Apolipoprotein E is an important component of circulating, cholesterol-rich lipoproteins. Defects in its metabolism have pronounced effects on plasma cholesterol levels and the development of atherosclerosis. A role for apo E has also been proposed in the etiology of Alzheimer s disease and other neurodegenerative disorders. The proposed work aims at the elucidation of two novel posttranscriptional regulatory steps in apo E expression in macrophages, discovered in this laboratory: a) translational down-regulation in response to stimuli that act through the protein kinase C signaling pathway, and b) rescue mechanism of newly-synthesized apo E, mediated by HDL and cAMP, which inhibits intracellular degradation and leads to enhanced secretion. First, we will test the hypothesis that protein kinase C activates a trans-acting repressor protein(s), which binds to the 5' or 3' untranslated region of the mature apo E transcript and inhibits translation. Studies are also proposed to identify the corresponding cis-acting elements within the transcript. The bulk of the proposed studies will utilize tools of molecular biology. These will include the synthesis of modified apo E transcripts, in vitro translation assays, coupled with gel mobility shift assays and UV cross-linking studies. Second, the mechanism of HDL/cAMP-mediated rescue of newly synthesized apo E from intracellular (lysosomal) degradation will be examined in primary macrophages. These studies will utilize biochemical and morphological approaches (immunoelectron microscopy) to test the hypothesis that retroendocytosis of HDL coupled with cAMP production results in the rescue of apo E in the endosomal compartment or the trans-Golgi network, followed by their co-secretion. HDL processing will also be examined in the J774 macrophage cell line (a cell line that appears defective in mobilization of intracellular cholesterol into an HDL-accessible pool) and a J774 cell line expressing human apo E3 (J774, E3), in order to examine HDL retroendocytosis and the extent and mechanism of HDL-mediated increase in apo E secretion, respectively. Macrophages or macrophage-like cells are responsible for apo E synthesis in the arterial wall and brain. The results of the proposed work will provide important information about the least-well known aspect of apo E metabolism-regulation of expression and processing within these cells.
