Cell death induced by the ER stressor thapsigargin involves death receptor 5, a non-autophagic function of MAP1LC3B, and distinct contributions from unfolded protein response components

Paula Lindner,Nikolai Engedal,Jesper Vuust Møller,Poul Nissen,Søren Brøgger Christensen

doi:10.1186/s12964-019-0499-z

Abstract

BackgroundCell death triggered by unmitigated endoplasmic reticulum (ER) stress plays an important role in physiology and disease, but the death-inducing signaling mechanisms are incompletely understood. To gain more insight into these mechanisms, the ER stressor thapsigargin (Tg) is an instrumental experimental tool. Additionally, Tg forms the basis for analog prodrugs designed for cell killing in targeted cancer therapy. Tg induces apoptosis via the unfolded protein response (UPR), but how apoptosis is initiated, and how individual effects of the various UPR components are integrated, is unclear. Furthermore, the role of autophagy and autophagy-related (ATG) proteins remains elusive.MethodsTo systematically address these key questions, we analyzed the effects of Tg and therapeutically relevant Tg analogs in two human cancer cell lines of different origin (LNCaP prostate- and HCT116 colon cancer cells), using RNAi and inhibitory drugs to target death receptors, UPR components and ATG proteins, in combination with measurements of cell death by fluorescence imaging and propidium iodide staining, as well as real-time RT-PCR and western blotting to monitor caspase activity, expression of ATG proteins, UPR components, and downstream ER stress signaling.ResultsIn both cell lines, Tg-induced cell death depended on death receptor 5 and caspase-8. Optimal cytotoxicity involved a non-autophagic function of MAP1LC3B upstream of procaspase-8 cleavage. PERK, ATF4 and CHOP were required for Tg-induced cell death, but surprisingly acted in parallel rather than as a linear pathway; ATF4 and CHOP were independently required for Tg-mediated upregulation of death receptor 5 and MAP1LC3B proteins, whereas PERK acted via other pathways. Interestingly, IRE1 contributed to Tg-induced cell death in a cell type-specific manner. This was linked to an XBP1-dependent activation of c-Jun N-terminal kinase, which was pro-apoptotic in LNCaP but not HCT116 cells. Molecular requirements for cell death induction by therapy-relevant Tg analogs were identical to those observed with Tg.ConclusionsTogether, our results provide a new, integrated understanding of UPR signaling mechanisms and downstream mediators that induce cell death upon Tg-triggered, unmitigated ER stress.1yG1LZYrokkFLLYyz3UJEsVideo Graphical abstract

Highlights

Cell death triggered by unmitigated endoplasmic reticulum (ER) stress plays an important role in physiology and disease, but the death-inducing signaling mechanisms are incompletely understood
death receptor 5 (DR5) and caspase-8 are main initiators of Tg-induced cell death in LNCaP and HCT116 cells ER stress-induced apoptosis has been linked to increased expression of DR5 and activation of caspase-8 in multiple cell types and conditions [9, 13, 17,18,19,20,21,22,23,24], but may involve death receptor 1 (DR1) (TNFR1) [26], death receptor 2 (DR2) (Fas) [27], and death receptor 4 (DR4) (TRAIL-R1) [19, 20]
To elucidate the putative involvement of different death receptors in Tg-induced cell death in LNCaP and HCT116 cells, we knocked down DR4, DR5 or Fas, or the adaptor proteins FADD and TRADD, which are crucial for cell death induction by death receptors 2, 4, and 5 (FADD) or death receptors 1, 3, and 6 (TRADD), and assessed the effects on Tg-induced cell death

Summary

Introduction

Cell death triggered by unmitigated endoplasmic reticulum (ER) stress plays an important role in physiology and disease, but the death-inducing signaling mechanisms are incompletely understood. Cell death induced by unresolved ER stress is implicated in a growing list of pathophysiological conditions, including neurological and cardiovascular diseases, ophthalmology disorders, viral infections, and diabetes [2,3,4]. In many of these cases, the pathophysiology is linked to decreased levels of ER Ca2+ [3], which leads to ER stress and UPR due to the dependency of the ER protein folding machinery on ER lumenal Ca2+ [5, 6]. If we can understand the molecular mechanisms that initiate cell death under such conditions, it can lay the ground for the development of novel treatment modalities for various diseases

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