The purpose of this proposal is to define the mechanism for the regulation of macrophage apo E production by cellular free cholesterol content. Firstly, the role of transcriptional regulation in mediating macrophage apo E cholesterol responsiveness will be probed. If transcriptional regulation is important, regulatory gene sequences and the potential role of transacting regulatory factors will be investigated. This will be accomplished using stable transfections of macrophage cell lines or by a gel retardation assay using cloned fragments of the 5' flanking region of the apo E gene. Secondly, the relationship of apo E regulation to the regulation of another protein intimately involved in cell cholesterol homeostasis, the LDL receptor, will be investigated. This will be done by comparing the time course of changes in cellular LDL receptor and apo E mRNA levels after incubations designed to alter cellular cholesterol balance. In addition, competition experiments will be performed using the cholesterol responsive sequence of the apo E gene, fused to the CAT expression sequences, co-transfected with increasing amounts of the cholesterol responsive sequences of the LDL receptor gene. These experiments may reveal that the cholesterol responsive sequences of these two genes share diffusible, transacting regulators. Thirdly, the subcellular distribution of free cholesterol in basal and cholesterol enriched macrophages will be determined. This will be done by fractionation of cells on sucrose density gradients to measure specific subcellular compartments. We will manipulate macrophage cholesterol content in ways that we have already shown regulate apo E production, using acetylated LDL, acyl Co-A: cholesterol acyl transferase inhibition and HDL3. The experiments outlined in this proposal should provide insight into a potential role for extrahepatic apo E, i.e. as a protein which functions as part of an integrated cellular response mechanism to maintain cholesterol homeostasis. The importance of these insights for understanding mechanisms of atherosclerosis derived from the central role played by cholesterol loaded macrophages in the atherosclerotic process. Macrophage apo E synthesis and its regulation could have important implications for cholesterol balance of normal and diseased vessel wall tissue.
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