No 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 sodiumdependent bile acid cotransporter (ntcp) gene expression, we isolated Ϸ30 kilobase pairs of rat genomic DNA in three overlapping phage 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 promoterdriven luciferase constructs (from Ϸ ؊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 liverenriched 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 liverenriched transcription factor hepatocyte nuclear factor 1, and an unknown liver-enriched factor that binds within a novel palindrome in footprint B.

No abstract

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that ∼75% of each protein was delivered to the basolateral surface compared with ∼90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 ± 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.

The ASBT promoter contains 2 distinct cis AP-1 elements; the 5' element binds homodimeric c-jun and mediates basal transcription. Inflammation is associated with up-regulation, phosphorylation, and nuclear translocation of c-fos, which then represses ASBT promoter activity via binding of the 3' AP-1 element by a c-fos/c-jun heterodimer.