Signaling cell death from the endoplasmic reticulum stress response

Inability to meet protein folding demands within the endoplasmic reticulum (ER) activates the unfolded protein response (UPR), a signaling pathway with both adaptive and apoptotic outputs. While some secretory cell types have a remarkable ability to increase protein folding capacity, their upper limits can be reached when pathological conditions overwhelm the fidelity and/or output of the secretory pathway. Irremediable 'ER stress' induces apoptosis and contributes to cell loss in several common human diseases, including type 2 diabetes and neurodegeneration. Researchers have begun to elucidate the molecular switches that determine when ER stress is too great to repair and the signals that are then sent from the UPR to execute the cell.     

Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis

8-Oxoguanine DNA glycosylase (Ogg1) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Ogg1 is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the β-Ogg1 isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Ogg1 functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human α-hOgg1 (mt-hOgg1) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial α-hOgg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOgg1 in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase, and apoptosis, suggesting that the protective effects of mt-hOgg1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOgg1 coprecipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOgg1 silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOgg1 acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity.     

Modulation of neuronal pentraxin 1 expression in rat pancreatic β-cells submitted to chronic glucotoxic stress

Insulin secretory granules are β-cell vesicles dedicated to insulin processing, storage, and release. The secretion of insulin secretory granule content in response to an acute increase of glucose concentration is a highly regulated process allowing normal glycemic homeostasis. Type 2 diabetes is a metabolic disease characterized by chronic hyperglycemia. The consequent prolonged glucose exposure is known to exert deleterious effects on the function of various organs, notably impairment of insulin secretion by pancreatic β-cells and induction of apoptosis. It has also been described as modifying gene and protein expression in β-cells. Therefore, we hypothesized that a modulation of insulin secretory granule protein expression induced by chronic hyperglycemia may partially explain β-cell dysfunction. To identify the potential early molecular mechanisms underlying β-cell dysfunction during chronic hyperglycemia, we performed SILAC and mass spectrometry experiments to monitor changes in the insulin secretory granule proteome from INS-1E rat insulinoma β-cells cultivated either with 11 or 30 mm of glucose for 24 h. Fourteen proteins were found to be differentially expressed between these two conditions, and several of these proteins were not described before to be present in β-cells. Among them, neuronal pentraxin 1 was only described in neurons so far. Here we investigated its expression and intracellular localization in INS-1E cells. Furthermore, its overexpression in glucotoxic conditions was confirmed at the mRNA and protein levels. According to its role in hypoxia-ischemia-induced apoptosis described in neurons, this suggests that neuronal pentraxin 1 might be a new β-cell mediator in the AKT/GSK3 apoptotic pathway. In conclusion, the modification of specific β-cell pathways such as apoptosis and oxidative stress may partially explain the impairment of insulin secretion and β-cell failure, observed after prolonged exposure to high glucose concentrations.     

Engineering responsiveness to cell culture stresses: growth arrest and DNA damage gene 153 (GADD153) and the unfolded protein response (UPR) in NS0 myeloma cells

The successful movement of a newly synthesized protein through the endoplasmic reticulum (ER) and associated membranous compartments is dependent on appropriate recognition by complex processing systems. Failure to perceive appropriately processed or modified intermediates in the pathway can initiate a series of cellular signaling events (ER stress or unfolded protein response, UPR) that can lead to cell apoptosis and loss of biomass in culture processes. We have shown that expression of growth arrest and DNA damage gene 153 (GADD153) is associated with recognition of damaged or mis-processed proteins within the secretory processes of CHO and NS0 myeloma cells. To directly characterize the roles of GADD153 in UPR-directed apoptosis, we have generated stable clones of NS0 myeloma cells with elevated (constitutive and inducible) and deleted GADD153 expression. Although GADD153 is a robust indicator of the onset of ER stress or the UPR, GADD153 expression alone is not sufficient to provoke NS0 myeloma apoptosis and it is not required for apoptosis to occur.     

Ubiquitin fold modifier 1 (UFM1) and its target UFBP1 protect pancreatic beta cells from ER stress-induced apoptosis

UFM1 is a member of the ubiquitin like protein family. While the enzymatic cascade of UFM1 conjugation has been elucidated in recent years, the biological function remains largely unknown. In this report we demonstrate that the recently identified C20orf116, which we name UFM1-binding protein 1 containing a PCI domain (UFBP1), and CDK5RAP3 interact with UFM1. Components of the UFM1 conjugation pathway (UFM1, UFBP1, UFL1 and CDK5RAP3) are highly expressed in pancreatic islets of Langerhans and some other secretory tissues. Co-localization of UFM1 with UFBP1 in the endoplasmic reticulum (ER) depends on UFBP1. We demonstrate that ER stress, which is common in secretory cells, induces expression of Ufm1, Ufbp1 and Ufl1 in the beta-cell line INS-1E. siRNA-mediated Ufm1 or Ufbp1 knockdown enhances apoptosis upon ER stress. Silencing the E3 enzyme UFL1, results in similar outcomes, suggesting that UFM1-UFBP1 conjugation is required to prevent ER stress-induced apoptosis. Together, our data suggest that UFM1-UFBP1 participate in preventing ER stress-induced apoptosis in protein secretory cells.     

Survivin is released from cancer cells via exosomes

Inhibitor of apoptosis (IAP) and Heat shock proteins (HSPs) provide assistance in protecting cells from stresses of hypoxia, imbalanced pH, and altered metabolic and redox states commonly found in the microenvironmental mixture of tumor and nontumor cells. HSPs are upregulated, cell-surface displayed and released extracellularly in some types of tumors, a finding that until now was not shared by members of the IAP family. The IAP Survivin has been implicated in apoptosis inhibition and the regulation of mitosis in cancer cells. Survivin exists in a number of subcellular locations such as the mitochondria, cytoplasm, nucleus, and most recently, the extracellular space. Our previous work showing that extracellular survivin was able to enhance cellular proliferation, survival and tumor cell invasion provides evidence that Survivin might be secreted via an unidentified exocytotic pathway. In the present study, we describe for the first time the exosome-release of Survivin to the extracellular space both basally and after proton irradiation-induced stress. To examine whether exosomes contributed to Survivin release from cancer cells, exosomes were purified from HeLa cervical carcinoma cells and exosome quantity and Survivin content were determined. We demonstrate that although proton irradiation does not influence the exosomal secretory rate, the Survivin content of exosomes isolated from HeLa cells treated with a sublethal dose of proton irradiation (3 Gy) is significantly higher than control. These data identify a novel secretory pathway by which Survivin can be actively released from cells in both the basal and stress-induced state.     

When ER stress reaches a dead end

Endoplasmic reticulum (ER) stress is a common feature of several physiological and pathological conditions affecting the function of the secretory pathway. To restore ER homeostasis, an orchestrated signaling pathway is engaged that is known as the unfolded protein response (UPR). The UPR has a primary function in stress adaptation and cell survival; however, under irreversible ER stress a switch to pro-apoptotic signaling events induces apoptosis of damaged cells. The mechanisms that initiate ER stress-dependent apoptosis are not fully understood. Several pathways have been described where we highlight the participation of the BCL-2 family of proteins and ER calcium release. In addition, recent findings also suggest that microRNAs and oxidative stress are relevant players on the transition from adaptive to cell death programs. Here we provide a global and integrated overview of the signaling networks that may determine the elimination of a cell under chronic ER stress. This article is part of a Special Section entitled: Cell Death Pathways. Guest Editors: Frank Madeo and Slaven Stekovic.     

Autophagy inhibition induces podocyte apoptosis by activating the pro-apoptotic pathway of endoplasmic reticulum stress

Podocyte apoptosis is a major factor inducing podocyte depletion that predicts the progressive course of glomerulosclerosis. However, the molecular mechanisms underlying podocyte apoptosis are still not well understood. Autophagy is a lysosomal degradation system involving the degradation and recycling of obsolete, damaged, or harmful cytoplasmic materials and organelles. Recent advances in the understanding of the molecular processes contributing to autophagy have provided insight into the relationship between autophagy and apoptosis. However, their cross-talk remains largely obscure until now. Here, we found that podocytes both in vivo and in vitro always exhibited high basal levels of autophagy, whereas autophagy inhibition could induce podocyte apoptosis, suggesting the pro-survival role of autophagy in podocytes. Besides, we found that autophagy inhibition by 3-methyladenine (3-MA) could induce the activation of endoplasmic reticulum stress even without any external stimulations, whereas knockdown of CHOP could effectively improve podocyte apoptosis and down-regulated expression of slit-diaphragm proteins induced by autophagy inhibition. Collectively, this study demonstrated that autophagy might act as a crucial regulatory mechanism of apoptotic cell death by modulating the balance between the pro-survival pathway and the pro-apoptotic pathway of endoplasmic reticulum stress, which might provide a novel mechanistic insight into the interface between autophagy and apoptosis in the progression of podocyte injury.     

MEK1 activation rescues Jurkat T cells from Fas-induced apoptosis.

Although the protease cascade initiated by Fas (CD95, Apo-1) is well characterized, there remains little known about how kinase pathways may impact on Fas-mediated apoptosis. We recently observed that in T lymphocytes Fas strongly induced activation of JNK (c-Jun N-terminal kinase) but not of second messengers leading to activation of ERK (extracellular regulated kinase). Additionally, Fas-mediated apoptosis was significantly inhibited with PMA, a potent activator of the ERK signaling pathway. This suggested a model whereby activation of the ERK pathway might attenuate Fas-mediated apoptosis. This was confirmed in the current study by showing that activation of MEK1, the upstream regulator of ERK, reduces Fas-mediated apoptosis, whereas inhibition of MEK1 augments apoptosis by Fas. Furthermore, Fas-mediated apoptosis of Jurkat T cells is not affected by constitutively active or dominant negative variants that modulate the JNK pathway. These results demonstrate that Fas-induced JNK activation is not required for apoptosis by Jurkat T cells, but rather is more likely secondary to cell stress during the early phases of apoptosis. This is supported by the ability of the caspase blocker zVAD to inhibit both apoptosis and JNK activation by Fas.     

Apoptosis and cell proliferation in proximal tubular cells exposed to apoptotic bodies. Novel pathophysiological implications in cisplatin-induced renal injury

The therapeutic efficacy of the antineoplastic drug cisplatin is limited by its nephrotoxicity, which affects particularly to proximal tubular cells (PTC). Cisplatin-induced cytotoxicity appears to be multifactorial and involves inflammation, oxidative stress as well as apoptosis. We have recently shown that the cyclo-oxygenase-2 (COX-2)/intracellular prostaglandin E₂ (iPGE₂)/EP receptor pathway mediates the apoptotic effect of cisplatin on human proximal tubular HK-2 cells. Here, we studied the effects on HK-2 cells of apoptotic bodies (ABs) generated after treatment of HK-2 cells with cisplatin. We found that ABs inhibited cell growth, induced apoptosis and increased COX-2 expression and iPGE₂ in ABs-recipient HK-2 cells. Inhibition of the COX-2/iPGE₂/EP receptor pathway in these cells prevented the effects of ABs without interfering with their internalization. Interestingly, 2nd generation ABs (i.e. ABs released by cells undergoing apoptosis upon treatment with ABs) did not trigger apoptosis in naïve HK-2 cells, and stimulated cell proliferation through the COX-2/iPGE₂/EP receptor pathway. These results suggest that ABs, through iPGE₂-dependent mechanisms, might have a relevant role in the natural history of cisplatin-induced acute kidney failure because they contribute first to the propagation of the noxious effects of cisplatin to non-injured PTC and then to the promotion of the proliferative tubular response required for proximal tubule repair. Since iPGE₂ also mediates both cisplatin-induced HK-2 cell apoptosis, intervention in the COX-2/iPGE₂/EP receptor pathway might provide us with new therapeutic avenues in patients with cisplatin-induced acute kidney injury.     

Role of Fanconi DNA repair pathway in neural stem cell homeostasis

Defects in DNA repair pathways have been involved in collapse of early neurogenesis leading to brain development abnormalities and embryonic lethality. However, consequences of DNA repair defects in adult neural stem and progenitor cells and their potential contribution in ageing phenotype are poorly understood. The Fanconi anaemia (FA) pathway, which functions primarily as a DNA damage response system, has been examined in neural stem and progenitor cells during developmental and adult neurogenesis. We have shown that loss of fanca and fancg specifically provokes neural progenitor apoptosis during forebrain development, related to DNA repair defects, which persists in adulthood leading to depletion of the neural stem cell pool with ageing. In addition, neural stem cells from FA mice had a reduced capacity to self-renew in vitro. Here, we expand upon our recent work and give further data examining possible implication of oxidative stress. Therefore, FA phenotype might be interpreted as a premature ageing of stem cells, DNA damages being among the driving forces of ageing.     

Inhibition of Endoplasmic Reticulum Stress-induced Apoptosis by Silkworm Storage Protein 1

The endoplasmic reticulum (ER) plays essential roles indispensable for cellular activity and survival, including functions such as protein synthesis, secretory and membrane protein folding, and Ca²⁺ release in cells. The ER is sensitive to stresses that can lead to the aggregation and accumulation of misfolded proteins, which eventually triggers cellular dysfunction; severe or prolonged ER stress eventually induces apoptosis. ER stress-induced apoptosis causes several devastating diseases such as atherosclerosis, neurodegenerative diseases, and diabetes. In addition, the production of biopharmaceuticals such as monoclonal antibodies requires the maintenance of normal ER functions to achieve and maintain the production of high-quality products in good quantities. Therefore, it is necessary to develop methods to efficiently relieve ER stress and protect cells from ER stress-induced apoptosis. The silkworm storage protein 1 (SP1) has anti-apoptotic activities that inhibit the intrinsic mitochondrial apoptotic pathway. However, the role of SP1 in controlling ER stress and ER stress-induced apoptosis has not been investigated. In this paper, we demonstrate that SP1 can inhibit apoptosis induced by a well-known ER stress inducer, thapsigargin, by alleviating the decrease in cell viability and mitochondrial membrane potential. Interestingly, SP1 significantly blocked increases in CHOP and GRP78 expression as well as ER Ca2+ leakage into the cytosol following ER stress induction. This indicates that SP1 protects cells from ER stressinduced apoptosis by functioning as an upstream inhibitor of apoptosis. Therefore, studying SP1 function can offer new insights into protecting cells against ER stress-induced apoptosis for future applications in the biopharmaceutical and medicine industries.     

内质网应激反应分子机理研究进展

内质网应激是导致心脑组织缺血梗塞、神经退行性疾病等发生的重要环节 .目前发现同型半胱氨酸、氧化应激、钙代谢紊乱等都能引起内质网应激级联反应 ,表现为蛋白质合成暂停、内质网应激蛋白表达和细胞凋亡等 .这些表现包括在未折叠蛋白反应 (UPR)、整合应激反应 (ISR)和内质网相关性死亡 (ERAD)三个相互关联的动态过程中 ,每一过程的分子机理现已逐步被揭示 .作为细胞保护性应对机制的内质网应激体系一旦遭到破坏 ,细胞将不能合成应有的蛋白质 ,亦不能发挥正常的生理功能 ,甚至会出现细胞凋亡 .掌握内质网应激过程对进一步理解多种疾病的发生机理有十分重要的理论意义     

Genome-wide RNAi identifies p53-dependent and -independent regulators of germ cell apoptosis in C. elegans

We used genome-wide RNA interference (RNAi) to identify genes that affect apoptosis in the C. elegans germ line. RNAi-mediated knockdown of 21 genes caused a moderate to strong increase in germ cell death. Genetic epistasis studies with these RNAi candidates showed that a large subset (16/21) requires p53 to activate germ cell apoptosis. Apoptosis following knockdown of the genes in the p53-dependent class also depended on a functional DNA damage response pathway, suggesting that these genes might function in DNA repair or to maintain genome integrity. As apoptotic pathways are conserved, orthologues of the worm germline apoptosis genes presented here could be involved in the maintenance of genomic stability, p53 activation, and fertility in mammals.     

Mechanisms underlying the pro-survival pathway of p53 in suppressing mitotic death induced by adriamycin

The p53 tumor suppressor responds to chemotherapeutic stress by triggering apoptosis or eliciting pro-survival pathway through arresting cell cycle progression for DNA damage repair. Here we examined the pro-survival activity of p53 on the adriamycin-induced stress using H1299 cells stably expressing tsp53 V143A, a temperature-sensitive mutant activating only the subset of p53 target genes related to growth arrest and DNA repair, but not apoptosis. At 38 degrees C, cells evaded from adriamycin-induced G2 arrest and died of apoptosis and mitotic catastrophe, which could be inhibited by Cdk inhibitors. Activation of functional tsp53 V143A at 32 degrees C led to suppression of Cdk1/2 activities and Cyclin B1/Cdk1 expression, cells exhibited prolonged G2 arrest, regained reproductive potential and were protected from mitotic catastrophe induced by adriamycin. Inhibition of mitotic catastrophe and Cyclin B1/Cdk1 expression was ablated upon silencing p21 Waf1 expression in tsp53 V143A-H1299 cells or in HCT116 cells. Together we show that p21 Waf1 is a key component of G2 checkpoint necessary and sufficient for protecting tumor cells against adriamycin-induced mitotic catastrophe.