Effects Of Endoplasmic Reticulum Stress Research Articles

Endoplasmic reticulum stress induces autophagy and apoptosis while inhibiting proliferation and drug resistance in multiple myeloma through the PI3K/Akt/mTOR signaling pathway.

We investigated the effects of endoplasmic reticulum stress (ERS) on autophagy, proliferation, apoptosis, and drug resistance in multiple myeloma (MM). MM patients enrolled in our study (n = 268) were classified into sensitive and resistant groups based on chemotherapy efficacy, and their serum levels of β2-MG, albumin (ALB), lactic dehydrogenase (LDH), Ca2+ and hemoglobin were determined. In addition, human MM U266 and MOLP-2/R cells were divided into blank, tunicamycin (TM), TM + insulin-like growth factor-1 (IGF-1), and TM + rapamycin groups, and measured expression of ERS-related, PI3K/Akt/mTOR pathway-related, and autophagy-related mRNA and proteins. Serum levels of β2-MG, LDH and Ca2+, and expression of PI3K, Akt, and mTOR were higher in the resistant than sensitive group. Serum levels of ALB and hemoglobin, and expression of glucose-regulated protein 78 (GRP78), GRP94, microtubule associated protein 1 light chain 3 (LC3), and Beclin1, were lower in the resistant than sensitive group. In U266 cells treated with TM and IGF-1 or rapamycin, ERS promoted autophagy and apoptosis while inhibiting proliferation through inhibition of PI3K/Akt/mTOR signaling. ERS also reversed drug resistance in MOLP-2/R cells via the PI3K/Akt/mTOR signaling pathway. These data suggest that ERS activation could be exploited for therapeutic benefits in the treatment of MM.

Effects of endoplasmic reticulum stress on the autophagy, apoptosis, and chemotherapy resistance of human breast cancer cells by regulating the PI3K/AKT/mTOR signaling pathway.

Nowadays, although chemotherapy is an established therapy for breast cancer, the molecular mechanisms of chemotherapy resistance in breast cancer remain poorly understood. This study aims to explore the effects of endoplasmic reticulum stress on autophagy, apoptosis, and chemotherapy resistance in human breast cancer cells by regulating PI3K/AKT/mTOR signaling pathway. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to detect the cell viability of six human breast cancer cell lines (MCF-7, ZR-75-30, T47D, MDA-MB-435s, MDA-MB-453, and MDA-MB-231) treated with tunicamycin (5 µM), after which MCF-7 cells were selected for further experiment. Then, MCF-7 cells were divided into the control (without any treatment), tunicamycin (8 µ), BEZ235 (5 µ), and tunicamycin + BEZ235 groups. Cell viability of each group was testified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Western blotting was applied to determine the expressions of endoplasmic reticulum stress and PI3K/AKT/mTOR pathway-related proteins and autophagy- and apoptosis-related proteins. Monodansylcadaverine and Annexin V-fluorescein isothiocyanate/propidium iodide staining were used for determination of cell autophagy and apoptosis. Furthermore, MCF-7 cells were divided into the control (without any treatment), tunicamycin (5 µM), cisplatin (16 µM), cisplatin (16 µM) + BEZ235 (5 µM), tunicamycin (5 µM) + cisplatin (16 µM), and tunicamycin (5 µM) + cisplatin (16 µM) + BEZ235 groups. Cell viability and apoptosis were also evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Annexin V-fluorescein isothiocyanate/propidium iodide staining. In MCF-7 cells treated with tunicamycin, cell viability decreased significantly, but PEAK, eIF2, and CHOP were upregulated markedly and p-PI3K, p-AKT, and p-MTOR were downregulated in dose- and time-dependent manners. In the tunicamycin + BEZ235 group, the cell viability was lower and the apoptosis rate was higher than those of the control and monotherapy groups. Compared with the cisplatin group, the tunicamycin + cisplatin group showed a relatively higher growth inhibition rate; the growth inhibition rate substantially increased in the tunicamycin + cisplatin + BEZ235 group than the tunicamycin + cisplatin group. The apoptosis rate was highest in tunicamycin + cisplatin + BEZ235 group, followed by tunicamycin + cisplatin group and then cisplatin group. Our study provide evidence that endoplasmic reticulum stress activated by tunicamycin could promote breast cancer cell autophagy and apoptosis and enhance chemosensitivity of MCF-7 cells by inhibiting the PI3K/AKT/mTOR signaling pathway.

Effects of Endoplasmic Reticulum Stress on Protein Quality Control

Protein quality control is critical for cellular health. The Endoplasmic Reticulum (ER)‐Associated Degradation (ERAD) protein quality control system degrades aberrant proteins at the ER membrane. ERAD can be divided into multiple pathways, including ERAD‐L (lumen), ‐M (membrane), ‐T (translocon), ‐C (cytosol), and ‐RA (ribosome associated). In general, each ERAD pathway degrades proteins with aberrations in specific positions relative to the ER membrane. In ERAD‐L and ‐M, the ubiquitin ligase Hrd1 recognizes proteins with aberrations in ER luminal or transmembrane segments. Hrd1 also functions in ERAD‐T, in which proteins that persistently or aberrantly associate with the translocon are targeted for destruction. In ERAD‐C, the ubiquitin ligase Doa10 recognizes soluble or transmembrane proteins with cytosolic aberrations. In ERAD‐RA, the ubiquitin ligase Rkr1/Ltn1 recognizes translationally stalled ER‐targeted proteins. ER stress occurs when misfolded proteins accumulate in the ER. Multiple human diseases, including Parkinson's disease and diabetes, are characterized by elevated levels of ER stress. The extent to which ER stress contributes to pathology is not clear. In this study, we systematically evaluate the effect of ER stress on ERAD in the model organism Saccharomyces cerevisiae by comparing the abundance and rate of degradation of a panel of model substrate proteins. Our data suggest that the ERAD pathways are differentially affected by ER stress. A better understanding of the effects of ER stress on protein quality control pathways may contribute to an improved understanding of the consequences of ER stress on cellular physiology and facilitate the discovery of therapeutic targets for diseases characterized by increased levels of ER stress.Support or Funding InformationNational Institutes of Health Grant R15 GM111713 (to EMR).Funds from the Ball State University Provost's Office and Department of Biology (to EMR).Ball State University ASPiRE research awards (to BWB).

Effects of endoplasmic reticulum stress on apoptosis induction in radioresistant macrophages.

Macrophages are important in the host's immune defense against pathogens. However, recent evidence has demonstrated that macrophages are also involved in the development of disease, including cancer. Therefore, it is important to regulate apoptosis in tumor‑related macrophages for effective cancer treatment. In the present study, the effect of endoplasmic reticulum (ER) stress on apoptosis induction was examined in human monocytic cell‑derived macrophages. Radiation therapy in cancer results in irradiating macrophages as well as cancer cells in the tumor microenvironment. Since ER stress has been demonstrated to sensitize cancer cells to radiation, it was hypothesized that ER stress may induce a similar effect in macrophages. Therefore, the effect of combination treatment with ER stress inducers and ionizing radiation on macrophage apoptosis was examined. Treatment of macrophages with ER stress inducers thapsigargin and tunicamycin, enhanced unfolded protein responses, including phosphorylation of eukaryotic initiation factor 2‑α and increased expression of binding immunoglobulin protein. Furthermore, treatment with thapsigargin and tunicamycin induced apoptosis in macrophages compared with untreated cells, although ionizing radiation did not. The thapsigargin-induced apoptosis in macrophages was demonstrated to be caspase‑3‑dependent. Finally, combination treatment with thapsigargin and ionizing radiation, did not result in any significant change in macrophage apoptosis. The present study demonstrated that ER stress regulated apoptosis in radioresistant macrophages and that ionizing radiation had no added effect on ER stress‑induced apoptosis in macrophages.

Causes and consequences of endoplasmic reticulum stress in rheumatic disease.

Rheumatic diseases represent a heterogeneous group of inflammatory conditions, many of which involve chronic activation of both innate and adaptive immune responses by multiple genetic and environmental factors. These immune responses involve the secretion of excessive amounts of cytokines and other signalling mediators by activated immune cells. The endoplasmic reticulum (ER) is the cellular organelle that directs the folding, processing and trafficking of membrane-bound and secreted proteins, including many key components of the immune response. Maintaining homeostasis in the ER is critical to cell function and survival. Consequently, elaborate mechanisms have evolved to sense and respond to ER stress through three main signalling pathways that together comprise the unfolded protein response (UPR). Activation of the UPR can rapidly resolve the accumulation of misfolded proteins, direct permanent changes in the size and function of cells during differentiation, and critically influence the immune response and inflammation. Recognition of the importance of ER stress and UPR signalling pathways in normal and dysregulated immune responses has greatly increased in the past few years. This Review discusses several settings in which ER stress contributes to the pathogenesis of rheumatic diseases and considers some of the therapeutic opportunities that these discoveries provide.

ER stress mediates mitochondrial dysfunction in human adipocytes which is exacerbated in obesity

The pathogenesis of obesity and T2DM mediates mitochondrial dysfunction which, in part, may arise as a consequence of endoplasmic reticulum stress (ERS). Therefore, we investigated whether induction of ERS contributes to mitochondrial dysfunction in human adipocytes. Chronic ERS was induced in post-differentiated human adipocyte cell line (Chub-S7) and primary lean and obese abdominal subcutaneous adipocytes (AbdSc Ad) using 0.25μg/ml and 0.75μg/ml tunicamycin. Key parameters of mitochondrial function were determined, including oxygen consumption rate (OCR), ATP concentration, mitochondrial membrane potential (MMP), mitochondrial number and dynamics. We observed dose-dependent increases of OCR in Chub-S7 adipocytes following treatment with Tn (p<0.01). This was accompanied by decreased elongation (16%↓P<0.05), decreased cellular area occupied by mitochondria (28%↓P<0.05), and no change to mitochondrial number, as observed by confocal microscopy. Moreover, MMP was significantly compromised (32%↓P=0.0001), whilst ATP levels were not affected. Consistent with increasing OCR levels in the cellular model, primary adipocytes from lean subjects revealed higher levels of OCR with Tn treatment, potentially as a mechanism to compensate for cellular stress; whereas, mitochondria from obese subjects displayed impaired respiratory function. In summary, these human data suggest that mitochondrial dysfunction occurs more readily in response to ERS, in adipocytes from obese subjects than their lean counterparts.

2-deoxyglucose inhibits induction of chemokine expression in 3T3-L1 adipocytes and adipose tissue explants.

To determine the influence of glycolytic inhibition on the adipocyte inflammatory response. To determine the effect of 2-deoxyglucose (2-DOG) on the inflammatory response, mature 3T3-L1 adipocytes were co-treated with 2-DOG and LPS or TNF. To determine the effect of endoplasmic reticulum stress on TNF-induced induction of chemokines, adipocytes were pretreated with thapsigargin or salubrinal. Chemokine mRNA levels were determined using quantitative real-time PCR, and secretion of CCL2 was determined by Western blot. 2-DOG treatment reduced the ability of LPS and TNF to induce CCL2 mRNA levels and reduced secreted CCL2 protein levels in a dose-dependent manner. A similar pattern of mRNA regulation was observed for other chemokines. The attenuation of TNF-induced CCL2 mRNA levels occurred regardless of whether glucose or pyruvate was present in the media, suggesting that mechanisms other than glycolysis might mediate the observed effects. Treatment with the endoplasmic reticulum stressor thapsigargin and the endoplasmic reticulum signaling activator salubrinal reduced chemokine mRNA levels similarly to 2-DOG. Collectively, our data indicate that 2-DOG suppresses inflammatory chemokine induction in adipocytes. The effects of 2-DOG do not seem to be linked to glycolysis but correlate with endoplasmic reticulum stress activation.

Abstract 285: Endoplasmic Reticulum Stress Induces Mitochondrial ROS Overproduction and Contributes to Electrical Remodeling in Heart Failure

Introduction: Heart failure (HF) is associated with endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), which has three branches (PERK, IRE1 and ATF6α). UPR inhibits protein translation, and we hypothesized UPR contributes to electrical remodeling in HF. Methods: HF was induced in C57BL/6 mice 6-7 weeks after unilateral nephrectomy, deoxycorticosterone acetate pellet implantation, and salt water substitution. Sham operated mice were used as controls. Isolated ventricular myocytes were utilized for whole-cell patch clamp recording and ROS measurement by DCFDA with confocal imaging. Heart tissue was used for mRNA and protein measurements. GSK2606414, a specific inhibitor of PERK, was applied to myocytes at 4-30 nM for 6-20 h. Results: The action potential duration was prolonged in HF myocytes (204±26 vs. 100±16 ms of sham, P&lt;0.05). The mRNA and protein levels of the UPR effectors indicated that the PERK and IRE1 branches were activated in HF heart tissue. The peak I Na , I to , I K1 , and I Kslow were decreased significantly in HF group, while I CaL and other K + currents were not affected. A PERK inhibitor, GSK2606414, restored I Na and I K,slow . Current changes were similar to those observed in myocytes under tunicamycin-induced ER stress. Significant reduction of channel protein levels were observed for K v 4.3, Kir2.1, K v 1.5, and Ca v 1.2, but not Na v 1.5, suggesting that I Na reduction was mediated by PERK but not by translational downregulation. Significant ROS production was observed in mouse cardiomyocytes under ER stress, which was suppressed by GSK2606414. Confocal imaging of ROS indicates the source to be mitochondria. Conclusions: TM-treated cardiomyocyte changes indicated that reductions of I Na , I to , I K1 and I K,slow were mediated by ER stress. Proportionally decreased protein and current levels of K v 4.3, Kir2.1, and K v 1.5 suggested direct effects of ER stress. The downregulation of Na v 1.5 and K v 1.5 could be prevented by PERK inhibition. Nevertheless, the effect of PERK inhibition on I Na was indirect and mediated by a PERK-dependent elevation in mitochondrial ROS. In conclusion, the UPR contributes to electrical remodeling in HF by direct and indirect means. Targeting the UPR may be a novel antiarrhythmic strategy.

Impact of activating transcription factor 4 signaling on lipogenesis in HepG2 cells.

Non-alcoholic fatty liver disease (NAFLD) is a rapidly growing health threat that has previously been associated with lipogenesis. The direct effect of endoplasmic reticulum stress (ERS) inhibition on the induction of lipogenesis has not been investigated in hepatocytes invitro. The impact of activating transcription factor‑4 (ATF4) on the lipogenic pathway and hepatic insulin transduction in liver cells also requires further investigation. In the present study, the triglyceride (TG) content of HepG2 cells stimulated with fructose was investigated using a commercially available enzymatic assay, and the expression levels of lipogenesis‑associated factors were determined by western blotting and reverse transcription‑quantitative polymerase chain reaction. Notably, the TG content of HepG2 cells was increased following incubation with fructose, which was accompanied by ERS. 4‑Phenylbutyric acid, an inhibitor of ERS, lowered the TG content by reducing the mRNA expression levels of sterol regulatory element‑binding protein 1 (SREBP‑1c) and carbohydrate‑responsive element‑binding protein (ChREBP), and the protein expression levels of fatty acid synthase (FAS), acetyl‑CoA carboxylase (ACC) and stearoyl‑CoA desaturase‑1 (SCD‑1). Conversely, tunicamycin, which is an inducer of ERS, increased the TG content and stimulated the expression of the above lipogeneic markers. ATF4 deficiency relieved TG accumulation and decreased the mRNA expression levels of SREBP‑1c and ChREBP, and protein expression levels of FAS, ACC and SCD‑1 in fructose‑treated HepG2 cells. Conversely, ATF4 overexpression increased the TG content by upregulating the mRNA expression levels of SREBP‑1c and ChREBP and protein expression levels of FAS, ACC and SCD‑1. Inhibition of ERS was shown to protect HepG2 cells against fructose‑induced TG accumulation, whereas induction of ERS stimulated hepatic lipogenesis. As a downstream transcription factor of the unfolded protein response, a deficiency in ATF4 attenuates fructose‑induced lipogenesis; while an overexpression of ATF4 can induce TG accumulation through stimulating hepatic lipogenesis. The results of the present study suggested that ATF4 may exert various physiological roles in lipid metabolism depending on the nutrient composition. In addition, these results suggested that ATF4 has a role in regulating lipogenesis and in the development of NAFLD; thus ATF4 may be considered a therapeutic target for NAFLD.

Endoplasmic reticulum stress is activated in acute pancreatitis.

Endoplasmic reticulum (ER) is one of the most important cell organelles in the body, regulating protein synthesis, folding and aggregation. Endoplasmic reticulum stress (ERS) is a particular subcellular pathological process involving an imbalance of homeostasis and ER disorder. In the early stage of ERS, cells show a protective unfolded protein response that changes the cellular transcriptional and translational programs to alleviate the process. Therefore, a certain degree of ERS can activate the protective adaptation of cells, whereas sustained severe ERS triggers an apoptotic signal and leads to apoptosis. Acute pancreatitis is a disease caused by trypsin digestion of the pancreas, although the pathogenesis is not completely understood. However, a close association has been suggested between pancreatitis and ERS. This article reviewed relevant research advances and discussed the effect of ERS on the development and progression of acute pancreatitis.

Effects of endoplasmic reticulum stress on the expression of inflammatory cytokines in patients with ulcerative colitis.

To explore the changes of X-box binding protein 1 splicing (XBP1s) and inflammatory cytokine expression in patients with ulcerative colitis (UC) in response to endoplasmic reticulum stress (ERS). Reverse transcription polymerase chain reaction and quantitative polymerase chain reaction were performed to detect the forms of XBP1s and the expression of interleukin (IL)-2, interferon (IFN)-γ, and IL-17α. Differences between patients with UC and normal subjects were then determined. Mononuclear cells of the peripheral blood of normal subjects and UC patients with were stimulated with no drugs (control), phytohemagglutinin (PHA), thapsigargin (TG), or both PHA and TG. XBP1s in patients with UC exhibited splicing, which was greater with co-stimulation than single stimulation. Co-stimulation increased the expression level of IL-2, IFN-γ, and IL-17α. The T lymphocytes of both normal subjects and patients with UC responded to ERS by activating the XBP1s-mediated signalling pathway, upregulating the expression of inflammatory cytokines, and increasing the occurrence of inflammation. The mononuclear cells in the peripheral blood of patients with UC were more sensitive to ERS than those in the peripheral blood of normal subjects.

Effect of endoplasmic reticulum stress on gastric cancer cell migration and invasion

Effect of endoplasmic reticulum stress on gastric cancer cell migration and invasion

Neuroprotection by salubrinal treatment in global cerebral ischemia.

Most common cerebrovascular accidents (CVAs) result from the occlusion of a blood vessel and are called ischemic stroke. The interruption of the blood flow reduces the oxygen and glucose levels in the neural parenchyma, which in turn decreases the energy of the cells and compromises their homeostasis. This condition has to be reverted for cell survival. One of the homeostatic processes impaired by cerebral ischemia is protein folding in the endoplasmic reticulum (ER) lumen where unfolded proteins are accumulated (Degracia and Monti, 2004). This situation, known as ER stress, elicits the so called unfolded protein response (UPR), whereby the cell tries to restore homeostasis to prevent its death. ER stress activates protein sensors that trigger the three different pathways described so far for UPR. Early UPR signaling events are initiated by the protein kinase RNA-like endoplasmic reticulum kinase (PERK). This UPR pathway is characterized by the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), which blocks most of protein translation and activates the transcription factor 4 (ATF4). This factor regulates the activation of genes that protect the cell against metabolic consequences of ER stress. PERK-ATF4 UPR pathway activation enhances the protein folding capacity of the ER and is essential for reducing ER stress. This pathway is turned off by protein phosphatase 1 (PP1) which dephosphorylates p-eIF2α (reviewed by Godin et al., 2016). The use of the PP1 blocker, salubrinal (Sal), which is able to penetrate into brain tissue in vivo (Sokka et al., 2007), provides a mechanism to maintain activated the PERK-UPR pathway (Ohri et al., 2013).

Effects of ER stress on unfolded protein responses, cell survival, and viral replication in primary effusion lymphoma

Primary effusion lymphoma (PEL), a subtype of non-Hodgkin's B-lymphoma, is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. Endoplasmic reticulum (ER) stress induces activation of the unfolded protein response (UPR), which induces expression of ER chaperones, which in turn decrease ER stress, leading to ER homeostasis. The UPR is necessary for not only ER homeostasis but also persistent infection by, and replication of, many viruses. However, the precise roles and regulation of the UPR in KSHV infection remain poorly understood. Here, we found that IRE1α and PERK were significantly downregulated in PEL cells cultured under normal conditions, compared with KSHV-uninfected B-lymphoma cells. IRE1α and PERK mRNA levels were decreased in PEL cells, and KSHV-encoded LANA and v-cyclin D led to suppressed IRE1α transcription. Thapsigargin-induced ER stress activated the UPR and increased the mRNA levels of UPR-related molecules, including IRE1α and PERK, in PEL cells. However the IRE1α and PERK mRNA levels in PEL cells were lower than those in KSHV-uninfected cells. Furthermore, ER stress induced by brefeldin A and thapsigargin dramatically reduced the viability of PEL cells, compared with KSHV-uninfected cells, and induced apoptosis of PEL cells via the pro-apoptotic UPR through expression of CHOP and activation of caspase-9. In addition to the pro-apoptotic UPR, thapsigargin-induced ER stress enhanced transcription of lytic genes, including RTA, K-bZIP and K8.1, and viral production in PEL cells resulted in induction of the lytic cycle. Thus, we demonstrated downregulation of IRE1α and PERK in PEL cells, transcriptional suppression of IRE1α by LANA and v-cyclin D, apoptosis induction in PEL cells by ER stress, and potentiation of lytic replication by ER stress.

Endoplasmic Reticulum (ER) Stress Induces Sirtuin 1 (SIRT1) Expression via the PI3K-Akt-GSK3β Signaling Pathway and Promotes Hepatocellular Injury

Sirtuin 1 (SIRT1), an NAD(+)-dependent histone deacetylase, plays crucial roles in various biological processes including longevity, stress response, and cell survival. Endoplasmic reticulum (ER) stress is caused by dysfunction of ER homeostasis and exacerbates various diseases including diabetes, fatty liver, and chronic obstructive pulmonary disease. Although several reports have shown that SIRT1 negatively regulates ER stress and ER stress-induced responses in vitro and in vivo, the effect of ER stress on SIRT1 is less explored. In this study, we showed that ER stress induced SIRT1 expression in vitro and in vivo. We further determined the molecular mechanisms of how ER stress induces SIRT1 expression. Surprisingly, the conventional ER stress-activated transcription factors XBP1, ATF4, and ATF6 seem to be dispensable for SIRT1 induction. Based on inhibitor screening experiments with SIRT1 promoter, we found that the PI3K-Akt-GSK3β signaling pathway is required for SIRT1 induction by ER stress. Moreover, we showed that pharmacological inhibition of SIRT1 by EX527 inhibited the ER stress-induced cellular death in vitro and severe hepatocellular injury in vivo, indicating a detrimental role of SIRT1 in ER stress-induced damage responses. Collectively, these data suggest that SIRT1 expression is up-regulated by ER stress and contributes to ER stress-induced cellular damage.

Abstract LB-A13: Cellular characterization of the selective inhibition of UBA5 by organometallic, adenosine-based inhibitors

Abstract Cells that undergo higher protein turnover, such as pancreatic secretory cells and cancerous cells, have the propensity to undergo endoplasmic reticulum (ER) stress. If left uncorrected, ER stress can result in the initiation of apoptosis. To avoid these fates, cells have developed support systems to counteract the apoptotic effects of ER stress, such as conjugation of certain stress-associated proteins with the ubiquitin-fold modifier 1 (UFM1) ubiquitin-like protein. Our research has recently focused on the discovery and in vitro validation of the first selective inhibitor of the UFM1 pathway, 5C-Z, which selectively targets the UFM1 E1 enzyme (UBA5) over other related enzymes and a panel of over 90 human kinases. This novel strategy of inhibiting UBA5 and subsequent UFMylation would make cancer cells that are highly dependent on this system more susceptible to pharmacological disturbances in ER homeostasis, which could lead to the use of milder drug dosing strategies. Our current efforts are focused on studying the effects of 5C-Z on intracellular signalling, the cellular distribution of proteins in the UFM1 pathway, and phenotypic changes within a lung cancer cell lung line (Sk-Luci6). Treatment of lung cancer cells that exhibit high levels of UBA5 protein expression results in decreased cell proliferation (EC50 = 216.9 μM, 95% C.I. = 211.5 - 222.5 μM), yet does not induce cell death in other diseased (A549) or healthy (MRC9) lung cells (up to 200 μM). The decreased cellular proliferation upon 5C-Z treatment mirrors the effect of treatment with UBA5 siRNA. Using immunocytochemistry, it appears that treatment with 5C-Z also induces changes in both cellular morphology and the distribution of UBA5-like staining within the cell. Furthermore, there is an apparent decrease in UFM1-like co-localization with its E2 conjugating enzyme (UFC1) after prolonged incubation with 5C-Z, although further intracellular analysis is required to confirm this observation. Preliminary work also indicates that these cellular effects may also be selective against the UFM1 pathway compared to related ubiquitin-like labelling pathways. We are currently evaluating the effects of 5C-Z on ER stress signalling pathways connected to the UFM1 labelling system. Citation Format: Sara R. da Silva, Mulu Geletu, Fiza Javed, Stacey-Lynn Paiva, Andrew M. Lewis, Honglin Li, Patrick T. Gunning. Cellular characterization of the selective inhibition of UBA5 by organometallic, adenosine-based inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A13.

Effect of endoplasmic reticulum stress on the expression and osteogenic differentiation of periodontal ligament stem cells

To determine the activity of endoplasmic reticulum stress (ERS) and its effect on osteogenic differentiation of periodontal ligament stem cells (PDLSC) in inflammatory microenvironment. PDLSC were obtained from the primary culture of the human tooth and cloned with limited diluted method. Real-time reverse transcription (RT)-PCR was used to examine the different expression of thapsigargin (TG) treated PDLSC and lipopolysaccharide (LPS) treated PDLSC. Real-time RT-PCR, alizarin red staining and cetyl pyridine chloride quantitative analyze were used to examine the osteogenic differentiation of PDLSC, TG + PDLSC, LPS + PDLSC and LPS + PDLSC + 4-PBA. Protein kinase receptor like endoplasmic reticulum kinase (PERK), glucose regulated protein 78 (GRP78), transcription activation factor 4(ATF4), CCAAT/enhancer-binding protein-homologous protein (CHOP) mRNA expression in group PDLSC + TG in 6 h were respectively 1.49 ± 0.24, 2.77 ± 0.60, 1.75 ± 0.16, 2.16 ± 0.32, which were all greater than that in group PDLSC (P < 0.05). PERK, CHOP mRNA expression reached the peak at 6 h (1.76 ± 0.08, 2.31 ± 0.17) and were greater than group PDLSC (P < 0.05). ERS could suppress osteogenic differentiation of TG + PDLSC and LPS + PDLSC. The runt-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN) mRNA expression of group TG + PDLSC was respectively 0.73 ± 0.06, 0.01 ± 0.00, 0.20 ± 0.06 (P < 0.05). The RUNX2, ALP, OCN mRNA expression of group LPS + PDLSC was respectively 0.80 ± 0.06, 0.48 ± 0.05, 0.29 ± 0.04 (P < 0.05). The RUNX2, ALP, OCN mRNA expression of group PDLSC + TG + 4-PBA was respectively 1.10 ± 0.09, 0.74 ± 0.05, 0.67 ± 0.13, which were greater higher than that of group LPS + PDLSC (P < 0.05). ERS was activated in PDLSC and suppressed osteogenic differentiation of PDLSC, which can simulate inflammatory microenvironment in vitro. This effect can be recovered by using ERS inhibitor 4-PBA.

Differential Effects of Endoplasmic Reticulum Stress on Dipsogenic and Blood Pressure Responses to DOCA‐Salt

Endoplasmic reticulum (ER) stress in the subfornical organ has been identified as an important mechanism mediating the hypertensive effects of angiotensin II (Ang‐II). We examined the role of ER stress in the polydipsic and pressor responses to DOCA‐salt. ICV delivery of the ER stress‐reducing chemical chaperone TUDCA had no effect on the magnitude of hypertension, but markedly decreased saline intake to DOCA‐salt. Saline intake increased one day after TUDCA was stopped. Similarly, decreased saline intake was observed after ICV 4‐phenylbutyrate, another chemical chaperone. CCAAT‐enhancer‐binding protein homologous protein (CHOP, a marker of chronic ER stress) immunoreactivity was increased in the SFO of DOCA‐salt mice, but the signal was absent in control and CHOP‐/‐ mice. Electron micrographic studies revealed abnormalities in ER structure (decrease in ER membrane length, distended ER membranes, and decreased ribosome numbers) in the SFO consistent with the induction of ER stress. SFO‐targeted injection of AdGRP78, a molecular chaperone, decreased DOCA‐salt‐induced saline intake. Like the effects of TUDCA, the increase in saline intake in response to DOCA‐salt was blunted in CHOP‐/‐ mice. However, CHOP‐/‐ mice exhibited a similar hypertensive response to DOCA‐salt. These data mechanistically implicate ER stress in saline intake, but not blood pressure responses to DOCA‐salt, and further suggest that CHOP may play a functional role in DOCA‐salt‐induced saline intake. The results suggest a distinction between the importance of ER stress in mediating effects of DOCA‐salt on polydipsia and hypertension.

Stress of endoplasmic reticulum modulates differentiation and lipogenesis of human adipocytes

BackgroundAdipocytes are cells specialized for storage of neutral lipids. This storage capacity is dependent on lipogenesis and is diminished in obesity. The reason for the decline in lipogenic activity of adipocytes in obesity remains unknown. Recent data show that lipogenesis in liver is regulated by pathways initiated by endoplasmic reticulum stress (ERS). Thus, we aimed at investigating the effect of ERS on lipogenesis in adipose cells. MethodsPreadipocytes were isolated from subcutaneous abdominal adipose tissue from obese volunteers and in vitro differentiated into adipocytes. ERS was induced pharmacologically by thapsigargin (TG) or tunicamycin (TM). Activation of Unfolded Protein Response pathway (UPR) was monitored on the level of eIF2α phosphorylation and mRNA expression of downstream targets of UPR sensors. Adipogenic and lipogenic capacity was evaluated by Oil Red O staining, measurement of incorporation of radio-labelled glucose or acetic acid into lipids and mRNA analysis of adipogenic/lipogenic markers. ResultsExposition of adipocytes to high doses of TG (100 nM) and TM (1 μg/ml) for 1–24 h enhanced expression of several UPR markers (HSPA5, EDEM1, ATF4, XBP1s) and phosphorylation of eIF2α. This acute ERS substantially inhibited expression of lipogenic genes (DGAT2, FASN, SCD1) and glucose incorporation into lipids. Moreover, chronic exposure of preadipocytes to low dose of TG (2.5 nM) during the early phases of adipogenic conversion of preadipocytes impaired both, lipogenesis and adipogenesis. On the other hand, chronic low ERS had no apparent effect on lipogenesis in mature adipocytes. ConclusionsAcute ERS weakened a capacity of mature adipocytes to store lipids and chronic ERS diminished adipogenic potential of preadipocytes.

Proteomic analysis of INS-1 rat insulinoma cells: ER stress effects and the protective role of exenatide, a GLP-1 receptor agonist.

Beta cell death caused by endoplasmic reticulum (ER) stress is a key factor aggravating type 2 diabetes. Exenatide, a glucagon-like peptide (GLP)-1 receptor agonist, prevents beta cell death induced by thapsigargin, a selective inhibitor of ER calcium storage. Here, we report on our proteomic studies designed to elucidate the underlying mechanisms. We conducted comparative proteomic analyses of cellular protein profiles during thapsigargin-induced cell death in the absence and presence of exenatide in INS-1 rat insulinoma cells. Thapsigargin altered cellular proteins involved in metabolic processes and protein folding, whose alterations were variably modified by exenatide treatment. We categorized the proteins with thapsigargin initiated alterations into three groups: those whose alterations were 1) reversed by exenatide, 2) exaggerated by exenatide, and 3) unchanged by exenatide. The most significant effect of thapsigargin on INS-1 cells relevant to their apoptosis was the appearance of newly modified spots of heat shock proteins, thimet oligopeptidase and 14-3-3β, ε, and θ, and the prevention of their appearance by exenatide, suggesting that these proteins play major roles. We also found that various modifications in 14-3-3 isoforms, which precede their appearance and promote INS-1 cell death. This study provides insights into the mechanisms in ER stress-caused INS-1 cell death and its prevention by exenatide.