Endoplasmic Reticulum Stress Marker Genes Research Articles

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

BackgroundResolvin 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.ResultsThe 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).ConclusionsCollectively, 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.

Seasonal changes in hepatic lipid metabolism and apoptosis in Chinese soft-shelled turtle (Pelodiscus sinensis)

Chinese soft-shelled turtle (Pelodiscus sinensis) hibernates without eating and drinking when the ambient temperature is very low. To better understand the characteristics of energy utilization during hibernation, the turtles in the physiological phases of summer active (SA), Pre-Hibernation (Pre-H), Mid-Hibernation (Mid-H) and early arousal (EA) were sampled. The results showed that the levels of serum triglyceride and hepatic lipid droplet were markedly increased in Pre-H and decreased in Mid-H compared with that in SA, indicating that P. sinensis experiences lipid accumulation in Pre-H and lipid is the predominant energy reserve during hibernation. The mRNA expression levels of genes (FABP and CPT-2) involved in lipolysis and lipid oxidation were up-regulated in Mid-H, while the genes related to lipid synthesis (FAS, ACSL-1, ACC, elovl5, and SCD1) were inhibited in Mid-H. Meanwhile, the mRNA expression levels of endoplasmic reticulum stress marker gene Bip and key genes (ATF4, ATF6, and IRE1α) involving the unfolded protein response were significantly increased in Mid-H and EA. Also, the expression levels of genes (ASK1, JNK1, and Bax) associated with cell apoptosis increased in Mid-H and EA, however, the expression of Bcl2 was inhibited in Mid-H. Therefore, hibernation can cause endoplasmic reticulum stress and apoptosis. The findings will provide a theoretical framework for an animal's cold adaptation and offer insights into preventing and managing metabolic syndrome.

Hepatotoxicity of cyanotoxin microcystin-LR in human: Insights into mechanisms of action in the 3D culture model Hepoid-HepaRG

Microcystin-LR (MC-LR) is a potent hepatotoxin produced by harmful cyanobacterial blooms (CyanoHABs). MC-LR targets highly differentiated hepatocytes expressing organic anion transporting polypeptides OATP1B1 and OATP1B3 that are responsible for hepatocellular uptake of the toxin. The present study utilized an advanced 3D in vitro human liver model Hepoid-HepaRG based on the cultivation of collagen-matrix embedded multicellular spheroids composed of highly differentiated and polarized hepatocyte-like cells. 14-d-old Hepoid-HepaRG cultures showed increased expression of OATP1B1/1B3 and sensitivity to MC-LR cytotoxicity at concentrations >10 nM (48 h exposure, EC20 = 26 nM). MC-LR induced neither caspase 3/7 activity nor expression of the endoplasmic reticulum stress marker gene BiP/GRP78, but increased release of pro-inflammatory cytokine IL-8, indicating a necrotic type of cell death. Subcytotoxic (10 nM) and cytotoxic (≥100 nM) MC-LR concentrations disrupted hepatocyte functions, such as xenobiotic metabolism phase-I enzyme activities (cytochrome P450 1A/1B) and albumin secretion, along with reduced expression of CYP1A2 and ALB genes. MC-LR also decreased expression of HNF4A gene, a critical regulator of hepatocyte differentiation and function. Genes encoding hepatobiliary membrane transporters (OATP1B1, BSEP, NTCP), hepatocyte gap junctional gene connexin 32 and the epithelial cell marker E-cadherin were also downregulated. Simultaneous upregulation of connexin 43 gene, primarily expressed by liver progenitor and non-parenchymal cells, indicated a disruption of tissue homeostasis. This was associated with a shift in the expression ratio of E-cadherin to N-cadherin towards the mesenchymal cell marker, a process linked to epithelial-mesenchymal transition (EMT) and hepatocarcinogenesis. The effects observed in the human liver cell in vitro model revealed mechanisms that can potentially contribute to the MC-LR-induced promotion and progression of hepatocellular carcinoma (HCC). Hepoid-HepaRG cultures provide a robust, accessible and versatile in vitro model, capable of sensitively detecting hepatotoxic effects at toxicologically relevant concentrations, allowing for assessing hepatotoxicity mechanisms, human health hazards and impacts of environmental hepatotoxins, such as MC-LR.

Antagonistic effect of selenium on programmed necrosis of testicular Leydig cells caused by cadmium through endoplasmic reticulum stress in chicken.

Cadmium (Cd) is a widely distributed environmental contaminant that is highly toxic to animals and humans. However, detailed reports on Cd-induced programmed necrosis have not been seen in chicken testicular Leydig cells. Selenium (Se) is a trace element in the human body that has cytoprotective effects in a variety of pathological damages caused by heavy metals. This study investigated the potential mechanisms of Cd-induced programmed cell necrosis and the antagonistic effect of Se on Cd toxicity. Chicken testis Leydig cells were divided into six groups, namely, control, Se (5µmol/L Na2SeO3), Cd (20µmol/L CdCl2), Se + Cd (5µmol/L Na2SeO3 and 20µmol/L CdCl2), 4-phenylbutyric acid (4-PBA) + Cd (10mmol/L 4-phenylbutyric acid and 20µmol/L CdCl2), and Necrostatin-1 (Nec-1) + Cd (60µmol/L Necrostatin-1 and 20µmol/L CdCl2). The results showed that Cd exposure decreased the activity of CAT, GSH-Px, and SOD and the concentration of GSH, and increased the concentration of MDA and the content of ROS. Relative mRNA and protein expression of GRP78, PERK, ATF6, IRE1, CHOP, and JNK increased in the Cd group, and mRNA and protein expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 significantly increased in the Cd group, while Caspase-8 mRNA and protein expression significantly decreased. The abnormal expression of endoplasmic reticulum stress-related proteins was significantly reduced by 4-PBA pretreatment; the increased expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 caused by Cd toxicity was alleviated; and the expression of caspase-8 was upregulated. Conversely, the increased mRNA and protein expression of endoplasmic reticulum stress marker genes (GRP78, ATF6, PERK, IRE1, CHOP, JNK) caused by Cd was not affected after pretreatment with Nec-1. We also found that these Cd-induced changes were significantly attenuated in the Se + Cd group. We clarified that Cd can cause programmed necrosis of chicken testicular Leydig cells through endoplasmic reticulum stress, and Se can antagonize Cd-induced programmed necrosis of chicken testicular Leydig cells.

ER Stress is Activated and Involved in Disuse-Induced Muscle Atrophy.

Muscle atrophy resulting wholly or partially from disuse represents a serious medical complication that decreases quality of life and increases morbidity and mortality. The accumulation of misfolded/unfolded proteins disrupts endoplasmic reticulum (ER) homeostasis and thus causes ER stress. Growing evidence indicates that ER stress plays an essential role in skeletal muscle remodeling under various physiological or pathophysiological conditions. However, whether ER stress is involved in disuse-induced muscle atrophy remains unclear. To induce muscle atrophy, 8-week-old C57BL/6JNifdc male mice were subjected to 3, 7, or 14 days of hindlimb unloading (HU), and rhesus macaques (Macaca mulatta) were subjected to 10∘ head-down tilted bed rest (HDBR) for 6 weeks. Tauroursodeoxycholic acid (TUDCA) (500 mg/kg/d) was orally administered to mice during HU to inhibit ER stress. Quantitative PCR, Western blotting, and immunohistochemistry were conducted to evaluate gene, protein, and structural changes, respectively. ER stress marker genes were rapidly induced by HU in a similar trend to that observed with atrophy-related genes such as Atrogin-1, muscle RING finger 1 (MuRF1), and muscle ubiquitin ligase of SCF complex in atrophy-1 (MUSA1). Inhibition of ER stress with TUDCA, a pan-ER stress inhibitor, attenuated HU-induced muscle atrophy and the upregulation of ubiquitin ligases via the AKT/forkhead box O3a pathway. In addition, the oxidative-to-glycolytic myofiber type transition caused by HU was also inhibited by TUDCA treatment. ER stress activation was also confirmed in HDBR-induced rhesus soleus muscle atrophy. The strong positive correlation between ER stress activation and both HU- and HDBR-induced muscle atrophy indicates that ER stress activation is ubiquitously involved in disuse-induced muscle atrophy, regardless of species. Thus, inhibiting ER stress may be an effective therapeutic strategy to prevent muscle atrophy during disuse.

Multi-walled carbon nanotube induced liver injuries possibly by promoting endoplasmic reticulum stress in Cyprinus carpio

The mass production and discharge of carbon nanotubes (CNTs) to the water environment are of great concern since they threaten the health of organisms in the aquatic ecosystem. CNTs induce multi-organ injuries in fish, but limited literature is available regarding the mechanisms involved. In the present study, juvenile common carp (Cyprinus carpio) were exposed to multi-walled carbon nanotubes (MWCNTs) (0.25 mg L−1 and 2.5 mg L−1) for four weeks. MWCNTs caused dose-dependent alterations in the pathological morphology of liver tissues. Ultrastructural changes manifested as nuclear deformation, chromatin condensation, endoplasmic reticulum (ER) disorderly arrangement, mitochondria vacuolation, and mitochondrial membrane destruction. TUNEL analysis indicated that the apoptosis rate in hepatocytes markedly increased upon exposure to MWCNTs. Moreover, the apoptosis was confirmed by significant upregulation of mRNA levels of apoptosis-related genes (Bcl-2, XBP1, Bax, and caspase3) in MWCNTs-exposure groups, except for Bcl-2 expression which was not significantly changed in HSC groups (2.5 mg L−1 MWCNTs). Furthermore, real-time PCR assay indicated the increased expression of ER stress (ERS) marker genes (GRP78, PERK, and eIF2α) in the exposure groups compared to the control groups, suggesting that the PERK/eIF2α signaling pathway involved in the injuries of the liver tissue. Overall, the results above indicate that MWCNTs induce ERS by activating the PERK/eIF2α pathway in the liver of common carp, and resulted in the initiation of apoptosis procedure.

Fine particulate matter induces heart defects via AHR/ROS-mediated endoplasmic reticulum stress

Accumulating body of evidence indicates that exposure to fine particulate matter (PM2.5) is closely associated with congenital heart disease in the offspring, but the underlying molecular mechanisms remain to be elucidated. We previously reported that extractable organic matter (EOM) from PM2.5 induces reactive oxygen species (ROS) overproduction by activating aromatic hydrocarbon receptor (AHR), leading to heart defects in zebrafish embryos. We hypothesized that endoplasmic reticulum (ER) stress might be elicited by the excessive ROS production and thereby contribute to the cardiac developmental toxicity of PM2.5. In this study, we examined the effects of EOM on endoplasmic reticulum (ER) stress, apoptosis, and Wnt signal pathway in zebrafish embryos, and explored their roles in EOM-induced heart defects. Our results showed that 4-Phenylbutyric acid (4-PBA), a pharmaceutical inhibitor of ER stress, significantly attenuated the EOM-elevated heart malformation rates. Moreover, EOM upregulated the expression levels of ER stress marker genes including CHOP and PDI in the heart of zebrafish embryos, which were counteracted by genetic or pharmaceutical inhibition of AHR activity. The ROS scavenger N-Acetyl-l-cysteine (NAC) also abolished the EOM-induced ER stress. We further demonstrated that both 4-PBA and CHOP genetic knockdown rescued the PM2.5-induced ROS overproduction, apoptosis and suppression of Wnt signaling. In conclusion, our results indicate that PM2.5 induces AHR/ROS-mediated ER stress, which leads to apoptosis and Wnt signaling inhibition, ultimately resulting in heart defects.

Ammonia exposure induces endoplasmic reticulum stress and apoptosis in Chinese striped-necked turtle (Mauremys sinensis).

Ammonia is a major pollutant in the water environment, which could cause severe harm to aquatic organisms. To explore the pathological and physiological effects of ammonia in Chinese striped-necked turtles (Mauremys sinensis), the individuals (body mass: 218.26±12.65g) were divided into two groups: control group and ammonia exposed group (6.25mM total ammonia), then the expression levels of signaling factors involved in the endoplasmic reticulum stress and apoptotic pathways were determined. The results showed that ammonia exposure up-regulated the transcriptional and protein levels of endoplasmic reticulum stress marker gene Bip. Meanwhile, the relative mRNA levels of key genes (PERK, ATF6, eIF2α, ATF4, IRE1α and XBP1) involved in unfolded protein response up-regulated, and the phosphorylation levels of PERK, eIF2α and IRE1α increased correspondingly. In addition, the protein and transcriptional levels of CHOP and JNK related to apoptotic pathway induced by unfolded protein reaction increased under ammonia exposure. Moreover, Bcl-2 mRNA expression levels and protein levels decreased, whereas BAX and caspase-3 showed an opposite trend, and the cleaved protein of caspase-3 appeared when the turtles in the elevated ammonia. Furthermore, the apoptotic cells in liver increased after ammonia exposure. These results suggested ammonia exposure induced endoplasmic reticulum stress, then activated unfolded protein response, followed by apoptosis in M. sinensis. The results will contribute to a better understanding of the toxicity mechanism of ammonia to aquatic turtles.

Microtubule Acetylation Controls MDA-MB-231 Breast Cancer Cell Invasion through the Modulation of Endoplasmic Reticulum Stress.

During aggressive cancer progression, cancer cells adapt to unique microenvironments by withstanding various cellular stresses, including endoplasmic reticulum (ER) stress. However, the mechanism whereby cancer cells overcome the ER stress to survive remains to be elucidated. Herein, we demonstrated that microtubule acetylation in cancer cells grown on a stiff matrix promotes cancer progression by preventing excessive ER stress. Downregulation of microtubule acetylation using shRNA or CRSIPR/Cas9 techniques targeting ATAT1, which encodes α-tubulin N-acetyltransferase (αTAT1), resulted in the upregulation of ER stress markers, changes in ER morphology, and enhanced tunicamycin-induced UPR signaling in cancer cells. A set of genes involved in cancer progression, especially focal adhesion genes, were downregulated in both ATAT1-knockout and tunicamycin-treated cells, whereas ATAT1 overexpression restored the gene expression inhibited by tunicamycin. Finally, the expression of ATAT1 and ER stress marker genes were negatively correlated in various breast cancer types. Taken together, our results suggest that disruption of microtubule acetylation is a potent therapeutic tool for preventing breast cancer progression through the upregulation of ER stress. Moreover, ATAT1 and ER stress marker genes may be useful diagnostic markers in various breast cancer types.

A New Hypothesis for Type 1 Diabetes Risk: The At-Risk Allele at rs3842753 Associates With Increased Beta-Cell INS Messenger RNA in a Meta-Analysis of Single-Cell RNA-Sequencing Data

ObjectivesType 1 diabetes is characterized by the autoimmune destruction of insulin-secreting beta cells. Genetic variants upstream at the insulin (INS) locus contribute to ∼10% of type 1 diabetes heritable risk. Previous studies showed an association between rs3842753 C/C genotype and type 1 diabetes susceptibility, but the molecular mechanisms remain unclear. To date, no large-scale studies have looked at the effect of genetic variation at rs3842753 on INS mRNA at the single-cell level. MethodsWe aligned all human islet single-cell RNA sequencing data sets available to us in year 2020 to the reference genome GRCh38.98 and genotyped rs3842753, integrating 2,315 β cells and 1,223 β-like cells from 13 A/A protected donors, 23 A/C heterozygous donors and 35 C/C at-risk donors, including adults without diabetes and with type 2 diabetes. ResultsINS expression mean and variance were significantly higher in single β cells from females compared with males. On comparing across β cells and β-like cells, we found that rs3842753 C‒containing cells (either homozygous or heterozygous) had the highest INS expression. We also found that β cells with the rs3842753 C allele had significantly higher endoplasmic reticulum stress marker gene expression compared with the A/A homozygous genotype. ConclusionsThese findings support the emerging concept that inherited risk of type 1 diabetes may be associated with inborn, persistent elevated insulin production, which may lead to β-cell endoplasmic reticulum stress and fragility.

The involvement of gamma-aminobutyric acid shunt in the endoplasmic reticulum stress response of Arabidopsis thaliana

The endoplasmic reticulum (ER) is the main site of secretory protein production and folding and its homeostasis under environmental stress is vital for the maintenance of the protein secretory pathway. The loss of homeostasis and accumulation of unfolded proteins in the ER is referred to as ER stress. Although, γ-aminobutyric acid (GABA) is an important regulator of stress response in plants, its roles during ER stress remains unclear. This study investigated the involvement of GABA in the ER stress response of plants. For this, changes in GABA metabolism under ER stress was analysed in Arabidopsis thaliana, then to study the response of the ER-folding machinery, plants were treated with exogenous GABA under ER stress. The antibiotic tunicamycin, which inhibits N-glycosylation was used to specifically induce ER stress. This stress up-regulated the expression of five glutamate decarboxylase (GAD) genes except GAD2 and GABA content of A. thaliana plants increased with an increasing concentration of tunicamycin (0.1 μg ml−1 and 0.25 μg ml−1). Moreover, expressions of genes involved in the conversion of GABA to succinate was also induced, while genes involved in transport across plasma and mitochondrial membrane showed no response to ER stress. The exogenous treatment of plants with 1-and 5-mM GABA increased plant performance under ER stress but 0.1 mM proved ineffective. Plants treated with GABA under ER stress had decreased expression of ER stress marker genes such as BIP1, BIP3 or CNX, but the expression of genes related to ER stress perception or ER-associated protein degradation showed no changes with respect to GABA treatments.

Erratum. Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK.

Study question Does metformin inhibit excessive androgen-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) in vivo and in vitro? Summary answer Metformin inhibits testosterone-induced ER stress and unfolded protein response (UPR) activation by suppressing p38 MAPK phosphorylation in ovarian GCs. What is known already Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism. Excessive testosterone induces ER stress and UPR activation in human cumulus cells, leading to cell apoptosis. Metformin has potential inhibitory effects on ER stress and UPR activation, as demonstrated in human pancreatic beta cells and obese mice. Study design, size, duration Cumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our IVF centre. A dihydrotestosterone (DHT)-induced PCOS mouse model was constructed and treated with or without metformin. Primary mouse GCs and cumulus-oocyte complexes (COCs) were cultured with testosterone, metformin, a p38 MAPK inhibitor, or p38 MAPK small interfering RNA. Participants/materials, setting, methods The levels of UPR sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients by real-time quantitative PCR (qPCR) and western blot. The ovaries, oocytes, GCs and COCs were collected from PCOS mice treated with metformin and controls. The expressions of ER stress markers and p38 MAPK phosphorylation were assessed by qPCR, western blot and immunofluorescence. A subsequent in vitro analysis with primary cultured GCs and COCs was used to confirm the influence of metformin on ER stress activation by qPCR and western blot. Finally, the effects of ER stress activation on GCs and COCs in relation to LH responsiveness were examined by qPCR and COC expansion. Main results and the role of chance The expression of the ER stress markers GRP78, CHOP and XBP1s in the cumulus cells was higher in PCOS patients than in control patients, as were the levels of the UPR sensor proteins p-IRE1α, p-EIF2α and GRP78. Compared to those of control mice, the ovaries, GCs and COCs of DHT-treated PCOS mice showed increased levels of ER stress marker genes and proteins. Hyperandrogenism in PCOS mouse ovaries also induced p38 MAPK phosphorylation in COCs and GCs. Metformin inhibited ER stress activation was associated with decreased p-p38 MAPK levels. In vitro experiments, testosterone-induced ER stress was mitigated by metformin or p38 MAPK inhibition in primary cultured GCs and COCs. COCs expanded rapidly in the presence of testosterone during LH administration, and ovulation-related genes, namely, Areg, Ereg, Ptgs2, Sult1e1, Ptx3 and Tnfaip6, were strongly expressed in the COCs and GCs. These effects were reversed by treatment with metformin, an ER stress inhibitor or by knockdown of p38 MAPK. Limitations, reasons for caution The number of PCOS patients in this study was small. Wider implications of the findings This study provides further evidence for metformin as a PCOS treatment. Study funding/competing interest(s) This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800), the Key Research and Development Program of Zhejiang Province (2017C03022), the Zhejiang Province Medical Science and Technology Plan Project (2017KY085, 2018KY457), the National Natural Science Foundation of China (31701260, 81401264, 81701514), and the Special Funds for Clinical Medical Research of the Chinese Medical Association (16020320648). The authors report no conflict of interest in this work and have nothing to disclose. Trial registration number N/A.

Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK.

STUDY QUESTIONDoes metformin inhibit excessive androgen-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) in vivo and in vitro?SUMMARY ANSWERMetformin inhibits testosterone-induced ER stress and unfolded protein response (UPR) activation by suppressing p38 MAPK phosphorylation in ovarian GCs.WHAT IS KNOWN ALREADYPolycystic ovary syndrome (PCOS) is associated with hyperandrogenism. Excessive testosterone induces ER stress and UPR activation in human cumulus cells, leading to cell apoptosis. Metformin has potential inhibitory effects on ER stress and UPR activation, as demonstrated in human pancreatic beta cells and obese mice.STUDY DESIGN, SIZE, DURATIONCumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our IVF centre. A dihydrotestosterone (DHT)-induced PCOS mouse model was constructed and treated with or without metformin. Primary mouse GCs and cumulus-oocyte complexes (COCs) were cultured with testosterone, metformin, a p38 MAPK inhibitor, or p38 MAPK small interfering RNA.PARTICIPANTS/MATERIALS, SETTING, METHODSThe levels of UPR sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients by real-time quantitative PCR (qPCR) and western blot. The ovaries, oocytes, GCs and COCs were collected from PCOS mice treated with metformin and controls. The expressions of ER stress markers and p38 MAPK phosphorylation were assessed by qPCR, western blot and immunofluorescence. A subsequent in vitro analysis with primary cultured GCs and COCs was used to confirm the influence of metformin on ER stress activation by qPCR and western blot. Finally, the effects of ER stress activation on GCs and COCs in relation to LH responsiveness were examined by qPCR and COC expansion.MAIN RESULTS AND THE ROLE OF CHANCEThe expression of the ER stress markers GRP78, CHOP and XBP1s in the cumulus cells was higher in PCOS patients than in control patients, as were the levels of the UPR sensor proteins p-IRE1α, p-EIF2α and GRP78. Compared to those of control mice, the ovaries, GCs and COCs of DHT-treated PCOS mice showed increased levels of ER stress marker genes and proteins. Hyperandrogenism in PCOS mouse ovaries also induced p38 MAPK phosphorylation in COCs and GCs. Metformin inhibited ER stress activation was associated with decreased p-p38 MAPK levels. In vitro experiments, testosterone-induced ER stress was mitigated by metformin or p38 MAPK inhibition in primary cultured GCs and COCs. COCs expanded rapidly in the presence of testosterone during LH administration, and ovulation-related genes, namely, Areg, Ereg, Ptgs2, Sult1e1, Ptx3 and Tnfaip6, were strongly expressed in the COCs and GCs. These effects were reversed by treatment with metformin, an ER stress inhibitor or by knockdown of p38 MAPK.LIMITATIONS, REASONS FOR CAUTIONThe number of PCOS patients in this study was small.WIDER IMPLICATIONS OF THE FINDINGSThis study provides further evidence for metformin as a PCOS treatment.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800), the Key Research and Development Program of Zhejiang Province (2017C03022), the Zhejiang Province Medical Science and Technology Plan Project (2017KY085, 2018KY457), the National Natural Science Foundation of China (31701260, 81401264, 81701514), and the Special Funds for Clinical Medical Research of the Chinese Medical Association (16020320648). The authors report no conflict of interest in this work and have nothing to disclose.TRIAL REGISTRATION NUMBERN/A.

Melatonin attenuates palmitic acid-induced mouse granulosa cells apoptosis via endoplasmic reticulum stress

BackgroundPalmitic acid (PA), the main component of dietary saturated fat, causes apoptosis in many cell types, including mouse granulosa cell. Melatonin, an important endogenous hormone, has beneficial effects on female reproductive processes. Since elevated PA levels are present in follicular fluid (FF) of patients with infertility and are shown to be toxic for granulosa cells, we investigated the molecular mechanisms of PA toxicity in mouse granulosa cells and explored the effects of melatonin on PA-induced apoptosis.MethodsGranulosa cells from immature female mice were cultured for 24 h in medium containing PA and/or melatonin. Then, the effects of PA alone or combined with melatonin on viability, apoptosis and endoplasmic reticulum (ER) stress in granulosa cells were detected by methyl thiazolyl tetrazolium (MTT) assay, flow cytometry assay and western blot. After 48 h of PA and/or melatonin treatment, the concentrations of estradiol (E2) and progesterone (P4) in the culture supernatants were measured with ELISA kits.ResultsIn this study, we explored the effects of melatonin on cell viability and apoptosis in PA-treated mouse granulosa cells and uncovered the signaling pathways involved in these processes. Our results showed that 200-800 μM PA treatment reduces cell viability, induces cell apoptosis, enhances the expression of apoptosis-related genes (Caspase 3 and B-cell lymphoma-2 (BCL-2) associated X protein (BAX)), and activates the expression of ER stress marker genes (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)). Melatonin treatment (1-10 μM) suppresses 400 μM PA-induced cell viability decrease, cell apoptosis, Caspase 3 activation, and BAX, CHOP, and GRP78 expression. In addition, we found that 10 μM melatonin successfully attenuated the 400 μM PA-induced estrogen (E2) and progesterone (P4) decreases.ConclusionsThis study suggests that PA triggers cell apoptosis via ER stress and that melatonin protects cells against apoptosis by inhibiting ER stress in mouse granulosa cells.

42 Nrf2 and nuclear factor kappa B cross-talk in bovine granulosa cells under lead challenge

Lead (Pb2+), one of the pervasive and protracted environmental heavy metals, is known to affect the female reproductive system by inducing oxidative stress. The Nrf2 and NF-κB are two key transcriptional factors (TF) known to regulate cellular redox status and response against stress and inflammation, respectively. Evidence showed that these TF undergo functional cross-talk. Although the properties of Pb2+ have been extensively studied, little is known about its effect on bovine granulosa cells with respect to Nrf2/NF-κB interaction. The aim of this study was to investigate the impact of Pb2+ on the response of bovine granulosa cells with regard to the cross-talk between Nrf2 and NF-κB. For this, bovine granulosa cells aspirated from small growing follicles (3-5mm of diameter) from ovaries obtained from a local slaughterhouse were in vitro cultured at 37°C and 5% CO2. Sub-confluent cells were exposed to different doses of lead acetate [Pb (C2H3O2)2] (1, 2, or 3µg mL−1) for 2h. Following this, the accumulation of intracellular reactive oxygen species was measured using the H2DCFDA fluorescent probe (Life Technologies, Darmstadt, Germany). The cell viability was assessed using Cell Counting Kit-8 (CCK-8, Dojindo, Munich, Germany) 24h after treatments. Moreover, cells were harvested and subjected to total RNA isolation using miRNeasy Mini Kit (Qiagen, Hilden, Germany) followed by cDNA synthesis using First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) and the gene expression was determined using quantitative PCR. Data were analysed using comparative threshold cycle (ΔΔCT) method and normalized against the expression of GAPDH and β-actin genes. Furthermore, the protein lysate was isolated from cells to assess carbonylated protein using OxyBlot protein oxidation detection kit (abcam, Cambridge, MA, USA). Results showed that exposure of granulosa cells to lead acetate provoked intracellular reactive oxygen species accumulation and protein carbonylation. This was accompanied by cell cycle arrest at G0/G1 phase, reduction in cell viability, and decrease in the expression of cell proliferation marker genes (CCND2 and PCNA). Moreover, lead acetate elicited down-regulation of both Nrf2 and NF-κB and their downstream antioxidant genes (SOD and CAT), whereas no significant difference was shown in the level of other genes involved in both TF pathways (Keap-1, HO-1, Trx, IKK, and TNF-α). In addition, lead acetate challenge increased the expression of endoplasmic reticulum stress marker genes (GRP78 and CHOP) and the pro-apoptotic gene (Caspase-3), whereas it reduced the anti-apoptotic gene (BCL-2). Our findings suggest that Pb+2 driven-oxidative stress deregulates granulosa cell viability and proliferation, enhances endoplasmic reticulum stress, induces cell cycle arrest, and mediates apoptosis probably via disruption of Nrf2/NF-κB cross-talk.

Selenate Prevents Adipogenesis through Induction of Selenoprotein S and Attenuation of Endoplasmic Reticulum Stress.

The conversion of preadipocytes to adipocytes (adipogenesis) is a potential target to treat or prevent obesity. Selenate, an inorganic form of selenium, elicits diverse health benefits, mainly through its incorporation into selenoproteins. The individual roles of selenium and certain selenoproteins have been reported. However, the effects of selenate treatment on selenoproteins in adipocytes are unclear. In this study, the effects of selenate pretreatment on selenoprotein and endoplasmic reticulum (ER) stress during adipogenesis were examined in vitro. The selenate pretreatment dose-dependently suppressed the adipogenesis of 3T3-L1 preadipocytes. The selenate pretreatment at 50 μM for 24 h almost completely suppressed adipogenesis without cytotoxic effects. The expression of the adipogenic genes peroxisome proliferator-activated receptor gamma, CCAAT-enhancer binding protein alpha, and leptin was suppressed by selenate. This pretreatment also upregulated selenoprotein S (SEPS1), an ER resident selenoprotein that reduces ER stress, and prevented dexamethasone-induced SEPS1 degradation during the early stage of adipogenesis. The selenate-inhibited adipogenesis was associated with an attenuation of ER stress. The expression of the ER stress marker genes was upregulated during the early stage of differentiation, whereas the selenate pretreatment suppressed the mRNA expression of the XBP1 and C/EBP homologous protein. The collective data suggest a preventive role of selenate and SEPS1 in adipogenesis, and support a novel dietary approach to prevent obesity.

Neuroprotective effect of quercetin against radiation-induced endoplasmic reticulum stress in neurons.

The endoplasmic reticulum (ER) plays an important role in the regulation and maintenance of cellular homeostasis. However, unresolved ER stress leads to deleterious effects by inducing the accumulation of unfolded proteins in the cell. Here we have demonstrated the protective aspects of quercetin against radiation-induced ER stress and against inflammation in primary cultured dorsal root ganglion (DRG) neurons. The mature DRG neurons were pretreated with different concentrations of quercetin (5-100 μM) for 24 hours before 2 Gy gamma radiation exposure and then subjected to a cytotoxicity assay, quantitative real-time polymerase chain reaction and Western blot analysis. The results showed that quercetin decreased the expression of BiP and C/EBP-homologous protein, the ER stress marker genes along with downregulation of tumor necrosis factor-α, JNK in irradiated DRG neurons. Furthermore, quercetin pretreatment significantly increased the cytoskeletal protein Tuj1 and the neurotrophin brain-derived neurotrophic factor in the neuron. These results indicate that quercetin plays a neuroprotective role against radiation-mediated ER stress and inflammatory responses.

Palmitic acid induces human osteoblast-like Saos-2 cell apoptosis via endoplasmic reticulum stress and autophagy.

Palmitic acid (PA) is the most common saturated long-chain fatty acid in food that causes cell apoptosis. However, little is known about the molecular mechanisms of PA toxicity. In this study, we explore the effects of PA on proliferation and apoptosis in human osteoblast-like Saos-2 cells and uncover the signaling pathways involved in the process. Our study showed that endoplasmic reticulum (ER) stress and autophagy are involved in PA-induced Saos-2 cell apoptosis. We found that PA inhibited the viability of Saos-2 cells in a dose- and time-dependent manner. At the same time, PA induced the expression of ER stress marker genes (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)), altered autophagy-related gene expression (microtubule-associated protein 1 light chain 3 (LC3), ATG5, p62, and Beclin), promoted apoptosis-related gene expression (Caspase 3 and BAX), and affected autophagic flux. Inhibiting ER stress with 4-PBA diminished the PA-induced cell apoptosis, activated autophagy, and increased the expression of Caspase 3 and BAX. Inhibiting autophagy with 3-MA attenuated the PA and ER stress-induced cell apoptosis and the apoptosis-related gene expression (Caspase 3 and BAX), but seemed to have no obvious effects on ER stress, although the CHOP expression was downregulated. Taken together, our results suggest that PA-induced Saos-2 cell apoptosis is activated via ER stress and autophagy, and the activation of autophagy depends on the ER stress during this process.

Dietary 18:3 and Long Chain n‐3 PUFA Protected Rats from Fructose‐Induced Oxidative and Endoplasmic Reticulum Stress in Visceral Adipose Tissue

Effect of dietary n‐6/n‐3 polyunsaturated fatty acids (PUFA) ratio on the fructose‐induced adipose tissue oxidative and endoplasmic reticulum (ER) stress was not explored previously. Here, we studied the effect of diets containing n‐6 PUFA & α‐linolenic acid (ALA, 18:3 n‐3 PUFA) and n‐6 PUFA & long chain n‐3 PUFA (docosahexanoic acid and eicosapentaenoic acid, DHA and EPA) in the ratio of 2:1 and 5:1 respectively on fructose‐induced oxidative and ER stress in adipose tissue.Weanling Wistar rats were divided in to four groups and fed with diets containing starch (n‐6/n‐3 PUFA ratio 215:1), fructose (n‐6/n‐3 PUFA ratio 215:1) and fructose‐with n‐6/n‐3 PUFA ratio 2:1 (18:3 n‐3 PUFA) and 5:1 (long chain n‐3 PUFA) diets for twenty‐four weeks. Activities of glucose‐6‐phosphate dehydrogenase (G6PDH), glutathione peroxidase (GPx), and catalase and superoxide dismutase (SOD) were measured in visceral adipose tissue along with glutathione levels and lipid peroxidation. Expression of p22 phox gene related oxidative stress was quantified along with ER stress marker gene, GRP78.ALA and long chain (LC) n‐3 PUFA feeding significantly reduced fructose‐induced G6PDH activity to similar extent by 28% (p≤0.01). Both ALA and LC n‐3 PUFA significantly reduced the fructose‐induced SOD activity by 15% and 12% respectively (p≤0.01), whereas they reduced fructose‐induced catalase activity to similar extent by 23.5% (p≤0.05). ALA and long chain (LC) n‐3 PUFA feeding significantly elevated the GPx activity that is reduced by high fructose‐ diet feeding by 14.6% and 12% respectively (p≤0.05) without altering the tissue glutathione levels. Lipid peroxidation, an important determinant of tissue oxidative stress is significantly reduced by both ALA‐ and LC n‐3 PUFA‐enriched diets by 12% and 35% respectively (p≤0.01). p22 phox gene expression is significantly reduced by both ALA and LC n‐3 PUFA by 12% and 33% respectively (p≤0.05) along with ER stress marker, GRP78 by 40% and 30% respectively (p≤0.05).We conclude that replacement of dietary n‐6 PUFA with ALA and LC n‐3 PUFA ameliorates fructose‐induced oxidative and ER stress in visceral adipose tissue. Though both ALA and LC n‐ 3 PUFA significantly reduced fructose‐induced oxidative stress, considering beneficial effects LC n‐3 PUFA on lipid peroxidation and LC n‐3 PUFA exhibited protective nature at less quantity than ALA, we propose that diets rich in LC n‐3 PUFA may protect against fructose‐induced oxidative and ER stress in adipose tissue.Support or Funding InformationThe present study was carried out with the financial support of Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, and Indian Council of Medical Research (ICMR), Government of India. AS was supported by Council of Scientific and Industrial Research (CSIR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification and functional study of the endoplasmic reticulum stress sensor IRE1 in Chlamydomonas reinhardtii.

In many eukaryotes, endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) via the transmembrane endoribonuclease IRE1 to maintain ER homeostasis. The ER stress response in microalgae has not been studied in detail. Here, we identified Chlamydomonas reinhardtii IRE1 (CrIRE1) and characterized two independent knock-down alleles of this gene. CrIRE1 is similar to IRE1s identified in budding yeast, plants, and humans, in terms of conserved domains, but differs in having the tandem zinc-finger domain at the Cterminus. CrIRE1 was highly induced under ER stress conditions, and the expression of a chimeric protein consisting of the luminal N-terminal region of CrIRE1 fused to the cytosolic C-terminal region of yeast Ire1p rescued the yeast ∆ire1 mutant. Both allelic ire1 knock-down mutants ire1-1 and ire1-2 were much more sensitive than their parental strain CC-4533 to the ER stress inducers tunicamycin, dithiothreitol and brefeldinA. Treatment with a low concentration of tunicamycin resulted in growth arrest and cytolysis in ire1 mutants, but not in CC-4533 cells. Furthermore, in the mutants, ER stress marker gene expression was reduced, and reactive oxygen species (ROS) marker gene expression was increased. The survival of ire1 mutants treated with tunicamycin improved in the presence of the ROS scavenger glutathione, suggesting that ire1 mutants failed to maintain ROS levels under ER stress. Together, these results indicate that CrIRE1 functions as an important component of the ER stress response in Chlamydomonas, and suggest that the ER stress sensor IRE1 is highly conserved during the evolutionary history.