Hepatocyte nuclear factor 4α (HNF4α, NR2A1) is required for development of the liver and for controlling the expression of many genes specifically expressed in the liver and associated with a number of critical metabolic pathways. Among the genes regulated by HNF4α are the xenobiotic-metabolizing cytochromes P450, UDP-glucuronosyltransferases and sulfotransferases thus making this transcription factor critical in the control of drug metabolism. HNF4α, a member of the nuclear receptor superfamily, binds as a homodimer to direct repeat elements upstream of target genes. However, in contrast to many other nuclear receptors, there is no convincing evidence that HNF4α is activated by exogenous ligands, at least in the classic mechanism used by other steroid and metabolic nuclear receptors. X-ray crystallographic studies revealed that HNF4α has a fatty acid embedded in its putative ligand binding site that may not be easily released or displaced by exogenous ligands. HNF4α, as a general rule, controls constitutive expression of many hepatic genes but under certain circumstances can be subjected to regulation by differential co-activator recruitment, by phosphorylation and by interaction with other nuclear receptors. The ability of HNF4α to be regulated offers hope that it could be a drug target.
Nuclear receptors constitutive active/androstane receptor (CAR) and pregnane X receptor (PXR) were originally characterized as transcription factors regulating the hepatic genes that encode drug metabolizing enzymes. Recent works have now revealed that these nuclear receptors also play the critical roles in modulating hepatic energy metabolism. While CAR and PXR directly bind to their response sequences phenobarbital-responsive enhancer module (PBREM) and xenobiotic responsive enhancer module (XREM) in the promoter of target genes to increase drug metabolism, the receptors also cross talk with various hormone responsive transcription factors such as forkhead box O1 (FoxO1), forkhead box A2 (FoxA2), cAMP-response element binding protein, and peroxisome proliferator activated receptor γ coactivator 1α (PGC 1α) to decrease energy metabolism through down-regulating gluconeogenesis, fatty acid oxidation and ketogenesis and up-regulating lipogenesis. In addition, CAR modulates thyroid hormone activity by regulating type 1 deiodinase in the regenerating liver. Thus, CAR and PXR are now placed at the crossroad where both xenobiotics and endogenous stimuli co-regulate liver function.
Human body needs to protect itself from a diverse array of harmful chemicals. These chemicals are also involved in drug metabolism, enzyme induction, and can cause adverse drug-drug interactions. Being a member of nuclear receptors (NRs), pregnane X receptor (PXR) has recently emerged as transcriptional regulators of cytochrome P450 (CYP) and transporters expression so as to against xenobiotics exposure. This review describes some common nuclear receptors, i.e. farnesoid X receptor (FXR), small heterodimer partner (SHP), hepatocyte nuclear factor-4α (HNF-4α), liver X receptor (LXR), glucocorticoid receptor (GR), constitutive androstane receptor (CAR) that crosstalk with PXR and involvement of coregulators thus control target genes expression.
Pairs of forward and reverse primers and TaqMan probes specific to each of 173 human solute carrier (SLC) transporters were prepared. The mRNA expression level of each target transporter was analyzed in total RNA from single and pooled specimens of various human tissues (adrenal gland, bladder, bone marrow, brain, colon, heart, kidney, liver, lung, mammary gland, ovary, pancreas, peripheral leukocytes, placenta, prostate, retina, salivary gland, skeletal muscle, small intestine, smooth muscle, spinal cord, spleen, stomach, testis, thymus, thyroid gland, trachea, and uterus) by real-time reverse transcription PCR using an Applied Biosystems 7500 Fast Real-Time PCR System. Individual differences in the mRNA expression of human SLC transporters in the liver were also evaluated. These newly determined expression profiles were used to study the gene expression in the 28 different human tissues listed above, and tissues with high transcriptional activity for human SLC transporters were identified. These results are expected to be valuable for research concerning the clinical diagnosis of disease.
We investigated the change of the mRNA levels of sulfotransferase and UDP-glucuronosyltransferase isoforms by the prototypical microsomal enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) in primary cultures of cryopreserved human and cynomolgus monkey hepatocytes. Real-time RT-PCR analysis was performed using primers and TaqMan probes. Rif, Dex, and Ome increased SULT2A1 mRNA level in both human and cynomolgus monkey hepatocytes in dose-dependent manner, but not SULT1A1 mRNA level. Rif, Dex, and Ome increased the mRNA level of UGT1A1 in both human and cynomolgus monkey hepatocytes, Ome more potently in humans and Rif and Ome more potently in monkeys. They also increased the mRNA levels of UGT1A6 and UGT1A9 in cynomolgus monkey hepatocytes, though the extent of elevation of UGT1A6 and UGT1A9 mRNA levels was smaller than that of UGT1A1 mRNA level. Furthermore, these inducers scarcely affected UGT1A6 and UGT1A9 in human hepatocytes. Rif, Dex, and Ome also showed no remarkable effect on the mRNA levels of UGT2Bs in human or cynomolgus monkey hepatocytes. We also studied in detail the time course of mRNA expression of these enzymes in primary cultures of hepatocytes. In conclusion, the results of the present study show that primary cultures of hepatocytes isolated from the cynomolgus monkey liver are as useful as human hepatocytes for evaluating the induction of drug-metabolizing enzymes in preclinical studies.
We reported the human flavin-containing monooxygenase 3 (FMO3) haplotypes (Pharmacogenet. Genomics: 17, 827, 2007). The objective was to gain the insight into transcriptional regulation in a Japanese population. The wild-type FMO3 reporter plasmids carrying 5′-flanking sequence from the transcriptional initiation site of the FMO3 haplotype 1 (prepared from three individuals) showed higher luciferase activities in HepG2 cells than those from the FMO3 haplotypes 2 and 3, with the wild-type coding region. Several deletion mutants of the FMO3 haplotype 1 (extending from −5,167 to −1,764, numbered relative to the A of the ATG translational initiation codon) revealed that the region of −2,064 to −1,804 contained an important cis-acting element(s) for activation of the FMO3 gene expression. Putative hepatocyte nuclear factor-4 (HNF-4) binding site and CCAAT box, but not Yin Yang 1 element, could be responsible cis-acting elements of the FMO3 gene, by site-directed mutagenesis analysis. The unknown suppressive cis-element(s) at the 5′-upstream region from −2,064 might show genetic polymorphism, because the FMO3 haplotypes 2 and 3 had three and ten mutations, respectively. These results suggest that the putative HNF-4 binding site and CCAAT box could be responsible cis-acting elements of the FMO3 gene in Japanese.
Pregnane X receptor (PXR; NR1I2), a key transcriptional factor that regulates genes encoding drug-metabolizing enzymes and drug transporters, is abundantly expressed in the human liver. However, studies on the molecular mechanism of human PXR gene regulation are limited. In this study, we examined the involvement of hepatocyte nuclear factor 4alpha (HNF4α; NR2A1) in the transcriptional regulation of the human PXR gene in the human liver. The activities of the human PXR promoter containing the direct repeat 1 (DR1) element located at −88/−76 of the promoter were significantly increased by co-expression of HNF4α in the human hepatocellular carcinoma cell line. In addition, introduction of mutation into the DR1 element abolished the transcriptional activation of the human PXR promoter by exogenous HNF4α. The results of gel mobility shift assays and chromatin immunoprecipitation assays showed that HNF4α was bound to the promoter region containing the DR1 element. A knock-down of HNF4α by siRNA significantly decreased expression levels of endogenous PXR mRNA in HepG2 cells. Furthermore, expression levels of PXR mRNA positively correlated with those of HNF4α mRNA in 18 human liver samples. These results suggested that HNF4α transactivated the human PXR gene by binding to the DR1 element located at −88/−76 of the promoter and was involved in the expression of PXR in the human liver.
Since rat organic cation transporter 1 (Oct1, Slc22a1) is expressed mainly in the liver and mediates drug transport, its activity may determine the hepatic handling of cationic drugs. Here, we studied the regulation mechanism of the expression of rat Oct1, focusing on the nuclear receptors. Various nuclear receptors are considered to regulate expressions of many genes for membrane transporters and enzymes that are involved in the drug absorption and disposition. Previously, we demonstrated that some ligands of nuclear receptors affected the transcriptional regulation of rat Oct1 when examined in the primary cultured rat hepatocytes. In the present study, dexamethasone, a ligand of glucocorticoid receptor, down-regulated the expression of rat Oct1. In addition, the transport activity of rat Oct1, evaluated by the uptake of substrates of rat Oct1, was decreased by treatment of dexamethasone in comparison with untreated rat hepatocytes, showing a good agreement with the change in mRNA level. In conclusion, these observations suggested that the expression of rat Oct1 gene and the apparent organic cation uptake activity of rat hepatocytes are down-regulated by dexamethasone presumably via a glucocorticoid receptor.
Mammalian carboxylesterases comprise a multigene family, the gene products of which are localized in the endoplasmic reticulum. The carboxylesterases catalyze the hydrolysis of various xenobiotics and endogenous substrates such as ester, amide and thioester bonds and are thought to function mainly in drug metabolism. We have suggested the possibility that individual variation of human liver carboxylesterase activity causes the difference in expression levels of CES1A isozymes. However, little is known about the transcriptional regulation of human carboxylesterase genes. In the present study, we isolated two CES genes encoding human carboxylesterase CES1A, which were designated as CES1A1 (AB119997) and CES1A2 (AB119998). These genes were identical except for exon 1 and the 5′ regulatory element. We investigated the transcriptional regulation of these two CES genes. A reporter gene assay and electrophoretic mobility shift assay demonstrated that Sp1 and C/EBPα could bind to each responsive element of the CES1A1 promoter but that the Sp1 and C/EBP could not bind to the responsive element of the CES1A2 promoter. Thus, CES1A1 mRNA expression level is much higher than the expression level of CES1A2 mRNA in the liver and lung. It is thought that these results provide information on individual variation of human carboxylesterase isozymes.