Autophagy is activated for cell survival after endoplasmic reticulum stress

Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 “dots”), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.     

Roles of CHOP/GADD153 in endoplasmic reticulum stress

Endoplasmic reticulum (ER) is the site of synthesis and folding of secretory proteins. Perturbations of ER homeostasis affect protein folding and cause ER stress. ER can sense the stress and respond to it through translational attenuation, upregulation of the genes for ER chaperones and related proteins, and degradation of unfolded proteins by a quality-control system. However, when the ER function is severely impaired, the organelle elicits apoptotic signals. ER stress has been implicated in a variety of common diseases such as diabetes, ischemia and neurodegenerative disorders. One of the components of the ER stress-mediated apoptosis pathway is C/EBP homologous protein (CHOP), also known as growth arrest- and DNA damage-inducible gene 153 (GADD153). Here, we summarize the current understanding of the roles of CHOP/GADD153 in ER stress-mediated apoptosis and in diseases including diabetes, brain ischemia and neurodegenerative disease.     

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.     

Signaling and induction of chaperone-mediated autophagy by the endoplasmic reticulum under stress conditions

Chaperone-mediated autophagy (CMA), a form of selective autophagy, maintains cellular proteostasis in response to diverse stress conditions. Whether and how endoplasmic reticulum (ER) stress triggers CMA remains elusive. In our recent study, we demonstrate that various types of ER stress activate the CMA pathway via an EIF2AK3/PERK-MAP2K4/MKK4-MAPK14/p38-dependent manner. We term this process ERICA for ER stress-induced chaperone-mediated autophagy. This pathway is activated in response to stress associated with Parkinson disease and is required for the viability of the SNc dopaminergic neurons in an animal model of Parkinson disease.     

The human endoplasmic reticulum molecular chaperone BiP is an autoantigen for rheumatoid arthritis and prevents the induction of experimental arthritis

Rheumatoid arthritis (RA) is the most common, crippling human autoimmune disease. Using Western blotting and tandem mass spectroscopy, we have identified the endoplasmic reticulum chaperone BiP, a 78-kDa glucose-regulated protein, as a possible autoantigen. It preferentially stimulated increased proliferation of synovial T cells from patients with RA but not from patients with other arthritides. Mice with established collagen- or pristane-induced arthritis developed IgG Abs to BiP. Although BiP injected in CFA failed to induce arthritis in several strains of rats and mice, including HLA-DR4(+/-)- and HLA-DR1(+/+)-transgenic animals, it completely inhibited the development of arthritis when given i.v. 1 wk before the injection of type II collagen arthritis. Preimmunization with BiP suppressed the development of adjuvant arthritis in Lewis rats in a similar manner. This is the first report of a mammalian chaperone that is an autoantigen in human RA and in experimental arthritis and that can also prevent the induction of experimental arthritis. These findings may stimulate the development of new immunotherapies for the treatment of RA.     

GLP-1 receptor activation improves beta cell function and survival following induction of endoplasmic reticulum stress

Perturbation of endoplasmic reticulum (ER) homeostasis impairs insulin biosynthesis, beta cell survival, and glucose homeostasis. We show that a murine model of diabetes is associated with the development of ER stress in beta cells and that treatment with the GLP-1R agonist exendin-4 significantly reduced biochemical markers of islet ER stress in vivo. Exendin-4 attenuated translational downregulation of insulin and improved cell survival in purified rat beta cells and in INS-1 cells following induction of ER stress in vitro. GLP-1R agonists significantly potentiated the induction of ATF-4 by ER stress and accelerated recovery from ER stress-mediated translational repression in INS-1 beta cells in a PKA-dependent manner. The effects of exendin-4 on the induction of ATF-4 were mediated via enhancement of ER stress-stimulated ATF-4 translation. Moreover, exendin-4 reduced ER stress-associated beta cell death in a PKA-dependent manner. These findings demonstrate that GLP-1R signaling directly modulates the ER stress response leading to promotion of beta cell adaptation and survival.     

Heightened induction of proapoptotic signals in response to endoplasmic reticulum stress in primary fibroblasts from a mouse model of longevity

Previous work from our laboratory has shown that primary fibroblasts from long-lived Snell dwarf mice display a higher sensitivity to the lethal effects of endoplasmic reticulum (ER) stressors, such as thapsigargin, than cells from normal mice. Here we show that thapsigargin induces higher expression of CHOP, enhanced cleavage of caspase-12, higher caspase-3 activity, and increased phosphorylation of c-JUN, all indicators of enhanced apoptosis, in dwarf fibroblasts. Dwarf and normal fibroblasts show no genotypic difference in up-regulating BiP, GRP94, and ERp72 proteins after exposure to thapsigargin. However, dwarf fibroblasts express lower basal levels of a number of putative XBP1 target genes including Armet, Edem1, Erdj3, p58(IPK) and Sec61a1, as well as Ire1α itself. Furthermore, when exposed to thapsigargin, dwarf fibroblasts display attenuated splicing of Xbp1, but similar phosphorylation of eIF2α, in comparison to normal fibroblasts. These data support the notion that IRE1/XBP1 signaling is set at a lower level in dwarf fibroblasts. Diminished Xbp1 splicing in dwarf-derived fibroblasts may tilt the balance between prosurvival and proapoptotic signals in favor of apoptosis, thereby leading to higher induction of proapoptotic signals in these cells and ultimately their increased sensitivity to ER stressors. These results, together with recent findings in Caenorhabditis elegans daf-2 mutants, point to a potential interplay between insulin/IGF-1 signals and unfolded protein response signaling.     

Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP

Brain ischemia induces apoptosis in neuronal cells, but the mechanism is not well understood. When wild-type mice were subjected to bilateral common carotid arteries occlusion (BCCAO) for 15 min, apoptosis-associated morphological changes and appearance of TUNEL-positive cells were observed in the striatum and in the hippocampus at 48 h after occlusion. RT-PCR analysis revealed that mRNAs for ER stress-associated proapoptotic factor CHOP and an ER chaperone BiP are markedly induced at 12 h after BCCAO. Immunohistochemical analysis showed that CHOP protein is induced in nuclei of damaged neurons at 24 h after occlusion. In contrast, ischemia-associated apoptotic loss of neurons was decreased in CHOP(-/-) mice. Primary hippocampal neurons from CHOP(-/-) mice were more resistant to hypoxia-reoxygenation-induced apoptosis than those from wild-type animals. These results indicate that ischemia-induced neuronal cell death is mediated by the ER stress pathway involving CHOP induction.     

Monocyte chemotactic protein-induced protein (MCPIP) promotes inflammatory angiogenesis via sequential induction of oxidative stress, endoplasmic reticulum stress and autophagy

Major diseases such as cardiovascular diseases, rheumatoid arthritis, diabetes, obesity and tumor growth are known to involve inflammation. Inflammatory molecules such as MCP-1, TNF-α, IL-1β and IL-8 are known to promote angiogenesis. MCP-induced protein (MCPIP), originally discovered as a novel zinc finger protein induced by MCP-1, is also induced by other inflammatory agents. MCPIP was shown to mediate MCP-1-induced angiogenesis. Whether angiogenesis induced by other inflammatory agents is mediated via MCPIP is unknown and the molecular mechanisms involved in angiogenesis induced by MCPIP have not been elucidated. The aim of this study was to bridge this gap and delineate the sequential processes involved in angiogenesis mediated via MCPIP. siRNA knockdown of MCPIP was used to determine whether different inflammatory agents, MCP-1, TNF-α, IL-1β and IL-8, mediate angiogenesis via MCPIP in human umbilical vein endothelial cells (HUVECs). Chemical inhibitors and specific gene knockdown approach were used to inhibit each process postulated. Oxidative stress was inhibited by apocynin or cerium oxide nanoparticles or knockdown of NADPH oxidase subunit, phox47. Endoplasmic reticulum (ER) stress was blocked by tauroursodeoxycholate or knockdown of ER stress signaling protein IRE-1 and autophagy was inhibited by the use of 3'methyl adenine, or LY 294002 or by specific knockdown of beclin1. Matrigel assay was used as a tool to study angiogenic differentiation induced by inflammatory agents or MCPIP overexpression in HUVECs. Tube formation induced by inflammatory agents, TNF-α, IL-1β, IL-8 and MCP-1 was inhibited by knockdown of MCPIP. Forced MCPIP-expression induced oxidative stress, ER stress, autophagy and angiogenic differentiation in HUVECs. Inhibition of each step caused inhibition of each subsequent step postulated. The results reveal that angiogenesis induced by inflammatory agents is mediated via induction of MCPIP that causes oxidative and nitrosative stress resulting in ER stress leading to autophagy required for angiogenesis. The sequence of events suggested to be involved in inflammatory angiogenesis by MCPIP could serve as possible targets for therapeutic intervention of angiogenesis-related disorders.     

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.     

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.     

Altered localization of amyloid precursor protein under endoplasmic reticulum stress

Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.     

Hypoxia triggers endothelial endoplasmic reticulum stress and apoptosis via induction of VLDL receptor

Endothelial cells express very low density lipoprotein receptor (VLDLr). Beyond the function as peripheral lipoprotein receptor, other roles of VLDLr in endothelial cells have not been completely unraveled. In the present study, human umbilical vein endothelial cells were subjected to hypoxia, and VLDLr expression, endoplasmic reticulum (ER) stress, and apoptosis were assessed. Hypoxia triggered endothelial ER stress and apoptosis, and induced VLDLr expression. Silencing or stabilization of HIF-1α reduced and enhanced VLDLr expression, respectively. HIF-1α affected vldlr promoter activity by interacting with a hypoxia-responsive element (HRE). Knockdown or overexpression of VLDLr alleviated and exacerbated hypoxia-induced ER stress and apoptosis, respectively. Thus, hypoxia induces VLDLr expression through the interaction of HIF-1α with HRE at the vldlr promoter. VLDLr then mediates ER stress and apoptosis.     

Taurine alleviates endoplasmic reticulum stress in the chondrocytes from patients with osteoarthritis

Osteoarthritis (OA), characterized by pain and stiffness, swelling, deformity and dysfunction of joints, affects large numbers of population. The purpose of this study was to discover the effects of taurine in human OA chondrocytes and explore the underlying mechanisms. 46 patients with different grades of OA were recruited. Of these patients, 24 underwent total knee replacement and cartilages were harvested. The mRNA expressions of type II collagen (Collagen II) and endoplasmic reticulum (ER) stress markers (GRP78, GADD153 and Caspase-12) in cartilages were quantified by qRT-PCR. Cell viability and apoptosis of patient-derived chondrocytes were assessed by the CCK-8 assay and flow cytometry assay, respectively. Meanwhile, protein levels of Collagen II and ER stress markers both in cartilages and chondrocytes were evaluated by Western blot. The mRNA and protein levels of Collagen II decreased as OA progressed, while the expressions of ER stress markers increased dramatically. H₂O₂ induced ER stress in chondrocytes, as shown by the significant increase in the expression of ER stress markers, inhibited chondrocyte viability and Collagen II synthesis, promoted apoptosis. However, taurine treatment inhibited these above phenomena. These results indicated that taurine exhibited anti-OA effect by alleviating H₂O₂ induced ER stress and subsequently inhibiting chondrocyte apoptosis.     

Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway

Accumulation of saturated fatty acids in the liver can cause nonalcoholic fatty liver disease (NAFLD). This study investigated saturated fatty acid induction of endoplasmic reticulum (ER) stress and apoptosis in human liver cells and the underlying causal mechanism. Human liver L02 and HepG2 cell lines were exposed to the saturated fatty acid sodium palmitate. MTT assay was used for cell viability, flow cytometry and Hoechst 33258 staining for apoptosis, RT-PCR for mRNA expression, and Western blot for protein expression. Silence of PRK-like ER kinase (PERK) expression in liver cells was through transient transfection of PERK shRNA. Treatment of L02 and HepG2 cells with sodium palmitate reduced cell viability through induction of apoptosis. Sodium palmitate also induced ER stress in the cells, indicated by upregulation of PERK phosphorylation and expression of BiP, ATF4, and CHOP proteins. Sodium palmitate had little effect on activating XBP-1, a common target of the other two canonical sensors of ER stress, ATF6, and IRE1. Knockdown of PERK gene expression suppressed the PERK/ATF4/CHOP signaling pathway during sodium palmitate-induced ER stress and significantly inhibited sodium palmitate-induced apoptosis in L02 and HepG2 cells. Saturated fatty acid-induced ER stress and apoptosis in these human liver cells were enacted through the PERK/ATF4/CHOP signaling pathway. Future study is warranted to investigate the role of these proteins in mediating saturated fatty acid-induced NAFLD in animal models.