Basic transcriptional regulation and cytokine responsiveness of the rat ileal bile salt transporter (ASBT) gene is mediated through AP-1

Abstract

Introduction: Transcriptional regulation of the ileal bile salt transporter (ASBT) appears to be involved in a wide range of regulated phenomenon including development, tissue specificity and cytokine responsiveness. The following studies were directed at understanding the molecular mechanisms of the regulation. MethodslResults:The rat ASBT gene, which was cloned from a PI library, extends over approximately 20 kb and contains five introns. Primer extension analysis identified two sites of transcription initiation. A 3.2 kb of 5' flanking region was cloned, and sequenced and 3.2, 2.0, 1.2, 0.5 and 0.3 kb fragments were subcloned into a luciferase expression vector. Promoter activity was greatest with the 3.2 kb fragment, although all fragments imparted promoter activity. The promoter activity of the fragments was cell line specific as it was observed in Caco-2, but not HepG2, MDCK and HeLa cells. Gel shift and cross linking studies with cellular extracts from Caco-2 and HepG2 cells revealed four distinct protein:DNA complexes, one of which was specific for Caco-2 and HepG2 cell extracts. These DNA:protein interactions were specific and could be competed. Sequence analysis revealed three potential AP-I sites in the proximal promoter sequence, one of which specifically bound protein elements from Caco-2 cells. It also competed for the specific complex seen in Caco-2 cells. Supershifting of the complex could be observed using c-jun antibodies. Mutation of a single nucleotide in the consensus AP-I site of the 15 bp oligonucleotide disrupted these specific DNA:protein interactions. TNF-aand IL-I {3, but not IL-6 down-regulated promoter activity in a dose-dependent fashion, and was mediated through the same AP-I sites as determined by gel shift. Corticosteroids up-regulated promoter activity in a dose-dependent fashion, through a different site, which was found only in the 3.2 and 2.0 kb fragments. Conclusion: Basic transcriptional activity and cytokine-mediated down-regulation of the ASBT gene appears to be mediated at least in part through a proximal AP-I site, while corticosteroidmediated up-regulation is mediated through distinct upstream elements.