In disease states, unfolded proteins accumulate in the endoplasmic reticulum because the folding capacity is exceeded, initiating a cellular stress response (the unfolded protein response or UPR). Our long-term interest is to understand the molecular mechanisms that allow cells to withstand stress and that contribute to pathologies during prolonged stress. Regulation of gene expression by transcriptional activators and repressors is a key feature of the stress response. During the previous grant period we found that the adaptive response to nutrient starvation increases expression of amino acid transporter genes, which can facilitate the recovery from stress. We also found that transcription of the arginine/lysine transporter gene, Cat-1, and of other genes involved in amino acid metabolism is attenuated during prolonged ER stress, mediated by the CCAAT/enhancer binding protein family transcription factor, C/EBP2. Regulated translation of the C/EBP2 mRNA produces both LAP, a transcriptional activator, and LIP a repressor. The LAP/LIP ratio plays a critical role in cell fate and metabolism. We found that the LAP/LIP ratios change during the UPR via mechanisms that involve proteasomal degradation of the proteins and translational control of the C/EBP2 mRNA;this provides the driving force behind this proposal. The regulation of transcription factor levels during ER stress via the proteasome pathway is a novel mechanism to modulate the cellular stress response. We hypothesize that the LAP/LIP ratio plays a role in controlling transcription of stress-response genes. We also hypothesize that the regulation of LIP levels promotes expression of prosurvival genes early in the stress response and restricts expression of proapoptotic genes during prolonged stress. In this proposal, we will study the mechanisms that regulate LIP synthesis and degradation during ER stress in cultured cells and in mice. Experiments using stress-inducing drugs and models of human disease will reveal the physiological significance of this regulation.
Our Specific Aims are: (i) Determine the mechanism for diminished LIP levels during the early (prosurvival) phase of ER stress (ii) Investigate the signaling pathways that regulate proteasome-mediated degradation of LIP during the prosurvival phase of ER stress. (iii) Determine the mechanisms that increase LIP levels during the late (proapoptotic) phase of ER stress. (iv) Determine the physiological significance of the LAP/LIP ratio during ER stress using MEFs defficient in C/EBP2 (v) Determine the effect of disruption of the C/EBP2 gene in animal models of ER stress-mediated disease. Our long term goal is to generate transgenic mice expressing only LAP and only LIP by knock-in mutations in the C/EBP2 gene, using the state of the art system of Bacterial Artificial Chromosomes. The knock-in mice will be a valuable tool to determining the functions of LAP and LIP in ER- stress mediated apoptosis and enable us to test the findings of Aims 1-5 in a physiological context with relevance to human disease. Cellular stress is important in a large number of diseases, such as diabetes, neurodegeneration, cancer and complications of obesity. A common feature of these diseases is the accumulation of damaged secretory proteins in the endoplasmic reticulum (ER), a vital organelle responsible for proper cellular function and metabolism. During the previous grant period we found that the adaptive response to nutrient starvation increases expression of amino acid transporter genes, which can facilitate the recovery from stress. PROJECT

Public Health Relevance

This proposal will study C/EBP2, an important regulator of the stress response and how this regulator controls the balance between cellular survival and death. Our studies will generate new therapeutic targets for the many stress-mediated diseases and provide a novel mechanism that regulates the balance between survival and death during these diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK053307-15
Application #
8306945
Study Section
Special Emphasis Panel (ZRG1-EMNR-G (02))
Program Officer
Maruvada, Padma
Project Start
1998-01-01
Project End
2013-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
15
Fiscal Year
2012
Total Cost
$428,725
Indirect Cost
$155,652
Name
Case Western Reserve University
Department
Nutrition
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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Guan, Bo-Jhih; Krokowski, Dawid; Majumder, Mithu et al. (2014) Translational control during endoplasmic reticulum stress beyond phosphorylation of the translation initiation factor eIF2?. J Biol Chem 289:12593-611
Balkrishna, Sarojini; Bröer, Angelika; Welford, Scott M et al. (2014) Expression of glutamine transporter Slc38a3 (SNAT3) during acidosis is mediated by a different mechanism than tissue-specific expression. Cell Physiol Biochem 33:1591-606
Han, Jaeseok; Back, Sung Hoon; Hur, Junguk et al. (2013) ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat Cell Biol 15:481-90
Majumder, Mithu; Huang, Charlie; Snider, Martin D et al. (2012) A novel feedback loop regulates the response to endoplasmic reticulum stress via the cooperation of cytoplasmic splicing and mRNA translation. Mol Cell Biol 32:992-1003
Mounir, Zineb; Krishnamoorthy, Jothi Latha; Wang, Shuo et al. (2011) Akt determines cell fate through inhibition of the PERK-eIF2? phosphorylation pathway. Sci Signal 4:ra62
Komar, Anton A; Hatzoglou, Maria (2011) Cellular IRES-mediated translation: the war of ITAFs in pathophysiological states. Cell Cycle 10:229-40
Basu, Abhijit; Das, Priyanka; Chaudhuri, Sujan et al. (2011) Requirement of rRNA methylation for 80S ribosome assembly on a cohort of cellular internal ribosome entry sites. Mol Cell Biol 31:4482-99
Krokowski, Dawid; Gaccioli, Francesca; Majumder, Mithu et al. (2011) Characterization of hibernating ribosomes in mammalian cells. Cell Cycle 10:2691-702
Hsieh, Chang-Wen; Huang, Charles; Bederman, Ilya et al. (2011) Function of phosphoenolpyruvate carboxykinase in mammary gland epithelial cells. J Lipid Res 52:1352-62

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