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Title: Signalling pathways in the regulation of gene expression by the proinflammatory cytokine interferon-gamma
Author: Harvey, Elizabeth Jane
ISNI:       0000 0004 2749 4098
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2006
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The cytokine IFN-gamma regulates the expression of numerous genes in macrophages, including many that are implicated in atherosclerotic processes. Studying the signalling mechanisms involved in the mediation of IFN-gamma responses may lead to the identification of future therapeutic targets for the treatment of atherosclerosis. Previous work in our laboratory revealed a role for the kinase CK2 in modulating the expression of the LPL and ICER genes in response to IFN-gamma. Experiments detailed in this report show that the CK2 inhibitor apigenin inhibited the IFN-gamma-mediated induction of each gene in the group selected for study (ICER, MCP-1, SOCS-1, IP-10, Mig, l-TAC, CCR2), suggesting that there may be a common role for CK2 in the regulation of macrophage gene expression by the cytokine. The PI3K inhibitor LY294002 has also previously been found in our laboratory to prevent the IFN-gamma-mediated suppression of LPL expression. In this study LY294002 selectively inhibited the induction of MCP-1, IP-10, Mig, l-TAC, and CCR2 expression by IFN-gamma but not that of ICER or SOCS-1. Extension of this line of investigation through microarray analysis provided further evidence for a gene-specific requirement for PI3K in mediating IFN-gamma responses. The signalling pathways involved in the regulation of MCP-1 and ICER gene expression by IFN-gamma were chosen for more detailed study. The JAK-STAT pathway is the most widely accepted mechanism of IFN-gamma signalling. However, recent studies have suggested the existence of potential STATl-independent pathways. The functions of CK2 and PI3K were investigated, in relation to a STATl-dependent or - independent mechanism of IFN-gamma signalling, in the regulation of MCP-1 and ICER expression. A role for the JAK-STAT pathway in the regulation of MCP-1 expression by IFN-gamma was revealed, in macrophages, through co-transfection studies with inactive mutant forms of JAK1, JAK2 and STAT1. Promoter binding analyses demonstrated that IFN- y induced the binding of STAT1 to a GAS site in the MCP-1 promoter. PKB was found to act as a downstream effector for PI3K in the IFN-gamma-mediated induction of MCP-1. Both CK2 and PI3K/PKB were found to be involved in the IFN-gamma-mediated activation of STAT1 through phosphorylation at Serine 727. Additionally, constitutive Spl protein binding to the MCP-1 promoter was demonstrated. Co- immunoprecipitation revealed an IFN-gamma-inducible interaction between Spl and STAT1 that may function in the cytokine response. In our laboratory CK2 has previously been shown to phosphorylate CREB in response to IFN-gamma. A luciferase reporter construct containing four tandem CRE consensus elements, used to model the ICER promoter that contains four CRE-like sites, showed inducible activation by IFN-gamma, through CK2. The JAK2 inhibitor AG490, and dominant negative forms of JAK1, JAK2 and STAT1, did not attenuate increases in ICER expression in response to IFN-gamma. These findings indicate that CREs represent a novel class of IFN-gamma-responsive element and that the regulation of ICER expression may occur through CK2 in a JAK-STAT-independent pathway. Together these studies have demonstrated novel roles for the kinases CK2 and PI3K in the regulation of macrophage gene expression by IFNy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available