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18 pages, 2425 KiB

Open AccessFeature PaperArticle

Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain

by Josef Skoda, Jan Dusek, Martin Drastik, Alzbeta Stefela, Klara Dohnalova, Karel Chalupsky, Tomas Smutny, Stanislav Micuda, Sabine Gerbal-Chaloin and Petr Pavek

Cells 2020, 9(12), 2532; https://doi.org/10.3390/cells9122532 - 24 Nov 2020

Cited by 7 | Viewed by 2657

Abstract

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR [...] Read more.

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice. Full article

(This article belongs to the Special Issue The Xenobiotic Receptors CAR and PXR in Health and Disease)

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14 pages, 1968 KiB

Open AccessArticle

CITCO as an Adjuvant Facilitates CHOP-Based Lymphoma Treatment in hCAR-Transgenic Mice

by Ritika Kurian, William Hedrich, Bryan Mackowiak, Linhao Li and Hongbing Wang

Cells 2020, 9(11), 2520; https://doi.org/10.3390/cells9112520 - 21 Nov 2020

Cited by 4 | Viewed by 2342

Abstract

Non-Hodgkin’s lymphoma (NHL) is a malignant cancer originating in the lymphatic system with a 25–30% mortality rate. CHOP, consisting of cyclophosphamide (CPA), doxorubicin, vincristine, and prednisone, is a first-generation chemotherapy extensively used to treat NHL. However, poor survival rates among patients in advanced [...] Read more.

Non-Hodgkin’s lymphoma (NHL) is a malignant cancer originating in the lymphatic system with a 25–30% mortality rate. CHOP, consisting of cyclophosphamide (CPA), doxorubicin, vincristine, and prednisone, is a first-generation chemotherapy extensively used to treat NHL. However, poor survival rates among patients in advanced stages of NHL shows a need to improve this standard of care treatment. CPA, an integral component of CHOP, is a prodrug that requires CYP2B6-mediated bioactivation to 4-hydroxy-CPA (4-OH-CPA). The expression of CYP2B6 is transcriptionally regulated by the constitutive androstane receptor (CAR, NRi13). We have previously demonstrated that the induction of hepatic CYP2B6 by CITCO, a selective human CAR (hCAR) agonist, results in CHOP’s enhanced antineoplastic effects in vitro. Here, we investigate the in vivo potential of CITCO as an adjuvant of CPA-based NHL treatment in a hCAR-transgenic mouse line. Our results demonstrate that the addition of CITCO to the CHOP regimen leads to significant suppression of the growth of EL-4 xenografts in hCAR-transgenic mice accompanied by reduced expression of cyclin-D1, ki67, Pcna, and increased caspase 3 fragmentation in tumor tissues. CITCO robustly induced the expression of cyp2b10 (murine ortholog of CYP2B6) through hCAR activation and increased plasma concentrations of 4-OH-CPA. Comparing to intraperitoneal injection, oral gavage of CITCO results in optimal hepatic cyp2b10 induction. Our in vivo studies have collectively uncovered CITCO as an effective facilitator for CPA-based NHL treatment with a pharmacokinetic profile favoring oral administration, promoting CITCO as a promising adjuvant candidate for CPA-based regimens. Full article

(This article belongs to the Special Issue The Xenobiotic Receptors CAR and PXR in Health and Disease)

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14 pages, 2146 KiB

Open AccessFeature PaperArticle

PXR Functionally Interacts with NF-κB and AP-1 to Downregulate the Inflammation-Induced Expression of Chemokine CXCL2 in Mice

by Maya Okamura, Ryota Shizu, Taiki Abe, Susumu Kodama, Takuomi Hosaka, Takamitsu Sasaki and Kouichi Yoshinari

Cells 2020, 9(10), 2296; https://doi.org/10.3390/cells9102296 - 15 Oct 2020

Cited by 34 | Viewed by 3669

Abstract

Pregnane X receptor (PXR) is a liver-enriched xenobiotic-responsive transcription factor. Although recent studies suggest that PXR shows anti-inflammatory effects by suppressing nuclear factor kappa B (NF-κB), the detailed mechanism remains unclear. In this study, we aimed to elucidate this mechanism. Mice were treated [...] Read more.

Pregnane X receptor (PXR) is a liver-enriched xenobiotic-responsive transcription factor. Although recent studies suggest that PXR shows anti-inflammatory effects by suppressing nuclear factor kappa B (NF-κB), the detailed mechanism remains unclear. In this study, we aimed to elucidate this mechanism. Mice were treated intraperitoneally with the PXR agonist pregnenolone 16α-carbonitrile (PCN) and/or carbon tetrachloride (CCl₄). Liver injury was evaluated, and hepatic mRNA levels were determined via quantitative reverse transcription polymerase chain reaction. Reporter assays with wild-type and mutated mouse Cxcl2 promoter-containing reporter plasmids were conducted in 293T cells. Results showed that the hepatic expression of inflammation-related genes was upregulated in CCl₄-treated mice, and PCN treatment repressed the induced expression of chemokine-encoding Ccl2 and Cxcl2 among the genes investigated. Consistently, PCN treatment suppressed the increased plasma transaminase activity and neutrophil infiltration in the liver. In reporter assays, tumor necrosis factor-α-induced Cxcl2 expression was suppressed by PXR. Although an NF-κB inhibitor or the mutation of an NF-κB-binding motif partly reduced PXR-dependent suppression, the mutation of both NF-κB and activator protein 1 (AP-1) sites abolished it. Consistently, AP-1-dependent gene transcription was suppressed by PXR with a construct containing AP-1 binding motifs. In conclusion, the present results suggest that PXR exerts anti-inflammatory effects by suppressing both NF-κB- and AP-1-dependent chemokine expression in mouse liver. Full article

(This article belongs to the Special Issue The Xenobiotic Receptors CAR and PXR in Health and Disease)

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19 pages, 2019 KiB

Open AccessArticle

Toxicoproteomic Profiling of hPXR Transgenic Mice Treated with Rifampicin and Isoniazid

by Christopher Trent Brewer, Kiran Kodali, Jing Wu, Timothy I. Shaw, Junmin Peng and Taosheng Chen

Cells 2020, 9(7), 1654; https://doi.org/10.3390/cells9071654 - 09 Jul 2020

Cited by 8 | Viewed by 3001

Abstract

Tuberculosis is a global health threat that affects millions of people every year, and treatment-limiting toxicity remains a considerable source of treatment failure. Recent reports have characterized the nature of hPXR-mediated hepatotoxicity and the systemic toxicity of antitubercular drugs. The antitubercular drug [...] Read more.

Tuberculosis is a global health threat that affects millions of people every year, and treatment-limiting toxicity remains a considerable source of treatment failure. Recent reports have characterized the nature of hPXR-mediated hepatotoxicity and the systemic toxicity of antitubercular drugs. The antitubercular drug isoniazid plays a role in such pathologic states as acute intermittent porphyria, anemia, hepatotoxicity, hypercoagulable states (deep vein thrombosis, pulmonary embolism, or ischemic stroke), pellagra (vitamin B₃ deficiency), peripheral neuropathy, and vitamin B₆ deficiency. However, the mechanisms by which isoniazid administration leads to these states are unclear. To elucidate the mechanism of rifampicin- and isoniazid-induced liver and systemic injury, we performed tandem mass tag mass spectrometry-based proteomic screening of mPxr^−/− and hPXR mice treated with combinations of rifampicin and isoniazid. Proteomic profiling analysis suggested that the hPXR liver proteome is affected by antitubercular therapy to disrupt [Fe–S] cluster assembly machinery, [2Fe–2S] cluster-containing proteins, cytochrome P450 enzymes, heme biosynthesis, homocysteine catabolism, oxidative stress responses, vitamin B₃ metabolism, and vitamin B₆ metabolism. These novel findings provide insight into the etiology of some of these processes and potential targets for subsequent investigations. Data are available via ProteomeXchange with identifier PXD019505. Full article

(This article belongs to the Special Issue The Xenobiotic Receptors CAR and PXR in Health and Disease)

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11 pages, 1248 KiB

Open AccessArticle

The Anti-Cancer Drug Dabrafenib Is a Potent Activator of the Human Pregnane X Receptor

by Nicolas Creusot, Matthieu Gassiot, Elina Alaterre, Barbara Chiavarina, Marina Grimaldi, Abdelhay Boulahtouf, Lucia Toporova, Sabine Gerbal-Chaloin, Martine Daujat-Chavanieu, Alice Matheux, Roger Rahmani, Céline Gongora, Alexandre Evrard, Philippe Pourquier and Patrick Balaguer

Cells 2020, 9(7), 1641; https://doi.org/10.3390/cells9071641 - 08 Jul 2020

Cited by 12 | Viewed by 2794

Abstract

The human pregnane X receptor (hPXR) is activated by a large set of endogenous and exogenous compounds and plays a critical role in the control of detoxifying enzymes and transporters regulating liver and gastrointestinal drug metabolism and clearance. hPXR is also involved in [...] Read more.

The human pregnane X receptor (hPXR) is activated by a large set of endogenous and exogenous compounds and plays a critical role in the control of detoxifying enzymes and transporters regulating liver and gastrointestinal drug metabolism and clearance. hPXR is also involved in both the development of multidrug resistance and enhanced cancer cells aggressiveness. Moreover, its unintentional activation by pharmaceutical drugs can mediate drug–drug interactions and cause severe adverse events. In that context, the potential of the anticancer BRAF inhibitor dabrafenib suspected to activate hPXR and the human constitutive androstane receptor (hCAR) has not been thoroughly investigated yet. Using different reporter cellular assays, we demonstrate that dabrafenib can activate hPXR as efficiently as its reference agonist SR12813, whereas it does not activate mouse or zebrafish PXR nor hCAR. We also showed that dabrafenib binds to recombinant hPXR, induces the expression of hPXR responsive genes in colon LS174T-hPXR cancer cells and human hepatocytes and finally increases the proliferation in LS174T-hPXR cells. Our study reveals that by using a panel of different cellular techniques it is possible to improve the assessment of hPXR agonist activity for new developed drugs. Full article

(This article belongs to the Special Issue The Xenobiotic Receptors CAR and PXR in Health and Disease)

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Review

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13 pages, 1089 KiB

Open AccessReview

PXR and 4β-Hydroxycholesterol Axis and the Components of Metabolic Syndrome

by Janne Hukkanen and Jukka Hakkola

Cells 2020, 9(11), 2445; https://doi.org/10.3390/cells9112445 - 09 Nov 2020

Cited by 11 | Viewed by 2905

Abstract

Pregnane X receptor (PXR) activation has been found to regulate glucose and lipid metabolism and affect obesity in response to high-fat diets. PXR also modulates vascular tone. In fact, PXR appears to regulate multiple components of metabolic syndrome. In most cases, the effect [...] Read more.

Pregnane X receptor (PXR) activation has been found to regulate glucose and lipid metabolism and affect obesity in response to high-fat diets. PXR also modulates vascular tone. In fact, PXR appears to regulate multiple components of metabolic syndrome. In most cases, the effect of PXR action is harmful to metabolic health, and PXR can be hypothesized to play an important role in metabolic disruption elicited by exposure to endocrine-disrupting chemicals. The majority of the data on the effects of PXR activation on metabolic health come from animal and cell culture experiments. However, randomized, placebo-controlled, human trials indicate that the treatment with PXR ligands impairs glucose tolerance and increases 24-h blood pressure and heart rate. In addition, plasma 4β-hydroxycholesterol (4βHC), formed under the control of PXR in the liver, is associated with lower blood pressure in healthy volunteers. Furthermore, 4βHC regulates cholesterol transporters in peripheral tissues and may activate the beneficial reverse HDL cholesterol transport. In this review, we discuss the current knowledge on the role of PXR and the PXR–4βHC axis in the regulation of components of metabolic syndrome. Full article

(This article belongs to the Special Issue The Xenobiotic Receptors CAR and PXR in Health and Disease)

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18 pages, 841 KiB

Open AccessReview

Constitutive Androstane Receptor: A Peripheral and a Neurovascular Stress or Environmental Sensor

by Fabiana Oliviero, Céline Lukowicz, Badreddine Boussadia, Isabel Forner-Piquer, Jean-Marc Pascussi, Nicola Marchi and Laila Mselli-Lakhal

Cells 2020, 9(11), 2426; https://doi.org/10.3390/cells9112426 - 06 Nov 2020

Cited by 10 | Viewed by 3682

Abstract

Xenobiotic nuclear receptors (NR) are intracellular players involved in an increasing number of physiological processes. Examined and characterized in peripheral organs where they govern metabolic, transport and detoxification mechanisms, accumulating data suggest a functional expression of specific NR at the neurovascular unit (NVU). [...] Read more.

Xenobiotic nuclear receptors (NR) are intracellular players involved in an increasing number of physiological processes. Examined and characterized in peripheral organs where they govern metabolic, transport and detoxification mechanisms, accumulating data suggest a functional expression of specific NR at the neurovascular unit (NVU). Here, we focus on the Constitutive Androstane Receptor (CAR), expressed in detoxifying organs such as the liver, intestines and kidneys. By direct and indirect activation, CAR is implicated in hepatic detoxification of xenobiotics, environmental contaminants, and endogenous molecules (bilirubin, bile acids). Importantly, CAR participates in physiological stress adaptation responses, hormonal and energy homeostasis due to glucose and lipid sensing. We next analyze the emerging evidence supporting a role of CAR in NVU cells including the blood–brain barrier (BBB), a key vascular interface regulating communications between the brain and the periphery. We address the emerging concept of how CAR may regulate specific P450 cytochromes at the NVU and the associated relevance to brain diseases. A clear understanding of how CAR engages during pathological conditions could enable new mechanistic, and perhaps pharmacological, entry-points within a peripheral–brain axis. Full article

(This article belongs to the Special Issue The Xenobiotic Receptors CAR and PXR in Health and Disease)

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40 pages, 614 KiB

Open AccessReview

Regulation of CAR and PXR Expression in Health and Disease

by Martine Daujat-Chavanieu and Sabine Gerbal-Chaloin

Cells 2020, 9(11), 2395; https://doi.org/10.3390/cells9112395 - 31 Oct 2020

Cited by 48 | Viewed by 5594

Abstract

Pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are members of the nuclear receptor superfamily that mainly act as ligand-activated transcription factors. Their functions have long been associated with the regulation of drug metabolism and disposition, and it is now [...] Read more.

Pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are members of the nuclear receptor superfamily that mainly act as ligand-activated transcription factors. Their functions have long been associated with the regulation of drug metabolism and disposition, and it is now well established that they are implicated in physiological and pathological conditions. Considerable efforts have been made to understand the regulation of their activity by their cognate ligand; however, additional regulatory mechanisms, among which the regulation of their expression, modulate their pleiotropic effects. This review summarizes the current knowledge on CAR and PXR expression during development and adult life; tissue distribution; spatial, temporal, and metabolic regulations; as well as in pathological situations, including chronic diseases and cancers. The expression of CAR and PXR is modulated by complex regulatory mechanisms that involve the interplay of transcription factors and also post-transcriptional and epigenetic modifications. Moreover, many environmental stimuli affect CAR and PXR expression through mechanisms that have not been elucidated. Full article

(This article belongs to the Special Issue The Xenobiotic Receptors CAR and PXR in Health and Disease)

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28 pages, 887 KiB

Open AccessReview

Metabolism-Disrupting Chemicals and the Constitutive Androstane Receptor CAR

by Jenni Küblbeck, Jonna Niskanen and Paavo Honkakoski

Cells 2020, 9(10), 2306; https://doi.org/10.3390/cells9102306 - 15 Oct 2020

Cited by 23 | Viewed by 4925

Abstract

During the last two decades, the constitutive androstane receptor (CAR; NR1I3) has emerged as a master activator of drug- and xenobiotic-metabolizing enzymes and transporters that govern the clearance of both exogenous and endogenous small molecules. Recent studies indicate that CAR participates, together with [...] Read more.

During the last two decades, the constitutive androstane receptor (CAR; NR1I3) has emerged as a master activator of drug- and xenobiotic-metabolizing enzymes and transporters that govern the clearance of both exogenous and endogenous small molecules. Recent studies indicate that CAR participates, together with other nuclear receptors (NRs) and transcription factors, in regulation of hepatic glucose and lipid metabolism, hepatocyte communication, proliferation and toxicity, and liver tumor development in rodents. Endocrine-disrupting chemicals (EDCs) constitute a wide range of persistent organic compounds that have been associated with aberrations of hormone-dependent physiological processes. Their adverse health effects include metabolic alterations such as diabetes, obesity, and fatty liver disease in animal models and humans exposed to EDCs. As numerous xenobiotics can activate CAR, its role in EDC-elicited adverse metabolic effects has gained much interest. Here, we review the key features and mechanisms of CAR as a xenobiotic-sensing receptor, species differences and selectivity of CAR ligands, contribution of CAR to regulation hepatic metabolism, and evidence for CAR-dependent EDC action therein. Full article

(This article belongs to the Special Issue The Xenobiotic Receptors CAR and PXR in Health and Disease)

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12 pages, 699 KiB

Open AccessReview

Associations between Pregnane X Receptor and Breast Cancer Growth and Progression

by Bradley A. Creamer, Shelly N. B. Sloan, Jennifer F. Dennis, Robert Rogers, Sidney Spencer, Andrew McCuen, Purnadeo Persaud and Jeff L. Staudinger

Cells 2020, 9(10), 2295; https://doi.org/10.3390/cells9102295 - 15 Oct 2020

Cited by 8 | Viewed by 3498

Abstract

Pregnane X receptor (PXR, NR1I2) is a member of the ligand-activated nuclear receptor superfamily. This receptor is promiscuous in its activation profile and is responsive to a broad array of both endobiotic and xenobiotic ligands. PXR is involved in pivotal cellular detoxification processes [...] Read more.

Pregnane X receptor (PXR, NR1I2) is a member of the ligand-activated nuclear receptor superfamily. This receptor is promiscuous in its activation profile and is responsive to a broad array of both endobiotic and xenobiotic ligands. PXR is involved in pivotal cellular detoxification processes to include the regulation of genes that encode key drug-metabolizing cytochrome-P450 enzymes, oxidative stress response, as well as enzymes that drive steroid and bile acid metabolism. While PXR clearly has important regulatory roles in the liver and gastrointestinal tract, this nuclear receptor also has biological functions in breast tissue. In this review, we highlight current knowledge of PXR’s role in mammary tumor carcinogenesis. The elevated level of PXR expression in cancerous breast tissue suggests a likely interface between aberrant cell division and xeno-protection in cancer cells. Moreover, PXR itself exerts positive effect on the cell cycle, thereby predisposing tumor cells to unchecked proliferation. Activation of PXR also plays a key role in regulating apoptosis, as well as in acquired resistance to chemotherapeutic agents. The repressive role of PXR in regulating inflammatory mediators along with the existence of genetic polymorphisms within the sequence of the PXR gene may predispose individuals to developing breast cancer. Further investigations into the role that PXR plays in driving tumorigenesis are needed. Full article

(This article belongs to the Special Issue The Xenobiotic Receptors CAR and PXR in Health and Disease)

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25 pages, 957 KiB

Open AccessReview

The Connection of Azole Fungicides with Xeno-Sensing Nuclear Receptors, Drug Metabolism and Hepatotoxicity

by Philip Marx-Stoelting, Constanze Knebel and Albert Braeuning

Cells 2020, 9(5), 1192; https://doi.org/10.3390/cells9051192 - 11 May 2020

Cited by 33 | Viewed by 3811

Abstract

Azole fungicides, especially triazole compounds, are widely used in agriculture and as pharmaceuticals. For a considerable number of agricultural azole fungicides, the liver has been identified as the main target organ of toxicity. A number of previous studies points towards an important role [...] Read more.

Azole fungicides, especially triazole compounds, are widely used in agriculture and as pharmaceuticals. For a considerable number of agricultural azole fungicides, the liver has been identified as the main target organ of toxicity. A number of previous studies points towards an important role of nuclear receptors such as the constitutive androstane receptor (CAR), the pregnane-X-receptor (PXR), or the aryl hydrocarbon receptor (AHR), within the molecular pathways leading to hepatotoxicity of these compounds. Nuclear receptor-mediated hepatic effects may comprise rather adaptive changes such as the induction of drug-metabolizing enzymes, to hepatocellular hypertrophy, histopathologically detectable fatty acid changes, proliferation of hepatocytes, and the promotion of liver tumors. Here, we present a comprehensive review of the current knowledge of the interaction of major agricultural azole-class fungicides with the three nuclear receptors CAR, PXR, and AHR in vivo and in vitro. Nuclear receptor activation profiles of the azoles are presented and related to histopathological findings from classic toxicity studies. Important issues such as species differences and multi-receptor agonism and the consequences for data interpretation and risk assessment are discussed. Full article

(This article belongs to the Special Issue The Xenobiotic Receptors CAR and PXR in Health and Disease)

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