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Farabaugh Thesis 2019 Final v2.pdf (6 MB)

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Insights into a Novel Signaling Pathway that Determines Cell Fate in Response to Hyperosmotic Stress

Farabaugh, Kenneth Thomas, kt
http://rave.ohiolink.edu/etdc/view?acc_num=case1571692276973131

Abstract Details

2019, Doctor of Philosophy, Case Western Reserve University, Pharmacology.
Hyperosmotic stress is relevant in both physiological and pathological conditions. The response to hyperosmotic stress includes a vast body of research that with each passing year becomes more diverse. Aspects of this multifaceted response include the mechanism (or mechanisms) by which cells sense extracellular hyperosmolarity, the capacity with which various cell types respond, the relationship between hyperosmolarity and inflammation, and mechanisms by which hyperosmolarity contributes to the pathology of disease. We used the response to hyperosmotic stress in cell culture as a tool to study potential mechanisms of disease pathology in tissues affected by hyperosmolarity. In response to an increased concentration of extracellular osmolytes, cells will undergo transcriptional, translational, and signaling responses that either enable adaptation and survival, or initiate apoptosis. This decision will depend on the intensity of stress. The Rel family of transcription factors, including the Tonicity Enhancer Binding Protein (TonEBP) and Nuclear Factor-κB (NF-κB), play critical roles in the switch from osmoadaptive homeostasis to inflammation. Lower intensity hyperosmotic conditions will promote TonEBP and NF-κB family member c-Rel-dependent transcriptional induction of osmoadaptive genes SNAT2 and GADD34. As stress intensity increases, however, the inability of cells to adapt to higher environmental tonicity can lead to inflammatory gene expression and pathogenesis. High extracellular osmolarity results in activation of protein kinase R (PKR) via interaction with the PKR-Activating protein (PACT). Here we identified PACT-mediated PKR activation as a marker of the termination of adaptation and initiation of inflammation. We found that high stress-induced PACT-mediated PKR activation inhibits the interaction between NF-κB c-Rel and TonEBP essential for the increased expression of TonEBP-dependent osmoprotective genes; this resulted in enhanced formation of TonEBP/NF-κB p65 complexes and enhanced transcription of proinflammatory genes, including inducible nitric oxide synthase (iNOS) and IL6. PKR mediated-hyperinduction of iNOS decreased cell survival in mouse embryonic fibroblasts via mechanisms involving nitric oxide (NO) synthesis and post-translational modification of proteins. Moreover, we demonstrate that treatment with the PKR inhibitor C16 dampens both iNOS hyperamplification and phenotypic breakdown of the intestinal epithelial barrier caused by an increase in extracellular osmolarity induced by dextran sodium sulfate (DSS) in vivo. In the following pages, we describe a unique signaling pathway that is activated upon hyperosmotic stimulation that mediates the switch between adaptation and inflammation. Collectively, these findings indicate that PKR functions as a signal transducer in the sensing of extracellular hyperosmolarity. These data demonstrate a novel role of c-Rel in the adaptive response to hyperosmotic stress, which is inhibited via a PACT/PKR-dependent reorganization of Rel family transcription factors. Our results suggest that inhibiting PACT-PKR signaling may prove a novel target for alleviating stress-induced inflammatory diseases.
Youwei Zhang, PhD (Committee Chair)
Maria Hatzoglou, PhD (Advisor)
Parameswaran Ramakrishnan, PhD (Committee Member)
Taylor Derek, PhD (Committee Member)
Buchner David, PhD (Committee Member)
170 p.
Biomedical Research; Cellular Biology; Genetics; Molecular Biology; Pharmacology
PKR; PACT; NF-kB; p65; c-Rel; hyperosmotic stress; osmoadaptation

Recommended Citations

Citations

  • Farabaugh, kt, K. T. (2019). Insights into a Novel Signaling Pathway that Determines Cell Fate in Response to Hyperosmotic Stress [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1571692276973131

    APA Style (7th edition)

  • Farabaugh, kt, Kenneth. Insights into a Novel Signaling Pathway that Determines Cell Fate in Response to Hyperosmotic Stress. 2019. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1571692276973131.

    MLA Style (8th edition)

  • Farabaugh, kt, Kenneth. "Insights into a Novel Signaling Pathway that Determines Cell Fate in Response to Hyperosmotic Stress." Doctoral dissertation, Case Western Reserve University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1571692276973131

    Chicago Manual of Style (17th edition)

case1571692276973131
179
© 2019, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.