Abstract

Cytochrome P450 3A4 (CYP3A4) is the major cytochrome P450 present in adult human liver and is involved in the metabolism of over 50% of therapeutic compounds currently in use. Since expression levels of CYP3A4 are regulated by many of these compounds, this raises the potential for drug-drug interactions and subsequent altered efficacy or toxicity of the individual compounds at the dose prescribed. Hence, understanding the molecular mechanisms of CYP3A4 regulation is of key importance in predicting and understanding such interactions. To examine this we have used DNase I footprinting and bioinformatic analysis to identify putative transcription factor binding sites within the 250 base pairs of promoter proximal to the transcription start site. We identified several protected fragments within this region that corresponded to putative binding sites for Sp1, AP2, CCAAT/enhancer binding protein (C/EBPα), and hepatic nuclear factor-3 (HNF3), as well as confirming previously identified C/EBPα, pregnane X receptor (PXR), and HNF3 binding sites. Sequential site-directed mutagenesis of C/EBPα, Sp1, HNF3, and PXR binding sites was next used to examine the role of these sites in basal CYP3A4 expression. Disruption of the C/EBPα, HNF3, and PXR binding sites all affected basal expression. Finally, the role of these sites was examined in activation of CYP3A4 expression by rifampicin, metyrapone, clotrimazole, and phenobarbital. Disruption of any of these sites either led to an altered pattern of activation by the xenobiotic, as altered maximal activation, or altered the EC₅₀ value of activation. Such effects were xenobiotic-specific, with each disrupted site playing a role in the activation of some of the xenobiotics.