ABSTRACT

Selective degradation of the endoplasmic reticulum (ER; reticulophagy) is a type of autophagy involved in the removal of ER fragments. So far, amino acid starvation as well as ER stress have been described as inducers of reticulophagy, which in turn restores cellular energy levels and ER homeostasis. Here, we explored the autophagy-inducing mechanisms that underlie the autophagic cell death (ACD)-triggering compound loperamide (LOP) in glioblastoma cells. Interestingly, LOP triggers upregulation of the transcription factor ATF4, which is accompanied by the induction of additional ER stress markers. Notably, knockout of ATF4 significantly attenuated LOP-induced autophagy and ACD. Functionally, LOP also specifically induces the engulfment of large ER fragments within autophagosomes and lysosomes as determined by electron and fluorescence microscopy. LOP-induced reticulophagy and cell death are predominantly mediated through the reticulophagy receptor RETREG1/FAM134B and, to a lesser extent, TEX264, confirming that reticulophagy receptors can promote ACD. Strikingly, apart from triggering LOP-induced autophagy and ACD, ATF4 is also required for LOP-induced reticulophagy. These observations highlight a key role for ATF4, RETREG1 and TEX264 in response to LOP-induced ER stress, reticulophagy and ACD, and establish a novel mechanistic link between ER stress and reticulophagy, with possible implications for additional models of drug-induced ER stress.

Abbreviations: ACD: autophagic cell death; ATF6: activating transcription factor 6; ATL3: atlastin 3; BafA₁: bafilomycin A₁; CCPG1: cell cycle progression gene 1; co-IP: co-immunoprecipitation; DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; GABARAP: GABA type A receptor-associated protein; GBM: glioblastoma multiforme; HSPA5/BiP: heat shock protein family (Hsp70) member 5; LOP: loperamide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; RETREG1/FAM134B: reticulophagy regulator 1; RTN3L: reticulon 3 long; SEC62: SEC62 homolog, protein translocation factor; TEX264: testis-expressed 264, reticulophagy receptor; UPR: unfolded protein response.

摘要

内质网的选择性降解（ER；网状吞噬）是一种参与去除 ER 片段的自噬。到目前为止，氨基酸饥饿和 ER 应激已被描述为网状吞噬的诱导剂，进而恢复细胞能量水平和 ER 稳态。在这里，我们探索了胶质母细胞瘤细胞中自噬细胞死亡 (ACD) 触发化合物洛哌丁胺 (LOP) 的自噬诱导机制。有趣的是，LOP 会触发转录因子 ATF4 的上调，这伴随着额外 ER 应激标记的诱导。值得注意的是，ATF4 的敲除显着减弱了 LOP 诱导的自噬和 ACD。在功能上，LOP 还特异地诱导自噬体和溶酶体中的大 ER 片段被吞噬，这由电子和荧光显微镜确定。LOP 诱导的网状吞噬和细胞死亡主要通过网状吞噬受体 RETREG1/FAM134B 和在较小程度上通过 TEX264 介导，证实了网状吞噬受体可以促进 ACD。引人注目的是，除了触发 LOP 诱导的自噬和 ACD，ATF4 也是 LOP 诱导的网状吞噬所必需的。这些观察结果突出了 ATF4、RETREG1 和 TEX264 在响应 LOP 诱导的 ER 应激、网状吞噬和 ACD 中的关键作用，并在 ER 应激和网状吞噬之间建立了一种新的机制联系，可能对药物诱导的其他模型产生影响 ER 应激。除了触发 LOP 诱导的自噬和 ACD 外，ATF4 也是 LOP 诱导的网状吞噬所必需的。这些观察结果突出了 ATF4、RETREG1 和 TEX264 在响应 LOP 诱导的 ER 应激、网状吞噬和 ACD 中的关键作用，并在 ER 应激和网状吞噬之间建立了一种新的机制联系，可能对药物诱导的其他模型产生影响 ER 应激。除了触发 LOP 诱导的自噬和 ACD 外，ATF4 也是 LOP 诱导的网状吞噬所必需的。这些观察结果突出了 ATF4、RETREG1 和 TEX264 在响应 LOP 诱导的 ER 应激、网状吞噬和 ACD 中的关键作用，并在 ER 应激和网状吞噬之间建立了一种新的机制联系，可能对药物诱导的其他模型产生影响 ER 应激。

缩写：ACD：自噬细胞死亡；ATF6：激活转录因子 6；ATL3：atlastin 3；BafA ₁ : 巴弗洛霉素 A ₁; CCPG1：细胞周期进程基因1；co-IP：免疫共沉淀；DDIT3/CHOP：DNA 损伤诱导转录本 3；ER：内质网；EIF2A/eIF2α：真核翻译起始因子 2A；EIF2AK3/PERK：真核翻译起始因子 2 α 激酶 3；ERN1/IRE1α：内质网到核信号 1；GABARAP：GABA A 型受体相关蛋白；GBM：多形性胶质母细胞瘤；HSPA5/BiP：热休克蛋白家族 (Hsp70) 成员 5；LOP：洛哌丁胺；MAP1LC3/LC3：微管相关蛋白 1 轻链 3；RETREG1/FAM134B：网状吞噬调节剂 1；RTN3L：网状3长；SEC62：SEC62同源物，蛋白质易位因子；TEX264：睾丸表达的 264，网状吞噬受体；UPR：未折叠蛋白反应。