It is well known that the risk of developing cardiovascular diseases is inversely related to plasma concentrations of high-density lipoprotein (HDL), the so-called "good cholesterol". This protective mechanism is thought to be due to the ability of an HDL particle to transport excess cholesterol from cells within the arterial wall to the liver for disposal as bile acids (reverse cholesterol transport). HDL cholesterol is delivered to the liver and steroidogenic tissues by a mechanism called "selective lipid uptake" which is mediated by the HDL receptor (HDL-R), or scavenger receptor class B type I (SR-BI). SR-BI expression in the liver, but not in other tissues, has been shown to be the main determining factor that regulates plasma HDL levels. Estrogen is thought to protect against cardiovascular diseases by enhancing plasma HDL levels and promoting reverse cholesterol transport. Since SR-BI is the major determinant of plasma HDL levels, direct regulation of the SR-BI gene by estrogen is theorized. The long-term objective of our research is to characterize the molecular mechanisms involved in the estrogen regulation of the SR-BI gene. We have previously shown that estradiol (E2) acting via the estrogen receptors (ER; alpha and beta) positively regulate the SR-BI promoter and that sterol regulatory element binding protein-1a (SREBP-1a) may be involved in this regulatory process as a co-activator. Until recently, this E2-dependent increase in SR-BI promoter activity did not correlate with reports showing that hepatic SR-BI protein levels disappeared after E2-treatment. An interesting explanation for these contradictory results came from the finding that an alternatively spliced product of the SR-BI gene, designated SR-BII, was significantly increased after E2-treatment in liver cells. This suggests that further characterization of estrogen's effect on the SR-BI/II expression in the liver is required to define the molecular mechanisms that mediate the regulation of this gene. One potential factor that may be involved in the estrogen-dependent regulation of the SR-B gene is the recently identified PDZK1 protein. This adaptor protein has been found to regulate the cell surface expression of SR-BI in the liver. When PDZK1 protein levels are eliminated, either by treatment or by genetic ablation, hepatic levels of SR-BI decrease by 95%. We hypothesize that estrogen regulates the expression of the SR-B gene by at least two distinct mechanisms: (1) directly by controlling the alternative splicing of the SR-B pre-mRNA and (2) indirectly by affecting SR-B protein stability mediated by PDZK1. In this proposal, we will test these hypotheses through the following specific aims: Aim I: To identify genomic regions involved in estrogen-mediated alternative splicing of the SR-B pre-mRNA, and Aim II: To determine whether estrogen regulates the stability of SR-BI and SR-BII isoforms and to determine whether PDZK1 is involved in this regulatory process.