The overall aim of the proposed studies is to achieve a better understanding of the regulation of apoE expression in tissue macrophages by physiological stimuli, such as HDL or endotoxin. Specifically, the hypothesis that second messengers of the cAMP- dependent protein kinase and protein kinase C pathways are involved in the positive and negative regulation of apoE secretion and synthesis by HDL or endotoxin, respectively, will be examined in cholesterol- loaded mouse peritoneal, thioglycolate- elicited macrophages. The role of adenylate cyclase in mediating the stimulation of apoE secretion by HDL will be examined in cell cultures. The mechanism of adenylate cyclase activation by HDL through GTP- binding proteins will be examined in detail in macrophage membrane preparations, using both, biochemical and immunological techniques. The second portion of the proposed work is aimed at elucidating the mechanism of endotoxin-mediated suppression of apoE synthesis and secretion through activation of protein kinase C. The effects of phorbol ester, endotoxin and exogenous phospholipase C on macrophage protein kinase C activities, IP3 generation and cytosolic C++ accumulation will be correlated to changes in rates of apoE synthesis and secretion in cultured macrophages. The apparent, protein kinase C- mediated translational regulation of apoE synthesis will be examined by in vitro translation assays. The extent of apoE synthesis and secretion by cultured cells or in translation assays will be determined by immunoprecipitation. The proposed studies will provide insight into the process of reverse cholesterol transport and further our understanding of the protective role HDL in the development of atherosclerosis. Useful information about the mechanisms of endotoxin-induced hyperlipidemia and regulation of immune response will also be obtained. These studies will be the foundation of future studies on the interaction of the two signalling pathways in the regulation of apoE expression.
