Transcriptional Regulation of CYP2B6 Expression by Hepatocyte Nuclear Factor 3β in Human Liver Cells.

Linhao Li,Daochuan Li,Scott Heyward,Hongbing Wang,Makoto Makishima

doi:10.1371/journal.pone.0150587

Abstract

CYP2B6 plays an increasingly important role in xenobiotic metabolism and detoxification. The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) have been established as predominant regulators for the inductive expression of CYP2B6 gene in human liver. However, there are dramatic interindividual variabilities in CYP2B6 expression that cannot be fully explained by the CAR/PXR-based modulation alone. Here, we show that expression level of CYP2B6 was correlated with that of hepatocyte nuclear factor 3β (HNF3β) in human primary hepatocytes prepared from 35 liver donors. Utilizing recombinant virus-mediated overexpression or knockdown of HNF3β in HepG2 cells, as well as constructs containing serial deletion and site-directed mutation of HNF3β binding motifs in CYP2B6 luciferase reporter assays, we demonstrated that the presence or lack of HNF3β expression markedly correlated with CYP2B6 gene expression and its promoter activity. Novel enhancer modules of HNF3β located upstream of the CYP2B6 gene transcription start site were identified and functionally validated as key elements governing HNF3β-mediated CYP2B6 expression. Chromatin immunoprecipitation assays in human primary hepatocytes and surface plasmon resonance binding affinity experiments confirmed the essential role of these enhancers in the recruitment of HNF3β to the promoter of CYP2B6 gene. Overall, these findings indicate that HNF3β represents a new liver enriched transcription factor that is involved in the transcription of CYP2B6 gene and contributes to the large interindividual variations of CYP2B6 expression in human population.

Highlights

Traditionally thought to be of minor significance in pharmacology and toxicology [1], the clinical importance of CYP2B6 was recently established with the identification of increasing numbers of CYP2B6 substrates, including clinically important drugs such as the anticancer agents, cyclophosphamide, ifosfamide [2, 3] and tamoxifen [4], the anti-retrovirals efavirenz and PLOS ONE | DOI:10.1371/journal.pone.0150587 March 1, 2016Transcriptional Regulation of CYP2B6 by hepatocyte nuclear factor 3β (HNF3β)
These results indicate that besides the known role of constitutive androstane receptor (CAR), hepatic nuclear factor 4α (HNF4α) and CCAAT/enhancer-binding protein α (C/EBPα) in CYP2B6 expression, HNF3β may represent another liver enriched transcription factors (LETFs) that contributes to the transcription of CYP2B6 gene in human liver
Expression of CYP2B6 is controlled by many transcription factors including drug and hormone responsive nuclear receptors such as CAR, pregnane X receptor (PXR), the vitamin D and glucocorticoid receptors, and constitutively activated LETFs including HNF4α and C/EBPα [21, 36,37,38]

Summary

Introduction

Traditionally thought to be of minor significance in pharmacology and toxicology [1], the clinical importance of CYP2B6 was recently established with the identification of increasing numbers of CYP2B6 substrates, including clinically important drugs such as the anticancer agents, cyclophosphamide, ifosfamide [2, 3] and tamoxifen [4], the anti-retrovirals efavirenz and PLOS ONE | DOI:10.1371/journal.pone.0150587 March 1, 2016Transcriptional Regulation of CYP2B6 by HNF3β. Traditionally thought to be of minor significance in pharmacology and toxicology [1], the clinical importance of CYP2B6 was recently established with the identification of increasing numbers of CYP2B6 substrates, including clinically important drugs such as the anticancer agents, cyclophosphamide, ifosfamide [2, 3] and tamoxifen [4], the anti-retrovirals efavirenz and PLOS ONE | DOI:10.1371/journal.pone.0150587.

Methods

Results

Conclusion