Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor.

S M Son,I Mook-Jung,S J Kim,J Byun,S-E Roh

doi:10.1038/cddis.2014.129

Abstract

The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca2+. Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer's and Parkinson diseases. One key regulator that underlies cell survival and Ca2+ homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca2+ dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca2+ concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca2+ through the InsP3 receptor (InsP3R). The Ca2+ efflux in IRE1α-deficient cells correlates with dissociation of the Ca2+-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α–TRAF2–ASK1 complex. The increased cytosolic concentration of Ca2+ induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca2+ dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca2+ influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca2+ homeostasis and cell survival during ER stress and reveal a previously unknown Ca2+-mediated cell death signaling between the IRE1α–InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.

Highlights

The endoplasmic reticulum (ER) is an intracellular organelle responsible for protein synthesis and quality control and serves as a Ca2 þ store to maintain intracellular Ca2 þ levels.[1,2] Most integral membrane proteins and secretory proteins are synthesized at the ER, where they fold and, when necessary, become covalently modified and assembled into high-quality, functional proteins
After SH-SY5Y cells were transfected with IRE1aspecific siRNA for 48 h, total inositol-requiring enzyme 1a (IRE1a) levels were reduced by 40–60% versus control siRNA-transfected cells, without changes in b-actin expression (Figures 1a–c and Supplementary Figures S1a and b)
As IRE1a is localized in the ER membrane[26] and the ER structure undergoes dramatic changes upon cellular damage,[27,28] we examined ER morphology under IRE1a KD

Summary

Introduction

The endoplasmic reticulum (ER) is an intracellular organelle responsible for protein synthesis and quality control and serves as a Ca2 þ store to maintain intracellular Ca2 þ levels.[1,2] Most integral membrane proteins and secretory proteins are synthesized at the ER, where they fold and, when necessary, become covalently modified and assembled into high-quality, functional proteins. MRNA encodes a transcriptional activator that induces the transcription of chaperone protein-encoding genes, whose products have a role in ER protein folding.[7] Under prolonged stress, IRE1a interacts with TNF receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1. (ASK1) or activates caspase-12, an ER resident caspase, to cause cell death in neuronal cells.[8,9] PERK is a transmembrane kinase that phosphorylates eukaryotic translation initiation factor 2 subunit alpha (eIF2a), thereby reducing protein synthesis and counteracting ER protein overload.[10]. EIF2a phosphorylation allows selective translation of certain mRNA molecules that contain small open reading frames in their 50 untranslated regions, which in turn leads to the production of transcriptional activators, such as ATF4.11. ATF6 is a membrane-bound transcription factor that drives transcription in the ER stress response.

Methods

Results

Conclusion