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Murray, James Thomas Crawford
(1999).
DOI: https://doi.org/10.21954/ou.ro.00010206
Abstract
Aberrant regulation of macrophage recruitment and activation by M-CSF is integral to inflammatory disease and cancer. M-CSF stimulated responses are mediated by the c-fms gene product found on monocytes and macrophages. A key component of signalling pathways immediately downstream of c-fms is Phosphatidylinositol 3-kinase. The research data presented in this thesis examined the role of PI 3-kinase in various M-CSF stimulated responses in a murine macrophage cell line, BAC1.2F5. Analysis of the expression profile for PI 3-kinase identified p85α/p110α as the predominant PI 3-kinase isoforms present in BAC1.2F5 cells which was activated upon M-CSF stimulation. The PI 3-kinase specific inhibitors wortmannin and LY294002 were used to characterise the M-CSF stimulated responses in BAC1.2F5 cells. It was found that PI 3-kinase activity is required for protection of BAC1.2F5 macrophages against apoptosis, but not for the proliferation signal. It was observed that PI 3-kinase inhibitors prevented die correct transport of internalised c-fms from the plasma membrane to the endosomal/lysosomal compartments. In addition, PI 3-kinase inhibition also resulted in aberrant reorganisation of the actin cytoskeleton, characterised by the absence of fine actin cables within the cytoplasm of BAC1.2F5 cells. In addition to the characterisation of c-fms signalling with respect to PI 3-kinase, this thesis also reports the production of human c-fms expression constructs containing mutations at critical PI 3-kinase binding sites and possessing C-terminal epitope tags for identification and purification. These constructs were expressed in HEK 293 cells and demonstrated to be recognised by both gene and epitope specific antibodies. Thus these constructs represent novel tools for the investigation of PI 3-kinase signalling.
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