Rheumatoid arthritis as a hyper-endoplasmic reticulum-associated degradation disease

We introduce Synoviolin as a novel pathogenic factor in rheumatoid arthritis (RA). Experimental studies indicate that this endoplasmic reticulum (ER)-resident E3 ubiquitin ligase has important functions in the ER-associated degradation (ERAD) system, an essential system for ER homeostasis. Overexpression of Synoviolin in mice causes arthropathy with synovial hyperplasia, whereas heterozygous knockdown results in increased apoptosis of synovial cells and resistance to collagen-induced arthritis in mice. On the basis of these experimental data, we propose that excess elimination of unfolded proteins (that is, 'hyper-ERAD') by overexpression of Synoviolin triggers synovial cell overgrowth and hence a worsening of RA. Further analysis of the hyper-ERAD system may permit the complex pathomechanisms of RA to be uncovered.     

Bax inhibitor-1 regulates endoplasmic reticulum stress-associated reactive oxygen species and heme oxygenase-1 expression

The Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that is located in endoplasmic reticulum (ER) membranes and protects cells from ER stress-induced apoptosis. The ER is associated with generation of reactive oxygen species (ROS) through oxidative protein folding. This study examined the role of BI-1 in the regulation of ER stress-induced accumulation of ROS and expression of unfolded protein response-associated proteins. BI-1 reduced the expression levels of glucose response protein 78, C/EBP homologous protein, phospho-eukaryotic initiation factor 2alpha, IRE1alpha, XBP-1, and phospho-JNK and inhibited the cleavage of ATF-6alpha p-90, leading to the inhibition of ROS. Although ROS scavengers offer some protection against ER stress-induced apoptosis, the expression of pro-apoptotic ER stress proteins was not affected. This study shows that the response of unfolded proteins is followed by ROS accumulation under ER stress, which is regulated in BI-1 cells. The mechanism for these BI-1-associated functions involves the expression of heme oxygenase-1 (HO-1) through nuclear factor erythroid 2-related factor 2. In BI-1 cells, the transfection of HO-1 small interfering RNA completely abolished the BI-1-induced protection. The endogenous expression of HO-1 through ER stress-initiated ROS is believed to be as a protection signal. In conclusion, these observations suggest that BI-1 can inhibit the ER stress proteins as well as the accumulation of ROS, thereby protecting the cells. Moreover, HO-1 plays an important role in the BI-1-associated protection against ER stress.     

Essential role of synoviolin in embryogenesis

We recently reported the importance of Synoviolin in quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system and its involvement in the pathogenesis of arthropathy through its anti-apoptotic effect. For further understanding of the role of Synoviolin in vivo, we generated in this study synoviolin-deficient (syno(-/-)) mice by genetargeted disruption. Strikingly, all fetuses lacking syno died in utero around embryonic day 13.5, although Hrd1p, a yeast orthologue of Synoviolin, is non-essential for survival. Histologically, hypocellularity and aberrant apoptosis were noted in the syno(-/-) fetal liver. Moreover, definitive erythropoiesis was affected in non-cell autonomous manner in syno(-/-) embryos, causing death in utero. Cultured embryonic fibroblasts derived from syno(-/-) mice were more susceptible to endoplasmic reticulum stress-induced apoptosis than those from syno(+/+) mice, but the susceptibility was rescued by overexpression of synoviolin. Our findings emphasized the indispensable role of the Synoviolin in embryogenesis.     

Parecoxib Suppresses CHOP and Foxo1 Nuclear Translocation, but Increases GRP78 Levels in a Rat Model of Focal Ischemia

Parecoxib, a novel COX-2 inhibitor, functions as a neuroprotective agent and rescues neurons from cerebral ischemic reperfusion injury-induced apoptosis. However, the molecular mechanisms underlying parecoxib neuroprotection remain to be elucidated. There is growing evidence that endoplasmic reticulum (ER) stress plays an important role in neuronal death caused by brain ischemia. However, very little is known about the role of parecoxib in mediating pathophysiological reactions to ER stress induced by ischemic reperfusion injury. Therefore, in the present study, we investigated whether delayed administration of parecoxib attenuates brain damage via suppressing ER stress-induced cell death. Adult male Sprague–Dawley rats were administered parecoxib (10 or 30 mg kg^?1, IP) or isotonic saline twice a day starting 24 h after middle cerebral artery occlusion (MCAO) for three consecutive days. The expressions of glucose-regulated protein 78 (GRP78) and oxygen-regulated protein 150 (ORP150) and C/EBP-homologous protein (CHOP) and forkhead box protein O 1 (Foxo1) in cytoplasmic and nuclear fraction were determined by Western blotting. The levels of caspase-12 expression were checked by immunohistochemistry analysis, served as a marker for ER stress-induced apoptosis. Parecoxib significantly suppressed cerebral ischemic injury-induced nuclear translocation of CHOP and Foxo1 and attenuated the immunoreactivity of caspase-12 in ischemic penumbra. Furthermore, the protective effect of delayed administration of parecoxib was accompanied by an increased GRP78 and ORP150 expression. Therefore, our study suggested that elevation of GRP78 and ORP150, and suppression of CHOP and Foxo1 nuclear translocation may contribute to parecoxib-mediated neuroprotection during ER stress responses.     

The Roles of Synoviolin in Crosstalk Between Endoplasmic Reticulum Stress-Induced Apoptosis and p53 Pathway

Endopalsmic reticulum (ER) is specialized organelle to maintain the integrity of secreted and membranous proteins. ER also senses so-called “ER stress”, which is a resulted from a various internal and external stresses, and triggers apoptosis when the diverse attempts to accommodate with the stress are in fail. The impairment these ER functions has been implicated in several human diseases, in which aberrant ER stress induced apoptosis is observed. We discuss about another disease model related with ER mediated apoptosis based on the recent studies about Synoviolin, an E3 ubiquitin ligase inherently utilized for ER associated degradation (ERAD). In addition to its canonical role in ERAD, Synoviolin targets tumor suppressor gene p53 for proteasomal degradation, suggesting the crosstalk between ERAD and p53 mediated apoptotic pathway under ER stress. Together with the anti-apoptotic property of Synoviolin previously elucidated by both in vitro and in vivo analyses, its new function in p53 regulation may provide a new insight into the pathomechanism of proliferative diseases such as cancer or rheumatoid arthritis.     

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.     

Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death

Recent studies have suggested that neuronal death in Alzheimer's disease or ischemia could arise from dysfunction of the endoplasmic reticulum (ER). Although caspase-12 has been implicated in ER stress-induced apoptosis and amyloid-beta (Abeta)-induced apoptosis in rodents, it is controversial whether similar mechanisms operate in humans. We found that human caspase-4, a member of caspase-1 subfamily that includes caspase-12, is localized to the ER membrane, and is cleaved when cells are treated with ER stress-inducing reagents, but not with other apoptotic reagents. Cleavage of caspase-4 is not affected by overexpression of Bcl-2, which prevents signal transduction on the mitochondria, suggesting that caspase-4 is primarily activated in ER stress-induced apoptosis. Furthermore, a reduction of caspase-4 expression by small interfering RNA decreases ER stress-induced apoptosis in some cell lines, but not other ER stress-independent apoptosis. Caspase-4 is also cleaved by administration of Abeta, and Abeta-induced apoptosis is reduced by small interfering RNAs to caspase-4. Thus, caspase-4 can function as an ER stress-specific caspase in humans, and may be involved in pathogenesis of Alzheimer's disease.     

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.     

Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.     

Progranulin is required for proper ER stress response and inhibits ER stress-mediated apoptosis through TNFR2

Progranulin (PGRN) was reported to be a stress-response factor in response to hypoxia and acidosis. Here we present evidences demonstrating that PGRN is also an endoplasmic reticulum (ER) stress responsive factor: PGRN expression was induced and its activation of Erk1/2 and Akt signaling enhanced in response to ER stress; Normal ER stress response was lost in PGRN deficient cells and PGRN deficient cells became hypersusceptible to ER stress-induced apoptosis; additionally, recombinant PGRN could rescue the defects in ER-stress responses seen in PGRN deficient cells. Mechanistic studies indicated that PGRN/TNFR2 was critical for PGRN mediated regulation of ER stress response: similar to PGRN, the expression of TNFR2, but not TNFR1, was also induced in the course of ER stress; in addition, the association between PGRN and TNFR2 was markedly enhanced following ER stress; More importantly, PGRN protection of ER stress induced apoptosis was abolished when TNFR2 signaling was blocked. In addition, the 2nd and 3rd cysteine-rich domains (CRD) in the extracellular portion of TNFR2 (CRD2CRD3), known to directly bind to PGRN, disturbed the interaction of PGRN with TNFR2, and in turn abolished PGRN-mediated activation of Erk1/2 and Akt signaling and protection against apoptosis in response to ER-stress. Collectively, PGRN plays an important role in ER stress and regulates ER stress response through interacting with TNFR2. This study provides new insight into PGRN regulation of stress response and may also present PGRN as a potential molecular target for treating stress-associated disorders.     

The Orphan Nuclear Receptor NR4A1 Protects Pancreatic β-Cells from Endoplasmic Reticulum (ER) Stress-mediated Apoptosis

The role of NR4A1 in apoptosis is controversial. Pancreatic β-cells often face endoplasmic reticulum (ER) stress under adverse conditions such as high free fatty acid (FFA) concentrations and sustained hyperglycemia. Severe ER stress results in β-cell apoptosis. The aim of this study was to analyze the role of NR4A1 in ER stress-mediated β-cell apoptosis and to characterize the related mechanisms. We confirmed that upon treatment with the ER stress inducers thapsigargin (TG) or palmitic acid (PA), the mRNA and protein levels of NR4A1 rapidly increased in both MIN6 cells and mouse islets. NR4A1 overexpression in MIN6 cells conferred resistance to cell loss induced by TG or PA, as assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and TUNEL assays indicated that NR4A1 overexpression also protected against ER stress-induced apoptosis. This conclusion was further confirmed by experiments exploiting siRNA to knockdown NR4A1 expression in MIN6 cells or exploiting NR4A1 knock-out mice. NR4A1 overexpression in MIN6 cells reduced C/EBP homologous protein (CHOP) expression and Caspase3 activation induced by TG or PA. NR4A1 overexpression in MIN6 cells or mouse islets resulted in Survivin up-regulation. A critical regulatory element was identified in Survivin promoter (−1872 bp to −1866 bp) with a putative NR4A1 binding site; ChIP assays demonstrated that NR4A1 physically associates with the Survivin promoter. In conclusion, NR4A1 protects pancreatic β-cells against ER stress-mediated apoptosis by up-regulating Survivin expression and down-regulating CHOP expression, which we termed as “positive and negative regulation.”