Effects of Constitutive Androstane Receptor (CAR) on PBRU Transactivation of CYP2B Gene in Different Culture Cell Types: Comparison Between Hep G2 and COS-cells

배양세포의 Type에 따른 Constitutive Androstane 수용체 (CAR)의 CYP2B PBRU 전사활성 효과: Hep G2와 COS 세포의 비교

  • 민계식 (진주산업대학교 미생물공학과)
  • Published : 2003.06.01


The objective of this study was to examine if transient transfection of CAR can transactivate CYP2B1 PBRU reporter gene in COS cells in which the endogenous CYP2B1 gene is not induced by PB. In non-transfeced cells of both Hep G2 and COS, the endogeneous expression of CAR was not detected by antibody against CAR. When cultured cells were transfected with CAR expression plasmid, mCAR1-GFP, both cell types expressed high levels of CAR protein and could allow to examine the effect of CAR in PBRU transactivation. Both cell types expressed endogenous RXR and transfection of RXR expression plasmid dramatically increased its protein expression. Whereas CAR transactivated PBRU2C1Luciferase about 12 fold as compared to 2C1Luciferase in Hep G2 cells, it did not stimulate the luciferase activity of the PBRU reporter gene in COS cells. These results indicate that Hep G2 cells can respond to CAR differently from COS cells, and suggest that factors other than CAR and RXR may be required in inducing PBRU activation and the expression of these factors may be different between liver and kidney.


  1. Axelrod, J. 1955. The enzymatic deamination of am-phetamine (benzedrine). J. Bioi. Chem. 214, 753-763
  2. Baes, M., T. Gulick, H. S. Choi, M. G. Martinoli, D. Simha and D. D. Moore. 1994. A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response ele-ments. Mol. Cell Bioi. 14, 1544-1551
  3. Bhagwat, S. V., B. C. Leelavathi, S. K. Shankar, M. R. Boyd and V. avindranath. 1995. Cytochrome P450 and associated monooxygenase activities in the rat and human spinal cord-induction, immunological char-acterization, and immunocytochemical localization. Neurosci. 68, 593-601
  4. Brodie, B. B., J. Axelrod, J. R. Cooper, L. Gaudette, B. N. LaDu, C. Mitoma and S. Udenfriend. 1955. Detox-ication of drugs and other foreign compounds by liver microsomes. Science 121, 603-604
  5. Choi, H-S., M. Chung, I. Tzameli, D. Simha, Y-K. Lee, W. Seol and D. Moore. 1997. Differential trans-activation by two isoforms of the orphan nuclear hormone receptor CAR. J. BioI. Chem. 272, 23565-23571
  6. Conney, A. H., C. Davison, R. Gasten and J. J. Burns. 1960. Adaptive increase in drug-metabolizing enzymes induced by phenobarbital and other drugs. J. Pharmacal. Exp. Therap. 130, 1-8
  7. Cooper, D. Y., S. Levine, S. Narasimhulu, O. Ro-senthal and R. W. Estabrook. 1965. Photochemical action spectrum of the terminal oxidase of 'mixed function oxidase system. Science. 147, 400-402
  8. Forman, B. M., I. Tzameli, H. S. Choi, J. Chen, D. Simha, W. Seol, R. M. Evans and D. D. Moore. 1998. Androstane metabolites bind to and deactivate the nuclear receptor CAR-beta. Nature. 395, 612-615
  9. Honkakoski, P., A. Kojo and M. A. Lang. 1992. Reg-ulation of the mouse liver cytochrome P450 2B subfamily by sex hormones and phenobarbital. Biochemical. J. 285, 979-983
  10. Honkakoski, P., R. Moore, K. Washburn and M. Negishi. 1988. Activation by diverse xenochemicals of the 51-base pair phenobarbital-response enhancer module in the CYP2B10 gene. Mol. Pharmacal. 53, 597-601
  11. Honkakoski, P. and M. Negishi. 1997. Character-ization of a phenobarbital-responsive enhancer mod-ule in mouse P450 Cyp2blO gene. J. BioI. Chem. 272, 14943-14949
  12. Honkakoski, P. and M. Negishi. 2000. Regulation of cytochrome P450 (CYP) genes by nuclear receptors. Biochem. J. 347, 321-337
  13. Honkakoski, P., I. Zelko, T. Sueyoshi and M. Negishi. 1998. The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene. Mol. Cell Bioi. 18, 5652-5658
  14. Jean, A., A. Reiss, M. Desrochers, S. Dubois, E. Trottier, Y. Trottier, L. Wirtanen, M. Adesnik, D. J. Waxman and A. Anderson. 1994. Rat liver cyto-chrome P450 2B3: structure of the CYP2B3 gene and immunological identification of a constitutive P450 2B3-like protein in rat liver. DNA Cell BioI. 13, 781-792
  15. Johnson, E. F., K. J. Griffin and U. S. Keith. 1999. Molecular mechanisms of cytochrome P-450 induction by xenobiotics: An expanded role for nuclear hormone receptors. Molecular Pharmacology. 56, 851-857
  16. Kawamoto, T., T. Sueyoshi, I. Zelko, R. Moore, K. Washburn and M. Negish. 1999. Phenobarbital responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Mol. Cell Bioi. 19, 6318-6322
  17. Kim, J. and B. Kemper. 1997. Phenobarbital alters protein binding to the CYP2Bl/2 phenobarbital-responsive unit in native chromatin. J. Bioi. Chem. 272, 29423-29426
  18. Kim, J., G. Min and B. Kemper. 2001. Chromatin assembly enhances binding to the CYP2B1 phenobar-bital-responsive unit (PBRD) of nuclear factor-1, which binds simultaneously with constitutive andro-stane receptor (CAR)/retinoid $\times$receptor (RXR) and enhances CAR/RXR-mediated activation of the PBRU. J. Biol. Chem. 276, 7559-7567
  19. Kim, J., I. Rivera-Rivera and B. Kemper. 2000. Tissue-specific chromatin structure of the CYP2B1 phenobar-bital-responsive unit and proximal promotor and modulation by phenobarbital. Nucleic Acids Res. 28, 1126-1132
  20. Liu, S., Y. Park, I. Rivera-Rivera, H. Li and B. Kemper. 1998. Nuclear factor-1 motif and redundant regulatory elements comprise phenobarbital-responsive enhancer in CYP2B1/2. DNA Cell Bioi. 17, 461-470
  21. Min, G., J. K. Kemper and B. Kemper. 2002. Glucocorticoid receptor-interacting protein 1 mediates ligand-independent nuclear translocation and activation of constitutive androstane receptor in uiuo. J. BioI. Chem. 277, 26356-26363
  22. Moore, D. and I. Tzameli. 2001. Role reversal: new insights from new ligands for the xenobiotic receptor CAR. TRENDS in Endocrinology and Metabolism 12, 7-10
  23. Moore, L. B., D. J. Parks, S. A. Jones, R. K. Bledsoe, T. G. Consler, J. B. Stimmel, B. Goodwin, C. Liddle, S. G. Blanchard, T. M. Willson, J. L. Collins and S. A. Kliewer. 2000. Orphan nuclear receptors con-stitutive androstane receptor and pregnene X receptor share xenobiotic and steroid ligands. J. Biol. Chern. 275, 15122-15127
  24. Mueller, G. C. and J. A. Miller. 1949. The reductive cleavage of 4-demethylaminoazobenzene by rat liver: The intracellular distribution of the enzyme system and the requirement for triphosphopyridine nucleotide. J. BioI. Chem. 180, 1125-1136
  25. Negishi, M. and P. Honkakoski. 2000. Regulation of cytochrome P450 (CYP) genes by nuclear receptors. Biochem. J. 347, 321-337
  26. Negishi, M. and T. Sueyoshi. 2001. Phenobarbital response elements of Cytochrome P450 genes and nuclear receptors. Annu. Rev. Pharmacol. Toxicol. 41,123-143
  27. Park, Y., H. Li and B. Kemper. 1996. Phenobarbital induction mediated by a distal CYP2B2 sequence in rat liver transiently transfected in situ. J. Biol. Chem. 271, 23725-23728
  28. Ramsden, R., N. B. Beck, K. M. Sommer and C. J. Omiecinski. 1999. Phenobarbital responsiveness con-ferred by the 5-flanking region of the rat CYP2B2 gene in transgenic mice. Gene. 228, 169-179
  29. Ramsden, R., K. M. Sommer and C. J. Omiecinski. 1993. Phenobarbital induction and tissue-specific ex-pression of the rat CYP2B2 gene in transgenic mice. J. BioI. Chem. 268, 21722-21726
  30. Savas, U., K. J. Griffin and E. F. Johnson. 1999. Molecular mechanisms of cytochrome P-450 induction by xenobiotics: An expanded role for nuclear hormone receptors. Molecular Pharmacology. 56, 851-857
  31. Shervington, A. 1999. CYP2B2 gene expression and phenobarbital induction in kidneys using an in vitro transcription system. Biochem. Mol. BioI. Int. 47, 233-237
  32. Strobel, H. W., A. Y H. Lu, J. Heidma and M. J. Coon. 1970. Phosphotidylcholine requirement in the enzymatic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation. J. Biol. Chem. 245, 4851-4954
  33. Sueyoshi, T., T. Kawamoto, I. Zelko, P. Honkakoski and M. Negishi. 1999. The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene. J. Biol. Chem. 274, 6043-6046
  34. Sueyoshi, T. and M. Negishi. 2001. Phenobarbital response elements of cytochrome P450 genes and nuclear receptors. Annu. Rev. Pharmacol. Toxicol. 41, 123-143
  35. Suwa, Y, Y Mizukami, K. Sogawa and Y. FujiiKuriyama. 1985. Gene structure of a major form of phenobarbital-inducible cytochrome P-450 in rat liver. J. Biol. Chem. 260, 7980-7984
  36. Trottier, E., A. Belzil, C. Stoltz and A. Anderson. 1995. Localization of a phenobarbital-responsive el-ement (PBRE) in the 5'-flanking region of the rat CYP2B2 gene. Gene 158, 263-268
  37. Tzameli, I., P. Pissios, E. G. Schuetz and D. D. Moore. 2000. The xenobiotic compound 1,4-bis[2-(3,5dichloropyridyloxy)] benzene is an agonist ligand for the nuclear receptor CAR. Mol. Cell Bioi. 20, 2951-2958
  38. Waxman, D. J. 1999. P450 gene induction by struc-turally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR. Archives of Biochemistry and Biophysics 369, 11-23
  39. Waxman, D. J. and L. Azaroff. 1992. Phenobarbital induction of cytochrome P-450 gene expression. Biochem. J. 281, 577-592
  40. Wei, P., J. Zhang, M. Egan-Hafley, S. Liang and D. D. Moore. 2000. The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature. 407, 920-923

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