- Research article
- Open Access
Endoplasmic reticulum stress response in an INS-1 pancreatic β-cell line with inducible expression of a folding-deficient proinsulin
© Hartley et al; licensee BioMed Central Ltd. 2010
- Received: 11 November 2009
- Accepted: 26 July 2010
- Published: 26 July 2010
Cells respond to endoplasmic reticulum stress (ER) stress by activating the unfolded protein response. To study the ER stress response in pancreatic β-cells we developed a model system that allows for pathophysiological ER stress based on the Akita mouse. This mouse strain expresses a mutant insulin 2 gene (C96Y), which prevents normal proinsulin folding causing ER stress and eventual β-cell apoptosis. A double-stable pancreatic β-cell line (pTet-ON INS-1) with inducible expression of insulin 2 (C96Y) fused to EGFP was generated to study the ER stress response.
Expression of Ins 2 (C96Y)-EGFP resulted in activation of the ER stress pathways (PERK, IRE1 and ATF6) and caused dilation of the ER. To identify gene expression changes resulting from mutant insulin expression we performed microarray expression profiling and real time PCR experiments. We observed an induction of various ER chaperone, co-chaperone and ER-associated degradation genes after 24 h and an increase in pro-apoptotic genes (Chop and Trib3) following 48 h of mutant insulin expression. The latter changes occurred at a time when general apoptosis was detected in the cell population, although the relative amount of cell death was low. Inhibiting the proteasome or depleting Herp protein expression increased mutant insulin levels and enhanced cell apoptosis, indicating that ER-associated degradation is maintaining cell survival.
The inducible mutant insulin expressing cell model has allowed for the identification of the ER stress response in β-cells and the repertoire of genes/proteins induced is unique to this cell type. ER-associated degradation is essential in maintaining cell survival in cells expressing mutant insulin. This cell model will be useful for the molecular characterization of ER stress-induced genes.
- Endoplasmic Reticulum Stress
- Unfold Protein Response
- EGFP Expression
- Endoplasmic Reticulum Stress Response
- Induce Endoplasmic Reticulum Stress
An increase in post-prandial blood glucose stimulates pancreatic β-cells to secrete insulin, which mediates glucose disposal into tissues such as muscle and fat. Glucose also acutely stimulates insulin translation, which replenishes the β-cell granules that have been depleted during secretion . The effect of glucose on translation increases the protein folding load in the endoplasmic reticulum (ER) of the β-cell, which may transiently induce ER stress, a condition in which the folding capacity of the ER is insufficient for the level of newly synthesized secretory proteins and results in the accumulation of misfolded proteins. Such conditions are sensed by ER localized stress sensors that mediate the unfolded protein response (UPR) . This results in a transient reduction in translation efficiency to reduce secretory protein load and induces chaperone capacity and ER-associated degradation (ERAD). ER stress is likely a physiological situation the β-cell transiently experiences following feeding.
ER stress is also a feature of pathological conditions associated with obesity and diabetes. Excess nutrients (free fatty acids, glucose), amyloid deposits and inflammatory cytokines have all been shown to induce ER stress in pancreatic β-cells . These conditions induce the UPR pathways that attempt to counteract ER stress. However, chronic ER stress can lead to apoptosis induction , particularly in cell types with high secretory capacity such as the β-cell. This is evident from the fact that perturbation of the UPR signaling pathways in vivo leads to pancreatic β-cell death in animal models [5–7] and humans  and mutations in the insulin molecule in Akita mice results in ER stress-induced β-cell apoptosis [9, 10]. In addition, it has been hypothesized that chronic conditions associated with obesity over time may lead to chronic ER stress-induced β-cell death and depletion of β-cell mass that results in type 2 diabetes .
Thus, understanding how pancreatic β-cells deal with, and respond to ER stress, is vital for understanding β-cell biology under physiological and pathological conditions. A detailed temporal analysis of the ER stress response in β-cells has not been conducted. In addition the molecular mechanism of ER stress-induced apoptosis is still, for the most part, unclear. As mentioned, expression of folding-deficient proinsulin in the Akita mouse leads to pancreatic β-cell death as a result of ER stress and apoptosis . Thus, the Akita mouse provides a model of β-cell dysfunction resulting from ER stress and affords a system to study the ER stress response independently of using commonly used toxins such as thapsigargin or tunicamycin.
To study the ER stress response in pancreatic β-cells we developed a pancreatic β-cell culture system based on the Akita mouse. Inducible expression of mutant insulin 2 (C96Y) fused to EGFP [Ins2 (C96Y)-EGFP] caused ER stress and apoptosis. In this study we examined the temporal gene expression response resulting from induction of Ins2 (C96Y)-EGFP expression in INS-1 β-cells and the role of ERAD in mediating cell survival.
Cloning of the pTRE-Tight Insulin 2 (C96Y)-EGFP construct
Full-length wild-type mouse insulin 2 cDNA was amplified by PCR from pIns2 (WT)-EGFP, a fusion construct obtained from Dr. Seiichi Oyadomari (New York University School of Medicine) . The PCR fragment was cloned into pCRII-TOPO vector (Invitrogen). Using the QuikChange II XL Site-Directed Mutagenesis kit (Stratagene), a C96Y mutation was introduced into the insulin 2 cDNA. The mutation was confirmed by DNA sequencing. Ins2 (C96Y) was cloned into the pEGFP-N1 vector (BD Biosciences), the stop codon was removed and the resulting fusion construct pIns2 (C96Y)-EGFP was cloned into the NheI/NotI site of a pTRE-Tight vector (Clontech).
Generation of an inducible Insulin 2 (C96Y)-EGFP expressing INS-1 β-cell line
Initially a stable pTet-ON INS-1 clone was generated by transfecting INS-1 cells (obtained from Dr. Claes Wollheim, University of Geneva)  with the pTet-ON plasmid and stable clones were selected following the protocol provided (Clontech). Several clones were isolated and tested by transient transfection using a luciferase reporter assay and the clone with the lowest basal expression and highest doxycyline induction (INS1 pTet-ON #46) was used for generation of the double stable cell line.
Double-stable Ins2 (C96Y)-EGFP expressing cells were generated by co-transfection with a hygromycin resistance plasmid and the pTRE -Ins2 (C96Y)-EGFP plasmid into pTet-ON INS-1 #46 cells and selection by addition of antibiotics: 200 μg/ml geneticin and 50 μg/ml hygromycin B (Invitrogen). Individual clones were isolated and tested for Ins2 (C96Y)-EGFP expression by western blotting and immunofluorescence before and after induction with doxycycline (2 μg/ml) for 24 h. A positive clone was identified and fluorescence-activated cell sorting was used to isolate the highest 20% of EGFP-positive cells in this clonal population after induction with doxycycline for 48 h. The cells were expanded in media without doxycycline and referred to as clone #4S2.
Clone #4S2 cells were maintained in RPMI 1640 (11.1 mM glucose, 1 mM sodium pyruvate, 10 mM HEPES) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 55 μM β-mercaptoethanol with antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) and selection drugs (200 μg/ml G418 and 50 μg/ml hygromycin).
Cell lysis and western blot analysis
Cells were washed in PBS and lysed in ice-cold lysis buffer (1% Triton X-100, 20 mM HEPES, pH 7.4, 100 mM KCl, 2 mM EDTA, 1 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin, 10 mM NaF, 2 mM Na3VO4, and 10 nM okadaic acid) for 15-20 min on ice. Lysates were centrifuged at 13,000 rpm for 10 min at 4°C and the protein concentration in the supernatant was determined using the BCA protein assay (Pierce). Equal amounts of protein were resolved by SDS-PAGE and immunoblotted as described previously . For detection of insulin and cleaved caspase-3 by immunoblotting, the samples were resolved using 4-12% NuPAGE gels (Invitrogen). The following primary antibodies were used: Phospho-eIF2α (Cell Signaling, #9721), GADD153/CHOP (Santa Cruz, sc-575), KDEL (StressGen, SPA-827), insulin (Santa Cruz, sc-9168), insulin (Dako, A0564), γ-tubulin (Sigma, T6557), polyclonal anti-GFP (obtained from Dr. James E. Rothman, Yale University), monoclonal anti-GFP (Clontech, 632381), cleaved caspase 3 (Cell Signaling, #9661S), ubiquitin (Dako, Z0458), GM130 (Transduction Laboratories, G65120), Hsp90 (Transduction Laboratories, 610418). The polyclonal Herp antibody was provided by Dr. Linda Hendershot (St. Jude Children's Hospital, Memphis, TN).
Cells were treated as described in the figure legends, washed with PBS and fixed with 4% paraformaldehyde in PBS for 20 minutes, washed with PBS and mounted on glass coverslips using Fluoromount G (EM Sciences). GFP fluorescence was visualized with a Zeiss laser scanning confocal microscope. Clone #4S2 INS-1 cells were treated as described in the figure legend, washed with PBS and fixed with 4% paraformaldehyde in PBS for 20 minutes. The samples were processed for immunofluorescence microscopy as reported previously  and immunostained with anti-ATF6 (TO13/14) antibody at a 1:100 dilution . Quantitation of ATF6 staining in the nucleus (marked by DAPI staining) was measured using ImageJ Software. The average intensity in arbitrary units in the nuclear area was determined. A minimum of 18 cells for all experimental condition were quantified and the mean relative nuclear intensity is reported.
Following doxycycline treatment for 72 hours, clone #4S2 cells were washed with PBS and resuspended in homogenization buffer: (0.25 M Sucrose, 4 mM HEPES, 1 mM MgCl2, 1.5 mM EDTA containing a complete protease inhibitor tablet (Roche), 1 mM PMSF, 10 mM NaF, 2 mM Na3VO4, 10 nM okadaic acid). Cells were homogenized on ice with 12 strokes using a ball-bearing cell homogenizer. The resulting homogenate was either centrifuged at 100 000×g for 1.5 h to generate membrane (pellet) and cytosol (SN) fractions, or subjected to a 0.45 M to 2 M sucrose gradient for 18 h at 30 000 rpm, 4°C as outlined in . After ultracentrifugation, 0.5 ml fractions were taken from the top of the gradient.
Clone #4S2 cells were fixed in 2% glutaraldehyde (EM Sciences) in 0.15 M Sorensen's Phosphate Buffer (pH 7.4) (EM Sciences) for 30 min at room temperature. The cells were washed twice with PBS and collected into new tubes. Samples were prepared for transmission electron microscopy by the electron microscopy facility at Mount Sinai Hospital (Toronto). Briefly, the cell pellets were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 2 h, rinsed with the same buffer for 10 min, and post-fixed for 1.5 h in 1% OsO4. After rinsing with the same buffer for 10 min, the cells were dehydrated through a graded ethanol series up to 100% ethanol. The cells were then embedded in Spurr resin in an oven at 65°C overnight. Thin sections (~100 nm) were cut with an RMC MT6000 ultramicrotome. The sections were placed on copper grids and stained with uranyl acetate (20 min) and lead citrate (10 min). The grids were examined in a FEI Tecnai 20 transmission electron microscope and images were captured using a Gatan Dualview digital camera.
RNA isolation and real-time PCR analysis
For real-time PCR analysis, total RNA was isolated from cells untreated or treated with 2 μg/ml doxycycline for the indicated times using TRIzol Reagent (Invitrogen) followed by purification with RNeasy Mini Kit (Qiagen). Total RNA was reverse transcribed to single-stranded cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The resulting cDNA was used for real-time PCR analysis by the TaqMan Gene Expression system (Applied Biosystems) as described previously . The following primers were obtained from Applied Biosystems: Chop (Rn00492098_g1), Atf4 (Rn00824644_g1), Grp78 (Rn00565250_m1), Pdi (Rn00564459_m1), Ero1α (Rn00593473_m1), Ero1β (Rn01520960_m1), Sel1 (Rn00710081_m1), Erdj4 (Rn00562259_m1), Erdj5 (Rn01486444_m1), β-actin (4352931E).
XBP1 splicing assay
Total RNA was isolated and rat XBP-1 cDNA was amplified by RT-PCR (QIAGEN OneStep RT-PCR kit) using primers that flank the intron excised by IRE1 exonuclease activity as described previously .
Clone #4S2 cells were reverse transfected using Lipofectamine RNAiMAX with 10 nM Herp siRNA (Invitrogen) or a control siRNA (directed to bacterial β-galactosidase) as described previously . Twenty-four hours later the cells were incubated or not with doxycycline for 48 h then either lysed for western blot analysis or for measurement of apoptosis using the cell death ELISA kit (Roche).
Cell apoptosis was measured using the cell death detection ELISA kit (Roche) according to the instructions provided in the kit. Briefly, clone #4S2 cells were seeded in 24-well plates (200 000 cells/well) and treated as indicated in the figure legends. Following the treatments, the cells were lysed and oligonucleosomes in the cytoplasm (indicative of apoptosis-associated DNA degradation) were quantified according to the manufacturer's instructions.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays were performed using the APO-BrdU TUNEL Assay Kit (Invitrogen). Clone #4S2 cells were treated as described in the figure legend, washed with warm PBS, trypsinized, then stained with Fluor-647 dye-labeled anti-BrdU antibody, as described by the manufacturer's instructions. After staining, the samples were analyzed in a FACSCalibur flow cytometer (Becton Dickinson). To standardize each sample and set the quandrants, a negative control (INS-1 cells) and a positive control (provided by the APO-BrdU TUNEL assay kit) were used. GFP and the Fluor-647 dye-labeled anti-BrdU antibody were excited using the FL1 (530/30) and FL4 (661/16) lasers, respectively.
Microarray expression profiling was used to assess the global transcriptional changes in response to Ins2 (C96Y)-EGFP expression in clone #4S2 cells. Cells were induced or not with doxycycline for 24 h, 48 h or 5 days and total RNA was isolated using TRIzol Reagent (Invitrogen) followed by isolation using an RNeasy Mini Kit (Qiagen). Assessment of RNA quality and microarray analysis was performed at the University Health Network (Toronto) microarray centre. Briefly, following RNA quality assessment with an Agilent BioAnalyzer, samples were reverse transcribed to cDNA. cDNA was purified with a cDNA purification module from Affymetrix. Biotin was incorporated during in vitro transcription and purified cRNA was then fragmented with a chemical reaction. 15 μg of labeled and fragmented cRNA was hybridized to Rat Genome 230 2.0 arrays (Affymetrix Genechip) for 17 h at 45°C at 60 rpm. The arrays were stained and washed using fluidic stations with antibody and streptavidin phycoerythrin conjugate solutions. All arrays are scanned using an Affymetrix 3000 7G scanner. The data sets were analyzed using GeneSpring software (Version 7.1) (Agilent Technologies) and statistical analysis was performed using GeneSpring software according to the manufacturer's specifications. Genes with a minimum 2-fold difference between control and doxycycline-treated cells in at least two independent experiments at 24 h, 48 h and 5 days are listed in the Additional file 1 and Additional file 2. The raw microarray data has been deposited in the Gene Expression Omnibus (GEO) database (GSE22537).
Results are presented as mean ± SE. Statistical significance between two experimental conditions was analyzed using a two-sample t-test assuming equal variance. Data from several or more groups was analyzed by ANOVA, followed by Tukey post hoc test. P < 0.05 was considered statistically significant.
Generation and characterization of the Insulin 2 (C96Y)-EGFP stable INS-1 cell line
The ER stress response to Insulin 2 (C96Y)-EGFP expression
To examine the global transcriptional response to mutant insulin expression we performed microarray analysis of the gene expression changes following Ins2 (C96Y)-EGFP expression. Total RNA was prepared after 24 h, 48 h and 5 days of doxycycline treatment from three independent experiments, reverse transcribed and hybridized to Affymetrix rat genome arrays. Gene expression changes were examined and those that were upregulated or downregulated by 2-fold after doxycyline-induced expression of Ins2 (C96Y)-EGFP are listed in the Additional files 1 and 2. Following 24 h induction 45 genes were upregulated and 5 were downregulated. After 48 h induction 86 genes were upregulated and 37 downregulated, whereas 68 genes were induced and 56 reduced after 5 days induction. These numbers include genes (both well-substantiated and transcribed loci) that were identified as statistically significant in at least two of three experiments.
Selected list of induced genes comparing uninduced cells to cells expressing insulin 2 (C96Y)-EGFP for 24 h, 48 h and 5 days identified by Affymetrix rat genome arrays.
Entrez Gene ID
PDI family, isomerase
PDI family, isomerase
PDI family, isomerase
ER Protein folding
PDI family, isomerase
PDI family, isomerase
ER Protein folding?
RNA polymerase II-like 3
Following 48 h of mutant protein expression a greater number of genes were induced compared to 24 h (Table 1 and Additional files 1 and 2). Additional chaperone genes (Pdia2, Pdia3 and Pdia4) were upregulated. The levels of the Chop transcription factor were also significantly higher than at the 24 h time point. However, the most abundantly induced gene at 48 h of mutant protein expression was Trib3 (Trb3), a gene whose protein product has been implicated in apoptosis among other cellular processes [18, 19]. At this time point other genes including protein transport genes and transcription factors were also upregulated. By 5 days of mutant protein expression the gene expression changes as monitored by microarray was more variable between experiments than at the 48 h time point. However, all the chaperone genes observed at earlier time points were still elevated and additional PDI family member genes (Pdia6 and Txndc4) were induced. The levels of Trib3 were lower compared to the 48 h time point, but still elevated. Interestingly, the levels of Sdf2l1, Creld2 and Armet remained highly induced throughout the time course of mutant protein expression.
Overall, the microarray and real-time PCR results show that Ins2 (C96Y)-EGFP expression causes the induction of many established ER stress response genes, although there is clearly some selectivity in the genes induced.
Induction of apoptosis following Insulin 2 (C96Y)-EGFP expression
The UPR is an essential process by which cells respond to the accumulation of misfolded protein in the ER. This can occur either physiologically, as a result of acute secretory protein load, or as a result of pathological insults that may impede protein folding capacity. The basic ER stress response is well established involving an acute translational attenuation followed by the upregulation of a transcriptional profile that includes ER chaperone genes, ERAD components, protein transport genes, among others . However, it is probable that different cell types have a unique profile of upregulated genes that are dependent on a particular cells function and the secretory proteins it produces. The goal of the present study was to examine the ER stress response in the insulin producing pancreatic β-cell. To do this we developed a pancreatic β-cell culture model with inducible expression of a folding-deficient insulin fusion protein based on the proinsulin mutation found in the Akita mouse . The rationale behind this approach was that regulated expression of a single misfolded protein would allow for a temporal characterization of the UPR in β-cells. This may reflect the ER stress response to physiological conditions (acute insulin synthesis) and potentially a chronic response induced by pathological conditions occurring in type 2 diabetes (chronic insulin biosynthetic demands coupled with the effects of chronic free fatty acids, glucose and cytokines that induce ER stress).
Using the tetracycline/doxycycline-regulated expression system we created a double stable INS-1 cell clone with stable integration of the pTet-ON regulatory plasmid and the Ins2 (C96Y)-EGFP plasmid driven by the Tet/Dox-responsive promoter. The EGFP tag was used in order to facilitate selection of stable clones, as well as for sorting of the positive clones for cells with high expression of the Ins2 (C96Y)-EGFP fusion protein. In the clone selected for the studies basal expression of the mutant insulin is low, although detectable. Following doxycyline addition the mutant insulin is markedly induced by 24 h, reaching maximal expression by ~72 h and expression is maintained for several days. Expression of the mutant insulin fusion protein leads to a swollen ER lumen that is readily detectable in many cells and is indicative of misfolded protein accumulation and a stressed ER. This result is similar to the phenotype observed in the β-cells of Akita mice . As expected, expression of folding-deficient Ins 2 (C96Y)-EGFP fusion protein resulted in the induction of UPR pathways (PERK, IRE1 and ATF6).
To analyze the ER stress response in the cell line we performed microarray expression profiling and real time PCR analysis at various times following mutant protein expression. We chose time-points of Ins2 (C96Y)-EGFP expression when no significant apoptosis was observed (24 h) and longer expression time points (48 h and 5 days), when apoptotic cell death was detectable. After 24 h, 45 genes were induced, while the number of induced genes was substantially increased after 72 h (86) and 5 days (68). Interestingly, the number of downregulated genes was also increased at the longer incubation times (37 and 56 after 48 h and 5 d of doxycycline induction, respectively). This may reflect the observation that prolonged activation of IRE1α can result in relaxed specificity and cleavage of cellular mRNAs , resulting in the downregulation of some genes as a result of prolonged ER stress.
After 24 h of Ins2 (C96Y)-EGFP expression the most consistently induced genes were ER resident chaperones and co-chaperones (Grp78, Erdj4, P58 IPK , Erdj3, Fkbp11) and an ERAD component (Herp). The J-domain containing co-chaperones (Erdj4, P58 IPK ) have been shown to be induced by ER stress in various cell types [20, 25–32]. Recently, P58IPK and ERdj3 have been found to interact with misfolded proteins in the ER prior to recruitment of the chaperone GRP78 [26, 29, 30, 32]. In addition, these proteins have also been implicated in targeting misfolded proteins for degradation via the ERAD system [20, 28]. Thus, expression of misfolded insulin causes the induction of chaperone proteins early in an effort to fold the mutant insulin. These chaperones are maintained at high levels throughout the time course of mutant insulin induction and some may assist in degradation of the misfolded molecule once folding is found to be futile (discussed below). The importance of P58IPK function in normal pancreatic β-cells in vivo is evidenced by increased pancreatic β-cell apoptosis and hyperglycemia in knock-out mice . However, the detailed molecular function of these co-chaperones in β-cells has not been examined to date.
Interestingly, although there are six mammalian DNAJ domain-containing proteins, we only found a subset to be induced by mutant insulin expression. Notable among those that were not upregulated is ERdj5. Recently, expression of a folding-deficient version of the secreted protein surfactant protein C in HEK293 cells caused the induction of various ER stress response genes, including Erdj4 and Erdj5, which were shown to be required for degradation of the folding-deficient surfactant protein C . Surfactant protein C, like proinsulin, contains disulfide bonds, but is not glycosylated. Thus, it is interesting that we do not observe induction of Erdj5 in β-cells expressing folding-deficient proinsulin. A recent report, however, has shown that ERdj5 can interact with EDEM proteins and is required for reducing disulfide bonds on misfolded, glycosylated proteins prior to retrotranslocation . EDEM genes were not detected in the microarray analysis, suggesting that proinsulin may not require EDEM or ERdj5 for its degradation.
Comparing the UPR response observed in our study to ER stress responses in other cell types shows that some of the early chaperone genes induced by mutant protein expression are not observed in other systems. For example, tunicamycin treatment of neuroblastoma cells  or expression of a retroviral protein in astrocytes, which induces ER stress , did not cause an upregulation of Erdj4, P58 IPK , Erdj3, or Fkbp11. Importantly, all of these genes have been shown to be induced by ER stress caused by prolonged palmitate exposure in cultured mouse MIN6 cells . Saturated FFAs have been shown to induce ER stress in pancreatic β-cells and this may contribute to β-cell dysfunction [15, 36, 37]. In addition, a recent proteomic study examining islets from a diabetic insulin resistant mouse model has shown that the protein (and mRNA levels) of some of these (P58IPK , ERdj3, Fkbp11) are upregulated . Thus, the induction of these genes/proteins are likely important cell protective ER stress response proteins in β-cells and a detailed biochemical analysis of their function in mediating proinsulin folding and/or degradation is warranted.
In addition to ER chaperone genes and ERAD components, several other genes were found to be induced early as a result of Ins2 (C96Y)-EGFP expression, including Sdf2l1, Armet and Creld2. All three were abundantly upregulated at both early and late time points. The exact functions of the protein products of these genes in the ER stress response is largely undefined, although these proteins have also been reported to be induced by ER stress-inducing compounds such as tunicamycin in other cell types [39–41].
The appearance of prominent lower migrating bands recognized by anti-GFP antibodies indicates that the Ins2 (C96Y)-EGFP fusion protein is being degraded. Similar cleavage products have been detected with wild-type proinsulin tagged with GFP at the C-terminus and was suggested to occur from non-specific cleavage of the fusion protein at some point along the secretory pathway . In the case of the Ins2 (C96Y)-EGFP the cleavage products are not efficiently immunoprecipitated by anti-GFP antibodies and are not detected in ER fractions. Thus, the smaller migrating bands recognized by GFP antibodies are likely degradation products of proteolysis occurring in the cytoplasm. The full-length mutant insulin fusion protein appears to be inaccessible to the secretory pathway and is retained in the ER, which is consistent with a report showing that the C96Y mutation in proinsulin results in misfolding of the molecule and retention in the ER .
The Ins2 (C96Y)-EGFP fusion protein is likely degraded by the ubiquitin-dependent ERAD system. The ERAD-associated genes Herp and Sel1 were induced by mutant insulin expression, which is consistent with the finding that Sel1 is upregulated in Akita islets . Inhibiting the 26S proteasome resulted in an increase in the levels of Ins2 (C96Y)-EGFP and sensitized the cells to cell death. This was also the effect observed by reducing Herp expression using siRNA, a protein recently shown to be required for the degradation of a disulfide bond-containing mutant version of the kappa light chain . The process of Ins2 (C96Y)-EGFP degradation however, may be quite complex. If the proteasome was responsible for generating the degradation fragments observed (Figure 2) than inhibiting the proteasome would be expected to not affect the steady-state levels of these fragments; new fragments would be prevented from being generated from Ins2 (C96Y)-EGFP and fragments already present prevented from being degraded. However, most degradation fragments were actually increased by proteasome inhibition (Figure 8B), suggesting that other proteases may be responsible for generating the degradation fragments that are then targeted for ubiquitin-dependent degradation.
It should be pointed out that although expression of the Ins2 (C96Y)-EGFP fusion protein induces ER stress and degradation of the fusion protein, due to the presence of the EGFP tag the degradation of the fusion construct may not be identical to the degradation of untagged proinsulin C96Y. Furthermore, it is possible that the ER stress response elicited by untagged proinsulin C96Y is different compared to that of Ins2 (C96Y)-EGFP. These possibilities should be explored in future studies.
This model system also allowed us to examine potential gene expression changes associated with ER stress-induced cell death. An upregulation of the pro-apoptotic transcription factor Chop and one of its target genes (Trib3) was evident by 24 h of Ins2 (C96Y)-EGFP expression, which is indicative of PERK pathway activation and is likely to be a normal physiological response. At this time point no significant apoptosis is detected in the population. After 48 h of mutant insulin expression however, the levels of Chop and Trib3 were even higher, which coincided with detection of apoptosis in the population. Thus, a sustained increase in the levels of CHOP and TRIB3 proteins may tilt cells towards apoptosis induction. Pancreatic β-cells of CHOP-deficient heterozygous Akita mice are partially protected from cell death , although clearly there are other factors also involved. In the mutant insulin expressing cell line the most highly induced gene after 48 h was Trib3. Recently, the TRIB3 protein has been shown to be induced by the PERK-ATF4-CHOP pathway and has been implicated in mediating apoptosis [18, 19] and in hyperglycemia-induced pancreatic β-cell death . The mechanism of this effect, however, is unclear. Whether the TRIB3 protein is responsible for apoptosis induction and how much of the apoptosis is TRIB3-dependent in this model system requires future study.
Despite the abundant upregulation of Chop and Trib3 in response to mutant insulin expression, the level of apoptotic cells in the population remained rather low, even after long term expression of the mutant construct. This is likely because one of the main cell protective effects induced by the UPR, ERAD, is also upregulated. Inhibition of ERAD by inhibiting the cytosolic proteasome or inhibiting Herp upregulation sensitizes the cells to apoptosis, supporting the notion that the ERAD pathway may support cell survival by degradation of misfolded insulin.
The β-cell model of ER stress described in this study has allowed for a detailed characterization of the ER stress response induced by misfolded Ins2 (C96Y)-EGFP expression and for the identification of both cell protective and possible cell destructive genes. The repertoire of genes/proteins induced is unique to this cell type compared to the UPR induced in other cell types, perhaps reflecting the unique role of β-cells to produce insulin. The Herp protein and ER-associated degradation is essential for maintaining cell survival in cells expressing mutant insulin. The cell model developed here will be useful in studies examining the function of ER stress-induced genes.
We thank Dr. Claes Wollheim, University of Geneva for providing the insulinoma cell line INS-1, Dr. Seiichi Oyadomari for providing the insulin-EGFP plasmid, Dr. Linda Hendershot for the anti-Herp antibody and Dr. Jonathon Rocheleau for help with Image J fluorescence intensity quantitation. We thank Monika Sharma at the UHN microarray facility for the microarray analysis. This work was supported, in part, by operating grants to A.V. from the Canadian Diabetes Association (GA-2-06-2138-AV) and from the Canadian Institutes for Health Research (86641). Infrastructure support for the A.V. laboratory was provided by funding from the Canadian Foundation for Innovation and the Ontario Innovation Trust. AV is the recipient of a Tier II Canada Research Chair award. TH, MS and EL were supported in part by Banting and Best Diabetes Centre (University of Toronto) graduate studentships. TT was supported by a graduate studentship from NSERC.
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