Food Biochemistry Laboratory, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan.
Nutri-Life Science Laboratory, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan.
J Biol Chem. 2021 Aug;297(2):100989. doi: 10.1016/j.jbc.2021.100989. Epub 2021 Jul 21.
Insulin-induced genes (INSIGs) encode endoplasmic reticulum-resident proteins that regulate intracellular cholesterol metabolism. Oxysterols are oxygenated derivatives of cholesterol, some of which orchestrate lipid metabolism via interaction with INSIGs. Recently, it was reported that expression of activating transcription factor-4 (ATF4) was induced by certain oxysterols; the precise of mechanism is unclear. Herein, we show that INSIGs mediate ATF4 upregulation upon interaction with oxysterol. Oxysterols that possess a high affinity for INSIG, such as 27- and 25-hydroxycholesterol (25HC), markedly induced the increase of ATF4 protein when compared with other oxysterols. In addition, ATF4 upregulation by these oxysterols was attenuated in INSIG1/2-deficient Chinese hamster ovary cells and recovered by either INSIG1 or INSIG2 rescue. Mechanistic studies revealed that the binding of 25HC to INSIG is critical for increased ATF4 protein via activation of protein kinase RNA-activated-like ER kinase and eukaryotic translation initiation factor 2α. Knockout of INSIG1 or INSIG2 in human hepatoma Huh7 cells attenuated ATF4 protein upregulation, indicating that only one of the endogenous INSIGs, unlike overexpression of intrinsic INSIG1 or INSIG2, was insufficient for ATF4 induction. Furthermore, ATF4 proactively upregulated the cell death-inducible gene expression, such as Chop, Chac1, and Trb3, thereby markedly reducing cell viability with 25HC. These findings support a model whereby that INSIGs sense an increase in oxysterol in the endoplasmic reticulum and induce an increase of ATF4 protein via the protein kinase RNA-activated-like ER kinase-eukaryotic translation initiation factor 2α pathway, thereby promoting cell death.
胰岛素诱导基因（INSIGs）编码内质网驻留蛋白，调节细胞内胆固醇代谢。氧化固醇是胆固醇的含氧衍生物，其中一些通过与 INSIGs 的相互作用来协调脂质代谢。最近，有报道称某些氧化固醇诱导激活转录因子-4（ATF4）的表达；确切的机制尚不清楚。本文表明，INSIGs 通过与氧化固醇相互作用介导 ATF4 的上调。与其他氧化固醇相比，具有与 INSIG 高亲和力的氧化固醇，如 27- 和 25-羟胆固醇（25HC），明显诱导 ATF4 蛋白的增加。此外，在 INSIG1/2 缺陷型中国仓鼠卵巢细胞中，这些氧化固醇对 ATF4 的上调作用减弱，而 INSIG1 或 INSIG2 的恢复则恢复了这种上调作用。机制研究表明，25HC 与 INSIG 的结合对于通过激活蛋白激酶 RNA 激活样内质网激酶和真核翻译起始因子 2α 增加 ATF4 蛋白是至关重要的。在人肝癌 Huh7 细胞中敲除 INSIG1 或 INSIG2 减弱了 ATF4 蛋白的上调，表明只有内源性 INSIG 之一，与内在 INSIG1 或 INSIG2 的过表达不同，不足以诱导 ATF4。此外，ATF4 主动上调细胞死亡诱导基因的表达，如 Chop、Chac1 和 Trb3，从而在用 25HC 处理时显著降低细胞活力。这些发现支持这样一种模型，即 INSIGs 在内质网中感知氧化固醇的增加，并通过蛋白激酶 RNA 激活样内质网激酶-真核翻译起始因子 2α 途径诱导 ATF4 蛋白的增加，从而促进细胞死亡。
