Reduced Expression of Oligodendrocyte Linage-Enriched Transcripts During the Endoplasmic Reticulum Stress/Integrated Stress Response.

Endoplasmic reticulum (ER) stress in oligodendrocyte (OL) linage cells contributes to several CNS pathologies including traumatic spinal cord injury (SCI) and multiple sclerosis. Therefore, primary rat OL precursor cell (OPC) transcriptomes were analyzed using RNASeq after treatments with two ER stress-inducing drugs, thapsigargin (TG) or tunicamycin (TM). Gene ontology term (GO) enrichment showed that both drugs upregulated mRNAs associated with the general stress response. The GOs related to ER stress were only enriched for TM-upregulated mRNAs, suggesting greater ER stress selectivity of TM. Both TG and TM downregulated cell cycle/cell proliferation-associated transcripts, indicating the anti-proliferative effects of ER stress. Interestingly, many OL lineage-enriched mRNAs were downregulated, including those for transcription factors that drive OL identity such as Olig2. Moreover, ER stress-associated decreases of OL-specific gene expression were found in mature OLs from mouse models of white matter pathologies including contusive SCI, toxin-induced demyelination, and Alzheimer's disease-like neurodegeneration. Taken together, the disrupted transcriptomic fingerprint of OL lineage cells may facilitate myelin degeneration and/or dysfunction when pathological ER stress persists in OL lineage cells.

The ER stress response compromises the transcriptomic identity of the OL lineage. Therefore, persistent, pathological ER stress may have a negative impact on structural and/or functional integrity of the white matter.

Impaired myoblast differentiation and muscle IGF-1 receptor signaling pathway activation after N-glycosylation inhibition.

The role of N-glycosylation in the myogenic process remains poorly understood. Here, we evaluated the impact of N-glycosylation inhibition by Tunicamycin (TUN) or by phosphomannomutase 2 (PMM2) gene knockdown, which encodes an enzyme essential for catalyzing an early step of the N-glycosylation pathway, on C2C12 myoblast differentiation. The effect of chronic treatment with TUN on tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of WT and MLC/mIgf-1 transgenic mice, which overexpress muscle Igf-1Ea mRNA isoform, was also investigated. TUN-treated and PMM2 knockdown C2C12 cells showed reduced ConA, PHA-L, and AAL lectin binding and increased ER-stress-related gene expression (Chop and Hspa5 mRNAs and s/uXbp1 ratio) compared to controls. Myogenic markers (MyoD, myogenin, and Mrf4 mRNAs and MF20 protein) and myotube formation were reduced in both TUN-treated and PMM2 knockdown C2C12 cells. Body and TA weight of WT and MLC/mIgf-1 mice were not modified by TUN treatment, while lectin binding slightly decreased in the TA muscle of WT (ConA and AAL) and MLC/mIgf-1 (ConA) mice. The ER-stress-related gene expression did not change in the TA muscle of WT and MLC/mIgf-1 mice after TUN treatment. TUN treatment decreased myogenin mRNA and increased atrogen-1 mRNA, particularly in the TA muscle of WT mice. Finally, the IGF-1 production and IGF1R signaling pathways activation were reduced due to N-glycosylation inhibition in TA and EDL muscles. Decreased IGF1R expression was found in TUN-treated C2C12 myoblasts which was associated with lower IGF-1-induced IGF1R, AKT, and ERK1/2 phosphorylation compared to CTR cells. Chronic TUN-challenge models can help to elucidate the molecular mechanisms through which diseases associated with aberrant N-glycosylation, such as Congenital Disorders of Glycosylation (CDG), affect muscle and other tissue functions.

Investigation of the influence of pH and temperature on melanization and survival under oxidative stress in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic pathogenic fungus that produces melanin during infection, an important virulence factor in Cryptococcal infections that enhances the ability of the fungus to resist immune defense. This fungus can synthesize melanin from a variety of substrates, including L-DOPA (L-3,4-dihydroxyphenylalanine). Since melanin protects the fungus from various stress factors such as oxidative, nitrosative, extreme heat and cold stress; we investigated the effects of environmental conditions on melanin production and survival. In this study, we investigated the effects of different pH values (5.6, 7.0 and 8.5) and temperatures (30 °C and 37 °C) on melanization and cell survival using a microtiter plate-based melanin production assay and an oxidative stress assay, respectively. In addition, the efficacy of compounds known to inhibit laccase involved in melanin synthesis, i.e., tunicamycin, ß-mercaptoethanol, dithiothreitol, sodium azide and caspofungin on melanization was evaluated and their sensitivity to temperature and pH changes was measured. The results showed that melanin content correlated with pH and temperature changes and that pH 8.5 and 30 °C, were best for melanin production. Besides that, melanin production protects the fungal cells from oxidative stress induced by hydrogen peroxide. Thus, changes in pH and temperature drastically alter melanin production in C. neoformans and it correlates with the fungal survival. Due to the limited antifungal repertoire and the development of resistance in cryptococcal infections, the investigation of environmental conditions in the regulation of melanization and survival of C. neoformans could be useful for future research and clinical phasing.

TXNDC5 Plays a Crucial Role in Regulating Endoplasmic Reticulum Activity through Different ER Stress Signaling Pathways in Hepatic Cells.

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is influenced by a number of variables, including endoplasmic reticulum stress (ER). Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family and acts as an endoplasmic reticulum (ER) chaperone. Nevertheless, the function of TXNDC5 in hepatocytes under ER stress remains largely uncharacterized. In order to identify the role of TXNDC5 in hepatic wild-type (WT) and TXNDC5-deficient (KO) AML12 cell lines, tunicamycin, palmitic acid, and thapsigargin were employed as stressors. Cell viability, mRNA, protein levels, and mRNA splicing were then assayed. The protein expression results of prominent ER stress markers indicated that the ERN1 and EIF2AK3 proteins were downregulated, while the HSPA5 protein was upregulated. Furthermore, the ATF6 protein demonstrated no significant alterations in the absence of TXNDC5 at the protein level. The knockout of TXNDC5 has been demonstrated to increase cellular ROS production and its activity is required to maintain normal mitochondrial function during tunicamycin-induced ER stress. Tunicamycin has been observed to disrupt the protein levels of HSPA5, ERN1, and EIF2AK3 in TXNDC5-deficient cells. However, palmitic acid has been observed to disrupt the protein levels of ATF6, HSPA5, and EIF2AK3. In conclusion, TXNDC5 can selectively activate distinct ER stress pathways via HSPA5, contingent on the origin of ER stress. Conversely, the absence of TXNDC5 can disrupt the EIF2AK3 cascade.

ATF4-mediated different mode of interaction between autophagy and mTOR determines cell fate dependent on the level of ER stress induced by Cr(VI).

Hexavalent chromium [Cr(VI)] exists widely in occupational environments. The mechanistic target of rapamycin (mTOR) has been well-documented to regulate autophagy negatively. However, we found that low concentration of Cr(VI) (0.2 µM) elevated both mTOR and autophagy and promote cell survival. Conversely, high concentration of Cr(VI) (6 µM) caused cell death by inhibiting mTOR and subsequently inducing autophagy. Tunicamycin (Tm), as an Endoplasmic reticulum (ER) stress activator was used to induce mild ER stress at 0.1 µg/ml and it activated both autophagy and mTOR, which also caused cell migration in a similar manner to that observed with low concentration of Cr(VI). Severe ER stress caused by Tm (2 µg/ml) decreased mTOR, increased autophagy and then inhibited cell migration, which was the same as 6 µM Cr(VI) treatment, although Cr(VI) in high concentration inhibited ER stress. Activating transcription factor 4 (ATF4), a downstream target of ER stress, only increased under mild ER stress but decreased under severe ER stress and 6 µM Cr(VI) treatment. Chromatin immunoprecipitation (ChIP) experiment indicated that ATF4 could bind to the promoter of ATG4B and AKT1. To sum up, our data revealed that mild ER stress induced by low concentration of Cr(VI) could enhance transcriptional regulation of ATG4B and AKT1 by ATF4, which induced both autophagy and mTOR to promote cell viability.

Drug-induced ER stress leads to induction of programmed cell death pathways of the malaria parasite.

The rapid emergence of drug resistance against the mainstream antimalarial drugs has increased the need for development of novel drugs. Recent approaches have embarked on the repurposing of existing drugs to induce cell death via programmed cell death pathways. However, little is known about the ER stress response and programmed cell death pathways of the malaria parasite. In this study, we treated ex vivo Plasmodium berghei cultures with tunicamycin, 5-fluorouracil, and chloroquine as known stress inducer drugs to probe the transcriptional changes of autophagy and apoptosis-related genes (PbATG5, PbATG8, PbATG12, and PbMCA2). Treatments with 5-fluorouracil and chloroquine resulted in the upregulation of all analyzed markers, yet the levels of PbATG5 and PbATG12 were dramatically higher in chloroquine-treated ex vivo cultures. In contrast, tunicamycin treatment resulted in the downregulation of both PbATG8 and PbATG12, and upregulation of PbMCA2. Our results indicate that the malaria parasite responds to various ER stressors by inducing autophagy- and/or apoptosis-like pathways.

Tunicamycins from Marine-Derived Streptomyces bacillaris Inhibit MurNAc-Pentapeptide Translocase in Staphylococcus aureus.

Four tunicamycin class compounds, tunicamycin VII (1), tunicamycin VIII (2), corynetoxin U17a (3), and tunicamycin IX (4), were isolated from the culture broth of the marine-derived actinomycete Streptomyces sp. MBTG32. The strain was identified using the 16S rDNA sequencing technique, and the isolated strain was closely related to Streptomyces bacillaris. The structures of the isolated compounds were elucidated based on spectroscopic data and comparisons with previously reported NMR data. Compounds 1-4 showed potent antibacterial activities against Gram-positive bacteria, especially Staphylococcus aureus, with MIC values of 0.13-0.25 µg/mL. Through a recombinant enzyme assay and overexpression analysis, we found that the isolated compounds exerted potent inhibitory effects on S. aureus MurNAc-pentapeptide translocase (MraY), with IC50 values of 0.08-0.21 µg/mL. The present results support that the underlying mechanism of action of tunicamycins isolated from marine-derived Streptomyces sp. is also associated with the inhibition of MraY enzyme activity in S. aureus.

ER stress-induced transcriptional response reveals tolerance genes in yeast.

Saccharomyces cerevisiae is important for protein secretion studies, yet the complexities of protein synthesis and secretion under endoplasmic reticulum (ER) stress conditions remain not fully understood. ER stress, triggered by alterations in the ER protein folding environment, poses substantial challenges to cells, especially during heterologous protein production. In this study, we used RNA-seq to analyze the transcriptional responses of yeast strains to ER stress induced by reagents such as tunicamycin (Tm) or dithiothreitol (DTT). Our gene expression analysis revealed several crucial genes, such as HMO1 and BIO5, that are involved in ER-stress tolerance. Through metabolic engineering, the best engineered strain R23 with HMO1 overexpression and BIO5 deletion, showed enhanced ER stress tolerance and improved protein folding efficiency, leading to a 2.14-fold increase in α-amylase production under Tm treatment and a 2.04-fold increase in cell density under DTT treatment. Our findings contribute to the understanding of cellular responses to ER stress and provide a basis for further investigations into the mechanisms of ER stress at the cellular level.

Quantitative proteomics reveals cellular responses to individual mAb expression and tunicamycin in CHO cells.

Chinese hamster ovary (CHO) cells are popular in the pharmaceutical industry for their ability to produce high concentrations of antibodies and their resemblance to human cells in terms of protein glycosylation patterns. Current data indicate the relevance of CHO cells in the biopharmaceutical industry, with a high number of product commendations and a significant market share for monoclonal antibodies. To enhance the production capabilities of CHO cells, a deep understanding of their cellular and molecular composition is crucial. Genome sequencing and proteomic analysis have provided valuable insights into the impact of the bioprocessing conditions, productivity, and product quality. In our investigation, we conducted a comparative analysis of proteomic profiles in high and low monoclonal antibody-producing cell lines and studied the impact of tunicamycin (TM)-induced endoplasmic reticulum (ER) stress. We examined the expression levels of different proteins including unfolded protein response (UPR) target genes by using label-free quantification techniques for protein abundance. Our results show the upregulation of proteins associated with protein folding mechanisms in low producer vs. high producer cell line suggesting a form of ER stress related to specific protein production. Further, Hspa9 and Dnaja3 are notable candidates activated by the mitochondria UPR and play important roles in protein folding processes in mitochondria. We identified significant upregulation of Nedd8 and Lgmn proteins in similar levels which may contribute to UPR stress. Interestingly, the downregulation of Hspa5/Bip and Pdia4 in response to tunicamycin treatment suggests a low-level UPR activation. KEY POINTS: • Proteome profiling of recombinant CHO cells under mild TM treatment. • Identified protein clusters are associated with the unfolded protein response (UPR). • The compared cell lines revealed noticeable disparities in protein expression levels.

bZIP60 and Bax inhibitor 1 contribute IRE1-dependent and independent roles to potexvirus infection.

IRE1, BI-1, and bZIP60 monitor compatible plant-potexvirus interactions though recognition of the viral TGB3 protein. This study was undertaken to elucidate the roles of three IRE1 isoforms, the bZIP60U and bZIP60S, and BI-1 roles in genetic reprogramming of cells during potexvirus infection. Experiments were performed using Arabidopsis thaliana knockout lines and Plantago asiatica mosaic virus infectious clone tagged with the green fluorescent protein gene (PlAMV-GFP). There were more PlAMV-GFP infection foci in ire1a/b, ire1c, bzip60, and bi-1 knockout than wild-type (WT) plants. Cell-to-cell movement and systemic RNA levels were greater bzip60 and bi-1 than in WT plants. Overall, these data indicate an increased susceptibility to virus infection. Transgenic overexpression of AtIRE1b or StbZIP60 in ire1a/b or bzip60 mutant background reduced virus infection foci, while StbZIP60 expression influences virus movement. Transgenic overexpression of StbZIP60 also confers endoplasmic reticulum (ER) stress resistance following tunicamycin treatment. We also show bZIP60U and TGB3 interact at the ER. This is the first demonstration of a potato bZIP transcription factor complementing genetic defects in Arabidopsis. Evidence indicates that the three IRE1 isoforms regulate the initial stages of virus replication and gene expression, while bZIP60 and BI-1 contribute separately to virus cell-to-cell and systemic movement.

SIRT1 regulates hepatic vldlr levels.

BACKGROUND: Endoplasmic reticulum (ER) stress-mediated increases in the hepatic levels of the very low-density lipoprotein (VLDL) receptor (VLDLR) promote hepatic steatosis by increasing the delivery of triglyceride-rich lipoproteins to the liver. Here, we examined whether the NAD(+)-dependent deacetylase sirtuin 1 (SIRT1) regulates hepatic lipid accumulation by modulating VLDLR levels and the subsequent uptake of triglyceride-rich lipoproteins. METHODS: Rats fed with fructose in drinking water, Sirt1-/- mice, mice treated with the ER stressor tunicamycin with or without a SIRT1 activator, and human Huh-7 hepatoma cells transfected with siRNA or exposed to tunicamycin or different inhibitors were used. RESULTS: Hepatic SIRT1 protein levels were reduced, while those of VLDLR were upregulated in the rat model of metabolic dysfunction-associated steatotic liver disease (MASLD) induced by fructose-drinking water. Moreover, Sirt1-/- mice displayed increased hepatic VLDLR levels that were not associated with ER stress, but were accompanied by an increased expression of hypoxia-inducible factor 1α (HIF-1α)-target genes. The pharmacological inhibition or gene knockdown of SIRT1 upregulated VLDLR protein levels in the human Huh-7 hepatoma cell line, with this increase abolished by the pharmacological inhibition of HIF-1α. Finally, SIRT1 activation prevented the increase in hepatic VLDLR protein levels in mice treated with the ER stressor tunicamycin. CONCLUSIONS: Overall, these findings suggest that SIRT1 attenuates fatty liver development by modulating hepatic VLDLR levels.

Hhatl ameliorates endoplasmic reticulum stress through autophagy by associating with LC3.

Endoplasmic reticulum (ER) stress, a common cellular stress response induced by various factors that interfere with cellular homeostasis, may trigger cell apoptosis. Autophagy is an important and conserved mechanism for eliminating aggregated proteins and maintaining protein stability of cells, which is closely associated with ER stress and ER stress-induced apoptosis. In this paper, we report for the first time that Hhatl, an ER-resident protein, is downregulated in response to ER stress. Hhatl overexpression alleviated ER stress and ER stress induced apoptosis in cells treated with tunicamycin or thapsigargin, whereas Hhatl knockdown exacerbated ER stress and apoptosis. Further study showed that Hhatl attenuates ER stress by promoting autophagic flux. Mechanistically, we found that Hhatl promotes autophagy by associating with autophagic protein LC3 (microtubule-associated protein 1A/1B-light chain 3) via the conserved LC3-interacting region motif. Noticeably, the LC3-interacting region motif was essential for Hhatl-regulated promotion of autophagy and reduction of ER stress. These findings demonstrate that Hhatl ameliorates ER stress via autophagy activation by interacting with LC3, thereby alleviating cellular pressure. The study indicates that pharmacological or genetic regulation of Hhatl-autophagy signaling might be potential for mediating ER stress and related diseases.

Pleiotropic role of endoplasmic reticulum stress in the protection of psoralidin against sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is a severe complication that affects the central nervous system and is a leading cause of increased morbidity and mortality in intensive care units. Psoralidin (PSO), a coumarin compound isolated from the traditional Chinese medicine Psoralea corylifolia L., can penetrate the blood-brain barrier and has various pharmacological activities, including anti-inflammation, anti-oxidation and anti-depression. This study aims to explore whether PSO alleviates SAE and delve into the underlying mechanisms. We found that PSO treatment significantly reduced sepsis scores, aspartate transaminase (AST) and aspartate transaminase (LDH), while increased anal temperature and neurological scores in CLP-injured mice. Moreover, PSO treatment ameliorated sepsis-associated cognitive impairment, mood, anxiety disorders, inhibited inflammatory responses, as well as attenuated endoplasmic reticulum stress (ERS). These results were also validated in vitro experiments, PSO treatment reduced ROS, inflammation response, and attenuated ERS in LPS-injured N2a cells. Importantly, tunicamycin (TUN), as ERS agonist, significantly reversed the protective effect of PSO on LPS-injured N2a cells, as evidenced by increased expression levels of IL-6, NLRP3, CHOP, and ATF6. Likewise, ATF6 overexpression also reversed the protective effect of PSO. In conclusion, these results confirmed that PSO has a protective effect on SAE, which was largely attributed to neuroinflammation and ERS. These findings provide new insights into the neuroprotective role of PSO and suggest that PSO is a new therapeutic intervention of SAE.

Thapsigargin and Tunicamycin Block SARS-CoV-2 Entry into Host Cells via Differential Modulation of Unfolded Protein Response (UPR), AKT Signaling, and Apoptosis.

BACKGROUND: SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in host cells, which may contribute to the pathogenesis of COVID-19. To understand the complex interplay between SARS-Co-V2 infection and UPR signaling, we examined the effects of acute pre-existing ER stress on SARS-Co-V2 infectivity. METHODS: Huh-7 cells were treated with Tunicamycin (TUN) and Thapsigargin (THA) prior to SARS-CoV-2pp transduction (48 h p.i.) to induce ER stress. Pseudo-typed particles (SARS-CoV-2pp) entry into host cells was measured by Bright GloTM luciferase assay. Cell viability was assessed by cell titer Glo® luminescent assay. The mRNA and protein expression was evaluated by RT-qPCR and Western Blot. RESULTS: TUN (5 µg/mL) and THA (1 µM) efficiently inhibited the entry of SARS-CoV-2pp into host cells without any cytotoxic effect. TUN and THA's attenuation of virus entry was associated with differential modulation of ACE2 expression. Both TUN and THA significantly reduced the expression of stress-inducible ER chaperone GRP78/BiP in transduced cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated with THA treatment, but not TUN in transduced cells. Insulin-mediated glucose uptake and phosphorylation of Ser307 IRS-1 and downstream p-AKT were enhanced with THA in transduced cells. Furthermore, TUN and THA differentially affected lipid metabolism and apoptotic signaling pathways. CONCLUSIONS: These findings suggest that short-term pre-existing ER stress prior to virus infection induces a specific UPR response in host cells capable of counteracting stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.

Aß1-42 stimulates an increase in autophagic activity through tunicamycin-induced endoplasmic reticulum stress in HTR-8/SVneo cells and late-onset pre-eclampsia.

Environmental changes can trigger endoplasmic reticulum (ER) stress and misfolded protein accumulation, potentially leading to pre-eclampsia (PE). Amyloid-ß (Aß) is a crucial misfolded protein that can overactivate autophagy. Our study assessed the expression of Aß1-42 and autophagic activity in PE placental tissues and trophoblasts under ER stress. Placental tissues were surgically collected from normal pregnant women (NP) and pregnant women with late-onset PE (LOPE) delivering through cesarean section. The expression levels of Aß1-42 were detected in both PE and NP placental tissues, as well as in tunicamycin (TM)-induced HTR-8/SVneo cells. Autophagy-related proteins, such as Beclin-1, the ratio of LC3-II to LC3-I, ATG5, and SQSTM1/p62 in the placental tissues and HTR-8/SVneo cells were measured by Western blot. The number and morphology of autophagosomes were observed using transmission electron microscopy (TEM). Potential targets associated with the unfolded protein response (UPR) in the placental tissues of NP and PE cases were screened using PCR Arrays. The misfolded protein was significantly upregulated in the PE group. In both PE placental tissues and TM-induced HTR-8/SVneo cells, not only was Aß1-42 upregulated, but also Beclin-1, ATG5, and LC3BII/I were significantly increased, accompanied by an increase in autophagosome count, while SQSTM1/P62 was downregulated. A total of 17 differentially expressed genes (DEGs) associated with the UPR were identified, among which elevated calnexin (CANX) was validated in the placenta from both PE and TM-induced HTR-8/SVneo cells. Autophagy is significantly upregulated in PE cases due to ER stress-induced Aß1-42 accumulation, likely mediated by autophagy-related proteins involved in the UPR.

Endoplasmic Reticulum Stress Induces Vasodilation in Liver Vessels That Is Not Mediated by Unfolded Protein Response.

There is a growing body of evidence that ER stress and the unfolded protein response (UPR) play a key role in numerous diseases. Impaired liver perfusion and ER stress often accompany each other in liver diseases. However, the exact impact of ER stress and UPR on the hepatic perfusion is not fully understood. The aim of this study was to disclose the effect of ER stress and UPR on the size of liver vessels and on the levels of Ca2+ and nitric oxide (NO), critical regulators of vascular tonus. This study was carried out in precisely cut liver tissue slices. Confocal microscopy was used to create 3D images of vessels. NO levels were determined either using either laser scan microscopy (LSM) in cells or by NO-analyser in medium. Ca2+ levels were analysed by LSM. We show that tunicamycin, an inducer of ER stress, acts similarly with vasodilator acetylcholine. Both exert a similar effect on the NO and Ca2+ levels; both induce significant vasodilation. Notably, this vasodilative effect persisted despite individual inhibition of UPR pathways-ATF-6, PERK, and IRE1-despite confirming the activation of UPR. Experiments with HUVEC cells showed that elevated NO levels did not result from endothelial NO synthase (eNOS) activation. Our study suggests that tunicamycin-mediated ER stress induces liver vessel vasodilation in an NO-dependent manner, which is mediated by intracellular nitrodilator-activatable NO store (NANOS) in smooth muscle cells rather than by eNOS.

Ghrelin regulates the endoplasmic reticulum stress signalling pathway in gestational diabetes mellitus.

OBJECTIVE: We aimed to investigate the effects and mechanisms of the ghrelin-regulated endoplasmic reticulum stress (ERS) signalling pathway in gestational diabetes mellitus (GDM). METHODS: Pregnant female C57BL/6 mice were randomly divided into a normal group, GDM group (high-fat diet + STZ), GDM + ghrelin group (acyl ghrelin), and GDM + ghrelin + ghrelin inhibitor group ([D-lys3]-GHRP-6). We measured body weight, the intake of water and food, glucose, cholesterol, triglyceride and fasting insulin levels in each group. HE staining was used to observe the morphological changes in the pancreas. The TUNEL method was used to detect the apoptosis rate of islet cells. qPCR and Western boltting were performed to detect the relative expression levels of PERK, ATF6, IREIα, GRP78, CHOP and caspase-12, which are related to the ERS signalling pathway in the pancreas. Then, NIT-1 cells were cultured to verify whether ghrelin regulates ERS under high-glucose or tunicamycin conditions. RESULTS: Compared with the GDM group, the GDM + ghrelin group showed improved physical conditions and significantly decreased the fasting blood glucose, glucose tolerance, cholesterol, triglyceride and fasting insulin levels. Damaged islet areas were inhibited by ghrelin in the GDM group. The GDM + ghrelin group showed reduced ß-cell apoptosis compared to the GDM and GDM + ghrelin + ghrelin inhibitor groups. ERS-associated factors (PERK, ATF6, IREIα, GRP78, CHOP and caspase-12) mRNA and protein levels were obviously lower in the GDM + ghrelin group than in the GDM group, while expression levels were restored in the inhibitor group. Ghrelin treatment improved the high-glucose or tunicamycin-induced apoptosis, increased insulin levels and upregulation of GRP78, CHOP and caspase-12 in NIT-1 cells. CONCLUSION: Ghrelin suppressed ERS signalling and apoptosis in GDM mice and in NIT-1 cells. This study established a link between ghrelin and GDM, and the targeting of ERS with ghrelin represents a promising therapeutic strategy for GDM.

Resolvin D1 alleviates apoptosis triggered by endoplasmic reticulum stress in IPEC-J2 cells.

BACKGROUND: Resolvin D1 (RvD1), a specialized pro-resolving lipid mediator (SPM), is derived from docosahexaenoic acid (DHA). It plays a key role in actively resolving inflammatory responses, which further reduces small intestinal damage. However, its regulation of the apoptosis triggered by endoplasmic reticulum (ER) stress in intestinal epithelial cells is still poorly understood. The intestinal porcine epithelial cells (IPEC-J2) were stimulated with tunicamycin to screen an optimal stimulation time and concentration to establish an ER stress model. Meanwhile, RvD1 (0, 1, 10, 20, and 50 nM) cytotoxicity and its impact on cell viability and the effective concentration for reducing ER stress and apoptosis were determined. Finally, the effects of RvD1 on ER stress and associated apoptosis were furtherly explored by flow cytometry analysis, AO/EB staining, RT-qPCR, and western blotting. RESULTS: The ER stress model of IPEC-J2 cells was successfully built by stimulating the cells with 1 µg/mL tunicamycin for 9 h. Certainly, the increased apoptosis and cell viability inhibition also appeared under the ER stress condition. RvD1 had no cytotoxicity, and its concentration of 1 nM significantly decreased cell viability inhibition (p= 0.0154) and the total apoptosis rate of the cells from 14.13 to 10.00% (p= 0.0000). RvD1 at the concentration of 1 nM also significantly reduced the expression of glucose-regulated protein 78 (GRP-78, an ER stress marker gene) (p= 0.0000) and pro-apoptotic gene Caspase-3 (p= 0.0368) and promoted the expression of B cell lymphoma 2 (Bcl-2, an anti-apoptotic gene)(p= 0.0008). CONCLUSIONS: Collectively, the results shed light on the potential of RvD1 for alleviating apoptosis triggered by ER stress, which may indicate an essential role of RvD1 in maintaining intestinal health and homeostasis.

ATF6 supports lysosomal function in tumor cells to enable ER stress-activated macroautophagy and CMA: impact on mutant TP53 expression.

The inhibition of the unfolded protein response (UPR), which usually protects cancer cells from stress, may be exploited to potentiate the cytotoxic effect of drugs inducing ER stress. However, in this study, we found that ER stress and UPR activation by thapsigargin or tunicamycin promoted the lysosomal degradation of mutant (MUT) TP53 and that the inhibition of the UPR sensor ATF6, but not of ERN1/IRE1 or EIF2AK3/PERK, counteracted such an effect. ATF6 activation was indeed required to sustain the function of lysosomes, enabling the execution of chaperone-mediated autophagy (CMA) as well as of macroautophagy, processes involved in the degradation of MUT TP53 in stressed cancer cells. At the molecular level, by pharmacological and genetic approaches, we demonstrated that the inhibition of ATF6 correlated with the activation of MTOR and with TFEB and LAMP1 downregulation in thapsigargin-treated MUT TP53 carrying cells. We hypothesize that the rescue of MUT TP53 expression by ATF6 inhibition, could further activate MTOR and maintain lysosomal dysfunction, further inhibiting MUT TP53 degradation, in a vicious circle. The findings of this study suggest that the presence of MUT TP53, which often exerts oncogenic properties, should be considered before approaching treatments combining ER stressors with ATF6 inhibitors against cancer cells, while it could represent a promising strategy against cancer cells that harbor WT TP53.

Melatonin alleviates endoplasmic reticulum stress and follicular granulosa cell apoptosis by regulating ATF4 to activate mTOR signaling pathway in chickens.

Follicular atresia in chickens reduces the number of follicles that can further develop, leading to decrease egg laying. Endoplasmic reticulum stress (ERS) can initiate a unique pathway inducing the apoptosis of follicular granulosa cells, thus reducing egg laying. Melatonin (MEL) is involved in the regulation of follicle development, ovulation, and oocyte maturation, and is closely related to follicle fate. Mammalian target of Rapamycin (mTOR) signaling pathway plays an important role in cell growth regulation, and that there is a possible crosstalk between melatonin and mTOR activity in granular cells maturation and ovulation. This study aimed to investigate whether MEL inhibits ERS and follicular granulosa cell apoptosis by regulating ATF4 to activate mTOR signaling pathway in chickens. Frist, we established an in vitro ERS cell model using tunicamycin (TM). The results showed that different concentrations of TM exhibited dose-dependent inhibition of cell activity and induction of granulosa cells (P<0.01). Therefore, we chose 5 µg/mL of TM and a treatment time for 6 h as the optimal concentration for the following experiments. Then we investigate whether melatonin can inhibit ERS. TM treatment decreased the cell viability and Bcl-2 expression, increasing ROS levels and the mRNA expression of Grp78, ATF4, CHOP, PERK, eIF-2α, and BAX (P<0.01), whereas TM+MEL treatment significantly inhibited these changes (P<0.01). Then we explored whether melatonin protects follicular granulosa cells from ERS-induced apoptosis through the mammalian target of rapamycin (mTOR) signaling pathway by regulating ATF4, we found that ATF4 knockdown inhibited ERS by decreasing the expression of ERS-related genes and proteins and activating mTOR signaling pathway by increasing the protein expression of p4E-BP1 and pT389-S6K (P<0.001), while these changes were promoted by TM+si-ATF4+MEL treatment (P<0.01). These results indicate that MEL could alleviate TM-induced ERS by regulating ATF4 to activate mTOR signaling pathway in follicular granulosa cells, thus providing a new perspective for prolonging the laying cycle in chickens.