Integrated Stress Response Modulates Cellular Redox State via Induction of Cystathionine γ-Lyase: CROSS-TALK BETWEEN INTEGRATED STRESS RESPONSE AND THIOL METABOLISM

Jeffrey G Dickhout,Rachel E Carlisle,Danielle E Jerome,Zahraa Mohammed-Ali,Hua Jiang,Guangdong Yang,Sarathi Mani,Sanjay K Garg,Ruma Banerjee,Randal J Kaufman,Kenneth N Maclean,Rui Wang,Richard C Austin

doi:10.1074/jbc.m111.304576

Abstract

The integrated stress response mediated by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation maintains cellular homeostasis under endoplasmic reticulum (ER) stress. eIF2α phosphorylation induces activating transcription factor 4 (ATF4), a basic leucine zipper transcription factor that regulates the expression of genes responsible for amino acid metabolism, cellular redox state, and anti-stress responses. Cystathionine γ-lyase (CSE) and cystathionine β-synthase are critical enzymes in the transsulfuration pathway, which also regulate cellular redox status by modulating glutathione (GSH) levels. To determine the link between the integrated stress response and the transsulfuration pathway, we used homocysteine (Hcy) as an inducer of eIF2α phosphorylation and ATF4 gene induction. Mouse embryonic fibroblasts (MEFs) lacking ATF4 (ATF4(-/-)) had reduced GSH levels and increased reactive oxygen species and were susceptible to apoptotic cell death under normal culture conditions. Further, ATF4(-/-) MEFs were more sensitive to Hcy-induced cytotoxicity and showed significantly reduced intracellular GSH levels associated with apoptosis. ATF4(-/-) MEFs could be rescued from l-Hcy-induced apoptosis by β-mercaptoethanol medium supplementation that increases cysteine levels and restores GSH synthesis. ATF4(-/-) MEFs showed little or no CSE protein but did express cystathionine β-synthase. Further, ER stress-inducing agents, including tunicamycin and thapsigargin, induced the expression of CSE in ATF4(+/+) MEFs. Consistent with ATF4(-/-) MEFs, CSE(-/-) MEFs showed significantly greater apoptosis when treated with tunicamycin, thapsigargin, and l-Hcy, compared with CSE(+/+) MEFs. Liver and kidney GSH levels were also reduced in CSE(-/-) mice, suggesting that CSE is a critical factor in GSH synthesis and may act to protect the liver and kidney from a variety of conditions that cause ER stress.

Highlights

The integrated stress response (ISR) maintains cellular homeostasis during aberrant protein folding (ER stress)
The integrated stress response mediated by eukaryotic translation initiation factor 2␣ phosphorylation maintains cellular homeostasis under endoplasmic reticulum (ER) stress. eIF2␣ phosphorylation induces activating transcription factor 4 (ATF4), a basic leucine zipper transcription factor that regulates the expression of genes responsible for amino acid metabolism, cellular redox state, and anti-stress responses
4 The abbreviations used are: Hcy, homocysteine; HHcy, hyperhomocysteinemia; CBS, cystathionine ␤-synthase; Cystathionine ␥-lyase (CSE), cystathionine ␥-lyase; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; LDH, lactate dehydrogenase; Mouse embryonic fibroblasts (MEFs), mouse embryonic fibroblast; NEAA, non-essential amino acids; PERK, protein kinase R-like ER kinase; PPG, propargylglycine; ROS, reactive oxygen species; UPR, unfolded protein response; H2DCF, 2Ј,7Јdichlorofluorescein; ISR, integrated stress response; Tg, thapsigargin; Tm, tunicamycin; ␤-Mer, ␤-mercaptoethanol; xCT, cystine/glutamate antiporter system; DMϩϩ, Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 55 ␮M ␤-Mer and 1ϫ NEAA

Summary

Background

The integrated stress response (ISR) maintains cellular homeostasis during aberrant protein folding (ER stress). 2 Supported by Canadian Institutes of Health Research operating Grant MOP-86708. ER stress-inducing agents, including tunicamycin and thapsigargin, induced the expression of CSE in ATF4؉/؉ MEFs. Consistent with ATF4؊/؊ MEFs, CSE؊/؊ MEFs showed significantly greater apoptosis when treated with tunicamycin, thapsigargin, and L-Hcy, compared with CSE؉/؉ MEFs. Liver and kidney GSH levels were reduced in CSE؊/؊ mice, suggesting that CSE is a critical factor in GSH synthesis and may act to protect the liver and kidney from a variety of conditions that cause ER stress. Elevated levels of homocysteine (Hcy), termed hyperhomocysteinemia (HHcy), are positively associated with coronary heart disease and atherosclerosis [1, 2] This relationship has proven to be dose-dependent, independent of other risk factors, and biologically plausible [1, 2]. Large randomized controlled trials in patients with elevated risk for cardiovascular disease who experienced Hcy lowering through B-vitamin and folic acid therapy showed no

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION