Endoplasmic Reticulum Stress Inducer Thapsigargin Research Articles

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.

The Role of SLIT3-ROBO4 Signaling in Endoplasmic Reticulum Stress-Induced Delayed Corneal Epithelial and Nerve Regeneration.

In the present study, we aim to elucidate the underlying molecular mechanism of endoplasmic reticulum (ER) stress induced delayed corneal epithelial wound healing and nerve regeneration. Human limbal epithelial cells (HLECs) were treated with thapsigargin to induce excessive ER stress and then RNA sequencing was performed. Immunofluorescence, qPCR, Western blot, and ELISA were used to detect the expression changes of SLIT3 and its receptors ROBO1-4. The role of recombinant SLIT3 protein in corneal epithelial proliferation and migration were assessed by CCK8 and cell scratch assay, respectively. Thapsigargin, exogenous SLIT3 protein, SLIT3-specific siRNA, and ROBO4-specific siRNA was injected subconjunctivally to evaluate the effects of different intervention on corneal epithelial and nerve regeneration. In addition, Ki67 staining was performed to evaluate the proliferation ability of epithelial cells. Thapsigargin suppressed normal corneal epithelial and nerve regeneration significantly. RNA sequencing genes related to development and regeneration revealed that thapsigargin induced ER stress significantly upregulated the expression of SLIT3 and ROBO4 in corneal epithelial cells. Exogenous SLIT3 inhibited normal corneal epithelial injury repair and nerve regeneration, and significantly suppressed the proliferation and migration ability of cultured mouse corneal epithelial cells. SLIT3 siRNA inhibited ROBO4 expression and promoted epithelial wound healing under thapsigargin treatment. ROBO4 siRNA significantly attenuated the delayed corneal epithelial injury repair and nerve regeneration induced by SLIT3 treatment or thapsigargin treatment. ER stress inhibits corneal epithelial injury repair and nerve regeneration may be related with the upregulation of SLIT3-ROBO4 pathway.

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.

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.

Lupenone attenuates thapsigargin-induced endoplasmic reticulum stress and apoptosis in pancreatic beta cells possibly through inhibition of protein tyrosine kinase 2 activity

AimsProlonged high levels of cytokines, glucose, or free fatty acids are associated with diabetes, elevation of cytosolic Ca2+ concentration ([Ca2+]C), and depletion of Ca2+ concentration in the endoplasmic reticulum (ER) of pancreatic beta cells. This Ca2+ imbalance induces ER stress and apoptosis. Lupenone, a lupan-type triterpenoid, is beneficial in diabetes; however, its mechanism of action is yet to be clarified. This study evaluated the protective mechanism of lupenone against thapsigargin-induced ER stress and apoptosis in pancreatic beta cells. Materials and methodsMIN6, INS-1, and native mouse islet cells were used. Western blot for protein expressions, measurement of [Ca2+]C, and in vivo glucose tolerance test were mainly performed. Key findingsThapsigargin increased the protein levels of cleaved caspase 3, cleaved PARP, and the phosphorylated form of JNK, ATF4, and CHOP. Thapsigargin increased the interaction between stromal interaction molecule1 (Stim1) and Orai1, enhancing store-operated calcium entry (SOCE). SOCE is further activated by protein tyrosine kinase 2 (Pyk2), which is Ca2+-dependent and phosphorylates the tyrosine residue at Y361 in Stim1. Lupenone inhibited thapsigargin-mediated Pyk2 activation, suppressed [Ca2+]C, ER stress, and apoptosis. Lupenone restored impaired glucose-stimulated insulin secretion effectuated by thapsigargin and glucose intolerance in a low-dose streptozotocin-induced diabetic mouse model. SignificanceThese results suggested that lupenone attenuated thapsigargin-induced ER stress and apoptosis by inhibiting SOCE; this may be due to the hindrance of Pyk2-mediated Stim1 tyrosine phosphorylation. In beta cells that are inevitably exposed to frequent [Ca2+]C elevation, the attenuation of abnormally high SOCE would be beneficial for their survival.

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.

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.

MiRNAs associated with endoplasmic reticulum stress and unfolded protein response during decidualization

Do microRNAs (miRNAs) play a role in regulating endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) in decidualized cells and endometrium associated with reproductive failures? Endometrial stromal cell line St-T1b was decidualized in vitro with 8-Br-cAMP over 5 days, or treated with the ERS inducer thapsigargin. Expression of ERS sensors, UPR markers and potential miRNA regulators was analysed by quantitative PCR. Endometrial biopsies from patients with recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) were investigated for the location of miRNA expression. Decidualization of St-T1b cells resulted in increased expression of ERS sensors including ATF6α, PERK and IRE1α, and the UPR marker, CHOP. TXNIP, which serves as a link between the ERS pathway and inflammation, as well as inflammasome NLRP3 and interleukin 1β expression increased in decidualized cells. An in-silico analysis identified miR-17-5p, miR-21-5p and miR-193b-3p as miRNAs potentially involved in regulation of the ERS/UPR pathways and inflammation associated with embryo implantation. Their expression decreased significantly (P ≤ 0.0391) in non-decidualized cells in the presence of thapsigargin. Finally, expression of the selected miRNAs was localized by in-situ hybridization in stromal and glandular epithelial cells in endometrial samples from patients with RPL and RIF. Expression in stroma cells from patients with RPL was lower in comparison with stroma cells from patients with RIF. Decidualization in St-T1b cells is accompanied by ERS/UPR processes, associated with an inflammatory response that is potentially influenced by miR-17-5p, miR-21-5p and miR-193b-3p. These miRNAs are expressed differentially in stromal cells from patients with RPL and RIF, indicating an alteration in regulation of the ERS/UPR pathways.

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.

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.

Calreticulin nuclear translocalization alleviates CaM/CaMKII/CREB signaling pathway to enhance chemosensitivity in HDAC inhibitor-resistant hepatocellular carcinoma cells.

Calreticulin (CRT) is located in the endoplasmic reticulum (ER), it helps proteins fold correctly inside the ER, and acts as a modulator of Ca2+ homeostasis. Aberrant expression of CRT is implicated in several cancer types, qualifying CRT as a potential therapeutic target. However, it remains unclear how CRT affects specific oncogenic pathways. In this study, we used histone deacetylase inhibitors (HDACis) to establish drug-resistant liver cancer cells and further analyzed the molecular mechanism of development of drug resistance in those cells. The 2D gel electrophoresis and RT-PCR data showed that CRT was downregulated in HDACis-resistant cells by comparing with HA22T parental cells. We previously elucidated the development of drug-resistance in HCC cells via activation of PP1-eIF2α pathway, but not via ER stress pathway. Here, we show that thapsigargin induced ER stress through mechanism other than ER stress downstream protein GRP78-PERK to regulate CRT expression in HDACis-R cells. Moreover, the expression level of CRT was not the main cause of apoptosis in HDACis-resistant cells. Mechanistic studies identified the apoptosis factors in the nucleus—the HDACis-mediated overexpression of CRT, CRT translocation to the cell nucleus, and reduced CaM/CaMKII/CREB pathway—that led to chemosensitivity in HDACis-R HCC cells.

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.

Downregulation of RIP3 Improves the Protective Effect of ATF6 in an Acute Liver Injury Model.

Background Activating transcription factor 6 (ATF6) and receptor-interacting protein 3 (RIP3) are important signaling proteins in endoplasmic reticulum (ER) stress and necroptosis, respectively. However, their regulatory relationship and clinical significance are unknown. We investigate the impact of ATF6 on RIP3 expression, and its role in hepatocyte necroptosis in an acute liver injury model. Methods In vivo and in vitro experiments were carried out. LO2 cells were treated with thapsigargin (TG). In vivo, male BALB/c mice were treated with carbon tetrachloride (CCl4, 1 mL/kg) or tunicamycin (TM, 2 mg/kg). Then, the impact of ATF6 or RIP3 silencing on liver injury, hepatocyte necroptosis, and ER stress-related protein expression was examined. Results TG induced ER stress and necroptosis and ATF6 and RIP3 expression in LO2 cells. The knockdown of ATF6 significantly decreased RIP3 expression (p < 0.05) and increased ER stress and necroptosis. The downregulation of RIP3 significantly reduced necroptosis and ER stress (p < 0.05). Similar results were observed in CCl4 or the TM-induced mouse model. The knockdown of ATF6 significantly decreased CCl4-induced RIP3 expression and increased liver injury, necroptosis, and ER stress in mice livers (p < 0.05). In contrast, the downregulation of RIP3 significantly reduced liver injury, hepatocyte necroptosis, and ER stress. Conclusions Hepatocyte ATF6 has multiple roles in acute liver injury. It reduces hepatocyte necroptosis via negative feedback regulation of ER stress. In addition, ATF6 can upregulate the expression of RIP3, which is not helpful to the recovery process. However, downregulating RIP3 reduces hepatocyte necroptosis by promoting the alleviation of ER stress. The findings suggest that RIP3 could be a plausible target for the treatment of liver injury.

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.

Sestrin2 suppression aggravates oxidative stress and apoptosis in endothelial cells subjected to pharmacologically induced endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is an inflammatory response that contributes to endothelial cell dysfunction, a hallmark of cardiovascular diseases, in close interplay with oxidative stress. Recently, Sestrin2 (SESN2) emerged as a novel stress-inducible protein protecting cells from oxidative stress. We investigated here, for the first time, the impact of SESN2 suppression on oxidative stress and cell survival in human endothelial cells subjected to pharmacologically (thapsigargin)-induced ER stress and studied the underlying cellular pathways. We found that SESN2 silencing, though did not specifically induce ER stress, it aggravated the effects of thapsigargin-induced ER stress on oxidative stress and cell survival. This was associated with a dysregulation of Nrf-2, AMPK and mTORC1 signaling pathways. Furthermore, SESN2 silencing aggravated, in an additive manner, apoptosis caused by thapsigargin. Importantly, SESN2 silencing, unlike thapsigargin, caused a dramatic decrease in protein expression and phosphorylation of Akt, a critical pro-survival hub and component of the AMPK/Akt/mTORC1 axis. Our findings suggest that patients with conditions characterized by ER stress activation, such as diabetes, may be at higher risk for cardiovascular complications if their endogenous ability to stimulate and/or maintain expression levels of SESN2 is disturbed or impaired. Therefore, identifying novel or repurposing existing pharmacotherapies to enhance and/or maintain SESN2 expression levels would be beneficial in these conditions.

Non-alcoholic fatty liver disease in mice with hepatocyte-specific deletion of mitochondrial fission factor

Aims/hypothesisMitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred to as mitochondrial dynamics, to adapt to nutritional demands. Evidence suggests that impaired mitochondrial dynamics leads to metabolic abnormalities such as non-alcoholic steatohepatitis (NASH) phenotypes. However, how mitochondrial dynamics are involved in the development of NASH is poorly understood. This study aimed to elucidate the role of mitochondrial fission factor (MFF) in the development of NASH.MethodsWe created mice with hepatocyte-specific deletion of MFF (MffLiKO). MffLiKO mice fed normal chow diet (NCD) or high-fat diet (HFD) were evaluated for metabolic variables and their livers were examined by histological analysis. To elucidate the mechanism of development of NASH, we examined the expression of genes related to endoplasmic reticulum (ER) stress and lipid metabolism, and the secretion of triacylglycerol (TG) using the liver and primary hepatocytes isolated from MffLiKO and control mice.ResultsMffLiKO mice showed aberrant mitochondrial morphologies with no obvious NASH phenotypes during NCD, while they developed full-blown NASH phenotypes in response to HFD. Expression of genes related to ER stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic TG secretion was downregulated, with reduced hepatic TG secretion in MffLiKO mice in vivo and in primary cultures of MFF-deficient hepatocytes in vitro. Furthermore, thapsigargin-induced ER stress suppressed TG secretion in primary hepatocytes isolated from control mice.Conclusions/interpretationWe demonstrated that ablation of MFF in liver provoked ER stress and reduced hepatic TG secretion in vivo and in vitro. Moreover, MffLiKO mice were more susceptible to HFD-induced NASH phenotype than control mice, partly because of ER stress-induced apoptosis of hepatocytes and suppression of TG secretion from hepatocytes. This study provides evidence for the role of mitochondrial fission in the development of NASH.Graphical abstract

Extracellularly Released Calreticulin Induced by Endoplasmic Reticulum Stress Impairs Syncytialization of Cytotrophoblast Model BeWo Cells.

The pregnancy-specific syndrome preeclampsia is a major cause of maternal mortality throughout the world. The initial insult resulting in the development of preeclampsia is inadequate trophoblast invasion, which may lead to reduced maternal perfusion of the placenta and placental dysfunction, such as insufficient trophoblast syncytialization. Endoplasmic reticulum (ER) stress has been implicated in the pathology of preeclampsia and serves as the major risk factor. Our previous studies suggested critical roles of calreticulin (CRT), which is an ER-resident stress response protein, in extravillous trophoblast invasion and cytotrophoblast syncytialization. Here, we studied the mechanism by which ER stress exposes the placenta to the risk of preeclampsia. We found that CRT was upregulated in the serum samples, but not in the placental specimens, from preeclamptic women. By using BeWo cells, an established model of cytotrophoblasts that syncytialize in the presence of forskolin, we demonstrated that thapsigargin-induced ER stress caused extracellular release of CRT from BeWo cells and that the extracellular CRT suppressed forskolin-induced release of β-human chorionic gonadotropin and altered subcellular localization of E-cadherin, which is a key adhesion molecule associated with syncytialization. Our results together provide evidence that induction of ER stress leads to extracellular CRT release, which may contribute to placental dysfunction by suppressing cytotrophoblast syncytialization.