United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
Graduate School of Natural Science and Technology, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
Mol Cell Biochem. 2020 Feb;465(1-2):53-64. doi: 10.1007/s11010-019-03666-w. Epub 2019 Dec 13.
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.
IRE1 是最保守的内质网（ER）驻留应激传感器。其激活不仅剪接 XBP1，还参与多种细胞信号转导。我们通过建立 IRE1α 缺陷细胞并应用四种 IRE1 抑制剂，阐明了 IRE1α 在 Neuro2a 细胞中的作用。用 thapsigargin（Tg）和衣霉素（Tm）处理时，IRE1α 缺陷几乎阻止了所有剪接的 XBP1（sXBP1）蛋白的表达；这些现象与我们的两种基于 Nanoluciferase 的 IRE1 测定值相吻合。然而，IRE1α 缺陷细胞的细胞活力和其他 ER 应激反应因子的蛋白表达与 Tm 处理前后的亲本野生型细胞相当。接下来，我们阐明了四种化合物：STF083010、KIRA6、4μ8C 和 toyocamycin 的 IRE1 抑制作用和细胞毒性。KIRA6 以剂量依赖性方式减弱 IRE1 活性，但即使在低浓度下，在 IRE1α 缺陷细胞中也表现出严重的细胞毒性。在存在和不存在 ER 应激的情况下，IRE1α 缺陷细胞对 0.1μM 的 KIRA6 略有抗性；然而，在 0.02μM 时没有观察到抗性。仅用 0.1μM 的 KIRA6 处理 12 小时会显著诱导两种亲本和 IRE1α 缺陷细胞中的自噬标记物 LC3 II。KIRA6 和 Tm 的共同处理诱导 LC3 II、裂解的 caspase-9 和裂解的 caspase-3；然而，IRE1α 缺陷并没有消除这两种裂解 caspase 的表达。另一方面，KIRA6 以 IRE1 非依赖性方式阻止 Tm 诱导的 ATF4 诱导；然而，KIRA6 和 Tm 的共同处理也诱导了 ATF4 缺陷的 Neuro2a 细胞中的 LC3 II 和两种裂解的 caspase。因此，我们证明 IRE1α 缺陷对 Neuro2a 细胞的细胞活力和 ER 应激反应因子的表达几乎没有影响，并且 KIRA6 的药理作用包括 IRE1 非依赖性途径。
