Multiple factors regulate the rat liver basolateral sodium-dependent bile acid cotransporter gene promoter

Abstract

The hepatic uptake of bile acids from the portal circulation is primarily dependent upon a sodium-dependent basolateral membrane transporter. In order to begin to investigate the factors controlling rat liver sodium-dependent bile acid cotransporter (ntcp) gene expression, we isolated approximately 30 kilobase pairs of rat genomic DNA in three overlapping lambdaphage clones. The rat ntcp gene is distributed over 16.5 kilobase pairs as five exons. Primer extension analysis revealed two closely spaced transcription initiation sites, 27 and 41 nucleotides downstream of a TATA sequence. Regulation of transcription was investigated first by transfection of primary rat hepatocytes by a series of 5'-deleted rat ntcp promoter-driven luciferase constructs (from approximately -6 kilobase pairs to -59 base pairs of upstream sequences, terminating at nucleotide +47), identifying a minimal promoter element: nucleotide -158 to +47. This minimal promoter was active in transfected HepG2, but inactive in NIH3T3, Caco-2, and Madin-Darby canine kidney cells, indicating that the determinants of hepatocyte-specific expression reside within this region. The individual elements within the minimal promoter were investigated via transfection of HepG2 cells by a series of 20 mutant plasmids, each containing a 10-base pair sequential block mutation. Eight mutant constructs profoundly suppressed promoter activity; encompassing sequences from -66 to +4 nt, and +15 to +24 nucleotides, while no other 10-base pair mutation significantly interfered with minimal promoter activity. Deoxyribonuclease I footprint analysis of the minimal promoter revealed three bound regions; -92 to -74 (footprint C), -50 to -37 (footprint B), and -17 to +12 (footprint A). Gel mobility shift assays provided evidence for hepatocyte nuclear factor 1 binding within footprint A and a liver-enriched factor(s) that binds within a novel palindrome in footprint B. These studies indicate that three elements direct the basal and tissue-restricted expression of the rat ntcp promoter; a TATA element, the liver-enriched transcription factor hepatocyte nuclear factor 1, and an unknown liver-enriched factor that binds within a novel palindrome in footprint B.