The Peripheral Benzodiazepine Receptor Ligand 1-(2-Chlorophenyl-methylpropyl)-3-isoquinoline-carboxamide Is a Novel Antagonist of Human Constitutive Androstane Receptor
Abstract:As a promiscuous xenobiotic sensor, the constitutive androstane receptor (CAR; NR1I3) regulates the expression of multiple drugmetabolizing enzymes and transporters in liver. The constitutively activated nature of CAR in the cell-based transfection assays has hindered its use as a predictor of metabolism-based drugdrug interactions. Here, we have identified 1-(2-chlorophenylmethylpropyl)-3-isoquinoline-carboxamide (PK11195), a typical peripheral benzodiazepine receptor (PBR) ligand, as a selective and potent i… Show more
“…dmd.aspetjournals.org looping, possibly enabling the promoter to recruit RNA polymerase II for effective promoter activation Negishi, 2008, 2009). In addition to early growth response 1, various other transcription factors were shown to interact with and activate the CYP2B promoters together with CAR, such as peroxisome proliferator-activated receptor -g coactivator-1 alpha (PGC-1a), steroid receptor coactivator-1 (SRC-1), HNF-4a, and CCAAT/enhancer-binding protein alpha (Shiraki et al, 2003;Li et al, 2008;Benet et al, 2010). Since p38 MAPK can activate these factors (Lim et al, 1998;Knutti et al, 2001;Guo et al, 2007;Qiao et al, 2006;Fernandez-Marcos and Auwerx, 2011;Antoon et al, 2012), it may enable CAR to bind to PBREM and to loop the promoter by phosphorylating one or more of them.…”
Nuclear receptor constitutive androstane receptor (CAR, NR1I3), which regulates hepatic drug and energy metabolisms as well as cell growth and death, is sequestered in the cytoplasm as its inactive form phosphorylated at threonine 38. CAR activators elicit dephosphorylation, and nonphosphorylated CAR translocates into the nucleus to activate its target genes. CAR was previously found to require p38 mitogen-activated protein kinase (MAPK) to transactivate the cytochrome P450 2B (CYP2B) genes. Here we have demonstrated that p38 MAPK forms a complex with CAR, enables it to bind to the response sequence, phenobarbital-responsive enhancer module (PBREM), within the CYP2B promoter, and thus recruits RNA polymerase II to activate transcription. Subsequently, p38 MAPK elicited rephosphorylation of threonine 38 to inactivate CAR and exclude it from the nucleus. Thus, nuclear p38 MAPK exerted dual regulation by sequentially activating and inactivating CAR-mediated transcription through phosphorylation of threonine 38.
“…dmd.aspetjournals.org looping, possibly enabling the promoter to recruit RNA polymerase II for effective promoter activation Negishi, 2008, 2009). In addition to early growth response 1, various other transcription factors were shown to interact with and activate the CYP2B promoters together with CAR, such as peroxisome proliferator-activated receptor -g coactivator-1 alpha (PGC-1a), steroid receptor coactivator-1 (SRC-1), HNF-4a, and CCAAT/enhancer-binding protein alpha (Shiraki et al, 2003;Li et al, 2008;Benet et al, 2010). Since p38 MAPK can activate these factors (Lim et al, 1998;Knutti et al, 2001;Guo et al, 2007;Qiao et al, 2006;Fernandez-Marcos and Auwerx, 2011;Antoon et al, 2012), it may enable CAR to bind to PBREM and to loop the promoter by phosphorylating one or more of them.…”
Nuclear receptor constitutive androstane receptor (CAR, NR1I3), which regulates hepatic drug and energy metabolisms as well as cell growth and death, is sequestered in the cytoplasm as its inactive form phosphorylated at threonine 38. CAR activators elicit dephosphorylation, and nonphosphorylated CAR translocates into the nucleus to activate its target genes. CAR was previously found to require p38 mitogen-activated protein kinase (MAPK) to transactivate the cytochrome P450 2B (CYP2B) genes. Here we have demonstrated that p38 MAPK forms a complex with CAR, enables it to bind to the response sequence, phenobarbital-responsive enhancer module (PBREM), within the CYP2B promoter, and thus recruits RNA polymerase II to activate transcription. Subsequently, p38 MAPK elicited rephosphorylation of threonine 38 to inactivate CAR and exclude it from the nucleus. Thus, nuclear p38 MAPK exerted dual regulation by sequentially activating and inactivating CAR-mediated transcription through phosphorylation of threonine 38.
“…Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA) and reverse-transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems) per manufacturers' instructions. Primers and probes for CYP2B6, CYP3A4, UGT1A1, and MDR1 genes (Table 1) were designed using Primer Express version 2.0 (Applied Biosystems) and entered into the National Center for Biotechnology Information BLAST to ensure specificity as described previously (Maglich et al, 2002;Smith et al, 2005;Faucette et al, 2007;Li et al, 2008). The mRNA expression of CYP2B6, CYP3A4, UGT1A1, and MDR1 was normalized against that of human -actin, which was detected using a predeveloped primer/probe mixture (Applied Biosystems).…”
ABSTRACT:Methadone (MD) is the most established substance abuse pharmacotherapy of choice for the management of heroin dependence. To date, drug-drug interactions involving MD have been characterized asymmetrically among existing reports, which describe how other drugs affect the metabolic or pharmacokinetic profiles of MD; however, limited information is available regarding the potential for MD to influence similar fates of coadministered drugs. Moreover, little to no mechanistic evidence has been explored. Here, we show that MD induces hepatic drug-metabolizing enzymes (
“…Secondly, the effects of Andro, CLT, artemisinin (Simonsson et al, 2006), and PK11195 (Li et al, 2008), all ligands of CAR, on the transactivation potential of GAL4/DBD-hCAR/LBD(+3a.a.) were evaluated.…”
Section: The Establishment and Confirmation Of A Novel Car Ligand Scrmentioning
-The expression of the CAR gene is lost in most cultured cell lines, and exogenously expressed CAR accumulates spontaneously in the nuclear compartment, where CAR is constitutively active. Therefore, the assessment of CAR-dependent transcriptional activation has to be evaluated using either animal models or primary cultured hepatocytes. Furthermore, due to the species-specific response of CAR to individual compounds, the results obtained with animal models are not directly applicable to human cases. We established a novel screening system for hCAR ligands (including agonists and inverse agonists) by expressing GAL4/DBD-fused hCAR/LBD, in which three consecutive alanine residues were inserted between helix 11 and helix 12 of LBD, and a commercially obtained plasmid consisting of the firefly luciferase gene downstream of the GAL4 responsive element in a cultured cell line. Androstenol (Andro) and clotrimazole (CLT), which are both inverse agonists of hCAR, were classified as an antagonist and weak agonist, respectively, in our novel assay system. Among DDT and its metabolites DDE and DDD, only DDT worked as an agonist of the hCAR mutant, although all of them were enrolled as agonists of SXR. Using this system, the screening for hCAR ligands can be conducted without sacrificing animals.
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