Chronic inflammation is a major contributor to the onset and progression of atherosclerosis and other vascular disorders. Studies of inflammation have focused primarily on the initiating processes and less attention has been given to the mechanisms by which inflammation is terminated. During the last several years a new paradigm has been invoked in which endogenous mediators limit or reduce these responses as part of an active """"""""resolution of chronic inflammation"""""""" process. In our own studies we have described the discovery of a novel translational control system that has the features of an endogenous regulator of the inflammatory response in myeloid cells. We have shown that interferon (IFN)-gamma, a classic activator of monocyte/macrophages, induces assembly of the IFN-Gamma-Activated Inhibitor of Translation (GAIT) complex that binds a specific RNA element in the 3'untranslated region (UTR) of target mRNAs, e.g., vascular endothelial growth factor (VEGF)-A, and inhibits their translation. Remarkably, the GAIT complex consists of four abundant, """"""""house-keeping"""""""" proteins especially recruited for this function: Glu-Pro-tRNA synthetase (EPRS), ribosomal protein L13a, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and NS1-associated protein (NSAP1 or hnRNP Q). The complex assembles in two stages: An early, pre-GAIT complex of unknown function that contains EPRS and NSAP1, and a late, functional GAIT complex that contains all four proteins. We have elucidated the critical phosphorylation sites essential for GAIT complex activation, namely, Ser77 in L13a and Ser999 in EPRS. Also, we have reported that interaction of NSAP1 acts as an early negative regulator of EPRS binding to target RNAs, and that the inhibition is overcome by late joining of L13a and GAPDH to form an active GAIT complex. In new Preliminary Studies we show that the GAIT pathway is functional in mouse macrophages;however, the mouse GAIT complex is heterotrimeric, lacking NSAP1 and early negative regula- tion. We propose to take advantage of this new information to generate a comprehensive ensemble of genetically-altered mice with mutations in key phosphorylation sites of GAIT components that inactivate, constitutively activate, or modulate, GAIT function in vivo. We will investigate gene expression and the role of the GAIT pathway in atherosclerosis using the well-established apoE-null mouse model. Generation of these mouse models will permit us and others to investigate the role of post-transcriptional gene regulation in the resolution of inflammation, and also the pathological consequences resulting from its dysregulation. These experiments will reveal insights into post-transcriptional mechanisms that regulate the protein expression pro- file of inflammatory macrophages, and the models will provide a unique resource for rigorous analysis of the causes of inflammation-resolution and the consequences of its dysregulation.
Chronic inflammation is a major contributor to the onset and progression of atherosclerosis and other vascular disorders. Our laboratory has discovered a new pathway, the interferon-gamma-activated inhibitor of translation (GAIT) pathway, that functions to reduce or resolve inflammation by inhibiting the synthesis of pro-inflammatory proteins. We propose to develop genetically-modified mice to test the role of the GAIT pathway in preventing chronic inflammatory, cardiovascular disorders such as atherosclerosis. These models may reveal alternative targets for novel anti-inflammatory therapeutic strategies that, because of their specificity, may exhibit minimal adverse side-effects.