DNA elements for constitutive androstane receptor- and pregnane X receptor-mediated regulation of bovine CYP3A28 gene

• DOI: 10.1371/journal.pone.0214338
• Publication Date: 2019-03-25T17:26:36Z
• Authors: Mery Giantin; Jenni Küblbeck; Vanessa Zancanella; Viktoria Prantner; Fabiana Sansonetti; Axel Schoeniger; Roberta Tolosi; Giorgia Guerra; Silvia Da Ros; Mauro Dacasto; Paavo Honkakoski
• Keywords: 0303 health sciences; Binding Sites; Science; Q; R; Pregnane X Receptor; Receptors, Cytoplasmic and Nuclear; Hep G2 Cells; Sequence Analysis, DNA; Transfection; 03 medical and health sciences; Gene Expression Regulation; Biochemistry, Genetics and Molecular Biology (all); Agricultural and Biological Sciences (all); Cell Line, Tumor; Medicine; Animals; Cytochrome P-450 CYP3A; Humans; Cattle; Cloning, Molecular; Promoter Regions, Genetic; Constitutive Androstane Receptor; Research Article; Transcription Factors

The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter (PP; -284/+71 bp) contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.