Stewart Claire, Estrada Andrea, Kim Paul, Wang Dong, Wei Yuren, Gentile Chris, Pagliassotti Michael
Colorado State University, Fort Collins, Colorado, United States.
Endoplasmic Reticulum Stress Dis. 2017 Apr;4(1):1-10. doi: 10.1515/ersc-2017-0001. Epub 2017 Apr 27.
The unfolded protein response (UPR) is activated in response to impairments of the folding environment in the endoplasmic reticulum (ER). The most conserved arm of the UPR, inositol-requiring ER-to-nucleus signaling protein (IRE1α), has been linked to the regulation of a diverse array of cellular processes including ER-associated degradation, inflammatory signaling, cell proliferation and membrane biogenesis. Recent studies have utilized the selective, small molecule inhibitor, 4μ8c, to examine the role of IRE1α endoribonuclease (RNase) activity in various cell types including multiple myeloma, mouse embryonic fibroblasts and pancreatic beta cells [1-5]. In the present study we utilized this inhibitor to examine the role of IRE1α RNase activity in hepatoma cells (H4IIE), specifically concentrating on cell proliferation and the identification of potential off target effects under both unstressed and stressed conditions. Experiments were performed in H4IIE hepatoma cells in the absence (control conditions (LG)) or presence (LG + Thapsigargin (Thap)) of ER stress. The presence of 4μ8c decreased IRE1α RNase activity, based on reduced splicing of X-box binding protein-1 (XBP1s) and regulated IRE1α-dependent decay of mRNA in both treatments and at concentrations ranging from 10-90 μM. Cell proliferation was significantly reduced at higher concentrations (> 60 μM 4μ8c) in unstressed cells and displayed a dose-response relationship with 4μ8c in both treatments. The presence of 4μ8c did not promote cytoxicity in either of the treatment conditions but higher concentrations of the inhibitor (60 μM) were associated with apparent off-target or compensatory responses that were not observed at 10 μM. Overall, the small-molecule inhibitor, 4μ8c is an effective inhibitor of IRE1α RNase activity in H4IIE cells. Potential off-target effects associated with this inhibitor require the use of multiple inhibitor concentrations in all experiments.
未折叠蛋白反应（UPR）在内质网（ER）折叠环境受损时被激活。UPR中最保守的分支，肌醇需求性内质网到细胞核信号蛋白（IRE1α），已与多种细胞过程的调节相关联，包括内质网相关降解、炎症信号传导、细胞增殖和膜生物合成。最近的研究利用选择性小分子抑制剂4μ8c，来研究IRE1α核糖核酸酶（RNase）活性在多种细胞类型中的作用，包括多发性骨髓瘤、小鼠胚胎成纤维细胞和胰腺β细胞[1-5]。在本研究中，我们利用这种抑制剂来研究IRE1α RNase活性在肝癌细胞（H4IIE）中的作用，特别关注细胞增殖以及在未应激和应激条件下潜在的脱靶效应的鉴定。实验在无内质网应激（对照条件（LG））或有内质网应激（LG + 毒胡萝卜素（Thap））的H4IIE肝癌细胞中进行。基于X盒结合蛋白1（XBP1s）剪接减少，4μ8c 的存在降低了IRE1α RNase活性，并且在两种处理中以及浓度范围为10 - 90μM时调节了mRNA的IRE1α依赖性降解。在未应激细胞中，较高浓度（> 60μM 的4μ8c）时细胞增殖显著降低，并且在两种处理中均与4μ8c呈现剂量反应关系。在任何一种处理条件下，4μ8c 的存在均未促进细胞毒性，但较高浓度的抑制剂（60μM）与明显的脱靶或补偿反应相关，而在10μM时未观察到这些反应。总体而言，小分子抑制剂4μ8c是H4IIE细胞中IRE1α RNase活性的有效抑制剂。在所有实验中，与该抑制剂相关的潜在脱靶效应需要使用多种抑制剂浓度。
