Pathology - Theses

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    Molecular characterisation of IFN-γ induced Stat1-independent and Erk1/2- and AP-1-dependent signalling pathway and genes that respond to IFN-γ
    Arthur, Helen Anne ( 2013)
    Interferon (IFN)-γ regulates a diverse range of biological activities that includes anti-pathogenic, anti-cancer and immunoregulatory effects. IFN-γ mediates these activities by regulating changes in gene expression via intracellular pathways. Although the canonical IFN-γ-induced Jak/Stat1 signalling pathway is the primary mechanism regulating gene expression, IFN-γ activates multiple intracellular cascades. There is evidence suggesting that Stat1-independent pathways are important for IFN-γ activity since a third of IFN-γ regulated genes (IRGs) were regulated in the absence of Stat1 (Gil, Bohn et al. 2001) and Stat1-/- mice were more resistant to viral challenge than mice deficient in type I and II IFN receptors (Gil, Bohn et al. 2001; Shresta, Sharar et al. 2005). Our laboratory identified the downstream components of a novel Stat1-independent signalling pathway that activated Erk1/2 and AP-1 and regulated transcription of IRGs (Gough, Sabapathy et al. 2007). However, the upstream components of this pathway have not been characterised. Since Stat1 was not required for IFN-γ-induced activation of Erk1/2 and AP-1 I hypothesised that this pathway is entirely independent of canonical IFN-γ Jak/Stat1 signalling. In support of this, studies have shown that IFN-γ-induced engaged multiple membrane-proximal kinases such as the PI-3K/Akt and adapter proteins such as MyD88 independent of Stat1 and/or Jak proteins. Therefore I hypothesised that Jak1 and Jak2 were not required for activation of the IFN-γ induced Stat1-independent Erk1/2 and AP-1 pathway or to regulate transcription of IRGs. Therefore, alternate kinases or membrane associated proteins would be recruited to the IFN-γ receptor (Ifngr) to activate Erk1/2 and AP-1 and regulate gene expression. The most appropriate method to investigate IFN-γ-induced Jak1 and Jak2-independent signalling is the use of a doubly deficient Jak1/Jak2 model. However, since Jak1-deficient and Jak2-deficient mice are not viable, Jak1-/-/Jak2-/- mice are not viable either and there is no published literature on a doubly deficient Jak1/Jak2 model. The studies in this thesis developed and fully characterised the first reported doubly deficient Jak1/Jak2 model. In a novel finding the signalling analyses performed using doubly deficient Jak1/Jak2 cells demonstrated that IFN-γ-induced activation of Erk1/2 and likely AP-1 was independent of Jak1 and Jak2. Therefore building on previous findings by our laboratory the data in this thesis established that IFN-γ-induced a Jak1/Jak2/Stat1-independent and Erk1/2/AP-1-dependent pathway. Since Jak1 and 2 were not involved IFN-γ must engage another mechanism to active downstream Erk1/2. The studies in this thesis excluded a role for c-Src; therefore alternate signalling molecules must be recruited to the Ifngr. Gene expression analyses showed that transcription of the Immediate Early Genes c-Jun and Nfkbia appeared to be independent of Jak2 but not Jak1. Further analyses performed using a larger gene subset of genome wide studies are required to identify a subset of IRGs potentially regulated by Jak1/Jak2/Stat1-independent and Erk1/2/AP-1-dependent pathway. The studies in this thesis suggest that the Erk1/2 and AP-1 pathway may represent a primary yet transient response to IFN-γ that regulates gene expression independent of canonical Jak/Stat1 signal transduction. Determination of the cellular and biological effects regulated by the non-canonical Erk1/2/AP-1 pathway may have important implications for our understanding of the anti-viral, anti-cancer and immunomodulatory effects of IFN-γ.