Insulin-induced genes INSIG1 and INSIG2 mediate oxysterol-dependent activation of the PERK–eIF2α–ATF4 axis

Yuichi Watanabe,Takashi Sasaki,Shoko Miyoshi,Makoto Shimizu,Yoshio Yamauchi,Ryuichiro Sato

doi:10.1016/j.jbc.2021.100989

Abstract

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.

Highlights

Severe acute respiratory syndrome coronavirus 2 infection [6, 7]
When parental Chinese hamster ovary (CHO)-7 and SRD-15 cells were incubated with an endoplasmic reticulum (ER) stress inducer, thapsigargin, a highly increased level of activating transcription factor-4 (ATF4) protein was observed in both cells, suggesting that deficiency of insulininduced gene (INSIG) has no effects on protein kinase RNA-activated–like ER kinase (PERK)–ATF4 pathway (Fig. 1B and Fig. S1A)
These cells were incubated with seven types of oxysterols (Fig. 1A) with higher or lower INSIG-binding capacity for 24 h to clarify the need for INSIG to bind to oxysterols in order to elevate ATF4 protein

Summary

Introduction

Severe acute respiratory syndrome coronavirus 2 infection [6, 7]. certain oxysterols have been reported as stressors that induce cytotoxicity or cell death and are implicated in various diseases [8,9,10,11]. When parental CHO-7 and SRD-15 cells were incubated with an ER stress inducer, thapsigargin, a highly increased level of ATF4 protein was observed in both cells, suggesting that deficiency of INSIGs has no effects on PERK–ATF4 pathway (Fig. 1B and Fig. S1A).

Results

Conclusion