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

The integrated stress response mediated by eukaryotic translation initiation factor 2 alpha (eIF2 alpha) phosphorylation maintains cellular homeostasis under endoplasmic reticulum (ER) stress. eIF2 alpha 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 gamma-lyase (CSE) and cystathionine beta-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 eIF2 alpha 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 beta-mercaptoethanol medium supplementation that increases cysteine levels and restores GSH synthesis. ATF4(-/-) MEFs showed little or no CSE protein but did express cystathionine beta-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.