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

Abstract

The formation of ␤-D-glucopyranosides (glucuronides) by the UDP-glucuronosyltransferases (UGTs) is a significant metabolic pathway that facilitates the elimination of small hydrophobic molecules such as drugs, dietary constituents, steroids, and bile acids. We elucidate here that an anti-oxidative response leads to induction of UGT1A1 through the Nrf2-Keap1 pathway. When human HepG2 cells were treated with the prooxidants tert-butylhydroquinone and ␤-naphthoflavone, cellular UGT1A1 glucuronidation activities were increased. The induction of UGT1A1 proceeded following the overexpression of Nrf2 and was blocked following overexpression of Keap1, demonstrating that Keap1 suppresses Nrf2 activation of the UGT1A1 gene. Loss of function analysis for Nrf2 conducted by small interfering RNA revealed that induction of UGT1A1 was not seen in Nrf2 knock-out cells. To examine the contribution of oxidants toward the regulation of human UGT1A1 in vivo, transgenic mice bearing the human UGT1 locus (Tg-UGT1) were treated with tert-butylhydroquinone. Human UGT1A1 was markedly increased in small and large intestines as well as in liver. Gene mapping experiments including transfections of UGT1A1 reporter gene constructs into HepG2 cells coupled with functional analysis of Nrf2 expression and binding to antioxidant-response elements (ARE) resulted in identification of an ARE in the phenobarbital-response enhancer module region of the UGT1A1 gene. The ARE flanks the recently identified Ah receptor xenobiotic-responsive element. The results suggest that Nrf2-Keap1-dependent UGT1A1 induction by prooxidants might represent a key adaptive response to cellular oxidative stress that defends against a variety of environmental insults, including electrophile attacks and chemical carcinogenesis.

Coordinate Control of Expression of Nrf2-Modulated Genes in the Human Small Airway Epithelium Is Highly Responsive to Cigarette Smoking

“…Specificity was further confirmed by the disappearance of the gel shift band in the presence of an antiNrf2 affinity-purified polyclonal antibody (lane 7), whereas no disappearance was seen with the use of a corresponding IgG control (lane 8). Instead of a supershift, the disappearance of the gel shift band suggests a competition for binding between the Nrf2 antibody and NQO1 oligonucleotide, similar to that reported for Nrf2 binding to an antioxidant response element of UGT1A1 with nuclear extracts from HepG2 cells after oxidative stress (38). Figure 3.…”

“…Newly identified Nrf2-modulated genes were validated with electrophoretic mobility shift assay (37,38). We incubated 10 μg of nuclear extract from small airway epithelium with a 32 P-labeled antioxidant response element oligonucleotide from NAD(P)H dehydrogenase, quinone 1 (NQO1), a known Nrf2-modulated gene (10,37).…”

Transcriptional expression analysis of ABC efflux transporters and xenobiotic-metabolizing enzymes in the Chinese rare minnow

“…In line with this, the role of Nrf2 in mediating the co-regulation of the phase II enzymes and ABC transporters was further confirmed by the discovery of AREs (antioxidant response elements) in many of these genes. For example, the AREs have been detected in many genes of the phase II enzymes including the GST (Wasserman and Fahl, 1997;Kwak et al, 2003), GCLC (Kwak et al, 2003), and UGT1A1 genes (Yueh and Tukey, 2007); ARE-like sequences were also identified in the mouse ABCCs (Vollrath et al, 2006). These finding suggest that a common regulatory mechanism is possible for the GST, GCLC and ABCC genes (Meijerman et al, 2008).…”

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

“…It is tempting to speculate that evolution of the gtPBREM cluster of binding sites for a number of LATFs in the promoter of UGT1A1 [46,47] may be related to the need for perinatal UGT1A1 induction in primates. This cluster contains binding sites for CAR and PXR [47], AhR [33], Nrf2 [76], PPARα [51] and for the glucocorticoid receptor [47]. It is obvious that perinatal induction represents a stressful condition.…”