Abstract
Despite the established interindividual variability and ontogeny of the CYP3A enzymes, the most abundant phase I drug-metabolizing enzymes in human liver and intestine, the mechanisms that regulate basal expression remain poorly understood. Electrophoretic mobility shift assays using nuclear proteins extracted from human prenatal and postnatal liver samples identified multiple, developmentally distinct nuclear factor I (NFI)-containing protein complexes from human liver bound to sequences from the CYP3A4 (−243/−220) and CYP3A7 (−242/−219) proximal promoters. In addition, a hepatocyte nuclear factor (HNF) 3γ-containing complex from prenatal liver interacted with CYP3A7−242/−219 but not CYP3A4−243/−220. Cotransfection of HepG2 cells with a CYP3A4 proximal promoter construct and expression vectors for the NFI isoforms NFIA1.1, NFIB2, NFIC1, NFIC2, and NFIX1 enhanced the expression of luciferase activity. In contrast, cotransfection of NFIB2, NFIC1, NFIC2, NFIX1, and NFIX2 reduced the expression of luciferase under the control of the CYP3A7 gene promoter. Mutagenesis of the NFI/HNF3γ binding sites in the CYP3A7 and CYP3A4 proximal promoters suggests that regulation of basal promoter activity by members of the NFI transcription factor family occur via multiple mechanisms. These results demonstrate that members of the NFI transcription factor family regulate CYP3A4 and CYP3A7 basal expression in an isoform- and promoter-specific manner.
- P450, cytochrome P450
- C/EBP, CCAAT/enhancer binding protein
- Sp, specificity protein
- NFI, nuclear factor I
- HNF, hepatocyte nuclear factor
- NF-Y, nuclear transcription factor Y
- YY, yin-yang
- USF, upstream stimulatory factor
- EMSA, electrophoretic mobility shift assay
- MOPS, 3-(N-morpholino)propanesulfonic acid
- TNT, Tris/NaCl/Tween 20
- RT-PCR, reverse transcription-polymerase chain reaction
- PCR, polymerase chain reaction
- RXR, retinoid X receptor
- luc, luciferase
- DMEM, Dulbecco's modified Eagle's medium
- FBS, fetal bovine serum.
Footnotes
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This work was supported by the Katherine Berry Richardson Foundation [Grant 01.4223].
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.055699
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ABBREVIATIONS:
- Received February 17, 2009.
- Accepted August 25, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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