The endoplasmic reticulum (ER) is a vital organelle involved in the synthesis and maintenance of secretory membrane proteins. It also houses a number of chaperone proteins that ensure quality control by ensuring proper protein folding. During periods of stress, the ER initiates a series of coping mechanisms termed the unfolded protein response (UPR) in an attempt to resolve the pathological alterations in protein folding (1, 2). Upon activation, the UPR restructures the cellular transcriptional, translational, and degradation pathways to help resolve the defects in protein folding (3–5). These actions are accomplished through the activation of three trans-membrane ER proteins, namely protein kinase RNA-dependent-like ER kinase (PERK), inositol requiring ER-to-nucleus signal kinase 1 (IRE-1), and activating transcription factor 6 (ATF6) (1, 3–5). The activation of PERK halts protein translation through the phosphorylation of eukaryotic translation initiator factor 2α (eIF2α) (6). While IRE-1 activation initiates the degradation of mis-folded proteins (4), under instances where ER stress is prolonged, the UPR initiates programmed cell death through the actions of C/EBP homologous protein (CHOP) and ATF6 (7).
Interestingly, chronic ER stress has been implicated in the pathology of a number of conditions like diabetes, traumatic brain injury, sepsis, and neurodegenerative diseases (8–10). Therefore, studying the ER stress response in both an in vitro and in vivo context can help uncover and shed light on the pathology and development of these diseases. Presently, there are a number of ER stress-inducing drugs that recapitulate the pathological alterations mediated by ER stress. Among these are the well-characterized thapsigargin (TG), an inhibitor of Ca2+-ATPase pump and tunicamycin (TUN), an inhibitor of protein glycosylation. However, currently, there is a lack of literature characterizing the optimal dose and specificity of each drug. Therefore, we examined the effectiveness of two distinct prominent ER stress inducers TG and TUN using an in vitro and in vivo model. Thus, our findings help to provide a closer look at each ER stress inducing drug and how they manifest differently in the context of in vitro and in vivo research.
Induction of ER stress in vitro. HepG2 cells were cultured in Dulbecco Modified Eagle Medium (DMEM low glucose, 1 g/L glucose) (Wisent, Toronto, ON, Canada) supplemented with 10% fetal bovine serum (FBS) and 1% Antibiotic/Antimicotic (Wisent). HepG2 cells were seeded in wells 24 to 48 h prior to treatments, and treated when cells reached 70% confluence. Medium was removed and replaced with treatments consisting of thapsigargin (25 nM, 50 nM, 100 nM, 200 nM) or tunicamycin (2.5 ug/mL, 5 ug/mL, 10 ug/mL, 20 ug/mL) (Sigma Aldrich, St. Louis, Mo) diluted in medium. 3T3L1 and C3H/10T1/2 adipocytes were maintained in DMEM (4.5 g/L glucose) supplemented with FBS and differentiated for 10 days following incubation with a differentiation protocol previously described (11). Differentiation was initiated 2 days post-confluency in DMEM containing 10% FBS in the presence of a differentiation cocktail (insulin, Dexamethasone, IBMX) for 2 days. In the two following days, dexamethasone and IBMX were removed. In the following days, the cells were maintained in DMEM, 10% FBS until full differentiation was achieved (day 8) (11). Following differentiation, the medium was removed and replaced with treatments consisting of thapsigargin (25 nM, 50 nM, 100 nM) or tunicamycin (2.5 ug/mL, 5 ug/mL, 10 ug/mL) (Sigma Aldrich) diluted in the medium.
Induction of ER stress in vivo. Male Balb/c mice (Taconics, New York) were housed and cared in accordance with the Guide for the Care and Use of Laboratory Animals. All procedures performed in this study were approved by the Sunnybrook Research Institute Animal Care Committee (Toronto, ON, Canada). Tunicamycin and thapsigargin were purchased from Sigma and dissolved in dimethyl sulfoxide (DMSO) and diluted in sterile 150 mM dextrose to obtain a concentration of 10 μg/μL. Male Balb/c mice (20–25 g) were injected intraperitoneally with tunicamycin solution (1 μg/g body mass), as described previously. For thapsigargin solution a dose response was conducted using (0.25 ug/g, 0.5 ug/g, and 1 ug/g body mass). As controls, mice were injected intraperitoneally with control buffer (150 mM dextrose containing 1% DMSO). Adipose and liver tissues were harvested 24 h post-treatment, whereas liver tissues utilized for Oil Red O staining were harvested 5 days post-treatment with the ER stress inducing agents.
Immunoblotting. Tissues were homogenized in RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM NaF and protease inhibitors) incubated on ice for 20 min and centrifuged at 10,000 × g for 10 min at 4°C. The infranatant was removed and proteins quantified using the BCA Protein Assay Kit (Pierce, Waltham, Mass). Proteins were resolved by SDS-PAGE followed by Western blotting using antibodies recognizing GRP78 (BiP), inositol-requiring enzyme (IRE1α), eukaryotic initiation factor (eIF2α), CCAAT-enhancer-binding protein (CHOP), alpha/ beta tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), isocitrate dehydrogenases (IDH1), and activating transcription factor 6 (ATF-6) (Novus Biologicals, Littleton, Colo). All antibodies were purchased from Cell Signaling unless otherwise stated. Species appropriate secondary antibodies conjugated to horse radish peroxidise (BioRad, Mississauga, ON, Canada) were used and proteins visualized by enhanced chemiluminescence using the BioRad ChemiDoc MP Imaging System.
Semiquantitative polymerase chain reaction (PCR). Total RNA was extracted from mouse liver using TRIzol-chloroform (Life Technologies, Carlsbad, Calif) with subsequent purification using the RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA (2 μg) was transcribed to cDNA using the high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, Calif). Real-time quantitative PCR was performed using the Applied Biosystems StepOnePlus Real-Time PCR System. The sequences of all primers are listed in Supplementary Table 1, https://links.lww.com/SHK/A494.
Oil Red O Staining. Tissues were perfused with phosphate-buffered saline, coated with optimal cutting temperature compound (Tissue-Tek), placed on dry ice and stored at −80°C until further analysis. Frozen tissue blocks were sectioned 10 μm thick, mounted on slides, and fixed in formaldehyde (40%) for 1 min. The slides were stained with Oil Red O for 10 min at room temperature, rinsed with water and stained using Gill hematoxylin for 1 min. The slides were washed with water, mounted with aqueous mounting medium, and imaged using the Leica DM 2000 LED microscope.
Immunofluorescence staining and microscopy. Cells on eight chamber slides were fixed in paraformaldehyde, blocked with 1% serum bovine serum albumin in PBS, and were stained with antibodies against cleaved-ATF6, BiP at a dilution ratio of 1:200 overnight. Slides were washed and incubated for 1 h at room temperature with the appropriate secondary antibodies (each at a dilution factor of 1:500). The ER tracker used was from Invitrogen and used according to the manufacturer's protocol. Slides were washed and counterstained with DAPI before mounting. Slides were stored in 4°C. Slides were imaged using a Zeiss spinning disk confocal microscope.
Cytokine/inflammatory profile. Rodent sera was collected and utilized to compare inflammatory profiles between control, thapsigargin, and tunicamycin treated samples. Using the Multiplex platform (Millipore, Mass), a panel of pro-inflammatory cytokines, chemokines, and growth factors were all analyzed. Raw data was processed using Millipore Analyst software and all values are presented as mean ± SEM for the respective cytokine concentration, expressed in μg/mL.
Statistical analysis. All the mouse data was analyzed using Student t test, whereas the in vitro data a one-way ANOVA with a Bonferroni post hoc test was used in comparing the groups. Heatmaps are represented as the mean cytokine concentration for each of the control, TG, and TUN groups respectively using GNU Octave. All other graphs were created using Graphpad Prism 5.0 (San Diego, Calif) and analyzed statistically using SPSS 20 (IBM Corp, NY), with significance accepted at P < 0.05.
Characterizations of ER stress inducers TG and TUN in vitro
To assess which of drug, TG, or TUN is more effective at inducing ER stress in an in vitro modeling system, we treated human hepatocellular carcinoma cells (HepG2) and primary mouse embryonic adipocytes (3T3-L1 and C3H/10T1/2) with both drugs and performed a comparative analysis. The selection of these specific cell lines was based on the criteria that they have been widely used in the in vitro study of ER stress in numerous disease models. Treatment of hepatocytes with TUN yielded a consistent heightened ER stress response as compared with TG at the treated concentrations. Up-regulation of binding immunoglobulin protein (BiP) and C/EBP homology protein (CHOP) gene expression was seen with TUN doses tested (Fig. 1, A and B). Additionally, key unfolded protein response (UPR) markers DNAj homologue (DNAjb9) and protein Disulfide Isomerase Family A Member 3 (PDAI3) transcript were consistently induced with TUN relative to control (Fig. 1, C and D). Thapsigargin treatment induced ER stress gene expression consistently only at the elevated concentrations of 50 nM and 100 nM (Fig. 1, A–D). We also confirmed the effects of TG to induce ER stress at 100 nM by assessing ATF-6 shuttling, which upon ER stress is cleaved and transported to the ER from the Golgi (Fig. 1E). To eliminate the possibility of cytotoxicity effects of these ER stress inducers, we performed a cell viability assay to further assess their efficacy at the preferred doses. Viability was quantifiably reduced with both ER stress-inducing agents relative to control (Fig. 1F). However, significant effects on cell viability were noticed with the higher TG (200 nM) and TUN (10 and 20 ug) (Fig. 1F). In summary, TG at a dose of 100 nM and TUN at a dose of 5 ug both yielded robust ER stress induction with limited adverse effects on cell viability relative to control and other doses tested.
Similarly to hepatocytes, differentiated mature adipocytes showed a heightened sensitivity to TUN as assessed by enhanced gene expression of ER stress and UPR markers BiP, CHOP, DNAjb9, and PDAI3 when compared with TG in 3T3-L1 (Fig. 2, A–E). In fact, TUN at a dose of 5 ug consistently yielded robust ER stress induction, which was also confirmed by immunofluorescence staining of cleaved-ATF6 (Fig. 2F). Similar findings were also observed in C3H/10T1/2 mature adipocytes, confirming our 3T3-L1 adipocyte data (Supplementary Figure 1, https://links.lww.com/SHK/A495). Additionally, TUN at a dose of 5 ug/mL compared with 10 ug has previously been shown not to adversely affect cell viability (12, 16). Interestingly, TG doses lower than 100 nM was effective also in activating ER stress in these mature adipocytes, although not to the same degree as TUN at a dose 5 ug/mL (Fig. 2, B–E). We believe that 100 nM of TG was toxic to the adipocytes as gross observation of these cells indicated unhealthy morphology, which explains why lower doses of TG (25 mM, 50 mM) were able to induce ER stress. Collectively, this data indicates that the level of ER stress induction by these two drugs is casually linked to cell type and further demonstrates selective sensitivity in expression. Nevertheless, since both cells types responded well to TUN at a dose of 5 ug consistently, we suggest that this dose and agent is superior when it comes to the in vitro study of ER stress.
Characterizations of ER stress inducers TG and TUN in vivo
To assess ER stress in vivo, we performed interperitoneal injection of either TG (0.5 ug/g/body weight) or TUN (1 ug/g/body weight) in mice. The dose selected for TUN in vivo is in the mid-range of doses used by others and does not elicit cytotoxic effects as assessed by mouse survival from previous studies (13–15). Unfortunately, choosing a TG dose in vivo has been difficult as the literature of TG use in vivo is scarce. In order to proceed with using TG in a mouse model to evaluate the effectiveness of TG in ER stress induction, we needed to demonstrate that our dose of TG (0.5 ug/g/body) did not compromise mouse survival. In fact, we found that using TG at a dose of 0.5 ug/g/body weight was safe and did not elicit any adverse effects on survival in these mice (Supplementary Figure 2, https://links.lww.com/SHK/A495). We then examined the induction of ER stress in the liver and adipose tissue of mice treated with either TG or TUN, by assessing the expression of key ER stress and UPR markers.
We focused all our analysis on the adipose and liver tissue, which have been shown to exhibit a significant ER stress response following disease or injury. Both TG and TUN treatments in mice resulted in significant expression of ER stress markers ATF6 and eIF2α in adipose tissue (P < 0.05) (Fig. 3, A and B). We next investigated the level of ER stress and UPR activation in hepatic tissue. Tunicamycin treated mice showed significant up-regulation at the mRNA level of Dnajb1, CHOP, PDIA, BiP, and Xbp1s well-established markers of ER stress and UPR activation (P < 0.05) (Fig. 4, A and B). These findings were confirmed with the results of western blot analysis (Fig. 4, C–E). However, TG treatment in mice failed to induce the expression of most ER stress and UPR proteins in the liver (Fig. 4, A–E).
It has been widely reported that the ER is the site of triglyceride synthesis and early lipid droplet formation (16). In addition to this, activation of the ER stress response has been shown to elicit significant lipolysis in adipose tissue as well as hepatic de novo lipogenesis (16). The unexpected and striking differences in liver appearance of mice treated with TUN and TG (Fig. 5A) led us to question whether ER stress-mediated hepatic steatosis was also evident under these conditions. Hepatic steatosis is characterized by accumulation of lipids, mainly triglycerides, in the cytoplasm of hepatocytes. Using oil red O staining, TUN-treated mice showed an abundance of fat infiltration in the liver (Fig. 5B). In contrast, the livers of both sham and TG-treated mice showed no evidence of hepatic steatosis, indicating that they likely do not suffer from a substantial ER stress response, and in agreement with the evaluations of ER stress in this tissue earlier (Fig. 4). Collectively, these results indicate that TUN is superior to TG with regards to the activation of hepatic ER stress; in addition, it is able to capture the ER stress mediated fatty liver phenotype in vivo.
Hepatic steatosis is one of the key features of inflammation. In fact, there is growing interest in the contributions of both inflammation and ER stress to metabolic diseases associated with obesity-like diabetes (17). As such, measurement of inflammation was conducted to evaluate ER stress activation in mice treated with either TG or TUN. In an attempt to characterize inflammation associated with ER stress, we conducted an immune-screening cytokine assay in the serum of mice treated with either TG or TUN. The multi-analyte analysis of systemic inflammation revealed that TG had a 2 to 5-fold significant increase in chemokine and pro-inflammatory expression, relative to controls (Supplemental Figure 3, https://links.lww.com/SHK/A495). When specifically comparing both TG and TUN treatments, TG had increased cytokine protein expression for a multitude of cytokines including GM-CSF, RANTES, IFN-γ, IL-1α, TNF-α, IL-2, IL-4, IL-5, and IL-12 (Supplemental Figure 2, https://links.lww.com/SHK/A495). Although TUN did result in a slight increase in chemokine and pro-inflammatory cytokine expression relative to controls, notable increases were only reported for immune mediator GM-CSF, systemic inflammatory indicator IL-6, and hepatic IL-1β induction (Supplemental Figure 2, https://links.lww.com/SHK/A495). Collectively, these findings suggest that after treatment, TG is more sensitive to inducing a systemic immune response characterized by the aforementioned mediators.
The ability to accurately model ER stress in vitro and in vivo is critical for uncovering the pathology and development of such conditions as diabetes, burns, traumatic brain injury, and sepsis all of which ER stress has been implicated (8, 9, 12). By using two different approaches or models of study (in vitro and in vivo), we sought to establish the effectiveness of two key ER stress inducers in recapitulating the ER stress response. The current study used thapsigargin and tunicamycin as our ER stress-inducing agents because of being both readily available and has widely used in research involving the ER. Our in vitro experiments showed that tunicamycin worked as a rapid and efficacious inducer of ER stress in hepatocytes and adipocytes. The experiments in vivo showed that tunicamycin was superior in not only inducing ER stress but also recapturing the metabolic alterations associated with ER stress.
The well-characterized thapsigargin and tunicamycin act by triggering ER stress through the inhibition of sarco/ER Ca2+ ATPase (SERCA) activity to deplete calcium storage from the ER and protein glycosylation, respectively. TUN treatment and TG only at 100 nM in hepatocytes was found to serve as a rapid and potent inducer of key ER stress proteins CHOP, BiP, and UPR markers (DNAJb9, PDAI3). Furthermore, treatment of adipocytes with TUN was more effective in the expression of ER stress proteins CHOP and BiP as well as UPR markers (DNAJb9, PDAI3). It is, of course, possible that the differential response of our hepatocyte and adipocyte cells to the two ER stress-inducing drugs may be explained by dosing uniformity. This is unlikely, as the doses of the two drugs we have used covered a wide range and characterized to maximize cell viability (16, 17). As such, we believe that our cell culture findings support that TUN is the more effective ER stress inducer overall, whereas TG is a reliable ER stress inducer only in hepatocytes.
We next studied the effects of TG and TUN on their ability to induce ER stress in vivo using a mouse model. Both TG and TUN showed a similar ER stress profile in adipose tissue compared with sham mice. However, in respect to adipose tissue more specific assessment of ER stress output such as lipolysis or adipose tissue remodeling might be warranted to conclude the level of ER stress activation by these two drugs. Upon assessing the effects of these two drugs in the liver, TUN treatment appeared to be liver specific, as hepatic ER stress was significantly up regulated compared with other tissues assessed. The liver-specific effects of TUN-induced ER stress were corroborated by our gross pathological and histological analysis of hepatic lipid accumulation. Collectively, our animal model experiments supported TUN as a potent and effective in vivo inducer of acute ER stress in comparison to TG.
It is well known that ER stress activation disturbs lipid homeostasis in both the liver and adipose tissue (13, 15). Though we observed a mild activation of ER stress in the adipose tissue of TG-treated mice, the absence of the hepatic steatosis phenotype in mice treated with TG compared with TUN indicates that the ER stress response was likely not evoked by this drug (18). With regards to the cause of the hepatic steatosis phenotype seen in the TUN-treated mice, two explanations may be considered. For one, the hepatic steatosis phenotype is likely attributed to TUN-mediated activation of de novo-lipogenesis by the way of activating the ER stress response (19, 20). This is in line with studies that have reported ER stress induction leads to the accumulation of mature SREBP1a and SREBP1c, regulators of de novo lipogenesis (20, 21). Alternatively, the hepatic lipid accumulation can be explained by ER stress mediated lipolysis activation in adipose tissue (15, 16). However, the latter is less likely as TG treatment resulted in ER stress activation in adipose tissue without the fatty liver phenotype seen in TUN-treated mice, likely suggesting that ER stress mediated hepatic steatosis is a result of de novo lipogenesis not lipolysis.
Our findings here provide unique insights and valuable information as to the potency profile of these two commonly used ER stress agents in hopes of standardizing an in vitro and in vivo ER stress model. Misguided use of either class of ER stress-inducing agents in vitro or in vivo could potentially result in the lack of observed efficacy or even toxicity. The lack of significant progress in uncovering ER stress-mediated disease progression has often been blamed on flawed agents that aim to fully recapture the pathology of ER stress. Thus, we hope that the data presented here will be a valuable guide for those researchers investigating ER stress pathology in diverse experimental setups and contexts, which could contribute to the identification of future points of intervention in many important human diseases affected by ER stress.
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