The UDP–glucuronosyltransferase, UGT1A1, is the critical enzyme responsible for detoxification of the potentially neurotoxic bilirubin by conjugating it with glucuronic acid. For decades, phenobarbital (PB) treatment for hyperbilirubinemia has been known to increase expression of the UGT1A1 gene in liver. We have now delineated the PB response activity to a 290–bp distal enhancer sequence (-3483/-3194) of the UGT1A1 gene. The enhancer contains 3 putative nuclear receptor motifs, and it was activated by the nuclear orphan receptor, human constitutive active receptor (hCAR), in cotransfected HepG2 cells. Bacterially expressed hCAR, acting as a heterodimer with in vitro-translated retinoid X receptor (RXRα), only bound to 1 of the 3 NR motifs, named gtNR1 in a gel–shift assay. Consistently, mutations of the gtNR1 site significantly decreased the activation by hCAR of the 290–bp DNA in transfection assays. Moreover, the 290–bp DNA was effectively activated in mouse primary hepatocytes in response to PB, offering an excellent clinical test for the examination of the responsiveness of the UGT1A1 to PB in the human population, particularly individuals with hyperbilirubinemia. (Hepatology 2001;33:1232–1238.)

UDP-glucuronosyltransferase (UGT) 1A1 glucuronidates endogenous metabolites, such as bilirubin, and exogenous substances, and plays a critical role in their detoxification and excretion. In a previous article, we described the phenobarbital response activity to a 290-base pair (bp) distal enhancer sequence (–3499/–3210) of the human UGT1A1 gene that is activated by the constitutive androstane receptor (CAR). Here, we show that dexamethasone at submicromolar concentrations enhances the pregnane X receptor (PXR) activator-mediated expression of the UGT1A1 gene and protein in HepG2 cells. We investigated the molecular mechanism of UGT1A1 induction by glucocorticoids at submicromolar concentrations and PXR activators and the functional cross-talk between the glucocorticoid receptor (GR) and CAR/PXR. The glucocorticoid-response element (GRE) was characterized by cotransfection experiments, site-directed mutagenesis, and electrophoretic mobility shift assays. Analysis of the human UGT1A1 promoter revealed GREs at –3404/–3389 and –3251/–3236 close to the CAR/PXR response element gtNR1 (–3382/–3367). Furthermore, in an in vitro reporter gene assay, dexamethasone effectively enhanced CAR/PXR-mediated transactivation of the 290-bp distal enhancer module in HepG2 cells and CV-1 cells in the presence of exogenously expressed GR and glucocorticoid receptor-interacting protein 1 (GRIP1). In glutathione S-transferase pull-down experiments, CAR and PXR interacted with GRIP1. Together, these results demonstrate a rational mechanistic basis for UGT1A1 induction by glucocorticoids and PXR activators, showing that activated GR enhances CAR/PXR-mediated UGT1A1 regulation with the transcriptional cofactor GRIP1 and that GR may be involved synergistically in the xenobiotic-responsive regulation of UGT1A1 by CAR/PXR.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is an important transcription factor in hepatic gene expression. Here, we have investigated the role of HNF4alpha in the expression of drug-metabolizing enzymes and transporters in human hepatocytes using an adenovirus expressing human HNF4alpha-small interfering RNA (hHNF4alpha-siRNA). The hHNF4alpha-siRNA effectively reduced the mRNA and nuclear protein levels of hHNF4alpha in a concentration-dependent manner. The hHNF4alpha-siRNA also decreased the mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A9, SULT2A1, ABCB1, ABCB11, ABCC2, OATP1B1 and OCT1, as well as those of PXR and CAR. To discern the role of these nuclear receptors, we co-infected hepatocytes with hHNF4alpha-siRNA and PXR- or CAR-expressing adenovirus. The hHNF4alpha-siRNA-induced reductions of the enzyme and transporter mRNA levels were not restored except CYP2B6 mRNA levels, which were returned to the control level by overexpressing CAR. Furthermore, although hHNF4alpha-siRNA did not significantly affect the fold-induction of CYP2B6, CYP2C8, CYP2C9, or CYP3A4 mRNA levels following treatment with CYP inducers, the levels in hHNF4alpha-suppressed cells fell significantly compared to the control. These results suggest that HNF4alpha plays a dominant role in the expression of drug-metabolizing enzymes and transporters in human hepatocytes, and that HNF4alpha expression levels is a possible determinant for inter-individual variations in the expression of these enzymes and transporters.