Abstract
The constitutive androstane receptor (CAR, NR1I3) controls the transcription of numerous hepatic drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both mechanisms require the translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active and spontaneously localized in the nucleus of most immortalized cell lines. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the high basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify human CAR modulators through the employment of a double stable cell line. Using this line, we can identify activators, as well as deactivators, of the challenging nuclear receptor, CAR.
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References
Honkakoski P, Zelko I, Sueyoshi T, Negishi M (1998) The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene. Mol Cell Biol 18(10):5652–5658
Wang H, LeCluyse E (2003) Role of orphan nuclear receptors in the regulation of drug-Metabolising enzymes. Clin Pharmacokinet 42(15):1331–1357. https://doi.org/10.2165/00003088-200342150-00003
Lynch C, Pan Y, Li L, Heyward S, Moeller T, Swaan PW, Wang H (2014) Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes. Toxicol Appl Pharmacol 279(1):33–42. https://doi.org/10.1016/j.taap.2014.05.009
Dong B, Saha PK, Huang W, Chen W, Abu-Elheiga LA, Wakil SJ, Stevens RD, Ilkayeva O, Newgard CB, Chan L, Moore DD (2009) Activation of nuclear receptor CAR ameliorates diabetes and fatty liver disease. Proc Natl Acad Sci U S A 106:18831–18836. https://doi.org/10.1073/pnas.0909731106
De Mattia E, Cecchin E, Roncato R, Toffoli G (2016) Pregnane X receptor, constitutive androstane receptor and hepatocyte nuclear factors as emerging players in cancer precision medicine. Pharmacogenomics 17(14):1547–1571. https://doi.org/10.2217/pgs-2016-0095
Hedrich WD, Xiao J, Heyward S, Zhang Y, Zhang J, Baer MR, Hassan HE, Wang H (2016) Activation of the constitutive Androstane receptor increases the therapeutic index of CHOP in lymphoma treatment. Mol Cancer Ther 15(3):392–401. https://doi.org/10.1158/1535-7163.MCT-15-0667
Shmarakov IO, Lee YJ, Jiang H, Blaner WS (2019) Constitutive androstane receptor mediates PCB-induced disruption of retinoid homeostasis. Toxicol Appl Pharmacol 381:114731. https://doi.org/10.1016/j.taap.2019.114731
Mackowiak B, Li L, Lynch C, Ziman A, Heyward S, Xia M, Wang H (2019) High-content analysis of constitutive androstane receptor (CAR) translocation identifies mosapride citrate as a CAR agonist that represses gluconeogenesis. Biochem Pharmacol 168:224–236. https://doi.org/10.1016/j.bcp.2019.07.013
Li H, Chen T, Cottrell J, Wang H (2009) Nuclear translocation of adenoviral-enhanced yellow fluorescent protein-tagged-human constitutive androstane receptor (hCAR): a novel tool for screening hCAR activators in human primary hepatocytes. Drug Metab Dispos 37:1098–1106. https://doi.org/10.1124/dmd.108.026005
Kawamoto T, Sueyoshi T, Zelko I, Moore R, Washburn K, Negishi M (1999) Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Mol Cell Biol 19(9):6318–6322. https://doi.org/10.1128/mcb.19.9.6318
Chen T, Tompkins LM, Li L, Li H, Kim G, Zheng Y, Wang H (2010) A single amino acid controls the functional switch of human constitutive androstane receptor (CAR) 1 to the xenobiotic-sensitive splicing variant CAR3. J Pharmacol Exp Ther 332(1):106–115
Zelko I, Sueyoshi T, Kawamoto T, Moore R, Negishi M (2001) The peptide near the C terminus regulates receptor CAR nuclear translocation induced by Xenochemicals in mouse liver. Mol Cell Biol 21(8):2838–2846. https://doi.org/10.1128/mcb.21.8.2838-2846.2001
Huang W, Zhang J, Wei P, Schrader WT, Moore DD (2004) Meclizine is an agonist ligand for mouse constitutive Androstane receptor (CAR) and an inverse agonist for human CAR. Mol Endocrinol 18(10):2402–2408. https://doi.org/10.1210/me.2004-0046
Li L, Chen T, Stanton JD, Sueyoshi T, Negishi M, Wang H (2008) The peripheral benzodiazepine receptor ligand 1-(2-Chlorophenyl-methylpropyl)-3-isoquinoline-carboxamide is a novel antagonist of human constitutive androstane receptor. Mol Pharmacol 74:443–453. https://doi.org/10.1124/mol.108.046656
Moore LB, Parks DJ, Jones SA, Bledsoe RK, Consler TG, Stimmel JB, Goodwin B, Liddle C, Blanchard SG, Willson TM, Collins JL, Kliewer SA (2000) Orphan nuclear receptors constitutive Androstane receptor and Pregnane X receptor share xenobiotic and steroid ligands. J Biol Chem 275(20):15122–15127. https://doi.org/10.1074/jbc.M001215200
Lau AJ, Yang G, Rajaraman G, Baucom CC, Chang TKH (2011) Differential effect of meclizine on the activity of human Pregnane X receptor and constitutive Androstane receptor. J Pharmacol Exp Ther 336(3):816–826. https://doi.org/10.1124/jpet.110.175927
Mäkinen J, Frank C, Jyrkkärinne J, Gynther J, Carlberg C, Honkakoski P (2002) Modulation of mouse and human phenobarbital-responsive enhancer module by nuclear receptors. Mol Pharmacol 62(2):366–378. https://doi.org/10.1124/mol.62.2.366
Toell A, Kröncke K-D, Kleinert H, Carlberg C (2002) Orphan nuclear receptor binding site in the human inducible nitric oxide synthase promoter mediates responsiveness to steroid and xenobiotic ligands. J Cell Biochem 85(1):72–82. https://doi.org/10.1002/jcb.10104
Mackowiak B, Li L, Welch MA, Li D, Jones JW, Heyward S, Kane MA, Swaan PW, Wang H (2017) Molecular basis of metabolism-mediated conversion of PK11195 from an antagonist to an agonist of the constitutive Androstane receptor. Mol Pharmacol 92(1):75–87. https://doi.org/10.1124/mol.117.108621
Freitas J, Miller N, Mengeling BJ, Xia M, Huang R, Houck K, Rietjens IMCM, Furlow JD, Murk AJ (2014) Identification of thyroid hormone receptor active compounds using a quantitative high-throughput screening platform. Curr Chem Genom Transl Med 8:36–46. https://doi.org/10.2174/2213988501408010036
Shukla SJ, Sakamuru S, Huang R, Moeller TA, Shinn P, VanLeer D, Auld DS, Austin CP, Xia M (2011) Identification of clinically used drugs that activate Pregnane X receptors. Drug Metab Dispos 39(1):151–159. https://doi.org/10.1124/dmd.110.035105
Lynch C, Zhao J, Huang R, Xiao J, Li L, Heyward S, Xia M, Wang H (2015) Quantitative high-throughput identification of drugs as modulators of human constitutive Androstane receptor. Sci Rep 5. https://doi.org/10.1038/srep10405. http://www.nature.com/srep/2015/150520/srep10405/abs/srep10405.html#supplementary-information
Lynch C, Mackowiak B, Huang R, Li L, Heyward S, Sakamuru S, Wang H, Xia M (2019) Identification of modulators that activate the constitutive Androstane receptor from the Tox21 10K compound library. Toxicol Sci 167(1):282–292. https://doi.org/10.1093/toxsci/kfy242
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This work was supported, in part, by the Intramural research program of the NCATS, NIH.
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Lynch, C., Zhao, J., Wang, H., Xia, M. (2022). Identifying CAR Modulators Utilizing a Reporter Gene Assay. In: Zhu, H., Xia, M. (eds) High-Throughput Screening Assays in Toxicology. Methods in Molecular Biology, vol 2474. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2213-1_4
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DOI: https://doi.org/10.1007/978-1-0716-2213-1_4
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