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. 2001 Apr 1;355(Pt 1):71–78. doi: 10.1042/0264-6021:3550071

Xenobiotic induction of cytochrome P450 2B1 (CYP2B1) is mediated by the orphan nuclear receptor constitutive androstane receptor (CAR) and requires steroid co-activator 1 (SRC-1) and the transcription factor Sp1.

R Muangmoonchai 1, D Smirlis 1, S C Wong 1, M Edwards 1, I R Phillips 1, E A Shephard 1
PMCID: PMC1221713  PMID: 11256950

Abstract

The constitutive androstane receptor (CAR) activates the expression of a reporter gene attached to the phenobarbital-response element (PBRE) of the cytochrome P450 2B1 (CYP2B1) gene in response to the barbiturate phenobarbital and the plant product picrotoxin. The xenobiotic-mediated increase in transactivation occurs in transfected primary hepatocytes and in liver transfected by biolistic-particle-mediated DNA transfer, but not in the transformed cell lines HepG2, CV-1 and HeLa, which support only constitutive activation of gene expression by CAR. Steroid co-activator 1 (SRC-1) enhances both constitutive and xenobiotic-induced CAR-mediated transactivation via the CYP2B1 PBRE in transfected primary hepatocytes. The nuclear receptor 1 (NR1) site of the PBRE is sufficient for CAR-mediated transactivation, but additional sequences within the PBRE, and hence the proteins that bind to them, are required for the interaction of CAR with SRC-1. The NR2 site of the PBRE binds proteins other than CAR, including an unidentified nuclear receptor heterodimerized with retinoid X receptor alpha. By binding to the proximal promoter of CYP2B1, the transcription factor Sp1 increases both basal transcription and xenobiotic-induced expression via the PBRE. Thus induction of CYP2B1 expression by xenobiotics is mediated by the nuclear receptor CAR and, for optimal expression, requires SRC-1 and Sp1.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akrawi M., Rogiers V., Vandenberghe Y., Palmer C. N., Vercruysse A., Shephard E. A., Phillips I. R. Maintenance and induction in co-cultured rat hepatocytes of components of the cytochrome P450-mediated mono-oxygenase. Biochem Pharmacol. 1993 Apr 22;45(8):1583–1591. doi: 10.1016/0006-2952(93)90298-b. [DOI] [PubMed] [Google Scholar]
  2. Atchison M., Adesnik M. A cytochrome P-450 multigene family. Characterization of a gene activated by phenobarbital administration. J Biol Chem. 1983 Sep 25;258(18):11285–11295. [PubMed] [Google Scholar]
  3. Austin C. A., Shephard E. A., Pike S. F., Rabin B. R., Phillips I. R. The effect of terpenoid compounds on cytochrome P-450 levels in rat liver. Biochem Pharmacol. 1988 Jun 1;37(11):2223–2229. doi: 10.1016/0006-2952(88)90585-0. [DOI] [PubMed] [Google Scholar]
  4. Baes M., Gulick T., Choi H. S., Martinoli M. G., Simha D., Moore D. D. A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements. Mol Cell Biol. 1994 Mar;14(3):1544–1552. doi: 10.1128/mcb.14.3.1544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blumberg B., Sabbagh W., Jr, Juguilon H., Bolado J., Jr, van Meter C. M., Ong E. S., Evans R. M. SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev. 1998 Oct 15;12(20):3195–3205. doi: 10.1101/gad.12.20.3195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Choi H. S., Chung M., Tzameli I., Simha D., Lee Y. K., Seol W., Moore D. D. Differential transactivation by two isoforms of the orphan nuclear hormone receptor CAR. J Biol Chem. 1997 Sep 19;272(38):23565–23571. doi: 10.1074/jbc.272.38.23565. [DOI] [PubMed] [Google Scholar]
  7. Defranco D. B., Madan A. P., Tang Y., Chandran U. R., Xiao N., Yang J. Nucleocytoplasmic shuttling of steroid receptors. Vitam Horm. 1995;51:315–338. doi: 10.1016/s0083-6729(08)61043-2. [DOI] [PubMed] [Google Scholar]
  8. Dell H., Wong S. C., Phillips I. R., Shephard E. A. Identification of regulatory protein-binding sites in cytochrome P450 genes by means of the gel-retardation assay. Methods Mol Biol. 1998;107:381–394. doi: 10.1385/0-89603-519-0:381. [DOI] [PubMed] [Google Scholar]
  9. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dynan W. S., Tjian R. Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II. Cell. 1983 Mar;32(3):669–680. doi: 10.1016/0092-8674(83)90053-3. [DOI] [PubMed] [Google Scholar]
  11. Forman B. M., Tzameli I., Choi H. S., Chen J., Simha D., Seol W., Evans R. M., Moore D. D. Androstane metabolites bind to and deactivate the nuclear receptor CAR-beta. Nature. 1998 Oct 8;395(6702):612–615. doi: 10.1038/26996. [DOI] [PubMed] [Google Scholar]
  12. Frueh F. W., Zanger U. M., Meyer U. A. Extent and character of phenobarbital-mediated changes in gene expression in the liver. Mol Pharmacol. 1997 Mar;51(3):363–369. [PubMed] [Google Scholar]
  13. Glass C. K. Differential recognition of target genes by nuclear receptor monomers, dimers, and heterodimers. Endocr Rev. 1994 Jun;15(3):391–407. doi: 10.1210/edrv-15-3-391. [DOI] [PubMed] [Google Scholar]
  14. Gonzalez F. J. The molecular biology of cytochrome P450s. Pharmacol Rev. 1988 Dec;40(4):243–288. [PubMed] [Google Scholar]
  15. Hardwick J. P., Gonzalez F. J., Kasper C. B. Cloning of DNA complementary to cytochrome P-450 induced by pregnenolone-16 alpha-carbonitrile. Characterization of its mRNA, gene, and induction response. J Biol Chem. 1983 Aug 25;258(16):10182–10186. [PubMed] [Google Scholar]
  16. Honkakoski P., Moore R., Washburn K. A., Negishi M. Activation by diverse xenochemicals of the 51-base pair phenobarbital-responsive enhancer module in the CYP2B10 gene. Mol Pharmacol. 1998 Apr;53(4):597–601. doi: 10.1124/mol.53.4.597. [DOI] [PubMed] [Google Scholar]
  17. Honkakoski P., Negishi M. Regulation of cytochrome P450 (CYP) genes by nuclear receptors. Biochem J. 2000 Apr 15;347(Pt 2):321–337. doi: 10.1042/0264-6021:3470321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Honkakoski P., Zelko I., Sueyoshi T., Negishi M. The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene. Mol Cell Biol. 1998 Oct;18(10):5652–5658. doi: 10.1128/mcb.18.10.5652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Imataka H., Sogawa K., Yasumoto K., Kikuchi Y., Sasano K., Kobayashi A., Hayami M., Fujii-Kuriyama Y. Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene. EMBO J. 1992 Oct;11(10):3663–3671. doi: 10.1002/j.1460-2075.1992.tb05451.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Issemann I., Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature. 1990 Oct 18;347(6294):645–650. doi: 10.1038/347645a0. [DOI] [PubMed] [Google Scholar]
  21. Johnson E. F., Palmer C. N., Griffin K. J., Hsu M. H. Role of the peroxisome proliferator-activated receptor in cytochrome P450 4A gene regulation. FASEB J. 1996 Sep;10(11):1241–1248. doi: 10.1096/fasebj.10.11.8836037. [DOI] [PubMed] [Google Scholar]
  22. Kawamoto T., Sueyoshi T., Zelko I., Moore R., Washburn K., Negishi M. Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Mol Cell Biol. 1999 Sep;19(9):6318–6322. doi: 10.1128/mcb.19.9.6318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim J., Kemper B. Phenobarbital alters protein binding to the CYP2B1/2 phenobarbital-responsive unit in native chromatin. J Biol Chem. 1997 Nov 21;272(47):29423–29425. doi: 10.1074/jbc.272.47.29423. [DOI] [PubMed] [Google Scholar]
  24. Kim J., Rivera-Rivera I., Kemper B. Tissue-specific chromatin structure of the phenobarbital-responsive unit and proximal promoter of CYP2B1/2 and modulation by phenobarbital. Nucleic Acids Res. 2000 Mar 1;28(5):1126–1132. doi: 10.1093/nar/28.5.1126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kliewer S. A., Lehmann J. M., Willson T. M. Orphan nuclear receptors: shifting endocrinology into reverse. Science. 1999 Apr 30;284(5415):757–760. doi: 10.1126/science.284.5415.757. [DOI] [PubMed] [Google Scholar]
  26. Kliewer S. A., Moore J. T., Wade L., Staudinger J. L., Watson M. A., Jones S. A., McKee D. D., Oliver B. B., Willson T. M., Zetterström R. H. An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell. 1998 Jan 9;92(1):73–82. doi: 10.1016/s0092-8674(00)80900-9. [DOI] [PubMed] [Google Scholar]
  27. Kobayashi A., Sogawa K., Fujii-Kuriyama Y. Cooperative interaction between AhR.Arnt and Sp1 for the drug-inducible expression of CYP1A1 gene. J Biol Chem. 1996 May 24;271(21):12310–12316. doi: 10.1074/jbc.271.21.12310. [DOI] [PubMed] [Google Scholar]
  28. Liu S., Park Y., Rivera-Rivera I., Li H., Kemper B. Nuclear factor-1 motif and redundant regulatory elements comprise phenobarbital-responsive enhancer in CYP2B1/2. DNA Cell Biol. 1998 May;17(5):461–470. doi: 10.1089/dna.1998.17.461. [DOI] [PubMed] [Google Scholar]
  29. Meyer U. A., Hoffmann K. Phenobarbital-mediated changes in gene expression in the liver. Drug Metab Rev. 1999 May;31(2):365–373. doi: 10.1081/dmr-100101924. [DOI] [PubMed] [Google Scholar]
  30. Oñate S. A., Tsai S. Y., Tsai M. J., O'Malley B. W. Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science. 1995 Nov 24;270(5240):1354–1357. doi: 10.1126/science.270.5240.1354. [DOI] [PubMed] [Google Scholar]
  31. Park Y., Li H., Kemper B. Phenobarbital induction mediated by a distal CYP2B2 sequence in rat liver transiently transfected in situ. J Biol Chem. 1996 Sep 27;271(39):23725–23728. doi: 10.1074/jbc.271.39.23725. [DOI] [PubMed] [Google Scholar]
  32. Phillips I. R., Shephard E. A., Ashworth A., Rabin B. R. Cloning and sequence analysis of a rat liver cDNA coding for a phenobarbital-inducible microheterogenous cytochrome P-450 variant: regulation of its messenger level by xenobiotics. Gene. 1983 Dec;26(1):41–52. doi: 10.1016/0378-1119(83)90034-3. [DOI] [PubMed] [Google Scholar]
  33. Pike S. F., Shephard E. A., Rabin B. R., Phillips I. R. Induction of cytochrome P-450 by phenobarbital is mediated at the level of transcription. Biochem Pharmacol. 1985 Jul 15;34(14):2489–2494. doi: 10.1016/0006-2952(85)90531-3. [DOI] [PubMed] [Google Scholar]
  34. Ramsden R., Beck N. B., Sommer K. M., Omiecinski C. J. Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice. Gene. 1999 Mar 4;228(1-2):169–179. doi: 10.1016/s0378-1119(98)00612-x. [DOI] [PubMed] [Google Scholar]
  35. Ryan D. E., Wood A. W., Thomas P. E., Walz F. G., Jr, Yuan P. M., Shively J. E., Levin W. Comparisons of highly purified hepatic microsomal cytochromes P-450 from Holtzman and Long-Evans rats. Biochim Biophys Acta. 1982 Dec 20;709(2):273–283. doi: 10.1016/0167-4838(82)90470-8. [DOI] [PubMed] [Google Scholar]
  36. Seglen P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29–83. doi: 10.1016/s0091-679x(08)61797-5. [DOI] [PubMed] [Google Scholar]
  37. Stoltz C., Vachon M. H., Trottier E., Dubois S., Paquet Y., Anderson A. The CYP2B2 phenobarbital response unit contains an accessory factor element and a putative glucocorticoid response element essential for conferring maximal phenobarbital responsiveness. J Biol Chem. 1998 Apr 3;273(14):8528–8536. doi: 10.1074/jbc.273.14.8528. [DOI] [PubMed] [Google Scholar]
  38. Sueyoshi T., Kawamoto T., Zelko I., Honkakoski P., Negishi M. The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene. J Biol Chem. 1999 Mar 5;274(10):6043–6046. doi: 10.1074/jbc.274.10.6043. [DOI] [PubMed] [Google Scholar]
  39. Swanson H. I., Bradfield C. A. The AH-receptor: genetics, structure and function. Pharmacogenetics. 1993 Oct;3(5):213–230. doi: 10.1097/00008571-199310000-00001. [DOI] [PubMed] [Google Scholar]
  40. Trottier E., Belzil A., Stoltz C., Anderson A. Localization of a phenobarbital-responsive element (PBRE) in the 5'-flanking region of the rat CYP2B2 gene. Gene. 1995 Jun 9;158(2):263–268. doi: 10.1016/0378-1119(94)00916-g. [DOI] [PubMed] [Google Scholar]
  41. Tzameli I., Pissios P., Schuetz E. G., Moore D. D. The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the nuclear receptor CAR. Mol Cell Biol. 2000 May;20(9):2951–2958. doi: 10.1128/mcb.20.9.2951-2958.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Waxman D. J., Azaroff L. Phenobarbital induction of cytochrome P-450 gene expression. Biochem J. 1992 Feb 1;281(Pt 3):577–592. doi: 10.1042/bj2810577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Waxman D. J. Interactions of hepatic cytochromes P-450 with steroid hormones. Regioselectivity and stereospecificity of steroid metabolism and hormonal regulation of rat P-450 enzyme expression. Biochem Pharmacol. 1988 Jan 1;37(1):71–84. doi: 10.1016/0006-2952(88)90756-3. [DOI] [PubMed] [Google Scholar]
  44. Waxman D. J. P450 gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR. Arch Biochem Biophys. 1999 Sep 1;369(1):11–23. doi: 10.1006/abbi.1999.1351. [DOI] [PubMed] [Google Scholar]
  45. Whitlock J. P., Jr, Okino S. T., Dong L., Ko H. P., Clarke-Katzenberg R., Ma Q., Li H. Cytochromes P450 5: induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription. FASEB J. 1996 Jun;10(8):809–818. doi: 10.1096/fasebj.10.8.8666157. [DOI] [PubMed] [Google Scholar]
  46. Yamada H., Fujisaki H., Kaneko H., Ishii Y., Hamaguchi T., Oguri K. Picrotoxin as a potent inducer of rat hepatic cytochrome P450, CYP2B1 and CYP2B2. Biochem Pharmacol. 1993 May 5;45(9):1783–1789. doi: 10.1016/0006-2952(93)90434-x. [DOI] [PubMed] [Google Scholar]

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