BILE ACID METABOLISM AND PLASMA CHOLESTEROL REGULATION

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Hepatic cholesterol 7-alpha-hydroxylase catalyzes the initial and rate- limiting step in the major pathway where by cholesterol is converted to bile salts. As such, 7-alpha-hydroxylase plays a central role both in the maintenance of whole body sterol balance and, secondarily, in the regulation of serum lipoprotein concentrations. Hepatic 7-alpha- hydroxylase is regulated at the transcriptional level in response to bile salts, fatty acids and, in some species, cholesterol; however, the molecular mechanisms where by these major physiological regulators alter transcription of the 7-alpha-hydroxylase gene are completely unknown. This is due, in large part, to the lack of in vitro or cell culture systems that reproduce the major forms of regulation observed in vivo. Moreover, transgenic rats containing promoter/reporter constructs up to 4 kb of 7-alpha-hydroxylase 5 -flanking DNA manifested very low levels of transgene expression that were minimally regulated by bile slats or cholesterol indicating that sequences other than those present in the 4 kb of 5'-flanking DNA are required for normal expression and regulation in vivo. The overall goal of the proposed research is to understand how transcription of the 7-alpha- hydroxylase gene is regulated by bile salts and fatty acids and to determine the mechanism whereby cholesterol up regulates transcription of the 7-alpha-hydroxylase gene in some species (rats and mice) but not in others (hamsters). Using nuclei and nuclear extracts from animals fed bile salts, bile salt sequestrants, fatty acids or cholesterol, we propose a systematic evaluation of the rat and hamster 7 -alpha-hydroxylase gene loci starting with DNase I hypersensitive site mapping to identify important regulatory sequences followed by DNase I footprinting and methylation interference studies to identify nuclear factor binding sites. Selected footprints will be analyzed using gel mobility shift assays and competitor oligonucleotides to identify known transcription factors. Potential enhances identified in this manner will be cloned next to the 7-alpha-hydroxylase proximal promoter (or a heterologous promoter) and testing for enhancer activity and the ability to confer responsiveness to physiologic regulators in vivo using adenovirus-mediated gene transfer. In the unlikely event that the sequences mediating responsiveness to the major physiological regulators cannot be identified using the in vivo transient transfection studies outline above, we will use transgenic animals to map the sequences responsible for regulation. Transgenic mice will initially be generated using P1 clone of the rat 7-alpha-hydroxylase gene. Deletional mutagenesis analysis will then be carried out guided by the results of DNase I hypersensitive site mapping. These studies will provide critical information regarding the transcriptional regulation of the 7-alpha-hydroxylase gene in vivo and may open the door for the development of novel strategies aimed at enhancing the conversion of cholesterol to bile salts and reducing cardiovascular risk.