Regulation Of UDP-glucuronosyltransferases Research Articles

Regulation of Mammalian UDP-Glucuronosyltransferases.

UDP-glucuronosyltransferases (UGTs) are a class of important phase II drug metabolizing enzymes (DMEs), playing essential roles in the homeostasis of endobiotics as well as the dispositional behavior of exogenous compounds. The expression and enzyme activity of UGTs are regulated by multiple dimensions of mechanisms and can be influenced by diverse factors. Thus, the intensive research of its regulatory network is pivotal for better understanding about the physiological, pathological, and therapeutic significance of UGTs. Despite the lag to the research in cytochrome P450s, extensive efforts have been made to advance the understanding of the regulatory network of UGTs in recent years. This review presents a comprehensive summary and intensive discussion about the recent advancement on the regulatory network of UGTs. UGTs can be regulated at the epigenetic level via DNA methylation and histone modification. Various nuclear receptors can influence the mRNA levels of UGTs in a ligand dependent manner. Some general transcriptional factors such as AP-1, NF-κB, and p53, and some tissue specific transcriptional factors including HFN1α and HNF4α can also regulate UGTs at the transcriptional level. Multiple miRNAs have been found to be involved in the regulation of UGTs at post-transcriptional level. UGT proteins can be directly regulated via various post-translational modifications, protein-protein interactions, and protein-chemicals interactions, leading to the alternation of enzyme activities. In addition to the well-defined genetic polymorphism that induce individual variation of UGTs, this review reinforces the importance of other mechanisms that are critical for the regulation of UGTs.

Expression of hepatic cytochrome P450s and UDP-glucuronosyltransferases in PXR and CAR double humanized mice treated with rifampicin

Nuclear receptor humanized mice models have been developed to predict regulation of drug metabolizing enzyme by xenobiotics. However, limited information is available concerning xenobiotic-induced regulation of drug metabolizing enzymes in multiple nuclear receptor humanized mice. The present study investigated the hepatic regulation of cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs) in the pregnane X receptor (PXR) and the constitutive androstane receptor double humanized mice treated with rifampicin (RIF; 10mg/kg) for 4 days. RIF increased hepatic microsomal protein and total CYP contents, and CYP reductase activity in the humanized mice, but not in normal mice. Moreover, hepatic induction of Cyp2b10, Cyp2c, and Cyp3a11 were observed only in the RIF-treated humanized mice, suggesting that the humanized mice are sensitive to RIF with respect to the regulation of the hepatic CYP system. Hepatic UGT activities using estradiol, serotonin, and mefenamic acid, but not chenodeoxycholic acid as substrates, increased in the RIF-treated humanized mice, and the glucuronidation activities of estradiol and chenodeoxycholic acid increased in RIF-treated normal mice. These results raise the possibility that a PXR-independent mechanism may be involved in hepatic regulation of UGTs by RIF.

Interaction between Oxidative Stress Sensor Nrf2 and Xenobiotic-activated Aryl Hydrocarbon Receptor in the Regulation of the Human Phase II Detoxifying UDP-glucuronosyltransferase 1A10

The defense against oxidative stress is a critical feature that prevents cellular and DNA damage. UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of xenobiotics, mutagens, and reactive metabolites and thus act as indirect antioxidants. Aim of this study was to elucidate the regulation of UGTs expressed in the mucosa of the gastrointestinal tract by xenobiotics and the main mediator of antioxidant defense, Nrf2 (nuclear factor erythroid 2-related factor 2). Xenobiotic (XRE) and antioxidant (ARE) response elements were detected in the promoters of UGT1A8, UGT1A9, and UGT1A10. Reporter gene experiments demonstrated XRE-mediated induction by dioxin in addition to tert-butylhydroquinone (ARE)-mediated induction of UGT1A8 and UGT1A10, which are expressed in extrahepatic tissue in humans in vivo. The responsible XRE and ARE motifs were identified by mutagenesis. Small interfering RNA knockdown, electrophoretic mobility shifts, and supershifts identified a functional interaction of Nrf2 and the aryl hydrocarbon receptor (AhR). Induction of UGT1A8 and UGT1A10 requires Nrf2 and AhR. It proceeds by utilizing XRE- as well as ARE-binding motifs. In summary, we demonstrate the coordinated AhR- and Nrf2-dependent transcriptional regulation of human UGT1As. Cellular protection by glucuronidation is thus inducible by xenobiotics via AhR and by oxidative metabolites via Nrf2 linking glucuronidation to cellular protection and defense against oxidative stress.

Expression of UDP-glucuronosyltransferase 1A6 isoform in Caco-2 cells stimulated with lipopolysaccharide

Glucuronidation is an important metabolic process of detoxification in all vertebrates. The reaction is catalyzed by a multigene family of UDP-glucuronosyltransferases (UGTs) able to convert many xenobiotics and endobiotics (hydrophobic substances) to inactive, water-soluble glucuronides. The UGTs play a protective role, facilitating the elimination of potentially toxic metabolites via urine, bile and feces; therefore, impairment of UGTs may have important toxicological consequences. The regulation of UGTs during bacterial infection or inflammation is not well described. In this study, we investigated the in vitro effect of lipopolysaccharide (LPS) on the expression of the UGT1A6 isoform in human colon carcinoma Caco-2 cells. Results demonstrated a significant down-regulation of UGT1A6 expression, both in terms of mRNA and protein levels, and a reduced UGT activity after LPS exposure of cell cultures, suggesting a role for endotoxins on UGT regulation mechanisms.

Regulation of UDP-glucuronosyltransferase (UGT) 1A1 by progesterone and its impact on labetalol elimination

The authors recently reported the increased oral clearance of labetalol in pregnant women. To elucidate the mechanism of the elevated oral clearance, it was hypothesized that female hormones, at the high concentrations attainable during pregnancy, enhance hepatic metabolism of labetalol. Labetalol glucuronidation, which is the major elimination pathway of labetalol, was characterized by screening six recombinant human UGTs (UGT1A1, 1A4, 1A6, 1A9, 2B4, and 2B7) for their capacity to catalyse labetalol glucuronidation. The effect of female hormones (progesterone, oestradiol, oestriol, or oestrone) on the promoter activities of relevant UDP glucuronosyltransferases (UGT) was investigated using a luciferase reporter assay in HepG2 cells. The involvement of oestrogen receptor α (ERα) and pregnane X receptor (PXR) was examined by co-transfecting ERα- or PXR-constructs. UGT1A1 and UGT2B7 were identified as the major UGT enzymes producing labetalol glucuronides (trace amount of glucuronide conjugate was formed by UGT1A9). The activities of the UGT1A1 promoter containing PXR response elements were enhanced by progesterone, but not by oestrogens, indicating PXR-mediated induction of UGT1A1 promoter activity by progesterone. Results from semi-quantitative real-time polymerase chain reaction (PCR) assays are consistent with the above findings. This effect of progesterone on UGT1A1 promoter activities was concentration dependent. Promoter activities of UGT2B7 were not affected by either oestrogens or progesterone. The results suggest a potential role for progesterone in regulating labetalol elimination by modulating the expression of UGT1A1, leading to enhanced drug metabolism during pregnancy.

Phytochemical Regulation of UDP-Glucuronosyltransferases: Implications for Cancer Prevention

Uridine 5′-diphospho-glucuronosyltransferases (UGTs) are Phase II biotransformation enzymes that metabolize endogenous and exogenous compounds, some of which have been associated with cancer risk. Many phytochemicals have been shown to induce UGTs in humans, rodents, and cell culture systems. Because UGTs maintain hormone balance and facilitate excretion of potentially carcinogenic compounds, regulation of their expression and activity may affect cancer risk. Phytochemicals regulate transcription factors such as the nuclear factor-erythroid 2-related factor 2 (Nrf2), aryl hydrocarbon, and pregnane X receptors as well as proteins in several signal transduction cascades that converge on Nrf2 to stimulate UGT expression. This induction can be modified by several factors, including phytochemical dose and bioavailability and interindividual variation in enzyme expression. In this review, we summarize the knowledge of dietary modulation of UGTs, particularly by phytochemicals, and discuss the potential mechanisms by which phytochemicals regulate UGT transcription.

EXPRESSION OF UDP-GLUCURONOSYLTRANSFERASE ISOFORM mRNAS DURING INFLAMMATION AND INFECTION IN MOUSE LIVER AND KIDNEY

Inflammation or infection down-regulates the activity and expression of cytochrome P450 (P450) enzymes involved in hepatic drug clearance, possibly altering drug effectiveness and leading to toxicity. The regulation of UDP-glucuronosyltransferases (UGTs) in inflammation and infection is less well characterized. To determine the response of hepatic and renal UGTs during inflammation and infection, mice were administered either saline or 1 mg/kg lipopolysaccharide (LPS) (16 h), or Citrobacter rodentium by oral gavage (6 days). Hepatic mRNA expression of UGT1A1, 1A9, and 2B5 was similarly down-regulated after LPS exposure and C. rodentium infection, whereas UGT1A2 and 1A6 mRNAs were unchanged. Effects of C. rodentium infection did not require a functional Toll-like receptor 4. Conversely, renal UGT isoforms were relatively unaffected, except for UGT2B5 induction after LPS treatment. Regulation of UGTs during the inflammatory response exhibits similarities to and differences from regulation of P450s, and may be cytokine-mediated.

Glucuronidation and the UDP-glucuronosyltransferases in health and disease.

This article is an updated report of a symposium held at the June 2000 annual meeting of the American Society for Pharmacology and Experimental Therapeutics in Boston. The symposium was sponsored by the ASPET Divisions for Drug Metabolism and Molecular Pharmacology. The report covers research from the authors' laboratories on the structure and regulation of UDP-glucuronosyltransferase (UGT) genes, glucuronidation of xenobiotics and endobiotics, the toxicological relevance of UGTs, the role of UGT polymorphisms in cancer susceptibility, and gene therapy for UGT deficiencies.

Vitamin A modulates the effects of thyroid hormone on UDP-glucuronosyltransferase expression and activity in rat liver

We studied the influence of thyroid hormones and vitamin A status on the regulation of UDP-glucuronosyltransferase (UGT) expression and the glucuronidation of thyroid hormones by UGTs. For this, we used an original model of rats fed with different vitamin A diets and implanted subcutaneously by osmotic minipumps delivering vehicle or thyroid hormones, which permitted the control of plasma thyroid hormone concentrations. The activity and expression of family 1 UGTs are correlated and were significantly modified by both thyroid status and amounts of retinol in the diet. Dietary vitamin A did not perturbe the UGT1A expression in thyroidectomized animals. Thyroid hormones and dietary vitamin A did not affect the activity and expression of family 2 UGTs. We conclude that thyroid hormones and vitamin A are co-regulator of the UGT1 family expression, without affecting the UGT2 family; by modifying activity and expression of the bilirubin UOT isoform, a member of UGT1 family, thyroid hormone reduced the glucuronidation of T4 and rT3.

Tissue-specific regulation of canine intestinal and hepatic phenol and morphine UDP-glucuronosyltransferases by β-naphthoflavone in comparison with humans

UDP-glucuronosyltransferases (UGTs) are regulated in a species- and tissue-dependent manner by endogenous and environmental factors. The present study was undertaken to further our knowledge about regulation of UGTs in dogs, a species widely used in preclinical safety evaluation. β-Naphthoflavone (BNF) was selected as a known aryl hydrocarbon receptor agonist and antioxidant-type inducer. The latter group of inducers is intensively investigated as dietary chemoprotectants against colon cancer. Dog UGTs were investigated in comparison with related human UGTs by examples, (i) expression of dog UGT1A6, the first sequenced dog phenol UGT, and (ii) morphine UGT activities, responsible for intestinal and hepatic first-pass metabolism of morphine. The following results were obtained: (i) dog UGT1A6 was found to be constitutively expressed in liver and marginally increased by BNF treatment. Expression was low in small intestine but ca. 6-fold higher in colon than for example in jejunum. Conjugation of 4-methylumbelliferone, one of the substrates of dog UGT1A6, was also enhanced 7-fold in colonic compared to jejunal microsomes. (ii) Compared to the corresponding human tissues, canine 3-O- and 6-O-morphine UGT activities were found to be >10-fold higher in dog liver and ca. 10-fold lower in small intestinal microsomes. Small intestinal morphine and 4-hydroxybiphenyl UGT activities appeared to be moderately (2- to 3-fold) induced by oral treatment with BNF. (iii) In contrast to dogs, morphine UGT activities were found to be similar in homogenates from human enterocytes and liver. The results suggest marked differences in tissue-specific regulation of canine vs. human hepatic and intestinal phenol or morphine UGTs.

High specific activities and polymorphic regulation of UDP-glucuronosyltransferases characterize the metabolic barrier in human intestinal epithelium

High specific activities and polymorphic regulation of UDP-glucuronosyltransferases characterize the metabolic barrier in human intestinal epithelium

The effect of hormones on the expression of five isoforms of UDP-glucuronosyltransferase in primary cultures of rat hepatocytes.

To investigate the direct effects of sex hormones, growth hormone, thyroid hormones and dexamethasone on the regulation of UDP-glucuronosyltransferase (UGT). Rat hepatocytes were cultured on matrigel and treated with various hormones. Northern blot analysis was carried out using cDNA probes to family 1 and family 2 isoforms. Treatment with 10(-5) M testosterone increased the mRNA levels of UGT 2B1 by 29% and UGT2B3 by 32%. Incubation of growth hormone (10 mU) with hepatocytes suppressed the expression of UGT2B1 and UGT2B3 by 17% and 38%, respectively. T3 administration resulted in a time and dose-dependent effect on the expression of UGT 1 isoforms, with increased UGT1A6 by 70%, and decreased UGT1A1 by 38% and UGT1A5 by 35%. All UGT isoforms except UGT 1A6 studied in this assay were up-regulated by dexamethasone, but to different degrees. The regulation of UGT1A1 and UGT2B1 by dexamethasone was dose and time dependent, and the induction of dexamethasone in the expression of UGT1A1 and UGT2B1 was blocked by cycloheximide but not dichloro-1-D-ribofuranosylbenzimidazole. This study demonstrates that multiple hormones take part in the regulation of UGT mRNA expression in the rat and individual genes can be differentially modulated.

Characterization and regulation of UDP-glucuronosyltransferases in steroid target tissues

Conjugation of compounds by glucuronidation is a pathway found in all vertebrates studied to date. Although, it is widely recognized that the liver is a major site of glucuronidation, it is now clear that extrahepatic tissues are also involved in the conjugation of compounds to which these tissues are exposed. High levels of androsterone glucuronide and androstane-3 α,17 β-diol glucuronide found in the human prostate, breast cyst fluid and ovary follicular fluid suggest that glucuronidation of 5 α-reduced C 19 steroids occurs in these tissues. Recently, we have reported the tissue distribution of UGT2B15, which can conjugate steroids in several human extrahepatic steroid target tissues including the skin, breast and prostate. We have also isolated a new UGT2B cDNA encoding UGT2B17, that conjugates ADT which is the major 5 α-reduced C 19 steroid glucuronide in the circulation of humans. UGT2B17 is also widely distributed in several human steroid target tissues. This gene was mapped to human chromosome 4q13 and has an exon/intron structure similar to that of rat UGT2B1 and UGT2B2. Both UGT2B15 and UGT2B17, which are able to catalyze the glucuronidation of DHT, are expressed in LNCaP cells. Interestingly, glucuronidation of steroids is markedly regulated by several factors including androgens and growth factors. Treatment of LNCaP cells with dihydrotestosterone (DHT) and epidermal growth factor (EGF) caused a decrease of DHT glucuronidation and UGT2B mRNA levels. RNase protection assays showed a specific decrease of UGT2B17 transcript in LNCaP cells treated with DHT and EGF however, the level of UGT2B15 mRNA was not affected. As well, Western blot analysis demonstrated a diminution of UGT2B17 protein level in response to DHT and EGF. These results demonstrate a differential regulation of different isoforms of steroid conjugating UGTs present in human prostate LNCaP cells. In addition, UGT2B17 was shown to be more labile than UGT2B15 indicating that regulation of UGT2B17 expression would lead to a more rapid change in the level of glucuronidated steroids. Expression of exogenous UGT2B17 in LNCaP cells by gene transfer led to a significant decrease in the androgen response. This result indicates the ability of UGT enzymes to regulate the androgen response by conjugating androgens which abolishes their interaction with their receptor and facilitates their clearance from the cell. The glucuronidation of steroids by UGT enzymes is an important mechanism by which the levels of steroids is regulated in steroid target tissues.

Sex hormones differentially regulate isoforms of UDP-glucuronosyltransferase.

To investigate the role of sex hormones in the regulation of UDP-glucuronosyltransferase (UGT). We examined liver from adult, prepubertal, gonadectomised and gonadectomised plus hormone replaced rats of both sexes. Immunohistochemistry and immunoblots were performed using a polyclonal UGT antibody to a number of family 1 and family 2 UGT isoforms. Northern blot analysis was performed utilising cDNA probes to family 1 and family 2 isoforms. Immunohistochemistry demonstrated variations in intensity and distribution of staining in the hormonally manipulated rats. Immunoblots showed variations in individual band intensity between rat groups. Immunoblots using a more specific antibody (anti-17 beta-hydroxysteroid UGT, which recognises UGT2B3 and UGT2B2) demonstrated marked differences between male and female rats and significant alterations after gonadectomy and testosterone replacement in the male rats. In northern analysis, UGT2B3 and 2B1 mRNA were significantly higher in adult males than females, and in prepubertal males compared to prepubertal females. In male rats, gonadectomy resulted in a 45-53% reduction in UGT2B3 and 2B1 levels respectively, which increased significantly with testosterone treatment to greater than normal adult levels. No change in UGT2B3 or 2B1 occurred after gonadectomy in females. In contrast, UGT1*1 mRNA tended to be higher in adult female and prepubertal female rats than in their male counterparts. In females, gonadectomy resulted in significant up-regulation of UGT1*1, while gonadectomy plus oestradiol treatment resulted in markedly reduced levels. UGT1*1 mRNA was not significantly altered by gonadectomy in males. This study demonstrates the differential effects of sex hormones on the expression of isoforms from the two phylogenetically distinct UGT families.

Uridine diphosphoxylose enhances hepatic microsomal UDP-glucuronosyltransferase activity by stimulating transport of UDP-glucuronic acid across the endoplasmic reticulum membrane.

The UDP-glucuronosyltransferase (UGT) system fulfils a pivotal role in the biotransformation of potentially toxic endogenous and exogenous compounds. Here we report that the activity of UGT in rat liver is stimulated by UDP-xylose. This stimulation was found in native microsomal vesicles as well as in the intact endoplasmic reticulum (ER) membrane, as studied in permeabilized hepatocytes, indicating the potential physiological importance of UDP-xylose in the regulation of UGT. We present evidence that UDP-xylose enhances UGT activity by stimulation of (i) the uptake of UDP-glucuronic acid across the ER membrane and (ii) the elimination of the UDP and/or UMP reaction product out of the ER lumen. UDP-xyloe produced a marked trans-stimulation of microsomal UDP-glucuronic acid uptake when it was present within the lumen of the ER. When UDP-xylose was presented at the cytosolic side of the ER, it acted as a weak inhibitor of UDP-glucuronic acid uptake. Likewise, cytosolic UDP-glucuronic acid strongly trans-stimulated efflux of intravesicular UDP-xylose, whereas cytosolic UDP-xylose was inefficient in trans-stimulating efflux of UDP-glucuronic acid. Microsomal UDP-xylose influx was markedly stimulated by UMP and UDP. Such stimulation was only apparent when microsomes had been preincubated and thereby preloaded with UMP or UDP, indicating that UMP and UDP exeted their effect on UDP-xylose uptake by trans-stimulation from the luminal side of the ER membrane.

Heterogeneous alterations of UDP-glucuronosyltransferases in mouse hepatic foci

UDP-glucuronosyltransferase (UDPGT) was studied immunohistochemically in hepatic foci and nodules of N-nitrosomorpholine-treated mice. Serial sections were stained for glucose-6-phosphatase (G6Pase). It was found that a high percentage of G6Pase-negative liver foci and nodules were also UDPGT-negative (34%). In addition, G6Pase-negative foci without altered UDPGT phenotype (30%) and UDPGT-negative foci without altered G6Pase phenotype (8%) were detected. G6Pase-positive foci were also present (24%). Interestingly, most G6Pase-positive foci were UDPGT-positive (16%). Some G6Pase-positive lesions without altered UDPGT phenotype were also found (8%). The major phenotype observed in rat hepatocarcinogenesis models (UDPGT-positive/G6Pase-negative foci) was not detectable in the mouse model. These results demonstrate heterogeneous alterations of UDPGTs in mouse hepatic foci. They furthermore suggest marked differences between the mouse and the rat in the regulation of UDPGTs in similarly induced rat hepatic foci.

Regulation of UDP-glucuronosyltransferase by lipid-protein interactions: Comparison of the thermotropic properties of pure reconstituted enzyme with microsomal enzyme

The temperature dependence of two kinetic properties of the GT2P isoform of microsomal UDP-glucuronosyltransferase was studied for enzyme in intact microsomes and for pure enzyme reconstituted into different types of lipid bilayers. The properties studied were the non-Michaelis-Menten kinetics of the enzyme and activity at Vmax(app). For enzyme in intact microsomes, the pattern of non-Michaelis-Menten kinetics was seen at all temperatures in the range tested (23 to 48 degrees C), and the slopes of the Hill plots of the data were constant across this range of temperatures. Although non-Michaelis-Menten kinetics were present for pure enzyme in bilayers of 1,2-dimyristoylphosphatidylcholine or 1,2-dipalmitoylphosphatidylcholine only in the gel phase (Hockman, Y., Kelley, M., and Zakim, D. (1983) J. Biol. Chem. 258, 6509-6519), it was not possible to reconstitute this pattern of kinetics for enzyme at T greater than 40 degrees C. For example, GT2P displayed Michaelis-Menten kinetics in bilayers of 1,2-distearoylphosphatidylcholine at 44 degrees C. For enzyme in microsomes, activities at Vmax(app) increased with increasing temperature in the range 23 to 48 degrees C, with a discontinuity in the slope of the Arrhenius plot at 34 degrees C. This thermotropic property also could not be reconstituted with pure GT2P. Instead, activities at Vmax(app) for GT2P reconstituted in 1,2-dioleoylphosphatidylcholine, 1,2-distearoylphosphatidylcholine, or 1,2-stearoyl oleoylphosphatidylcholine increased in the range 23 to 37 degrees C, but then decreased at T greater than 37 degrees C. The fall in activity at T greater than 37 degrees C was reversible, indicating that GT2P undergoes a reversible change at 37 degrees C to a less active form of the enzyme. The differences between pure, reconstituted GT2P and microsomal GT2P indicate that the thermotropic properties of the microsomal enzyme are not properties of the enzyme per se but depend on interactions between it and other components in the microsome. The data suggest, therefore, that the properties of GT2P in microsomes results in part from an organization of components in the microsomal membrane.