Severe Endoplasmic Reticulum Stress Research Articles

Deciphering the Link Between ERUPR Signaling and MicroRNA in Pathogenesis of Alzheimer's Disease.

Alzheimer’s disease (AD) is a neurodegenerative proteinopathic disease. The deposits of misfolded Amyloid β and Tau proteins in the brain of patients with AD suggest an imbalance in endoplasmic reticulum (ER) proteostasis. ER stress is due to accumulation of aberrant proteins in the ER lumen, which then leads to activation of three sensor protein pathways that ultimately evokes the adaptive mechanism of the unfolded protein response (UPR). The UPR mechanism operates via adaptive UPR and the apoptotic UPR. Adaptive UPR tries to restore imbalance in ER hemostasis by decreasing protein production, enhanced chaperone involvement to restore protein folding, misfolded protein decay by proteasome, and suppression of ribosomal translation ultimately relieving the excessive protein load in the ER. Subsequently, apoptotic UPR activated under severe ER stress conditions triggers cell death. MicroRNAs (miRNAs) are small non-coding protein causing dysregulated translational of mRNAs in a sequential manner. They are considered to be critical elements in the maintenance of numerous cellular activities, hemostasis, and developmental processes. Therefore, upregulation or downregulation of miRNA expression is implicated in several pathogenic processes. Evidence from scientific studies suggest a strong correlation between ERUPR signaling and miRNA dysregulation but the research done is still dormant. In this review, we summarized the cross-talk between ER stress, and the UPR signaling processes and their role in AD pathology by scrutinizing and collecting information from original research and review articles.

A Redox-Silent Analogue of Tocotrienol May Break the Homeostasis of Proteasomes in Human Malignant Mesothelioma Cells by Inhibiting STAT3 and NRF1.

6-O-Carboxypropyl-alpha-tocotrienol (α-T3E) is a multi-target redox-silent analogue of tocotrienol that exhibits cytotoxicity against many cancer cells, including malignant mesothelioma (MM) cells. α-T3E has several molecular targets to effectively induce cytotoxicity against MM cells; however, the mechanisms underlying this cytotoxicity remain unclear. In the present study, we demonstrated that the α-T3E-dependent disruption of the homeostasis of proteasomes strongly induced endoplasmic reticulum (ER) stress, which resulted in effective cytotoxicity against MM cells. The α-T3E-dependent disruption of the homeostasis of proteasomes depended on decreases in proteasome subunits via the inactivation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor erythroid 2 related factor-1 (NRF1), which inhibited protease activity, such as chymotrypsin-like activity, in proteasomes. The α-T3E-dependent inhibition of this activity also induced severe ER stress and ultimately resulted in effective cytotoxicity against MM cells with chemoresistance. The present results indicate that α-T3E acts as an effective anti-mesothelioma agent by disrupting the homeostasis of proteasomes through the simultaneous inactivation of STAT3 and NRF1.

Drug Resistance and Endoplasmic Reticulum Stress in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common and deadly cancers worldwide. It is usually diagnosed in an advanced stage and is characterized by a high intrinsic drug resistance, leading to limited chemotherapeutic efficacy and relapse after treatment. There is therefore a vast need for understanding underlying mechanisms that contribute to drug resistance and for developing therapeutic strategies that would overcome this. The rapid proliferation of tumor cells, in combination with a highly inflammatory microenvironment, causes a chronic increase of protein synthesis in different hepatic cell populations. This leads to an intensified demand of protein folding, which inevitably causes an accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum (ER). This process is called ER stress and triggers the unfolded protein response (UPR) in order to restore protein synthesis or—in the case of severe or prolonged ER stress—to induce cell death. Interestingly, the three different arms of the ER stress signaling pathways have been shown to drive chemoresistance in several tumors and could therefore form a promising therapeutic target. This review provides an overview of how ER stress and activation of the UPR contributes to drug resistance in HCC.

Ryanodine receptor RyR1-mediated elevation of Ca2+ concentration is required for the late stage of myogenic differentiation and fusion

BackgroundCytosolic Ca2+ plays vital roles in myogenesis and muscle development. As a major Ca2+ release channel of endoplasmic reticulum (ER), ryanodine receptor 1 (RyR1) key mutations are main causes of severe congenital myopathies. The role of RyR1 in myogenic differentiation has attracted intense research interest but remains unclear.ResultsIn the present study, both RyR1-knockdown myoblasts and CRISPR/Cas9-based RyR1-knockout myoblasts were employed to explore the role of RyR1 in myogenic differentiation, myotube formation as well as the potential mechanism of RyR1-related myopathies. We observed that RyR1 expression was dramatically increased during the late stage of myogenic differentiation, accompanied by significantly elevated cytoplasmic Ca2+ concentration. Inhibition of RyR1 by siRNA-mediated knockdown or chemical inhibitor, dantrolene, significantly reduced cytosolic Ca2+ and blocked multinucleated myotube formation. The elevation of cytoplasmic Ca2+ concentration can effectively relieve myogenic differentiation stagnation by RyR1 inhibition, demonstrating that RyR1 modulates myogenic differentiation via regulation of Ca2+ release channel. However, RyR1-knockout-induced Ca2+ leakage led to the severe ER stress and excessive unfolded protein response, and drove myoblasts into apoptosis.ConclusionsTherefore, we concluded that Ca2+ release mediated by dramatic increase in RyR1 expression is required for the late stage of myogenic differentiation and fusion. This study contributes to a novel understanding of the role of RyR1 in myogenic differentiation and related congenital myopathies, and provides a potential target for regulation of muscle characteristics and meat quality.

Protective effect of endoplasmic reticulum stress inhibition on 5-fluorouracil-induced oral mucositis

5-Fluorouracil (5-FU)-induced oral mucositis has a severe negative impact on the patient's quality of life. This study aimed to investigate the role of endoplasmic reticulum stress (ERS) in the occurrence of 5-FU-induced oral mucositis in vivo and in the clinic. In vivo, 5-FU-induced oral mucositis model mice showed a higher level of glucose-regulated protein 78 kD (GRP78, a marker of ERS) than control mice. The inhibition of ERS could effectively reduce 5-FU-induced oxidative stress, inflammatory factor mRNA and cell apoptosis. Moreover, inhibition of ERS significantly decreased the activation of nuclear factor kappa-B (NF-κB) in 5-FU-induced oral mucositis model mice following tissue damage reduction. In the clinic, 5-FU could increase cell apoptosis and cause oral mucosa damage while increasing the expression of the ERS marker genes GRP78 and C/EBP-homologous protein (CHOP). Our study found that 5-FU could induce severe ERS, upregulate the expression of GRP78 and CHOP, raise oxidative stress and increase the expression of inflammatory factors by activating the NF-κB pathway, thus causing cell apoptosis and finally leading to oral mucosal injury.

In vitro and in vivo studies reveal the beneficial effects of chlorogenic acid against ER stress mediated ER-phagy and associated apoptosis in the heart of diabetic rat

Endoplasmic reticulum (ER) and associated signaling pathways are involved in diabetic cardiomyopathy (DCM) however, detailed studies are not available. The present study investigated the role of ER stress and related pathways such as ER-phagy, apoptosis and their underlying mechanisms using appropriate models. Beneficial effect of chlorogenic acid was also evaluated against ER stress mediated DCM. H9c2 cells with high glucose (33 mM, in vitro model of hyperglycemia) showed significant activation of ER stress response (GRP78, PERK, IRE1α, ATF6α) and altered its regulatory proteins (PDI, ERO1α). Also, it enhanced ER-phagy through upregulation of Sec62, RTN3 and downregulation of FAM134B. High glucose caused apoptosis via increased levels of CHOP, caspase 12 and calnexin. All these proteins (PERK, IRE1α, ATF6α, RTN3, Sec62 and FAM134B) have been found to have a significant role in the functioning of heart such as excitation contraction coupling and we expect these alterations to induce cardiomyopathy during diabetes. This was confirmed in in vivo study too. High fat, high fructose diet with mild streptozotocin induced diabetic rats showed an increased expression of BNP confirming cardiac injury. We also noticed severe ER stress in the heart of diabetic animals. All these have contributed significantly into alterations in histopathology and increase of weight of the hearts. These findings clearly show that ER stress plays a vital protagonist in the progression of DCM. We also found chlorogenic acid is effective against hyperglycemia induced pathological alteration both in vitro as well as in vivo.

Magnetite Fe3O4 Nanoparticles Enhance Mild Microwave Ablation of Tumor by Activating the IRE1-ASK1-JNK Pathway and Inducing Endoplasmic Reticulum Stress.

PurposeWith the development of nanomedicine, microwave ablation enhanced by multifunctional nanoplatforms has been widely studied for synergistic cancer therapy. Though scientists have got a lot of significant achievements in this field, the detailed molecular mechanisms and potential targets of microwave ablation enhanced by multifunctional nanoplatforms still need further exploration. In this study, we found that a kind of magnetite Fe3O4 nanoparticles (Fe3O4 NPs) could induce severe endoplasmic reticulum stress and activate cancer apoptosis under the irradiation of mild microwave.MethodsIn this study, plenty of studies including cell immunofluorescence, mitochondrial membrane potential, electron microscopy, atomic force microscopy and microwave ablation in vivo were conducted to explore the molecular mechanisms and potential targets of microwave ablation enhanced by the Fe3O4 NPs.ResultsThe IRE1-ASK1-JNK pathway was strongly activated in A375 cells treated with both Fe3O4 NPs and mild microwave. The endoplasmic reticulum of the A375 cells was significantly dilated and exhibited ballooning degeneration. By investigating the mitochondrial membrane potential (ΔΨm), we found that the mitochondria of cancer cells had been significantly damaged under microwave treatment coupled with Fe3O4 NPs. In addition, melanoma of B16F10-bearing mice had also been effectively inhibited after being treated with Fe3O4 NPs and microwave.ConclusionIn this study, we found that a kind of magnetite Fe3O4 nanoparticles could induce severe ER stress and activate cancer apoptosis under mild microwave irradiation. Apparent apoptosis had been observed in the A375 cells under a scanning electron microscope and transmission electron microscope. Moreover, melanoma had also been inhibited effectively in vivo. As a result, the endoplasmic reticulum stress is a promising target with clinical potential in nanomedicine and cancer therapy.

Thyroid hormone synthesis continues despite biallelic thyroglobulin mutation with cell death.

Complete absence of thyroid hormone is incompatible with life in vertebrates. Thyroxine is synthesized within thyroid follicles upon iodination of thyroglobulin conveyed from the endoplasmic reticulum (ER), via the Golgi complex, to the extracellular follicular lumen. In congenital hypothyroidism from biallelic thyroglobulin mutation, thyroglobulin is misfolded and cannot advance from the ER, eliminating its secretion and triggering ER stress. Nevertheless, untreated patients somehow continue to synthesize sufficient thyroxine to yield measurable serum levels that sustain life. Here, we demonstrate that TGW2346R/W2346R humans, TGcog/cog mice, and TGrdw/rdw rats exhibited no detectable ER export of thyroglobulin, accompanied by severe thyroidal ER stress and thyroid cell death. Nevertheless, thyroxine was synthesized, and brief treatment of TGrdw/rdw rats with antithyroid drug was lethal to the animals. When untreated, remarkably, thyroxine was synthesized on the mutant thyroglobulin protein, delivered via dead thyrocytes that decompose within the follicle lumen, where they were iodinated and cannibalized by surrounding live thyrocytes. As the animals continued to grow goiters, circulating thyroxine increased. However, when TGrdw/rdw rats age, they cannot sustain goiter growth that provided the dying cells needed for ongoing thyroxine synthesis, resulting in profound hypothyroidism. These results establish a disease mechanism wherein dead thyrocytes support organismal survival.

Endoplasmic Reticulum Stress Signaling and the Pathogenesis of Hepatocarcinoma.

Hepatocellular carcinoma (HCC), also known as hepatoma, is a primary malignancy of the liver and the third leading cause of cancer mortality globally. Although much attention has focused on HCC, its pathogenesis remains largely obscure. The endoplasmic reticulum (ER) is a cellular organelle important for regulating protein synthesis, folding, modification and trafficking, and lipid metabolism. ER stress occurs when ER homeostasis is disturbed by numerous environmental, physiological, and pathological challenges. In response to ER stress due to misfolded/unfolded protein accumulation, unfolded protein response (UPR) is activated to maintain ER function for cell survival or, in cases of excessively severe ER stress, initiation of apoptosis. The liver is especially susceptible to ER stress given its protein synthesis and detoxification functions. Experimental data suggest that ER stress and unfolded protein response are involved in HCC development, aggressiveness and response to treatment. Herein, we highlight recent findings and provide an overview of the evidence linking ER stress to the pathogenesis of HCC.

Unfolded Protein Response and Crohn’s Diseases: A Molecular Mechanism of Wound Healing in the Gut

Endoplasmic reticulum (ER) stress triggers a series of signaling and transcriptional events termed the unfolded protein response (UPR). Severe ER stress is associated with the development of fibrosis in different organs, including lung, liver, kidney, heart, and intestine. ER stress is an essential response of epithelial and immune cells in the pathogenesis of Inflammatory Bowel Disease (IBD), including Crohn’s disease (CD). Intestinal epithelial cells are susceptible to ER stress-mediated damage due to secretion of a large amount of proteins that are involved in mucosal defense. In other cells, ER stress is linked to myofibroblast activation, extracellular matrix production, macrophage polarization, and immune cell differentiation. This review focuses on the role of the UPR in the pathogenesis in IBD from an immunologic perspective. The roles of macrophage and mesenchymal cells in the UPR from in vitro and in vivo animal models are discussed. The links between ER stress and other signaling pathways, such as senescence and autophagy, are introduced. Recent advances in the understanding of the epigenetic regulation of the UPR signaling are also updated here. The future directions of development of the UPR research and therapeutic strategies to manipulate ER stress levels are also reviewed.

Chalcone suppresses tumor growth through NOX4-IRE1α sulfonation-RIDD-miR-23b axis

Chalcone is a polyphenolic compound found abundantly in natural plant components. They have been acclaimed as potential antitumor compounds in multiple tumor cells. However, not much attention has been paid to elucidate its antitumor mechanism of action. Here, chalcone was demonstrated to trigger endoplasmic reticulum (ER) stress-induced apoptosis through sulfonation of IRE1α by ER-localized NADPH oxidase 4 (NOX4). IRE1α-sulfonation at a cysteine residue was shown to induce “regulated IRE1α-dependent decay” (RIDD) of mRNA rather than specific splicing of XBP1. The IRE1α sulfonation-induced RIDD degraded miR-23b, enhancing the expression of NOX4. The expression of NOX4 was also upregulated in breast, and prostate cancer tissue. In chalcone-administered mice in vivo, tumor growth was regressed by the consistent mechanisms “NOX4-IRE1α sulfonation-RIDD”. Similarly, NOX4 activation and IRE1α sulfonation were also highly increased under severe ER stress conditions. Together, these findings suggest chalcone as a lead anticancer compound where it acts through NOX4-IRE1α-RIDD-miR-23b axis providing a promising vision of chalcone derivatives’ anticancer mechanism.

Chitosan oligosaccharide attenuates endoplasmic reticulum stress-associated intestinal apoptosis via the Akt/mTOR pathway.

Endoplasmic reticulum stress (ERS) and apoptosis are widely considered as essential factors associated with intestinal disorders, whereas nutritional therapeutic approaches targeting ERS may control disease activity. Thus, we focus on the potential benefit of chitosan oligosaccharide (COS) on repressing ERS and ERS-induced apoptosis. In this study, we used the ERS model with tunicamycin (TM)-induced IPEC-J2 cells in vitro and nutrient deprivation-induced ERS in piglets to evaluate the protective mechanism of COS against ERS and ERS-induced apoptosis. The results showed that cells were characterized by activation of the unfolded protein response (UPR) and increased epithelial apoptosis upon exposure to TM. However, these changes were significantly attenuated by COS and the expressions of Akt and mTORC1 were inhibited. Furthermore, a specific inhibitor of mTOR confirmed the suppression of Akt and reduced the activation of the UPR and apoptosis. In vivo, COS protected against nutrient deprivation-induced ERS in the jejunum of piglets, in which the overexpression of the UPR and apoptosis was rescued. Consistently, COS attenuated nutrient deprivation-induced disruption of intestinal barrier integrity and functional capacity. Together, we provided the first evidence that COS could protect against intestinal apoptosis through alleviating severe ERS, which may be related to the inhibition of the Akt/mTOR signaling pathway.

Thyrocyte cell survival and adaptation to chronic endoplasmic reticulum stress due to misfolded thyroglobulin

The large secretory glycoprotein thyroglobulin is the primary translation product of thyroid follicular cells. This difficult-to-fold protein is susceptible to structural alterations that disable export of the misfolded thyroglobulin from the endoplasmic reticulum (ER), which is a known cause of congenital hypothyroidism characterized by severe chronic thyrocyte ER stress. Nevertheless, individuals with this disease commonly grow a goiter, indicating thyroid cell survival and adaptation. To model these processes, here we continuously exposed rat PCCL3 thyrocytes to tunicamycin, which causes a significant degree of ER stress that is specifically attributable to thyroglobulin misfolding. We found that, in response, PCCL3 cells down-regulate expression of the tunicamycin transporter (major facilitator superfamily domain containing-2A, Mfsd2a). Following CRISPR/Cas9-mediated Mfsd2a deletion, PCCL3 cells could no longer escape the chronic effects of high-dose tunicamycin, as demonstrated by persistent accumulation of unglycosylated thyroglobulin; nevertheless, these thyrocytes survived and grew. A proteomic analysis of these cells adapted to chronic ER protein misfolding revealed many hundreds of up-regulated proteins, indicating stimulation of ER chaperones, oxidoreductases, stress responses, and lipid biosynthesis pathways. Further, we noted increased phospho-AMP-kinase, suggesting up-regulated AMP-kinase activity, and decreased phospho-S6-kinase and protein translation, suggesting decreased mTOR activity. These changes are consistent with conserved cell survival/adaptation pathways. We also observed a less-differentiated thyrocyte phenotype with decreased PAX8, FOXE1, and TPO protein levels, along with decreased thyroglobulin mRNA levels. In summary, we have developed a model of thyrocyte survival and growth during chronic continuous ER stress that recapitulates features of congenital hypothyroid goiter caused by mutant thyroglobulin.

Deficient Endoplasmic Reticulum Acetyl-CoA Import in Pancreatic Acinar Cells Leads to Chronic Pancreatitis.

Background & AimsMaintaining endoplasmic reticulum (ER) proteostasis is essential for pancreatic acinar cell function. Under conditions of severe ER stress, activation of pathogenic unfolded protein response pathways plays a central role in the development and progression of pancreatitis. Less is known, however, of the consequence of perturbing ER-associated post-translational protein modifications on pancreatic outcomes. Here, we examined the role of the ER acetyl-CoA transporter AT-1 on pancreatic homeostasis.MethodsWe used an AT-1S113R/+ hypomorphic mouse model, and generated an inducible, acinar-specific, AT-1 knockout mouse model, and performed histologic and biochemical analyses to probe the effect of AT-1 loss on acinar cell physiology.ResultsWe found that AT-1 expression is down-regulated significantly during both acute and chronic pancreatitis. Furthermore, acinar-specific deletion of AT-1 in acinar cells induces chronic ER stress marked by activation of both the spliced x-box binding protein 1 and protein kinase R-like ER kinase pathways, leading to spontaneous mild/moderate chronic pancreatitis evidenced by accumulation of intracellular trypsin, immune cell infiltration, and fibrosis. Induction of acute-on-chronic pancreatitis in the AT-1 model led to acinar cell loss and glad atrophy.ConclusionsThese results indicate a key role for AT-1 in pancreatic acinar cell homeostasis, the unfolded protein response, and that perturbations in AT-1 function leads to pancreatic disease.

Endoplasmic reticulum stress induces hepatic plasminogen activator inhibitor 1 in murine nonalcoholic steatohepatitis.

Plasminogen activator inhibitor 1 (PAI‐1) is a stress‐responsive gene that is highly induced in nonalcoholic steatohepatitis (NASH). Endoplasmic reticulum (ER) stress is a salient feature of NASH, yet it is unknown whether ER stress contributes to hepatic PAI‐1 induction in this disorder. Therefore, we aimed to (a) establish the role of ER stress in the regulation of hepatic Pai‐1 expression, and (b) determine whether induction of Pai‐1 in murine NASH is driven by ER stress. Hepatic Pai‐1 expression was measured in C57BL/6 J mice and human HepG2 cells subjected to acute or prolonged pharmacologic ER stress. We found that hepatic Pai‐1 expression was acutely suppressed in murine liver in response to severe ER stress followed by marked induction during the recovery phase of the ER stress response. Hepatic Pai‐1 expression was induced in response to prolonged low‐grade ER stress in mice. Induction of PAI‐1 by ER stress in HepG2 cells was prevented by pharmacologic inhibition of MEK1/ERK signaling or by siRNA‐mediated knockdown of XBP1, mediators of the recovery response to ER stress. Inhibiting ER stress with 4‐phenylbutyric acid prevented hepatic Pai‐1 induction in mice with diet‐induced steatohepatitis. We conclude that hepatic Pai‐1 is induced by ER stress via a pathway involving XBP1 and MEK1/ERK signaling, and induction of hepatic Pai‐1 in murine NASH is mediated by ER stress. These data implicate ER stress as a novel mechanistic link between Pai‐1 induction and NASH.

Natural activators of SIRT1 protect cardiac cells from severe ER stress

Perturbations of endoplasmic reticulum (ER) functions result in the accumulation of misfolded proteins inside the ER lumen, a condition known as ER stress. As a compensatory pro-survival response, the unfolded protein response (UPR) is activated to restore normal ER function. However, in the case of severe or chronic ER stress, apoptosis is triggered to eliminate damaged cells. Recently, ER stress was implicated in the pathogenesis of various heart diseases including heart failure (HF). Previously, we have shown that SIRT1 is cardioprotective against severe ER stress by regulating the PERK pathway of the UPR, suggesting that activation of SIRT1 could be effective to limit cardiac ER stress. Different natural polyphenols have been shown to activate SIRT1 in a variety of models. In this study, we thus evaluated the protective effect of 11 polyphenols (butein, fisetin, catechin…) that have been reported to activate SIRT1, against severe ER stress-induced cardiac cell death. Tunicamycin (TN), an inhibitor of N-glycosylation of proteins, was used to induce a severe ER stress in H9c2 cardiomyoblasts and adult rat ventricle myocytes (ARVM). Cell death was determined by fluorescence microscopy and flow cytometry and ER stress was analysed by western blot. As expected, TN induced an increase of the ER stress markers GRP78, GRP94 and the pro-apoptotic transcription factor CHOP associated with cardiac cell death. Three of the tested polyphenols were shown to protect H9c2 cells and ARVM against cell death induced by TN. Protection was abrogated when SIRT1 was inhibited by EX527. In addition, the level of the pro-apoptotic factor CHOP was reduced with polyphenols. These results indicated that among the 11 polyphenols tested, 3 were able to protect cardiac cells from ER stress-induced cell death in a SIRT1-dependent manner. Activation of SIRT1 by selected polyphenols thus appears as a promising strategy to modulate ER stress and protect cardiac cells.

Biological behaviors of mutant proinsulin contribute to the phenotypic spectrum of diabetes associated with insulin gene mutations

Insulin gene mutation is the second most common cause of neonatal diabetes (NDM). It is also one of the genes involved in maturity-onset diabetes of the young (MODY). We aim to investigate molecular behaviors of different INS gene variants that may correlate with the clinical spectrum of diabetes phenotypes. In this study, we concentrated on two previously uncharacterized MODY-causing mutants, proinsulin-p.Gly44Arg [G(B20)R] and p.Pro52Leu [P(B28)L] (a novel mutant identified in one French family), and an NDM causing proinsulin-p.(Cys96Tyr) [C(A7)Y]. We find that these proinsulin mutants exhibit impaired oxidative folding in the endoplasmic reticulum (ER) with blocked ER export, ER stress, and apoptosis. Importantly, the proinsulin mutants formed abnormal intermolecular disulfide bonds that not only involved the mutant proinsulin, but also the co-expressed WT-proinsulin, forming misfolded disulfide-linked proinsulin complexes. This impaired the intracellular trafficking of WT-proinsulin and limited the production of bioactive mature insulin. Notably, although all three mutants presented with similar defects in folding, trafficking, and dominant negative behavior, the degrees of these defects appeared to be different. Specifically, compared to MODY mutants G(B20)R and P(B28)L that partially affected folding and trafficking of co-expressed WT-proinsulin, the NDM mutant C(A7)Y resulted in an almost complete blockade of the ER export of WT-proinsulin, decreasing insulin production, inducing more severe ER stress and apoptosis. We thus demonstrate that differences in cell biological behaviors among different proinsulin mutants correlate with the spectrum of diabetes phenotypes caused by the different INS gene mutations.

Suppressing endoplasmic reticulum stress-related autophagy attenuates retinal light injury.

Excessive light exposure is a principal environmental factor, which can cause damage to photoreceptors and retinal pigment epithelium (RPE) cells and may accelerate the progression of age-related macular degeneration (AMD). In this study, oxidative stress, endoplasmic reticulum (ER) stress and autophagy caused by light exposure were evaluated in vitro and in vivo. Light exposure caused severe photo-oxidative stress and ER stress in photoreceptors (661W cells) and RPE cells (ARPE-19 cells). Suppressing either oxidative stress or ER stress was protective against light damage in 661W and ARPE-19 cells and N-acetyl-L-cysteine treatment markedly inhibited the activation of ER stress caused by light exposure. Moreover, suppressing autophagy with 3-methyladenine significantly attenuated light-induced cell death. Additionally, inhibiting ER stress either by knocking down PERK signals or with GSK2606414 treatment remarkably suppressed prolonged autophagy and protected the cells against light injury. In vivo experiments verified neuroprotection via inhibiting ER stress-related autophagy in light-damaged retinas of mice. In conclusion, the above results suggest that light-induced photo-oxidative stress may trigger subsequent activation of ER stress and prolonged autophagy in photoreceptors and RPE cells. Suppressing ER stress may abrogate over-activated autophagy and protect the retina against light injury.

Ultra-high α-linolenic acid accumulating developmental defective embryo was rescued by lysophosphatidic acid acyltransferase 2.

For decades, genetic engineering approaches to produce unusual fatty acids (UFAs) in crops has reached a bottleneck, including reduced seed oil production and seed vigor. Currently, plant models in the field of research are primarily used to investigate defects in oil production and seedling development, while the role of UFAs in embryonic developmental defects remains unknown. In this study, we developed a transgenic Arabidopsis plant model, in which the embryo exhibits severely wrinkled appearance owing to α-linolenic acid (ALA) accumulation. RNA-sequencing analysis in the defective embryo suggested that brassinosteroid synthesis, FA synthesis and photosynthesis were inhibited, while FA degradation, endoplasmic reticulum stress and oxidative stress were activated. Lipidomics analysis showed that ultra-accumulated ALA is released from phosphatidylcholine as a free FA in cells, inducing severe endoplasmic reticulum and oxidative stress. Furthermore, we identified that overexpression of lysophosphatidic acid acyltransferase 2 rescued the defective phenotype. In the rescue line, the pool capacity of the Kennedy pathway was increased, and the esterification of ALA indirectly to triacylglycerol was enhanced to avoid stress. This study provides a plant model that aids in understanding the molecular mechanism of embryonic developmental defects and generates strategies to produce higher levels of UFAs.

Effects of fatty acids on inducing endoplasmic reticulum stress in bovine mammary epithelial cells

Fatty acids play important roles in the regulation of endoplasmic reticulum (ER) stress-induced apoptosis in different cells. Currently, the effects of fatty acids on bovine mammary epithelial cells (MEC) remain unknown. Our study examined bovine MEC viability and measured unfolded protein response (UPR)-related gene and protein expressions following fatty acid treatments. To evaluate the role of fatty acids, we treated MAC-T cells (a line of MEC) with 100 to 400 μM of saturated (palmitic and stearic acid) and unsaturated (palmitoleic, oleic, linoleic, and linolenic acid) fatty acids and 1 to 5 mM of short- and medium-chain fatty acids (acetic, propionic, butyric, and octanoic acid). Thereafter, we determined UPR-related gene expression using quantitative real-time PCR. Palmitic acid stimulated expression of XBP1s, ATF4, ATF6A, and C/EBP homologous protein (CHOP). Stearic acid increased expression of XBP1s and CHOP and decreased expression of ATF4 and ATF6A. Results of Western blot analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that palmitic and stearic acid reduced MAC-T cell viability and induced extreme ER stress by increasing the protein expression of ER stress markers, such as phospho-PKR-like endoplasmic reticulum kinase, phospho-eIF2α, cleaved CASP-3, and CHOP. Among unsaturated long-chain fatty acids, palmitoleic acid increased expression of ATF4 and ATF6A. Oleic acid increased expression of XBP1s, ATF4, and ATF6A. Linoleic and linolenic acids increased expression of XBP1s, ATF4, and ATF6A but decreased expression of XBP1s and ATF6A at the highest dose. Although palmitoleic, oleic, and linoleic acid decreased CHOP expression, only palmitoleic acid increased MAC-T cell viability. Therefore, unsaturated long-chain fatty acids did not induce severe ER stress. Acetic, propionic, and butyric acids decreased expression of ATF4, ATF6A, and CHOP and increased XBP1s expression. Although only octanoic acid increased ATF4 and ATF6A expressions, it lowered expression of XBP1s and CHOP. Although fatty acid treatment did not increase the levels of ER stress proteins, butyric and octanoic acids reduced cell viability, possibly because of early differentiation. These results suggest that saturated fatty acids play important roles in MEC viability by inducing severe ER stress compared with unsaturated fatty acids. In addition, acetic and propionic acids (short- and medium-chain fatty acids) reduced ER stress. Therefore, the present study reflects the new insight that serum fatty acid concentration plays an important role in maintaining the lactation physiology of dairy cows.