Synergistically Enhanced CYP2B6 Inducibility between a Polymorphic Mutation in CYP2B6 Promoter and Pregnane X Receptor Activation

Abstract

CYP2B6 is a highly inducible and polymorphic enzyme involved in the metabolism of an increasing number of clinically important drugs. Significant interindividual variability in CYP2B6 expression has been attributed to either genetic polymorphisms or chemical-mediated induction through the activation of constitutive androstane receptor and/or pregnane X receptor (PXR). It was reported that the Ϫ82T3 C substitution within the CYP2B6*22 allele creates a functional CCAAT/enhancer-binding protein (C/EBP) binding site and enhances the basal expression of the CYP2B6 gene. Here, we explored whether this polymorphic mutation could affect drug-mediated induction of CYP2B6. Cell-based promoter reporter assays demonstrated that CYP2B6 luciferase activity was synergistically enhanced in the presence of both Ϫ82T3 C mutation and rifampicin (RIF)-activated PXR. On the other hand, this synergism was attenuated by disrupting the C/EBP binding site or knocking down C/EBP␣ expression. Mechanistic studies revealed that C/EBP␣ plays an important role in such synergism by directly interacting with PXR; enhancing RIF-mediated recruitment of PXR to the Ϫ82T3 C harboring CYP2B6 promoter; and looping the PXRbound distal phenobarbital-responsive enhancer module toward the proximal C/EBP binding site. Furthermore, the genotype-phenotype association was evaluated in cultured human primary hepatocytes from 44 donors. Interestingly, RIF-mediated induction of CYP2B6 in four Ϫ82T/C carriers was higher compared with that in the reference Ϫ82T/T homozygotes. Together, our results demonstrate, for the first time, a synergistic interplay between a CYP2B6 polymorphism and PXRmediated induction, which may contribute to the large individual variations and inducibility of CYP2B6 in humans.

Regulation of Human CYP2C9 Expression by Electrophilic Stress Involves Activator Protein 1 Activation and DNA Looping

“…In vitro 3C assays were performed essentially as previously described with minor modifications (Babu et al, 2008;Inoue and Negishi, 2009;Li et al, 2010). Nuclear extracts (100 mg) prepared from HepG2 cells transfected with empty vector or cFos expression vector were incubated with 50 ng linearized CYP2C9 plasmid (containing 23077 to 11 of the CYP2C9 promoter) at room temperature for 45 minutes.…”

Pharmacogenetics of cytochrome P450 2B6 (CYP2B6): advances on polymorphisms, mechanisms, and clinical relevance

“…It was shown that a -82T>C exchange alters the TATA-box into a functional CCAAT/enhancer-binding protein binding site that causes increased transcription from an alternative downstream initiation site (Zukunft et al, 2005). Interestingly, the -82T>C polymorphism also confers synergistically enhanced CYP2B6 inducibility by the PXR ligand rifampicin in human primary hepatocytes (Li et al, 2010). …”

Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR

“…Indeed, accumulating evidence demonstrates that a number of coactivators such as steroid receptor coactivator 1 (SRC-1), glucocorticoid receptor interacting protein 1 (GRIP-1), and peroxisome peroliferator-activated receptor coactivator 1 (PGC-1), play pivotal roles in determining the tissue specific induction of PXR target genes [60–63]. Most recently, we have shown that the interplay of the constitutive CCAAT/enhancer binding protein alpha (C/EBPα) and the xenobiotic PXR contributes to the synergistic CYP2B6 induction between a polymorphic mutation in CYP2B6 promoter and PXR activation [64]. In addition to the induction of CYPs, PXR activation also represses CYP7A1 expression as a protective feedback mechanism in response to the accumulation of bile acids in the liver, further reflecting the complexity of PXR in gene regulation [65,66].…”