Elevated levels of plasma low density lipoprotein (LDL) cholesterol are important in the development of atherosclerosis and coronary heart disease. Down-regulation of LDL receptor gene expression, in response to dietary intake of saturated fats and cholesterol, leads to an increase in plasma LDL cholesterol and contributes to the development of atherosclerosis. Non-sterol signals transduced during mitogenic stimulation apparently override the normal down-regulatory effects of exogenous LDL cholesterol on LDL receptor expression. The proposed studies are focused on understanding the molecular basis whereby these non-sterol mitogenic signals regulate LDL receptor gene transcription. The hypotheses to be tested in the proposed studies are that: 1). the mechanism(s) that increase LDL receptor gene transcription with mitogenic stimulation are distinct from those involved in sterol regulation; and 2). signals transmitted by growth factors and cytokines increase LDL receptor expression in a variety of cells involved in regulation of plasma LDL cholesterol levels and metabolism of LDL cholesterol in atherosclerotic lesions. The plan of approach entails initial utilization of Jurkat cells, a continuously proliferating T lymphocyte cell line that retains mitogen responsiveness. The elements that control LDL receptor gene expression with mitogenic stimulation will be identified by electrophoretic mobility shift analysis and confirmed by transfection of wild-type and mutant fragments from the 5' flanking region of the LDL receptor gene linked to a reporter gene. Additional studies will examine whether similar regions and mechanisms determine regulation of LDL receptor gene transcription in hepatocyte, monocyte/macrophage and fibroblast cell lines. DNA sequences that are important in regulation of LDL receptor gene expression but are not associated with known nuclear factors will be further characterized by gel mobility shift analysis and DNase protection assays. The results should provide insight into the means by which LDL receptor expression is increased by activation signals, suggest mechanisms whereby plasma LDL cholesterol levels might decrease during the acute phase response and also suggest possible approaches to regulate LDL cholesterol levels therapeutically.
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