Regulation and trafficking of the STAT3 transcription factor

Abstract

The Signal Transducer and Activator of Transcription (STAT) family of transcription factors plays crucial roles in regulating important cellular responses via the regulation of target gene expression. Of the seven members of the STAT family, STAT3 has attracted considerable research interest due to its activation by a range of extracellular stimuli and its involvement in a wide variety of human pathologies where it appears to show sustained activation. Despite the intense research on STAT3 activation in cancer and other diseases, few studies have directly addressed the regulatory mechanisms of STAT3, and in particular those that may contribute to its sustained activation that is a hallmark of disease conditions. Furthermore, the regulatory mechanisms and actions of the naturally occurring shorter STAT3 spliceform, STAT3β, have not been fully elucidated. Therefore, the aim of this thesis was to elucidate the differences between the regulations/regulatory mechanisms, trafficking and downstream transcriptional consequences of the individual STAT3 spliceforms, STAT3α and STAT3β. In addition, this thesis investigated how the combination of cytokine and abiotic stress can impact in different ways on the regulation of STAT3 phosphorylation, nuclear accumulation and transcriptional activity. The results here reveal for the first time that the STAT3 spliceforms, STAT3α and STAT3β, regulate a specific and unique subset of gene targets, exhibiting distinct nuclear import kinetics under cytokine-stimulated conditions. STAT3α exhibited transient phosphorylation and nuclear residency while STAT3β showed prolonged phosphorylation, nuclear residency, and the ability to modulate STAT3α phosphorylation and nuclear retention time. In addition, live cell imaging and fluorescence recovery after photobleaching (FRAP) revealed differences in STAT3 spliceform nuclear import kinetics due to the spliceform-specific C-terminal sequences and the fact that nuclear localisation of phosphorylated STAT3 dimers upon cytokine stimulation is a product of STAT3 nuclear accumulation and not due to an increase in its import rate as previously assumed. Lastly, disease-relevant models of persistent cytokine-driven STAT3 activation were examined, indicating that the deregulation of regulatory mechanisms by abiotic stresses such as oxidative stress and hyperosmotic stress did not necessarily correlate with STAT3 transcriptional output despite the observed persistent STAT3 activation. In summary, the studies presented in this thesis emphasize the importance of the STAT3α and STAT3β spliceforms in the regulation of specific gene targets, and the differential regulation of these spliceforms through the small differences in their C-terminal sequences. In addition, the impact of abiotic stresses on STAT3 signal transduction further emphasizes the importance of detailed characterisation of STAT3 phosphorylation and activation kinetics in true disease models. Only then will it be possible to embark on considered development of therapeutics to treat pathologies such as cancer, where persistent STAT3 activation is observed. Awards: Vice-Chancellor’s Commendation for Doctoral Thesis Excellence in 2014.