title: Analysis of the cytokine-induced signalling dynamics of STAT3 and NF-κB
Creators: name: Baldwin, Stephanie
Publisher: name: University of Liverpool Repository
Year: 2015

description: The transcription factors STAT3 and NF-κB play key roles in inflammation, immunity and cell fate. In the liver, they are responsible for transcribing hundreds of genes in response to combinations of IL-6, TNFα and IL-1β, and so together co-ordinate the acute phase response to infection. Dysregulated STAT3 and NF-κB signalling leads to chronic inflammation and is implicated in the development of many cancers. A variety of highly context-dependent intercellular and intracellular mechanisms have been discovered which facilitate both positive and negative cross-talk between STAT3 and NF-κB. Whilst the long-term signalling dynamics of NF-κB have been characterised in single cells, and were found to be oscillatory, imaging studies on STAT3 have focused upon the short-term mechanisms of nuclear transport rather than the long-term dynamics. STAT3 has been shown to oscillate in a population of synchronised cells so it is possible that STAT3 will exhibit oscillatory spatio-temporal signalling dynamics in response to cytokine stimulation. The primary aim of this thesis was to characterise the long-term signalling dynamics of STAT3 in response to IL-6, using fluorescent fusion protein reporters for STAT3 and its inhibitor SOCS3, in conjunction with live single cell fluorescence microscopy. Towards these aims, STAT3 and SOCS3 fluorescent fusion proteins were constructed. The responses of a candidate cell line to IL-6 and TNFα were investigated, and then the fluorescent reporters were characterised in that cell line. The N-terminal tagged EGFP-STAT3 reporter was found to be the most accurate reporter of IL-6 signalling. The EGFP-STAT3 was then used to investigate the single cell spatio-temporal dynamics of STAT3 in response to differently timed lengths of IL-6 stimulation. STAT3 was found to oscillate with a period of approximately 90 min in response to continuous IL-6 stimulation, but only underwent a transient nuclear translocation in response to a 30 min IL-6 pulse. Furthermore, the patterns of gene expression were characterised for the timed IL-6 treatments. The quantified single cell dynamics were used to constrain an existing generic model of STAT:SOCS signalling; the model was able to capture the observed single cell dynamics using a minimal ordinary differential equation approach. The secondary aim of the thesis was to study cross-talk between STAT3 and NF-κB using live cell microscopy techniques. The effects of co-stimulation of NF-κB and STAT3 were investigated using combinations of TNFα and IL-6 stimuli. Combinations of single or dual transfections, and single or dual stimulation were performed as controls in order to tease apart the effects of co-expression and co-stimulation. The importance of the timing of cytokine stimulation was also investigated. Finally, the effects of IL-1β upon IL-6 induction of STAT3 were investigated, as this was shown elsewhere to inhibit STAT3 signalling and so was expected to produce interesting spatio-temporal signalling effects. This preliminary study revealed distinct subpopulations of cells with different p65 and STAT3 response patterns. The STAT3 response was knocked down or significantly delayed in many cells but a small subset exhibited atypical oscillatory dynamics. Interestingly, the p65 dynamics were also significantly perturbed by IL-6 and IL-1β co-stimulation, indicating that there are cross-talk events occurring in both directions. Consequently these studies represent a very important area for future investigation.