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

Li, Haishan; Chen, Tao; Cottrell, John R.; Wang, Hongbing

doi:10.1124/dmd.108.026005

Cited by 72 publications

(86 citation statements)

References 37 publications

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“…Furthermore, there is evidence suggesting that lopinavir and abacavir can serve as ligands of hCAR in transfected human liver hepatocellular carcinoma cell line (HepG2) cells (22). In the present work, we performed a re- porter gene assay of primary cultures of mouse hepatocytes that has previously been identified to exhibit enhanced CARinducible signaling (i.e., nuclear translocation) compared to HepG2 cultures (35,36). We found that abacavir, which belongs to the nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) class, as well as efavirenz and nevirapine of the NNRTI class, can serve as a ligand of hCAR (Fig.…”

Section: Discussionmentioning

confidence: 99%

Induction of P-Glycoprotein by Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells

Chan

Patel

Cummins

et al. 2013

Antimicrob Agents Chemother

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The membrane-associated drug transporter P-glycoprotein (P-gp) plays an essential role in drug efflux from the brain. Induction of this protein at the blood-brain barrier (BBB) could further affect the ability of a drug to enter the brain. At present, P-gp induction mediated by antiretroviral drugs at the BBB has not been fully investigated. Since P-gp expression is regulated by ligand-activated nuclear receptors, i.e., human pregnane X receptor (hPXR) and human constitutive androstane receptor (hCAR), these receptors could represent potential pathways involved in P-gp induction by antiretroviral drugs. The aims of this study were (i) to determine whether antiretroviral drugs currently used in HIV pharmacotherapy are ligands for hPXR or hCAR and (ii) to examine P-gp function and expression in human brain microvessel endothelial cells treated with antiretroviral drugs identified as ligands of hPXR and/or hCAR. Luciferase reporter gene assays were performed to examine the activation of hPXR and hCAR by antiretroviral drugs. The hCMEC/D3 cell line, which is known to display several morphological and biochemical properties of the BBB in humans, was used to examine P-gp induction following 72 h of exposure to these agents. Amprenavir, atazanavir, darunavir, efavirenz, ritonavir, and lopinavir were found to activate hPXR, whereas abacavir, efavirenz, and nevirapine were found to activate hCAR. P-gp expression and function were significantly induced in hCMEC/D3 cells treated with these drugs at clinical concentrations in plasma. Together, our data suggest that P-gp induction could occur at the BBB during chronic treatment with antiretroviral drugs identified as ligands of hPXR and/or hCAR.

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Section: Discussionmentioning

confidence: 99%

Induction of P-Glycoprotein by Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells

Chan

Patel

Cummins

et al. 2013

Antimicrob Agents Chemother

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“…An adenovirus-expressing enhanced yellow fluorescent protein-tagged hCAR (Ad/EYFP-hCAR) construct was generated and functionally characterized, as previously reported (Li et al, 2009a). Cryo-HepaRG were thawed and seeded on 24-well BioCoat plates (Thermo Fisher) in WEM containing HepaRG Induction Media Supplements HPRG740 (serum-containing) and HPRG750 (serum-free).…”

Section: Methodsmentioning

confidence: 99%

Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures

Jackson

Chamberlain

et al. 2016

Drug Metabolism and Disposition

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Over the last decade HepaRG cells have emerged as a promising alternative to primary human hepatocytes (PHH) and have been featured in over 300 research publications. Most of these reports employed freshly differentiated HepaRG cells that require timeconsuming culture (∼28 days) for full differentiation. Recently, a cryopreserved, predifferentiated format of HepaRG cells (termed here "cryo-HepaRG") has emerged as a new model that improves global availability and experimental flexibility; however, it is largely unknown whether HepaRG cells in this format fully retain their hepatic characteristics. Therefore, we systematically investigated the hepatocyte functionality of cryo-HepaRG cultures in context with the range of interindividual variation observed with PHH in both sandwich-culture and suspension formats. These evaluations uncovered a novel adaptation period for the cryo-HepaRG format and demonstrated the impact of extracellular matrix on cryo-HepaRG functionality. Pharmacologically important drug-metabolizing alleles were genotyped in HepaRG cells and poor metabolizer alleles for CYP2D6, CYP2C9, and CYP3A5 were identified and consistent with higher frequency alleles found in individuals of Caucasian decent. We observed liver enzyme inducibility with aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor activators comparable to that of sandwich-cultured PHH. Finally, we show for the first time that cryo-HepaRG supports proper CAR cytosolic sequestration and translocation to hepatocyte nuclei in response to phenobarbital treatment. Taken together, these data reveal important considerations for the use of this cell model and demonstrate that cryo-HepaRG are suitable for metabolism and toxicology screening.

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“…The CYP2B6-phenobarbital-responsive enhancer module/xenobiotic-responsive enhancer module luciferase reporter plasmid was constructed as described previously elsewhere (Wang et al, 2003). The adenovirus expressing the EYFP-tagged hCAR (Ad/EYFP-hCAR) was generated as reported earlier elsewhere (Li et al, 2009). For mammalian two-hybrid assay, pG5-Luc, pACT, and pBind were obtained from Promega.…”

Section: Methodsmentioning

confidence: 99%

Metformin Represses Drug-Induced Expression of CYP2B6 by Modulating the Constitutive Androstane Receptor Signaling

et al. 2013

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Metformin is currently the most widely used drug for the treatment of type 2 diabetes. Mechanistically, metformin interacts with many protein kinases and transcription factors that alter the expression of numerous downstream target genes governing lipid metabolism, cell proliferation, and drug metabolism. The constitutive androstane receptor (CAR, NR1i3), a known xenobiotic sensor, has recently been recognized as a novel signaling molecule, in that its activation could be regulated by protein kinases in addition to the traditional ligand binding. We show that metformin could suppress drug-induced expression of CYP2B6 (a typical target gene of CAR) by modulating the phosphorylation status of CAR. In human hepatocytes, metformin robustly suppressed the expression of CYP2B6 induced by both indirect (phenobarbital) and direct CITCO [6-(4-chlorophenyl)imidazo[2,1-b]1,3thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime] activators of human CAR. Mechanistic investigation revealed that metformin specifically enhanced the phosphorylation of threonine-38 of CAR, which blocks CAR nuclear translocation and activation. Moreover, we showed that phosphorylation of CAR by metformin was primarily an AMP-activated protein kinase-and extracellular signal-regulated kinase 1/2-dependent event. Additional two-hybrid and coimmunoprecipitation assays demonstrated that metformin could also disrupt CITCO-mediated interaction between CAR and the steroid receptor coactivator 1 or the glucocorticoid receptorinteracting protein 1. Our results suggest that metformin is a potent repressor of drug-induced CYP2B6 expression through specific inhibition of human CAR activation. Thus, metformin may affect the metabolism and clearance of drugs that are CYP2B6 substrates.

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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

Abstract: ABSTRACT:The

Cited by 72 publications

References 37 publications

Induction of P-Glycoprotein by Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells

Induction of P-Glycoprotein by Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells

Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures

Metformin Represses Drug-Induced Expression of CYP2B6 by Modulating the Constitutive Androstane Receptor Signaling

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