The enterohepatic circulation of bile acids involves the coordinated interplay of bile acid synthesis, metabolism, and transport. Although the overall physiology is currently well-understood, the underlying regulatory control mechanisms are still largely unknown. The recent cloning of several genes, whose protein products are essential to the enterohepatic circulation of bile acids, has provided the opportunity to begin to systematically investigate the cellular and molecular factors governing this important pathway. The main objective of this proposal will be to test the hypothesis that common factors (including bile acids] control the transcription of these genes. These studies will initially focus on characterizing the cis-and trans-acting factors regulating the expression of the recently cloned rat liver basolateral sodium-dependent bile acid transporter (Ntcp] gene promoter. After a complete structural analysis of the Ntcp genomic organization, the transcriptional regulatory role of hormones, bile acids and related compounds will be assessed by transient transfection of primary rat hepatocyte cultures. The specific cis-acting DNA regulatory elements and cognate trans-acting DNA-binding proteins will be studied by a combination of standard DNA-binding protein investigations. The cis-acting region mediating bile acid responsiveness [Bile Acid Response Element, BARE] will be finely delineated, and the cognate specific BARE-binding protein cloned. Exploration beyond the initially-defined promoter region to surrounding regions will determine if any distantly-acting regulatory elements exist. Investigations will also be directed towards characterizing a new hepatocyte-derived cell line, WIF-B, that maintains polarized hepatocyte function in vitro, including vectorial transport of fluorescently-labeled bile acids, unlike any cell line characterized to date. The effects of bile acids on native Ntcp mRNA expression in hepatocytes and WIF-B cells will be tested by Northern blotting, ribonuclease protection and nuclear run-on assays. These findings will be applied to a focused evaluation of the regulatory role of bile acids and sterols on the promoter regions of several other key genes involved in the synthesis and transport of bile acids. Efforts will be initially directed towards exploring the factors regulating a canalicular bile acid transporter, the ectoATPase gene and subsequently expanded to include analyses of key genes in the cholesterol synthetic [HMGCoA synthase and HMGCoA reductase] and bile acid synthetic (cholesterol 7alpha-hydroxylase] pathways. It is anticipated that these studies will provide significant new information on the intrahepatocytic molecular control of the enterohepatic circulation of bile acids, and insight into the factors influencing cholesterol homeostasis and the pathophysiology of cholestatic liver diseases.
