The conversion of cholesterol to bile acids in the liver is the major pathway for the catabolism of cholesterol and plays an important role in the maintenance of cholesterol homeostasis. Enterohepatic recirculation of bile acids inhibits the first and rate-limiting enzyme in bile acid synthesis, cholesterol 7alpha-hydroxylase. Alterations of this mechanism could lead to pathological conditions such as hypercholesterolemia, atherosclerosis, cholelithiasis, sterol storage diseases and liver diseases in humans. Cholesterol 7alpha-hydroxylase cytochrome P450 (CYP7), specific antibodies and cDNA clones have been used as molecular probes to study molecular mechanisms of regulation. Many evidences suggest that this enzyme is regulated primarily at the level of gene transcription. Transient transfection assays of promoter activity of the rat CYP7 promoter/luciferase chimeric constructs in HepG2 cells revealed that most cis-acting elements conferring the repression by bile acids, phorbol esters (PMA), cAMP and insulin, and activation by glucocorticoid and retinoic acids are all localized in the region about 344 bp upstream the transcription start site. Multiple bile acid response elements and potential transcription factors binding sites have been mapped. We hypothesize that CYP7 gene is repressed by a strong repressor which binds to a unique repeating sequence in the proximal promoter. Bile acids may exert their negative effects through activations of protein kinases C, protein kinase A and insulin stimulated protein kinases in signal transduction pathways. To further identify and characterize the cis- regulatory elements and trans-acting protein factors involved in the CYP7 gene expression, the following three specific aims will be studied: 1). to characterize a novel bile acid responsive repressor region and to isolate nuclear DNA-binding protein by binding-site affinity column chromatography and by screening of liver expression libraries; 2). to delineate the response elements conferring the regulation by bile acids and other physiological stimuli by DNase I footprinting, linker scanning mutagenesis and electrophoretic mobility shift assay (EMSA); 3) to study the regulation of the rat CYP7 gene expression by signal transduction pathways involving PKC, PKA and insulin. Protein kinases and transcription factors responded to bile acids, PMA, cAMP and insulin will be identified by immunoblotting using specific antibodies, Southwestern blottings using oligonucleotide probes, EMSA, and transfection assays of CYP7 promoter activity in HepG2 cells over-expressing PKC isozymes. Our long term objective is to elucidate the molecular mechanisms of regulation of the cholesterol 7alpha-hydroxylase gene in bile acid biosynthesis and cholesterol homeostasis. Results obtained from this project would provide insights into the mechanism of human metabolic diseases. Understanding of transcriptional regulation of the CYP7 gene by physiological regulators is the first step toward the goals of designing therapeutic drugs to stimulate cholesterol 7alpha-hydroxylase activity in the liver and gene therapy targeted to regulation of CYP7 gene in the near future.
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