Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression

NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.     

Protective role of Gipie, a Girdin family protein, in endoplasmic reticulum stress responses in endothelial cells

Continued exposure of endothelial cells to mechanical/shear stress elicits the unfolded protein response (UPR), which enhances intracellular homeostasis and protect cells against the accumulation of improperly folded proteins. Cells commit to apoptosis when subjected to continuous and high endoplasmic reticulum (ER) stress unless homeostasis is maintained. It is unknown how endothelial cells differentially regulate the UPR. Here we show that a novel Girdin family protein, Gipie (78 kDa glucose-regulated protein [GRP78]-interacting protein induced by ER stress), is expressed in endothelial cells, where it interacts with GRP78, a master regulator of the UPR. Gipie stabilizes the interaction between GRP78 and the ER stress sensor inositol-requiring protein 1 (IRE1) at the ER, leading to the attenuation of IRE1-induced c-Jun N-terminal kinase (JNK) activation. Gipie expression is induced upon ER stress and suppresses the IRE1-JNK pathway and ER stress-induced apoptosis. Furthermore we found that Gipie expression is up-regulated in the neointima of carotid arteries after balloon injury in a rat model that is known to result in the induction of the UPR. Thus our data indicate that Gipie/GRP78 interaction controls the IRE1-JNK signaling pathway. That interaction appears to protect endothelial cells against ER stress-induced apoptosis in pathological contexts such as atherosclerosis and vascular endothelial dysfunction.     

Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

Disturbance of endoplasmic reticulum (ER) homeostasis causes ER stress and leads to activation of the unfolded protein response, which reduces the stress and promotes cell survival at the early stage of stress, or triggers cell death and apoptosis when homeostasis is not restored under prolonged ER stress. Here, we report that Cab45S, a member of the CREC family, inhibits ER stress-induced apoptosis. Depletion of Cab45S increases inositol-requiring kinase 1 (IRE1) activity, thus producing more spliced forms of X-box-binding protein 1 mRNA at the early stage of stress and leads to phosphorylation of c-Jun N-terminal kinase, which finally induces apoptosis. Furthermore, we find that Cab45S specifically interacts with 78-kDa glucose-regulated protein/immunoglobulin heavy chain binding protein (GRP78/BiP) on its nucleotide-binding domain. Cab45S enhances GRP78/BiP protein level and stabilizes the interaction of GRP78/BiP with IRE1 to inhibit ER stress-induced IRE1 activation and apoptosis. Together, Cab45S, a novel regulator of GRP78/BiP, suppresses ER stress-induced IRE1 activation and apoptosis by binding to and elevating GRP78/BiP, and has a role in the inhibition of ER stress-induced apoptosis.     

The role of thioredoxin-1 in suppression of endoplasmic reticulum stress in Parkinson disease

Endoplasmic reticulum (ER) stress has been implicated in Parkinson disease. We previously reported that thioredoxin 1 (Trx-1) suppressed the ER stress caused by 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine; however, its molecular mechanism remains largely unknown. In the present study, we showed that 1-methyl-4-phenylpyridinium ion (MPP⁺) induced ER stress by activating glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1α (IRE1α), tumor necrosis factor receptor-associated factor 2 (TRAF2), c-Jun N-terminal kinase (JNK), caspase-12, and C/EBP homologous protein (CHOP) in PC12 cells. The downregulation of Trx-1 aggravated the ER stress and further increased the expression of the above molecules induced by MPP⁺. In contrast, overexpression of Trx-1 attenuated the ER stress and repressed the expression of the above molecules induced by MPP⁺. More importantly, the overexpression of Trx-1 in transgenic mice suppressed ER stress by inhibiting the activation of these molecules. We present, for the first time, the molecular mechanism of Trx-1 suppression of endoplasmic reticulum stress in Parkinson disease in vitro and in vivo. Based on our findings, we conclude that Trx-1 plays a neuroprotective role in Parkinson disease by suppressing ER stress by regulating the activation of GRP78, IRE1α, TRAF2, JNK, caspase-12, and CHOP.     

Free cholesterol-induced macrophage apoptosis is mediated by inositol-requiring enzyme 1 alpha-regulated activation of Jun N-terminal kinase

Macrophage death in advanced atherosclerotic lesions leads to lesional necrosis, possible plaque rupture, and acute vascular occlusion. A likely cause of macrophage death is the accumulation of free cholesterol (FC) leading to activation of endoplasmic reticulum (ER) stress-induced apoptosis. Inositol-requiring enzyme 1 alpha (IRE1α) is an integral membrane protein of the ERthat is a key signaling step in cholesterol-induced apoptosis in macrophages, activated by stress in the ER. However, the role of IRE1α in the regulation of ER stress-induced macrophage death and the mechanism for this process are largely unclear. In this study, a cell culture model was used to explore the mechanisms involved in the ER stress pathway of FC-induced macrophage death. The results herein showed that FC loading of macrophages leads to an apoptotic response that is partially dependent on initiation by activation of IRE1α. Taken together, these results showed that the IRE1-apoptosis-signaling kinase 1-c-Jun NH2-terminal kinase cascade pathway was required in this process. Moreover, the data suggested a novel cellular mechanism for cholesterol-induced macrophage death in advanced atherosclerotic lesions. The critical function of this signaling cascade is indicated by prevention of ER stress-induced apoptosis after inhibition of IRE1α, or c-Jun NH2-terminal kinase.     

IRE1 directs proteasomal and lysosomal degradation of misfolded rhodopsin

Endoplasmic reticulum (ER) is responsible for folding of secreted and membrane proteins in eukaryotic cells. Disruption of ER protein folding leads to ER stress. Chronic ER stress can cause cell death and is proposed to underlie the pathogenesis of many human diseases. Inositol-requiring enzyme 1 (IRE1) directs a key unfolded protein response signaling pathway that controls the fidelity of ER protein folding. IRE1 signaling may be particularly helpful in preventing chronic ER stress and cell injury by alleviating protein misfolding in the ER. To examine this, we used a chemical-genetic approach to selectively activate IRE1 in mammalian cells and tested how artificial IRE1 signaling affected the fate of misfolded P23H rhodopsin linked to photoreceptor cell death. We found that IRE1 signaling robustly promoted the degradation of misfolded P23H rhodopsin without affecting its wild-type counterpart. We also found that IRE1 used both proteasomal and lysosomal degradation pathways to remove P23H rhodopsin. Surprisingly, when one degradation pathway was compromised, IRE1 signaling could still promote misfolded rhodopsin degradation using the remaining pathway. Last, we showed that IRE1 signaling also reduced levels of several other misfolded rhodopsins with lesser effects on misfolded cystic fibrosis transmembrane conductance regulator. Our findings reveal the diversity of proteolytic mechanisms used by IRE1 to eliminate misfolded rhodopsin.     

Tumour necrosis factor receptor 1 mediates endoplasmic reticulum stress-induced activation of the MAP kinase JNK

Tumour necrosis factor receptor (TNFR)1 is the main receptor responsible for TNF-induced diverse cellular events. In this study, we report that TNFR1 has a crucial role in endoplasmic reticulum (ER) stress-induced Jun amino-terminal kinase (JNK) activation. Although ER stress leads to JNK activation in wild-type mouse embryo fibroblasts, we failed to detect any JNK activation in TNFR1-/- cells. ER stress-induced JNK activation is restored in TNFR1-/- cells when TNFR1 expression is reconstituted. We also found that TNFR1 functions downstream of IRE1 and that IRE1 is present in the same complex with TNFR1 under ER stress condition. Therefore, our study shows a novel role of TNFR1 in mediating ER stress-induced JNK activation.     

HDLs inhibit endoplasmic reticulum stress and autophagic response induced by oxidized LDLs

The apoptotic effect of oxidized LDLs (oxLDLs) is mediated through a complex sequence of signaling events involving a deregulation of the cytosolic Ca(2+) homeostasis. OxLDLs also trigger ER stress that may lead to cellular dysfunction and apoptosis, through the activation of the IRE1α/c-Jun N-terminal kinase pathway. Moreover, ER stress and oxidized lipids have been shown to trigger autophagy. The antiatherogenic high-density lipoproteins (HDLs) display protective effects against oxLDLs toxicity. To more deeply investigate the mechanisms mediating the protective effects of HDLs, we examined whether ER stress and autophagy were implicated in oxLDLs-induced apoptosis and whether HDLs prevented these stress processes. We report that, in human endothelial cells, HDLs prevent the oxLDL-induced activation of the ER stress sensors IRE1α, eIF2α and ATF6 and subsequent activation of the proapoptotic mediators JNK and CHOP. OxLDLs also trigger the activation of autophagy, as assessed by LC3 processing and Beclin-1 expression. The autophagic process is independent of the proapoptotic arms of ER stress, but Beclin-1 contributes to PS exposure and subsequent phagocytosis of oxLDLs exposed cells. Induction of autophagy and PS exposure by oxLDLs is prevented by HDLs. Finally, the cytosolic Ca(2+) deregulation triggered by oxLDLs is a common signaling pathway that mediates ER stress-induced cell death and autophagy, all these events being blocked by HDLs.     

Inhibition of x‐box binding protein 1 reduces tunicamycin‐induced apoptosis in aged murine macrophages

Endoplasmic reticulum (ER) stress is induced by the accumulation of unfolded and misfolded proteins in the ER. Although apoptosis induced by ER stress has been implicated in several aging‐associated diseases, such as atherosclerosis, it is unclear how aging modifies ER stress response in macrophages. To decipher this relationship, we assessed apoptosis in macrophages isolated from young (1.5–2 months) and aged (16–18 months) mice and exposed the cells to the ER stress inducer tunicamycin. We found that aged macrophages exhibited more apoptosis than young macrophages, which was accompanied by reduced activation of phosphorylated inositol‐requiring enzyme‐1 (p‐IRE1α), one of the three key ER stress signal transducers. Reduced gene expression of x‐box binding protein 1 (XBP1), a downstream effector of IRE1α, enhanced p‐IRE1α levels and reduced apoptosis in aged, but not young macrophages treated with tunicamycin. These findings delineate a novel, age‐dependent interaction by which macrophages undergo apoptosis upon ER stress, and suggest an important protective role of IRE1α in aging‐associated ER stress‐induced apoptosis. This novel pathway may not only be important in our understanding of longevity, but may also have important implications for pathogenesis and potential treatment of aging‐associated diseases in general.     

Sustained IRE1 and ATF6 signaling is important for survival of melanoma cells undergoing ER stress

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with rapid attenuation of the IRE1α and ATF6 pathways but persistent activation of the PERK branch of the unfolded protein response (UPR) in cells. However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that the kinetics and durations of activation of the UPR pathways are deregulated in melanoma cells undergoing ER stress. We show here that the IRE1α and ATF6 pathways are sustained along with the PERK signaling in melanoma cells subjected to pharmacological ER stress, and that this is, at least in part, due to increased activation of the MEK/ERK pathway. In contrast to an initial increase followed by rapid reduction in activation of IRE1α and ATF6 signaling in control cells that were relatively sensitive to ER stress-induced apoptosis, activation of IRE1α and ATF6 by the pharmacological ER stress inducer tunicamycin (TM) or thapsigargin (TG) persisted in melanoma cells. On the other hand, the increase in PERK signaling lasted similarly in both types of cells. Sustained activation of IRE1α and ATF6 signaling played an important role in protecting melanoma cells from ER stress-induced apoptosis, as interruption of IRE1α or ATF6 rendered melanoma cells sensitive to apoptosis induced by TM or TG. Inhibition of MEK partially blocked IRE1α and ATF6 activation, suggesting that MEK/ERK signaling contributed to sustained activation of IRE1α and ATF6. Taken together, these results identify sustained activation of the IRE1α and ATF6 pathways of the UPR driven by the MEK/ERK pathway as an important protective mechanism against ER stress-induced apoptosis in melanoma cells.     

Modulating stress responses by the UPRosome: a matter of life and death

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR) through the activation of specialized sensors including inositol-requiring enzyme-1α (IRE1α). IRE1α signals by assembling a dynamic protein platform referred to as the UPRosome, where different modulator and adaptor proteins assemble to regulate the kinetics and amplitude of UPR effector responses. Conversely, chronic ER stress can cause apoptosis. Recent evidence indicates that several apoptosis-related proteins interact with IRE1α, regulating its prosurvival activities and performing a dual function in the regulation of cell death and adaptation to stress. Based on the increasing relevance of ER stress to the occurrence of diverse pathological conditions, strategies to target and modulate the assembly and composition of the UPRosome could have therapeutic benefits for disease intervention.     

Endoplasmic reticulum stress plays a role in the advanced glycation end product-induced inflammatory response in endothelial cells

Aims

Both advanced glycation end products (AGEs) and endoplasmic reticulum (ER) stress play important roles in the development of various diseases. This study aimed to clarify the consequence of AGE-induced ER stress and its underlying mechanisms in human umbilical venous endothelial cells (HUVECs).

Main methods

AGE-induced ER stress was assessed by the increased expression and activation of the ER stress marker proteins GRP78, IRE1α and JNK, which were detected using Western blot. NF-κB translocation was revealed using Western blot and immunofluorescent staining in IRE1α-knockdown HUVECs. The mechanism of AGE-induced ER stress was also explored by inhibiting the effect of reactive oxygen species (ROS) using NADPH oxidase 4 (Nox4) siRNA and the antioxidant reduced glutathione (GSH). The cellular ROS level was measured using flow cytometry.

Key findings

AGEs time- and dose-dependently enhanced the expression of GRP78 and increased the phosphorylation of IRE1α and its downstream signal JNK in HUVECs. siRNA-induced IRE1α down-regulation suppressed AGE-induced NF-κB p65 nuclear translocation. Inhibiting the ROS production using Nox4 siRNA or antagonizing ROS using GSH reduced cellular ROS level and attenuated AGE-induced GRP78 expression and IRE1α and JNK activation.

Significance

This study confirms that AGE-induced ER stress in HUVECs focuses on the ER stress-enhanced inflammatory response through JNK and NF-κB activation. It further reveals the involvement of ROS in the AGE-induced ER stress mechanism.