• BMB Reports
• /
• v.52 no.11
• /
• pp.665-670
• /
• 2019

Nucleotide-binding oligomerization domain protein 2 (NOD2), an intracellular pattern recognition receptor, plays important roles in inflammation and cell death. Previously, we have shown that NOD2 is expressed in vascular smooth muscle cells (VSMCs) and that NOD2 deficiency promotes VSMC proliferation, migration, and neointimal formation after vascular injury. However, its role in endoplasmic reticulum (ER) stress-induced cell death in VSMCs remains unclear. Thus, the objective of this study was to evaluate ER stress-induced viability of mouse primary VSMCs. NOD2 deficiency increased ER stress-induced cell death and expression levels of apoptosis mediators (cleaved caspase-3, Bax, and Bak) in VSMCs in the presence of tunicamycin (TM), an ER stress inducer. In contrast, ER stress-induced cell death and expression levels of apoptosis mediators (cleaved caspase-3, Bax, and Bak) were decreased in NOD2-overexpressed VSMCs. We found that the $IRE-1{\alpha}-XBP1$ pathway, one of unfolded protein response branches, was decreased in NOD2-deficient VSMCs and reversed in NOD2-overexpressed VSMCs in the presence of TM. Furthermore, NOD2 deficiency reduced the expression of XBP1 target genes such as GRP78, PDI-1, and Herpud1, thus improving cell survival. Taken together, these data suggest that the induction of ER stress through NOD2 expression can protect against TM-induced cell death in VSMCs. These results may contribute to a new paradigm in vascular homeostasis.

• The Korean Journal of Physiology and Pharmacology
• /
• v.23 no.2
• /
• pp.95-102
• /
• 2019

Endoplasmic reticulum (ER) stress is mediated by disturbance of $Ca^{2+}$ homeostasis. The store-operated calcium (SOC) channel is the primary $Ca^{2+}$ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on $Ca^{2+}$ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular $Ca^{2+}$ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.

• Molecules and Cells
• /
• v.47 no.1
• /
• pp.100004.1-100004.11
• /
• 2024

Insulin is essential for maintaining normoglycemia and is predominantly secreted in response to glucose stimulation by β-cells. Incretin hormones, such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide, also stimulate insulin secretion. However, as obesity and type 2 diabetes worsen, glucose-dependent insulinotropic polypeptide loses its insulinotropic efficacy, whereas GLP-1 receptor (GLP-1R) agonists continue to be effective owing to its signaling switch from Gs to Gq. Herein, we demonstrated that endoplasmic reticulum (ER) stress induced a transition from Gs to Gq in GLP-1R signaling in mouse islets. Intriguingly, chemical chaperones known to alleviate ER stress, such as 4-PBA and TUDCA, enforced GLP-1R's Gq utilization rather than reversing GLP-1R's signaling switch induced by ER stress or obese and diabetic conditions. In addition, the activation of X-box binding protein 1 (XBP1) or activating transcription factor 6 (ATF6), 2 key ER stress-associated signaling (unfolded protein response) factors, promoted Gs utilization in GLP-1R signaling, whereas Gq employment by ER stress was unaffected by XBP1 or ATF6 activation. Our study revealed that ER stress and its associated signaling events alter GLP-1R's signaling, which can be used in type 2 diabetes treatment.

• Journal of Embryo Transfer
• /
• v.32 no.1
• /
• pp.25-31
• /
• 2017

This study was conducted to investigate the effect of activation method on the endoplasmic reticulum (ER) stress induction, apoptosis and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by four activation methods; 1) electric stimulus (ES) (E), 2) $ES+10{\mu}M$ Ca-ionophore (A23187) treatment (EC), 3) ES+2 mM 6-dimethylaminopurine (6-DMAP) treatment (ED), or 4) ES+A23187 and 6-DMAP treatments (ECD). Parthenogenetic embryos were sampled to analyze x-box binding protein 1 (Xbp1) mRNA, ER stress-associated genes and apoptosis genes at 3 h after ES and the 1-cell and blastocyst stages. In the EC group, the band intensity of spliced Xbp1 (Xbp1s) mRNA was higher than those of the other groups at the 3 h and 1-cell stage, and higher than that of the E group at the blastocyst stage. Four ER stress-associated genes were expressed at the highest level in the EC group and weakly expressed in the ED group at 3 h after activation. However, most of the genes were highly expressed at the 1-cell and blastocyst stages with some variation in the EC and ECD groups. Expression of Bcl-2-associated X protein (Bax) and caspase-3 mRNA was significantly higher in the EC group than in the other groups at all development stages. The developmental rates to the blastocyst stage were higher in the ED and ECD groups than in the E and EC groups. These results suggest that the intracellular ER stress of parthenogenetic porcine embryos is affected by the activation method and subsequently lead to the apoptosis of embryos.

• The Korean Journal of Physiology and Pharmacology
• /
• v.20 no.4
• /
• pp.387-397
• /
• 2016

Neurofibrillary tangles (NFTs) of microtubule-associated protein tau are a pathological hallmark of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress has been known to be involved in the pathogenesis of AD. However, the exact role of ER stress in tau pathology has not yet been clearly elucidated. In present study, the possible relationship between tau pathology and ER stress was examined in terms of sorcin, which is a calcium binding protein and plays an important role in calcium homeostasis. Our previous yeast two hybrid study showed that sorcin is a novel tau interacting protein. Caspase-3-cleaved tau (T4C3) showed significantly increased tau-sorcin interaction compared to wild type tau (T4). Thapsigargin-induced ER stress and co-expression of constitutively active $GSK3{\beta}$ ($GSK3{\beta}-S9A$) also exhibited significantly increased tau-sorcin interactions. T4C3-expressing cells showed potentiated thapsigargin -induced apoptosis and disruption of intracellular calcium homeostasis compared to T4-expressing cells. Overexpression of sorcin significantly attenuated thapsigargin-induced apoptosis and disruption of calcium homeostasis. In contrary, siRNA-mediated knock-down of sorcin showed significantly increased thapsigargin-induced apoptosis and disruption of calcium homeostasis. These data strongly suggest that sequestration of sorcin by aberrant forms of tau compromises the function of sorcin, such as calcium homeostasis and cellular resistance by ER stress, which may consequently result in the contribution to the progression of AD.

• Molecules and Cells
• /
• v.47 no.1
• /
• pp.100001.1-100001.8
• /
• 2024

In eukaryotes, a primary protein quality control (PQC) process involves the destruction of conformationally misfolded proteins through the ubiquitin-proteasome system. Because approximately one-third of eukaryotic proteomes fold and assemble within the endoplasmic reticulum (ER) before being sent to their destinations, the ER plays a crucial role in PQC. The specific functions and biochemical roles of several E3 ubiquitin ligases involved in ER-associated degradation in mammals, on the other hand, are mainly unknown. We identified 2 E3 ligases, ubiquitin protein ligase E3 component N-recognin 1 (UBR1) and ubiquitin protein ligase E3 component N-recognin 2 (UBR2), which are the key N-recognins in the N-degron pathway and participate in the ER stress response in mammalian cells by modulating their stability. Cells lacking UBR1 and UBR2 are hypersensitive to ER stress-induced apoptosis. Under normal circumstances, these proteins are polyubiquitinated through Lys48-specific linkages and are then degraded by the 26S proteasome. In contrast, when cells are subjected to ER stress, UBR1 and UBR2 exhibit greater stability, potentially as a cellular adaptive response to stressful conditions. Although the precise mechanisms underlying these findings require further investigation, our findings show that cytoplasmic UBR1 and UBR2 have anti-ER stress activities and contribute to global PQC in mammals. These data also reveal an additional level of complexity within the mammalian ER-associated degradation system, implicating potential involvement of the N-degron pathway.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.12
• /
• pp.1785-1790
• /
• 2013

The synthetic machinery of ATF4 (activating transcription factor 4) is activated in response to various stress conditions involved in nutrient restriction, endoplasmic reticulum homeostasis, and oxidation. Stress-induced inhibition of proteasome activity triggers the unfolded protein response and endoplasmic reticulum stress, where ATF4 is crucial for consequent biological events. In the current study, we showed that the $NAD^+$-dependent deacetylase, SIRT1, suppresses ATF4 synthesis during proteasome inhibition. SIRT1 depletion via transfection of specific siRNA into HeLa cells resulted in a significant increase in ATF4 protein, which was observed specifically in the presence of the proteasome inhibitor MG132. Consistent with SIRT1 depletion data, transient transfection of cells with SIRT1-overexpressing plasmid induced a decrease in the ATF4 protein level in the presence of MG132. Interestingly, however, ATF4 mRNA was not affected by SIRT1, even in the presence of MG132, indicating that SIRT1-induced suppression of ATF4 synthesis occurs under post-transcriptional control. Accordingly, we propose that SIRT1 serves as a negative regulator of ATF4 protein synthesis at the post-transcriptional level, which is observed during stress conditions, such as proteasome inhibition.

• International Journal of Oral Biology
• /
• v.45 no.4
• /
• pp.190-196
• /
• 2020

Several factors, including genetic and environmental insults, impede protein folding and secretion in the endoplasmic reticulum (ER). Accumulation of unfolded or mis-folded protein in the ER manifests as ER stress. To cope with this morbid condition of the ER, recent data has suggested that the intracellular event of an unfolded protein response plays a critical role in managing the secretory load and maintaining proteostasis in the ER. Tauroursodeoxycholic acid (TUDCA) is a chemical chaperone and hydrophilic bile acid that is known to inhibit apoptosis by attenuating ER stress. Numerous studies have revealed that TUDCA affects hepatic diseases, obesity, and inflammatory illnesses. Recently, molecular regulation of ER stress in tooth development, especially during the secretory stage, has been studied. Therefore, in this study, we examined the developmental role of ER stress regulation in tooth morphogenesis using in vitro organ cultivation methods with a chemical chaperone treatment, TUDCA. Altered cellular events including proliferation, apoptosis, and dentinogenesis were examined using immunostaining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. In addition, altered localization patterns of the formation of hard tissue matrices related to molecules, including amelogenin and nestin, were examined to assess their morphological changes. Based on our findings, modulating the role of the chemical chaperone TUDCA in tooth morphogenesis, especially through the modulation of cellular proliferation and apoptosis, could be applied as a supporting data for tooth regeneration for future studies.

• The Korean Journal of Physiology and Pharmacology
• /
• v.24 no.6
• /
• pp.473-479
• /
• 2020

Endothelial cell injury is a major contributor to cardiovascular diseases. The 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-Glucoside (TSG) contributes to alleviate human umbilical vein endothelial cells (HUVECs) injury through mechanisms still know a little. This study aims to clarify the TSG effects on gene expression (mRNA and microRNA) related to oxidative stress and endoplasmic reticulum stress induced by H₂O₂ in HUVECs. We found that TSG significantly reduced the death rate of cells and increased intracellular superoxide dismutase activity. At qRT-PCR, experimental data showed that TSG significantly counteracted the expressions of miR-9-5p, miR-16, miR-21, miR-29b, miR-145-5p, and miR-204-5p. Besides, TSG prevented the expression of ATF6 and CHOP increasing. In contrast, TSG promoted the expression of E2F1. In conclusion, our results point to the obvious protective effect of TSG on HUVECs injury induced by H₂O₂, and the mechanism may through miR16/ATF6/ E2F1 signaling pathway.

• The Korean Journal of Physiology and Pharmacology
• /
• v.27 no.1
• /
• pp.39-48
• /
• 2023

Spinal nerve injury causes mechanical allodynia and structural imbalance of neurotransmission, which were typically associated with calcium overload. Storeoperated calcium entry (SOCE) is considered crucial elements-mediating intracellular calcium homeostasis, ion channel activity, and synaptic plasticity. However, the underlying mechanism of SOCE in mediating neuronal transmitter release and synaptic transmission remains ambiguous in neuropathic pain. Neuropathic rats were operated by spinal nerve ligations. Neurotransmissions were assessed by whole-cell recording in substantia gelatinosa. Immunofluorescence staining of STIM1 with neuronal and glial biomarkers in the spinal dorsal horn. The endoplasmic reticulum stress level was estimated from qRT-PCR. Intrathecal injection of SOCE antagonist SKF96365 dose-dependently alleviated mechanical allodynia in ipsilateral hind paws of neuropathic rats with ED₅₀ of 18 ㎍. Immunofluorescence staining demonstrated that STIM1 was specifically and significantly expressed in neurons but not astrocytes and microglia in the spinal dorsal horn. Bath application of SKF96365 inhibited enhanced miniature excitatory postsynaptic currents in a dosage-dependent manner without affecting miniature inhibitory postsynaptic currents. Mal-adaption of SOCE was commonly related to endoplasmic reticulum (ER) stress in the central nervous system. SKF96365 markedly suppressed ER stress levels by alleviating mRNA expression of C/ EBP homologous protein and heat shock protein 70 in neuropathic rats. Our findings suggested that nerve injury might promote SOCE-mediated calcium levels, resulting in long-term imbalance of spinal synaptic transmission and behavioral sensitization, SKF96365 produces antinociception by alleviating glutamatergic transmission and ER stress. This work demonstrated the involvement of SOCE in neuropathic pain, implying that SOCE might be a potential target for pain management.