Macrophage Ceruloplasmin Expression in Atherosclerosis

Cleveland Clinic Lerner Col/Med-Cwru, Cleveland OH

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Abstract

Our long-term goal is to understand the role of ceruloplasmin (Cp) in normal and pathological vascular processes, particularly atherosclerosis. Cp is an acute phase, inflammatory protein produced by the liver and by activated monocyte/ macrophages. Clinical studies indicate that elevated serum Cp is an important risk factor for atherosclerosis and other cardiovascular diseases. Cp is a multifunctional enzyme with several activities that may contribute to pathological vascular processes including LDL oxidase activity due to a surface copper and a ferroxidase activity that regulates tissue iron homeostasis. Besides the constitutive, systemic production by hepatocytes, Cp is secreted locally by mac-rophages in inflammatory sites, including in human atherosclerotic lesions. We have shown that interferon (IFN)-gamma, a contributor to lesion progression in murine models of atherosclerosis, stimulates Cp mRNA and protein expression by myeloid cells, including U937 cells and peripheral blood monocytes. Cp synthesis abruptly stops about 16 h after IFN-gamma treatment, despite the continued presence of high levels of Cp mRNA. We have described a novel translational silencing mechanism for this inhibition of Cp expression. Silencing depends on end-to-end circularization of the Cp transcript via interaction of the Cp 3'-terminus with the 5'-translation-initiation complex. In new preliminary studies we show that silencing is driven by a 29-nt element in the Cp 3'-untranslated region (UTR). We also show that cytosolic extracts from IFN-gamma-treated U937 cells contain a protein or complex, denoted IFN-Gamma-Activated Inhibitor of Translation (GAIT), that binds to the 29-nt GAIT element (GAITE) and blocks Cp translation in vitro. By RNA/protein interaction- cloning with the yeast 3-hybrid system we have identified candidate proteins that specifically bind to the GAIT element and block Cp translation in vitro. These results have led us to propose the following hypothesis: IFN-gamma-stimulated production of Cp by macrophages is a critical contributor to atherogenesis. In specific, IFN-gamma induces myeloid Cp synthesis, but it also causes a delayed activation or induction of cellular regulatory proteins that bind the Cp 3'-UTR GAITE, and subsequently silence translation of Cp (and possibly other related proteins). Dysregulation of this silencing mechanism may lead to prolonged production of Cp, accumulation of Cp to injurious levels, and accelerated atherosclerotic lesion progression. We will test this hypothesis by pursuing the following Specific Aims: (1) identify Cp 3'-UTR-binding proteins and determine their function in translational silencing of Cp by IFN-gamma, (2) determine the cellular function of translational silencing of Cp, and (3) determine the role of Cp in atherosclerotic lesion progression in genetically-altered mice. We believe that these studies will yield important information on the basic mechanisms of translational control relevant to IFN-gamma-activated macrophages, and in particular, on the endogenous mechanisms involved in the termination of inflammation. The studies will also help to elucidate the vascular function of macrophage-derived Cp and its role in atherosclerotic lesion progression.