TRANSCRIPTIONAL REGULATION OF OXIDATIVE STRESS IN THE ALCOHOLIC LUNG

1. Victor Tseng, MD Division of Pulmonary, Allergy, and Critical Care Medicine Mentors: ViranujSueblinvong, MD and David M. Guidot, MD TRANSCRIPTIONAL REGULATION OF OXIDATIVE STRESS IN THE ALCOHOLIC LUNG Keywords: Alcohol, Oxidative Stess, ATF-3, NRF-2, TGF-β

2. CLINICAL PROBLEM • Alcohol results in severe lung derangements at the cellular level1-3 • Decreased alveolar macrophage viability • Decreased neutrophil chemotaxis • Increased permeability of alveolar epithelium • Oxidized intracellular state (low GSH) • Alcohol renders the lung more susceptible to the following acute disease processes4,5 • ALI/ARDS • Bacterial Pneumonia • Pulmonary TB (Primary Infection, Reactivation)

3. OXIDATIVE STRESS PATHWAY Oxidative Stress6 Proteasome ACUTE Sepsis VILI Pneumonia Hyperoxia CHRONIC Alcohol HIV Cigarettes Aging ROS● Keap1 GSH Glutathione Nrf2 Cytosol Nucleus 5’ GCLC GST NADH-q 3’ Nrf2 binds the Anti-Oxidant Response Element (ARE)

4. ALCOHOL HOW? • Decreased Transcription? • Epigenetic • Repression • Decreased Protein Synthesis? • RNA interference • Post-Transcriptional Increases TGFβ17 Increases Nuclear Translocation of Nrf2 Decreases Nrf2 Expression Causes Profound Glutathione Depletion

5. TGFβ1 ATF3 Ubiquitous transcription factor known to be upregulated8 by TGFβ1 and then to suppress Nrf2-ARE activity in airway smooth muscle cells9, whole mouse lung10 and pulmonary fibrosis models11. ATF3 Nrf2

6. QUESTION 1 What transcriptional changes are seen in acute and chronic phenotypes of alcohol-induced stress? QUESTION 2 More specifically, is ATF3 involved in this process?

7. METHODS (CELLS) • NIH 3T3 (Mouse Lung Fibroblasts) • Grown to 80% confluent monolayer with standard tissue culture flask Incubated for 72 hours with 60 mM ethanol • Whole-cell lysate purified • Total mRNA extracted by Qiagen kit • Total protein isolated by RIPA buffer Quantified by RT-qPCR Normalized to 18S rRNA ATF3, Nrf2, and TGFβ1 cDNA created Nrf2 probed on Western blot TGFβ1 by flow cytometry Quantified by CCD Normalized to β-actin

8. METHODS (MICE) 3-month old C57 BL/6 mouse colony, approved by Emory IACUC Fed EtOH in water, increased by +5%/wk until final concentration 20% v/V. Standard chow Lungs harvested at 8 weeks and prepared for nucleic acid/protein analysis

9. RESULTS (Cells, Part 1) Downregulated Nrf2 For mRNA, alcohol treatment in vitro was associated with… Upregulated Tgfβ-1 Upregulated Atf3

10. RESULTS (Cells, Part 2) For protein, alcohol treatment in vitro was associated with… Downregulated GSTT2 Decreased NRF2

11. RESULTS (Mouse Lung) DownregulatedNrf2 For mRNA, alcohol treatment in vivo was associated with… Decreased NRF2 Downregulated Atf3

12. RESULTS SUMMARY EFFECTS OF ALCOHOL TREATMENT a Data not shown b n = 2 – 4, so unable to determine statistical significance

13. LIMITATIONS • Temporal alcohol exposure is difficult to model • No good model of chronic alcohol exposure in vitro • No good model of acute alcohol exposure in vivo • Variation in ATF3 expression can be explained by myriad factors • Genetic regulation of redox homeostasis is pleiotropic • More experiments are needed to confirm trend seen in ATF3

14. CONCLUSION • Alcohol exposure generates oxidative stress via an array of transcriptional changes • TGFβ1 expression is upregulated in both 3T3 cells and whole mouse lung • ATF3 expression is upregulated in 3T3 cells, but not in whole mouse lung • As a result, Nrf2-ARE activity and NRF2 protein levels are reduced • These changes may suggest potential therapeutic targets in alcohol-mediated ARDS

15. ACKNOWLEDGEMENTS RESIDENCY PROGRAM Dominique Cosco, MD Daniel Dressler, MD Megan DeMoss, MD FUNDING NIH K08 AA021404-01 NIH P50 AA013757 NIH R01 AA 017627 MENTORS: Viranuj Sueblinvong, MD David Guidot, MD • GUIDOT LAB • David Guidot, MD • Stephen Mills, MS • Bashar Staitieh, MD • Xian Fan, MD • Wendy Neveu, MD PhD

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