Characterization of IRE1α in Neuro2a cells by pharmacological and CRISPR/Cas9 approaches

  • 128 Accesses


IRE1 is the most conserved endoplasmic reticulum (ER)-resident stress sensor. Its activation not only splices XBP1 but also participates in a variety of cell signaling. We elucidated the role of IRE1α in Neuro2a cells by establishing IRE1α-deficient cells and applying four IRE1 inhibitors. IRE1α deficiency prevented almost all spliced XBP1 (sXBP1) protein expression by treatment with thapsigargin (Tg) and tunicamycin (Tm); these phenomena paralleled the values measured by our two Nanoluciferase-based IRE1 assays. However, cell viability and protein expression of other ER stress-responsive factors in the IRE1α-deficient cells were comparable to those in the parental wild-type cells with or without Tm treatment. Next, we elucidated the IRE1 inhibitory actions and cytotoxicity of four compounds: STF083010, KIRA6, 4μ8C, and toyocamycin. KIRA6 attenuated IRE1 activity in a dose-dependent manner, but it showed severe cytotoxicity even in the IRE1α-deficient cells at a low concentration. The IRE1α-deficient cells were slightly resistant to KIRA6 at 0.1 μM in both the presence and absence of ER stress; however, resistance was not observed at 0.02 μM. Treatment with only KIRA6 at 0.1 μM for 12 h remarkably induced LC3 II, an autophagic marker, in both parental and IRE1α-deficient cells. Co-treatment with KIRA6 and Tm induced LC3 II, cleaved caspase-9, and cleaved caspase-3; however, IRE1α-deficiency did not abolish the expression of these two cleaved caspases. On the other hand, KIRA6 prohibited Tm-induced ATF4 induction in an IRE1-independent manner; however, co-treatment with KIRA6 and Tm also induced LC3 II and two cleaved caspases in the ATF4-deficient Neuro2a cells. Thus, we demonstrate that IRE1α deficiency has little impact on cell viability and expression of ER stress-responsive factors in Neuro2a cells, and the pharmacological actions of KIRA6 include IRE1-independent ways.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6



Activating transcription factor 4


Activating transcription factor 6


Endoplasmic reticulum


Growth arrest and DNA damage inducible gene 153


78 kDa glucose-regulated protein


94 kDa glucose-regulated protein


Glyceraldehyde 3-phosphate dehydrogenase


Inositol-requiring enzyme-1


PKR-like endoplasmic reticulum kinase


Regulated IRE1-dependent mRNA decay


X-box binding protein 1


  1. 1.

    Helenius A, Marquardt T, Braakman I (1992) The endoplasmic reticulum as protein-folding compartment. Trends Cell Biol 2:227–231

  2. 2.

    Gething MJ, Sambrook J (1992) Protein folding in the cell. Nature 355:33–45

  3. 3.

    Morito D, Nagata K (2015) Pathogenic hijacking of ER-associated degradation: is ERAD flexible? Mol Cell 59:335–344

  4. 4.

    Hwang J, Qi L (2018) Quality control in the endoplasmic reticulum: crosstalk between ERAD and UPR pathways. Trends Biochem Sci 43:593–605

  5. 5.

    Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K (2001) XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107:881–891

  6. 6.

    Zhu C, Johansen FE, Prywes R (1997) Interaction of ATF6 and serum response factor. Mol Cell Biol 17:4957–4966

  7. 7.

    Harding HP, Zhang Y, Ron D (1999) Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397:271–274

  8. 8.

    Kratochvílová K, Moráň L, Paďourová S, Stejskal S, Tesařová L, Šimara P, Hampl A, Koutná I, Vaňhara P (2016) The role of the endoplasmic reticulum stress in stemness, pluripotency and development. Eur J Cell Biol 95:115–123

  9. 9.

    Kim I, Xu W, Reed JC (2008) Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov 7:1013–1030

  10. 10.

    Lee AH, Iwakoshi NN, Glimcher LH (2003) XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol 23:7448–7459

  11. 11.

    Okada T, Yoshida H, Akazawa R, Negishi M, Mori K (2002) Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response. Biochem J 366:585–594

  12. 12.

    Lange PS, Chavez JC, Pinto JT, Coppola G, Sun CW, Townes TM, Geschwind DH, Ratan RR (2008) ATF4 is an oxidative stress-inducible, prodeath transcription factor in neurons in vitro and in vivo. J Exp Med 205:1227–1242

  13. 13.

    Ohoka N, Yoshii S, Hattori T, Onozaki K, Hayashi H (2005) TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death. EMBO J 24:1243–1255

  14. 14.

    Tanaka T, Tsujimura T, Takeda K, Sugihara A, Maekawa A, Terada N, Yoshida N, Akira S (1998) Targeted disruption of ATF4 discloses its essential role in the formation of eye lens fibres. Genes Cells 3:801–810

  15. 15.

    Pasini S, Corona C, Liu J, Greene LA, Shelanski ML (2015) Specific downregulation of hippocampal ATF4 reveals a necessary role in synaptic plasticity and memory. Cell Rep 11:183–191

  16. 16.

    Oh-hashi K, Sugiura N, Amaya F, Isobe KI, Hirata Y (2018) Functional validation of ATF4 and GADD34 in Neuro2a cells by CRISPR/Cas9-mediated genome editing. Mol Cell Biochem 440:65–75

  17. 17.

    Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP, Clark SG, Ron D (2002) IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415:92–96

  18. 18.

    Tam AB, Koong AC, Niwa M (2014) Ire1 has distinct catalytic mechanisms for XBP1/HAC1 splicing and RIDD. Cell Rep 9:850–858

  19. 19.

    Nishitoh H, Matsuzawa A, Tobiume K, Saegusa K, Takeda K, Inoue K, Hori S, Kakizuka A, Ichijo H (2002) ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev 16:1345–1355

  20. 20.

    Ghosh R, Wang L, Wang ES, Perera BG, Igbaria A, Morita S, Prado K, Thamsen M, Caswell D, Macias H, Weiberth KF, Gliedt MJ, Alavi MV, Hari SB, Mitra AK, Bhhatarai B, Schürer SC, Snapp EL, Gould DB, German MS, Backes BJ, Maly DJ, Oakes SA, Papa FR (2014) Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress. Cell 158:534–548

  21. 21.

    Papandreou I, Denko NC, Olson M, Van Melckebeke H, Lust S, Tam A, Solow-Cordero DE, Bouley DM, Offner F, Niwa M, Koong AC (2011) Identification of an Ire1alpha endonuclease specific inhibitor with cytotoxic activity against human multiple myeloma. Blood 117:1311–1314

  22. 22.

    Bond PJ, Sadowski PG, Jha BK, Zak J, Goodman JM, Silverman RH, Neubert TA, Baxendale IR, Ron D, Harding HP (2012) The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule. Proc Natl Acad Sci USA 109:E869–E878

  23. 23.

    Ri M, Tashiro E, Oikawa D, Shinjo S, Tokuda M, Yokouchi Y, Narita T, Masaki A, Ito A, Ding J, Kusumoto S, Ishida T, Komatsu H, Shiotsu Y, Ueda R, Iwawaki T, Imoto M, Iida S (2012) Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing. Blood Cancer J 2:e79

  24. 24.

    Esvelt KM, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Church G (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826

  25. 25.

    Norisada J, Fujimura K, Amaya F, Kohno H, Hirata Y, Oh-hashi K (2019) Application of NanoBiT for monitoring dimerization of the null Hong Kong variant of α-1-antitrypsin, NHK, in living cells. Mol Biotech 60:539–549

  26. 26.

    Hikiji T, Norisada J, Hirata Y, Okuda K, Nagasawa H, Ishigaki S, Sobue G, Kiuchi K, Oh-hashi K (2015) A highly sensitive assay of IRE1 activity using the small luciferase NanoLuc: evaluation of ALS-related genetic and pathological factors. Biochem Biophys Res Commun 463:881–887

  27. 27.

    Oh-hashi K, Furuta E, Fujimura K, Hirata Y (2017) Application of a novel HiBiT peptide tag for monitoring ATF4 protein expression in Neuro2a cells. Biochem Biophys Rep 12:40–45

  28. 28.

    Schwinn MK, Machleidt T, Zimmerman K, Eggers CT, Dixon AS, Hurst R, Hall MP, Encell LP, Binkowski BF, Wood KV (2018) CRISPR-mediated tagging of endogenous proteins with a luminescent peptide. ACS Chem Biol 13:467–474

  29. 29.

    Yoshida H, Uemura A, Mori K (2009) pXBP1(U), a negative regulator of the unfolded protein response activator pXBP1(S), targets ATF6 but not ATF4 in proteasome-mediated degradation. Cell Struct Funct 34:1–10

  30. 30.

    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674

  31. 31.

    Fu Y, Lee AS (2006) Glucose regulated proteins in cancer progression, drug resistance and immunotherapy. Cancer Biol Ther 5:741–744

  32. 32.

    Bi M, Naczki C, Koritzinsky M, Fels D, Blais J, Hu N, Harding H, Novoa I, Varia M, Raleigh J, Scheuner D, Kaufman RJ, Bell J, Ron D, Wouters BG, Koumenis C (2005) ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth. EMBO J 24:3470–3481

  33. 33.

    Pyrko P, Schonthal AH, Hofman FM, Chen TC, Lee AS (2007) The unfolded protein response regulator GRP78/BiP as a novel target for increasing chemosensitivity in malignant gliomas. Cancer Res 67:9809–9816

  34. 34.

    Rzymski T, Milani M, Pike L, Buffa F, Mellor HR, Winchester L, Pires I, Hammond E, Ragoussis I, Harris AL (2010) Regulation of autophagy by ATF4 in response to severe hypoxia. Oncogene 29:4424–4435

  35. 35.

    Luhr M, Torgersen ML, Szalai P, Hashim A, Brech A, Staerk J, Engedal N (2019) The kinase PERK and the transcription factor ATF4 play distinct and essential roles in autophagy resulting from tunicamycin-induced ER stress. J Biol Chem 294:8197–8217

  36. 36.

    Mahameed M, Wilhelm T, Darawshi O, Obiedat A, Tommy WS, Chintha C, Schubert T, Samali A, Chevet E, Eriksson LA, Huber M, Tirosh B (2019) The unfolded protein response modulators GSK2606414 and KIRA6 are potent KIT inhibitors. Cell Death Dis 10:300

  37. 37.

    Zou X, Michael B (2017) Targeting p38 MAP kinase signaling in cancer through post-translational modifications. Cancer Lett 384:19–26

  38. 38.

    Abdelatef SA, El-Saadi MT, Amin NH, Abdelazeem AH, Omar HA, Abdellatif KR (2018) Design, synthesis and anticancer evaluation of novel spirobenzo [h] chromene and spirochromane derivatives with dual EGFR and B-RAF inhibitory activities. Eur J Med Chem 150:567–578

Download references


This work is, in part, is supported by Grant-in-aid from the Japan Society for the Promotion of Science (JSPS, Japan, KAKENHI, Nos. 17K19901 and 19H04030 to K.O.). We are grateful to Dr. George Church for providing the hCas9 gene.

Author information

KO and MK discussed and designed the research; KO and HK performed experiments; KO and YH wrote the manuscript.

Correspondence to Kentaro Oh-hashi.

Ethics declarations

Conflict of interest

There was no conflict of interest in this study.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PPTX 287 kb)

Supplementary material 2 (DOCX 83 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Oh-hashi, K., Kohno, H., Kandeel, M. et al. Characterization of IRE1α in Neuro2a cells by pharmacological and CRISPR/Cas9 approaches. Mol Cell Biochem 465, 53–64 (2020) doi:10.1007/s11010-019-03666-w

Download citation


  • ER stress
  • IRE1
  • XBP1