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Title: Redox role of Stat3 in cellular survival during oxidative stress
Author: Palmer , Carolyn
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2016
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A role for Signal Transducer and Activator of Transcription 3 (Stat3) in the maintenance of cardiomyocyte homeostasis under basal conditions as well as in response to ischaemia/reperfusion (l/R) is well established although the underlying mechanism by which Stat3 exerts cardioprotection is unresolved. Stat3 is a key effector molecule in signalling pathways activated by cytokines such as interleukin (IL) 6, and when tyrosine phosphorylated it coordinates the expression of multiple target genes by binding to consensus and nonconsensus serum-inducible elements (SIEs). Stat3 is also reported to activate transcription in a tyrosine phosphorylation-independent manner. Aside from its role as a nuclear transcription factor, Stat3 also operates within mitochondria, where it regulates the activity of the electron transport chain (ETC) and influences mitochondrial permeability transition pore (MPTP) opening, a hallmark event in the intrinsic apoptotic pathway. This function of Stat3 is dependent on serine and not tyrosine phosphorylation. Stat3 contains redox-sensitive cysteines and in the nucleus, Stat3 oxidation has been shown to decrease Stat3 binding to consensus SIEs but increase Stat3 binding to nonconsensus SIEs. Furthermore, expression of a Stat3 mutant substantially resistant to redox modification, Stat3-C3S, in BR293 breast cancer cells increased sensitivity of the cells to hydrogen peroxide (H2O2)-induced cell death and Stat3-C3S, unlike wild type (WT) Stat3, was unable to sustain ATP production in mitochondria isolated from P19CL6 cardiomyocytes subjected to hypoxia (unpublished, Shaw lab). The aim of this thesis was to determine whether redox-sensitive cysteines within the DNA binding domain (DBD) of Stat3 mediate the cytoprotective functions of Stat3. Murine embryonic fibroblast (MEF) cell lines were used to study the effect of Stat3 deletion on mitochondrial function. Firstly, oxygen consumption rate and mitochondrial membrane potential (AU^m) were measured in Stat^' V\rfa> and Stat3/(a> cells. In these cells, Stat3 deletion had no significant effect on mitochondrial function. As an alternative to Stat3 and Stat3 / cells, Stat3fl/fllb> and Stat3'/,b> MEFs, generated more recently, were used to assess the effect of Stot3 deletion on cellular oxygen consumption rate. Consistent with the results obtained with StatS'7'V\rfo> and Stat3/tal cells, no significant difference in mitochondrial function could be detected between Stat3fl/flfb> and Stat3'/ MEFs. Stat3 was then deleted from freshly isolated Stat3fl/fl,c> MEFs, forming Stat3'/Ic> MEFs. Immediately after Stat3 was deleted from freshly isolated Stat3fl/fl MEFs, a modest decrease in complex I (Cl) and complex II (Cll) stimulated respiration could be detected. A modest decrease in the maximal enzymatic activity of Cl was also detected as well as a small increase in superoxide (O2*-) production. However, with increased passage these differences were lost suggesting that cultured MEFs are not a suitable model to probe how redox-sensitive cysteines within Stat3 influence Stat3's ability to regulate mitochondrial function. Introduction of either WT-Stat3 or Stat3-C3S into freshly generated Stat3'/Ic> MEFs, however, produced cell lines that differed in morphology, proliferation and resistance to oxidative stress. In a global gene expression screen for genes regulated by an oxidation-competent Stat3, novel Stat3 target genes with links to pathophysiological remodelling of the heart were identified. In addition, gene set enrichment analysis (GSEA) showed that Stat3 oxidation is associated with the up-regulation of nuclear genes with roles in the mitochondria, programmed cell death, DNA repair, cell cycle and cellular response to oxidative stress. Furthermore, quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) showed that Stat3 oxidation is required for even the baseline expression of genes from each of these categories. This thesis provides new insights into the mechanisms by which Stat3 regulates gene expression. Understanding how Stat3 regulates gene expression during oxidative stress and the downstream targets of oxidised Stat3 will be important for the development of therapeutics for the treatment of ischaemic heart disease in the future.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available