Induction Of Endoplasmic Reticulum Stress Research Articles

Upregulation of mesencephalic astrocyte-derived neurotrophic factor (MANF) expression offers protection against alcohol neurotoxicity.

Alcohol exposure has detrimental effects on both the developing and mature brain. Endoplasmic reticulum (ER) stress is one of the mechanisms that contributes to alcohol-induced neuronal damages. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress-responsive protein and is neuroprotective in multiple neuronal injury and neurodegenerative disease models. MANF deficiency has been shown to exacerbate alcohol-induced ER stress and neurodegeneration. However, it is unknown whether MANF supplement is sufficient to protect against alcohol neurotoxicity. Alcohol alters MANF expression in the brain, but the mechanisms underlying alcohol modulation of MANF expression remain unclear. This study was designed to determine how alcohol alters MANF expression in neuronal cells and whether exogeneous MANF can alleviate alcohol neurotoxicity. We showed that alcohol increased MANF transcription and secretion without affecting MANF mRNA stability and protein degradation. ER stress was necessary for alcohol-induced MANF upregulation, as pharmacological inhibition of ER stress by 4-PBA diminished alcohol-induced MANF expression. In addition, the presence of ER stress response element II (ERSE-II) was required for alcohol-stimulated MANF transcription. Mutations or deletion of this sequence abolished alcohol-regulated transcriptional activity. We generated MANF knockout (KO) neuronal cells using CRISPR/Cas9. MANF KO cells exhibited increased unfolded protein response (UPR) and were more susceptible to alcohol-induced cell death. On the other hand, MANF upregulation by the addition of recombinant MANF protein or adenovirus gene transduction protected neuronal cells against alcohol-induced cell death. Further studies using early postnatal mouse pups demonstrated that enhanced MANF expression in the brain by intracerebroventricular (ICV) injection of MANF adeno-associated viruses ameliorated alcohol-induced cell death. Thus, alcohol increased MANF expression through inducing ER stress, which could be a protective response. Exogenous MANF was able to protect against alcohol-induced neurodegeneration.

PAQR9 regulates glucose homeostasis in diabetic mice and modulates insulin secretion in β cells in vitro under stress conditions

Progesterone and adipoQ receptor 9 (PAQR9) is an endoplasmic reticulum (ER)-localized membrane protein that is involved in protein quality control of ER by interacting with BAG6. One of the physiological functions of PAQR9 is regulation of fasting-induced ketogenesis and fatty acid oxidation in the liver via modulating protein degradation of PPARα. However, it is currently unknown whether or not PAQR9 impacts glucose homeostasis. We addressed this question using a Paqr9-deleted mouse model in which type 1 diabetes was induced by streptozotocin injection and type 2 diabetes was induced by high-fat diet (HFD) with streptozotocin injection. Paqr9 deletion improved hyperglycemia and glucose tolerance in both of the diabetic mouse models. In the pancreatic islets, Paqr9 deletion reduced apoptosis of β cells in type 2 diabetic mice. Paqr9 deletion also reduced HFD-induced hepatic steatosis and adiposity of white adipose tissue. In Min6 cells, overexpression of DUF3538 domain of BAG6 to block the interaction of PAQR9 with BAG6 was able to enhance glucose-stimulated insulin secretion upon treatment with inflammatory factors or thapsigargin, an ER stress inducer. Thapsigargin-induced ER stress markers were also reduced by overexpression of DUF3538 domain. Collectively, these results indicate that PAQR9 has a modulatory role in glucose homeostasis, associated with regulation on insulin secretion of β cells in vitro under stress conditions.

Fluorination‐Induced Multi‐Enhancement of a Nano‐Photosensitizer for Deep Photodynamic Therapy against Hypoxia Tumor

AbstractOrganic dyes hold great promise for application in photodynamic therapy (PDT). However, they currently face challenges such as inadequate photodynamic activity, limited tumor penetration, and constraints imposed by tumor hypoxia. Here, a facile and efficient strategy is presented for multi‐enhanced PDT through the fluorination of a squarylium indocyanine dye‐based photosensitizer (FCy). The amphiphilic FCy features perfluorooctane and PEG‐biotin moieties conjugated to a squarylium indocyanine core. In aqueous environments, FCy spontaneously self‐assembles into stable nano‐sized photosensitizers (FCy NPs), demonstrating a high oxygen loading ability attributable to the presence of perfluoroalkyl groups. Consequently, the aggregation of squarylium indocyanine dyes remarkably boosts the photodynamic effect, yielding a 15‐fold improvement in singlet oxygen quantum yield. Owing to the perfluoroalkyl group, FCy NPs exhibit increased endoplasmic reticulum (ER)‐ accumulating abilities, which further induce ER stress upon laser irradiation and enhance the PDT effect. Furthermore, the superior deep tumor penetration ability of FCy NPs is confirmed through both in vitro and in vivo studies. With efficient oxygen supply to the deep tumor regions, FCy NPs demonstrate potent imaging‐guided PDT against hypoxia tumors. The study substantiates the enhanced ER‐accumulating ability of the perfluoroalkyl group and presents a facile fluorination strategy for the multi‐enhancement of photosensitizers.

Uranium affects nitrogen metabolism and endoplasmic reticulum protein homeostasis in plants

The toxicity of uranium (U) to plants makes the phytoremediation effect of U contaminated soil unsatisfactory, and the understanding of the mechanism of U toxicity to plants is limited. In the present study, the effects of U on nitrogen (N) metabolism and endoplasmic reticulum (ER) protein homeostasis in radish (Raphanus sativus) and broad bean (Vicia faba) were analyzed. The results showed that the content of amine N was significantly increased in R. sativus root after U treatment, but significantly decreased in both root and leaf of V. faba, indicating that U had an adverse effect on the N nutrition in V. faba. After U treatment, the nitrate reductase (NR) activity in R. sativus roots and leaves was increased to 2.9 and 4.8 times of that in control group, but decreased by 26.9% and 74.3% in V. faba, respectively. The transcriptome sequencing results showed that 3 NR genes were up-regulated in R. sativus root, while 1 was down-regulated in V. faba after U treatment. In addition, U interfered with ER protein homeostasis and induced ER stress. The bZIP17/28 and bZIP60-mediated unfolded protein response (UPR) pathways were activated in R. sativus root. This promoted downstream ER associated degradation (ERAD) pathway, facilitating the degradation of unfolded (or misfolded) proteins, thereby alleviating ER stress. While in V. faba, U triggered severe programmed cell death through the induction of ER stress, which was significantly alleviated by exogenous addition of the chemical chaperone 4-phenylbutyric acid. Overall, more efficient N assimilation activity and ER protein homeostasis maintenance mechanism (especially ERAD) may be one of the key mechanisms for R. sativus tolerance to U stress. This study for the first time revealed the toxicological mechanism of U in plants in terms of N metabolism and ER protein homeostasis.

ER stress increases expression of intracellular calcium channel RyR1 to modify Ca2+ homeostasis in pancreatic beta cells

Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects oftunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these resultsshowchanges in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.

Emodin, an Emerging Mycotoxin, Induces Endoplasmic Reticulum Stress-Related Hepatotoxicity through IRE1α-XBP1 Axis in HepG2 Cells.

Emodin, an emerging mycotoxin, is known to be hepatotoxic, but its mechanism remains unclear. We hypothesized that emodin could induce endoplasmic reticulum (ER) stress through the inositol-requiring enzyme 1 alpha (IRE1α)-X-box-binding protein 1 (XBP1) pathway and apoptosis, which are closely correlated and contribute to hepatotoxicity. To test this hypothesis, a novel IRE1α inhibitor, STF-083010, was used. An MTT assay was used to evaluate metabolic activity, and quantitative PCR and western blotting were used to investigate the gene and protein expression of ER stress or apoptosis-related markers. Apoptosis was evaluated with flow cytometry. Results showed that emodin induced cytotoxicity in a dose-dependent manner in HepG2 cells and upregulated the expression of binding immunoglobulin protein (BiP), C/EBP homologous protein (CHOP), IRE1α, spliced XBP1, the B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 ratio, and cleaved caspase-3. Cotreatment with emodin and STF-083010 led to the downregulation of BiP and upregulation of CHOP, the Bax/Bcl-2 ratio, and cleaved caspase-3 compared with single treatment with emodin. Furthermore, the apoptosis rate was increased in a dose-dependent manner with emodin treatment. Thus, emodin induced ER stress in HepG2 cells by activating the IRE1α-XBP1 axis and induced apoptosis, indicating that emodin can cause hepatotoxicity.

ER Stress Decreases Gene Expression Of Transmembrane Protein 117 Via Activation of PKR-like ER Kinase.

Stress response is an inherent mechanism in the endoplasmic reticulum (ER). The inducers of ER cause a specific cascade of reactions, leading to gene expression. Transmembrane protein 117 (TMEM117) is in the ER and plasma membrane. In our previous study, TMEM117 protein expression was found to be decreased by an ER stress inducer. However, the mechanism underlying this decrease in TMEM117 protein expression remains unclear. This study aimed to elucidate the mechanism underlying the decrease in TMEM117 protein expression during ER stress and identify the unfolded protein response (UPR) pathway related to decreased TMEM117 protein expression. We showed that the gene expression levels of TMEM117 were decreased by ER stress inducers and were regulated by PKR-like ER kinase (PERK), indicating that TMEM117 protein expression was regulated by the signaling pathway. Surprisingly, gene knockdown of activating transcription factor 4 (ATF4) downstream of PERK did not affect the gene expression of TMEM117. These results suggest that TMEM117 protein expression during ER stress is transcriptionally regulated by PERK but not by ATF4. TMEM117 has a potential to be a new therapeutic target against ER stress-related diseases.

Exposure to copper induces endoplasmic reticulum (ER) stress-mediated apoptosis in chicken (Gallus gallus) myocardium.

Copper (Cu), an omnipresent environmental pollutant, can cause potential harm to the public and ecosystems. In order to study the cardiotoxicity caused by Cu, molecular biology techniques were used to analyze the effect of Cu on ER stress-mediated cardiac apoptosis. In vivo investigation, 240 1-day-old chickens were fed with Cu (11, 110, 220, and 330mg/kg) diet for 7 weeks. The consequence showed that high-Cu can induce ER stress and apoptosis in heart tissue. The vitro experiments, the Cu treatment for 24h could provoke ultrastructural damage and upregulate the apoptosis rate. Meanwhile, GRP78, GRP94, eIF2α, ATF6, XBP1, CHOP, Bax, Bak1, Bcl2, Caspase-12 and Caspase-3 genes levels, and GRP78, GRP94 and Caspase-3 proteins levels were increased, which indicated that ER stress and apoptosis in cardiomyocytes. But the mRNA level of Bcl2 were decreased after Cu exposure. Conversely, Cu-induced ER stress-mediated apoptosis can be alleviated by treatment with 4-PBA. These findings generally showed that Cu exposure can contribute to ER stress-mediated apoptosis in chicken myocardium, which clarifies the important mechanism link between ER stress and apoptosis, and provides a new perspective for Cu toxicology.

Marchf6 E3 ubiquitin ligase critically regulates endoplasmic reticulum stress, ferroptosis, and metabolic homeostasis in POMC neurons

The metabolic prohormone pro-opiomelanocortin (POMC) is generally translocated into the endoplasmic reticulum (ER) for entry into the secretory pathway. Patients with mutations within the signal peptide (SP) of POMC or its adjoining segment develop metabolic disorders. However, the existence, metabolic fate, and functional outcomes of cytosol-retained POMC remain unclear. Here, we show that SP-uncleaved POMC is produced in the cytosol of POMC neuronal cells, thus inducing ER stress and ferroptotic cell death. Mechanistically, the cytosol-retained POMC sequesters the chaperone Hspa5 and subsequently accelerates degradation of the glutathione peroxidase Gpx4, a core regulator of ferroptosis, via the chaperone-mediated autophagy. We also show that the Marchf6 E3 ubiquitin ligase mediates the degradation of cytosol-retained POMC, thereby preventing ER stress and ferroptosis. Furthermore, POMC-Cre-mediated Marchf6-deficient mice exhibit hyperphagia, reduced energy expenditure, and weight gain. These findings suggest that Marchf6 is a critical regulator of ER stress, ferroptosis, and metabolic homeostasis in POMC neurons.

Botulinum toxin type A activates protective autophagy by modulating endoplasmic reticulum stress in hypoxia/reoxygenation-treated endothelial cells

Botulinum toxin type A (BTXA) previously protected endothelial cells in free skin flaps from ischemia/reperfusion injury by inducing autophagy. Endoplasmic reticulum (ER) stress-autophagy activation may have a role in BTXA-antagonized ischemia/reperfusion damage in human dermal microvascular endothelial cells (HDMECs), however, this has yet to be proven. HDMECs were pretreated with BTXA at various concentrations for 12 h before being subjected to hypoxia and reoxygenation (H/R). Cell Count Kit 8 (CCK8) and Western blot (WB) data showed that H/R treatment significantly increased the expression of ER stress (GRP78, CHOP) and autophagy (LC3II/I, Beclin-1) proteins. The optimal BTXA pretreatment concentration was 1.6 U/mL, which resulted in the highest levels of cell survival and expression of ER stress and autophagy. Following pretreatment with 1.6 U/mL BTXA and the addition of the ER stress inducer Thapsigargin (Tg), the ER stress inhibitor 4-phenylbutyrate (4-PBA), and the inhibitor of autophagy Bafilomycin A1 (Baf A1), respectively, HDMECs were then subjected to H/R treatment. Cell survival and the percentage of ethynyldeoxyuridine-labeled cells in the BTXA pretreatment groups were reduced by the addition of Tg, 4-PBA, and Baf A1. While the expression of GRP78, CHOP, and LC3 was upregulated by Tg and Baf A1, it was downregulated by 4-PBA. The findings showed that ER stress produced by BTXA pretreatment triggers protective autophagy and protects HDMECs from H/R damage. There were no cytoprotective effects from either excessive activation or reduction of ER stress. Our results are consistent with the notion that autophagy and moderate ER stress are critical for cellular longevity and, consequently, functional integrity and may represent a potential therapeutic target.

Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress.

The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease. We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells. Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines. We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.

Persistent ER stress causes GPI anchor deficit to convert a GPI-anchored prion protein into pro-PrP via the ATF6–miR449c-5p–PIGV axis

Endoplasmic reticulum (ER) stress and unfolded protein response are cells' survival strategies to thwart disruption of proteostasis. Tumor cells are continuously being challenged by ER stress. The prion protein, PrP, normally a glycosylphosphatidylinositol (GPI)-anchored protein exists as a pro-PrP retaining its GPI-peptide signal sequence in human pancreatic ductal cell adenocarcinoma (PDAC). Higher abundance of pro-PrP indicates poorer prognosis in PDAC patients. The reason why PDAC cells express pro-PrP is unknown. Here, we report that persistent ER stress causes conversion of GPI-anchored PrP to pro-PrP via a conserved ATF6-miRNA449c-5p-PIGV axis. Mouse neurons and AsPC-1, a PDAC cell line, express GPI-anchored PrP. However, continuous culture of these cells with the ER stress inducers thapsigargin or brefeldin A results in the conversion of a GPI-anchored PrP to pro-PrP. Such a conversion is reversible; removal of the inducers allows the cells to re-express a GPI-anchored PrP. Mechanistically, persistent ER stress increases the abundance of an active ATF6, which increases the level of miRNA449c-5p (miR449c-5p). By binding the mRNA of PIGV at its 3'-UTRs, miR449c-5p suppresses the level of PIGV, a mannosyltransferase pivotal in the synthesis of the GPI anchor. Reduction of PIGV leads to disruption of the GPI anchor assembly, causing pro-PrP accumulation and enhancing cancer cell migration and invasion. The importance of ATF6-miR449c-5p-PIGV axis is recapitulated in PDAC biopsies as the higher levels of ATF6 and miR449c-5p and lower levels of PIGV are markers of poorer outcome for patients with PDAC. Drugs targeting this axis may prevent PDAC progression.

Effects of Different Spectrum of LEDs on Retinal Degeneration Through Regulating Endoplasmic Reticulum Stress.

To evaluate the impact of full-spectrum light-emitting diodes (LEDs) on albino guinea pigs' retina and investigate the roles of short-wavelength opsin (S-opsin) and endoplasmic reticulum (ER) stress in light-induced retinal degeneration (LIRD). Three-week-old albino guinea pigs (n = 30) were distributed into five groups under 12/12 light/dark cycles with indoor natural light (NC; 300-500 lux, n = 6), full-spectrum LEDs (FL; 300 lux, n = 6; 3000 lux, n = 6), and commercial cold-white LEDs (CL; 300 lux, n = 6; 3000 lux, n = 6) and raised for 28 days. Hematoxylin and eosin staining and transmission electron microscopy evaluated the morphological changes of retinas. The immunofluorescence and real-time quantitative polymerase chain reaction (RT-qPCR) measured the expression and content of S-opsin and ER stress-related genes and proteins. We found that albino guinea pigs exposed to FL at either 300 lux or 3000 lux developed less severe retinal morphological damage than animals exposed to the CL light, which emerged as a significant characteristic of LIRD. Meanwhile, the damage on the ventral retina was more serious, mainly due to its ability to absorb the blue light in the LEDs more easily. Compared to the FL-exposed groups, the CL light increased the aggregation of S-opsin and the expression of ER stress-related factors. Commercial cold-white LEDs can induce ER stress and unfolded protein response in LIRD, and full-spectrum LED attenuates LIRD by regulating ER stress in albino guinea pig retinas in vivo. Full-spectrum LEDs offer specific eye protection and eye adaptability that can well replace commercial cold-white LEDs in both clinical practice and research. It should be further developed for lighting used in health care facilities.

Mitofusin2 Ameliorated Endoplasmic Reticulum Stress and Mitochondrial Reactive Oxygen Species Through Maintaining Mitochondria-Associated Endoplasmic Reticulum Membrane Integrity in Cisplatin-Induced Acute Kidney Injury.

Aims: This study investigated the regulatory effect of Mitofusin2 (Mfn2) on mitochondria-associated endoplasmic reticulum membrane (MAM) integrity and cellular injury in cisplatin-induced acute kidney injury (CP-AKI). Results: CP-AKI mice exhibited decreased expression of Mfn2, increased expression of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), abnormal mitochondrial morphology, and reduced MAMs integrity, accompanied by the activation of mitochondrial reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress (inositol-requiring enzyme 1 [IRE1] and PERK pathways). In in vitro studies, CP-induced mitochondrial ROS, ER-stress activation, and increased apoptosis were accompanied by the downregulation of Mfn2 and MAMs integrity reduction in Boston University mouse proximal tubular cells (BUMPT) and human proximal tubular epithelial cells (HK-2). Pretreatment of BUMPT cells with the Mfn2 plasmid partially restored the integrity of MAMs, negatively controlled IRE1 and PERK pathways, and inhibited cell apoptosis. In contrast, ER-stress and MAMs integrity violations were increased after Mfn2 small-interfering RNA (siRNA) treatment in HK-2 cells under CP treatment. Coimmunoprecipitation analysis demonstrated that Mfn2 interacted with PERK and IRE1. Furthermore, the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), acadesine (AICAR), had a similar effect to Mfn2 plasmid in the regulation of ER stress and MAMs. Conversely, the ER-stress inhibitor, 4-phenylbutyric acid (4-PBA), had no effect on the expression of Mfn2 and MAMs integrity. Innovation and Conclusion: This is the first study to explore the association between MAMs, ER stress, and Mfn2 in CP-AKI. Downregulation of Mfn2 expression abolished the MAMs integrity, and induced ER stress, mitochondrial ROS, and tubular cell apoptosis. This suggests that the Mfn2-MAMs pathway is a potential therapeutic target in CP-AKI. Antioxid. Redox Signal. 40, 16-39. The Ethical Registration number of animal experiment in this study was CSU-2022-01-0095.

A novel heteroleptic Cu(II)-phenanthroline-UDCA complex as lipoxygenase inhibitor and ER-stress inducer in cancer cell lines

A new heteroleptic copper(II) compound named C0-UDCA was prepared by reaction of [Cu(phen)2(OH2)](ClO4)2 (C0) with the bile ursodeoxycholic acid (UDCA). The resulting compound is able to inhibit the lipoxygenase enzyme showing more efficacy than the precursors C0 and UDCA. Molecular docking simulations clarified the interactions with the enzyme as due to allosteric modulation. The new complex shows antitumoral effect on ovarian (SKOV-3) and pancreatic (PANC-1) cancer cells at the Endoplasmic Reticulum (ER) level by activating the Unfolded Protein Response. In particular, the chaperone BiP, the pro-apoptotic protein CHOP and the transcription factor ATF6 are upregulated in the presence of C0-UDCA. The combination of Intact Cell MALDI-MS and statistical analysis have allowed us to discriminate between untreated and treated cells based on their mass spectrometry fingerprints.

275-LB: Mitochondrial Fission Factor Can Be a Key Factor in the Regulation of Hepatic Lipid Metabolism

Nonalcoholic fatty liver disease (NAFLD) develops into nonalcoholic steatohepatitis (NASH) when fat accumulates in the liver, leading to hepatocyte apoptosis, inflammation and fibrosis. No effective treatments for NASH have been established. Mitochondria are important organelles that play a central role in de novo lipogenesis and fatty acid β-oxidation. We have shown that mitochondrial fission is enhanced by short-term high-fat diet (HFD) loading, but is impaired by long-term. We speculated that mitochondrial fission inhibition is associated with the development of NASH and generated mice deletion of hepatocyte-specific mitochondrial fission factor (KO) mice. Conditioning HFD, KO mice showed inhibition of hepatocyte mitochondrial fission, increased ER stress and decreased triglyceride secretion from the liver. The hepatic triglyceride contents had significantly increased in KO mice compared to control (CONT), and histological analysis showed a marked increase in large lipid droplets (LDs). In addition to marked fat accumulation, hepatic apoptosis, inflammation and fibrosis were increased in the livers of KO mice, indicating the development of NASH. We noted that large LDs were increased in KO mice and added an additional analysis to determine how mitochondrial fission inhibition is involved in LDs formation in the liver. Oleic acid is a representative monounsaturated fatty acid that promotes LDs deposition in hepatocytes and is mildly cytotoxic by inducing ER stress and apoptosis. Loading primary cultured hepatocytes (PH) with oleic acid increased the number of smaller LDs in PH of CONT mice, whereas LDs in cells of KO mice became larger. Gene expression of Cidec, which acts on the fusion of LDs, was increased in the liver of KO mice, suggesting that inhibition of mitochondrial fission may induce the fusion of LDs and the enlargement of intracellular LDs in the liver. Further analysis is planned, as inhibition of mitochondrial fission may be involved in changes of LDs morphology. Disclosure Y. Takeichi: None. T. Miyazawa: None. Y. Ogawa: None.

269-LB: Modeling Alpha-Cell Dysfunction Using Stem Cell–Derived Alpha Cells

Type 1 Diabetes (T1D) therapies have focused on restoring beta cell function via exogenous insulin or islet transplant. While promising, these approaches fail to account for the role of alpha cells and intra-islet communication in disease etiology. Alpha cells secrete glucagon and maintain effective paracrine signaling with beta cells to maintain glucose homeostasis and metabolic control. Mounting evidences suggest that impaired alpha cell function is a significant contributor to disease progression. Understanding triggers of alpha cell dysfunction and the resulting effects will be crucial for restoring both beta and alpha cell function in T1D. Lack of human alpha cell models have limited progress towards this goal. We recently reported a protocol to generate human alpha cells from stem cell sources (SC-alpha). Using these SC-alpha cells, we have established a T1D disease model to identify the triggers that induce alpha cell dysfunction. Importantly, we have identified defects in proglucagon processing that mimic those observed in diabetes and have identified conditions that modulate proglucagon processing and secretion. Here we demonstrate that exposure of SC-alpha cells to diabetogenic stressors such as hyperglycemia, hyperlipidemia, and ER stress impacts the processing of proglucagon and ultimately results in alpha cell dysfunction. We show that upon exposure to these stressors, SC-alpha cells secrete elevated glucagon compared to the control conditions and show a loss of glucose responsiveness. We also observed changes in the secretion of alternate proglucagon processing products, GLP-1, GLP-2, and glicentin. Interestingly, glicentin levels were affected by ER stress induction, but were unaffected by other stressors, suggesting that ER stress plays a direct role in the disruption of proglucagon processing, leading to alpha cell dysfunction. Identifying triggers of alpha cell dysfunction and assessing impacts in proglucagon processing will be a step towards restoring islet paracrine signaling. Disclosure S. Shrestha: None. Q. P. Peterson: None.

A decrease of mitochondrial ubiquitin ligase increases the secretion of matrix metalloproteinase-1 by dermal fibroblasts through the induction of ER stress.

We previously reported that the level of mitochondrial ubiquitin ligase (MITOL) protein in fibroblasts was decreased by UVA and that the knock-down (KD) of MITOL increased the secretion of matrix metalloprotease-1 (MMP-1) by fibroblasts. A recent study reported that MITOL suppresses endoplasmic reticulum (ER) stress by stabilizing the interaction between ER and mitochondria (MT) through the ubiquitination of mitofusin 2. These facts suggest that a decrease of MITOL would increase the secretion of MMP-1 through ER stress, but the detailed mechanism of that process in dermal fibroblasts remains unclear. Thus, this study was conducted to clarify the involvement of ER stress in the oversecretion of MMP-1 induced by the decreased MT quality caused by MITOL-KD. MITOL-KD normal human dermal fibroblast (NHDFs) were prepared by treating them with MITOL-small interfering RNA, after which their MMP-1 protein levels were measured. ER stress in NHDFs was evaluated by measuring the mRNA levels of spliced X-box binding protein 1 (sXBP1) and the protein levels of inositol-requiring enzyme 1α (IRE1α). MITOL-KD NHDFs enhanced the secretion of MMP-1 via interleukin-6 (IL-6) elicited by the activation of nuclear factor-kappa B (NF-κB). The secretion of MMP-1 could be abrogated by a neutralizing IL-6 antibody and by JSH23, which is an inhibitor of NF-κB activation. Furthermore, MITOL-KD NHDFs as well as UVA-irradiated NHDFs showed increased ER stress levels. In addition, tunicamycin, which is an inducer of ER stress, also increased MMP-1 secretion. These results suggested that the decrease of MITOL caused the oversecretion of MMP-1 via NF-κB-IL-6 signaling through the activation of ER stress in fibroblasts.

Oridonin promotes endoplasmic reticulum stress via TP53-repressed TCF4 transactivation in colorectal cancer

BackgroundThe incidence of colorectal cancer and cancer death rate are increasing every year, and the affected population is becoming younger. Traditional Chinese medicine therapy has a unique effect in prolonging survival time and improving the prognosis of patients with colorectal cancer. Oridonin has been reported to have anti-cancer effects in a variety of tumors, but the exact mechanism remains to be investigated.MethodsCell Counting Kit-8 assay (CCK8) and 5-Ethynyl-2'-deoxyuridine (EdU) staining assay, Tranwell, and Wound healing assays were performed to measure cell proliferation, invasion, and migration capacities, respectively. The protein and mRNA expression levels of various molecules were reflected by Western blot and Reverse Transcription quantitative Polymerase Chain Reaction (qRT-PCR). Transcription Factor 4 (TCF4) and its target genes were analyzed by Position Weight Matrices (PWMs) software and the Gene Expression Omnibus (GEO) database. Immunofluorescence (IF) was performed to visualize the expression and position of Endoplasmic Reticulum (ER) stress biomarkers. The morphology of the ER was demonstrated by the ER tracker-red. Reactive Oxygen Species (ROS) levels were measured using a flow cytometer (FCM) or fluorescent staining. Calcium ion (Ca2+) concentration was quantified by Fluo-3 AM staining. Athymic nude mice were modeled with subcutaneous xenografts.ResultsOridonin inhibited the proliferation, invasion, and migration of colorectal cancer, and this effect was weakened in a concentration-dependent manner by ER stress inhibitors. In addition, oridonin-induced colorectal tumor cells showed increased expression of ER stress biomarkers, loose morphology of ER, increased vesicles, and irregular shape. TCF4 was identified as a regulator of ER stress by PWMs software and GEO survival analysis. In vitro and in vivo experiments confirmed that TCF4 inhibited ER stress, reduced ROS production, and maintained Ca2+ homeostasis. In addition, oridonin also activated TP53 and inhibited TCF4 transactivation, further exacerbating the elevated ROS levels and calcium ion release in tumor cells and inhibiting tumorigenesis in colorectal cancer cells in vivo.ConclusionsOridonin upregulated TP53, inhibited TCF4 transactivation, and induced ER stress dysregulation in tumor cells, promoting colorectal cancer cell death. Therefore, TCF4 may be one of the important nodes for tumor cells to regulate ER stress and maintain protein synthesis homeostasis. And the inhibition of the TP53/TCF4 axis plays a key role in the anti-cancer effects of oridonin.

Inhibition of TRPA1, Endoplasmic Reticulum Stress, Human Airway Epithelial Cell Damage, and Ectopic MUC5AC Expression by Vasaka (Adhatoda vasica; Malabar Nut) Tea.

This study tested whether a medicinal plant, Vasaka, typically consumed as a tea to treat respiratory malaise, could protect airway epithelial cells (AECs) from wood smoke particle-induced damage and prevent pathological mucus production. Wood/biomass smoke is a pneumotoxic air pollutant. Mucus normally protects the airways, but excessive production can obstruct airflow and cause respiratory distress. Vasaka tea pre- and co-treatment dose-dependently inhibited mucin 5AC (MUC5AC) mRNA induction by AECs treated with wood smoke particles. This correlated with transient receptor potential ankyrin-1 (TRPA1) inhibition, an attenuation of endoplasmic reticulum (ER) stress, and AEC damage/death. Induction of mRNA for anterior gradient 2, an ER chaperone/disulfide isomerase required for MUC5AC production, and TRP vanilloid-3, a gene that suppresses ER stress and wood smoke particle-induced cell death, was also attenuated. Variable inhibition of TRPA1, ER stress, and MUC5AC mRNA induction was observed using selected chemicals identified in Vasaka tea including vasicine, vasicinone, apigenin, vitexin, isovitexin, isoorientin, 9-oxoODE, and 9,10-EpOME. Apigenin and 9,10-EpOME were the most cytoprotective and mucosuppressive. Cytochrome P450 1A1 (CYP1A1) mRNA was also induced by Vasaka tea and wood smoke particles. Inhibition of CYP1A1 enhanced ER stress and MUC5AC mRNA expression, suggesting a possible role in producing protective oxylipins in stressed cells. The results provide mechanistic insights and support for the purported benefits of Vasaka tea in treating lung inflammatory conditions, raising the possibility of further development as a preventative and/or restorative therapy.