CAR activation mechanism: What is unique about this system is the fact that xenobiotics do not directly bind to CAR to activate it. We previously determined that threonine 48 of endogenous CAR is phosphorylated in mouse primary hepatocytes and that phenobarbital treatment de-phosphorylates this threonine, activating CAR and translocating it into the nucleus. We identified protein phosphatase 2A as the enzyme that de-phosphorylates threonine 48 of CAR. Moreover, receptor for activated C-kinase1 (RACK1) was characterized as the essential regulatory subunit that activates the PP2A core enzyme to de-phosphorylate threonine 48 of CAR. Phenobarbital indirectly activates nuclear receptor CAR by de-phosphorylating threonine 38. Here we have now determined the underlying mechanism through which phenobarbital activates CAR. Phenobarbital binding to EGFR initiates cell signaling to de-phosphorylate tyrosine 52 of RACK1. Non-phosphorylated RACK1, acting as the regulatory subunit, stimulates protein phosphatase 2A to de-phosphorylate threonine 38 of CAR for its activation. CAR contains the intra-molecular peptide XRS to regulates phenobarbital-initiated RACK1signaling, thereby de-phosphorylating threonine 38 for its activation. In the absence of the XRS, RACK1 is unable to bind to CAR and stimulates de-phosphorylation by PP2Ac. Our investigations have defined the phenobarbital-EGFR-RACK1/PP2Ac-XRS as the principle mechanism for CAR activation. Threonine 38 of CAR is conserved as a phosphorylation motif in the majority of human nuclear receptors such as estrogen receptors. We examined serine 212 of human estrogen receptor (serine 216 in mouse ER) and found that this serine residue is phosphorylated in ER in leukocytes such as neutrophils, eosinophils and monocytes. Mutation analysis of serine 212 revealed that phosphorylated ER regulates a unique set of the genes. Therefore, this phosphorylation motif may confer distinct functions to a given nuclear receptor far beyond that of CAR. Xenobiotic-signal crosstalk mechanism: Upon activation by xenobiotics, CAR regulates genes differently from one another, conferring specificity to CAR-regulated gene expression. CAR acquires this specificity via crosstalk with cell signaling. We have identified various endogenous cell signals as the essential regulator of CAR activation and function: p38 MAPK, SGK2 and the GADD45 (growth arrest and DNA-damage inducible 45). Given these findings, we are investigating the molecular mechanisms by which these signaling regulate CAR activation and functions. CAR-mediated diseases: Chronic treatment with drugs, such as phenobarbital, is known to activate CAR and cause hepatocellular carcinoma (HCC) in rodents. We have now characterized KCNK1 and GADD45 as a CAR target for phenobarbital promotion of HCC development: CAR interacts with GADD45 protein and this interaction inhibits phosphorylation of JNK1, thus repressing apoptosis and possibly promoting tumor genesis. KCNK1 is specifically induced in male livers and attenuates hepatic hyperplasia. 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treatment is known to cause severe liver injury and proliferate postnatal hepatic progenitor oval cells. Utilizing CAR KO mice, we have determined that DDC activates CAR and this activation is essential for the developments of liver injury and oval cell proliferation.

Project Start
Project End
Budget Start
Budget End
Support Year
28
Fiscal Year
2013
Total Cost
$2,352,234
Indirect Cost
City
State
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Gotoh, Saki; Negishi, Masahiko (2014) Serum- and glucocorticoid-regulated kinase 2 determines drug-activated pregnane X receptor to induce gluconeogenesis in human liver cells. J Pharmacol Exp Ther 348:131-40
Sueyoshi, Tatsuya; Li, Linhao; Wang, Hongbing et al. (2014) Flame retardant BDE-47 effectively activates nuclear receptor CAR in human primary hepatocytes. Toxicol Sci 137:292-302
Timsit, Yoav E; Negishi, Masahiko (2014) Coordinated regulation of nuclear receptor CAR by CCRP/DNAJC7, HSP70 and the ubiquitin-proteasome system. PLoS One 9:e96092
Saito, Kosuke; Moore, Rick; Negishi, Masahiko (2013) Nuclear receptor CAR specifically activates the two-pore K+ channel Kcnk1 gene in male mouse livers, which attenuates phenobarbital-induced hepatic hyperplasia. Toxicol Sci 132:151-61
Kodama, Susumu; Negishi, Masahiko (2013) PXR cross-talks with internal and external signals in physiological and pathophysiological responses. Drug Metab Rev 45:300-10
Saito, Kosuke; Moore, Rick; Negishi, Masahiko (2013) p38 mitogen-activated protein kinase regulates nuclear receptor CAR that activates the CYP2B6 gene. Drug Metab Dispos 41:1170-3
Saito, Kosuke; Negishi, Masahiko; James Squires, E (2013) Sexual dimorphisms in zonal gene expression in mouse liver. Biochem Biophys Res Commun 436:730-5
Mutoh, Shingo; Sobhany, Mack; Moore, Rick et al. (2013) Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling. Sci Signal 6:ra31
Konno, Yoshihiro; Kodama, Susumu; Moore, Rick et al. (2009) Nuclear xenobiotic receptor pregnane X receptor locks corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) onto the CYP24A1 promoter to attenuate vitamin D3 activation. Mol Pharmacol 75:265-71
Adair, Jennifer E; Stober, Vandy; Sobhany, Mack et al. (2009) Inter-alpha-trypsin inhibitor promotes bronchial epithelial repair after injury through vitronectin binding. J Biol Chem 284:16922-30

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