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.
KeywordsEndoplasmic reticulum stress ER-associated degradation (ERAD) Proinsulin biosynthesis Pancreatic beta-cells
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).
- Walter P, Ron D: The unfolded protein response: from stress pathway to homeostatic regulation. Science. 2011, 334: 1081-1086.View ArticlePubMedGoogle Scholar
- Harding HP, Zhang Y, Ron D: Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature. 1999, 397: 271-274.View ArticlePubMedGoogle Scholar
- Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D: Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell. 2000, 6: 1099-1108.View ArticlePubMedGoogle Scholar
- Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP, Clark SG, Ron D: IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature. 2002, 415: 92-96.View ArticlePubMedGoogle Scholar
- Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K: XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell. 2001, 107: 881-891.View ArticlePubMedGoogle Scholar
- Haze K, Yoshida H, Yanagi H, Yura T, Mori K: Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endplasmic reticulum stress. Mol Biol Cell. 1999, 10: 3787-3799.PubMed CentralView ArticlePubMedGoogle Scholar
- Powers ET, Morimoto RI, Dillin A, Kelly JW, Balch WE: Biological and chemical approaches to diseases of proteostasis deficiency. Annu Rev Biochem. 2009, 78: 959-991.View ArticlePubMedGoogle Scholar
- Oslowski CM, Hara T, O’Sullivan-Murphy B, Kanekura K, Lu S, Hara M, Ishigaki S, Zhu LJ, Hayashi E, Hui ST, Greiner D, Kaufman RJ, Bortell R, Urano F: Thioredoxin-interacting protein mediates ER stress-induced beta-cell death through initiation of the inflammasome. Cell Metab. 2012, 16: 265-273.PubMed CentralView ArticlePubMedGoogle Scholar
- Lerner AG, Upton JP, Praveen PV, Ghosh R, Nakagawa Y, Igbaria A, Shen S, Nguyen V, Backes BJ, Heiman M, Heintz N, Greengard P, Hui S, Tang Q, Trusina A, Oakes SA, Papa FR: IRE1alpha induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress. Cell Metab. 2012, 16: 250-264.PubMed CentralView ArticlePubMedGoogle Scholar
- Han D, Lerner AG, Vande Walle L, Upton JP, Xu W, Hagen A, Backes BJ, Oakes SA, Papa FR: IRE1alpha kinase activation modes control alternate endoribonuclease outputs to determine divergent cell fates. Cell. 2009, 138: 562-575.PubMed CentralView ArticlePubMedGoogle Scholar
- Zinszner H, Kuroda M, Wang X, Batchvarova N, Lightfoot RT, Remotti H, Stevens JL, Ron D: CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes Dev. 1998, 12: 982-995.PubMed CentralView ArticlePubMedGoogle Scholar
- Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, Harding HP, Ron D: Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science. 2000, 287: 664-666.View ArticlePubMedGoogle Scholar
- Tabas I, Ron D: Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol. 2011, 13: 184-190.PubMed CentralView ArticlePubMedGoogle Scholar
- Stoy J, Steiner DF, Park SY, Ye H, Philipson LH, Bell GI: Clinical and molecular genetics of neonatal diabetes due to mutations in the insulin gene. Rev Endocr Metab Disord. 2010, 11: 205-215.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang J, Takeuchi T, Tanaka S, Kubo S-K, Kayo T, Lu D, Takata K, Koizumi A, Izumi T: A mutation in the insulin 2 gene induces diabetes with severe pancreatic beta-cell dysfunction in the Mody mouse. J Clin Invest. 1999, 103: 27-37.PubMed CentralView ArticlePubMedGoogle Scholar
- Delepine M, Nicolino M, Barrett T, Golamaully M, Lathrop GM, Julier C: EIF2AK3, encoding translation initiation factor 2-alpha kinase 3, is mutated in patients with Wolcott-Rallison syndrome. Nat Genet. 2000, 25: 406-409.View ArticlePubMedGoogle Scholar
- Harding HP, Zeng H, Zhang Y, Jungries R, Chung P, Plesken H, Sabatini DD, Ron D: Diabetes Mellitus and exocrine pancreatic dysfunction in Perk−/− mice reveals a role for translational control in secretory cell survival. Mol Cell. 2001, 7: 1153-1163.View ArticlePubMedGoogle Scholar
- Huang CJ, Haataja L, Gurlo T, Butler AE, Wu X, Soeller WC, Butler PC: Induction of endoplasmic reticulum stress-induced beta-cell apoptosis and accumulation of polyubiquitinated proteins by human islet amyloid polypeptide. Am J Physiol Endocrinol Metab. 2007, 293: E1656-1662.View ArticlePubMedGoogle Scholar
- Laybutt DR, Preston AM, Akerfeldt MC, Kench JG, Busch AK, Biankin AV, Biden TJ: Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes. Diabetologia. 2007, 50: 752-763.View ArticlePubMedGoogle Scholar
- Sachdeva MM, Claiborn KC, Khoo C, Yang J, Groff DN, Mirmira RG, Stoffers DA: Pdx1 (MODY4) regulates pancreatic beta cell susceptibility to ER stress. PNAS. 2009, 106: 19090-19095.PubMed CentralView ArticlePubMedGoogle Scholar
- Yusta B, Baggio LL, Estall JL, Koehler JA, Holland DP, Li H, Pipeleers D, Ling Z, Drucker DJ: GLP-1 receptor activation improves beta cell function and survival following induction of endoplasmic reticulum stress. Cell Metab. 2006, 4: 391-406.View ArticlePubMedGoogle Scholar
- Teodoro-Morrison T, Schuiki I, Zhang L, Belsham DD, Volchuk A: GRP78 overproduction in pancreatic beta cells protects against high fat diet-induced diabetes in mice. Diabetologia. 2013, 56: 1057-1067.View ArticlePubMedGoogle Scholar
- Hartley T, Siva M, Lai E, Teodoro T, Zhang L, Volchuk A: Endoplasmic reticulum stress response in an INS-1 pancreatic beta-cell line with inducible expression of a folding-deficient proinsulin. BMC Cell Biol. 2010, 11: 59-PubMed CentralView ArticlePubMedGoogle Scholar
- Cross BCS, Bond PJ, Sadowski PG, Jha BK, Zak J, Goodman JM, Silverman RH, Neubert TA, Baxendale IR, Ron D, Harding HP: The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule. PNAS. 2012, 109: E869-E878.PubMed CentralView ArticlePubMedGoogle Scholar
- Mimura N, Fulciniti M, Gorgun G, Tai Y-T, Cirstea D, Santo L, Hu Y, Fabre C, Minami J, Ohguchi H, Kiziltepe T, Ikeda H, Kawano Y, French M, Blumenthal M, Tam V, Kertesz NL, Malyankar UM, Hokenson M, Pham T, Zeng Q, Patterson JB, Richardson PG, Munshi NC, Anderson KC: Blockade of XBP1 splicing by inhibition of IRE1alpha is a promising therapeutic option in multiple myeloma. Blood. 2012, 119: 5772-5781.PubMed CentralView ArticlePubMedGoogle Scholar
- Asfari M, Janjic D, Meda P, Li G, Halban PA, Wollheim CB: Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology. 1992, 130: 167-178.PubMedGoogle Scholar
- Hohmeier HE, Mulder H, Chen G, Henkel-Rieger R, Prentki M, Newgard CB: Isolation of INS-1 derived cell lines with robust ATP-sensitive K + channel-dependent and -independent glucose-stimulated insulin secretion. Diabetes. 2000, 49: 424-430.View ArticlePubMedGoogle Scholar
- Zhang L, Lai E, Teodoro T, Volchuk A: GRP78, but not protein-disulfide isomerase, partially reverses hyperglycemia-induced inhibition of insulin synthesis and secretion in pancreatic beta-cells. J Biol Chem. 2009, 284: 5289-5298.View ArticlePubMedGoogle Scholar
- Guerriero CJ, Brodsky JL: The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology. Physiol Rev. 2012, 92: 537-576.PubMed CentralView ArticlePubMedGoogle Scholar
- Chou T-F, Brown SJ, Minond D, Nordin BE, Li K, Jones AC, Chase P, Porubsky PR, Stoltz BM, Schoenen FJ, Patricelli MP, Hodder P, Rosen H, Deshaies RJ: Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways. PNAS. 2011, 108: 4834-4839.PubMed CentralView ArticlePubMedGoogle Scholar
- Hetz C, Martinon F, Rodriguez D, Glimcher LH: The unfolded protein response: integrating stress signals through the stress sensor IRE1alpha. Physiol Rev. 2011, 91: 1219-1243.View ArticlePubMedGoogle Scholar
- Lee A-H, Iwakoshi NN, Glimcher LH: XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol. 2003, 23: 7448-7459.PubMed CentralView ArticlePubMedGoogle Scholar
- Prudente S, Sesti G, Pandolfi A, Andreozzi F, Consoli A, Trischitta V: The mammalian tribbles homolg TRIB3, glucose homeostasis, and cardiovascular diseases. Endocrine Rev. 2012, 33: 526-546.View ArticleGoogle Scholar
- Iwakoshi NN, Lee AH, Vallabhajosyula P, Otipoby KL, Rajewsky K, Glimcher LH: Plasma cell differentiation and the unfolded protein response intersect at the transcription factor XBP-1. Nat Immunol. 2003, 4: 321-329.View ArticlePubMedGoogle Scholar
- Lee AH, Chu GC, Iwakoshi NN, Glimcher LH: XBP-1 is required for biogenesis of cellular secretory machinery of exocrine glands. EMBO J. 2005, 24: 4368-4380.PubMed CentralView ArticlePubMedGoogle Scholar
- Iwakoshi NN, Pypaert M, Glimcher LH: The transcription factor XBP-1 is essential for the development and survival of dendritic cells. J Exp Med. 2007, 204: 2267-2275.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee A-H, Heidtman K, Hotamisligil GS, Glimcher LH: Dual and opposing roles of the unfolded protein response regulated by IRE1alpha and XBP1 in proinsulin processing and insulin secretion. PNAS. 2011, 108: 8885-8890.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.