The constitutive androstane receptor (CAR) is a nuclear receptor involved mainly in xenobiotic and endobiotic metabolism regulation. CAR is activated directly by its ligands via the ligand binding domain (LBD) or indirectly by inhibition of the epidermal growth factor (EGF) signaling. We found that leflunomide (LEF) and its main metabolite teriflunomide (TER), both used for autoimmune diseases treatment, induce the prototype CAR target gene CYP2B6 in primary human hepatocytes. As TER was discovered to be an EGF receptor antagonist, we sought to determine if TER is an indirect activator of CAR. In primary human hepatocytes and in differentiated HepaRG cells, we found that LEF and TER up-regulate CAR target genes CYP2B6 and CYP3A4 mRNAs and enzymatic activities. TER stimulated CAR+A mutant translocation into the nucleus but neither LEF nor TER activated the CAR LBD, CAR3 variant or pregnane X receptor (PXR) in gene reporter assays. Interestingly, TER significantly up-regulated CAR mRNA expression, a result which could be a consequence of both EGF receptor and ELK-1 transcription factor inhibition by TER or by TER-mediated activation of glucocorticoid receptor (GR), an upstream hormonal regulator of CAR. We can conclude that TER is a novel indirect CAR activator which through EGF inhibition and GR activation controls both detoxification and some intermediary metabolism genes.

Department of Pharmaceutical Technology Faculty of Pharmacy Charles University Prague Czechia

Department of Pharmacology and Toxicology Faculty of Pharmacy Charles University Prague Czechia

Department of Social and Clinical Pharmacy Faculty of Pharmacy Charles University Prague Czechia

Division of Rheumatology 2nd Department of Internal Medicine Gastroenterology Faculty of Medicine University Hospital in Hradec Kralove Charles University Prague Czechia

Faculty of Medicine and Dentistry Institute of Molecular and Translational Medicine Palacky University Olomouc Czechia

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Baes M., Gulick T., Choi H. S., Martinoli M. G., Simha D., Moore D. D. (1994). A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements. Mol. Cell. Biol. 14 1544–1552. 10.1128/MCB.14.3.1544 PubMed DOI PMC

Carazo A., Pavek P. (2015). The use of the LanthaScreen TR-FRET CAR Coactivator assay in the characterization of Constitutive Androstane Receptor (CAR) inverse agonists. Sensors 15 9265–9276. 10.3390/s150409265 PubMed DOI PMC

Carazo Fernandez A., Smutny T., Hyrsova L., Berka K., Pavek P. (2015). Chrysin, baicalein and galangin are indirect activators of the human constitutive androstane receptor (CAR). Toxicol. Lett. 233 68–77. 10.1016/j.toxlet.2015.01.013 PubMed DOI

Chai S. C., Cherian M. T., Wang Y. M., Chen T. (2016). Small-molecule modulators of PXR and CAR. Biochim. Biophys. Acta 1859 1141–1154. 10.1016/j.bbagrm.2016.02.013 PubMed DOI PMC

Chen T., Tompkins L. M., Li L., Li H., Kim G., Zheng Y., et al. (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. 332 106–115. 10.1124/jpet.109.159210 PubMed DOI PMC

de Boussac H., Gondeau C., Briolotti P., Duret C., Treindl F., Romer M., et al. (2018). Epidermal growth factor represses constitutive androstane receptor expression in primary human hepatocytes and favors regulation by pregnane X receptor. Drug Metab. Dispos. 46 223–236. 10.1124/dmd.117.078683 PubMed DOI

di Masi A., De Marinis E., Ascenzi P., Marino M. (2009). Nuclear receptors CAR and PXR: molecular, functional, and biomedical aspects. Mol. Aspects Med. 30 297–343. 10.1016/j.mam.2009.04.002 PubMed DOI

Dong B., Saha P. K., Huang W., Chen W., Abu-Elheiga L. A., Wakil S. J., et al. (2009). Activation of nuclear receptor CAR ameliorates diabetes and fatty liver disease. Proc. Natl. Acad. Sci. U.S.A. 106 18831–18836. 10.1073/pnas.0909731106 PubMed DOI PMC

Dusek J., Carazo A., Trejtnar F., Hyrsova L., Holas O., Smutny T., et al. (2017). Steviol, an aglycone of steviol glycoside sweeteners, interacts with the pregnane X (PXR) and aryl hydrocarbon (AHR) receptors in detoxification regulation. Food Chem. Toxicol. 109 130–142. 10.1016/j.fct.2017.09.007 PubMed DOI

Faucette S. R., Zhang T. C., Moore R., Sueyoshi T., Omiecinski C. J., LeCluyse E. L., et al. (2007). Relative activation of human pregnane X receptor versus constitutive androstane receptor defines distinct classes of CYP2B6 and CYP3A4 inducers. J. Pharmacol. Exp. Ther. 320 72–80. 10.1124/jpet.106.112136 PubMed DOI PMC

Gao J., He J., Zhai Y., Wada T., Xie W. (2009). The constitutive androstane receptor is an anti-obesity nuclear receptor that improves insulin sensitivity. J. Biol. Chem. 284 25984–25992. 10.1074/jbc.M109.016808 PubMed DOI PMC

Gao J., Yan J., Xu M., Ren S., Xie W. (2015). CAR suppresses hepatic gluconeogenesis by facilitating the ubiquitination and degradation of PGC1alpha. Mol. Endocrinol. 29 1558–1570. 10.1210/me.2015-1145 PubMed DOI PMC

Ghosh S., Narla R. K., Zheng Y., Liu X. P., Jun X., Mao C., et al. (1999). Structure-based design of potent inhibitors of EGF-receptor tyrosine kinase as anti-cancer agents. Anticancer Drug Des. 14 403–410. PubMed

Ghosh S., Zheng Y., Jun X., Narla R. K., Mahajan S., Navara C., et al. (1998). Alpha-cyano-beta-hydroxy-beta-methyl-N-[4-(trifluoromethoxy)phenyl] propenamide: an inhibitor of the epidermal growth factor receptor tyrosine kinase with potent cytotoxic activity against breast cancer cells. Clin. Cancer Res. 4 2657–2668. PubMed

Hardesty J. E., Al-Eryani L., Wahlang B., Falkner K. C., Shi H., Jin J., et al. (2018). Epidermal growth factor receptor signaling disruption by endocrine and metabolic disrupting chemicals. Toxicol. Sci. 162 622–634. 10.1093/toxsci/kfy004 PubMed DOI PMC

Hardesty J. E., Wahlang B., Falkner K. C., Clair H. B., Clark B. J., Ceresa B. P., et al. (2017). Polychlorinated biphenyls disrupt hepatic epidermal growth factor receptor signaling. Xenobiotica 47 807–820. 10.1080/00498254.2016.1217572 PubMed DOI PMC

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 5652–5658. 10.1128/MCB.18.10.5652 PubMed DOI PMC

Hyrsova L., Smutny T., Carazo A., Moravcik S., Mandikova J., Trejtnar F., et al. (2016). The pregnane X receptor down-regulates organic cation transporter 1 (SLC22A1) in human hepatocytes by competing for (“squelching”) SRC-1 coactivator. Br. J. Pharmacol. 173 1703–1715. 10.1111/bph.13472 PubMed DOI PMC

Kanno Y., Suzuki M., Miyazaki Y., Matsuzaki M., Nakahama T., Kurose K., et al. (2007). Difference in nucleocytoplasmic shuttling sequences of rat and human constitutive active/androstane receptor. BBA Mol. Cell Res. 1773 934–944. 10.1016/j.bbamcr.2007.03.020 PubMed DOI

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 6318–6322. 10.1128/MCB.19.9.6318 PubMed DOI PMC

Keen H. I., Conaghan P. G., Tett S. E. (2013). Safety evaluation of leflunomide in rheumatoid arthritis. Expert Opin. Drug Saf. 12 581–588. 10.1517/14740338.2013.798299 PubMed DOI

Krausova L., Stejskalova L., Wang H., Vrzal R., Dvorak Z., Mani S., et al. (2011). Metformin suppresses pregnane X receptor (PXR)-regulated transactivation of CYP3A4 gene. Biochem. Pharmacol. 82 1771–1780. 10.1016/j.bcp.2011.08.023 PubMed DOI PMC

Li D., Mackowiak B., Brayman T. G., Mitchell M., Zhang L., Huang S. M., et al. (2015). Genome-wide analysis of human constitutive androstane receptor (CAR) transcriptome in wild-type and CAR-knockout HepaRG cells. Biochem. Pharmacol. 98 190–202. 10.1016/j.bcp.2015.08.087 PubMed DOI PMC

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. 10.1124/dmd.108.026005 PubMed DOI PMC

Li L., Sinz M. W., Zimmermann K., Wang H. (2012). An insulin-like growth factor 1 receptor inhibitor induces CYP3A4 expression through a pregnane X receptor-independent, noncanonical constitutive androstane receptor-related mechanism. J. Pharmacol. Exp. Ther. 340 688–697. 10.1124/jpet.111.188854 PubMed DOI PMC

Lynch C., Pan Y., Li L., Ferguson S. S., Xia M., Swaan P. W., et al. (2013). Identification of novel activators of constitutive androstane receptor from FDA-approved drugs by integrated computational and biological approaches. Pharm. Res. 30 489–501. 10.1007/s11095-012-0895-1 PubMed DOI PMC

Mackowiak B., Wang H. (2016). Mechanisms of xenobiotic receptor activation: direct vs. indirect. Biochim. Biophys. Acta 1859 1130–1140. 10.1016/j.bbagrm.2016.02.006 PubMed DOI PMC

Miao J., Fang S., Bae Y., Kemper J. K. (2006). Functional inhibitory cross-talk between constitutive androstane receptor and hepatic nuclear factor-4 in hepatic lipid/glucose metabolism is mediated by competition for binding to the DR1 motif and to the common coactivators, GRIP-1 and PGC-1alpha. J. Biol. Chem. 281 14537–14546. 10.1074/jbc.M510713200 PubMed DOI

Molnar F., Kublbeck J., Jyrkkarinne J., Prantner V., Honkakoski P. (2013). An update on the constitutive androstane receptor (CAR). Drug Metabol. Drug. Interact. 28 79–93. 10.1515/dmdi-2013-0009 PubMed DOI

Moore L. B., Parks D. J., Jones S. A., Bledsoe R. K., Consler T. G., Stimmel J. B., et al. (2000). Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands. J. Biol. Chem. 275 15122–15127. 10.1074/jbc.M001215200 PubMed DOI

Mutoh S., Osabe M., Inoue K., Moore R., Pedersen L., Perera L., et al. (2009). Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3). J. Biol. Chem. 284 34785–34792. 10.1074/jbc.M109.048108 PubMed DOI PMC

Mutoh S., Sobhany M., Moore R., Perera L., Pedersen L., Sueyoshi T., et al. (2013). Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling. Sci. Signal. 6:ra31. 10.1126/scisignal.2003705 PubMed DOI PMC

Oh K. J., Han H. S., Kim M. J., Koo S. H. (2013). CREB and FoxO1: two transcription factors for the regulation of hepatic gluconeogenesis. BMB Rep. 46 567–574. 10.5483/BMBRep.2013.46.12.248 PubMed DOI PMC

Osabe M., Negishi M. (2011). Active ERK1/2 protein interacts with the phosphorylated nuclear constitutive active/androstane receptor (CAR; NR1I3), repressing dephosphorylation and sequestering CAR in the cytoplasm. J. Biol. Chem. 286 35763–35769. 10.1074/jbc.M111.284596 PubMed DOI PMC

Pascussi J. M., Gerbal-Chaloin S., Fabre J. M., Maurel P., Vilarem M. J. (2000). Dexamethasone enhances constitutive androstane receptor expression in human hepatocytes: consequences on cytochrome P450 gene regulation. Mol. Pharmacol. 58 1441–1450. 10.1124/mol.58.6.1441 PubMed DOI

Rezen T., Tamasi V., Lovgren-Sandblom A., Bjorkhem I., Meyer U. A., Rozman D. (2009). Effect of CAR activation on selected metabolic pathways in normal and hyperlipidemic mouse livers. BMC Genomics 10:384. 10.1186/1471-2164-10-384 PubMed DOI PMC

Rojas M., Yao S., Lin Y. Z. (1996). Controlling epidermal growth factor (EGF)-stimulated Ras activation in intact cells by a cell-permeable peptide mimicking phosphorylated EGF receptor. J. Biol. Chem. 271 27456–27461. 10.1074/jbc.271.44.27456 PubMed DOI

Rulcova A., Prokopova I., Krausova L., Bitman M., Vrzal R., Dvorak Z., et al. (2010). Stereoselective interactions of warfarin enantiomers with the pregnane X nuclear receptor in gene regulation of major drug-metabolizing cytochrome P450 enzymes. J. Thromb. Haemost. 8 2708–2717. 10.1111/j.1538-7836.2010.04036.x PubMed DOI

Sapio L., Gallo M., Illiano M., Chiosi E., Naviglio D., Spina A., et al. (2017). The natural cAMP elevating compound forskolin in cancer therapy: is it time? J. Cell. Physiol. 232 922–927. 10.1002/jcp.25650 PubMed DOI

Shizu R., Osabe M., Perera L., Moore R., Sueyoshi T., Negishi M. (2017). Phosphorylated nuclear receptor CAR forms a homodimer to repress its constitutive activity for ligand activation. Mol. Cell. Biol. 37:e00649-16. 10.1128/MCB.00649-16 PubMed DOI PMC

Tolson A. H., Li H., Eddington N. D., Wang H. (2009). Methadone induces the expression of hepatic drug-metabolizing enzymes through the activation of pregnane X receptor and constitutive androstane receptor. Drug Metab. Dispos. 37 1887–1894. 10.1124/dmd.109.027854 PubMed DOI PMC

Tzameli I., Pissios P., Schuetz E. G., Moore D. D. (2000). The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the nuclear receptor CAR. Mol. Cell. Biol. 20 2951–2958. 10.1128/MCB.20.9.2951-2958.2000 PubMed DOI PMC

Wang H., Faucette S., Moore R., Sueyoshi T., Negishi M., LeCluyse E. (2004). Human constitutive androstane receptor mediates induction of CYP2B6 gene expression by phenytoin. J. Biol. Chem. 279 29295–29301. 10.1074/jbc.M400580200 PubMed DOI

Yan J., Chen B., Lu J., Xie W. (2015). Deciphering the roles of the constitutive androstane receptor in energy metabolism. Acta Pharmacol. Sin. 36 62–70. 10.1038/aps.2014.102 PubMed DOI PMC

Yarushkin A. A., Kachaylo E. M., Pustylnyak V. O. (2013). The constitutive androstane receptor activator 4-[(4R,6R)-4,6-diphenyl-1,3-dioxan-2-yl]-N,N-dimethylaniline inhibits the gluconeogenic genes PEPCK and G6Pase through the suppression of HNF4alpha and FOXO1 transcriptional activity. Br. J. Pharmacol. 168 1923–1932. 10.1111/bph.12090 PubMed DOI PMC

Yu L., Wang Z., Huang M., Li Y., Zeng K., Lei J., et al. (2016). Evodia alkaloids suppress gluconeogenesis and lipogenesis by activating the constitutive androstane receptor. Biochim. Biophys. Acta 1859 1100–1111. 10.1016/j.bbagrm.2015.10.001 PubMed DOI

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