Macrophages are highly inducible and extremely potent cells of the innate immune system. Macrophage activation by invading pathogens initiates signalling cascades and a gene expression programme that can have dire consequences for the host if left unchecked. As well as signalling cascades and gene expression stimulated by macrophage recognition of pathogen products, secretory products of activated macrophages feed-back on macrophages themselves to further the inflammatory response and ensure its timely resolution. The attenuation of inflammation is already comprehended in normal responses to infection and this is most simply seen in the transient expression of inflammatory genes in a defined temporal sequence. Transient expression of inflammatory response genes is ensured in part by transcriptional induction and repression, and in part by sequential cycles of stabilisation and destabilisation of transcripts. This post-transcriptional regulation is an important control point in inflammatory responses, as well as in other periodic and specialised cellular functions, and the regulatory pathways, mechanisms and molecules operating at this level are as yet poorly characterised. The project described in this thesis originated from the observation that a candidate post-transcriptional regulator, G3BP2, was induced in macrophages during inflammatory responses (in this case to bacterial Lipopolysaccharide). It was thus sought to investigate the role of G3BP2 in macrophage biology. As a parallel project, a cDNA microarray experiment was designed to explore the contribution of posttranscriptional regulation to the macrophage inflammatory response, with a view to identifying regulated, and co-regulated, genes which might ultimately facilitate the characterisation of novel regulatory factors such as the G3BPs.
The GSBPs constitute a small family of widely expressed proteins which probably function in signal transduction and RNA metabolism. G3BP2, in particular, has been implicated in the regulation of a fundamental inflammatory response - NFKB activation. The mechanisms by which GSBPs regulate mRNAs are not well characterised, and nor are their physiological ligand/s; although a role in the regulation of mRNA stability has been proposed. All three G3BPs were expressed in macrophages, and both G3BP2a and 2b mRNAs were inducible by macrophage activating agents. This thesis is the first report implicating GSBPs in the inflammatory response, and the first demonstration of inducible GSBP2 expression. GSBP2b was the most highly expressed GSBP2 transcript in macrophages and in a range of mouse tissues examined. Stable over-expression of epitopetagged GSBP2b or GSBP1 in RAW264.7 macrophages was not compatible with cell survival, and both proteins were subject to proteolytic cleavage when expressed at high levels. Both G3BP2b and GSBP1 co-precipitated with the insoluble, cytoskeletal fraction in macrophages. Data presented in this thesis suggests that GSBP2b may directly interact with keratin intermediate filaments. GSBP2b localisation and solubility was not altered by macrophage activating agents, nor did GSBP2b over-expression affect the accumulation of the inflammatory products TNFα or nitric oxide. It is proposed that GSBP1 and GSBP2b act as cytoskeletal adaptor proteins, which may relay distinct signal transduction events via interaction with other proteins and/or RNAs.
Modulation of mRNA stability is a major mechanism of post-transcriptional regulation. Research into mRNA stability has mainly focused on an important stability-determining sequence, the AU-rich element (ARE), and associated regulatory proteins. ARE-independent mechanisms also exist, and these may involve novel regulatory factors. The stability of mRNAs in activated macrophages was investigated by microarray in Chapter 5 of this thesis. The strategy employed here is one of many that could be used to ask specific questions about the regulation of mRNA stability in an inducible system. Several insights were gained into the general stability characteristics of LPS-inducible transcripts. The majority of unstable transcripts in this experiment did not possess an ARE instability element, underlining the importance of studying factors and mechanisms involved in the regulation of non-ARE mRNAs.