IRE1 inhibition perturbs the unfolded protein response in a pancreatic β-cell line expressing mutant proinsulin, but does not sensitize the cells to apoptosis
© Zhang et al.; licensee BioMed Central Ltd. 2014
Received: 9 April 2014
Accepted: 1 July 2014
Published: 10 July 2014
The Akita mutation (C96Y) in the insulin gene results in early onset diabetes in both humans and mice. Expression of mutant proinsulin (C96Y) causes endoplasmic reticulum (ER) stress in pancreatic β-cells and consequently the cell activates the unfolded protein response (UPR). Since the proinsulin is terminally misfolded ER stress is irremediable and chronic activation of the UPR eventually activates apoptosis in some cells. Here we analyzed the IRE1-dependent activation of genes in response to misfolded proinsulin production in an inducible mutant proinsulin (C96Y) insulinoma cell line.
The IRE1 endoribonuclease inhibitors 4μ8c and MKC-3946 prevented the splicing of the XBP1 mRNA in response to ER stress caused by mutant proinsulin production. Microarray expression analysis and qPCR validation of select genes revealed that maximal upregulation of many UPR genes in response to mutant proinsulin production required IRE1, although most were still increased above control. Interestingly, neither degradation of misfolded proinsulin via ER-associated degradation (ERAD), nor apoptosis induced by prolonged misfolded proinsulin expression were affected by inhibiting IRE1.
Although maximal induction of most UPR genes requires IRE1, inhibition of IRE1 does not affect ERAD of misfolded proinsulin or predispose pancreatic β-cells expressing misfolded proinsulin to chronic ER stress-induced apoptosis.
Accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER) leads to the activation of the unfolded protein response (UPR) that serves to counteract this situation by transiently attenuating protein translation, followed by induction of a transcriptional response that increases the levels of genes involved in ER and secretory pathway function . The UPR is an adaptive program that in metazoans is mediated by three ER stress response sensors, PERK, IRE1 and ATF6. These are ER-localized transmembrane proteins that sense the accumulation of misfolded proteins in the ER and initiate signal transduction cascades that mediate the output of the UPR. The PERK pathway reduces global translation via phosphorylation of eIF2α , that in turn enhances translation of the ATF4 transcription factor . IRE1 activation in response to ER stress leads to the splicing of the XBP1 mRNA and translation of the XBP1 transcription factor in mammalian cells [4, 5], while ATF6 is an ER-localized protein that is activated by regulated intramembrane proteolysis in the Golgi to release an active transcription factor . Each of these transcription factors regulates genes involved in the UPR, although there is overlap in the genes controlled by these proteins. Furthermore, there is wide variability in the expression and relative abundance of various ER chaperone and co-chaperone proteins in different eukaryotic cells , likely due to the nature of the protein products produced by different cell types. Thus, highly specialized cells such as insulin-secreting pancreatic β-cells have a unique chaperone expression profile compared to other cell types and likely have a unique UPR output .
In addition to the cell survival output of the UPR, if ER stress remains persistent and these pathways remain active for prolonged periods then apoptosis can be initiated that involves a number of potential pathways, including prolonged expression of pro-apoptotic transcription factors such as CHOP, JNK stress kinase activation, and the IRE1-dependent degradation (or RIDD) activity of IRE1 that non-selectively degrades mRNAs in the vicinity of the ER membrane [8–13].
ER stress has been implicated in contributing to pancreatic β-cell dysfunction and death resulting in the development of diabetes. This is evident in rodents and human patients with certain mutations in the insulin gene that cause misfolding of proinsulin in the ER [14, 15] and in rodents and patients with mutations in the PERK gene [16, 17]. ER stress has also been implicated in contributing to pancreatic β-cell dysfunction in more common forms of diabetes associated with obesity. Several studies have reported increased ER stress markers in pancreatic islets in rodent models of obesity and diabetes and in humans with type 2 diabetes [18–21]. Furthermore, we recently showed that enhanced chaperone capacity in pancreatic β-cells can improve β-cell function and protect C57Bl/6 mice from developing glucose intolerance in response to a high fat diet . Thus, understanding how pancreatic β-cells respond to ER stress may prove beneficial in developing strategies to improve cell function and survival as potential treatment options for the disease.
To elucidate the UPR in pancreatic β-cells we recently identified gene expression changes resulting from the expression of a mutant proinsulin in an insulinoma cell culture model . Expression of the Akita mutant insulin 2 (C96Y) resulted in induction of various genes involved in ER and secretory pathway function. Furthermore, prolonged expression of the misfolded proinsulin also leads to detection of cell apoptosis in the population . Here we have taken advantage of recently described inhibitors of IRE1 endoribonuclease activity [24, 25] to analyse the role of the IRE1/XBP1 pathway in the UPR in this cell line and the effect on ER stress-induced apoptosis. We find that the IRE1 pathway is required for maximal induction of most UPR target genes, but unexpectedly does not sensitize the cells against chronic ER stress-induced apoptosis.
Rat INS-1 insulinoma cells were obtained from Dr. Claus Wollheim (University of Geneva) . INS1 832/13 insulinoma cells were obtained from Dr. Chris Newgard (Duke University) . INS-1 (Insulin 2 C96Y-GFP) cells (clone #4S2) were generated as described . These cell lines were maintained as described in the respective references.
INS-1 (Insulin 2 C96Y-GFP) cells (clone #4S2) were treated with or without Dox (2 μg/ml), Dox with 4μ8c (5 μM), or 4μ8c (5 μM) alone for 48 h. Two independent experiments were performed 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 Microarray Centre as described previously .
Genes with multiple probesets were averaged to produce a single fold change value for each gene. Fold change values for both Dox/Untreated and Dox4μ8c/Untreated were log2 transformed. These were then plotted. All analysis was done in R (http://www.r-project.org/).
RNA isolation and real-time PCR analysis
Total RNA was isolated from rat INS-1 (Insulin 2 C96Y-GFP) cells or mouse islets using TRIzol (Invitrogen) and real-time PCR analysis was performed using the TaqMan Gene Expression system (Life Technologies) as described previously . Gene-specific primers and control β-actin primers were obtained from Life Technologies: Trib3: Rn00595314_m1; HERP: Rn00585371_m1; SDF2L1: Rn01404682_m1; DNAJB9: Rn00562259_m1; GRP78/BiP: Rn01435771_g1; CHOP: Rn00492098_g1; EDEM1: Rn01421307_m1; TXNIP: Rn01533891_g1. The XBP1 splicing assay was performed as described previously .
Cell apoptosis assay
Cell apoptosis was measured using the cell death detection ELISA kit (Roche) according to the instructions provided in the kit and in reference . The ELISA assay detects oligonucleosomes in the cytosol, as an indicator of apoptotic cells.
MTS cell viability assay
INS-1 (Insulin 2 C96Y-GFP) cells (clone #4S2) cells were either left untreated or treated with 2 μg/ml doxycycline, 2 μg/ml doxycycline and 5 μM 4μ8C or 5 μM 4μ8C alone. After 48 h 50,000 cells/100 μl of media from each treatment well were seeded into a 96-well plate in duplicates. The CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay MTS (Promega, #G5421) was performed according to the instructions provided in the kit. Briefly, 20 μl of the combined PMS/MTS mixture was added to each well and incubated for 4 h at 37°C and 5% CO2. The absorbance at 490 nm was then measured with a plate reader.
Western blot analysis
Proteins were resolved using 10% SDS-PAGE gels or 4-12% NuPAGE gels (Invitrogen) and transferred to nitrocellulose membranes as described in . Antibodies: γ-tubulin, Sigma-Aldrich (T6557); GM130, BD Biosciences (G65120); GFP, Clontech, (632381); KDEL, StressGen, (SPA-827); Insulin, Santa Cruz Biotech. (SC-9168); cleaved caspase 3, Cell Signaling, (9661); Phospho-eIF2α, Cell Signaling, (9721); Herp (provided by Dr. Linda Hendershot, St. Jude Children’s Hospital, Memphis, TN).
In this study we examined the effect of IRE1 pathway inhibition on the UPR in a cell culture model of ER stress caused by expression of a misfolded mutant proinsulin. We found that inhibition of IRE1 endoribonuclease activity using selective inhibitors resulted in a generally blunted gene expression output, although no effect was observed on the kinetics of mutant proinsulin degradation, nor the sensitivity of the cells to apoptosis.
IRE1/XBP-1 has been shown to regulate a variety of genes in various cell types in response to ER stress, mostly related to ER function and the secretory pathway, although the target genes vary depending on the cell type and nature of the stress stimuli . In the proinsulin C96Y-GFP model of ER stress numerous genes related to ER function, the secretory pathway and ER-associated degradation are increased. Here we show that some genes such as GRP78 are completely IRE-1 independent, which is consistent with GRP78 not requiring XBP-1 for its induction . However, most other genes induced require IRE1 at least for maximal induction in response to mutant proinsulin-induced ER stress.
Previously we showed that perturbation of the ERAD pathway either by Herp knock-down or proteasome inhibition significantly perturbs mutant proinsulin degradation and significantly enhances susceptibility to apoptosis . Although the extent of the increase in gene expression was reduced for most genes in the presence of the inhibitor, genes such as those coding for ERAD components are still increased. This may explain the lack of effect of the inhibitor on the degradation of the mutant proinsulin and indicates that IRE1 output is not essential for maintaining ERAD capacity.
Perhaps not surprisingly then, the inhibitor did not increase susceptibility to apoptosis caused by mutant proinsulin expression. Several possibilities could contribute to a lack of effect on cell apoptosis, including reduced RIDD activity in response to chronic stress caused by the misfolded proinsulin, in addition to less induction of some pro-apoptotic genes such as Trb3  and TxNIP [8, 9]. Combined with no compromise in ERAD or ability to induce the main ER chaperone BiP/GRP78, cells are no worse off if the IRE1 pathway is inhibited in the context of chronic ER stress caused by mutant proinsulin expression. Our results are consistent with the effect of the inhibitor in other secretory cells where inhibition of IRE1 reduced expansion of secretory capacity, but did not sensitize the cells to ER stress .
IRE1 activation results in the production of the XBP1 transcription factor that in vivo is required for the development of various secretory cells including pancreatic cells [34–36]. Indeed, disruption of the XBP1 gene in pancreatic β-cells in mice using the RIP-Cre system resulted in hyperglycemia and abnormal β-cell function caused by decreased insulin secretion, decreased insulin granule content and impaired insulin processing . In addition, depletion of XBP1 resulted in constitutive hyperactivation of IRE1 including its RIDD activity . Thus, although inhibition of IRE1 in the context of the Akita insulin mutation does not sensitize the cells to increased apoptosis, it is possible that inhibition of IRE1 in vivo in a physiological context might be detrimental to pancreatic β-cell survival.
In summary, although inhibition of IRE1 compromised the full extent of UPR output in response to chronic ER stress caused by misfolded proinsulin expression, inhibition of IRE1 did not significantly affect ERAD or sensitize the cells to apoptosis. Future studies need to examine the effect of IRE1 inhibition in Akita mice and other more common models of rodent diabetes to determine whether targeting the IRE1 pathway could be of benefit to reducing pancreatic cell death caused by chronic ER stress.
Availability of supporting data
All supporting data are included as additional files. Microarray data is deposited in the GEO repository, accession number GSE58866.
We thank Dr. David Ron and Dr. Heather Harding from Cambridge University for providing the 4μ8c inhibitor and comments on the manuscript. We thank Dr. John Patterson from MannKind Corporation for providing the MKC-3946 inhibitor. AV is a recipient of a Canada Research Chair in Diabetes Research. The study was funded by operating grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) (326823–2009) and the Canadian Institutes for Health Research (MOP-114922).
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