Influenza A viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (IRE1) stress pathway

Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications.     

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.     

The unknown face of IRE1α – Beyond ER stress

IRE1α (Inositol Requiring kinase Enzyme 1 alpha), a transmembrane protein localized to the endoplasmic reticulum (ER) is a master regulator of the unfolded protein response (UPR) pathway. The fate determining steps during ER stress-induced apoptosis are greatly attributed to IRE1α’s endoribonuclease and kinase activities. Apart from its role as a chief executioner in ER stress, recent studies have shown that upon activation in the presence or absence of ER stress, IRE1α executes multiple cellular processes such as differentiation, immune response, progression and repression of the cell cycle. Besides its crucial role in protein misfolding, the versatile contributions of IRE1α in other cellular functions are greatly unknown. In this review, we have discussed the structural conservation of IRE1 among eukaryotes, the mechanisms underlying its activation and the recent understandings of the non-apoptotic functions of IRE1 other than ER stress-induced cell death.     

RNF13, a RING Finger Protein,Mediates Endoplasmic Reticulum Stress-induced Apoptosis through the Inositol-requiring Enzyme (IRE1α)/c-Jun NH2-terminal Kinase Pathway

Disturbance of homeostasis at endoplasmic reticulum (ER) causes stress to cells that in turn triggers an adaptive signaling pathway termed unfolded protein response for the purpose of restoring normal cellular physiology or initiating signaling events leading to apoptosis. Identification of those genes that are involved in the unfolded protein response-mediated apoptotic signaling pathway would be valuable toward elucidating the molecular mechanism underlying the relationship between ER stress and apoptosis. We initiated a genetic screen by using the retroviral insertion mutation system to search for genes whose inactivation confers resistance to apoptosis induction by staurosporine. Using this approach, RING finger protein 13 (RNF13) was identified. Interestingly, RNF13 is highly enriched in ER. RNF13 knockdown cells are resistant to apoptosis and JNK activation triggered by ER stress. Conversely, overexpression of RNF13 induces JNK activation and caspase-dependent apoptosis. The RING and transmembrane domains of RNF13 are both required for its effects on JNK activation and apoptosis. Moreover, systematic analysis of the involvement of individual signaling components in the ER stress pathway using knockdown approach reveals that RNF13 acts upstream of the IRE1α-TRAF2 signaling axis for JNK activation and apoptosis. Finally, RNF13 co-immunoprecipitates with IRE1α, and the intact RING domain is also required for mediating its interaction. Together, our data support a model that RNF13 is a critical mediator for facilitating ER stress-induced apoptosis through the activation of the IRE1α-TRAF2-JNK signaling pathway.     

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.     

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.     

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.     

Nck-dependent activation of extracellular signal-regulated kinase-1 and regulation of cell survival during endoplasmic reticulum stress

In response to stress, the endoplasmic reticulum (ER) signaling machinery triggers the inhibition of protein synthesis and up-regulation of genes whose products are involved in protein folding, cell cycle exit, and/or apoptosis. We demonstrate that the misfolding agents azetidine-2-carboxylic acid (Azc) and tunicamycin initiate signaling from the ER, resulting in the activation of Jun-N-terminal kinase, p44(MAPK)/extracellular signal-regulated kinase-1 (ERK-1), and p38(MAPK) through IRE1alpha-dependent mechanisms. To characterize the ER proximal signaling events involved, immuno-isolated ER membranes from rat fibroblasts treated with ER stress inducers were used to reconstitute the activation of the stress-activated protein kinase/mitogen-activate protein kinase (MAPK) pathways in vitro. This allowed us to demonstrate a role for the SH2/SH3 domain containing adaptor Nck in ERK-1 activation after Azc treatment. We also show both in vitro and in vivo that under basal conditions ER-associated Nck represses ERK-1 activation and that upon ER stress this pool of Nck dissociates from the ER membrane to allow ERK-1 activation. Moreover, under the same conditions, Nck-null cells elicit a stronger ERK-1 activation in response to Azc stress, thus, correlating with an enhanced survival phenotype. These data delineate a novel mechanism for the regulation of ER stress signaling to the MAPK pathway and demonstrate a critical role for Nck in ER stress and cell survival.     

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.     

JAB1 participates in unfolded protein responses by association and dissociation with IRE1

Recent papers have reported that neuronal death in patients with Alzheimer's disease, Parkinson's disease, and cerebral ischemia has its origin in the endoplasmic reticulum (ER). IRE1alpha is one of the ER stress transducers that detect the accumulation of unfolded proteins in the ER. IRE1alpha mediates two major cellular responses, which are the unfolded protein response (UPR), a defensive response, and apoptosis that leads to cell death. However, little is known about the regulatory mechanisms that select between the UPR and apoptosis. We identified Jun activation domain-binding protein-1 (JAB1) as a molecule that interacts with IRE1alpha using a yeast two-hybrid system. We demonstrated that JAB1 binds to IRE1alpha in the absence of stress, but that binding is decreased by ER stress inducers. Moreover, mutant JAB1 down-regulates the UPR signaling pathway through tight binding with IRE1alpha. These results suggested that JAB1 may act as a key molecule in selecting the UPR or cell death by association and dissociation with IRE1alpha.     

The unfolded protein response is shaped by the NMD pathway

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD''s ability to regulate normal mRNAs.