inhibitory autophosphorylation on Ser(308) by a PP2A-like phosphatase. Thus, DAPk constitutes a critical integration point in ER stress signaling, transmitting these signals into two distinct directions, caspase activation and autophagy, leading to cell death.     

Opposing roles of prion protein in oxidative stress- and ER stress-induced apoptotic signaling

Although the prion protein is abundantly expressed in the CNS, its biological functions remain unclear. To determine the endogenous function of the cellular prion protein (PrP(c)), we compared the effects of oxidative stress and endoplasmic reticulum (ER) stress inducers on apoptotic signaling in PrP(c)-expressing and PrP(ko) (knockout) neural cells. H(2)O(2), brefeldin A (BFA), and tunicamycin (TUN) induced increases in caspase-9 and caspase-3, PKCdelta proteolytic activation, and DNA fragmentation in PrP(c) and PrP(ko) cells. Interestingly, ER stress-induced activation of caspases, PKCdelta, and apoptosis was significantly exacerbated in PrP(c) cells, whereas H(2)O(2)-induced proapoptotic changes were suppressed in PrP(c) compared to PrP(ko) cells. Additionally, caspase-12 and caspase-8 were activated only in the BFA and TUN treatments. Inhibitors of caspase-9, caspase-3, and PKCdelta significantly blocked H(2)O(2)-, BFA-, and TUN-induced apoptosis, whereas the caspase-8 inhibitor attenuated only BFA- and TUN-induced cell death, and the antioxidant MnTBAP blocked only H(2)O(2)-induced apoptosis. Overexpression of the kinase-inactive PKCdelta(K376R) or the cleavage site-resistant PKCdelta(D327A) mutant suppressed both ER and oxidative stress-induced apoptosis. Thus, PrP(c) plays a proapoptotic role during ER stress and an antiapoptotic role during oxidative stress-induced cell death. Together, these results suggest that cellular PrP enhances the susceptibility of neural cells to impairment of protein processing and trafficking, but decreases the vulnerability to oxidative insults, and that PKCdelta is a key downstream mediator of cellular stress-induced neuronal apoptosis.     

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca(2+)-independent phospholipase A(2) (iPLA(2)β), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.     

GPR177 promotes gastric cancer proliferation by suppressing endoplasmic reticulum stress-induced cell death

Gastric cancer is the fourth most common cancer worldwide. Despite the high incidence of gastric cancer, efficient chemotherapy treatments still need to be developed. In this study, we examined the anticancer effects of endoplasmic reticulum (ER) stress inducer tunicamycin in gastric cancer. Previously, we found that overexpression of WLS1/GPR177 correlated with poor prognosis in patients with gastric cancer. Furthermore, tunicamycin treatment downregulated GPR177 expression in a dose-dependent manner. GPR177 transports WNT ligand from ER to the plasma membrane, mediating its secretion to the extracellular matrix. In gastric cancer cells, GPR177 preferentially localizes to the ER. Small interfering RNA-mediated knockdown of GPR177 leads to sensitization to ER stress and induces apoptosis of cancer cells along with tunicamycin treatment. GPR177 suppression promoted the ER stress-mediated proapoptotic pathway, such as PERK-CHOP cascade. Furthermore, fluorouracil treatment combined with tunicamycin dramatically reduced cancer cell proliferation. Efficacy of tunicamycin chemotherapy treatments depended on GPR177 expression in gastric cancer cell lines. Together, our results indicate that ER stress can potentiate anticancer effects and suggest GPR177 as a potential gastric cancer therapeutic target.     

Comprehensive proteomic analysis of the effects of purine analogs on human Raji B-cell lymphoma

Cladribine (CdA) and fludarabine (FdAMP) are purine analogs that induce apoptosis in chronic lymphocytic leukemia and non-Hodgkin's lymphoma, but the mechanisms are undefined. The effects of CdA and fludarabine nucleoside (FdA) on the cytosolic, mitochondrial, and nuclear proteomes in human Raji lymphoma cells have been determined using two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry. Differentially abundant proteins have provided new insights into CdA- and FdA-induced apoptosis. Treatment with these purine analogs induced changes in proteins involved with intermediary metabolism, cell growth, signal transduction, protein metabolism, and regulation of nucleic acids. Differentially abundant mitochondrial 39S ribosomal protein L50, mTERF domain-containing protein 1, Chitinase-3 like 2 protein, and ubiquinone biosynthesis protein COQ9 have been identified in cells undergoing apoptosis. Up-regulation of several stress-associated proteins found in the endoplasmic reticulum (ER) including GRP78, ERp57, and ORP150 suggests that purine analog-induced apoptosis may result from ER stress and unfolded protein response. While mitochondria-dependent apoptosis has been associated with purine analog cytotoxicity, the likely involvement of the ER stress pathway in CdA- and FdA-induced apoptosis has been shown here for the first time.