The objectives are to define the molecular events that serve to regulate the synthesis of cholesterol and LDL receptors in human fetal liver (HFL). Estrogen appears to be a major factor in simulating these processes. Since the fetal liver is a site of of aromatase as well as 16Alpha-hydroxylase activity, our objectives also include the study of the regulation of estrogen metabolism. To approach these objectives, we will utilize HFL cells maintained in primary culture. Cells will be incubated in the presence of various hormones that have been shown to stimulate lipoprotein binding and metabolism as well as cholesterol synthesis in HFL. We propose to test the effects of these putative agents on the rates of synthesis of HMG-CoA reductase in (35S)methionine-labeled cell lysates after immunoprecipitation and SDS-PAGE using an antibody directed against HMG-CoA reductase. We will determine the effect of these putative agents on the amount of LDL receptor in liver cells after SDS-PAGE and ligand blotting using biotin-modified LDL. We shall also determine the cellular content of the mRNA for HMG-CoA reductase and LDL receptor by hybridization studies utilizing specific cDNA's. We have previously demonstrated that glucocorticosteroids, dbcAMP and other factors stimulate aromatase and 16Alpha-hydroxylase activity in HFL, and these enzymes are involved in the formation and metabolism of estrogen in HFL. We propose to purify HFL 16Alpha-hydroxylase (cytochrome P-450 16Alpha) and to develop antibodies specific to this enzyme. We intend to study the mechanisms whereby putative agents regulate the synthesis of aromatase (cytochrome P-450AROM) and cytochrome P-450AROM) and cytochrome P-450 16Alpha in HFL by use of antibodies directed against these enzymes with immunoblotting as well as by (35S)methionine labeling of cells followed by immunoisolation and SDS-PAGE. We will compare the ability of these putative agents to stimulate the rates of synthesis of these enzymes with our previous findings with respect to the rates of increase in aromatase and 16Alpha-hydroxylase activities. Finally, we propose to use a cDNA insert specific for cytochrome P-450AROM to investigate the synthesis of mRNA for cytochrome P-450AROM by hybridization studies. These findings will provide insight into the mechanisms of the regulation of cholesterol synthesis, LDL receptor content, estrogen formation and metabolism in human fetal liver. These processes are of extraordinary importance in the maintenance of the normal hormonal milieu of pregnancy.
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