Stress Inducer Thapsigargin Research Articles

TIGAR suppresses ER stress-induced neuronal injury through targeting ATF4-signaling in cerebral ischemia/reperfusion.

Endoplasmic reticulum (ER) stress is crucial in cerebral ischemia/reperfusion injury by triggering cellular apoptosis and exacerbating neuronal damage. This study elucidates the dynamics of TP53-induced glycolysis and apoptosis regulator (TIGAR) translocation and its role in regulating neural fate during cerebral ischemia-induced ER stress, specifically in male mice. We found enhanced nuclear localization of TIGAR in neurons after transient middle cerebral artery occlusion/reperfusion (tMCAO/R) in male mice, as well as oxygen glucose deprivation/reperfusion (OGD/R) and treatment with ER stress inducer (tunicamycin and thapsigargin) in neuronal cells. Conditional neuronal knockdown of Tigar aggravated the injury following ischemia-reperfusion, whereas overexpression of Tigar attenuated cerebral ischemic injury and ameliorated intra-neuronal ER stress. Additionally, TIGAR overexpression reduced the elevation of ATF4 target genes and attenuated ER stress-induced cell death. Notably, TIGAR co-localized and interacted with ATF4 in the nucleus, inhibiting its downstream pro-apoptotic gene transcription, consequently protecting against ischemic injury. In vitro and in vivo experiments revealed that ATF4 overexpression reversed the protective effects of TIGAR against cerebral ischemic injury. Intriguingly, our study identified the Q141/K145 residues of TIGAR, crucial for its nuclear translocation and interaction with ATF4, highlighting a novel aspect of TIGAR's function distinct from its known phosphatase activity or mitochondrial localization domains. These findings reveal a novel neuroprotective mechanism of TIGAR in regulating ER stress through ATF4-mediated signaling pathways. These insights may guide targeted therapeutic strategies to protect neuronal function and alleviate the deleterious effects of cerebral ischemic injury.Significance statement TIGAR (TP53-induced glycolysis and apoptosis regulator) is one of the downstream target genes of p53, and its encoded protein exerts Fru-2, 6-BPase activity to promote glucose metabolic flux to pentose phosphate pathway. However, the non-enzymatic function of TIGAR has been gradually discovered. Here, we demonstrate that TIGAR translocates to the nucleus to interact with ATF4 in neurons after cerebral ischemia/reperfusion induced ER stress via its Q141/K145 residues. Then TIGAR inhibits ATF4's downstream pro-apoptotic genes expression, reduces ER stress-dependent apoptosis, consequently alleviating neuronal damage. This study uncovered a novel neuroprotective mechanism of TIGAR by regulating ER stress via ATF4-mediated signaling pathway. The Q141/K145 residues of TIGAR are critical for its interaction with ATF4 and inhibition of ATF4 target genes.

Krüppel-like factor 6 involvement in the endoplasmic reticulum homeostasis of extravillous trophoblasts

IntroductionTrophoblast homeostasis and differentiation require a proper endoplasmic reticulum (ER) function. The Krüppel-like factor-6 (KLF6) transcription factor modulates trophoblast migration, differentiation, and reactive oxygen species (ROS) production. Since ROS may impact on ER homeostasis, we assessed whether downregulation of KLF6 altered the unfolded protein response (UPR) and cellular process associated with ER homeostasis. Materials and methodsProtein and RNA expression were analyzed by Western blot and qRT-PCR, respectively, in extravillous trophoblast HTR-8/SVneo cells silenced for KLF6. Apoptosis was detected by flow cell cytometry using Annexin V Apoptosis Detection Kit. Protein trafficking was assessed by confocal microscopy of a reporter fluorescent protein whose release from the ER was synchronized. ResultsKLF6 downregulation reduced the expression of BiP, the master regulator of the UPR, at protein, mRNA, and pre-mRNA levels. Ire1α protein, XBP1 splicing, and DNAJB9 mRNA levels were also reduced in KLF6-silenced cells. Instead, PDI, Ero1α, and the p-eIF2α/eIF2α ratio as well as autophagy and proteasome dependent protein degradation remained unchanged while intracellular trafficking was increased. Under thapsigargin-induced stress, KLF6 silencing impaired BiP protein and mRNA expression increase, as well as the activation of the Ire1α pathway, but it raised the p-eIF2α/eIF2α ratio and CHOP protein levels. Nevertheless, apoptosis was not increased. DiscussionResults provide the first evidence of KLF6 as a modulator of the UPR components. The increase in protein trafficking and protection from apoptosis, observed in KLF6-silenced cells, are consistent with its role in extravillous trophoblast migration and differentiation.

Cyclophilin D induces necrotic core formation by mediating mitochondria-associated macrophage death in advanced atherosclerotic lesions

Background and aimsIn advanced atherosclerotic lesions, macrophage deaths result in necrotic core formation and plaque vulnerability. Cyclophilin D (CypD) is a mitochondria-specific cyclophilin involved in the process of cell death after organ ischemia-reperfusion. However, the role of CypD in atherosclerosis, especially in necrotic core formation, is unknown. Therefore, this experiment aims to clarify the role of CypD in necrotic core formation. MethodsTo clarify the specific role of CypD, encoded by Ppif in mice, apolipoprotein-E/CypD-double knockout (Apoe−/−Ppif−/−) mice were generated. These mice were fed a high-fat diet containing 0.15 % cholesterol for 24 weeks to accelerate atherosclerotic lesion development. ResultsDeletion of CypD decreased the necrotic core size, accompanied by a reduction of macrophage apoptosis compared to control Apoe−/− mice. In RAW264.7 cells, siRNA-mediated knockdown of CypD attenuated the release of cytochrome c from the mitochondria to the cytosol induced by endoplasmic reticulum stress inducer thapsigargin. In addition, necroptosis, induced by TNF-α and caspase inhibitor, was attenuated by knockdown of CypD. Ly-6Chigh inflammatory monocytes in peripheral blood leukocytes and mRNA expression of Il1b in the aorta were decreased by deletion of CypD. In contrast, siRNA-mediated knockdown of CypD did not significantly decrease Il1b nor Ccl2 mRNA expression in RAW264.7 cells treated with LPS and IFN-γ, suggesting that inhibition of inflammation in vivo is likely due to decreased cell death in the atherosclerotic lesions rather than a direct action of CypD deletion on the macrophage. ConclusionsThese results indicate that CypD induces macrophage death and mediates necrotic core formation in advanced atherosclerotic lesions. CypD could be a novel therapeutic target for treating atherosclerotic vascular diseases.

Social defeat stress induces liver injury by modulating endoplasmic reticulum stress in C57BL/6J mice

Social defeat stress is associated with endoplasmic reticulum (ER) stress, inflammation and apoptosis. ER stress is thought to contribute to many lifestyle diseases such as liver injury, cardiovascular dysfunction and depression. We investigated the expression of the ER stress markers RNA-dependent protein kinase-like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α) and C/EBP homologous protein (CHOP), as well as inflammatory and apoptotic factors, to assess how social defeat stress induces liver injury. Furthermore, we evaluated the effects of the ER stress inhibitor phenylbutyric acid (PBA) and ER stress inducer thapsigargin (TG) on liver injury. Adult mice were divided into the control, social defeat, social defeat + PBA, TG, PBA and TG + PBA groups. The social defeat and social defeat + PBA groups were simultaneously exposed to social defeat stress for 10 days. The social defeat + PBA, TG, PBA and TG + PBA groups were treated with PBA or TG via intraperitoneal injections. PBA was injected 1 h before the TG injection into the TG + PBA group. Liver samples from six groups of mice were analyzed by histological analysis and western blotting. Social defeat stress promoted ER stress, increased the expression of inflammatory factors and induced apoptosis in the liver of socially defeated mice, which was reversed by PBA. Moreover, ER stress induces TG-induced liver injury by initiating ER stress. Social defeat stress initiates ER stress, promotes the expression of inflammatory and apoptotic factors, and induces liver injury. PBA suppresses liver injury caused by social defeat stress and TG treatment.

The endoplasmic reticulum stress protein GRP94 modulates cathepsin L activity in M2 macrophages in conditions of obesity-associated inflammation and contributes to their pro-inflammatory profile.

Adipose tissue macrophages (ATM) are key actors in the pathophysiology of obesity-related diseases. They have a unique intermediate M2-M1 phenotype which has been linked to endoplasmic reticulum (ER) stress. We previously reported that human M2 macrophages treated with the ER stress inducer thapsigargin switched to a pro-inflammatory phenotype that depended on the stress protein GRP94. In these conditions, GRP94 promoted cathepsin L secretion and was co-secreted with complement C3. As cathepsin L and complement C3 have been reported to play a role in the pathophysiology of obesity, in this work we studied the involvement of GRP94 in the pro-inflammatory phenotype of ATM. GRP94, cathepsin L and C3 expression were analyzed in CD206 + ATM from mice, WT or obesity-resistant transgenic fat-1, fed a high-fat diet (HFD) or a standard diet. GRP94 colocalization with cathepsin L and C3 and its effects were analyzed in human primary macrophages using thapsigargin as a control to induce ER stress and palmitic acid (PA) as a driver of metabolic activation. In WT, but not in fat-1 mice, fed a HFD, we observed an increase in crown-like structures consisting of CD206 + pSTAT1+ macrophages showing high expression of GRP94 that colocalized with cathepsin L and C3. In vitro experiments showed that PA favored a M2-M1 switch depending on GRP94. This switch was prevented by omega-3 fatty acids. PA-induced GRP94-cathepsin L colocalization and a decrease in cathepsin L enzymatic activity within the cells (while the enzymatic activity in the extracellular medium was increased). These effects were prevented by the GRP94 inhibitor PU-WS13. GRP94 is overexpressed in macrophages both in in vivo and in vitro conditions of obesity-associated inflammation and is involved in changing their profile towards a more pro-inflammatory profile. It colocalizes with complement C3 and cathepsin L and modulates cathepsin L activity.

Synergistic lethality in chronic myeloid leukemia – targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.

TMEM16A deficiency in alveolar type 2 epithelial cells protected against endoplasmic reticulum stress-induced ferroptosis during acute lung injury

Transmembrane protein 16A (TMEM16A) is one of the members of the ten-member family of “transmembrane protein 16”, playing critical roles in infection and solid organ injury. Acute lung injury (ALI) is a devastating disease which could be triggered by sepsis, trauma, and ischemia reperfusion. However, molecular mechanisms contributing to ALI are poorly understood at presently. In this study, we investigated the role of TMEM16A in sepsis-induced ALI using TMEM16A-deficient mice. Sepsis-induced ALI model was established by intratracheal injection of lipopolysaccharide (LPS). Our results showed that LPS stimulation significantly upregulated the expression levels of TMEM16A in lung tissues and in alveolar epithelial type II (AT2) cells. Knockout of TMEM16A in AT2 cells significantly improved pulmonary function and alleviated lung pathological injury in LPS-treated mice. Meanwhile, TMEM16A deficiency also inhibited endoplasmic reticulum (ER) stress and ferroptosis in AT2 cells from LPS-treated mice. In vitro experiments further demonstrated that ER stress and ferroptosis were inhibited after TMEM16A was knocked out. Furthermore, we used ER stress inducer thapsigargin to induce ER stress in TMEM16A-null AT2 cells and found that the induction of ER stress abolished the inhibition of ferroptosis by TMEM16A deficiency in LPS-treated AT2 cells. Finally, we disclosed that pharmacological inhibition of TMEM16A by shikonin also showed similar therapeutic effect on LPS-induced ALI in vivo. In conclusion, TMEM16A deficiency in AT2 cells could alleviate sepsis-induced ALI by decreasing ER stress-induced ferroptosis during ALI.

Cigarette smoke restricts the ability of mesenchymal cells to support lung epithelial organoid formation.

Adequate lung epithelial repair relies on supportive interactions within the epithelial niche, including interactions with WNT-responsive fibroblasts. In fibroblasts from patients with chronic obstructive pulmonary disease (COPD) or upon in vitro cigarette smoke exposure, Wnt/β-catenin signalling is distorted, which may affect interactions between epithelial cells and fibroblasts resulting in inadequate lung repair. We hypothesized that cigarette smoke (CS), the main risk factor for COPD, interferes with Wnt/β-catenin signalling in fibroblasts through induction of cellular stress responses, including oxidative- and endoplasmic reticulum (ER) stress, and thereby alters epithelial repair support potential. Therefore, we assessed the effect of CS-exposure and the ER stress inducer Thapsigargin (Tg) on Wnt/β-catenin signalling activation in MRC-5 fibroblasts, and on their ability to support lung epithelial organoid formation. Exposure of MRC-5 cells for 15min with 5AU/mL CS extract (CSE), and subsequent 6h incubation induced oxidative stress (HMOX1). Whereas stimulation with 100nMTg increased markers of both the integrated stress response (ISR - GADD34/PPP1R15A, CHOP) and the unfolded protein response (UPR - XBP1spl, GADD34/PPP1R15A, CHOP and HSPA5/BIP), CSE only induced GADD34/PPP1R15A expression. Strikingly, although treatment of MRC-5 cells with the Wnt activator CHIR99021 upregulated the Wnt/β-catenin target gene AXIN2, this response was diminished upon CSE or Tg pre-exposure, which was confirmed using a Wnt-reporter. Furthermore, pre-exposure of MRC-5 cells to CSE or Tg, restricted their ability to support organoid formation upon co-culture with murine pulmonary EpCam+ cells in Matrigel at day 14. This restriction was alleviated by pre-treatment with CHIR99021. We conclude that exposure of MRC-5 cells to CSE increases oxidative stress, GADD34/PPP1R15A expression and impairs their ability to support organoid formation. This inhibitory effect may be restored by activating the Wnt/β-catenin signalling pathway.

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.

Cannabidiol Protects Striatal Neurons by Attenuating Endoplasmic Reticulum Stress.

Introduction: The aggregation of misfolded proteins in the endoplasmic reticulum (ER) is a pathological trait shared by many neurodegenerative disorders. This aggregation leads to the persistent activation of the unfolded protein response (UPR) and ultimately apoptosis as a result of ER stress. Cannabidiol (CBD) has been demonstrated to be neuroprotective in various cellular and animal models of neurodegeneration, which has been attributed to its antioxidant and anti-inflammatory properties. However, little is known about the role of CBD in the context of protein folding and ER stress. The purpose of this study was to investigate whether CBD is neuroprotective against an in vitro model of ER stress. Materials and Methods: Using different exposure models, mouse striatal STHdhQ7/Q7 cells were exposed to either the ER stress inducer thapsigargin (TG) and/or CBD. Cell viabilities assays were used to investigate the effect of CBD pre-treatment, co-treatment, and post-treatment on TG-induced cell death. Real-time quantitative polymerase chain reaction was used to measure changes in ER stress regulators and UPR genes such as glucose-regulated protein-78 (GRP78), mesencephalic astrocyte-derived neurotrophic factor (MANF), B cell lymphoma 2 (BCL-2), BCL-2 interacting mediator of cell death (BIM), and caspase-12. Results: Cell viability increased significantly when cells were pre-treated with CBD before TG exposure. An increase in the gene expression of pro-survival ER chaperone GRP78 and ER-resident neurotrophic factor MANF coincided with this effect and decreased ER-mediated pro-apoptotic markers such as BIM, and caspase-12 was observed. Conclusions: These data suggest that CBD pre-treatment is neuroprotective against TG-induced cell death. Understanding the role of ER stress in CBD-driven neuroprotection provides insight into the therapeutic potential of CBD and the role of ER dysfunction in neurodegenerative disorders.

Endoplasmic reticulum stress-regulated high temperature requirement A1 (HTRA1) modulates invasion and angiogenesis-related genes in human trophoblasts

The pathogenesis of hypertensive disorder of pregnancy (HDP), which affects about 10% of pregnant women, is still incompletely understood. Our previous study showed that endoplasmic reticulum (ER) stress influences high-temperature requirement A serine peptidase 1 (HTRA1) expression and trophoblast invasion. However, the involvement of ER stress in the regulation of HTRA subtype expression and pathophysiology of HDP has not been characterized in extravillous trophoblasts (EVTs). To investigate this, HTR8/SVneo EVTs cell line was treated with the ER stress inducers Thapsigargin (Thap) or Tunicamycin (Tuni). Treatment with either Thap or Tuni inhibited trophoblast invasion, reduced HTRA1 and HTRA3 expression, but did not alter HTRA2 or HTRA4 expression. Knockdown of HTRA1 or HTRA3 also inhibited trophoblast invasion. Furthermore, treatment with either ER stress inducer or HTRA1 silencing increased the ratio of soluble fms-like tyrosine kinase-1/placental growth factor (sFLT1/PlGF), which is a marker of HDP. Immunohistochemical analysis revealed that HTRA1 is localized to EVTs and the endometrial decidua in the placenta of patients with HDP. These results suggest that factors that cause ER stress could result in the inhibition of EVTs invasion via HTRA1.

1, 25-(OH)2D3 protects against ER stress and miRNA dysregulation in Mus musculus neurons.

The pathophysiology of neurodegenerative diseases (NDDs) is closely associated with cellular oxidative stress which can result in the accumulation of toxic proteins in the endoplasmic reticulum (ER) leading to ER stress and subsequent unfolded protein response (UPR) signaling, a mechanism that aggravate these disorders. Vitamin D has been suggested to have important neuroprotective role and its administration has been shown to reduce neuronal injury, neurotoxicity and oxidative stress in various animal systems. The current study was undertaken to examine the effect of vitamin D3 on UPR in ER stress induced Mus musculus neuronal cells. Mus musculus cortical and hippocampal primary neuronal cultures were pretreated with 1,25-dihydroxyvitamin D3 (1, 25-(OH)2D3), the active form of vitamin D, followed by ER stress induction with a chemical ER stress inducer thapsigargin and with an advanced glycated protein, AGE-BSA. The UPR genes and related microRNAs (miRNA) expressions were analyzed mainly using real-time PCR. The experiment resulted in the suppression of ER stress marker BiP and UPR pathway genes such as Perk, Ire1α, Chop and Puma which mediate cellular apoptosis indicating the protective effect of 1, 25-(OH)2D3 against neuronal ER stress. Further studies into the molecular aspects showed that ER stress mediated down-regulated expression of microRNAs (miRNAs) such as mmu-miR-24, 27b, 124, 224, 290, 351 and 488 which are known to regulate the UPR pathway genes were also reduced with vitamin pretreatment, of which the miRNAs miR-24 and 27b which shares the same cluster are potentially involved in various human diseases. This study emphasizes the therapeutic role of vitamin D in reducing neuronal ER stress and the need for maintaining sufficient amount of this vitamin in our diet.

Multi-level inhibition of coronavirus replication by chemical ER stress

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.

Abstract 2026: KDM1A inhibition augment ER stress inducers efficacy to reduce glioblastoma stemness

Abstract Background: Glioblastoma (GBM) is the most common malignant brain tumor with a dismal prognosis and median survival of 20 months. Standard therapy consists of surgical resection, external beam radiation therapy, adjuvant chemotherapy with temozolomide, and tumor-treating fields. Despite these therapies, GBM patients inevitably experience tumor progression and eventually succumb to their disease. Glioma stem cells (GSCs) play a central role in GBM development and contribute to treatment resistance. Recently we showed that lysine-specific histone demethylase 1A (KDM1A/LSD1) is essential for GSCs stemness, and inhibition of KDM1A induces unfolded protein response (UPR) in GSCs. In this study, we tested the hypothesis that inhibition of KDM1A sensitizes GSCs to ER stress inducers by inducing UPR. Methods: KDM1A knockdown (KDM1A-KD) cells were generated using KDM1A specific shRNA. We studied the effect of KDM1A-KD or pharmacological KDM1A inhibitors (NCD38 and NCL-1) in combination with ER stress inducers (thapsigargin and brefeldin A) on GSCs viability using CellTiter-Glo assay. Stemness was determined using extreme limiting dilution analysis (ELDA) and sphere formation assays. Mechanistic studies were conducted using RNA-seq, ChIP, RT-qPCR, and Western blotting analysis. Furthermore, the in vivo efficacy of KDM1A inhibitor and ER stress inducer was studied using orthotopic models of GBM. Results: Cell viability assays demonstrated that knockdown or inhibition of KDM1A sensitized GSCs to ER stress inducers thapsigargin and brefeldin A. Furthermore, KDM1A-KD, NCD38, or NCL-1, in combination with ER stress inducers, significantly decreased the stemness and sphere-forming ability of GSCs. RNA-seq analysis revealed that UPR was activated after knockdown or inhibition of KDM1A in GSCs. Western blot and RT-qPCR analysis showed that a combination of KDM1A inhibitors and ER stress inducers increased UPR signaling in GSCs. ChIP analysis indicated that KDM1A inhibition enriched the active histone methylation mark (H3K4me2) at the promoter of UPR target genes. In vivo studies showed that KDM1A inhibition activates UPR in tumors and improved overall survival. Conclusions: Our results support that KDM1A knockdown or inhibition sensitizes GSCs to ER stress inducers and that the use of KDM1A inhibitors in conjunction with ER stress inducers is a potential novel therapy for GBM patients. Citation Format: Yi He, Prabhakar Pitta Venkata, Salvador Alejo, Yihong Chen, Bridgitte Palacios, Gabrielle Gray, Uday P. Pratap, Suryavathi Viswanadhapalli, Siyuan Zheng, Rajeshwar R. Tekmal, Andrew J. Brenner, Gangadhara R. Sareddy. KDM1A inhibition augment ER stress inducers efficacy to reduce glioblastoma stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2026.

Abstract 1960: Mechanisms of therapeutic synergy between pattern recognition response agonists and cdk4 inhibitors

Abstract Cytoplasmic nucleic acid sensors for double-stranded (ds) RNA (RIG-I/MDA5) and DNA (cGAS-STING) are pattern recognition receptors (PRRs) key to intracellular anti-viral responses. Recent research has highlighted roles for PRR agonists, including oncolytic virotherapy agents, in anti-tumor immunotherapy. Reovirus type 3 Dearing (Rt3D) is an oncolytic dsRNA virus with limited single-agent activity in clinical studies, but potential for use in combination regimens. We sought synergistic drug-virotherapy combinations using an unbiased screening approach that highlighted the CDK4/6 inhibitor, palbociclib, as a leading hit. We found that, when combined with Rt3D, palbociclib augmented oncolytic virus-induced endoplasmic reticulum (ER) stress/unfolded protein response (UPR) signaling. Combined Rt3D-palbociclib treatment potently increased interferon signaling and endogenous retroviral transcripts. Knockdown (siRNA) studies indicated key UPR proteins and the RNA sensor, RIG-I, were essential to the phenotype observed. Mechanistically independent experiments, using canonical RIG-I agonists and the ER stress inducer (thapsigargin), confirmed cross-talk between RNA sensing and ER stress pathways that augment cancer cell death and interferon production. Combined Rt3D-palbociclib increased innate immune activation and effector function. Our findings demonstrate that UPR signaling and innate immune RNA sensor crosstalk can be exploited to enhance anti-cancer efficacy with pro-immunogenic consequences. This has implications for future clinical development of PRR agonists and oncolytic viruses, as well as broadening the therapeutic remit of CDK4/6 inhibitors to include their role as ER stress sensitizers. Citation Format: Victoria Roulstone, Joan Kyula, Richard Elliot, Christopher J. Lord, Nik Matthews, Vicki Jennings, Harriet Whittock, David Mansfield, Jyoti Choudhary, James Wright, Lu Yu, Alan Melcher, Richard Vile, Matt Coffey, Martin McLaughlin, Kevin Harrington. Mechanisms of therapeutic synergy between pattern recognition response agonists and cdk4 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1960.

Extracellular α-Synuclein Modulates Iron Metabolism Related Proteins via Endoplasmic Reticulum Stress in MES23.5 Dopaminergic Cells.

Alpha-synuclein plays a vital role in the pathology of Parkinson's disease (PD). Spreading of α-synuclein in neighboring cells was believed to contribute to progression in PD. How α-synuclein transmission affects adjacent cells is not full elucidated. Here, we used recombinant α-synuclein to mimic intercellular transmitted α-synuclein in MES23.5 dopaminergic cells, to investigate whether and how it could modulate iron metabolism. The results showed that α-synuclein treatment up-regulated divalent metal transporter 1 (DMT1) and down-regulated iron transporter (FPN), also up-regulated iron regulatory protein 1 (IRP1) protein levels and hepcidin mRNA levels. Endocytosis inhibitor dynasore pretreatment completely abolished and even reversed the upregulation of DMT1 and IRP1 induced by α-synuclein, however, FPN down-regulation was partially blocked by dynasore. Autophagy-inducing agent rapamycin reversed DMT1 up-regulation and FPN down-regulation, and fully blocked the upregulation of IRP1. Elevated hepcidin levels induced by α-synuclein was fully blocked by dynasore pretreatment, however, even higher with rapamycin pretreatment. Alpha-synuclein treatment triggered endoplasmic reticulum (ER) stress. ER stress inducer thapsigargin induced similar responses elicited by α-synuclein. ER stress inhibitor salubrinal blocked the up-regulation of IRP1 and hepcidin, as well as DMT1 up-regulation and FPN down-regulation, also dramatically abolished cAMP-response elements binding protein phosphorylation induced by α-synuclein. Taken together, these finding indicated that extracellular α-synuclein could regulate cellular iron metabolism, probably mediated by ER stress. It provides novel evidence to elucidate the relationships between transmitted α-synuclein and iron metabolism disturbance in PD.

Extracellular Vesicles from Thapsigargin-Treated Mesenchymal Stem Cells Ameliorated Experimental Colitis via Enhanced Immunomodulatory Properties.

Therapeutic applications of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have attracted considerable attention because of their immunomodulatory properties against immune-mediated, inflammatory diseases. Here, we demonstrated enhanced immunomodulatory properties of EVs secreted from endoplasmic reticulum (ER) stress inducer thapsigargin (TSG)-primed human Wharton’s jelly-derived MSCs (WJ-MSCs). EVs from TSG-primed WJ-MSCs (TSG-EV) showed increased yield and expression of immunomodulatory factors, such as transforming growth factor-β1 (TGFβ), cyclooxygenase-2 (COX2), and especially indoleamine 2,3-dioxygenase (IDO), compared to control EVs. TSG-EV showed a significantly enhanced immunosuppressive effect on human peripheral blood-derived T cell proliferation and Th1 and Th17 differentiation, whereas Treg and M2-type macrophage were enriched compared to a control EV-treated group. Furthermore, TSG-EV substantially mitigated mouse experimental colitis by reducing the inflammatory response and maintaining intestinal barrier integrity. A significant increase of Tregs and M2-type macrophages in colitic colons of a TSG-EV-treated mouse suggests an anti-inflammatory effect of TSG-EV in colitis model, possibly mediated by Treg and macrophage polarization. These data indicate that TSG treatment promoted immunomodulatory properties of EVs from WJ-MSCs, and TSG-EV may provide a new therapeutic approach for treatment of colitis.

Hypoxia and Hypoxia-Inducible Factor-1α Regulate Endoplasmic Reticulum Stress in Nucleus Pulposus Cells: Implications of Endoplasmic Reticulum Stress for Extracellular Matrix Secretion

Endoplasmic reticulum (ER) stress is shown to promote nucleus pulposus (NP) cell apoptosis and intervertebral disc degeneration. However, little is known about ER stress regulation by the hypoxic disc microenvironment and its contribution to extracellular matrix homeostasis. NP cells were cultured under hypoxia (1% partial pressure of oxygen) to assess ER stress status, and gain-of-function and loss-of-function approaches were used to assess the role of hypoxia-inducible factor (HIF)-1α in this pathway. In addition, the contribution of ER stress induction on the NP cell secretome was assessed by a nontargeted quantitative proteomic analysis by sequential windowed data independent acquisition of the total high-resolution mass spectra-mass spectrometry. NP cells exhibited a lower ER stress burden under hypoxia. Knockdown of HIF-1α increased C/EBP homologous protein, protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) levels, whereas HIF-1α stabilization decreased the expression of ER stress markers Ddit3, Hsp5a, Atf6, and Eif2a. Interestingly, ER stress inducers tunicamycin and thapsigargin induced HIF-1α activity under hypoxia while promoting the unfolded protein response. NP cell secretome analysis demonstrated an impact of ER stress induction on extracellular matrix secretion, with decreases in collagens and cell adhesion-related proteins. Moreover, analysis of transcriptomic data of NP tissues from aged mice and degenerated human discs showed higher levels of unfolded protein response markers and decreased levels of matrix components. Our study shows, for the first time, that hypoxia and HIF-1α attenuate ER stress responses in NP cells, and ER stress promotes inefficient extracellular matrix secretion under hypoxia.