The induction of human UDP-glucuronosyltransferase 1A1 mediated through a distal enhancer module by flavonoids and xenobiotics

Abstract

We identified the UDP-glucuronosyltransferase (UGT) 1A1 5′-upstream region that confers UGT1A1 induction by various agents, including flavonoids, on a luciferase reporter gene and has the properties of a transcriptional enhancer. Chrysin- and rifampicin-response activities were traced to the same element as a 290-bp distal enhancer module (−3483/−3194), in which the reporter activities were enhanced by activators of nuclear receptors [constitutive androstane receptor (CAR) and pregnane X receptor (PXR)] and transcription factor [aryl hydrocarbon receptor (AhR)]. Utilizing transactivation experiments with the UGT1A1 290-bp reporter gene, we assessed UGT1A1 induction by various flavonoids. 5,7-Dihydroxyflavones with varying substituents in the B-ring and gallocatechin dimers increased the reporter activity in a time- and dose-dependent manner. The treatment of HepG2 cells with the flavonoids for 24 hr elevated the expression of mRNAs and proteins of UGT1A1 and CYP1A1, while the mRNA levels of CYP2B6, CYP3A4, CAR, PXR and AhR was not altered. Chrysin and rifampicin induced the activation of the wild-type reporter gene and T–3263G-mutated gene to a similar extent in HepG2 cells cotransfected with expression vectors of CAR and PXR. Mutation of the AhR core binding region most prominently suppressed the activation of the 290-bp reporter gene by chrysin and baicalein, while mutations of four putative nuclear receptor motifs (DR4 element, PXRE, CARE and DR3 element) partly decreased its activation. Taken together, the results indicate that UGT1A1 was induced in response to flavonoids and xenobiotics through the transactivation of the 290-bp reporter gene, that was a multi-component enhancer containing CAR, PXR and AhR motifs.

Introduction

UDP-glucuronosyltransferase, UGT1A1, plays a critical role in the detoxification of potentially neurotoxic bilirubin by conjugating it with glucuronic acid [1], and conjugates many drugs and other xenobiotics [2], [3], [4]. The requirements for the glucuronidation of bilirubin for excretion were first elucidated on the basis of the finding that the severe form of unconjugated hyperbilirubinemia in the absence of hemolysis or other liver disease, known as Crigler–Najjar type I syndrome, can be fatal [5]. Reduced UGT1A1 activity causes unconjugated hyperbilirubinemia (Crigler–Najjar syndrome and Gilbert’s syndrome) [6] and decreases the glucuronidation of SN-38 (a pharmacologically active metabolite of the anticancer drug, irinotecan), leading to an increased risk for the development of severe irinotecan-associated toxicity [3], [7]. Phenobarbital is used as a therapeutic drug for patients with Crigler–Najjar type II syndrome, because it increases the expression of bilirubin glucuronosyltransferase and markedly reduces the incidence of unconjugated hyperbilirubinemia [8]. The 51-bp phenobarbital-responsive enhancer module, which is conserved between species, has been identified in phenobarbital-inducible CYP2B genes and is regulated by the nuclear receptor known as constitutive androstane receptor (CAR; NR1I3) in response to phenobarbital induction [9]. Furthermore, we have identified the module in the phenobarbital-inducible human UGT1A1 gene (UGT1A1 phenobarbital-responsive enhancer module, PBREM), and have identified CAR as a transcription factor regulating PBREM in response to phenobarbital induction [10]. PBREM is a composite 290-bp element consisting of four nuclear receptor motifs, named DR4 element, CAR response element (CARE) and DR3 element [10] and pregnane X receptor (PXR; NR1I2) response element (PXRE) [11], and aryl hydrocarbon receptor (AhR) response element (AhRE) [12]. The functional roles of DR4 and DR3 elements are still unknown, whereas CARE, PXRE and AhRE have been reported as the binding sites of CAR, PXR and AhR, respectively. Recently, a single nucleotide polymorphism changing T to G at nucleotide −3263 has been found in UGT1A1[13]. Since the T–3263G mutation in the direct-repeat spacer of the PBREM DR3 site significantly decreases the transcriptional activity (associated with increased plasma total bilirubin levels) [13], the DR3 site may play an important role in the regulation of UGT1A1 expression in human subjects.

The modulation of UGT1A1 expression by dietary factors is of considerable interest, because phenobarbital is a therapeutic drug for mild unconjugated hyperbilirubinemia but has the serious disadvantage of inducing sleep. Walle et al. have demonstrated that the flavonoid chrysin can induce UGT1A1 mRNA and protein in Caco-2 cells [14] and HepG2 cells [15], [16]. As the mechanism by which chrysin induces UGT1A1 expression has not been addressed, we focused on the induction of UGT1A1 in response to dietary flavonoids. First, we clarified the location of the chrysin-responsive enhancer element in the UGT1A1 gene using constructs in which various DNA fragments generated from a 11-kbp 5′-flanking region of UGT1A1 were placed in front of the reporter luciferase gene and examined for enhancer activity in HepG2 cells. Surprisingly, we traced the chrysin-response activity as well as the rifampicin-response activity to the 290-bp distal enhancer module, which is activated by CAR, PXR and AhR [10], [11], [12]. In this study, we further investigated the effects of dietary flavonoids and xenobiotics on human UGT1A1 induction, utilizing a screening system for assessing UGT1A1 induction with the 290-bp distal enhancer module, and the characterization of UGT1A1 induction by flavonoids and xenobiotics.