Advertisement

Transcriptional Regulation of UDP-Glucuronosyltransferases

  • Anna Radominska-Pandya
  • Peter I. Mackenzie
  • Wen Xie
Protocol
  • 837 Downloads
Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

Recent evidence has been provided that five nuclear receptors (NRs), (pregnane X receptor [PXR], constitutive androstane receptor [CAR], farnesoid X receptor [FXR] and peroxisome proliferator-activated receptor [PPARα and γ]) regulate the expression of UGT1A loci. This chapter presents an overview of the most recent developments in our understanding of transcriptional regulation of UDP-glucuronosyltransferase (UGT) genes, and specifically focuses on the regulation of UGTs via NRs, with a strong emphasis on the biochemical and molecular techniques applied to this area of investigation. Procedures are described for the isolation and analysis of UGT proximal and distal promoters. The use of transgenic animals in the study of the mechanism of regulation of UGT expression is described, as is the assessment of the effects of various gene inducers on expression of UGTs in HepG2 and Caco-2 cell culture. Techniques for transient transfection of HepG2 and Caco-2 cells with NRs and UGT promoter constructs are presented. In addition, general methods are provided for various molecular and biochemical methods used in the isolation and characterization of UGT promoters.

Key Words

Aryl hydrocarbon receptor bile acids bilirubin (bio)flavonoids CaCo-2 cells constitutive androgen receptor cytochrome P450 DNase 1 footprinting electrophoretic mobility shift assay glucuronidation assays HepG2 cells luciferase reporter constructs mouse: “humanized” mouse: transgenic Northern blot hybridization peroxisome proliferator-activated receptor pregnane X receptor retinoids reverse transcriptase-polymerase chain reaction steroid hormones transfection: transient UDP-glucuronosyltransferase UGT promoter constructs UGT transcriptional regulation xenobiotic response element 

References

  1. 1.
    Mackenzie PI, Gregory PA, Gardner-Stephen DA, et al. Regulation of UDP glucuronosyltransferase genes. Curr Drug Metab 2003;4:49–57.CrossRefGoogle Scholar
  2. 2.
    Barbier O, Villeneuve L, Bocher V, et al. The UDP-glucuronosyltransferase 1A9 enzyme is a peroxisome proliferator-acivated receptor α and γ target gene. J Biol Chem 2003;278:13975–13983.PubMedCrossRefGoogle Scholar
  3. 3.
    Sugatani J, Kojima H, Ueda A, et al. The phenobarbital response enhancer module in the human bilirubin UDP-glucuronosyltransferase UGT1A1 gene and regulation by the nuclear receptor CAR. Hepatology 2001;33:1232–1238.PubMedCrossRefGoogle Scholar
  4. 4.
    Xie W, Yeuh M-F, Radominska-Pandya A, et al. Control of steroid, heme and carcinogen metabolism by nuclear receptors PXR and CAR. Proc Natl Acad Sci USA 2003;100:4150–4156.PubMedCrossRefGoogle Scholar
  5. 5.
    Gardner-Stephen DA, Heydel J-M, Goyal A, et al. Human PXR variants and their differential effects on the fegualtion of human UDP-glucuronosyltransferases. Drug Metab Dispos;32:340–347.Google Scholar
  6. 6.
    Yueh MG, Huang YH, Hiller A, Chen S, Nguyen N, Tukey RH. Involvement of the xenobiotic response element (XRE) in Ah receptor-mediated induction of human UDP-glucuronosyltransferase 1A1. J Biol Chem 2003;278:15001–15006.PubMedCrossRefGoogle Scholar
  7. 7.
    Barbier O, Peneda Torra I, Sirvent A, et al. FXR induces the UGT2B4 enzyme in hepatocytes: a potential mechanism of negative feedback control of FXR activity. Gastroenterology 2003;124:1926–1940.PubMedCrossRefGoogle Scholar
  8. 8.
    Bock KW. Metabolic polymorphisms affecting activation of toxic and mutagenic arylamines. TiPS 1992:223–226.Google Scholar
  9. 9.
    Oelberg DG, Chari MV, Little JM, Adcock EW, Lester R. Lithocholate glucuronide is a cholestatic agent. J Clin Invest 1984;73:1507–1514.PubMedCrossRefGoogle Scholar
  10. 10.
    Vore M, Montgomery C, Meyers M. Steroid D-ring glucuronides: characterization of a new class of cholestatic agents. Drug Metab Rev 1983;14:1005–1019.PubMedCrossRefGoogle Scholar
  11. 11.
    Nebert DW. Drug-metabolizing enzymes in ligand-modulated transcription. Biochem Pharmacol 1994;47:25–37.PubMedCrossRefGoogle Scholar
  12. 12.
    Nebert DW. Proposed role of drug-metabolizing enzymes: regulation of steady state levels of the ligands that effect growth, homeostasis, differentiation, and neuroendocrine functions. Mol Endocrinol 1991;5:1203–1214.PubMedCrossRefGoogle Scholar
  13. 13.
    Ritter JK, Kessler FK, Thompson MT, Grove AD, Auyeung DJ, Fisher RA. Expression and inducibility of the human bilirubin UDP-glucuronosyltransferase UGT1A1 in liver and cultured primary hepatocytes: evidence for both genetic and environmental influences. Hepatology 1999;30:476–84.PubMedCrossRefGoogle Scholar
  14. 14.
    Walle UK, Walle T. Induction of human UDP-glucuronosyltransferase UGT1A1 by flavonoids-structural requirements. Drug Metab Dispos 2002;30:564–569.PubMedCrossRefGoogle Scholar
  15. 15.
    Galijatovic A, Otake Y, Walle UK, Walle T. Induction of UDP-glucuronosyltransferase UGT1A1 by the flavonoid chrysin in Caco-2 cells-poteial role in carcinogen bioinactivation. Pharmaceut Res 2001;18:374–379.CrossRefGoogle Scholar
  16. 16.
    Munzel PA, Lehmkoster T, Bruck M, Ritter JK, Bock KW. Aryl hydrocarbon receptor-inducible or constitutive expression of human UDP glucuronosyltransferase UGT1A6. Arch Biochem Biophys 1998;350:72–78.PubMedCrossRefGoogle Scholar
  17. 17.
    Bock KW, Eckle T, Ouzzine M, Fournel-Gigleux S. Coordinate induction by antioxidants of UDP-glucuronosyltransferase UGT1A6 and the apical conjugate export pump MRP2 (multidrug resistance protein 2) in Caco-2 cells. Biochem Pharmacol 2000;59:467–470.PubMedCrossRefGoogle Scholar
  18. 18.
    Munzel PA, Schmohl S, Heel H, Kälberer K, Bock-Hennig B, Bock KW. Induction of human UDP-glucuronosyltransferases (UGT1A6, UGT1A9, and UGT2B7) by t-butylhydroquinone and 2,3,7,8-tetrachlorodibenzo-p-dioxin in Caco-2 cells. Drug Metab Dispos 1999;27:569–573.PubMedGoogle Scholar
  19. 19.
    Sabolovic N, Magdalou J, Netter P, Abid A. Nonsteroidal anti-inflammatory drugs and phenols glucuronidation in Caco-2 cells. Identification of the UDP-glucuronosyltransferases UGT1A6, 1A3 and 2B7. Life Sci 2000;67:185–196.PubMedCrossRefGoogle Scholar
  20. 20.
    Abid A, Sablovic N, Magdalou J. Expression and inducibility of UDP-glucuronosyltransferases 1-naphthol in human cultured hepatocytes and hepatocarcinoma cell lines. Life Sci 1997;60:1943–1951.PubMedCrossRefGoogle Scholar
  21. 21.
    Hum DW, Belanger A, Levesque E, et al. Characterization of UDP-glucuronosyltransferases active on steroid hormones. J Steroid Biochem Mol Biol 1999;69: 413–423.PubMedCrossRefGoogle Scholar
  22. 22.
    Brandsch C, Friedl P, Lange K, Richter T, Mothes T. Primary culture and transfection of epithelial cells of human small intestine. Scand J Gastroenterol 1998;33: 833–838.PubMedCrossRefGoogle Scholar
  23. 23.
    Xie W, Barwick JL, Downes M, et al. Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature 2000;406:435–439.PubMedCrossRefGoogle Scholar
  24. 24.
    Xie W, Barwick JL, Simon CM, et al. Reciprocal activation of xenobiotic response genes by nuclear receptors SXR/PXR and CAR. Genes Dev 2000;14:3014–3023.PubMedCrossRefGoogle Scholar
  25. 25.
    Congiu M, Mashford ML, Slavin JL, Desmond PV. UDP-glucuronosyltransferase mRNA levels in human liver disease. Drug Metab Dispos 2002;30:129–134.PubMedCrossRefGoogle Scholar
  26. 26.
    Strassburg CP, Oldhaffer K, Manns MP, Tukey RH. Differential expression of the UGT1A locus in human liver, biliary, and gastric tissue: identification of UGT1A7 and UGT1A10 transcripts in extrahepatic tissue. Mol Pharmacol 1997;52:212–220.PubMedGoogle Scholar
  27. 27.
    Radominska-Pyrek A, Zimniak P, Irshaid YM, Lester R, Tephly TR, Pyrek JS. Glucuronidation of 6α-hydroxy bile acids by human liver microsomes. J Clin Invest 1987;80:234–241.PubMedCrossRefGoogle Scholar
  28. 28.
    Fisher MB, Campanale K, Ackermann BL, Vandenbranden M, Wrighton SA. In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin. Drug Metab Dispos 2000;28:560–566.PubMedGoogle Scholar
  29. 29.
    Little JM, Lehman PA, Nowell S, Samokyszyn V, Radominska A. Glucuronidation of all trans-retinoic acid and 5,6-epoxy-all trans-retinoic acid: activation of rat liver microsomal UDP-glucuronosyltranferase activity by alamethicin. Drug Metab Dispos 1997;25:5–11.PubMedGoogle Scholar
  30. 30.
    Little JM, Radominska A. Application of photoaffinity labeling with [11,12-3H]all trans-retinoic acid to characterization of rat liver microsomal UDP-glucuronosyltransferase(s) with activity toward retinoic acid. Biochem Biophys Res Commun 1997;230:497–500.PubMedCrossRefGoogle Scholar
  31. 31.
    Radominska-Pyrek A, Zimniak P, Chari M, Golunski E, Lester R, Pyrek JS. Glucuronides of monohydroxylated bile acids: specificity of microsomal glucuronyltransferase for the glucuronidation site, C-3 configuration, and side chain length. J Lipid Res 1986;27:89–101.PubMedGoogle Scholar
  32. 32.
    Radominska-Pandya A, Little JM, Pandya JT, et al. UDP-Glucuronosyltransferases in human intestinal mucosa. Biochim Biophys Acta 1998;1394:199–208.PubMedGoogle Scholar
  33. 33.
    Lemon B, Tijian R. Orchestrated response: a symphony of transcription factors for gene control. Genes Dev 2001;14:2551–2569.CrossRefGoogle Scholar
  34. 34.
    Shaefer B. Revolution in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full length cDNA ends. Anal Biochem 1995;227:255–276.CrossRefGoogle Scholar
  35. 35.
    Heinemeyer T, Wingender E, Reuter I, et al. Databases on transcriptional regulation: TRANSFAC, TRRD, and COMPEL. Nucleic Acids Res 1998;26: 364–370.CrossRefGoogle Scholar
  36. 36.
    Podvinec M, Kaufamm MR, Handschin C, Meyer UA. NUBIScan, an in silico approach for prediction of nuclear receptor response elements. Mol Endocrinol 2002;16:1269–1279.PubMedCrossRefGoogle Scholar
  37. 37.
    Hansen AJ, Lee YH, Gonzalez FJ, Mackenzie PI. HNF1 alpha activates the rat UDP glucuronosyltransferase UGT2B1 gene pomoter. DNA Cell Biol 1997;16:207–214.PubMedCrossRefGoogle Scholar
  38. 38.
    Gregory PA, Gardner-Stephen DA, Lewinsky RH, Duncliffe KN, Mackenzie PI. Cloning and characterization of the human UDP-glucuronosyltransferase 1A8, 1A9 and 1A10 gene promoters. Differential regulation through an initiator-like region. J Biol Chem 2003; 278:36107–36114.PubMedCrossRefGoogle Scholar
  39. 39.
    Kroeger KM, Abraham LJ. Magnetic bead purification of specific transcription factors using mutant competitor oligonucleotides. Anal Biochem 1997;250: 127–129.PubMedCrossRefGoogle Scholar
  40. 40.
    Schreiber E, Matthias P, Muller MM, Schaffner W. Rapid detection of octamer binding proteins with “mini-extracts” prepared from a small number of cells. Nucleic Acid Res 1989;17:6419–6420.PubMedCrossRefGoogle Scholar
  41. 41.
    Kostrubsky VE, Lewis LD, Strom SC, et al. Induction of cytochrome P4503A by taxol in primary cultures of human hepatocytes. Arch Biochem Biophys 1998;355: 131–136.PubMedCrossRefGoogle Scholar
  42. 42.
    Kostrubsky VE, Strom SC, Wood SG, Wrighton SA, Sinclair PR, Sinclair JF. Ethanol and isopentanol increase CYP3A and CYP2E in primary cultures of human hepatocytes. Arch Biochem Biophys 1995;322:516–520.PubMedCrossRefGoogle Scholar
  43. 43.
    Strom SC, Pisarov LA, Dorko K, Thompson MT, Schuetz JD, Schuetz EG. Use of human hepatocytes to study P450 gene induction. Methods Enzymol 1996;272: 388–401.PubMedCrossRefGoogle Scholar
  44. 44.
    Li P. Primary hepatocyte cultures as an in vitro experimental model for the evaluation of pharmacokinetic drug–drug interactions. Adv Pharmacol 1997;43: 103–130.PubMedCrossRefGoogle Scholar
  45. 45.
    Maurel P. The CYP3A family. In: Ioannides C, ed. Cytochrome P450: Metabolic and Toxicological Aspects. Boca Raton, FL: CRC Press, 1996:241–270.Google Scholar
  46. 46.
    Kocarek TA, Schuetz EG, Strom SC, Fisher RA, Guzelian PS. Comparative analysis of cytochrome P4503A induction in primary cultures of rat, rabbit, and human hepatocytes. Drug Metab Dispos 1995;23:415–421.PubMedGoogle Scholar
  47. 47.
    Schuetz EG, Schinkel AH, Relling MV, Schuetz JD. P-glycoprotein: a major determinant of rifampicin-inducible expression of cytochrome P4503A in mice and human. Proc Natl Acad Sci USA 1996;93:4001–4005.PubMedCrossRefGoogle Scholar
  48. 48.
    Schuetz EG, Schuetz JD, Strom SC, et al. Regulation of human liver cytochromes P-450 in family 3A in primary and continuous culture of human hepatocytes. Hepatology 1993;18:1254–1262.PubMedCrossRefGoogle Scholar
  49. 49.
    Watkins PB, Wrighton SA, Maurel P, Identification of an inducible form of cytochrome P-450 in human liver. Proc Natl Acad Sci USA 1985;82:6310–6314.PubMedCrossRefGoogle Scholar
  50. 50.
    Wrighton SA, Schuetz EG, Watkins PB, et al. Demonstration of multiple species of inducible hepatic cytochromes P-450 and their mRNAs related to the glucocorticoid-inducible cyrochrome P-450 of the rat. Mol Pharmacol 1985;28: 312–321.PubMedGoogle Scholar
  51. 51.
    Barwick JL, Quattrochi LC, Mills AS, Potenza C, Tukey RH, Guzelian PS. Transspecies gene transfer for analysis of glucocorticoid-inducible transcriptional activation of transiently expresed human CYP3A4 and rabbit CYP3A6 in primary cultures of adult rat and rabbit hepatocytes. Mol Pharmacol 1996;50:10–16.PubMedGoogle Scholar
  52. 52.
    Sonoda J, Xie W, Rosenfeld JM, Barwick JL, Guzelian PS, Evans RM. Regulation of a xenobiotic sulfonation cascade by nuclear pregnane X receptor (PXR). Proc Natl Acad Sci USA 2002;99:13801–13806.PubMedCrossRefGoogle Scholar
  53. 53.
    Xie W, Evans RM. Orphan nuclear receptors: the exotics of xenobiotics. J Biol Chem 2001;276:37739–37742.PubMedGoogle Scholar
  54. 54.
    Xie W, Evans RM. Pharmaceutical use of mouse models humanized for the xenobiotic receptor. Drug Discov Today 2002;7:509–517.PubMedCrossRefGoogle Scholar
  55. 55.
    Guicciardi ME, Gores GJ. Bile acid-mediated hepatocyte apoptosis and cholestatic liver disease. Digest Liver Dis 2002;34:387–392.CrossRefGoogle Scholar
  56. 56.
    Kullak-Ublick GA, Meier PJ. Mechanisms of cholestasis. Clin Liver Dis 2000;4: 357–385.PubMedCrossRefGoogle Scholar
  57. 57.
    Radominska A, Treat S, Little J. Bile acid metabolism and the pathophysiology of cholestasis. Semin Liver Dis 1993;13:219–234.PubMedCrossRefGoogle Scholar
  58. 58.
    Xie W, Radominska-Pandya A, Shi Y, et al. An essential role for SXR/PXR in detoxification of cholestatic bile acids. Proc Natl Acad Sci USA 2001;98:3375–3380.PubMedCrossRefGoogle Scholar
  59. 59.
    Watkins RE, Wisely GB, Moore LB, et al. The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity. Science 2001;292:2329–2333.PubMedCrossRefGoogle Scholar
  60. 60.
    Blumberg B, Sabbagh W Jr, Juguilon H, et al. SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev 1998;12:3195–3205.PubMedCrossRefGoogle Scholar
  61. 61.
    Pinkert CA, Ornitz DM, Brinster RL, Palmiter RD. An albumin enhancer located 10 kb upstream functions along with its promoter to direct efficient, liver-specific expression in transgenic mice. Genes Dev 1987;1:268–276.PubMedCrossRefGoogle Scholar
  62. 62.
    Ritter JK, Chen F, Sheen YY, et al. A novel complex locus UGT1 encodes human bilirubin, phenol, and other UDP-glucuronosyltransferase isozymes with identical carboxyl termini. J Biol Chem 1992;267:3257–3261.PubMedGoogle Scholar
  63. 63.
    Findlay KA, Kaptein E, Visser TJ, Burchell B. Characterization of the uridine diphosphate-glucuronosyltransferase-catalyzing thyroid hormone glucuruonidaion in man. J Clin Endocrinol Metab 2000;85:2879–2883.PubMedCrossRefGoogle Scholar
  64. 64.
    Nowell S, Massengill J, Williams S, et al. Glucuronidation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human microsomal proteins: Identification of the UGT1A isoforms involved. Carcinogenesis 1999;20:101–108.CrossRefGoogle Scholar
  65. 65.
    Broudy VC, Lin NL, Priestley GV, Nocka K, Wolf NS. Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen. Blood 1996;88:75–81.PubMedGoogle Scholar
  66. 66.
    Brock WJ, Durham S, Vore M. Characterization of the interaction between estrogen metabolites and taurocholate for uptake into isolated hepatocytes. Lack of correlation between cholestasis and inhibition of taurocholate uptake. J Steroid Biochem 1984;20:1181–1185.PubMedCrossRefGoogle Scholar
  67. 67.
    Garcia-Allen C, Lord PG, Loughlin JM, Orton TC, Sidaway JE. Identification of phenobarbitone-modulated genes in mouse liver by differential display. J Biochem Mol Toxicol 2000;14:65–72.CrossRefGoogle Scholar
  68. 68.
    Kikuchi S, Hata M, Fukumoto K, et al. Radixin deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile canalicular membranes. Nat Genet 2002;31:320–325.PubMedCrossRefGoogle Scholar
  69. 69.
    Smith GW, Aubry JM, Dellu F, et al. Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron 1998;20:1093–1102.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Anna Radominska-Pandya
    • 1
  • Peter I. Mackenzie
    • 2
  • Wen Xie
    • 3
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Arkansas for Medical SciencesLittle Rock
  2. 2.Department of Clinical PharmacologyFlinders UniversityBedford ParkAustralia
  3. 3.Center for Pharmacogenetics and Department of Pharmaceutical SciencesUniversity of PittsburghPittsburgh

Personalised recommendations