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.
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Activating transcription factor 4
Activating transcription factor 6
Growth arrest and DNA damage inducible gene 153
78 kDa glucose-regulated protein
94 kDa glucose-regulated protein
Glyceraldehyde 3-phosphate dehydrogenase
PKR-like endoplasmic reticulum kinase
Regulated IRE1-dependent mRNA decay
X-box binding protein 1
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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.
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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
- ER stress