Abstract
The integrated stress response (ISR), a defense mechanism cells employ when under stress (e.g., amino acid deprivation), causes suppression of global protein synthesis along with the paradoxical increased expression of a host of proteins that are useful in combating various stresses. Genes that were similarly differentially expressed under conditions of either leucine- or cysteine-depletion were identified. Many of the genes known to contain an amino acid response element and to be induced in response to eIF2α phosphorylation and ATF4 heterodimer binding (ATF3, C/EBPβ, SLC7A1, SLC7A11, and TRIB3), as well as others shown to be induced downstream of eIF2α phosphorylation (C/EBPγ, CARS, SARS, CLCN3, CBX4, and PPP1R15A) were among the upregulated genes. Evidence for the induction of the ISR in these cells also included the increased phosphorylation of eIF2α and increased protein abundance of ATF4, ATF3, and ASNS in cysteine- and leucine-depleted cells. Based on genes highly differentially expressed in both leucine- and cysteine-deficient cells, a list of 67 downregulated and 53 upregulated genes is suggested as likely targets of essential amino acid deprivation in mammalian cells.
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- ISR:
-
Integrated stress response
- AARE:
-
Amino acid response element
- ATF4:
-
Activating transcription factor 4
- GCN2:
-
General control nonderepressible 2, or eIF2α kinase 4
- ORF:
-
Open reading frame
References
Dang Do AN, Kimball SR, Cavener DR, Jefferson LS (2009) eIF2α kinases GCN2 and PERK modulate transcription and translation of distinct sets of mRNAs in mouse liver. Physiol Genomics 38:328–341
Deval C, Chaveroux C, Maurin AC, Cherasse Y, Parry L, Carraro V, Milenkovic D, Ferrara M, Bruhat A, Jousse C, Fafournoux P (2009) Amino acid limitation regulates the expression of genes involved in several specific biological processes through GCN2-dependent and GCN2-independent pathways. FEBS J 276:707–718
Gabauer F, Hentze M (2004) Molecular mechanisms of translational control. Mol Cell Biol 5:827–834
Guo F, Cavener DR (2007) The GCN2 eIF2α kinase regulates fatty-acid homeostasis in the liver during deprivation of an essential amino acid. Cell Metab 5:103–114
Hao S, Sharp JW, Ross-Inta CM, McDaniel BJ, Anthony TG, Wek RC, Cavener DR, McGrath BC, Rudell JB, Koehnle TJ, Gietzen DW (2005) Uncharged tRNA and sensing of amino acid deficiency in mammalian piriform cortex. Science 307:1776–1778
Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D (2000) Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell 6:1099–1108
Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, Sadri N, Yun C, Popko B, Paules R, Stojdl DF, Bell JC, Hettmann T, Leiden JM, Ron D (2003) An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell 11:619–633
Hinnebusch AG (2000) Mechanism and regulation of initiator methionyl-tRNA binding to ribosomes. In: Sonenberg N, Hershey JWB, Mathews MB (eds) Translational control of gene expression. Cold Spring Harbor Laboratory Press, Plainview, NY, pp 185–243
Hinnebusch AG (2005) Translational regulation of GCN4 and the general amino acidcontrol of yeast. Annu Rev Microbiol 59:407–450
Ishikawa F, Akimoto T, Yamamoto H, Araki Y, Yoshie T, Mori K, Hayashi H, Nose K, Shibanuma M (2009) Gene expression profiling identifies a role for CHOP during inhibition of the mitochondrial respiratory chain. J Biochem 146:123–132
Jousse C, Bruhat A, Ferrara M, Fafournoux P (2000) Evidence for multiple signaling pathways in the regulation of gene expression by amino acids in human cell lines. J Nutr 130:1555–1560
Kimball SR, Antonetti DA, Brawley RM, Jefferson LS (1991) Mechanism of inhibition of peptide chain initiation by amino acid deprivation in perfused rat liver. Regulation involving inhibition of eukaryotic initiation factor 2 α phosphatase activity. J Biol Chem 266:1969–1976
Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, Ezaki J, Mizushima N, Ohsumi Y, Uchiyama Y, Kominami E, Tanaka K, Chiba T (2005) Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol 169:425–434
Kubica N, Jefferson LS, Kimball SR (2006) Eukaryotic initiation factor 2B and its role in alterations in mRNA translation that occur under a number of pathophysiological and physiological conditions. Prog Nucleic Acid Res Mol Biol 81:271–296
Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, Ohsumi Y, Tokuhisa T, Mizushima N (2004) The role of autophagy during the early neonatal starvation period. Nature 432:1032–1036
Lee J-I, Dominy JE Jr, Sikalidis AK, Hirschberger LL, Wang W, Stipanuk MH (2008) HepG2/C3A cells respond to cysteine deprivation by induction of the amino acid deprivation/integrated stress response pathway. Physiol Genomics 33:218–229
Li X, Wang T, Zhao Z, Weinman SA (2002) The ClC-3 chloride channel promotes acidification of lysosomes in CHO-K1 and Huh-7 cells. Am J Physiol Cell Physiol 282:C1483–C1491
Lu SC, Huang HY (1994) Comparison of sulfur amino acid utilization for GSH synthesis between HepG2 cells and cultured rat hepatocytes. Biochem Pharmacol 47:859–869
Lu PD, Harding HP, Ron D (2004a) Translation reinitiation at alternative open reading frames regulate gene expression in an integrated stress response. J Cell Biol 167:27–33
Lu PD, Jousse C, Marciniak SJ, Zhang Y, Novoa I, Scheuner D, Kaufman RJ, Ron D, Harding HP (2004b) Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2. EMBO J 23:169–179
Mungrue IN, Pagnon J, Kohannim O, Gargalovic PS, Lusis AJ (2009) CHAC1/MGC4504 is a novel proapoptotic component of the unfolded protein response, downstream of the ATF4-ATF3-CHOP cascade. J Immunol 182:466–476
Okamoto F, Kajiya H, Toh K, Uchida S, Yoshikawa M, Sasaki S, Kido MA, Tanaka T, Okabe K (2008) Intracellular ClC-3 chloride channels promote bone resorption in vitro through organelle acidification in mouse osteoclasts. Am J Physiol Cell Physiol 294:C693–C701
Padyana AK, Qiu H, Roll-Mecak A, Hinnebusch AG, Burley SK (2005) Structural basis for autoinhibition and mutational activation of eukaryotic initiation factor 2α protein kinase GCN2. J Biol Chem 280:29289–29299
Palii SS, Kays CE, Deval C, Bruhat A, Fafournoux P, Kilberg MS (2009) Specificity of amino acid regulated gene expression: analysis of genes subjected to either complete or single amino acid deprivation. Amino Acids 37:79–88
Pan YX, Chen H, Thiaville MM, Kilberg MS (2007) Activation of the ATF3 gene through a co-ordinated amino acid-sensing response programme that controls transcriptional regulation of responsive genes following amino acid limitation. Biochem J 401:299–307
Patwari P, Higgins LJ, Chutkow WA, Yoshioka J, Lee RT (2006) The interaction of thioredoxin with Txnip. Evidence for formation of a mixed disulfide by disulfide exchange. J Biol Chem 281:21884–21891
Sato H, Nomura S, Maebara K, Sato K, Tamba M, Bannai S (2004) Transcriptional control of cystine/glutamate transporter gene by amino acid deprivation. Biochem Biophys Res Commun 325:109–116
Shan J, Ord D, Ord T, Kilberg MS (2009) Elevated ATF4 expression, in the absence of other signals, is sufficient for transcriptional induction via CCAAT enhancer-binding protein-activating transcription factor response elements. J Biol Chem 284:21241–21248
Su N, Thiaville MM, Awad K, Gjymishka A, Brant JO, Yang TP, Kilberg MS (2009) Protein or amino acid deprivation differentially regulates the hepatic forkhead box protein A (FOXA) genes through an activating transcription factor-4-independent pathway. Hepatology 50:282–290
Tallóczy Z, Jiang W, Virgin HW 4th, Leib DA, Scheuner D, Kaufman RJ, Eskelinen EL, Levine B (2002) Regulation of starvation- and virus-induced autophagy by the eIF2alpha kinase signaling pathway. Proc Natl Acad Sci USA 99:190–195
Thiaville MM, Pan YX, Gjymishka A, Zhong C, Kaufman RJ, Kilberg MS (2008) MEK signaling is required for phosphorylation of eIF2α following amino acid limitation of HepG2 human hepatoma cells. J Biol Chem 283:10848–10857
Vesely PW, Staber PB, Hoefler G, Kenner L (2009) Translational regulation mechanisms of AP-1 proteins. Mutat Res 682:7–12
Wek SA, Zhu S, Wek RC (1995) The histidyl-tRNA synthetase-related sequence in the eIF-2α a protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids. Mol Cell Biol 15:4497–4506
Wek RC, Jiang HY, Anthony TG (2006) Coping with stress: eIF2 kinases and translational control. Biochem Soc Trans 34:7–11
Acknowledgments
We gratefully acknowledge the kind gifts of ATF4 antibody from Dr. Michael S. Kilberg, University of Florida, Gainesville, FL. The microarray analyses were done by the Cornell University microarray core facility center (Cornell Big Red Spots) and were funded in part by the Cornell University Center for Vertebrate Genomics. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases through Public Health Service Grants DK056649 and DK0664303.
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Sikalidis, A.K., Lee, JI. & Stipanuk, M.H. Gene expression and integrated stress response in HepG2/C3A cells cultured in amino acid deficient medium. Amino Acids 41, 159–171 (2011). https://doi.org/10.1007/s00726-010-0571-x
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DOI: https://doi.org/10.1007/s00726-010-0571-x