25-Hydroxycholesterol Activates the Integrated Stress Response to Reprogram Transcription and Translation in Macrophages

Norihito Shibata,Casey E Romanoski,Douglas R Cavener,Michael T Lam,Kaoru Saijo,Minna U Kaikkonen,Aaron F Carlin,Donna Reichart,Edward A Dennis,Robert C Murphy,Nathanael J Spann,Jeffrey G Mcdonald,Shankar Subramaniam,Eoin Fahy,Christopher K Glass,Christopher S Morello,Alfred H Merrill,H Alex Brown,Deborah H Spector,Jesse N Fox,Andrea Crotti,Oswald Quehenberger,M Cameron Sullards,David W Russell,Mano Ram Maurya ,Christian R H Raetz

doi:10.1074/jbc.m113.519637

Abstract

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

Highlights

Interferons and viral infections stimulate the production of 25-hydroxycholesterol
A, Gene Ontology (GO) terms significantly enriched in up-regulated genes in bone marrow-derived macrophages (BMDMs) treated with 25OHC
A–C, protein levels of eIF2␣, phosphorylated eIF2␣ (p-eIF2␣), and CHOP in BMDMs treated with 25OHC (5 ␮M), DMSO, or indicated oxysterols (5 ␮M) for 24 h, or for the indicated times

Summary

Results

25-Hydroxycholesterol significantly alters cholesterol ester and sphingolipid levels and activates the integrated stress response. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/ eIF2␣/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity. Phosphorylation of eIF2␣ rapidly suppresses initiation of protein translation, but paradoxically there is increased production of the transcription factor ATF4 causing induction of certain stress-related genes [15, 16]. We found that 25OHC treatment significantly altered specific subsets of lipids and activated a robust integrated stress response that reprogrammed macrophage transcription and translation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION