Modeling Acute ER Stress in Vivo and in Vitro
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Abstract:
The endoplasmic reticulum (ER) is a critical organelle that synthesizes secretory proteins and serves as the main calcium storage site of the cell. The accumulation of unfolded proteins at the ER results in ER stress. Although the association between ER stress and the pathogenesis of many metabolic conditions have been well characterized using both in vivo and in vitro models, no standardized model concerning ER stress exists. Here, we report a standardized model of ER stress using two well-characterized ER stress-inducing agents, thapsigargin and tunicamycin. Our aim in this current study was 2-fold: to characterize and establish which agent is optimal for in vitro use to model acute ER stress and to evaluate which agent is optimal for in vivo use. To study the first aim we used two well-established metabolic cell lines; human hepatocellular carcinoma (HepG2s) and differentiated mouse adipocytes (3T3-L1). In the second aim we utilized C57BL/6J mice that were randomized into three treatment groups of sham, thapsigargin, and tunicamycin. Our in vitro results showed that tunicamycin worked as a rapid and efficacious inducer of ER stress in adipocytes consistently, whereas thapsigargin and tunicamycin were equally effective in inducing ER stress in hepatocytes. In regards to our in vivo results, we saw that tunicamycin was superior in not only inducing ER stress but also recapturing the metabolic alterations associated with ER stress. Thus, our findings will help guide and inform researchers as to which ER stress agent is appropriate with regards to their model.Keywords:
Tunicamycin
Thapsigargin
Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca2+ homeostasis. The store-operated calcium (SOC) channel is the primary Ca2+ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca2+ 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 Ca2+ 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.
Tunicamycin
Orai1
Thapsigargin
Endothelial Dysfunction
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Tunicamycin
Viability assay
Schizosaccharomyces
Proteostasis
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Objective To investigate if the Ca~(2+)/calmodulin-dependent protein kinase(CaMK)Ⅱinvolved in the ER-initiated cardiomyocyte death in rat.Methods Rat adult cardiomyocytes were used in this study.ER stress initiated agents,thapsigargin,tunicamycin and brefeldin A were used to induce ER stress.Cell viability was measured with propidium iodide staining.Western blot analysis was used to detect the expression of glucose-regulated protein(GRP) 78 and CHOP.Results Thapsigargin,tunicamycin and brefeldin A induced cardiomyocyte death in a dose and time-dependent manner(P0.01) and up-regulated ER stress-induced ER chaperone GRP-78 and CHOP(P0.05).While CaMKⅡblockers, KN93 and AIP,attenuated the ER stress,evidenced by down-regulation of expression of GRP78 and CHOP(P0.05),both inhibited the cell death rate(P0.05) induced by ER stress initiators.Conclusion These findings demonstrate that thapsigargin,tunicamycin and brefeldin A induced ER-initiated cardiomyocyte death via CaMKⅡ-dependent pathways.CaMKⅡmight become one of target to treat cardiac infarcts or prevent heart failure by blocking ER stress-induced apoptosis.
Thapsigargin
Tunicamycin
Brefeldin A
Propidium iodide
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Backgroud
Endoplasmic reticulum stress was induced by the accumulation and aggregation of unfolded proteins due to stresses that disturbed the cellular energy levels, the redox state, or Ca2+ concentration, and leading to the unfolded protein response (UPR) pathway. The hepatic UPR was activated in several forms of liver disease. Recent data showed that the role of the UPR in hepatic cells have identified molecular mechanisms that may underlie the association between UPR activation and liver disease. SERP1 was known as ribosome-associated membrane protein 4 (RAMP4), was homologous to yeast suppressor of SecY 6 protein (YSY6p) which suggested a role in pathways controlling membrane protein biogenesis at the ER level. Expression of SERP1 was enhanced during cellular stress, causing accumulation of unfolded proteins in the ER.By interaction with the molecular chaperone calnexin, SERP1/RAMP4 could control biogenesis of membrane proteins and take participate in the endoplasmic reticulum stress.Objective To study the effects of stress-associated endoplasmic reticulum protein 1(SERP1) on the endoplasmic reticulum stress induced by the tunicamycin in HepG2 cells.
Methods
The tunicamycin was used to induce endoplasmic reticulum stress in the HepG2 cells.We divided the cells into 5 groups: normal control group, tunicamycin treated group, tunicamycin + 0.25μg/μl SERP1 transfected group, tunicamycin + 0.25μg/μl SERP1 transfected group, tunicamycin + 0.5μg/μl SERP1 transfected group, tunicamycin + 1μg/μl SERP1 transfected group. Each experiment was repeated three times.MTT was used to detect the effect on the survival rate of the HepG2 cells and selected the optimal concentration and time of tunicamycin treatment.Western blot was used to detect the standard of expression of endoplasmic reticulum stress spcific mark proteins, glucose-regulated protein 78(GRP78), C/EBP homologous protein (CHOP) and calnexin.
Results
Compared with the control group, the expression levels of GRP78, CHOP and calnexin were significantly increased in the tunicaymicin treated group, which were 3.8 times, 1.3 times and 1.4 times respectively. With the increasing amount of transfection, SERP1 over expression was found to relieve the expression of GRP78 12%(1.838±0.29, 1.6±0.132, P>0.05), 24% and 30%(1.838±0.29, 1.40±0.11, 1.27±0.21, F=50.56, P 0.05)、24%和28%(1.20±0.18, 0.92±0.07, 0.87±0.18, F=8.116, P<0.01)respectively, which were induced by tunicamycin treatment.
Conclusion
SERP1 overexpression could attenuate the ER stress induced by tunicamycin, and may reduce the cell damage mediated by the ER stress.
Key words:
Endopasmic reticulum stress; Stress-associated Endoplasmic Reticulum Protein 1; Glucose-regulated protein 78; C/EBP homologous protein; Calnexin
Tunicamycin
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Endoplasmic reticulum stress occurs in a variety of patho-physiological mechanisms and there has been great interest in managing this pathway for the treatment of clinical diseases. Autophagy is closely interconnected with endoplasmic reticulum stress to counteract the possible injurious effects related with the impairment of protein folding. Studies have shown that glomerular podocytes exhibit high rate of autophagy to maintain as terminally differentiated cells. In this study, podocytes were exposed to tunicamycin and thapsigargin to induce endoplasmic reticulum stress. Thapsigargin/tunicamycin treatment induced a significant increase in endoplasmic reticulum stress and of cell death, represented by higher GADD153 and GRP78 expression and propidium iodide flow cytometry, respectively. However, thapsigargin/tunicamycin stimulation also enhanced autophagy development, demonstrated by monodansylcadaverine assay and LC3 conversion. To evaluate the regulatory effects of autophagy on endoplasmic reticulum stress-induced cell death, rapamycin (Rap) or 3-methyladenine (3-MA) was added to enhance or inhibit autophagosome formation. Endoplasmic reticulum stress-induced cell death was decreased at 6 h, but was not reduced at 24 h after Rap+TG or Rap+TM treatment. In contrast, endoplasmic reticulum stress-induced cell death increased at 6 and 24 h after 3-MA+TG or 3-MA+TM treatment. Our study demonstrated that thapsigargin/tunicamycin treatment induced endoplasmic reticulum stress which resulted in podocytes death. Autophagy, which counteracted the induced endoplasmic reticulum stress, was simultaneously enhanced. The salvational role of autophagy was supported by adding Rap/3-MA to mechanistically regulate the expression of autophagy and autophagosome formation. In summary, autophagy helps the podocytes from cell death and may contribute to sustain the longevity as a highly differentiated cell lineage.
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Thapsigargin
Autophagosome
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The endoplasmic reticulum (ER) is a critical organelle that synthesizes secretory proteins and serves as the main calcium storage site of the cell. The accumulation of unfolded proteins at the ER results in ER stress. Although the association between ER stress and the pathogenesis of many metabolic conditions have been well characterized using both in vivo and in vitro models, no standardized model concerning ER stress exists. Here, we report a standardized model of ER stress using two well-characterized ER stress-inducing agents, thapsigargin and tunicamycin. Our aim in this current study was 2-fold: to characterize and establish which agent is optimal for in vitro use to model acute ER stress and to evaluate which agent is optimal for in vivo use. To study the first aim we used two well-established metabolic cell lines; human hepatocellular carcinoma (HepG2s) and differentiated mouse adipocytes (3T3-L1). In the second aim we utilized C57BL/6J mice that were randomized into three treatment groups of sham, thapsigargin, and tunicamycin. Our in vitro results showed that tunicamycin worked as a rapid and efficacious inducer of ER stress in adipocytes consistently, whereas thapsigargin and tunicamycin were equally effective in inducing ER stress in hepatocytes. In regards to our in vivo results, we saw that tunicamycin was superior in not only inducing ER stress but also recapturing the metabolic alterations associated with ER stress. Thus, our findings will help guide and inform researchers as to which ER stress agent is appropriate with regards to their model.
Tunicamycin
Thapsigargin
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Objective: By using ox-LDL-induced macrophage foam cell model, we used different doses of tunicamycin to induce different degree of the endoplasmic reticulum stress, to observe the effects of it on autophagy. Methods: Different doses of tunicamycin were added to macrophage RAW264.7. The apoptosis rate was detected with TUNEL method. And the expression level of the endoplasmic reticulum stress marker protein GRP78 and autophagy marker protein P62 were detected by western blotting. Results: Compared with the ox-LDL group and the high-dose tunicamycin group, low-dose of tunicamycin significantly reduced the apoptosis of macrophage(P0.01); compared with ox-LDL group, the endoplasmic reticulum stress marker protein GRP78 expression was remarkable increased in low-dose tunicamycin group; meanwhile, the autophagy marker protein P62 moderately decreased(P0.01); in the high-dose tunicamycin group, there was more significant increase in the endoplasmic reticulum stress marker protein GRP78 expression, however the autophagy marker protein P62 also increased notably(P0.01). Conclusion: Low-dose of tunicamycin caused a certain degree of endoplasmic reticulum stress, which activated moderate autophagy, decreasing macrophage apoptosis. It may help to reduce the extent of the atherosclerosis.
Tunicamycin
Glucose-regulated protein
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We have investigated the impact of endoplasmic reticulum (ER) stress, which is often implicated in neurodegenerative diseases, on the expression of Hrd1, an E3 ubiquitin ligase that plays a central role in the process of ER-associated degradation (ERAD).SH-SY5Y neuroblastoma cells, a frequently used model for studying neurotoxicity in dopaminergic neurons and the mechanisms of neurodegeneration associated with Parkinson's disease, and parental SK-N-SH cells were studied.We demonstrate that ER stress, induced by thapsigargin or tunicamycin, correlates with the increased expression of Hrd1 in both SH-SY5Y and SK-N-SH cells. Inhibition of PERK does not significantly suppress the thapsigargin- or tunicamycin-induced expression of Hrd1. Nevertheless, PERK inhibition has a positive effect on the survival of SH-SY5Y cells treated with thapsigargin but not on those treated with tunicamycin. Inhibition of IRE1 associated with the inhibition of XBP1 splicing does not affect the survival of SH-SY5Y cells treated with either thapsigargin or tunicamycin but results in the complete suppression of both the thapsigargin- and tunicamycin-induced expression of Hrd1.Thus, the ER-stress-induced expression of Hrd1 in SH-SY5Y depends on Hrd1 transcription activation, which is a consequence of IRE1 but not of PERK activation.
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Thapsigargin
XBP1
Cyclopiazonic acid
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Unfolded protein response (UPR) is an adaptive response, allowing the endoplasmic reticulum (ER) responds to an accumulation of unfolded proteins in its lumen, also known as ER stress. The ER reacts to ER stress through ER transmembrane protein sensors, thus activating intracellular signal transduction pathways. The UPR is interconnected with inflammation through reactive oxygen species production, activation of nuclear factor-kB (NF-kB) and JUN N-terminal kinase (JNK) via inositol-requiring enzyme 1 (IRE1) and induction of acute-phase response. LCN2 is one of the acute phase proteins that are induced under inflammatory conditions and up-regulated during ER stress. We therefore examined the ER stress responses in LCN2-/- condition.
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