Involvement of the Xenobiotic Response Element (XRE) in Ah Receptor-mediated Induction of Human UDP-glucuronosyltransferase 1A1

Abstract

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays an important physiological role by contributing to the metabolism of endogenous substances such as bilirubin in addition to xenobiotics and drugs. The UGT1A1 gene has been shown to be inducible by nuclear receptors steroid xenobiotic receptor (SXR) and the constitutive active receptor, CAR. In this report, we show that in human hepatoma HepG2 cells the UGT1A1 gene is also inducible with aryl hydrocarbon receptor (Ah receptor) ligands such as 2,3,7,8-tetrachlodibenzo-p-dioxin (TCDD), ␤-naphthoflavone, and benzo[a]pyrene metabolites. Induction was monitored by increases in protein and catalytic activity as well as UGT1A1 mRNA. To examine the molecular interactions that control UGT1A1 expression, the gene was characterized and induction by Ah receptor ligands was regionalized to bases ؊3338 to ؊3287. Nucleotide sequence analysis of this UGT1A1 enhancer region revealed a xenobiotic response element (XRE) at ؊3381/؊3299. The dependence of the XRE on UGT1A1-luciferase activity was demonstrated by a loss of Ah receptor ligand inducibility when the XRE core region (CACGCA) was deleted or mutated. Gel mobility shift analysis confirmed that TCDD induction of nuclear proteins specifically bound to the UGT1A1-XRE, and competition experiments with Ah receptor and Arnt antibodies demonstrated that the nuclear protein was the Ah receptor. These observations reveal that the Ah receptor is involved in human UGT1A1 induction.

From differential induction of UDP-glucuronosyltransferases in rat liver to characterization of responsible ligand-activated transcription factors, and their multilevel crosstalk in humans

“…The first XRE was characterized in the promoter region of CYP1A1 [28]. In the case of UGTs, XREs were first identified in the regulatory region of rat liver UGT1A6 [29], human UGT1A6 [30,31] and human UGT1A1 [32]. In fact, all human UGT1 family members appear to be regulated by the AhR [33] in cooperation with subsequently discussed Nrf2.…”

Nrf2-Keap1 Signaling Pathway Regulates Human UGT1A1 Expression in Vitro and in Transgenic UGT1 Mice

“…Identification of the Response Element in the UGT1A1 Promoter Region-A 11-kb region of the UGT1A1 promoter region was amplified by PCR from a human BAC library, as described previously (11), and subcloned into either PGL basic vector or PGL promoter vectors. For transient transfections, HepG2 cells were seeded in a 12-well plate (2 ϫ 10 5 cells/well) 1 day before transfections and grown overnight to 50% confluence.…”

“…Following tBHQ treatment, induced luciferase activity was compromised significantly when the ARE-1 sites were mutated in comparison with mutation of the ARE-2 and ARE-3 sites. It is noted that this 60-bp fragment also consists of an XRE, an enhancer element responsible for Ah receptor-mediated transcription activation (11). We further examined the involvement of the Ah receptor in tBHQ-mediated transactivation.…”

“…The xenobiotic receptors pregnane X receptor (5), the constitutive androstane receptor (6), the peroxisome proliferator-activated receptors (7,8), the liver X receptor (9) as well as the Ah receptor (10,11) have been shown in tissue culture and Tg-UGT1 mice studies to regulate expression of the UGT genes (4). Recently, the transcription factor Nrf2 has been identified as the major regulator of cytoprotective genes encoding detoxification and antioxidant enzymes (12,13).…”

“…In tissue culture and Tg-UGT1 mice, UGT1A1 has been shown to be induced by activators of the Ah receptor, constitutive androstane receptor, pregnane X receptor, and the glucocorticoid receptor (4,11,23,24). The DNA binding domain of the UGT1A1 promoter for all of these activated transcriptional factors resides approximately Ϫ3300 bp from the start of transcription within a 300-bp region identified as the phenobarbital-responsive enhancer module.…”