Activation of caspase-12 by endoplasmic reticulum stress induced by transient middle cerebral artery occlusion in mice

We sought to clarify the involvement of caspase-12, a representative molecule related to endoplasmic reticulum (ER) stress-induced cell-death signaling pathways, in neuronal death resulting from ischemia/reperfusion in mice. Transient focal cerebral ischemia (1 h) was produced by intraluminal occlusion of the middle cerebral artery (MCA). We assessed the expression patterns of caspase-12, Bip/GRP78, an ER-resident molecular chaperone whose expression serves as a good marker of ER stress, and caspase-7 by Western blotting and/or immunohistochemistry. Double-fluorescent staining of caspase-12 immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) method was performed to clarify the involvement of caspase-12 in cell death. We confirmed that ER stress was induced during reperfusion in our model, as witnessed by up-regulated Bip/GRP78 expression in the MCA territory. Western blot analysis revealed that caspase-12 activation occurred at 5-23 h of reperfusion, and immunoreactivity for caspase-12 was enhanced mainly in striatal neurons on the ischemic side at the same time points. We found the co-localization of caspase-12 immunoreactivity and DNA fragmentation detectable by the TUNEL method. We did not detect the presence of caspase-7 in the ER fraction at the period of caspase-12 cleavage. Our results imply that cerebral ischemia/reperfusion induces ER stress and that caspase-12 activation concurred with ER stress. Caspase-12 seems to be involved in neuronal death induced by ischemia/reperfusion. Caspase-7 is not likely to contribute to the cleavage of caspase-12 in our experimental model.     

内质网应激在动脉粥样硬化发生、发展和防治中的作用

Endoplasmic reticulum (ER) is a multifunctional organelle responsible for the synthesis and folding of proteins and regulation of calcium homeostasis. Multiple stimuli, such as oxidative stress, glycosylation change and so on, lead to ER dysfunction characterized by the accumulation of unfolded and/or misfolded proteins and calcium homeostasis imbalance (ER stress). Mode-rate ER stress is an important cytoprotective mechanism against stressors. However, severe and/or prolonged ER stress can trigger apoptotic signaling including CHOP, caspase-12 and JNK pathways. Recent studies have shown that ER stress plays a critical role in the development of atherosclerosis and it can bring about inhibitory effects on the progression of atherosclerosis through the intervention of the relevant pathways, which may be a new therapeutic target for atherosclerosis.     

Endoplasmic reticulum stress-induced caspase-4 activation mediates apoptosis and neurodegeneration in INCL

Infantile neuronal ceroid lipofuscinosis (INCL), a neurodegenerative storage disorder of childhood, is caused by mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. PPT1 cleaves thioester linkages in S-acylated (palmitoylated) proteins and its mutation causes abnormal intracellular accumulation of fatty-acylated proteins and peptides leading to INCL pathogenesis. Although apoptosis is the suggested cause of neurodegeneration in INCL, the molecular mechanism(s) of apoptosis remains unclear. Using the PPT1-knockout (PPT1-KO) mice that mimic INCL, we previously reported that one mechanism of apoptosis involves endoplasmic reticulum (ER) stress-induced caspase-12 activation. However, the human caspase-12 gene contains several mutations, which make it functionally inactive. Thus, it has been suggested that human caspase-4 is the counterpart of murine caspase-12. Here we report that in the human INCL brain ER stress-induced activation of unfolded protein response (UPR) mediates caspase-4 and caspase-3 activation and apoptosis. Moreover, we show that the INCL brain contains high level of growth-associated protein-43 (GAP-43), which is known to undergo palmitoylation. We also demonstrate that transfection of cultured INCL cells with a green fluorescent protein-GAP-43 cDNA construct shows abnormal localization of this protein in the ER. Further, INCL cells manifest evidence of ER stress and UPR (elevated levels of Grp-78/Bip and GADD153), caspase-4 as well as caspase-3 activation and cleavage of poly(ADP)-ribose polymerase, a compelling sign of apoptosis. Most importantly, we show that inhibition of caspase-4 activity protects INCL cells from undergoing apoptosis. Our results provide insight into at least one of the molecular mechanisms of apoptosis in INCL and may allow the identification of potential targets for therapeutic intervention.     

Polyglutamine protein aggregation and toxicity are linked to the cellular stress response

Chronic exposure of cells to expanded polyglutamine proteins results in eventual cell demise. We constructed mouse cell lines expressing either the full-length androgen receptor (AR), or truncated forms of AR containing 25 or 65 glutamines to study the cellular consequences of chronic low-level exposure to these proteins. Expression of the polyglutamine-expanded truncated AR protein, but not the full-length expanded protein, resulted in the formation of cytoplasmic and nuclear aggregates and eventual cell death. Nuclear aggregates preferentially stained positive for heat shock protein (hsp)72, a sensitive indicator of a cellular stress response. Biochemical studies revealed that the presence of nuclear aggregates correlated with activation of the c-jun NH2-terminal kinase (JNK). Different metabolic insults, including heat shock treatment, and exposure to sodium arsenite or menadione, proved more toxic to those cells expressing the polyglutamine-expanded truncated protein than to cells expressing the non-expanded form. Cells containing cytoplasmic polyglutamine-protein aggregates exhibited a delayed expression of hsp72 after heat shock. Once expressed, hsp72 failed to localize normally and instead was sequestered within the protein aggregates. This was accompanied by an inability of the aggregate-containing cells to cease their stress response as evidenced by the continued presence of activated JNK. Finally, activation of the cellular stress response increased the overall extent of polyglutamine protein aggregation, especially within the nucleus. Inclusion of a JNK inhibitor reduced this stress-dependent increase in nuclear aggregates. Abnormal stress responses may contribute to enhanced cell vulnerability in cells expressing polyglutamine-expanded proteins and may increase the propensity of such cells to form cytoplasmic and nuclear inclusions.     

Endoplasmic reticulum stress and unfolded protein response in inclusion body myositis muscle

Proteins in the endoplasmic reticulum (ER) require an efficient system of molecular chaperones whose role is to assure their proper folding and to prevent accumulation of unfolded proteins. The response of cells to accumulation of unfolded proteins in the ER is termed "unfolded protein response" (UPR). UPR is a functional mechanism by which cells attempt to protect themselves against ER stress, resulting from the accumulation of the unfolded/misfolded proteins. Because intracellular inclusions, containing either amyloid-beta (Abeta) or phosphorylated tau, are the characteristic feature of sporadic inclusion body myositis (s-IBM) muscle biopsies, we studied expression and immunolocalization of five ER chaperones, calnexin, calreticulin, GRP94, BiP/GRP78, and ERp72, in s-IBM and control muscle biopsies. Physical interaction of the ER chaperones with amyloid-beta precursor protein (AbetaPP) was studied by a combined immunoprecipitation/immunoblotting technique in s-IBM and control muscle biopsies, and in AbetaPP-overexpressing cultured human muscle fibers. In all s-IBM muscle biopsies, all five of the ER chaperones were immunodetected in the form of inclusions that co-localized with amyloid-beta. By immunoblotting, expression of ER chaperones was greatly increased as compared to the controls. By immunoprecipitation/immunoblotting experiments, ER chaperones co-immunoprecipitated with AbetaPP. Our studies provide evidence of the UPR in s-IBM muscle and demonstrate for the first time that the ER chaperones calnexin, calreticulin, GRP94, BiP/GRP78, and ERp72 physically associate with AbetaPP in s-IBM muscle, suggesting their playing a role in AbetaPP folding and processing.     

Inhibition of JNK3 Promotes Apoptosis Induced by BH3 Mimetic S1 in Chemoresistant Human Ovarian Cancer Cells

Previous studies have suggested that the novel BH3 mimetic S1 could induce apoptosis in diverse tumor cell lines through endoplasmic reticulum (ER) stress or mitochondrial cell death pathways. The activation of c‐Jun N‐terminal kinase (JNK) through inositol requiring enzyme‐1 (IRE1) is closely connected to ER stress‐induced apoptosis. However, the role of JNK is complex, as there are different JNK subtypes and the function of each subtype is still not entirely clear. Here we found that the mRNA expression of JNK3 was continuously high in S1‐treated human ovarian cancer SKOV3/DDP cells using a human unfolded protein response (UPR) pathway PCR array. Pharmacological inhibition of JNK3 increased cell sensitivity to apoptosis induced by S1. Furthermore, inhibition of JNK3 induced accumulation of both acidic compartment and p62, and upregulated ROS production. Our results suggest that JNK3 plays a pro‐survival role during ER stress through preventing the block of autophagic flux and reducing oxidative stress in SKOV3/DDP cells. Inhibition of JNK3 may be a potential method to enhance the killing effect of the Bcl‐2 inhibitor S1. Anat Rec, 298:386–395, 2015. © 2014 Wiley Periodicals, Inc.     

Caspases involved in ER stress-mediated cell death

Caspases are cysteine proteases involved in apoptotic pathways. Excess endoplasmic reticulum (ER) stress, induced by the accumulation of unfolded or malfolded proteins, activates various apoptotic pathways. Crosstalk between the mitochondria and ER plays an essential role in ER stress-mediated cell death. The cytochrome c-dependent apoptotic pathway is activated by ER stress. On the other hand, caspase-12, which is located at the ER, is also activated by excess ER stress and results in cell death in the absence of the cytochrome c-dependent pathway. The predominant apoptotic pathway may differ among cell type and differentiation stage.     

HYPK, a Huntingtin interacting protein, reduces aggregates and apoptosis induced by N-terminal Huntingtin with 40 glutamines in Neuro2a cells and exhibits chaperone-like activity

Expansion of polymorphic glutamine (Q) numbers present at the protein Huntingtin (Htt) beyond 36Q results in its misfolding and aggregation, and the aggregates recruit several other proteins. Here we show that HYPK, initially identified as an Htt-interacting partner by yeast two-hybrid assay, physically interacts with N-terminal Htt in Neuro2A cells and alters the numbers and distribution of aggregates formed by N-terminal Htt with 40Q. HYPK also alters the kinetics of mutated N-terminal Htt-mediated aggregate formation. Fluorescence recovery after photobleaching studies reveal that over-expression of HYPK results in the appearance of Htt poly Q aggregates, which upon bleaching recovers approximately 80% of initial fluorescence intensity within 6 min. Fluorescence loss in photobleaching studies indicate loss off fluorescence intensity of the aggregates with time in presence of HYPK. Over-expression of this protein reduces poly Q-mediated caspase-2, caspase-3 and caspase-8 activations, whereas gamma ray-induced activations of these enzymes are not affected. In vitro and in vivo studies demonstrate that HYPK possesses a novel chaperone-like activity. We conclude that HYPK, without having any sequence similarity with known chaperones, plays an effective role in protecting neuronal cells against apoptosis induced by mutated N-terminal Htt by modulating the aggregate formation.     

Berberine ameliorates pro-inflammatory cytokine-induced endoplasmic reticulum stress in human intestinal epithelial cells in vitro

Endoplasmic reticulum stress (ER stress) in intestinal epithelial cells (IECs) plays an important role in the pathogenesis and perpetuation of inflammatory bowel disease (IBD). The aim of this study is to explore the potential of berberine (BBR) in regulating pro-inflammatory cytokine-induced ER stress and apoptosis in IECs. ER stress in cultured Caco-2 cells was induced by IFN-γ/TNF-α and tunicamycin, respectively. The experimental groups were pretreated with BBR. Cell viability was determined by MTT assay. In vitro apoptosis was examined by flow cytometry using annexin V-FITC labeling. The molecular markers of ER stress, including GRP-78, p-JNK, caspase-12, and cleaved caspase-3 were analyzed by Western blot. Xbp-1 mRNA splicing was detected by RT-PCR. Pretreatment of BBR helped to survive in cytokine-induced Caco-2 cells. BBR significantly attenuated cytokine-induced Caco-2 apoptosis. GRP78 expression and xbp-1 mRNA splicing were enhanced significantly in the presence of IFN-γ/TNF-α and tunicamycin, and they could be dampened by BBR. Further study revealed BBR could downregulate JNK phosphorylation, and the level of caspase-12 and cleaved caspase-3. Berberine can ameliorate pro-inflammatory cytokines induced ER stress in vitro, and it might be one of the targeted therapeutic agents for IBD.     

Cataracts: role of the unfolded protein response

Many cataracts are caused by unfolded protein aggregates in highly oxidized lenses, but the underlying mechanisms of their formation are poorly understood. A literature search has shown that many cataractogenic stressors are also endoplasmic reticulum (ER) stressors, which induce the unfolded protein response (UPR) in a wide range of cell types. Since the lumen of the ER is highly oxidized, ER stressors might generate unfolded protein aggregates, which activate the UPR leading to the production of reactive oxygen species (ROS) in lens epithelial cells (LECs). ROS decrease the amount of free glutathione from whole lenses and elicit a more oxidized environment, where unfolded protein aggregates are formed and grown to large protein aggregate particles to scatter light. Recently, we have shown that ER stressors, homocysteine, tunicamycin, Ca(2+) ionophore (A23187), and glucose deprivation induce the UPR in LECs. Here we hypothesize the cataractogenic stressors induce ER stress, initiate the UPR and ROS production in LECs with or without apoptosis and eventually resulted in cataracts.     

Aggregate formation inhibits proteasomal degradation of polyglutamine proteins

Insoluble protein aggregates are consistently found in neurodegenerative disorders caused by expanded polyglutamine [poly(Q)] repeats. The aggregates contain various components of the ubiquitin/proteasome system, suggesting an attempt of the cell to clear the aberrant substrate. To investigate the effect of expanded poly(Q) repeats on ubiquitin/proteasome-dependent proteolysis, we targeted these proteins for proteasomal degradation by the introduction of an N-end rule degradation signal. While soluble poly(Q) proteins were degraded, they resisted proteasomal degradation once present in the aggregates. Stabilization was also observed for proteins that are co-aggregated via interaction with the expanded poly(Q) domain. Introduction of a degradation signal in ataxin-1/Q92 reduced the incidence of nuclear inclusions and the cellular toxicity, conceivably by accelerating the clearance of the soluble substrate.     

Reactive oxygen species-triggered trophoblast apoptosis is initiated by endoplasmic reticulum stress via activation of caspase-12, CHOP, and the JNK pathway in Toxoplasma gondii infection in mice

Toxoplasma gondii infection in pregnant women may result in abortion or in fetal teratogenesis; however, the underlying mechanisms are still unclear. In this paper, based on a murine model, we showed that maternal infection with RH strain T. gondii tachyzoites induced elevated production of reactive oxygen species (ROS), local oxidative stress, and subsequent apoptosis of placental trophoblasts. PCR array analysis of 84 oxidative stress-related genes demonstrated that 27 genes were upregulated at least 2-fold and that 9 genes were downregulated at least 2-fold in the T. gondii infection group compared with levels in the control group. The expression of NADPH oxidase 1 (Nox1) and glutathione peroxidase 6 (Gpx6) increased significantly, about 25-fold. The levels of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) increased significantly with T. gondii infection, and levels of glutathione (GSH) decreased rapidly. T. gondii infection increased the early expression of endoplasmic reticulum stress (ERS) markers, followed by cleavage of caspase-12, activation of ASK1/JNK, and increased apoptosis of trophoblasts, both in vivo and in vitro. The apoptosis of trophoblasts, the activation of caspase-12 and the ASK1/JNK pathway, and the production of peroxides were dramatically inhibited by pretreatment with N-acetylcysteine (NAC). The upregulation of Nox1 was contact dependent and preceded the increase in levels of ERS markers and the activation of the proapoptosis cascade. Thus, we concluded that apoptosis in placental trophoblasts was initiated predominantly by ROS-mediated ERS via activation of caspase-12, CHOP, and the JNK pathway in acute T. gondii infection. Elevated ROS production is the central event in T. gondii-induced apoptosis of placental trophoblasts.     

Thiamine deficiency induces endoplasmic reticulum stress in neurons

Thiamine (vitamin B1) deficiency (TD) causes region selective neuronal loss in the brain; it has been used to model neurodegeneration that accompanies mild impairment of oxidative metabolism. The mechanisms for TD-induced neurodegeneration remain incompletely elucidated. Inhibition of protein glycosylation, perturbation of calcium homeostasis and reduction of disulfide bonds provoke the accumulation of unfolded proteins in the endoplasmic reticulum (ER), and cause ER stress. Recently, ER stress has been implicated in a number of neurodegenerative models. We demonstrated here that TD up-regulated several markers of ER stress, such as glucose-regulated protein (GRP) 78, growth arrest and DNA-damage inducible protein or C/EBP-homologus protein (GADD153/Chop), phosphorylation of eIF2alpha and cleavage of caspase-12 in the cerebellum and the thalamus of mice. Furthermore, ultrastructural analysis by electron microscopic study revealed an abnormality in ER structure. To establish an in vitro model of TD in neurons, we treated cultured cerebellar granule neurons (CGNs) with amprolium, a potent inhibitor of thiamine transport. Exposure to amprolium caused apoptosis and the generation of reactive oxygen species in CGNs. Similar to the observation in vivo, TD up-regulated markers for ER stress. Treatment of a selective inhibitor of caspase-12 significantly alleviated amprolium-induced death of CGNs. Thus, ER stress may play a role in TD-induced brain damage.     

The protection of Bcl-2 overexpression on rat cortical neuronal injury caused by analogous ischemia/reperfusion in vitro

Recent studies have suggested that neuronal apoptosis in cerebral ischemia could arise from dysfunction of endoplasmic reticulum (ER) and mitochondria. B-cell lymphoma/leukemia-2 gene (Bcl-2) has been described as an inhibitor both in programmed cell death (PCD) and ER dysfunction during apoptosis, and the Bcl-2 family play a key role in regulating the PCD, both locally at the ER and from a distance at the mitochondrial membrane. However, its signal pathways and concrete mechanisms in endoplasmic reticulum-initiated apoptosis remain incompletely understood. We therefore investigate whether ischemia/reperfusion (I/R) causes neuronal apoptosis in part via cross-talk between ER and mitochondria or not, and how the overexpression of Bcl-2 prevents this form of cell death. Here we show that analogous I/R-induced cell death occurs consequent to interactions of ER stress and mitochondrial death pathways. The participation of the mitochondrial pathway was demonstrated by the release of cytochrome C (cyt C) from mitochondrial into cytoplasmic fractions and caspase-9 cleavage. The involvement of ER stress was further supported by the observable increase of glucose-regulated protein 78(GRP78)/BiP expression and caspase-12 activity. Furthermore, prior to these changes, swelling of the ER lumen and dissociation of ribosomes from rough ER were detected by electron microscopy. Bcl-2 overexpression inhibits the release of cyt C and the activation of caspase-9/-8/-3 but not caspase-12 based on the results of Western blot. These suggest that cross-talk between ER and mitochondria participate in neuronal damage after ischemia/reperfusion. Bcl-2 overexpression could suppress I/R-induced neuronal apoptosis via influencing mitochondrial integrity.     

ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats

Expansion of CAG trinucleotide repeats that encode polyglutamine is the underlying cause of at least nine inherited human neurodegenerative disorders, including Huntington's disease and spinocerebellar ataxias. PolyQ fragments accumulate as aggregates in the cytoplasm and/or in the nucleus, and induce neuronal cell death. However, the molecular mechanism of polyQ-induced cell death is controversial. Here, we show the following: (1) polyQ with pathogenic repeat length triggers ER stress through proteasomal dysfunction; (2) ER stress activates ASK 1 through formation of an IRE1-TRAF2-ASK1 complex; and (3) ASK1(-/-) primary neurons are defective in polyQ-, proteasome inhibitor-, and ER stress-induced JNK activation and cell death. These findings suggest that ASK1 is a key element in ER stress-induced cell death that plays an important role in the neuropathological alterations in polyQ diseases.