Ph.D., 2019 Education University of Maryland, Baltimore

Metabolism-based Alterations of Constitutive Androstane Receptor (CAR) Activity and Downstream Effects

Item Type dissertation

Authors Mackowiak, Bryan

Publication Date 2019

Abstract The xenobiotic defense network in the liver has evolved so that many foreign compounds can activate xenobiotic receptors like the constitutive androstane receptor (CAR) and the pregnane x receptor (PXR), induce the expression of drug metabolizing enz...

Keywords pharmaceutical sciences; activator; CAR; constitutive androstane receptor; metabolism; PK11195; screening

Download date 06/10/2021 08:55:19

Link to Item http://hdl.handle.net/10713/12675 CURRICULUM VITAE

Bryan Robert Mackowiak

Degree and Date to be Conferred: Ph.D., 2019

Education

University of Maryland, Baltimore, 2019 Ph.D., Pharmaceutical Sciences

University of North Carolina, Chapel Hill, 2014 B.S., Biology Minors: Chemistry, History

Professional Experience

Graduate Research Assistant, School of Pharmacy, University of Maryland, Baltimore, Baltimore, MD

Principal Investigator: Dr. Hongbing Wang Thesis project studies the effect of metabolism on CAR activation and deactivation by FDA-approved drugs and how that contributes to toxicity or enhance the effects of these drugs

Summer Intern in DMPK, Biogen Idec, Cambridge, MA, May 2013 – August 2013

Evaluated Caco-2 cell monolayers as an in vitro model of the blood-brain barrier permeability of amyloid-β peptides

Undergraduate Researcher, Eshelman School of Pharmacy, University of North Carolina – Chapel Hill, Chapel Hill, NC, 2012 – 2014

Principal Investigator: Dr. Dhiren Thakker Elucidated the cation-selective transporters involved in the apical efflux of metformin in the small intestine which leads to metformin’s continued cycling and improved absorption.

Publications

Lynch, C.; Mackowiak, B.; Huang, R.; Li, L.; Heyward, S.; Sakamuru, S.; Wang, H.; Xia, M. Identification of Modulators that Activate the Constitutive Androstane Receptor from the Tox21 10K Compound Library. Toxicological Sciences. 2018. Epub ahead of print. PMID: 30247703

Mackowiak, B.; Hodge, J; Stern, S; Wang, H. The Roles of Xenobiotic Receptors: Beyond Chemical Disposition. Drug Metab Dispos. 2018. 46 (9) 1361-1371. PMID: 29759961 Mackowiak, B.; Li, L.; Welch, M.A.: Li, D.; Jones, J.; Heyward, S.; Kane, M.; Swaan, P.; Wang, H. Molecular basis of metabolism-mediated conversion of PK11195 from an antagonist to an agonist of the constitutive androstane receptor. Mol Pharm. 2017. 92 (1): 75-87. PMID: 28442602 Mackowiak, B. & Wang, H. Mechanisms of Xenobiotic Receptor Activation: Direct vs. Indirect. BBA Gene Regulatory Mechanisms. 2016. 1859 (9): 1130-1140. PMID: 26877237 Li, D.; Mackowiak, B.; Brayman, T.; Mitchell, M.; Zhang, L.; Huang, S.M.; Wang, H. Genome-w ide analysis of human constitutive androstane receptor (CAR) transcriptome in wild-type and CAR-knockout HepaRG cells. Biochemical Pharmacology, 2015. 98 (1): 190-202. PMID: 26275810

Selected Posters and Presentations

B Mackowiak, H Wang. High-content Analysis of CAR Nuclear Translocation Accurately Identifies Physiologically-Relevant. (Short-Course Presentation) Globalization of Pharmaceutics Education Network 2018. Singapore.

B Mackowiak, W Huang, Z Li, MA Kane, H Wang. Comparative Pharmacoproteomics of Phenobarbital and CITCO in Human Primary Hepatocytes. ISSX Annual Meeting 2017. Providence, RI.

B Mackowiak, D Li, M Welch, L Li, M Mitchell, S Heyward, P Swaan, H Wang. Molecular basis of metabolism-mediated conversion of PK11195 from an antagonist to an agonist of the constitutive androstane receptor. Society of Toxicology Annual Meeting 2017. Baltimore, MD.

B Mackowiak, D Li, M Welch, L Li, M Mitchell, S Heyward, P Swaan, H Wang. Prototypical CAR antagonist PK11195 is metabolized to a CAR activator in physiologically-relevant cellular systems. (Podium Presentation) Drug Metabolism Gordon Research Conference 2016. Holderness, NH.

B Mackowiak, D Li, L Li, M Mitchell, S Heyward, H Wang. Prototypical Inverse Agonists of CAR Induce CYP2B6 Expression Independent of PXR. ISSX Annual Meeting 2015. Orlando, FL.

B Mackowiak, C Costales, R Alluri, T Han, D Thakker. Evidence for the Role of an Unknown Cation Transporter in the Apical Efflux of Metforminin Caco-2 Cell Monolayers. AAPS Annual Meeting 2013. San Antonio, TX.

B Mackowiak, C Costales, R Alluri, T Han, R Everett, D Thakker. Apical Membrane Transporters in Human and Mouse Intestinal Epithelia Mediate Metformin Efflux. AAPS Workshop on Drug Transporters in ADME: From the Bench to the Bedside. Bethesda, MD, March 2014.

Fellowships and Awards

Department of Pharmaceutical Sciences Fellowship Award. University of Maryland School of Pharmacy, 2017-2018.

Travel Grant Award. ISSX Annual Meeting 2017 (Providence, RI).

Eagle Scout Award. 2007.

Memberships and Service

Pharmacy Student Graduate Association. First Year Representative (2014-2015), Vice- President (2015-2016), President (2016-2017)

Rho Chi Honor Society. Vice-President, Graduate Affairs (2016-2017)

Patents

(Pending, US Application # 16/315,753, January 7th, 2019) Lead Inventor: Bryan Mackowiak Co-inventors: Hongbing Wang, Linhao Li

ABSTRACT

Title of the Dissertation: Metabolism-based Alterations of Constitutive Androstane

Receptor (CAR) Activity and Downstream Effects

Bryan Mackowiak, Doctor of Philosophy, 2019

Dissertation Directed by: Dr. Hongbing Wang, Professor and Program Chair

(Experimental & Translational Therapeutics), Department of Pharmaceutical Sciences

The xenobiotic defense network in the liver has evolved so that many foreign compounds can activate xenobiotic receptors like the constitutive androstane receptor (CAR) and the pregnane x receptor (PXR), induce the expression of drug metabolizing enzymes, and enhance the clearance of drugs. Typically, autoinduction of a compound’s metabolism leads to its breakdown, disrupting the detoxification feedback loop. However, multiple lines of evidence suggest that metabolites of autoinducers can have diverse effects on xenobiotic receptors, including agonism and antagonism conversion, and cause unexpected consequences, including drug-drug interactions (DDIs) that can lead to liver toxicity. While the effect of xenobiotic receptor-mediated CYP induction on drug metabolism has been well-characterized, the effect of metabolism on the activity of xenobiotic receptors has received little attention.

Although the “traditional” role of CAR revolves around inducing xenobiotic metabolism and detoxification, evidence has accumulated that CAR also plays important roles in energy metabolism, cellular proliferation, and liver homeostasis, making it a potential drug target for various liver disorders. In addition, the effects of CAR activation in human primary hepatocytes (HPH) are not well understood and need further study to determine whether or not CAR is a potential drug target for different types of liver dysfunction, including cancer. The overall objectives of this proposal are to investigate the effect of drug metabolism on CAR activity, identify FDA-approved drugs and metabolites that alter CAR activity, and determine whether CAR activation is beneficial for liver disorders such as cancer. Using CAR as a model xenobiotic receptor, my studies have shown that potent CAR antagonist PK11195 is metabolized to a CAR agonist in metabolically-competent systems. Therefore, I hypothesize that hepatic metabolism capacity and CAR activity can form a regulatory feedback loop, altering the PK/PD profiles of drug substrates.

Successful completion of these studies has provided a model for metabolism-based changes in xenobiotic receptor activity, identified FDA-approved drugs that modulate

CAR activity, and determined the clinically-relevant downstream effects of CAR activation.

Metabolism-based Alterations of Constitutive Androstane Receptor (CAR) Activity and Downstream Effects

by Bryan Mackowiak

Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2019

DEDICATION

This dissertation is dedicated to my wonderful fiancé Kelsey, our dog Camden, my family and friends, and everyone who made me into the person I am today.

iii

TABLE OF CONTENTS

DEDICATION ...... iii LIST OF TABLES ...... vii LIST OF FIGURES ...... viii LIST OF ABBREVIATIONS ...... x

Chapter 1: Overview and objectives ...... 1 1.1 Xenobiotic Receptors ...... 1 1.2 Constitutive androstane receptor ...... 4 1.2.1 CAR Activation ...... 5 1.2.2 Direct CAR Activation ...... 8 1.2.3 Indirect activation of CAR ...... 13 1.2.4 CAR and Energy Homeostasis...... 17 1.2.5 CAR on Cell Proliferation and Cancer ...... 22 1.2.6 Additional Endobiotic Functions of CAR...... 26 1.3 Pregnane X Receptor ...... 28 1.3.1 Direct activation of PXR ...... 31 1.3.2 Indirect activation of PXR ...... 33 1.3.3 PXR and Energy Homeostasis ...... 36 1.3.4 PXR in cancer and cell proliferation ...... 41 1.3.5 Additional non-traditional functions of PXR ...... 43

Chapter 2: Metabolism-mediated alterations of constitutive androstane receptor activity46 2.1 Introduction ...... 46 2.2 Materials and Methods ...... 48 2.2.1 Chemicals and Biological Reagents ...... 48 2.2.2 Culture and treatment of HPH and HepaRG cells ...... 49 2.2.3 Real-time RT-PCR ...... 50 2.2.4 Western Blot Analysis ...... 51 2.2.5 HepG2 cell Transfection and HepG2/HPH co-culture ...... 51 2.2.6 LC-MS Measurement of PK11195 Metabolism ...... 52 2.2.7 Mammalian two-hybrid assay ...... 53 2.2.8 Nuclear Translocation of CAR in HPH ...... 53

2.2.9 Molecular Modeling...... 54 2.2.10 Statistical Analysis ...... 55 2.3 Results ...... 55 2.3.1 PK11195 induces the expression of CYP2B6 and CYP3A4 in HPH ...... 55 2.3.2 Induction of CYP2B6 and CYP3A4 by PK11195 in PXR-KO HepaRG cells 56 2.3.3 PK11195 is metabolized to a hCAR activator in HPH ...... 57 2.3.4 PK11195 is metabolized to ND-PK and COOH-PK in HPH ...... 59 2.3.5 The (R)-N-desmethyl metabolite of PK11195 mediates hCAR activation 61 2.3.6 ND-PK induces CYP2B6, CYP3A4 expression and nuclear translocation of hCAR in HPH ...... 64 2.3.7 Computational modeling of PK11195 and ND-PK ...... 66 2.4 Conclusion and Discussion ...... 69

Chapter 3: Identification of constitutive androstane receptor modulators with a high- throughput translocation assay ...... 75 3.1 Introduction ...... 75 3.2 Materials and Methods ...... 77 3.2.1 Chemicals and Biological Reagents ...... 77 3.2.2 Culture of HPH ...... 78 3.2.3 Ad/EYFP-hCAR Translocation Assay ...... 78 3.2.4 High-Content Imaging and Analysis ...... 79 3.2.5 Real-time RT-PCR ...... 81 3.2.6 Western Blot Analysis ...... 81 3.2.7 Glucose Production Assay ...... 82 3.2.8 Statistical Analysis ...... 82 3.3 Results ...... 83 3.3.1 High-Throughput EYFP-hCAR Translocation Assay Development and Validation ...... 83 3.3.2 Screening FDA-approved Drugs for CAR Translocation ...... 86 3.3.3 Mosapride Citrate potently induces CYP2B6 and CYP3A4 mRNA and protein in HPH ...... 94 3.3.4 Mosapride Citrate inhibits the expression of gluconeogenic genes and reduces glucose output from HPH ...... 95 3.4 Conclusion and Discussion ...... 97 v

Chapter 4: Examination of downstream effects of constitutive androstane receptor activation ...... 105 4.1 Introduction ...... 105 4.2 Materials and Methods ...... 107 4.2.1 Chemicals and Biological Reagents ...... 107 4.2.2 HPH Culture...... 108 4.2.3 Proteomics...... 108 4.2.4 Real-time RT-PCR ...... 109 4.2.5 Western Blot Analysis ...... 109 4.2.6 Statistical Analysis ...... 110 4.3 Results ...... 110 4.3.1 Differential protein expression in HPH after PB and CITCO treatment .. 110 4.3.2 Upstream regulators and pathways perturbed by PB and CITCO treatment 112 4.3.3 Functional consequences of PB and CITCO treatment ...... 114 4.4 Conclusion and Discussion ...... 115

Chapter 5: Conclusion and future directions ...... 121

REFERENCES ...... 124

LIST OF TABLES

Table 2.1 - Age and sex of liver donors...... 50

Table 3.1. CYP2B6 induction by selected hits in the luciferase and translocation assays...... 89

Table 3.2. Summary of CAR Luciferase Assay Agonist Data...... 91

Table 3.3. Summary of CAR Luciferase Assay Antagonist Data...... 91

Table 3.4. Summary of CAR Translocator Data...... 93

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LIST OF FIGURES

Figure 1.1 - Schematic illustration of mechanisms of CAR activation ...... 8

Figure 1.2 - CAR and Energy Homeostasis ...... 22

Figure 1.3 - Schematic illustration of Mechanisms of PXR Activation...... 31

Figure 1.4 - PXR and Energy Homeostasis ...... 41

Figure 2.1 - PK11195 induces CYP2B6 and CYP3A4 expression in HPH...... 56

Figure 2.2 - PK11195 induces CYP2B6 and CYP3A4 expression in HepaRG cells independent of

PXR ...... 57

Figure 2.3 - PK11195 is metabolized to a CAR activator in HepG2-HPH co-culture ...... 59

Figure 2.4 - PK11195 is metabolized to COOH-PK and ND-PK in HPH ...... 61

Figure 2.5 - The (R)-N-desmethyl (ND) metabolite of PK11195 mediates CAR activation ...... 63

Figure 2.6 - ND-PK induces CAR target genes in HPH ...... 65

Figure 2.7 - ND-PK induces CAR nuclear translocation in HPH ...... 66

Figure 2.8 - N-demethylation of PK11195 leads to reduced interaction with residues important to

CAR activity ...... 68

Figure 3.1 - Adapting the Ad/EYFP-hCAR nuclear translocation assay for high-throughput screening ...... 86

Figure 3.2 - Screening of FDA-approved drugs with the Ad/EYFP-hCAR translocation assay compared with the HepG2-hCAR-CYP2B6 luciferase assay ...... 88

Figure 3.3 - CAR antagonists can mediate CAR nuclear translocation ...... 94

Figure 3.4 - Mosapride citrate potently induces CYP2B6 expression over a range of concentrations in HPH ...... 95

Figure 3.5 - Mosapride citrate represses gluconeogenic gene expression in HPH ...... 96

Figure 3.6 - Mosapride citrate decreases glucose output from HPH ...... 97

Figure 3.7 - Proposed mechanisms for mosapride-dependent improvement in glycemic control103

viii

Figure 4.1 - Differential protein expression in HPH after PB and CITCO treatment ...... 112

Figure 4.2 - Upstream regulators and pathways perturbed by PB and CITCO treatment ...... 113

Figure 4.3 - PB and CITCO alter the EIF2 pathway via EIF5 upregulation ...... 114

Figure 4.4 - Functional consequences of PB and CITCO treatment ...... 115

LIST OF ABBREVIATIONS

5-HT4 5-Hydroxytryptamine receptor 4

Ad/EYFP-hCAR adenovirus-expressing enhanced yellow fluorescent protein-

tagged human CAR

AF2 activation function 2

AhR aryl hydrocarbon receptor

ARNT aryl hydrocarbon nuclear translocator

CAR constitutive androstane receptor

CCRP cytoplasmic retention protein

CINPA1 CAR inhibitor not PXR activator 1 cAMP cyclic adenosine monophosphate

CITCO 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-

O-(3,4-dichlorobenzyl)oxime

COOH-PK 1-(2-Chlorophenyl)isoquinoline-3-carboxylic acid

DAPI 4',6-diamidino-2-phenylindole

DBD DNA-binding domain

DDIs drug-drug interactions

DMEs drug-metabolizing enzymes

EGFR epidermal growth factor receptor

ERK extracellular signal-regulated kinase

FGF fibroblast growth factor

FOXO1 forkhead box protein O1

FRET fluorescence resonance energy transfer

G6Pase glucose-6-phosphatase

GRIP-1 glucocorticoid receptor interacting protein 1

HCC hepatocellular carcinoma

HNF-4α hepatic nuclear factor 4 alpha

HPH human primary hepatocytes

HSP heat-shock protein

KET ketoconazole

LBD ligand-binding domain

MEK mitogen-activated protein kinase kinase

NcoR1 nuclear receptor co-repressor 1

ND-PK (R)-N-Desmethyl PK11195

NLS nuclear localization signal

NR nuclear receptor

OA okadaic acid

PB phenobarbital

PEPCK phosphoenolpyruvate carboxykinase

PCN pregnenolone 16 α-carbonitrile

PGC1α peroxisome proliferator-activated receptor gamma coactivator-1α

PK11195 1-(2-Chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-

isoquinolinecarboxamide

PKA protein kinase A

PKC protein kinase C

PP protein phosphatase

PPAR peroxisome proliferator activated receptor

PXR pregnane X receptor

PXR-KO PXR-knockout

RACK1 receptor for activated C kinase 1

RIF rifampicin

RXR retinoid x receptor

SMRT silencing mediator of retinoid and thyroid receptors

SRC-1 steroid receptor coactivator 1

TCPOBOP 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene

TKI tyrosine-kinase inhibitor

XR xenobiotic receptors

xii

Chapter 1: Overview and objectives

1.1 Xenobiotic Receptors

Nuclear receptors (NRs) are important cellular proteins that govern the expression of numerous genes involved in a wide range of cellular processes including cell growth, differentiation, metabolism, and stress responses. Prototypical NRs exert their effects by acting as transcription factors, which sense both intracellular and extracellular signals and respond by inducing the transcription of their target genes (Forman and Evans, 1995).

Although the cellular impact of each NR is different, all NRs share a number of characteristic structures, functional domains, and sequence similarities. In general, NRs feature a variable N-terminal region with an activation function 1 (AF-1) domain, a highly conserved DNA-binding domain (DBD), a hinge region, and a ligand-binding domain (LBD) that contains the activation function 2 (AF-2) domain. The AF-1 domain mediates ligand-independent activation of the NR while AF-2 activation is ligand- dependent (Nagy and Schwabe, 2004). The hinge region connects the DBD to the LBD and upon ligand binding, helices in this region undergo a conformational change allowing coactivators to bind, ultimately leading to nuclear localization and activation (McKenna et al., 1999; Nagy and Schwabe, 2004). These features differentiate NRs from many other regulatory proteins and allow them to be dynamic sensors that respond to various stimuli and regulate cellular responses.

NRs can be roughly divided into two main classes based on their ligand specificity – endocrine NRs and orphan NRs (Sonoda et al., 2008). Endocrine NRs bind with nanomolar-affinity to specific endogenous ligands such as hormones and steroids that are present at low concentrations physiologically. Examples of endocrine NRs

1 include, but are not limited to, the thyroid hormone receptor, the retinoic acid receptor, the androgen receptor, and the estrogen receptor (Nagy and Schwabe, 2004; Sonoda et al., 2008). On the other hand, orphan NRs usually have no identified high-affinity endogenous ligands and are instead activated by abundant and low-affinity endogenous metabolites or xenobiotics. However, it is important to note that some NRs previously designated as orphan NRs have been “adopted” after discovering an endogenous ligand, such as the farnesoid x receptor, which was “adopted” after bile acids were identified as high-affinity ligands (Parks et al., 1999). A number of orphan NRs promiscuously bind to a wide range of both endogenous compounds and xenobiotics, often at micromolar concentrations, and are instrumental in mounting cellular responses to toxic compounds and their metabolites. This subset of orphan NRs, termed xenobiotic receptors (XRs), have become increasingly important in coordinating toxic or protective responses when humans are exposed to accumulated endotoxins or high concentrations of environmental chemicals (Wang and LeCluyse, 2003). XRs coordinate the gene transcription of numerous phase I and II drug-metabolizing enzymes and transporters in the liver and intestine, where XRs are also enriched (Tolson and Wang, 2010). To date, extensive investigations have been centered on three XRs; namely, the constitutive androstane receptor (CAR, NR1I3), the pregnane x receptor (PXR, NR1I2), and the aryl hydrocarbon receptor (AhR), due majorly to their predominance in regulating hepatic responses to drugs and environmental chemicals. Although AhR, a member of the Per-ARNT-Sim

(PAS) family of proteins, is not typically classified as a nuclear receptor, it has similar functionality and follows the same overall paradigm as other XRs in a pharmacological or toxicological perspective (Burbach et al., 1992). While all of these XRs are capable of

2 altering cellular metabolism and homeostasis individually, significant cross-talk exists between these receptors leading to multifaceted regulation of xenobiotic detoxification.

For example, previous studies have shown that PXR activation in human primary hepatocytes increases the conversion of AhR antagonist omeprazole-sulfide to a prototypical AhR activator, omeprazole. In addition, all three XRs are known to regulate the important drug-metabolizing enzyme UGT1A1 and the transporter BCRP at the transcriptional level (Tolson and Wang, 2010). Also, shared ligands such as non-coplanar polychlorinated biphenyls and flavonoids derived from Ginkgo biloba extract activate

CAR, PXR, and AhR, which can lead to complicated effects in the liver. As these XRs exhibit complex interplay that markedly alters cellular metabolism and homeostasis, understanding the types of compounds that activate these XRs and the underlying mechanical bases of XR activation are essential.

Traditionally, ligand-binding has been thought of as an essential component of XR activation and has been studied with methods such as mammalian two-hybrid assays, luciferase reporter assays, and more recently, fluorescence resonance energy transfer

(FRET) assays (Raucy and Lasker, 2013). However, recent evidence has shown that many compounds activate XRs in lieu of direct ligand binding; rather, they activate XRs via ligand-independent (indirect) mechanisms that have yet to be fully elucidated. This paradigm of XR activation mediated through both direct and indirect mechanisms raises new challenges in our understanding of how XR signaling is controlled when exposed to various cellular stresses and has profound implications on how XR modulators will be identified in the future.

1.2 Constitutive androstane receptor

Screening of a human liver library with a degenerate oligonucleotide based on the sequence of the conserved DNA binding domain of NRs led to the cloning of CAR, originally named MB67, in 1994 (Baes et al., 1994). CAR heterodimerizes with the retinoid x receptor (RXR) and transactivates genes that contain the retinoic acid response element “constitutively” in the absence of a ligand, leading to its early name as the constitutive activated receptor (Choi et al., 1997). The mouse and rat orthologs of CAR were cloned soon thereafter, exhibiting the same heterodimerization and constitutive activation features as human CAR (hCAR) (Choi et al., 1997; Yoshinari et al., 2001).

Under normal physiological conditions, CAR is sequestered in the cytoplasm in a complex with heat-shock protein (HSP) 90 and CAR cytoplasmic retention protein

(CCRP); and HSP70 has recently been shown to stabilize this complex in the inactive state (Kobayashi et al., 2003; Timsit and Negishi, 2014; Yoshinari et al., 2003).

Interestingly, although CAR is retained in the cytoplasm in the inactive state and translocates to the nucleus upon activation in physiologically-relevant primary hepatocytes, CAR is localized to the nucleus and constitutively active in immortalized cell lines (Kanno et al., 2005; Kawamoto et al., 1999b).

As a xenobiotic receptor, CAR governs the inductive expression of many phase I and phase II drug-metabolizing enzymes and transporter proteins, coordinating a defensive network against xenobiotic challenges in the liver (Qatanani and Moore, 2005; Stanley et al., 2006). Although the well-established xeno-sensing role of CAR continues to be important in predicting potential drug-drug interactions (DDIs) and pharmacokinetic profiles of drugs, emerging evidence reveals that CAR also affects physiological and

4 pathophysiological conditions including obesity, diabetes, and tumor development by modulating hepatic energy homeostasis, insulin signaling, and cell proliferation (Dong et al., 2009; Gao et al., 2009; Huang et al., 2005; Masuyama and Hiramatsu, 2012). For instance, in contrast to the up-regulation of genes encoding drug-metabolizing enzymes and transporters, activation of CAR by the selective mouse CAR (mCAR) activator 1,4- bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) significantly represses a cluster of genes associated with gluconeogenesis and lipogenesis, and attenuates high-fat diet

(HFD)-induced obesity and diabetes in wild-type but not CAR-knockout mice (Dong et al., 2009; Gao et al., 2009; Masuyama and Hiramatsu, 2012). Assuming such beneficial effects also occur in humans, CAR may function as a novel therapeutic target for metabolic disorders in addition to its known role as a xenobiotic sensor. Therefore, understanding of the mechanistic basis of CAR activation is pivotal, and these recent developments have stimulated much interest in identifying selective and potent activators of human CAR.

1.2.1 CAR Activation

The pharmacological importance of CAR was first appreciated when CAR activation was linked to the induction of CYP2b10 expression by phenobarbital (PB) in mouse liver

(Honkakoski et al., 1998). Subsequent studies revealed that numerous structurally unrelated PB-like compounds induce CYP2B genes in different species through the activation of CAR (Kawamoto et al., 1999b; Moore et al., 2000; Qatanani and Moore,

2005). The initial step of CAR activation involves cellular translocation of the receptor from the cytoplasm to the nucleus, where it interacts with its heterodimer partner RXR and other transcriptional proteins to stimulate the expression of target genes (Kawamoto

5 et al., 1999b; Li et al., 2009). Further analysis of the 5’ upstream regions of CAR target genes revealed key promoter elements often containing direct repeats of the hexamer

AGGTCA separated by 3-5 nucleotides which directly interact with the CAR/RXR heterodimer (Mäkinen et al., 2002). These findings led to the establishment of specific cell-based luciferase reporter assays, by which pharmacological modulation of CAR activity could be monitored efficiently. Initial ligands identified for CAR include two endogenous testosterone metabolites, 5α-androst-16-en-3α-ol (androstenol) and 5α- androstan-3α-ol (androstanol) (Forman et al., 1998). Both compounds repressed the constitutive activity of mCAR in vitro and disrupted its interactions with coregulatory proteins such as the nuclear receptor coactivator 1 (SRC-1) (Forman et al., 1998).

Notably, although this finding leads to the current name of CAR as the constitutive androstane receptor, these steroid metabolites are not likely to be “real” endogenous ligands of CAR in vivo because the concentrations needed to antagonize CAR in vitro are several magnitudes higher than their physiological levels. Subsequently, TCPOBOP was identified as a potent and selective agonist of mCAR with the ability to reverse the antagonism conferred by androstanes while promoting CAR interaction with coactivator

SRC-1 (Tzameli et al., 2000). Interestingly, although CAR demonstrates promiscuity in ligand binding, it also exhibits divergent activation profiles across species. For instance,

TCPOBOP activates mouse but not human CAR while androstanol represses mouse but not human CAR (Moore et al., 2000; Tzameli et al., 2000). The later discovery of a potent and selective hCAR agonist, 6-(4-chlorophenyl)imidazo[2,1-beta][1,3]-thiazole-5- carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO), has been instrumental in studying

6 the effects of CAR in human systems and conferring physiological relevance to many studies carried out in mice only (Maglich et al., 2003).

CAR displays unique activation mechanisms compared with typical nuclear receptors, requiring both nuclear translocation and nuclear activation. In HepG2 cells, transfected

CAR spontaneously accumulates in the nucleus in the absence of an inducer and exhibits constitutive activation of its target genes (Choi et al., 1997; Kawamoto et al., 1999b).

This would suggest that nuclear translocation alone is sufficient to confer CAR activation. However, several lines of evidence support nuclear activation as a distinct step in CAR-mediated gene regulation in primary hepatocytes and intact liver in vivo.

For example, pretreatment of primary hepatocytes with the protein phosphatase inhibitor okadaic acid (OA) inhibits PB-induced CAR nuclear translocation but does not repress

CAR-mediated activation of reporter genes in HepG2 cells since CAR is constitutively localized in the nuclei of HepG2 cells (Kawamoto et al., 1999b; Swales et al., 2005).

Additionally, pretreatment of primary mouse hepatocytes with KN-62, a calcium/calmodulin-dependent kinase inhibitor, repressed PB- and TCPOBOP-associated

CYP2b10 induction and reporter gene activation without affecting CAR nuclear accumulation (Yamamoto et al., 2003). Nevertheless, although these results indicate nuclear translocation is only one of the two required steps for target gene activation, nuclear localized CAR prefers interaction with coactivators instead of corepressors even in the absence of agonist binding in contrast to other nuclear receptors (Dussault et al.,

2002; Suino et al., 2004). Many compounds, like PB, have no CAR binding activity and indirectly activate CAR, most likely by facilitating nuclear translocation of the receptor instead (Fig. 1.1).

Figure 1.1 - Schematic illustration of mechanisms of CAR activation. Direct Activation – CAR is sequestered in a cytoplasmic complex containing HSP90 and CCRP. Upon ligand binding, these chaperones dissociate and CAR translocates to the nucleus where it heterodimerizes with RXR, recruits coactivators GRIP1 and SRC1, and binds to its response element to initiate gene transcription. Indirect Activation – When sequestered in the cytoplasm, the Thr38 residue of CAR is phosphorylated by PKC, and dephosphorylation of this residue for CAR activation can be inhibited by ERK1/2 upon activation of the EGFR signaling pathway. When a PB-like compound binds to the EGFR receptor and inhibits EGF-mediated signaling, RACK1 is dephosphorylated at Tyr52 and recruits PP2A to the CAR protein complex, where it dephosphorylates the Thr38 residue and induces CAR nuclear translocation and activation.

1.2.2 Direct CAR Activation

Although the basic structure of CAR is similar to other NRs, the structure-activity relationship of CAR is quite distinct and has been widely studied to understand the factors behind its constitutive activity. In general, the ligand-binding pocket of a NR is enclosed by 12 α-helices, where helix 12 (H12) containing the AF-2 domain is the master regulator of activation. Early mutagenesis studies showed that the AF-2 motif of CAR mediates ligand-independent coactivator binding and confers constitutive activity

(Andersin et al., 2003). Compared to other NRs, CAR has a shortened loop between helices 11 and 12 in addition to a shortened H12 helix which allows the negatively

8 charged carboxy-terminus to interact with the positive K195 (K205 for mCAR) residue, favoring the active conformation of H12 (Dussault et al., 2002; Suino et al., 2004). The crystal structures of TCPOBOP- and CITCO-bound mCAR:RXR and hCAR:RXR heterodimers have given much insight into how direct activators bind to CAR and impart activity (Suino et al., 2004; Xu et al., 2004). The direct activator TCPOBOP is often referred to as a “superagonist” of mCAR, as it drastically increases mCAR transcriptional activity above constitutive levels, unlike CITCO for hCAR (Xu et al., 2004). Most likely, this is due to the interactions of TCPOBOP with L353 on the AF-2 domain and L346 and

T350 on the linker helix of mCAR, directly stabilizing H12 in the active conformation

(Suino et al., 2004). In contrast, the structure of hCAR bound to CITCO is remarkably similar to the unbound constitutively active form, suggesting that the mechanism of hCAR activation by CITCO may result majorly in a conformational change that promotes nuclear localization (Xu et al., 2004). This is consistent with observations that CITCO cannot induce hCAR activity over its basal level in immortalized cell lines, but strongly induces translocation-dependent hCAR activation in human primary hepatocytes (Li et al., 2008).

Acquiring crystal structures of the CAR LBD bound to activators has facilitated computational screening for possible CAR activators based on both structural and pharmacophore models. This method is normally used to narrow down a compound library to likely activators, which can then be further screened in a less high-throughput manner (Lynch et al., 2013). Many different binding assays have been designed to find direct ligands of CAR over the years, and most of them feature the CAR LBD and its ability to interact with coactivators or heterodimerize with RXR. For instance, two-hybrid

9 mammalian cell assays have been extensively used to characterize CAR binding to coactivators in the presence of different compounds to determine possible ligands

(MÄKINEN et al., 2003). FRET assays are also frequently employed in this capacity, representing another method to assess CAR binding and activation (Carazo and Pavek,

2015). Once a potential activator is found to bind to the CAR LBD and induce association with coactivators, it is usually subjected to testing in luciferase reporter assays, normally in HepG2, Huh7, COS-1, or CV-1 cells (Raucy and Lasker, 2013). This type of assay determines whether a compound can induce CAR transcriptional activity, which is the hallmark of CAR activation. However, the basal constitutive activity and nuclear localization of CAR in immortalized cell lines provide challenges to this approach. As CAR is constitutively active, even the most potent hCAR inducers can only increase CAR transcriptional activity moderately over the high basal level in cellular systems. In addition, the constitutive nuclear localization of CAR in immortalized cells prevents the use of nuclear translocation assays when probing compounds as potential activators. Accordingly, many studies have sought to modify or supplement the basic luciferase assay hoping to more effectively identify CAR modulators in vitro. Among others, genetic variations within the CAR gene may alter the expression and function of the encoded protein. To date, more than 25 alternatively spliced transcripts of hCAR have been identified (Arnold et al., 2004; Lamba et al., 2004). Although their functional significance in vivo remains unknown, several splice variants exhibit altered enhancer binding and coregulatory recruitment, as well as differential chemical responses (Arnold et al., 2004; Auerbach et al., 2003; Lamba et al., 2004). The splicing variant hCAR3, which contains an in-frame insertion of five amino acids (APYLT) in the LBD, was

10 reported to exhibit significantly reduced basal, but potent ligand-induced activities in cell-based reporter assays (Auerbach et al., 2003). Further delineation of the five amino acid insertion found that retaining the alanine alone (hCAR1+A) is sufficient to convert the constitutively activated hCAR into its xenobiotic-sensitive surrogate in vitro (Chen et al., 2010). Importantly, hCAR1+A displays chemical-mediated activation superior to that of hCAR3 (Chen et al., 2010). A similar approach was taken by Kanno and Inouye by inserting three consecutive alanine residues between helices 11 and 12, which lowered the high basal activity of CAR in immortalized cells (Kanno and Inouye, 2010).

However, it is important to note that the addition of any residue to a protein, especially in the ligand-binding pocket, may alter the chemical binding specificity of the protein.

Indeed, the splice variant hCAR2 containing an additional four amino acids (SPTV) reshapes the ligand-binding pocket of CAR and recognizes the common plasticizer, di(2- ethylhexyl) phthalate as a specific and highly potent agonist for hCAR2 without affecting the activity of the reference hCAR1 (DeKeyser et al., 2011; DeKeyser et al., 2009).

Although androstanol and androstenol were among the first known inverse agonists of mCAR, discovery of potent inverse agonists/antagonists of hCAR has lagged behind.

Clotrimazole was one of the first inverse agonists discovered for hCAR (Moore et al.,

2000); however, studies since have shown complex outcomes regarding the effects of clotrimazole on CAR activity. In CV-1 cells, the maximal deactivation of CAR achieved by clotrimazole was approximately 50%. In contrast, in HEK293 and COS1 cells, clotrimazole failed to repress the constitutively activated hCAR1, but potently activated hCAR3 and the hCAR1+A construct (Chen et al., 2010; Omiecinski et al., 2011). A typical peripheral benzodiazepine receptor ligand, PK11195, was later established as a

11 potent hCAR antagonist that decreases the high basal activation of wild-type CAR in

HepG2 cells by directly competing with agonists such as CITCO for binding to the LBD

(Li et al., 2008). Interestingly, cell-based luciferase reporter assays showed that

PK11195-mediated repression of CAR activity can be efficiently recovered by CITCO (a direct activator) but not by PB (an indirect activator) (Li et al., 2008). Utilizing this model, Lynch et al. recently established a quantitative high-throughput screening (qHTS) assay for identification of hCAR modulators. By screening approximately 2800 compounds from the NIH Chemical Genomics Center Pharmaceutical Collection, 115 activators of hCAR were identified, which include both novel and known hCAR activators and CYP2B6 inducers (Lynch et al., 2015).

One of the concerns surrounding the identification of selective CAR activators lies in the fact that CAR and PXR share many xenobiotic ligands and have significant overlap in the regulation of their target genes (Moore et al., 2000; Tolson and Wang,

2010). Many drugs previously identified as PXR agonists are also activators of CAR, such as the antimalarial artemisinin (Burk et al., 2005). Notably, the CAR inverse agonists PK11195 and clotrimazole are also potent activators of human PXR, making the identification of selective hCAR activators extremely challenging (Li et al., 2008; Moore et al., 2000). Most recently, Cherian et al. reported CINPA1 as a more selective deactivator of hCAR that does not activate PXR (Cherian et al., 2015a). In HepG2 cells,

CINPA1 and PK11195 exhibit comparable deactivation of CAR, but CINPA1 does not activate PXR. More importantly, CINPA1 effectively repressed CITCO-induced

CYP2B6 expression in human primary hepatocyte cultures (Cherian et al., 2015a). This new compound could be used as a novel molecular tool for elucidating hCAR activators.

Overall, ligand-dependent modulation of CAR activity represents the fundamental mechanisms of direct chemical-protein interactions. Due to the relatively low sequence homology between LBD of human CAR and its rodent counterparts, direct modulators of

CAR (agonists and antagonists) such as CITCO and TCPOBOP often exhibit more species selectivity than indirect activators, such as PB. Identification of a human-specific

CAR modulator may eventually be of clinical importance. In the meantime, it is important to point out that the physiological condition and microenvironment of cells are crucial for the investigation of CAR activation. Many compounds that have shown robust induction of CAR transcriptional activity in immortalized cell lines fail to induce CAR and PXR target genes in human primary hepatocytes. This can be exemplified by the fact that buprenorphine was identified as an effective activator for both PXR and CAR in cell- based luciferase assays but failed to either translocate CAR to the nucleus or induce CAR and PXR target genes in human primary hepatocytes (Li et al., 2010). Further investigation revealed that buprenorphine experienced a dramatically different rate of elimination between primary hepatocytes and HepG2 cells, leading to the observed discrepancy and highlighting the need for cautious interpretation of data obtained from in vitro assays.

1.2.3 Indirect activation of CAR

Shortly after PB was discovered to be a CAR activator, Kawamoto et al. demonstrated that activation of CAR by PB was majorly related to nuclear translocation that could be blocked by the protein phosphatase inhibitor OA, suggesting that PB- mediated CAR nuclear translocation was associated with a signaling cascade that requires phosphatase-mediated protein dephosphorylation (Kawamoto et al., 1999b). It was later

13 confirmed by scintillation-proximity binding assays that PB does not directly bind to

CAR even though it effectively activates CAR in multiple species (Moore et al., 2000).

Hosseinpour et al., showed that when Ser202 of CAR was mutated to an aspartate mimicking the status of protein phosphorylation, CAR was sequestered in the cytoplasm of mouse primary hepatocytes and unable to translocate to the nucleus in response to PB treatment, while mutation of Ser202 to an alanine did not affect PB-mediated mCAR nuclear translocation (Hosseinpour et al., 2006). Furthermore, Western-blotting analysis revealed that an antibody specific for phospho-Ser202 only recognized cytoplasmic CAR protein but not nuclear-localized CAR protein in liver cells, suggesting that dephosphorylation of Ser202 is likely an early, essential step of PB-mediated CAR activation.

CAR resides in the cytoplasm of primary hepatocytes and non-induced liver in contrast to the nuclear localization seen in immortalized cell lines. In primary hepatocytes, cytoplasmic chaperones associated with CAR have been proposed as important determinants for CAR’s cytoplasmic retention before activation and nuclear translocation following PB treatment (Zelko et al., 2001b). The initial simplified CAR protein complex was reported by Yoshinari et. al in non-induced mouse liver, where

CAR forms a complex in the cytoplasm with heat-shock protein (HSP) 90 (Yoshinari et al., 2003). Pretreatment with geldanamycin, a compound known to interrupt the chaperoning function of HSP90, profoundly repressed CYP2b10 expression and mCAR nuclear translocation induced by PB-type activators. It appears that the CAR:HSP90 complex also transiently recruits protein phosphatase 2A (PP2A) in response to PB treatment, further validating that chemically-stimulated CAR nuclear translocation might

14 be a OA-sensitive, protein phosphatase-mediated process (Yoshinari et al., 2003).

Interestingly, although TCPOBOP does not bind hCAR when expressed in mouse liver, hCAR translocates into the nuclei of mice livers in response to TCPOBOP treatment, suggesting CAR nuclear translocation is not initiated by ligand binding (Zelko et al.,

2001b). Further work by Negishi and colleagues identified CCRP (a member of the

HSP40 family) as another protein that could bind to the mCAR:HSP90 complex important in the cellular localization of CAR (Kobayashi et al., 2003). When co- expressed with CCRP in HepG2 cells, mCAR protein levels dramatically increased in the cytosol, providing evidence that CCRP may stabilize the CAR:HSP90 complex and affect

CAR nuclear translocation. Additional studies revealed that CCRP is subjected to ubiquitination and proteasomal degradation in cells when exposed to PB-type CAR activators (Timsit and Negishi, 2014). In HepG2 cells overexpressing both CCRP and

CAR, MG132 (a proteasome inhibitor) treatment further increased the retention of CAR in cytoplasm (Timsit and Negishi, 2014). However, whether CAR can be directly ubiquitinated is debatable (Chen et al., 2014). Negishi and colleagues also identified

HSP70, another chaperone protein, as a novel component of the cytosolic CAR protein complex (Timsit and Negishi, 2014). When HepG2 cells expressing CAR and CCRP were exposed to thermal stress, the increase in HSP70 protein levels was correlated with increased cytoplasmic CAR levels, mimicking the effects of MG132. Collectively, these findings represent important steps towards a better understanding of how CAR is released from the cytoplasmic protein complex and translocated to the nucleus – the crucial step for CAR activation.

Although PB has long been known as a CYP2B inducer and represents a class of compounds that activate CAR without binding to the receptor, the hunt for the direct target of PB has been a lengthy journey. Early observations by Bauer et al. indicated that treatment with epidermal growth factor (EGF) could repress the induction of CYP2B1 by

PB in primary rat hepatocytes (Bauer et al., 2004). Subsequently, Joannard et al. revealed that activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen- activated protein kinase (MAPK) pathways could play a role in PB-mediated gene transcription (Joannard et al., 2006). These findings turned out to be important clues for elucidating the indirect activation mechanism of CAR (Figure 1.1). Negishi and colleagues went on to show that hepatocyte growth factor treatment increased ERK1/2 phosphorylation, leading to the inhibition of TCPOBOP-induced CAR nuclear translocation and transcription of CAR target genes, while inhibition of mitogen- activated protein kinase kinase (MEK) upstream of ERK by U0126 enhanced the induction of CYP2b10 (Koike et al., 2007). Subsequently, the Thr38 residue of hCAR was found to be phosphorylated by protein kinase C (PKC), dephosphorylation of Thr38 was found to be required for CAR nuclear translocation, and most importantly, PB treatment resulted in dephosphorylation of the Thr38 of hCAR and the corresponding

Thr48 residue of mCAR (Mutoh et al., 2009). Additional studies indicated that the

ERK1/2:CAR interaction was increased in response to EGF treatment, while inhibition and knockdown of MEK led to a decrease in ERK1/2:CAR interaction (Osabe and

Negishi, 2011). Most recently, Mutoh et al., defined the epidermal growth factor receptor

(EGFR) as the initial binding protein for PB to induce CAR activation in the liver. This binding potently inhibited EGF-mediated signaling and led to the dephosphorylation of

16 the downstream receptor for activated C kinase 1 (RACK1) at Tyr52, which promoted dephosphorylation of the Thr38 residue of hCAR by PP2A, leading to the nuclear translocation and activation of CAR (Mutoh et al., 2013). Subsequent studies with the flavonoids chrysin, baicalein, and galangin, which are known to activate CAR, found that these compounds do not bind to CAR and instead activate CAR through inhibition of

EGF-mediated signaling, further validating inhibition of the EGF signaling pathway as a common mechanism for indirect CAR activation (Carazo Fernández et al., 2015)

Unlike classical NRs that are often activated or deactivated in a ligand-dependent manner, CAR is activated by both direct and indirect activators through overlapping yet distinct mechanisms. This dual-mechanism paradigm for CAR activation presents challenges in studying CAR’s signaling and function, heightened by the fact that traditional ligand binding assays are essentially useless in finding indirect activators, which make up a large portion of currently available activators of CAR. The constitutive nuclear localization and activation of CAR in immortalized cell lines also renders extra difficulties in using cell-based luciferase reporter assays to identify activators. It is worth noting that even with all these challenges, our understanding of both the biological function of CAR and the mechanistic bases of CAR activation has expanded considerably in the past decade.

1.2.4 CAR and Energy Homeostasis

The involvement of CAR in energy homeostasis was first recognized over a decade ago when CAR activation by PB in mice resulted in downregulation of genes associated with gluconeogenesis and fatty acid synthesis (Ueda et al., 2002).

Subsequently, growing evidence supporting a role of CAR in energy homeostasis and

17 metabolic disorders has promoted investigation into the broad function of CAR beyond xenobiotic disposition. In 2004, Maglich et al. reported that under caloric restriction and fasting, CAR mediated a compensatory response to limit energy expenditure in mice by downregulation of serum levels of triiodothyronine (T3) and tetraiodothyronine (T4), two major thyroid hormones that control the basal metabolic rate (Maglich et al., 2004).

Notably, fasting stimulated a CAR-dependent induction of Sult1a1, Sult2a1, and Ugt1a1, which are important for the metabolic breakdown of T3 and T4. In CAR-/- mice, however, fasting failed to induce the expression of these enzymes and the serum concentrations of T3 and T4 remained high, which led to more weight loss under caloric restriction than in WT mice (Maglich et al., 2004). Interestingly, in another report, while the authors did not observe fasting-stimulated CAR activation, the study demonstrated that CAR is required for a PB-induced decrease of T3 and T4 levels in the serum; treatment with PB or TCPOBOP induced the expression of sulfotransferases and UGTs that are important for T3 and T4 metabolism in WT but not CAR-/- mice (Qatanani and

Moore, 2005). Given that decreased basal energy expenditure represents a major barrier for obese individuals trying to lose weight, antagonism of human CAR may potentially benefit patients under a weight loss program, if the above findings hold true in humans.

In contrast to these findings, two research groups independently showed that activation of

CAR by TCPOBOP markedly ameliorated symptoms of obesity, diabetes, and fatty liver induced by high-fat diet (HFD) in WT mice, while such effects were not observed in

TCPOBOP-treated CAR-/- mice (Dong et al., 2009; Gao et al., 2009). Further gene expression and biochemical analyses have revealed that the metabolic benefits of CAR

18 activation may involve the suppression of glucose and lipid production, the inhibition of triglyceride and VLDL export, and induction of β-oxidation and energy expenditure.

Mechanistically, CAR-mediated energy homeostasis appears to be involved in a combined repression of an array of genes associated with gluconeogenesis, fatty acid synthesis, and energy expenditure such as the phosphoenolpyruvate carboxykinase

(PEPCK), glucose-6-phosphatase (G6Pase), fatty acid synthase (FAS), and stearoyl-CoA desaturase-1 (SCD-1) (Yu et al., 2016). This downregulation is involved in the prevention of the forkhead box protein O1 (FOXO1) transcription factor from interacting with insulin response element binding sites located upstream of genes such as PEPCK1,

G6Pase, and insulin-like growth factor-binding protein 1 (Kodama et al., 2004). Through direct interaction between CAR and FOXO1, activated CAR acting as a corepressor downregulates FOXO1-mediated transcription of gluconeogenic genes. Binding of CAR to the direct repeat 1 site in the PEPCK promoter in place of hepatic nuclear factor-4α

(HNF4α), a key hepatic factor crucial for the expression of bile acid synthesis and gluconeogenic genes, has also been reported as a means of metabolic suppression by

CAR (Miao et al., 2006). The peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) is another key transcriptional coactivator that governs energy metabolism by regulating the expression of PEPCK and G6Pase (Herzig et al., 2001).

Gao et al. recently demonstrated that when bound by its ligand, CAR alters the subcellular localization and degradation of PGC1α through direct CAR-PGC1α interaction, by which the CAR-PGC1α complex was co-redistributed to the promyelocytic leukemia protein-nuclear bodies (PML-NBs), where activated CAR facilitates the ubiquitination and degradation of PGC1α by recruiting Cullin 1 E3 ligase

(Gao et al., 2015). This finding suggests that in addition to transcriptional repression, posttranslational modification of protein stability may also contribute to CAR-mediated suppression of hepatic gluconeogenesis.

In contrast with the relatively consistent repression of gluconeogenesis by CAR activation, more conflicting experimental results have been generated regarding the role of CAR in the regulation of lipogenesis. Activation of CAR in mice has been shown to mitigate hepatic steatosis, increase glucose tolerance and insulin sensitivity, and alleviate or prevent obesity in diabetic mouse models (Dong et al., 2009; Gao et al., 2009). CAR- mediated anti-lipogenic effects were also observed in hyperlipidemic HepG2 cell cultures treated with evodia alkaloid (Yu et al., 2016). Furthermore, the hormone irisin was recently identified as a direct target of CAR and protects HFD-induced obese mice through the CAR-irisin axis (Mo et al., 2016). Results of this study corroborated prior research on the effects of the hormone and demonstrated that hepatic expression of irisin suppresses lipogenesis (Mo et al., 2016; Polyzos et al., 2014; Zhang et al., 2013).

On the other hand, the majority of current studies were carried out under metabolic/nutritional challenges such as HFD-feed or caloric restriction. Interestingly,

Marmugi et al. reported that treatment with TCPOBOP provoked the expression of lipogenic and glycolytic genes and increased lipid levels in a CAR-dependent manner in the livers of healthy mice under physiological conditions (Marmugi et al., 2016). A novel

CAR target gene within the lipogenic category, Pnpla3, was identified that may contribute to the observed fatty liver phenotype (Romeo et al., 2008; Smagris et al.,

2015). Notably, when maintained on a normal diet, treatment with TCPOBOP (0.3 mg/kg ip once daily for 14 days) resulted in a 50% increase in serum triglycerides in WT but not

CAR-/- mice. This is in stark contrast to Gao’s observation where TCPOBOP (0.5 mg/kg ip once per week for 8 weeks) reduced serum triglyceride from 230 to 132 mg/dl in wild- type mice fed with a HFD regimen (Gao et al., 2009). It is possible that HFD-induced nutritional stress contributes significantly to the contradicting results in these studies, though factors such as the TCPOBOP treatment regimen and the genetic background of the mice used cannot be excluded. Additionally, the inherited species differences between human and mouse CAR may further complicate the dispute. In primary and immortalized human hepatocytes, activation of CAR promotes the expression of lipogenic genes such as SCD-1 and PNPLA3 (Marmugi et al., 2016). Another study using human primary hepatocytes found that CAR activation, while inhibiting gluconeogenesis, did not affect the expression of genes associated with hepatic lipogenesis (Lynch et al., 2014b).

Collectively, a correlation between CAR and energy homeostasis has been firmly established. Numerous studies have demonstrated that activation or deactivation of CAR can disturb the balance of energy metabolism/expenditure (Figure 1.2). However, the exact role of CAR in metabolic disorders continues to be uncertain or even controversial.

Information pertaining to humans in particular is limited.

Figure 1.2 - CAR and Energy Homeostasis. Summary of how CAR activation affects energy homeostasis though both direct gene regulation and inhibition of important energy regulation pathways in the liver.

1.2.5 CAR on Cell Proliferation and Cancer

The effect of CAR activation on mitogenesis has been the subject of intense inquiry since the discovery that CAR is responsible for the PB- and TCPOBOP-induced liver hypertrophic and hyperplastic responses in mice (Wei et al., 2000). This topic is intriguing from two standpoints: whereas a hyperplastic response might lead to the development of cancer in certain circumstances, a regenerative response following severe tissue insult or injury is often critical to survival. The essential role of CAR in PB- and

TCPOBOP-mediated tumor promotion was initially established by using CAR-/- and WT mice, in that activation of CAR is associated with both induction of DNA replication and suppression of apoptosis (Huang et al., 2005; Phillips et al., 2007; Yamamoto et al.,

2004). Subsequent studies have further confirmed that a class of rodent CAR activators exhibit their tumor-promoting activities in CAR-dependent manners (Maeda et al., 2015;

Okuda et al., 2017; Tamura et al., 2015; Tamura et al., 2016; Wang et al., 2017). 22

Although the underlying molecular mechanisms by which CAR stimulates tumor promotion are not fully elucidated, accumulating evidence reveals that activation of CAR alters the expression of the growth arrest and DNA damage-inducible 45 β (GADD45β), the murine double minute 2 (mdm2), the tubulin alpha 8 (TUBA8), the family with sequence similar 84, member A (FAM84A), and c-Myc which are all closely correlated with cell proliferation and oncogenic signaling (Blanco-Bose et al., 2008; Huang et al.,

2005; Kamino et al., 2011a; Kamino et al., 2011b; Yamamoto et al., 2010).

Recently, Dong and colleagues studied the relationship between mouse CAR activation and the Wnt/β-catenin pathway in the development of liver tumors (Dong et al., 2015). Although no evidence was found of direct interaction between CAR and β- catenin at the transcriptional level, results of the study showed that CAR activation prevented the senescence that would otherwise be triggered by Wnt/β-catenin activation over time and that the two act synergistically to promote liver cell proliferation and hepatocellular carcinoma (HCC) development. Another study by Braeuning et al., however, found that in Apc-/- mice (APC forms part of the protein complex that is essential to normal degradation of β-catenin), treatment with PB did not result in a persistent proliferative advantage (Braeuning et al., 2016). PB was shown to promote adenoma but inhibit carcinoma in liver cells of Apc-/- mice. Although mechanisms other than those directly involving CAR in the inhibition of HCC were not ruled out, the study points to the paradoxical properties of PB in tumor promotion and the need for additional investigation. Most recently, Tschuor and colleagues studied the regenerative effects of

CAR in mouse liver following extreme (91% of liver volume resected), extended (86% resected), and standard (70% resected) hepatectomy (Tschuor et al., 2016). Marked

23 impairment in mouse CAR activation following extended hepatectomy was observed, and liver dysfunction and lack of regeneration corresponded with similar phenomena in Car-/- mice that had undergone standard hepatectomy. Following administration of the mouse

CAR activator TCPOBOP, survival was significantly improved in WT but not CAR-/- mice. As a regenerative response is essential to avoid potential liver failure after significant resection in the setting of tumor invasion or following transplantation with reduced-size liver grafts, therapeutic human CAR intervention may play a role in recovery from compromising liver surgery in the future (Clavien et al., 2010; Tschuor et al., 2016).

MicroRNAs (miRNAs) are short noncoding RNAs that play important roles in the post-transcriptional regulation of genes associated with various diseases, including HCC. miR-122, the most abundant hepatic miRNA, has been established as a tumor suppressive miRNA in the liver (Coulouarn et al., 2009). Of note, the expression of miR-122 was markedly downregulated in C3H/HeN mice in vivo and HepG2 cells in vitro treated with

PB (Shizu et al., 2012). Kazantseva et al. further demonstrated that activated CAR represses the expression of miR-122 through direct competition with HNF4a for binding to the DR1 response element located upstream of the pri-miR-122 promoter, a mechanism by which CAR suppresses a number of other HNF4a target genes

(Kazantseva et al., 2015). Using deep sequencing approaches, Hao et al. profiled the global miRNA expression patterns in livers from C57BL/6J mice treated with TCPOBOP or DMSO as vehicle control (Hao et al., 2016). Among the 51 miRNAs significantly altered by TCPOBOP treatment in this study, known oncogenic miRNAs, such as miR-

148a, miR-let-7f, and miR-671, are upregulated, supporting the idea that CAR may

24 modulate a network of miRNAs in facilitating mouse hepatocyte proliferation. In addition to CAR-mediated regulation of miRNA expression, the expression of CAR itself can also be repressed by miRNA such as miR-137, which was observed in cellular models of hepatocellular and colon cancers (Takwi et al., 2013). More in-depth analysis of the rather comprehensive roles of miRNA in CAR-dependent hepatocarcinogenesis is warranted.

Another mechanism by which CAR may influence cancer development is through its involvement in circadian rhythm homeostasis. CAR expression has been shown to elevate during the night in mice, corresponding to their regular feeding patterns (Gachon et al.,

2006). More recent studies have expanded on the link between CAR activity and circadian rhythms. For example, the period circadian regulator 2 protein (PER2) has been found to directly interact with CAR, with implications that have yet to be explored

(Martini et al., 2017). Additionally, a shifted feeding schedule in rats (i.e., daytime rather than nighttime feeding) likewise caused a shift in CAR expression(de Vries et al., 2017).

Significant disruption in circadian rhythms over time, in turn, has recently been demonstrated by Kettner and colleagues to provoke non-alcoholic fatty liver disease, fibrosis, and hepatocellular carcinoma in correlation with elevated bile acid and CAR levels in mice. Increased levels of CAR were found to be related to disruption in sympathetic nervous system signaling and peripheral tissue clock activity (Kettner et al.,

2016).

Compared to what we have learned from rodent animals with regard to the role of

CAR in cancer development, the function of CAR in human hepatocarcinogenesis continues to be controversial, and in-depth studies are limited. Indeed, although PB

25 represents a prototypical CAR activator and known nongenotoxic carcinogen that promotes liver cancer in rodents, PB-induced replicative DNA synthesis and hepatocellular proliferation in rodents were not observed in either cultured human hepatocytes in vitro or in chimeric mice with humanized liver in vivo (Elcombe et al.,

2012; Haines et al., 2018; Soldatow et al., 2016; Yamada et al., 2014). Moreover, epidemiological studies have shown that PB and a number of PB-like nongenotoxic rodent carcinogens do not increase the incidence of liver tumors in humans, even after long therapeutic applications at doses producing plasma concentrations challenging those that are carcinogenic in rodents (Braeuning, 2014; La Vecchia and Negri, 2014). When the human CAR transcriptome was recently analyzed using WT and CAR-/- HepaRG cells, many cell proliferation-associated genes were upregulated in CAR-/- but not WT cells (Li et al., 2015a). Additionally, in human brain tumor stem cells, activation of CAR by CITCO (6-(4-chlorophenyl)imidazo [2,1-beta][1,3]thiazole-5-carbaldehyde-O-(3,4- dichlorobenzyl)oxime) was associated with cell cycle arrest and enhanced apoptosis both in vitro and in an in vivo xenograft model (Chakraborty et al., 2011). Collectively, these studies raise significant concerns regarding direct extrapolation of findings from rodents to humans, particularly with regard to the role of CAR in cancer development.

1.2.6 Additional Endobiotic Functions of CAR

In addition to the roles discussed above, CAR has important endobiotic metabolism functions, including its regulation of bilirubin and bile acid processing genes (Huang et al., 2003; Wagner et al., 2005). A 2017 study addressing a potential role of CAR in prevention of cholesterol gallstone disease found that CAR activation by TCPOBOP in lithogenic diet-fed mice prevented the development of cholesterol gallstones (Cheng et

26 al., 2017). Furthermore, although primarily studied in the liver, CAR has also been investigated in other organs, such as brain and intestine. Boussadia et al. recently explored the role of CAR in pathophysiological brain processes and found that CAR-/- mice displayed inferior memory function and greater levels of anxiety, as indicated by behavioral tests, when compared with WT mice (Boussadia et al., 2016).

Electroencephalographic changes in CAR-/- mice were found to correlate with memory impairment, and microvessels exhibited morphological changes that were suggestive of inflammatory processes. Additionally, when a seizure-inducing neurotoxin was peripherally administered, CAR-/- mice experienced quicker-onset and more prolonged seizure episodes than did WT mice, reinforcing the increased vascular permeability suggested by other experimental results (Boussadia et al., 2016). The effects of CAR in intestinal tissue were also studied by Hudson and colleagues recently. The expression of

CAR in inflamed, non-ulcerated intestinal mucosal tissue from patients with ulcerative colitis and Crohn’s disease was found to be markedly reduced when compared with corresponding tissue from healthy donors, results that were duplicated in intestinal mucosal samples from mice with chemically-induced inflammation (Hudson et al., 2017).

When intestinal tissue was collected from CAR-/- mice after a week’s recovery time following chemically-induced mucosal damage, mucosal tissue had failed to recover to the extent observed in WT mice in terms of both damage and inflammation. CAR activation by CITCO in Caco-2 intestinal epithelial cells was found to increase the migratory distance of these cells, an effect that was correlated with increased p38 MAP kinase activation, and to aid wound closure while having no effect on cell proliferation.

Most recently, Choi et al. demonstrated an interesting kidney-liver cross-talk in response

27 to acute kidney injury, where TCPOBOP alleviates serum IL-6 elevation induced by renal ischemia-reperfusion in a CAR-dependent manner (Choi et al., 2018).

Taken together, these findings demonstrate that the role of CAR has extended well beyond its traditional function in xenobiotic metabolism and transport. Endogenous roles involving energy homeostasis, cancer development and prevention, and tissue integrity and regeneration continue to be elucidated. New insight into the mechanisms by which

CAR exerts its effects and the precise conditions in which it does so will likely lead to therapeutic advances in many pathological conditions.

1.3 Pregnane X Receptor

The pharmacological importance of PXR, also referred to as steroid X receptor (SXR) and pregnane-activated receptor (PAR), was almost immediately recognized after its cloning in 1998 when its primary gene target was found to be CYP3A4, the most abundant human hepatic P450 (Bertilsson et al., 1998; Blumberg et al., 1998; Kliewer et al., 1998). As a promiscuous mediator for metabolism-based DDIs, PXR significantly induces the expression of numerous drug-metabolizing enzymes and transporters important in xenobiotic disposition, which can result in clinically important DDIs

(Stanley et al., 2006). To date, a large number of compounds, including drugs and environmental chemicals, have been established as PXR activators due in part to the unique structure of the PXR LBD, which contains a bulky and flexible ligand-binding pocket characterized by a 5-strand β-sheet in contrast to the 3-strand β-sheet exhibited by the majority of NRs (Ekins et al., 2009; Ngan et al., 2009; Watkins et al., 2001). Upon ligand binding and activation, PXR recruits many of the same coactivators/repressors as

CAR to carry out its transcriptional activities (Wang and LeCluyse, 2003). In fact, PXR

28 is the closest sister receptor of CAR in the whole NR superfamily tree, sharing many small molecule activators and participating in cross-talk over common target genes (di

Masi et al., 2009).

PXR is activated by a broad range of chemicals with no obvious common structural features, including endobiotics such as steroid hormone metabolites, vitamins, and bile acids, and xenobiotics such as herbals, macrolide antibiotics, antifungals, etc. (Chang and

Waxman, 2006; di Masi et al., 2009). Interestingly, although PXR is a promiscuous receptor, it exhibits divergent ligand activation profiles across species. Clearly, the low homology in the PXR LBD regions contributes to the observed species differences in

PXR activation and target gene induction (Ostberg et al., 2002). For instance, the anti- glucocorticoid pregnenolone 16α-carbonitrile (PCN) and DTBA activate rat PXR but not human PXR, while rifampin and SR12813 activate human but not rat and mouse PXR

(Orans et al., 2005). Remarkably, rabbit PXR was activated by both rifampin and DTBA, reflecting its evolutionary conservation (Orans et al., 2005; Savas et al., 2000). It was speculated that differences in diet and xenobiotic exposure among species were responsible for evolutionary changes in amino acids lining the ligand-binding pockets of different PXRs (Krasowski et al., 2011).

Currently, there is controversy regarding the cellular localization of PXR. Like CAR, human PXR (hPXR) is consistently localized in the nucleus in immortalized cell lines

(Saradhi et al., 2005). Nevertheless, such auto-accumulation in the nucleus does not exhibit spontaneous activation without chemical stimulation. In contrast, multiple studies have shown that mouse PXR (mPXR) is primarily localized in the cytoplasm of non- induced mouse liver and translocates to the nucleus following treatment with the

29 prototypical mPXR agonist PCN (Squires et al., 2004). In addition, mPXR can form a complex in the cytoplasm with HSP90 and CCRP similar to CAR, although its PCN- dependent translocation could not be reversed by overexpression of CCRP (Squires et al.,

2004). Studies have also demonstrated that the nuclear localization signal of hPXR is essential to its translocation from the cytoplasm to the nucleus and transcription of target genes in immortalized cells (Kawana et al., 2003). More studies in vivo or in physiologically-relevant cell culture systems are needed to determine the importance of nuclear translocation in hPXR activation. Regardless, PXR is not constitutively active like CAR and therefore, nuclear localization alone is not sufficient for PXR to induce transcription of its target genes. Although all PXR activators identified to date have either been proven or assumed to be directly binding to PXR, a number of studies have shown that signaling pathways can influence PXR activation status (Figure 1.3).

Figure 1.3 - Schematic illustration of Mechanisms of PXR Activation. Direct Activation – PXR is retained in a cytoplasmic complex with HSP90 and CCRP, which dissociate upon ligand binding leading to PXR nuclear accumulation. Unbound PXR in the nucleus is complexed to corepressors NcoR and SMRT, which dissociate upon ligand stimulation, leading to the recruitment of coactivators SRC1 and GRIP1 and the transcription of target genes. Indirect Activation – This is a less well-developed mechanism of PXR activation, although the Ser350, Thr248, Thr290, and Thr408 phosphorylation sites of PXR are known to affect its activation. Compounds disturbing signaling pathways may influence the phosphorylation, nuclear translocation, and target gene express of PXR.

1.3.1 Direct activation of PXR

Ligand-dependent activation of PXR has long been a focus of investigation, due to the extensive ligand promiscuity and robust target gene induction. Without ligand binding, PXR is constantly silenced by recruitment of corepressors instead of coactivators when residing in the nucleus. Agonist binding of PXR results in release of preoccupied corepressors, such as silencing mediator of retinoid and thyroid receptors (SMRT) and the nuclear receptor corepressor 1 (NcoR1), and recruitment of coactivators such as SRC-

1 and glucocorticoid receptor interacting protein 1 (GRIP1) (Figure 1.3) (di Masi et al.,

2009). Similar to CAR, PXR also dimerizes with RXR and binds to two AGGTCA

31 hexamers formed as DR 3-5, or ER6 motifs (Orans et al., 2005). Accumulating evidence reveals that the ligand promiscuity of PXR stems from the unique structure of its LBD.

The crystal structures of the hPXR LBD complexed with prototypical activators, including macrolide antibiotic rifampicin, St. John’s Wort component hyperforin, and cholesterol-lowering drug SR12813, have provided important structural information that has dramatically increased our understanding of the structure-activity relationship of PXR

(Chrencik et al., 2005; Watkins et al., 2003b; Watkins et al., 2001). The PXR LBD contains a rather large (1280-1600A), flexible spherical ligand-binding pocket, and several unique structural features allow it to fit different sizes and types of ligands

(Chrencik et al., 2005; Watkins et al., 2001). The ligand-binding pocket of PXR is mostly made up of hydrophobic residues, but contains a few important polar residues which participate in hydrogen-bonding interactions with ligands. Importantly, these features of the PXR ligand-binding pocket allow a single compound to dock in more than one orientation and contact different polar residues to exert its activity (Ostberg et al., 2002).

However, when the PXR LBD and a peptide of coactivator SRC-1 were co-crystallized with SR12813, the ligand was bound in only one conformation, showing the inherent limitations of crystallographic analysis (Watkins et al., 2003a).

Extensive efforts have been made to understand the structural basis that makes a compound a PXR activator. Although structural models based on the LBD of PXR have been developed, they have had limited success in predicting PXR activators. Diverse predictive models have also been developed for ligand-based computational methods, including machine learning techniques, pharmacophore modeling, and quantitative structure-activity relationship analysis, which have all been used to virtually screen

32 compound libraries with varying rates of success (Ekins et al., 2009). As with CAR, many of these computational models are used in combination with binding and luciferase assays to validate potential PXR activators (Raucy and Lasker, 2013; Xiao et al., 2011).

One HTS assay developed for PXR used time-resolved FRET between fluorescent-tagged

PXR LBD and coactivator SRC-1 to identify PXR activators, similar to those used in

CAR activator screening (Shukla et al., 2009). Stable HepG2 cell lines expressing a reporter plasmid have also been developed for HTS and successfully used to identify

PXR activators, but again, these immortalized cell lines do not exhibit metabolic capacity and therefore, will not necessarily predict physiologically-relevant PXR activators, showing the need for screening in human primary hepatocytes (Ratajewski et al., 2015).

1.3.2 Indirect activation of PXR

Although classical understanding of PXR activation necessitates that a ligand must be bound to impart transcriptional activity, emerging evidence indicates that the status of PXR activation can be influenced by different cellular signaling pathways. Ding and Staudinger observed that activation of protein kinase A (PKA) signaling by forskolin potentiates PXR-mediated induction of CYP3A gene expression in mouse hepatocytes

(Ding and Staudinger, 2005a). On the other hand, increase of PKC activity by cytokines dramatically repressed PXR activity in both luciferase reporter and target gene expression assays (Ding and Staudinger, 2005b). These initial findings suggest that PXR activity is altered by signaling pathways that influence the phosphorylation status of PXR and/or its associated protein partners. Subsequently, utilization of computational analysis and site- directed mutagenesis approaches predicted 18 consensus phosphorylation sites of PXR that were further characterized in vitro (Lichti-Kaiser et al., 2009). A number of

33 phosphomimetic mutants altered PXR transcriptional activity by affecting response element binding or coactivator binding, including a phosphomimetic mutation at Ser350 of PXR which inhibited its heterodimerization with RXR. Interestingly, mutation of

Thr248 to aspartate converts PXR to a constitutively active format that is irresponsive to ligand stimulation while mutations at Thr408 mimicking both phosphorylation (T408D) and lack thereof (T408A) abolished the ability of rifampicin to translocate PXR to the nucleus of CV-1 cells (Doricakova et al., 2013). Additionally, Sugatani et. al showed that the dephosphorylation of Thr290 by PP1 was essential to xenobiotic-induced nuclear translocation of PXR while phosphorylation of Thr290 by Ca2+/calmodulin-dependent protein kinase II led to repression of PXR nuclear translocation, suggesting that phosphorylation status may alter the activity of PXR by affecting its subcellular localization as well (Sugatani et al., 2014). PXR has also been shown to interact with a number of other signaling kinases and phosphatases, including PKA, PKC, cyclin- dependent kinases (Cdk), and the 70 kDa ribosomal protein S6 kinase (S6K), which all generate different effects on PXR activation (Ding and Staudinger, 2005a; Ding and

Staudinger, 2005b; Elias et al., 2014; Lin et al., 2008; Pondugula et al., 2015). For instance, PKC signaling represses PXR activity, potentially by strengthening PXR interaction with corepressor NcoR1, while PKA signaling enhances the recruitment of

SRC-1 to PXR, potentiating target gene transcription (Ding and Staudinger, 2005a; Ding and Staudinger, 2005b). Notably, the protein phosphatase inhibitor OA can completely abolish PXR activity in both reporter assays and cultured hepatocytes, providing further evidence that protein phosphatases play role in PXR activation (Ding and Staudinger,

2005b).

Indirect activation of PXR can also be exemplified by the Cdk signaling pathway.

Chen and colleagues found that the small-molecule inhibitors of Cdk2, kenpaullone and roscovitine, induced PXR-mediated gene expression in HepG2 luciferase assays, while activation of the Cdk2 pathway led to repression of PXR-mediated CYP3A4 activation

(Lin et al., 2008). Kinase assays showed that Cdk2 phosphorylates the Ser350 residue of

PXR in vitro. Notably, kenpaullone binds to PXR with moderate affinity compared to its robust activation of PXR activity, uggesting that direct ligand-binding may not be essential for PXR activity, particularly for chemicals that predominantly influence signaling pathways (Lin et al., 2008). Further studies found that the flavonoids luteolin and apigenin could bind to PXR only at concentrations ≥ 10 µM; however, these compounds significantly enhanced PXR-mediated CYP3A4 gene expression at concentrations lower than 10 µM, indicating that signaling pathways may be involved in flavonoid-mediated PXR activation (Dong et al., 2010). Mechanistic analysis revealed that Cdk5 phosphorylated PXR in vitro and expression of Cdk5 and its regulatory subunit repressed PXR activation by rifampicin in HepG2 cells, while this effect of Cdk5 on PXR was efficiently reversed by both the knockdown of Cdk5 as well as the addition of flavonoids that are known inhibitors of Cdk5 (Dong et al., 2010).

Collectively, accumulating evidence demonstrates that many factors can alter

PXR’s cellular localization and association with other proteins in addition to direct ligand-binding. Post-translational modification of PXR by signaling molecules clearly plays a crucial role in modulation of its biological function and represents a currently understudied area. Further studies are needed to fully characterize the mechanism of PXR indirect activation in more physiologically-relevant cell systems.

1.3.3 PXR and Energy Homeostasis

Previous studies reveal that activation of PXR by PCN results in decreased blood glucose levels in mice, an effect attributable to PXR-mediated repression of genes such as

PEPCK1 and G6Pase that are pivotal to hepatic gluconeogenesis (Bhalla et al., 2004;

Kodama et al., 2004). In human hepatocarcinoma Huh7 cells overexpressing transfected human PXR, addition of cAMP induced the expression of G6Pase and PEPCK mRNAs

13- and 20-fold, respectively, while the induction of these genes was markedly repressed by rifampicin, the prototypical activator of human PXR (Kodama et al., 2007).

Mechanistically, PXR acts as a corepressor of FOXO1 and FOXA2 and downregulates

FOXO1-mediated insulin response sequence (IRS) activation and transcription of gluconeogenic genes (Kodama et al., 2004). GST pull-down and co-immunoprecipitation assays demonstrated that PXR directly binds CREB, a cAMP-response element-binding protein, and represses cAMP-mediated expression of G6Pase thereafter (Kodama et al.,

2007). Further, this study showed that the binding affinity between PXR and CREB was strengthened by PCN treatment, which led to a decreased binding of CREB to the G6Pase promoter in mice. Additional studies investigating the effect of PXR on bile acid synthesis and gluconeogenesis in HepG2 cells found that human PXR interacts with the coactivator PGC1α in the presence of rifampicin (Bhalla et al., 2004). This ligand- dependent PGC1-PXR interaction prevents PGC1α from binding to HNF4α and forms a functionally inhibitory cross-talk between PXR and HNF4α, leading to the repression of

PEPCK1.

In contrast with aforementioned findings suggesting a potential glucose-lowering benefit of PXR activation, clinical studies have indicated that treatment with rifampicin

36 increases blood glucose levels in both tuberculosis patients and healthy volunteers (Rysa et al., 2013; Takasu et al., 1982). Such clinical observations correlate with a recent in vitro study using human primary hepatocytes and HepG2 cells stably expressing human

PXR, where activation of PXR by rifampicin and a statin significantly induced the expression of PEPCK1 and G6Pase in both hepatic cell systems (Gotoh and Negishi,

2014; Gotoh and Negishi, 2015). The serum/glucocorticoid regulated kinase 2 (SGK2) that was also upregulated by human PXR activators appears to be essential for this PXR- mediated induction of gluconeogenesis, and the drug-PXR-SGK2 signaling requires the recruitment of the protein phosphatase 2Cα (PP2Cα) by ligand-activated PXR to dephosphorylate SGK2 at Thr193, which in turn facilitates PXR-mediated transactivation of genes encoding gluconeogenesis, including PEPCK and G6Pase. Interestingly, this drug-PXR-SGK2 signaling is not present in mice, which may explain some of the discrepancies observed between murine and human studies (Gotoh and Negishi, 2014;

Gotoh and Negishi, 2015). Most recently, Gotoh et al. further demonstrated that rather than drug challenges, a low level of glucose induced the phosphorylation of PXR at

Ser350 and enhanced gluconeogenesis in cultured HepG2 cells (Gotoh et al., 2017).

Immunoprecipitation and in vitro kinase assays revealed that the vaccinia related kinase 1

(VRK1), a serine/threonine kinase, is responsible for the phosphorylation of PXR at

Ser350 under low glucose conditions, which enabled the phosphorylated PXR to scaffold

PP2Cα for subsequent dephosphorylation of SGK2 at Thr193. Knockdown of VRK1, on the other hand, markedly repressed the phosphorylation of PXR-Ser350, increased

SGK2-Thr193, and nearly abolished the expression of PEPCK in HepG2 cells cultured under low glucose. Importantly, this low glucose-stimulated VRK1-PXR-PP2C-SGK2

37 signaling was also observed in mice under fasting conditions, suggesting that this signaling pathway may represent a novel feedback mechanism in response to low glucose that is conserved in both humans and mice (Gotoh et al., 2017). In another study,

Oladimeji et al. observed that high glucose increased the expression and activity of PXR in HepG2 cells and that this induction was partially reversed by the activation of AMPK, suggesting that PXR activity can be modulated by the energy status of the cells

(Oladimeji et al., 2017).

Adding yet another layer of complexity to our understanding of the role of PXR in glucose homeostasis, recent studies revealed that PXR also alters the uptake and utilization of glucose. Studies in mice and rats found that activation of PXR with PCN downregulates the expression of glucose transporter 2 (GLUT2), the transporter responsible for glucose uptake into hepatocytes during the fed state, and glucokinase

(GCK), which deactivates G6Pase by phosphorylation (Hassani-Nezhad-Gashti et al.,

2018; Ling et al., 2016). Collectively, activation of PXR in various in vivo and in vitro models exhibiting different types of metabolic function has led to mixed outcomes, with

PXR activation improving glucose tolerance in some models while worsening glucose homeostasis in others (Hakkola et al., 2016). Multiple confounding factors including genetic variations and experimental conditions may contribute to the observed discrepancies. Clearly, the effects of PXR activation on glucose tolerance in humans require further evaluation.

Unlike the beneficial effects of CAR activation on lipid homeostasis that have been reported by several groups, activation of PXR has been shown to enhance lipogenesis while decreasing lipid oxidation, promoting a fatty liver phenotype (Bitter et

38 al., 2015; Nakamura et al., 2007; Zhou et al., 2006). Using PXR-/- and WT-mice,

Nakamura et al. reported that treatment with PCN resulted in downregulation of CPT1A

(β-oxidation) and mitochondrial 3-hydroxy-3-methylglutarate-CoA synthase 2 (Hmgcs2; ketogenesis) but upregulation of the stearoyl-CoA desaturase 1 (lipogenesis) in a PXR- dependent manner (Nakamura et al., 2007). At the molecular level, PXR affects the expression of these genes at least partly through cross-talk with the insulin response forkhead factor FoxA2. Unexpectedly, this study also found that untreated PXR-/- mice developed severe hepatic steatosis accompanied with induction of lipogenesis and repression of fatty acid β-oxidation reminiscent of those associated with the pharmacological activation of PXR (Nakamura et al., 2007). Whether unidentified endogenous ligands may contribute to this contradictory observation is largely unknown.

Studies using a combination of human PXR transgenic, PXR-/-, and WT mice found that both genetic and pharmacological activation of PXR in the liver resulted in elevated hepatic lipid accumulation, which is associated with induction of the fatty acid translocase protein CD36 without activation of the lipogenic transcriptional factor sterol regulatory element-binding protein-1c (Zhou et al., 2006). On the other hand, genetic

PXR ablation protected mice from HFD- and genetically induced obesity, hepatic steatosis, and insulin resistance (He et al., 2013). In addition, Ma et al. reported that activation of PXR by PCN prevents HFD-induced obesity in AKR/J mice (Ma and Liu,

2012). Potential factors contributing to this discrepancy may include different genetic backgrounds of mice used, C57BL/6J vs AKR/J, and PCN treatment dosage and schedules. Indeed, PXR-mediated alteration of lipid homeostasis may exhibit tissue, cell type, and species specificities. Activation of PXR in human primary hepatocytes with

39 rifampicin did not induce CD36 expression, and lipid accumulation in the hepatocytes was due to increased fatty acid synthesis and reduced fatty acid β-oxidation instead of increased free fatty acid uptake as observed in the mouse models (Moreau et al., 2009).

Another possible mechanism proposed for PXR-dependent increases in hepatic lipid accumulation is the induction of the novel PXR target gene SLC13A5, an uptake transporter that imports citrate from the circulation into the hepatocyte, where it facilitates de novo synthesis of lipids and cholesterol (Li et al., 2015b). Collectively, activation of PXR quite consistently leads to increased hepatic lipid accumulation, while the differences in mechanisms between preclinical species and humans require that caution be taken when attempting to define the physiological relevance of findings in animal models (Figure 1.4).

Figure 1.4 - PXR and Energy Homeostasis. Summary of how PXR activation affects energy homeostasis though both direct gene regulation and either activation or inhibition of important energy regulation pathways in the liver.

1.3.4 PXR in cancer and cell proliferation

PXR-mediated alterations in drug disposition have been known to play a significant role in chemotherapy resistance, as many anticancer agents are substrates of

DMEs and efflux transporters that can be upregulated by PXR activation (Oladimeji and

Chen, 2018; Zhuo et al., 2014). Although PXR in the liver and intestine accelerates drug clearance in general, tumor-specific expression of PXR becomes an additional barrier to the therapeutic efficacy of anticancer agents (Chen et al., 2012; Chen et al., 2007; Mani et al., 2005). This is exemplified by a recent study investigating the therapeutic efficacy of sorafenib in HCC treatment, where sorafenib was found to enhance its own clearance via

CYP3A4 and P-glycoprotein induction in HCC by the activation of PXR (Feng et al.,

2018). Outside of its traditional role of xenobiotic detoxification, accumulating evidence reveals that PXR can also regulate the expression of multiple genes associated with cell apoptosis and proliferation, which play pivotal roles in cancer progression (Chen et al.,

2009; Gupta et al., 2008; Masuyama et al., 2007; Pondugula et al., 2016).

Mounting cell-based evidence thus far supports that PXR plays a pleiotropic role in cell proliferation and cancer development in a cell-type specific manner. Treatment of hepatocytes with dexamethasone, a PXR activator, inhibited spontaneous apoptosis by upregulating B-cell leukemia 2 (Bcl-2), an antiapoptotic protein that inhibits p53- mediated apoptosis signaling, and this phenomenon was also confirmed using other PXR agonists in both rat and human hepatocytes (Bailly-Maitre et al., 2001; Zucchini et al.,

2005). Additionally, PXR inhibited apoptosis in LS180 colorectal adenocarcinoma cells by inducing Bcl-2 and MCL-1, another antiapoptotic protein, while downregulating

41 proapoptotic proteins such as Bcl-2 antagonist/killer 1 and p53 (Zhou et al., 2008).

Further studies probing the mechanistic interactions between PXR and p53 found that

WT-p53 can directly bind to PXR, and heterodimerization of PXR and p53 appears to form a mutually repressive cross-talk through which each inhibits the other’s transcriptional activity in HCT116 and LS180 colon cancer cells. This mutual inhibition protects them against chemotherapeutic-induced cell death by decreasing apoptosis and increasing malignant transformation (Elias et al., 2013; Robbins et al., 2016). In addition to its role in liver and colon cancers, PXR is also expressed in prostate cancer, breast cancer, and a number of other tumor tissues, with differential biological function and tissue and cell type/context-specific consequences (Miki et al., 2006).

In the case of colorectal cancers, Wang et al. reported that activation of PXR is sufficient to enhance neoplastic characteristics of LS174T cells and human primary colon tumor cells both in vitro and in xenografted mice in vivo, and pointed out that mechanistically this may involve a PXR-dependent induction of FGF19 expression in cancer (Wang et al., 2011). Using similar approaches, Ouyang et al., however, observed a

PXR-mediated anticancer activity in HT29 cells, another colorectal cancer cell line with relatively low expression of PXR. Stable transfection of PXR in HT29 cells led to repressed cell proliferation, migration, and xenograft growth, which is accompanied by cell-cycle arrest, elevated p21 expression, and inhibition of E2F1 (Ouyang et al., 2010).

Interestingly, this report also indicated that expression of PXR is reduced in human colon cancer tissues, albeit using a relatively small sample size (Ouyang et al., 2010). A tumor- suppressive role of PXR was further supported by another report where intestine-specific activation of PXR by rifaximin significantly reduced azoxymethane/dextran sulfate

42 sodium-induced colon cancer in human PXR transgenic but not WT or PXR-/- mice, possibly through the PXR-NF-kB axis (Cheng et al., 2014).

In addition to cancer development, PXR has been shown to be important in liver regeneration by augmenting the proliferation of hepatocytes (Dai et al., 2008; Elcombe et al., 2012). In fact, PXR was necessary for full liver regeneration in mice after a partial hepatectomy, with the PXR-/- mice exhibiting severe inhibition of hepatocyte proliferation 3 days after hepatectomy surgery (Dai et al., 2008). PXR-/- mice showed inactivation of signal transducer and activator of transcription protein 3 (STAT3) 5 days post-surgery, which was the most likely cause of hepatocyte quiescence (Dai et al.,

2008). While activation of PXR in WT mice did not enhance hepatocyte proliferation, co- treatment of PCN with activators of either CAR or PPARα led to a synergistic enhancement of hepatocyte proliferation (Shizu et al., 2013). Collectively, activation of

PXR perturbs the balance of cell proliferation and apoptosis in cell-, tissue-, and species- specific manners without an overarching phenotype, making the study of PXR in different cancer types complex.

1.3.5 Additional non-traditional functions of PXR

Studies have shown that PXR is also expressed in immune cells, such as T and B lymphocytes, and in the skin of mice and humans, where perturbation of PXR expression and activity alters the immune response (Dubrac et al., 2010; Elentner et al., 2015;

Haslam et al., 2013). Many patients with atopic dermatitis have compromised immune barrier function, which leads to an increase in the penetration of lipophilic pollutants

(Oetjen et al., 2018). This penetration has been shown to trigger PXR activation in keratinocytes and a subsequent hyper-responsive immune response, further impairing the

43 barrier function (Oetjen et al., 2018). Specifically, Elenter et al. reported that transgenic mice expressing constitutively activated human PXR display increased transepidermal water loss, abnormal stratum corneum lipids, focal epidermal hyperplasia, and increased expression of local T cells (Elentner et al., 2018).

The same compromise in barrier function exhibited in atopic dermatitis is also observed in the GI tract in diseases such as inflammatory bowel disease (IBD) and

Crohn’s disease, and PXR plays a role in both of these diseases by increasing epithelial permeability (Terc et al., 2014). Additionally, PXR has been shown to regulate the intestinal epithelial wound healing response, allowing mutations to reduce the healing response, leading to an increase in IBD risk factors. This has been shown by using PXR agonists as protective agents that prevent intestinal inflammation from occurring (Terc et al., 2014). It is suggested that PXR plays a role in the healing response by modulating gene transcription, thereby upregulating genes that are related to metabolic functioning, while hindering inflammatory genes (Mencarelli et al., 2010). Beyond the role of PXR in the disease state of the gastrointestinal tract, PXR activation plays a major role in the maintenance of homeostasis of bile acids, which can affect the potential progression of many cholesterol-related diseases.

As described throughout this review, many xenobiotics and endobiotics activate

PXR, leading to the regulation of key enzymes that have been implicated in a wide array of physiological activities. There is an abundance of knowledge on the role of PXR in xenobiotic metabolism, and recently the evidence has shifted to the more critical nontraditional role that PXR plays in the regulation of endogenous functions which has led to interest in uncovering the magnitude of PXR’s influence. However, more

44 information is needed to determine whether PXR may eventually be used as a target to prevent and treat diseases.

Chapter 2: Metabolism-mediated alterations of constitutive androstane receptor activity

2.1 Introduction The constitutive androstane receptor (CAR) is a xenobiotic sensor primarily expressed in the liver that regulates expression of genes associated with drug- metabolizing enzymes (DME) and transporters (Omiecinski et al., 2011; Qatanani and

Moore, 2005; Zelko and Negishi, 2000). Under physiological conditions, CAR is sequestered in the cytoplasm of hepatocytes and translocates to the nucleus upon activation, in which it dimerizes with the retinoid x receptor, recruits coactivators such as steroid receptor coactivator 1 (SRC-1), and induces the transcription of its target genes

(Kawamoto et al., 1999b). Activation of CAR leads to the coordinated induction of a cellular defensive network in the liver, which enables hepatocytes to metabolize and excrete foreign compounds. However, induction of important DME through CAR activation can also significantly alter the levels of toxic metabolites and lead to unexpected drug-drug interactions (DDIs) (Kobayashi et al., 2015; Tolson and Wang,

2010). Recently, novel functions of CAR beyond modulation of drug disposition have come to light, including its roles in energy metabolism and cell proliferation (Dong et al.,

2009; Gao et al., 2009; Yamamoto et al., 2004). Thus, understanding the molecular basis of CAR modulators would not only benefit early prediction of DDIs but also offer potential therapeutic choices for metabolic disorders and cancers.

Although CAR is sequestered in the cytoplasm and must translocate to the nucleus to transactivate gene expression in primary hepatocytes and intact livers, it is spontaneously localized in the nucleus and exhibits constitutive activity in immortalized cell lines

(Kawamoto et al., 1999b; Li et al., 2009). This unique feature makes the investigation of chemical-mediated CAR modulation rather challenging. We have previously identified 1-

(2-Chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a peripheral benzodiazepine receptor ligand, as a potent and selective antagonist of human CAR (hCAR) with negligible effects on its rodent counterparts (Li et al., 2008).

At low micromolar concentrations, PK11195 effectively represses hCAR-mediated gene expression in cell lines and has been used as a research tool for studying the molecular mechanisms of hCAR activation (Anderson et al., 2011; Lynch et al., 2015). Notably, although a potent antagonist of hCAR, PK11195 also activates the human pregnane X receptor (PXR) efficiently (Anderson et al., 2011; Li et al., 2008). Given that CAR and

PXR share an overlapping array of target genes, it is challenging to delineate the exact effects of PK11195 on CAR and PXR in cells that express both receptors(Moore et al.,

2000). To this end, the fact that PK11195 induces the expression of CYP2B6, the primary target of hCAR, and CYP3A4, the primary target of hPXR, in human primary hepatocytes (HPH) has been attributed to its activation of hPXR. Nevertheless, unlike the prototypical hPXR activator rifampicin, which preferentially induces the expression of

CYP3A4 over CYP2B6, PK11195 favorably enhances the expression of CYP2B6 over

CYP3A4, challenging this assumption.

It is well established that CAR plays a key role in determining xenobiotic metabolism and disposition through transactivation of numerous genes in the liver. However, whether and how hepatic metabolism can influence CAR action are not adequately understood. A previous study showed that although omeprazole represents a prototypical

47 activator of the aryl hydrocarbon receptor (AhR), its degradation metabolite omeprazole- sulfide is a pure antagonist of AhR (Gerbal-Chaloin et al., 2006). In screening of antimalarials and their major metabolites for CAR activation, Burk et al., found that artemisinin derivatives and metabolites differentially affect activities of CAR (Burk et al.,

2012). Clearly, the metabolic capacity of a cellular system can be a key determinant for the biological function of a given compound including its role in nuclear receptor activation. Thus, we hypothesize that PK11195 induction of CYP2B6 and CYP3A4 in

HPH is not fully mediated by the activation of PXR, instead, PK11195 is converted from an antagonist to an agonist of hCAR in the metabolically-competent HPH.

In the current study, we demonstrate that PK11195 is N-demethylated to a metabolite that robustly activates CAR in HPH. Using a PXR-knockout (PXR-KO) HepaRG cell line, we confirm PK11195 can induce the expression of CYP2B6/CYP3A4 independent of PXR. A co-culture system that adds metabolism to CAR luciferase reporter assays was used to determine how metabolism influences the agonist/antagonist feature of PK11195.

Mammalian two-hybrid assays and molecular modeling were used to elucidate the molecular basis and structural features of PK11195 and its metabolites in the modulation of CAR activity.

2.2 Materials and Methods

2.2.1 Chemicals and Biological Reagents

Phenobarbital (PB), rifampicin (RIF), ketoconazole, and PK11195 were obtained from

Sigma-Aldrich (St. Louis, MO). 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5- carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) was obtained from BIOMOL

Research Laboratories (Plymouth Meeting, PA). (R)-N-Desmethyl PK11195 (ND-PK) was obtained from ABX Advanced Biochemical Compounds (Radeberg, Germany). 1-(2-

Chlorophenyl)isoquinoline-3-carboxylic acid (COOH-PK) was obtained from Biogene

Organics (The Woodlands, TX). PCR primers were synthesized by Integrated DNA

Technologies (Coralville, IA). HepaRG WT and PXR-KO cells (Catalog #

MTOX1011P24) and the cell culture medium were obtained from Sigma-Aldrich.

Optima LC/MS grade water (H2O), acetonitrile (ACN), and formic acid were purchased from Fisher Scientific (Pittsburg, PA). All chemicals and reagents were used without further purification.

2.2.2 Culture and treatment of HPH and HepaRG cells

HPH were isolated using a modified two-step perfusion protocol (LeCluyse et al., 2005) from human liver specimens obtained from University of Maryland Medical Center with prior approval by the Institutional Review Board at the University of Maryland at

Baltimore or obtained from Bioreclamation In Vitro Technologies (Baltimore, MD). The age and sex of each liver donor used are detailed in Table 2.1. Hepatocytes with viability over 90% were seeded at 0.75 × 106 cells/well in 12-well collagen-coated plates as described previously (Faucette et al., 2006b). After attachment at 37 °C in a humidified atmosphere of 5% CO2, hepatocytes were cultured in serum-free William’s E Medium supplemented with insulin, transferrin, and selenium, 0.1 μM dexamethasone, 100 U/ml penicillin, and 100 μg/ml streptomycin, and overlaid with Matrigel (0.25 mg/ml). Thirty- six hours after seeding, HPH were treated with vehicle control (0.1% DMSO), CITCO (1

μM), RIF (10 μM), PB (1 mM), PK11195 (10 μM), ND-PK (10 μM), or COOH-PK (10

μM) for 24 or 72 h before harvesting cells to collect RNA or protein, respectively. In

49 separate experiments, wild-type and PXR-KO HepaRG cells were plated in 12-well plates (1 × 105 cells/well) and cultured for 21 days following Sigma-Aldrich’s instructions to induce differentiation before treatment with compounds as described above.

Liver Donor # Sex Age 107 Male 47 116* Female 69 117* Female 61 118* Male 45 119* Male 36 120 Male 45 121 Male 52 122 Male 10 123 Female 30 Table 2.1 - Age and sex of liver donors. *Denotes livers used for co-culture experiments in Figure 2.3.

2.2.3 Real-time RT-PCR

Total RNA was isolated from cells using TRIzol reagent (ThermoFisher, Rockford, IL) and reverse transcribed using a High Capacity cDNA Archive kit (Applied Biosystems,

Foster, CA) according to the manufacturer’s instructions. Real-time PCR assay was performed using SYBR Green PCR Mastermix (Qiagen) on an ABI StepOnePlus Real-

Time PCR system (Applied Biosystems). Primer sequences for CYP2B6, CYP3A4, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are: CYP2B6, 5’-

AGACGCCTTCAATCCTGACC-3’ and 5’-CCTTCACCAAGACAAATCCGC-3’;

CYP3A4, 5’-GTGGGGCTTTTATGATGGTCA-3’ and 5’-

GCCTCAGATTTCTCACCAACACA-3’; GAPDH, 5’-

CCCATCACCATCTTCCAGGAG-3’ and 5’-GTTGTCATGGATGACCTTGGC-3’.

Expression values were quantified using the equation: fold over control = 2ΔΔCt method, where ΔCt represents the differences in cycle threshold numbers between the target gene and GAPDH, and ΔΔCt represents the relative change in these differences between control and treatment groups.

2.2.4 Western Blot Analysis

Protein samples extracted from treated cells were electrophoretically separated on SDS-

PAGE gels (4-12%), and transferred to polyvinylidine fluoride membranes.

Subsequently, membranes were incubated with primary antibodies against CYP2B6

(1:200, Santa Cruz), CYP3A4 (1:5000, Sigma-Aldrich), or β-actin (1:50,000, Sigma-

Aldrich) at 4 °C overnight. Blots were developed with West Pico chemiluminescent substrate (ThermoFisher) after incubation with horse-radish peroxidase secondary antibodies.

2.2.5 HepG2 cell Transfection and HepG2/HPH co-culture

HepG2 or HepG2-CAR-CYP2B6 stable cells as described previously (Lynch et al., 2015) were cultured in 24-well plates (1 × 105 cells/well) at 37 °C, 5% CO2 for 24 h. HepG2 cells were co-transfected with CYP2B6-2.2k reporter (60 ng/well), hCAR1+A expression vector (30 ng/well), and pRL-TK (10 ng/well) by using X-tremeGENE 9 DNA

Transfection Reagent (Sigma-Aldrich) following the manufacturer’s instruction. Twenty- four hours after transfection, cells were treated with solvent (0.1% DMSO) or test compounds at indicated concentrations. Subsequently, cell lysates were assayed for firefly activities normalized against the activities of renilla using a Luciferase Kit

(Promega, WI). In separate experiments, HPH were plated on collagen-coated cover slips

51 with the corners bent upwards in a 24-well plate. After preincubation of vehicle control and test compounds with HPH for 4 h, both the media and HPH-containing cover slips were transferred to new 24-well plates containing transfected HepG2 cells or the HepG2-

CAR-2B6 stable line and incubated for 24 h before measurement of luciferase activities.

Data from a representative liver donor is shown and represent the mean ± S.D. of three individual transfections.

2.2.6 LC-MS Measurement of PK11195 Metabolism

Complete William’s E Medium containing 10 µM PK11195 was added to HPH cultured in 24-well plates 36 h after seeding. Media (450 µL) was collected from each well at 0 h and 1 h and frozen immediately at -80°C. Samples were thawed on ice before adding 50

µL of medium to 450 µL of ice-cold methanol/H2O (8:1, v/v). After centrifugation at

16,000g for 30 min, 200 µL of supernatant was transferred to a separate tube and dried down before resuspension in 200 µL H2O/ACN (1:1, v/v) with 0.1% formic acid.

Authentic standards were prepared to 1 µM in H2O/ACN (1:1, v/v) with 0.1% formic acid. LC-MS/MS analysis was performed on a TSQ Quantum Ultra Triple Stage

Quadrupole Mass Spectrometer coupled to an Ultimate 3000 RS Liquid Chromatogram system (Thermo Scientific). The LC separation was performed on a Waters BEH C18 column (2.1 x 50 mm, 1.7 µm) operated at 30°C. Solvent A and B consisted of 0.1 % formic acid in H2O and 0.1 % formic acid in ACN, respectively. The gradient program was 0.0-0.5 min, 50% B; 0.5-2.0 min, gradient to 95% B; 2.0-3.5 min, 95% B; 3.5-4.0 min, gradient to 50% B; 4.0-5.0 min, 50% B. The flow rate was to 0.5 mL/min and injection volume was 5 µL. Tandem Mass Spectrometry was performed in the positive- ion mode and the electrospray ionization (ESI) source parameters were as follows: spray

52 voltage, 3000; capillary temperature, 325; sheath gas pressure, 40; auxiliary gas pressure,

15; capillary offset, 35; tube lens offset, 80. Selected reaction monitoring (SRM) was used for mass detection with the following transitions: PK11195 (m/z 353.1 → 238.0),

ND-PK (m/z 339.1 → 238.0), and COOH-PK (m/z 284.0 → 238.0). Data collection and analysis was performed using Xcalibur V 2.1 (Thermo Scientific).

2.2.7 Mammalian two-hybrid assay

COS1 cells seeded in 24-well plates were transfected with 110 ng of the reporter gene plasmid pG5luc, 80 ng of expression plasmids encoding the respective VP16-AD/hCAR fusions, 40 ng of expression plasmids encoding GAL4-DBD/coregulatory fusions, and 20 ng of reference plasmid pRL-TK each well using X-tremeGENE 9 DNA Transfection

Reagent. Twenty-four hours after transfection, the cells were treated with solvent (0.1%

DMSO), CITCO (1 µM), PK11195 (10 µM), or ND-PK (10 µM) for 24 h. Luciferase activities were measured in cell lysates using the Dual-Luciferase kit. Data represent the mean ± S.D. of three individual transfections.

2.2.8 Nuclear Translocation of CAR in HPH

Human primary hepatocytes were plated in collagen-coated 24-well plates and infected with adenovirus-expressing enhanced yellow fluorescent protein-tagged hCAR

(Ad/EYFP-hCAR) as described previously (Li et al., 2009). Twenty-four hours after infection, HPH were treated with vehicle control (0.1% DMSO), PB (1 mM), PK11195

(10 µM), ND-PK (10 µM), or COOH-PK (10 µM) for another 8 h. After treatment, cells were fixed with 4% paraformaldehyde, stained with 1 µg/mL 4',6-diamidino-2- phenylindole (DAPI) (Sigma-Aldrich) for 30 min, and EYFP-hCAR localization in hepatocytes was visualized on a Nikon Eclipse TI fluorescent microscope. Quantitative

53 distribution of EYFP-hCAR was analyzed using General Analysis in the Nikon Elements

AR High Content Analysis software package (Version 4.50.00). Nuclear localization was defined and quantified as the percentage of total EYFP that overlaps with DAPI. Data from a representative liver donor is shown and represent the mean ± S.D. of five individual images for each treatment.

2.2.9 Molecular Modeling

The hCAR-ligand binding domain (LBD) protein crystal structure (PDB ID: 1XVP) was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org). The PK11195 and

ND-PK molecular structures were generated and obtained from ChemAxon Chemicalize

(http://chemicalize.com) and the CITCO and CINPA1 structures obtained from NCBI

PubChem (http://pubchem.ncbi.nlm.nih.gov/). Biovia Discovery Studio (v4.5.0.15071,

Biovia) was used to remove water and ligands from the crystallographic data and isolate the D chain protein which contains the crystal structure of CAR, which was subsequently protonated at pH 7.0. A binding site was defined based on the CITCO binding cavity and defined as an 11.5 Å radius sphere at 24.972 (x), 54.702 (y), and 29.512 (z). The receptor-ligand docking was performed in Discovery Studio using the CDOCKER protocol (Wu et al., 2003). Briefly, the docking parameter were set to generate 255 conformations for each ligand and return the top 10 docks with the lowest CDOCKER energy which is the sum of the receptor-ligand interaction energy and internal ligand strain. The ligand-receptor interactions of the docked molecules were analyzed and visualized in Discovery Studio.

2.2.10 Statistical Analysis

All data are expressed as the mean ± S.D. Statistical comparisons were made using a one-way ANOVA with a Dunnett’s post-test or a two-way ANOVA with a Bonferroni post-test as needed. Statistical significance was set at *: p < 0.05, **: p < 0.01, ***: p <

0.001.

2.3 Results

2.3.1 PK11195 induces the expression of CYP2B6 and CYP3A4 in HPH

We first examined the effects of PK11195, a known hCAR antagonist, on the expression of CYP2B6 and CYP3A4, two prototypical targets for human CAR and PXR, in HPH prepared from liver donors #107 and #122. As shown in Figure 2.1, PB, CITCO,

RIF, and PK11195 at selected concentrations robustly induced CYP2B6 and CYP3A4 at mRNA and protein levels in both liver donors. As expected, CITCO (a selective activator of hCAR) and RIF (a selective activator of hPXR) preferentially induced the expression of CYP2B6 and CYP3A4, respectively. Intriguingly, PK11195 at 10 µM, a concentration at which it represses the activity of hCAR by more than 85% in HepG2 cells (Li et al.,

2008), markedly induced the expression of both CYP2B6 and CYP3A4 without a clear preference mimicking PB, a dual activator of hCAR and hPXR. These findings call into question whether PK11195 is as an antagonist of hCAR in physiologically-relevant cells, and challenge the assumption that PK11195 induction of CYPs in HPH relies on PXR activation.

Figure 2.1 - PK11195 induces CYP2B6 and CYP3A4 expression in HPH. Primary hepatocytes from liver donors #107 (A, B) and #122 (C, D) were treated with 1mM PB, 1µM CITCO, 10µM RIF, or 10µM PK11195 for 24 or 72 hr to analyze mRNA or protein expression, respectively. Results are expressed as fold over control, mean ± S.D. (n = 3). ***, p < 0.001.

2.3.2 Induction of CYP2B6 and CYP3A4 by PK11195 in PXR-KO HepaRG cells

HepaRG cells have been validated as a promising surrogate for HPH and importantly, fully differentiated HepaRG cells exhibit proper CAR cellular localization and maintain physiologically-relevant metabolic capacity, which are not present in most immortalized cell models (Jackson et al., 2016). The PXR-KO HepaRG cell line obtained from Sigma-Aldrich is a newly generated cell line that doesn’t express functional PXR

(Williamson et al., 2016). As expected, PK11195 and other known CAR/PXR modulators induced the expression of CYP2B6 and CYP3A4 mRNA and protein in wild-type

HepaRG cells in a trend that mirrors what was observed in HPH (Figure 2.2 A-B).

Notably in PXR-KO HepaRG cells, although induction of CYP2B6 and CYP3A4 by RIF was fully abrogated, PK11195 significantly induced both CYP2B6 and 3A4 expression at mRNA and protein levels (Figure 2.2 C-D). These data suggest that differential metabolism of PK11195 in the physiologically-relevant HPH/HepaRG cells vs. the

56 immortalized HepG2 cells may contribute to the observed PXR-independent induction of

CYP2B6 and CYP3A4.

Figure 2.2 - PK11195 induces CYP2B6 and CYP3A4 expression in HepaRG cells independent of PXR. Wild-type and PXR-KO HepaRG cells were cultured for 21 days in accordance with Sigma-Aldrich instructions to induce differentiation. Differentiated HepaRG cells were treated with 1mM PB, 1µM CITCO, 10µM RIF, or 10µM PK11195 for 24 or 72 hr to analyze mRNA (A, C) or protein expression (B, D), respectively. Data represent the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

2.3.3 PK11195 is metabolized to a hCAR activator in HPH

Lack of metabolism is a major limitation of almost all studies using immortalized cell lines, including HepG2 cells. A HPH-HepG2 co-culture model was established as depicted in Figure 2.3A, which introduces the metabolism-competent HPH into the culture environment shared with HepG2 cells. In agreement with previous reports,

PK11195 concentration-dependently inhibits the constitutive hCAR activity in HepG2 cells without the presence of HPH (Figure 2.3B, open bars). Results from the co-culture with HPH from a representative liver donor indicate that PK11195 significantly increased the luciferase activity of the CYP2B6 reporter in the presence of hCAR (Figure 2.3B, black bars) or its low-basal alternative hCAR1+A (Figure 2.3C). These findings suggest

57 that PK11195 is converted from a CAR antagonist to an agonist in the presence of HPH.

To further explore the contribution of metabolism in this antagonism/agonism conversion, ketoconazole (KET), a potent inhibitor of CYP3A4, the most abundant hepatic CYP isoform in humans, was co-treated with different concentrations of

PK11195 in HepG2-CAR-2B6 cells with and without HPH co-culture. Repression of

CAR activity by PK11195 was not influenced by the presence of KET in HepG2-CAR-

2B6 cells alone (Figure 2.3D). On the other hand, when HPH were co-cultured with

HepG2-CAR-2B6 cells, KET significantly inhibited the conversion of PK11195 (1 and

10 µM) from a CAR antagonist to a CAR activator (Figure 2.3E). Taken together, these results validate the use of the co-culture system to investigate the mechanistic effects of metabolism on CAR activity, and suggest that CYP3A4 contributes to the biotransformation of PK11195 in HPH.

Figure 2.3 - PK11195 is metabolized to a CAR activator in HepG2-HPH co-culture. HPH were plated on plastic cover slips with the corners bent upwards and incubated with test compounds for 4 hr before the media and the cover slips were transferred to a 24-well plate containing HepG2 cells and incubated for 24 hr (A). HepG2-CAR-2B6 stable line (B) or transiently transfected with CAR1+A and CYP2B6-2.2K (C) were treated with PK11195 and CITCO at indicated concentrations with or without co-culture with HPH for 24 hr. HepG2-CAR- 2B6 cells alone (D) or co-cultured with HPH (E) were treated with PK11195 alone or co-treated with 8µM KET for 24 hr. Luciferase activity was measured as described in Materials and Methods. Data represent the mean ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001.

2.3.4 PK11195 is metabolized to ND-PK and COOH-PK in HPH

Previous studies postulated that PK11195 is metabolized into two major metabolites, ND-PK and COOH-PK, which are formed through N-demethylation and amide hydrolysis, respectively (Figure 2.4A) (Roivainen et al., 2008). To confirm that these metabolites were generated in our experimental system, PK11195 (10 µM) was

59 incubated with HPH and media was collected at 0 and 1 h. These samples were prepared and analyzed by LC-MS/MS as detailed in Materials and Methods to determine whether the proposed ND- and COOH-PK metabolites of PK11195 were formed in HPH. The 1

µM standard mix exhibited chromatographic separation and reproducible retention times of 1.81, 0.76, and 2.14 for PK11195, COOH-PK, and ND-PK, respectively (Figure 2.4B).

As expected PK11195 was abundantly present in HPH culture medium at 0 h while the amounts of COOH-PK and ND-PK were negligible (Figure 2.4C). However, the peaks for COOH-PK and ND-PK significantly increased in intensity after 1 h in HPH culture, indicating that PK11195 is metabolized by HPH to COOH-PK and ND-PK (Figure

2.4D). To our knowledge, this is the first report confirming that the COOH-PK and ND-

PK metabolites of PK11195 are generated by HPH, and these metabolites require further study to determine whether they mediate CAR activation.

Figure 2.4 - PK11195 is metabolized to COOH-PK and ND-PK in HPH. Proposed PK11195 metabolism pathway (A). An authentic 1 µM standard mix was run on LC-MS/MS as described in Materials and Methods and retention times for PK11195, COOH-PK, and ND-PK were determined to be 1.81, 0.76, and 2.14, respectively (B). Media containing 10 µM PK11195 was cultured with HPH and harvested at 0 h (C) and 1 h (D) to qualitatively determine whether PK11195 is metabolized by HPH to the metabolites of interest.

2.3.5 The (R)-N-desmethyl metabolite of PK11195 mediates hCAR activation

After confirming that COOH-PK and ND-PK were generated in our experimental system, we tested these metabolites for hCAR activation in HepG2-based luciferase

61 assays. As shown in Figure 2.5A, ND-PK activated hCAR in a concentration-dependent manner while COOH-PK had no effect on CAR activity. ND-PK activation of hCAR was further confirmed in the CAR1+A/CYP2B6-2.2k reporter assay, where ND-PK robustly activates hCAR to a level similar to the positive control (CITCO), while COOH-PK exhibits negligible CAR activation (Figure 2.5B). In separate experiments, the mammalian two-hybrid assay revealed that PK11195 could significantly repress the interaction between hCAR and SRC-1 or glucocorticoid receptor interacting protein 1

(GRIP1), while ND-PK moderately enhanced hCAR recruitment of these coactivators in a manner similar to that by CITCO (Figure 2.5 C-D). Notably, ND-PK markedly rescued

PK11195-mediated repression of SRC-1/CAR interaction, while only minimally affecting

PK11195-repressed binding of GRIP1 to hCAR. Furthermore, ND-PK significantly induced CYP2B6 and CYP3A4 mRNA and protein expression in the PXR-KO HepaRG cells, indicating that ND-PK can induce CYP expression independent of PXR (Figure 2.5

E-F). Overall, these results identify ND-PK as the metabolite of PK11195 that is responsible for PK11195-mediated CAR activation in metabolically-competent hepatic cells.

Figure 2.5 - The (R)-N-desmethyl (ND) metabolite of PK11195 mediates CAR activation. HepG2-CAR-2B6 cells (A) and HepG2 cells transfected with CAR1+A and CYP2B6-2.2k (B) were treated with vehicle control (CT), ND-PK, COOH PK, or CITCO at indicated concentrations for 24 hr before luciferase activities were determined. Mammalian two-hybrid assays were performed in COS1 cells measuring interaction between CAR/SRC-1 (C) or CAR/GRIP1 (D) as detailed in Materials and Methods. Transfected cells were treated with CITCO, PK11195, ND-PK, or ND-PK+PK1195 for 24 hr before determining luciferase activity. PXR-KO HepaRG cells were treated with vehicle control, PB (1 mM), CITCO (1 µM), RIF (10 µM), ND-PK (10 µM), or COOH-PK (10 µM) and harvested for mRNA (E) or protein (F) expression analysis. Data represent the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

2.3.6 ND-PK induces CYP2B6, CYP3A4 expression and nuclear translocation of

hCAR in HPH

To fully characterize the effect of ND-PK on hCAR activation and target gene induction, HPH from two liver donors (#120 and #121) were treated with multiple concentrations of PK11195, ND-PK and COOH-PK. Our results indicate that ND-PK concentration-dependently and potently induced CYP2B6 and CYP3A4 at both mRNA and protein levels, while COOH-PK only exhibited negligible effects on the expression of these genes (Figure 2.6 A-D). Nuclear translocation of CAR in HPH has been regarded as the essential step in its activation. To test the nuclear translocation of CAR by PK metabolites, HPH were infected with Ad/EYFP-hCAR overnight before treatment with compounds for 8 h. Without activation, EYFP-hCAR was predominantly expressed in the cytoplasm of HPH and the prototypical CAR activator PB, as well as PK11195, induced significant hCAR nuclear accumulation as expected (Figure 2.7 A-B). Importantly, ND-

PK at 10 µM robustly increased nuclear translocation of hCAR, while COOH-PK (10

µM) caused negligible CAR nuclear accumulation, which correlate well with their capacity for CYP induction. These data further support that ND-PK is the metabolite of

PK11195 that activates hCAR in physiologically-relevant systems.

Figure 2.6 - ND-PK induces CAR target genes in HPH. Primary hepatocytes from liver donors #120 (A, B) or #121 (C, D) were treated with PB (1 M), CITCO (1 µM), RIF (10 µM), or increasing concentrations (0.1, 1, 10µM) of PK11195, ND-PK or COOH-PK for 24 or 72 hrs to analyze mRNA (A, C) or protein (B, D) expression, respectively. Data represent the mean ± S.D. (n = 3)

Figure 2.7 - ND-PK induces CAR nuclear translocation in HPH. Primary hepatocytes from liver donor #123 were infected with Ad/EYFP-hCAR for 24hr and treated with 1 mM PB, 10 µM PK11195, 10 µM ND-PK, or 10 µM COOH-PK for 8 hr before being fixed with 4% paraformaldehyde, stained with 1 µg/mL DAPI, and visualized on a Nikon Eclipse TI fluorescent microscope. Quantitation of nuclear localization was determined using General Analysis in the Nikon Elements AR High Content Analysis software package and defined as the percentage of EYFP that overlaps with DAPI. Representative images (A) and quantitative analysis (B) of nuclear localization are shown. Data represent the mean ± S.D. of five individual images.

2.3.7 Computational modeling of PK11195 and ND-PK

The metabolism of a potent CAR antagonist (PK11195) and its conversion into a potent CAR activator (ND-PK) with a difference of only one methyl group provided a

66 unique opportunity to probe the structure-activity relationship for CAR through molecular modeling. Docking studies utilized the 1XVP crystal structure of CITCO bound to the CAR LBD to identify PK and ND-PK interactions with different residues in the binding pocket (Xu et al., 2004). Upon validation of our model by docking CITCO into the LBD, both PK and ND-PK were docked and found to interact with many of the same residues in the binding pocket, suggesting that they bind in similar conformations

(Figure 2.8A). However, PK11195 interacts with residues important to CAR activity such as V199, Y224, and Y326, including the N-methyl group interacting with H203, whereas the demethylated form of PK (ND-PK) does not (Figure 2.8 B-C). Notably, PK11195 and

CAR antagonist CINPA1 (Figure 2.8D) share these interactions, which may contribute to their antagonism of hCAR (Cherian et al., 2015a). The demethylation of PK11195 allows it move away and not interact with these important residues in the CAR binding pocket, which may explain how the difference of one methyl group between PK11195 and ND-

PK can have such a substantial effect on CAR activity. These docking studies determined the structure-activity relationship of PK11195 and ND-PK with CAR and demonstrated that even small alterations in ligand structure, like the demethylation of PK11195, have the potential to alter its interactions with amino acids in the CAR binding pocket and lead to large differences in CAR activity.

Figure 2.8 - N-demethylation of PK11195 leads to reduced interaction with residues important to CAR activity. The hCAR-LBD crystal structure (1XVP) was prepared in Biovia Discovery Studio and docking was carried out using the CDOCKER protocol as detailed in Materials and Methods. After model validation, PK11195 (yellow) and ND-PK (light blue) were docked into the CAR ligand-binding pocket (A). Interactions of PK11195 (B), ND-PK (C), CINPA1 (D), and CITCO (E) with residues in the CAR binding pocket from docking studies are shown above. Residues are color-coded as follows: dark blue - interact with all structures; light blue – PK11195 and ND-PK; orange – PK11195, CINPA1, and CITCO; red –PK11195 and CINPA1; green – CINPA1 and CITCO; yellow – CITCO only; purple – ND-PK only.

2.4 Conclusion and Discussion

The biological function of CAR is regulated by the interplay between specific cellular factors and small molecular modulators. PK11195, a well-known peripheral benzodiazepine receptor ligand, has been used as a potent hCAR deactivator in cell-based luciferase reporter assays. In contrast to CAR antagonism exhibited in immortalized cell lines, PK11195 robustly induces the expression of both CYP2B6 and CYP3A4 in HPH that are prototypical targets for hCAR and hPXR, respectively. The mechanistic basis for this observed discrepancy is largely unknown, although it was presumed that PK11195- mediated CYP induction in HPH was attributed to its activation of hPXR. Here, we show that PK11195 significantly induces the expression of CYP2B6 and CYP3A4 in PXR-KO

HepaRG cells, which demonstrates that PK11195 can stimulate CYP expression independent of PXR. Utilizing a HPH-HepG2 co-culture model, we show that introduction of metabolically-competent HPH is sufficient to convert PK11195 from an antagonist to an agonist of hCAR in HepG2 cells. In HPH, PK11195 is biotransformed to

ND-PK and COOH-PK. Further studies demonstrated that ND-PK is the active metabolite that potently activates hCAR and induces the expression of CYP2B6 and

CYP3A4. Moreover, structural-activity analysis reveals that N-demethylation of

PK11195 allows its side chain to rotate away from residues in the CAR ligand-binding pocket towards a conformation in favor of CAR activation.

CAR and PXR regulate an overlapping array of target genes and share many common chemical modulators (Hernandez et al., 2009; Mackowiak and Wang, 2016).

Although such cross-talk between CAR and PXR can be beneficial by forming a

69 defensive network against xenobiotics, it makes the delineation of specific function of each individual receptor extremely challenging, particularly in cells such as HPH where both CAR and PXR are abundant and functionally intact. Recently, the HepaRG cell line has emerged as a useful alternative for HPH. Differentiated HepaRG cells exhibit prototypical HPH morphology, inductive expression of major DMEs and transporters, and have been used for in vitro drug metabolism and toxicology studies (Andersson et al.,

2012; Josse et al., 2008). The PXR-KO HepaRG cell line provides an excellent model to determine the contribution of CAR/PXR to PK11195-mediated CYP induction. Our results uncover an unexpected induction of both CYP2B6 and CYP3A4 by PK11195 in the PXR-KO HepaRG cells, although induction by selective hPXR activator RIF was fully abrogated. These findings provide conclusive evidence that PK11195 can induce

CYP2B6/CYP3A4 expression in physiologically-relevant hepatic cells independent of hPXR. Indeed, previous and current studies in HPH have shown that PK11195 induces both CYP2B6 and CYP3A4 in a pattern that mimics that of PB, a dual activator of hCAR and hPXR (Anderson et al., 2011; Li et al., 2008). Together, these results indicate that

PK11195 modulates CAR differently in HPH and HepaRG cells versus in immortalized cell lines, and is most likely metabolized from an antagonist to an agonist in cells exhibiting physiologically-relevant metabolism.

Lack of metabolism capacity is a significant drawback associated with the use of immortalized cell lines in toxicity assessment and drug development. Cell-based luciferase reporter assays using immortalized cell lines in particular have been extensively used to investigate nuclear receptor activity and predict target gene

70 expression. However, proper interpretation of such data has become a heightened concern in both academia and pharmaceutical industry. Several lines of evidence indicate that introduction of metabolic capacity to cell cultures appears to be an attractive solution to overcome this issue. In this regard, we have previously used an HPH- leukemia/lymphoma cell co-culture model to show that presence of HPH markedly increases the biotransformation of cyclophosphamide, a chemotherapeutic prodrug, to its pharmacologically active metabolite and leads to enhanced anticancer activity in co- cultured HL-60 and SU-DHL-4 cells (Hedrich et al., 2016; Wang et al., 2013). Using a

HPH-HepG2 co-culture system in the current study, we observed that PK11195 concentration-dependently increased CAR activation in contrast to decreasing CAR activity when exposed to HepG2 cells only. More importantly, such agonistic effects of

PK11195 in the co-culture can be reversed by ketoconazole, suggesting CYP3A4 plays a key role in the metabolism-based conversion of PK11195 in HPH. It is not uncommon that metabolism can influence the pharmacological action of drugs. For instance, chrysin, a dietary flavonoid, markedly induces the expression and activity of UDP- glucuronosyltransferase 1A1 in HepG2 and Caco-2 cells, but not in HPH (Smith et al.,

2005). Buprenorphine, a potent activator of PXR in HepG2 cells, is not a physiologically relevant activator of PXR or an inducer of associated CYPs in HPH (Li et al., 2010). On the other hand, phenytoin, an antiepileptic agent, is a potent inducer of CYP2B6 and

CYP3A4 in HPH but does not activate CAR or PXR in cell-based reporter assays (Wang et al., 2004). Collectively, these studies demonstrate that the metabolic capacity of a cellular system can be a key determinant for the biological function of a given compound, including its role in nuclear receptor activation.

Previous reports have postulated that PK11195 is rapidly biotransformed into two major metabolites: the N-desmethyl metabolite, ND-PK, and the amide hydrolysis product, COOH-PK (Roivainen et al., 2008). In the current study, we have evaluated the activation of hCAR and induction of related CYPs by both metabolites. Notably, ND-PK significantly increased the luciferase activity of the CYP2B6 reporter by activating hCAR or hCAR1+A in HepG2 cells and induced the expression of CYP2B6 and CYP3A4 in

HPH, HepaRG, and PXR-KO HepaRG cells. In contrast, COOH-PK failed to activate hCAR in HepG2 cells and only marginally induced CYP2B6/CYP3A4 expression in

HPH. Consistent with these observations, fluorescent microscopy analysis of Ad/EYFP- hCAR infected HPH further confirmed that ND-PK but not COOH-PK efficiently translocates CAR from the cytoplasm to the nucleus of HPH, the first step in CAR activation. This discovery may also provide a mechanism for the previously observed

PK11195-mediated CAR nuclear translocation in HPH (Li et al., 2008). Together, these findings suggest that removing one methyl group from PK11195 changes it from an antagonist to an agonist of CAR and contributes to CYP induction in HPH.

Mechanistically, ligand binding changes the secondary structure of a nuclear receptor, influences the recruitment of co-regulators, and alters the target gene expression thereafter. Previous reports indicated that nuclear localized CAR can interact with coactivators such as SRC-1 and GRIP-1 without the presence of agonists (Min et al.,

2002; Muangmoonchai et al., 2001). Our mammalian two-hybrid results demonstrate that

PK11195-repressed CAR/SRC-1 interaction can be efficiently rescued by ND-PK.

However, this recovery is moderate in the CAR/GRIP-1 interaction, reflecting the

72 differential capacity between PK11195 and ND-PK in influencing the recruitment of

SRC-1 vs. GRIP-1 by CAR.

Computational modeling studies have shown that the constitutive activity of CAR is mediated by residues in the binding pocket interacting with and stabilizing the AF2 domain. (Andersin et al., 2003; Windshügel et al., 2005; Xu et al., 2004). Agonists tend to bind and further stabilize the AF2 domain in the active conformation, while antagonists disrupt its stability (Jyrkkärinne et al., 2008). To explore how the N- demethylation of PK11195 has such a drastic effect on CAR activity, we utilized docking studies to probe the structure-activity relationship of PK11195 and ND-PK with CAR.

Although both compounds bound in similar conformations, PK11195 interacted with residues important to CAR activation, such as V199, H203, Y224, and Y326, while the

ND-PK does not. Residues V199 and Y326 are thought to stabilize the CAR AF2 domain in the active conformation by interacting with H12, while Y224 may be involved in local protein folding. Mutating any of these residues abrogates the basal activity of CAR while mutating H203 reduces CAR activity by 50%, demonstrating the importance of these residues to CAR activity (Jyrkkärinne et al., 2005). Therefore, PK11195 interactions with these residues may destabilize the AF2 domain, while loss of the N-methyl group in ND-

PK could re-stabilize H12. Indeed, potent CAR antagonist CINPA1 also interacts with these residues, suggesting such interactions are important in CAR antagonism (Cherian et al., 2015a). CITCO interacts with V199, Y224, and Y326, but the interaction distances are generally greater than those of PK11195 or CINPA1 thus may not displace these residues enough to inhibit stabilization of H12 (Table S1). Although docking studies

73 provide a possible mechanism for this drastic change in CAR activity, further studies will utilize molecular dynamics and mutagenesis studies to further probe the detailed structure-activity relationship between PK11195 and ND-PK with CAR.

In conclusion, this study demonstrates that PK11195 is metabolically converted from an antagonist to an agonist of hCAR and this conversion contributes significantly to the observed induction of CYP2B6 and CYP3A4 in HPH and HepaRG cells. We show that ND-PK is the metabolite responsible for PK11195-mediated CAR activation by facilitating CAR interactions with SRC-1 and GRIP-1 and enhancing CAR nuclear translocation in HPH. The demethylation of PK11195 also disrupts its interaction with residues critical to CAR activity, providing a possible mechanism for the activity shift.

Additionally, this report highlights the importance of metabolic-competence when attempting to identify modulators of nuclear receptors and provides a possible solution to this problem with a novel HPH co-culture system.

Chapter 3: Identification of constitutive androstane receptor modulators with a high-throughput translocation assay

3.1 Introduction

The constitutive androstane receptor (CAR; NR1|3) is a xenobiotic receptor, primarily expressed in the liver, whose canonical role involves the upregulation of detoxification mechanisms such as drug metabolizing enzymes (DMEs) and transporters to protect the body from foreign chemicals. Upon activation, CAR induces prominent DMEs such as

CYP2B6, its main target gene, and CYP3A4; both of these enzymes metabolize important clinically-used drugs and are known to participate in drug-drug interactions

(DDIs) (Timsit and Negishi, 2007; Wang et al., 2012). More recently, the non-canonical roles of xenobiotic receptors, such as the pregnane X receptor (PXR; NR1|2), have been the subjects of intense research, and CAR is no exception. Studies have found that CAR plays a significant role in liver physiology, modulating cellular energy homeostasis and cell proliferation (Banerjee et al., 2015; Gao and Xie, 2012; Mackowiak et al., 2018).

This makes CAR an enticing target for metabolic disorders and has led to a search for novel CAR activators that may have therapeutic potential.

In the liver and primary hepatocytes, CAR is localized in the cytoplasm and upon activation, translocates to the nucleus where it heterodimerizes with the retinoid x receptor (RXR) and enhances the transcription of target genes (Kanno et al., 2005;

Kawamoto et al., 1999b; Li and Wang, 2010). However, CAR is localized in the nucleus and constitutively active in most immortalized cell lines, making mechanistic studies of

CAR difficult (Andersin et al., 2003; Xu et al., 2004). In addition, CAR can be activated

75 either directly through ligand binding or indirectly via signaling cascades, adding further complexity to our understanding of CAR activation (Mackowiak and Wang, 2016).

Despite these challenges, many efforts have been made to identify compounds that modulate CAR activity because of its ability to mediate DDIs and control important physiological processes in the liver.

A number of screening methods have been developed to identify CAR modulators, employing approaches from computational modeling to cell-based luciferase reporter assays with varying success(Imai et al., 2013; Küblbeck et al., 2011; Lynch et al., 2013;

Lynch et al., 2015; Marija et al., 2017). While computational and in situ approaches have had some success finding CAR ligands, cell-based assays are the most widely used to identify chemical modulators of this receptor. Modification of the CAR protein sequence and application of CAR inverse agonists/antagonists have been successfully used to lower the high basal activity and identify activators of CAR in cell-based luciferase reporter assays (Kanno and Inouye, 2010; Küblbeck et al., 2011; Lynch et al., 2014a). To date, the two largest screens for human CAR (hCAR) modulators have utilized HepG2 cells stably expressing full-length hCAR and a luciferase reporter driven by the CYP2B6 promoter (Lynch et al., 2018; Lynch et al., 2015). Out of the eight novel CAR activators chosen for further characterization in the most recent study, only four induced CYP2B6 or 3A4 in HPH (Lynch et al., 2018). Therefore, activation of CAR in the luciferase assay may not be highly predictive of physiologically-relevant CAR activation.

One assay that has shown success in predicting CAR activation is the EYFP-hCAR

76 nuclear translocation assay in HPH. This assay overexpresses EYFP-tagged hCAR in

HPH and monitors whether CAR, which is sequestered in the cytoplasm in the quiescent state, undergoes the first step of activation and translocates to the nucleus (Li et al.,

2009). In fact, this assay was able to accurately classify the 8 validated compounds from the previous Tox21 study (Lynch et al., 2018). In the current study, we established a high-throughput CAR translocation assay and have comprehensively validated the outcomes from screening an FDA-approved drug library containing 978 drugs. Through this approach, we were able to identify mosapride citrate (MOS), a selective 5-

Hydroxytryptamine receptor 4 (5-HT4) agonist used to increase gastric motility, as an activator of hCAR in HPH at physiological concentrations. In addition to its role as a gastroprokinetic agent, MOS has been shown to significantly lower fasting glucose levels in human clinical studies (Ueno et al., 2002; Ueno and Satoh, 2009). Here, we offer evidence that MOS represses gluconeogenic genes and lowers the glucose output of

HPH, providing a novel, liver-specific mechanism through which MOS can lower blood sugar levels in humans.

3.2 Materials and Methods

3.2.1 Chemicals and Biological Reagents

The FDA Approved Drug Screening Library L1300 (96-well-Z151018-200uL) was obtained from Selleck Chemicals (Houston, TX). Phenobarbital (PB), rifampicin (RIF), mosapride citrate (MOS), PK11195, dibutyryl cyclic-AMP (cAMP), dimethyl sulfoxide

(DMSO) and 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4- dichlorobenzyl)oxime (CITCO) were obtained from Millipore Sigma (St. Louis, MO).

CAR inhibitor not PXR activator 1 (CINPA1) was obtained from Tocris Bioscience

(Minneapolis, MN). PCR primers were synthesized by Integrated DNA Technologies

(Coralville, IA). Data from the Tox21 10K HepG2-CYP2B6-hCAR luciferase assay screen in agonist (AID: 1224892) and antagonist (AID: 1224893) modes were obtained from the Tox21 public database (https://tripod.nih.gov/tox21/assays).

3.2.2 Culture of HPH

HPH were obtained from BIOIVT (Baltimore, MD) and seeded at 7.5 × 105 cells/well for

12-well, 3.75 × 105 cells/well for 24-well, or 6.0 × 104 cells/well for 96-well collagen- coated plates as described previously (Faucette et al., 2006a). After cell attachment, HPH were cultured in serum-free William’s E Medium (WEM) supplemented with ITS+

(insulin, transferrin, and selenium, 100x; BD Biosciences, Bedford, MA), 0.1 μM dexamethasone (DEX; Millipore Sigma), 100 U/ml penicillin, and 100 μg/ml streptomycin (Pen-Strep; ThermoFisher Scientific, Waltham, MA), and 2mM L- glutamine (Invitrogen, Carlsbad, CA). Cells used for RNA or protein expression were overlaid with 0.25 mg/mL Matrigel 24 h after seeding (Corning, Corning, NY). Overlaid

HPH were treated with compounds in Complete WEM (CYP2B6 and CYP3A4 studies) or WEM with Pen-Strep and L-glutamine with or without 10 μM dibutyryl cAMP and 50 nM DEX (gluconeogenic gene studies) for 24 h or 48 h to isolate RNA or protein, respectively.

3.2.3 Ad/EYFP-hCAR Translocation Assay

HPH plated in collagen-coated, black-walled, 96-well plates (Corning Cat. #3603) on

Day 0 were infected with Ad/EYFP-hCAR on Day 1 at a concentration of 3 μL/mL of media and incubated for 16 h overnight. On Day 2, the media for each plate was changed and cells were treated with compounds for 8 h. To validate the assay, positive (PB,

CITCO, and PK11195) and negative controls (DMSO and RIF) were added to the plate in triplicate, results were analyzed as detailed below, and the z-factor was calculated according to Zhang et al. (1999) (Zhang et al., 1999). For screening, compounds from the

FDA Approved Drug Screening Library L1300 (1mM in DMSO) were dispensed into each well using the Biomek FXP Laboratory Automation Workstation with Dual Arm

System, Multichannel and Span-8 Pipettors (Beckman Coulter, Indianapolis, IN) for a final DMSO concentration of 1% and a compound concentration of 10 μM. DMSO, PB,

CITCO, and PK11195 were dosed in duplicate in column 12 for each plate. After an 8 h incubation, cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 15 min, stained with 1 µg/mL 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for

30min, and stored at 4°C protected from light until imaging.

3.2.4 High-Content Imaging and Analysis

Imaging of plates was completed on a Nikon Eclipse Ti-E inverted microscope (Nikon,

Edgewood, NY) equipped with a TI-SH-W Well Plate Holder (Nikon),

CFP/YFP/mCherry Filter Cube (Nikon), CFI S Plan Fluor ELWD ADM 20x Objective

(Nikon), SpectraX Multi-Spectral Solid-State Excitation Source (Lumencor, Beaverton,

OR), and an ORCA-Flash4.0 Camera (Hamamatsu, Hamamatsu City, Japan). A custom acquisition method was developed in the Nikon Elements AR High Content Analysis software package (Version 4.50.00) for plate imaging of DAPI (Ex: 390 nm; Em: 461 nm) and YFP (Ex: 513 nm; Em: 575 nm) channels. Images were acquired in a regular pattern at five points in each well using YFP autofocus with the 20x objective.

A high-content analysis method was established using General Analysis in the Nikon

Elements AR High Content Analysis software package. The goal of this analysis method

79 was to calculate the number of cells where hCAR was nuclear localized and normalize to the number of cells expressing EYFP-hCAR to determine percent CAR Translocation.

First, an image mask was created for DAPI to define the “Nuclear” region of interest

(ROI). Preprocessing utilized the Smooth, Rolling Ball Correction, and Local Contrast functions and only objects ≥ 9 µm in size were allowed while binary processing steps included Filter on Fill Area, Morpho Separate Objects, and Erode functions to accurately portray nuclei. Next, an image mask for YFP was created to define the “hCAR” ROI using Smooth, Rolling Ball Correction, Autocontrast, and Local Contrast functions during pre-processing and Filter on Fill Area and Erode functions during binary processing. A “Cells” ROI image mask was created by dilating the “Nuclear” ROI by 2

µm and then an “Expressing Cells” ROI was created by determining the “Cells” that have

“hCAR” to calculate the number of cells expressing hCAR. To calculate the number of cells where hCAR is nuclear localized, a “Combined” ROI was made by including

“Nuclear” objects that are ≥ 50% covered by “hCAR”. Then, a “Nuclear Localized” mask was created by including the “Expressing Cells” having “Combined” and the number of

“Nuclear Localized” objects was divided by the number of “Expressing Cells” objects and multiplied by 100 to obtain percent translocation. All values were then corrected by subtracting the average baseline translocation for each plate (1% DMSO wells) and normalized to the PB translocation to obtain normalized CAR translocation. Each compound was screened in two separate livers and the average normalized CAR translocation values were obtained.

3.2.5 Real-time RT-PCR

After 24h treatment, HPH were washed and TRIzol reagent (ThermoFisher, Rockford,

IL) was added for total RNA isolation using a phenol-chloroform extraction. Extracted

RNA (1 μg) was reverse transcribed to cDNA using the High Capacity cDNA Archive kit according to manufacturer’s instructions (Applied Biosystems, Foster, CA). To determine gene expression changes, real-time PCR was performed on an ABI StepOnePlus Real-

Time PCR system (Applied Biosystems) using Fast SYBR Green Master Mix

(Thermofisher, Rockford, IL). Primer sequences for CYP2B6, CYP3A4, Glucose 6- phosphatase (G6Pase), Phosphoenolpyruvate carboxykinase (PEPCK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are: CYP2B6, 5’-

AGACGCCTTCAATCCTGACC-3’ and 5’-CCTTCACCAAGACAAATCCGC-3’;

CYP3A4, 5’-GTGGGGCTTTTATGATGGTCA-3’ and 5’-

GCCTCAGATTTCTCACCAACACA-3’; G6Pase, 5’- GTGTCCGTGATCGCAGACC-

3’ and 5’- GACGAGGTTGAGCCAGTCTC-3’; PEPCK, 5’-

GCAAGACGGTTATCGTCACCC-3’ and 5’- GGCATTGAACGCTTTCTCAAAAT-3’;

GAPDH, 5’-CCCATCACCATCTTCCAGGAG-3’ and 5’-

GTTGTCATGGATGACCTTGGC-3’. Expression values were quantified by the 2ΔΔCt method, normalizing to GAPDH as the housekeeping gene and determining the fold change compared to control values (n = 3).

3.2.6 Western Blot Analysis

HPH were washed 1x with PBS and harvested using cell lysis buffer (Cell Signaling

Technology, Danvers, MA) with cOmplete protease inhibitor cocktail (Roche,

Mannheim, Germany) before sonication and extraction of soluble protein. Samples were

81 electrophoretically separated on NuPAGE Bis-Tris gels (4-12%) and transferred to polyvinylidine fluoride (PVDF) membranes. Subsequently, membranes were blocked with 5% milk in Tris-buffered saline with Tween 20 (TBST) and then incubated with primary antibodies against CYP2B6 (1:500, Abcam), CYP3A4 (1:5000, Sigma-Aldrich), or β-actin (1:50,000, Sigma-Aldrich) at 4 °C overnight. Membranes were washed with

TBST 3x before incubating with horseradish peroxidase-linked secondary antibodies for

1h at room temperature. Membranes were washed and probed with West Pico chemiluminescent substrate (ThermoFisher) and detected using film.

3.2.7 Glucose Production Assay

Overlaid HPH plated in 24-well plates were switched to William’s E Media with only

100 U/ml penicillin, and 100 μg/ml streptomycin and 2 mM L-glutamine added and treated with compounds overnight in triplicate. The next morning, cells were washed 3x with PBS and starved in glucose-free DMEM supplemented with 10 mM HEPES buffer

(pH 7.4) with treatments and with/without 10 μM dibutyl cAMP and 50 nM dexamethasone for 2h. HPH were washed again 3 times with PBS and the same treatments as above were added to glucose-free DMEM supplemented with 10 mM

HEPES buffer (pH 7.4) and 2 mM sodium pyruvate (Invitrogen). Samples of media (100

μL) were taken at 5 h and 24 h to analyze glucose content. The relative amount of glucose in each sample was measured with the Amplex Red Glucose Kit (Invitrogen) after diluting samples 5-100 times according to the manufacturer’s instructions.

3.2.8 Statistical Analysis

All data are expressed as the mean ± S.D. Statistical comparisons were made using a two-way ANOVA with a Bonferroni post-test. Statistical significance was set at *: p <

0.05, **: p < 0.01, ***: p < 0.001.

3.3 Results

3.3.1 High-Throughput EYFP-hCAR Translocation Assay Development and

Validation

CAR translocation, from the cytoplasm to the nucleus of hepatocytes, has been used in numerous studies over the years as a marker of CAR activation (Li et al., 2009; Lynch et al., 2013; Lynch et al., 2015). Studies have used either nuclear/cytosolic extracts of CAR protein to study its translocation, or assays that employ fluorescently-labeled CAR protein to study its localization (Kawamoto et al., 1999a; Li et al., 2009; Zelko et al.,

2001a). These methods are relatively low-throughput, and until now, have only been used to study CAR localization or characterize small numbers of CAR activators. In this study, we have adapted the Ad/EYFP-hCAR translocation assay in HPH from its traditional 24- well plate format to a 96-well screening platform where cells expressing EYFP-hCAR are treated with drugs from a compound library, undergo automated high-content microscopy, and images are evaluated by an analysis method to determine the extent of

CAR translocation (Fig. 3.1A, B). The first step in adapting this assay to a high- throughput platform was optimization of the experimental time course, HPH plating density, and amount of adenovirus added (Data not shown). From the assay optimization,

8 hour compound treatment, 6,000 cells per well, and 3 μL of high-titer adenovirus per 1 mL of media were selected in the current study. Once the experimental system was in place, the automated microscopy and analysis methods were developed using several sets of HPH. While there are numerous ways to define the nuclear localization of a protein, we opted to define CAR translocation as the percent of EYFP-hCAR expressing cells

83 where more than 50% of the DAPI-stained nucleus overlaps with EYFP-hCAR. This analysis approach gave consistent results over multiple liver donors and was validated in

HPH136 and 139 using known CAR translocators phenobarbital (PB), CITCO, and

PK11195 as positive controls, while DMSO and rifampicin (RIF) were used as negative controls. The analysis method was able to successfully differentiate between the positive and negative controls, shown by Z-factors >0.5 for all positive controls in two different liver donors (Fig. 3.1C).

Figure 3.1 - Adapting the Ad/EYFP-hCAR nuclear translocation assay for high-throughput screening. Inactive CAR is sequestered in the nucleus and translocates to the nucleus upon activation by 1 mM PB in a representative liver donor, and the analysis method accurately identifies EYFP-hCAR and the DAPI-stained nucleus (A). Workflow for the high-throughput Ad/EYFP-hCAR nuclear translocation assay where HPH are plated in 96-well plates and infected with virus before they are treated with compounds, imaged, and analyzed (B). HPH from liver donors #136 and #139 were treated with negative controls (0.1% DMSO and 10 µM RIF) or positive controls (1 mM PB, 1 µM CITCO, or 10 µM PK11195) in triplicate using 96-well plates for 8h, cells were fixed and stained with DAPI, five images were taken in each well. CAR translocation (% of expressing cells with 50% of nucleus overlapping with EYFP-hCAR) was then calculated and Z- factors were obtained for each positive control compared to DMSO (C). Outline for comparison of the CAR nuclear translocation assay with the Tox21 compound library screened with the HepG2-CYP2B6-hCAR luciferase assay (D).

3.3.2 Screening FDA-approved Drugs for CAR Translocation

To identify physiologically-relevant CAR activators, we screened the FDA-Approved

Drug Library containing 978 compounds with each compound at a 10 μM concentration using the validated CAR translocation assay. Any compounds that exhibited CAR translocation over 50% of the positive control (PB) were considered “hits”. In addition, we wanted to compare our translocation results with results from the Tox21 10K HepG2-

CYP2B6-hCAR luciferase assay screen in agonist (AID: 1224892) and antagonist (AID:

1224893) modes to better understand the similarities and differences between the two systems using the 642 overlapping compounds (Fig. 3.1D).

In the combined dataset, there were 31 hits in the translocation assay (>50% PB translocation) compared with 66 agonists and 32 antagonists identified in the CAR luciferase assay (Fig. 3.2A). There were 13 agonists and 4 antagonists in the luciferase assay that also translocated CAR, while 14 translocators were either inconclusive or negative in the CAR luciferase assay (Fig. 3.2A). The efficacy for each agonist in the luciferase assay was plotted against the average translocation for each compound to

86 identify any trends (Fig. 3.2B). While only 13 compounds were both agonists and translocators by our cutoff (50% of PB), a total of 31 agonists in the luciferase assay exhibited over 20% translocation. To determine how the assays performed at predicting physiologically-relevant CAR activators, we completed a literature search focused on the hits in the translocation, agonist, or antagonist assays, for CYP2B6 or CYP3A4 induction

(mRNA, protein, or activity) in HPH or in vivo as an indicator of CAR activation. We also selected 10 potentially novel CAR activators and deactivators, including several with potent EC50s in the luciferase assay, to evaluate in HPH (Table 3.1). Overall, we were able to find CYP2B6 or CYP3A4 induction data for 45 compounds (Tables 3.2-4, Figure

3.2C). The point biserial correlation (rpb) was calculated comparing average translocation and luciferase assay agonist efficacy with CYP2B6 and CYP3A4 induction in HPH.

Through this study, we found that CAR translocation is more tightly correlated with

CYP2B6 and CYP3A4 induction in HPH (rpb = 0.46) than CAR activation of the

CYP2B6 reporter plasmid in the HepG2-based luciferase assay (rpb = -0.16).

Interestingly, some compounds that mediated CAR nuclear translocation potently repressed CYP2B6 expression in HPH, such as rimonabant and sorafenib (Fig. 3.3A-D,

Table 3.1). To determine whether it is possible for CAR antagonists to translocate CAR to the nucleus, we subjected a CAR antagonist, CINPA1, to the CAR translocation assay.

CINPA1 has been shown to bind to CAR and repress its activity, while its metabolites exhibit either weak antagonism or no activity for CAR (Cherian et al., 2015b; Cherian et al., 2016). Here, CINPA1 mediates potent nuclear translocation of CAR in multiple HPH

87 donors, demonstrating that antagonists can also mediate CAR translocation while repressing its activity in the nucleus (Fig. 3.3E).

Figure 3.2 - Screening of FDA-approved drugs with the Ad/EYFP-hCAR translocation assay compared with the HepG2-hCAR-CYP2B6 luciferase assay. Each compound in the FDA-approved drug library was screened in two separate livers (four donors for the study). Overall, there were 86 hits in the translocation assay, compared with 66 agonists and 32 antagonists identified in the CAR luciferase assay. Thirty translocators overlapped

88 with the agonist mode, five overlapped with antagonists, and fifty-one were either inconclusive or negative in the CAR luciferase assay (A). Agonist efficacy (% of CITCO) in the CAR luciferase assay was plotted against average translocation (% of PB) from the CAR translocation assay, displaying agonists only (green, triangle), agonists and translocators (yellow, circle), and translocators only (grey, diamond) (B). Literature data for CYP2B6 or CYP3A4 induction was overlaid onto B, with known inducers in red, known repressors in blue, and non-modulators in black squares (C).

Table 3.1. CYP2B6 induction by selected hits in the luciferase and translocation assays. HPH145 and 147 were treated with compounds selected from the pool of hits in either the translocation assay or the luciferase assay (with an emphasis on low EC50 values or high efficacy) at 10 μM to determine CYP2B6 mRNA expression.

Table 3.2. Summary of CAR Luciferase Assay Agonist Data. The efficacy values, average translocation, and literature data for each agonist identified by the CAR luciferase assay are summarized.

Table 3.3. Summary of CAR Luciferase Assay Antagonist Data. The efficacy values, average translocation, and literature data for each antagonist identified by the CAR luciferase assay are summarized.

Table 3.4. Summary of CAR Translocator Data. The efficacy values from the CAR luciferase assay in both the agonist and antagonist mode, average translocation, and literature data for each for each translocator identified by the CAR translocation assay that were not agonists or antagonists in the luciferase assay are summarized.

Interestingly, some compounds that mediated CAR nuclear translocation potently repressed CYP2B6 expression in HPH such as rimonabant and sorafenib (Figure 3.3 A-

D). While CAR antagonist PK11195 is known to translocate CAR, it is metabolized into an agonist and induces CYP2B6 expression in HPH. To determine whether it is possible for CAR antagonists to translocate CAR to the nucleus, we subjected CAR antagonist

CINPA1 to the CAR translocation assay. CINPA1 has been shown to bind to CAR and repress its activity, and its metabolites exhibit either weak antagonism or no activity for

CAR (Cherian et al., 2015a; Cherian et al., 2016). CINPA1 mediates potent nuclear translocation of CAR in multiple HPH donors, demonstrating that antagonists can also mediate CAR translocation while repressing its activity in the nucleus (Figure 3.3E).

Figure 3.3 - CAR antagonists can mediate CAR nuclear translocation. Antagonist efficacy (% of PK11195) in the CAR luciferase assay was plotted against average translocation (% of PB) in the CAR translocation assay, displaying antagonists only (purple, triangle), antagonists and translocators (pink, circle), and translocators only (grey, diamond) (B). Literature data for CYP2B6 or CYP3A4 induction was overlaid onto A, with known inducers in red, known repressors in blue, and non-modulators in black squares (B). Rimonabant, an antagonist in the CAR luciferase assay that represses CYP2B6 expression translocates CAR to the nucleus (C). Sorafenib also mediates potent CAR translocation while repressing CYP2B6 mRNA expression in HPH (D). Known CAR antagonist CINPA1 also potently translocates CAR to the nucleus of HPH in multiple liver donors (E).

3.3.3 Mosapride Citrate potently induces CYP2B6 and CYP3A4 mRNA and

protein in HPH

One compound we found intriguing in the validation dataset was mosapride citrate, which was an agonist in the CAR luciferase dataset and a hit in the CAR translocation assay (Table 3.2). Recently, several clinical studies have found that mosapride can lower blood glucose levels in subjects with Type 2 diabetes, which correlates with CAR activation having been shown to lower blood glucose levels and improve glucose tolerance in obese mice (Dong et al., 2009; Gao et al., 2009; Ueno et al., 2002; Ueno and

Satoh, 2009). The Cmax of mosaspride in humans ranges from ~25-280 ng/mL (~50-600 nM) after oral doses between 5 and 40 mg. Using concentrations ranging from 100 nM to

10 μM, we investigated whether mosapride could induce CYP2B6 expression at physiologically-relevant concentrations (Sakashita et al., 1993). Indeed, mosapride was able to induce CAR target genes CYP2B6 and CYP3A4 at the RNA and protein levels at concentrations as low as 500 nM in three separate livers, indicating that CAR activation by mosapride is possible at physiologically-relevant concentrations (Fig. 3.4).

Figure 3.4 - Mosapride citrate potently induces CYP2B6 expression over a range of concentrations in HPH. HPH from liver donors #147 (A), #148 (B), and #149 (C) were treated with 0.1% DMSO, 1 mM PB, 1 µM CITCO, 10 µM RIF, or mosapride citrate at 0.1, 0.5, 1, 5, or 10 µM for 24h or 48h and harvested for mRNA or protein expression analysis, respectively.

3.3.4 Mosapride Citrate inhibits the expression of gluconeogenic genes and

reduces glucose output from HPH

While multiple mechanisms for the reduction of blood glucose levels by mosapride have been proposed, we sought to investigate whether mosapride could act on the liver to affect glucose levels. Previous studies have shown that CAR activation, in both mice and

HPH, leads to decreases in the expression of gluconeogenic genes such as glucose-6- phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) (Gao et al.,

2009; Lynch et al., 2014b). Therefore, we investigated the effect of mosapride on these gluconeogenic genes with or without stimulation of gluconeogenesis by cyclic AMP

(cAMP). Mosapride treatment of cAMP-stimulated HPH at 5 and 10 μM led to the repression of G6Pase and PEPCK expression in two separate livers (Fig. 3.5).

Accordingly, mosapride was able to significantly decrease the glucose output of cAMP-

95 stimulated HPH at both 5 and 24 h time points, indicating that mosapride may be able to reduce glucose output from the liver under fasting conditions (Fig. 3.6).

Figure 3.5 - Mosapride citrate represses gluconeogenic gene expression in HPH. HPH from liver donors #150 (A, C) or #151 (B, D) were treated with 0.1% DMSO, 1 µM CITCO, or MOS at 1, 5, or 10 µM in ITS+ and dexamethasone-free media with or without 10 µM dibutyl cAMP and 50 nM dexamethasone to induce gluconeogenic gene expression. The mRNA expression of G6Pase (A, B) and PEPCK (C, D) were measured after 24h treatment.

Figure 3.6 - Mosapride citrate decreases glucose output from HPH. HPH from liver donors #149 (A, C) or #151 (B, D) were treated with 0.1% DMSO, 1 µM CITCO, or MOS at 1, 5, or 10 µM in ITS+ and dexamethasone-free media overnight, incubated in glucose-free media with treatments with or without 10 µM dibutyl cAMP and 50 nM dexamethasone for 2h to deplete intracellular glucose and glycogen, and incubated for 5h (A, B) or 24h (C, D) before taking media samples and measuring glucose content. Data represent the mean ± S.D. (n = 3)

3.4 Conclusion and Discussion

CAR is a xenobiotic receptor, highly expressed in the liver, whose primary function is to upregulate detoxification mechanisms in the liver. However, recent studies have shown that CAR also plays an important role in vital cellular functions such as cell proliferation, energy homeostasis, and lipid metabolism, making it a potential drug target for treating diseases such as diabetes (Dong et al., 2009; Gao et al., 2009; Mackowiak et al., 2018).

Due to its therapeutic importance, accurately identifying physiologically-relevant modulators of CAR is essential. Most current screening assays for xenobiotic receptors employ immortalized cell lines overexpressing the protein of interest coupled to a luciferase reporter system containing a binding element for the receptor. While this is a relatively straightforward assay for other xenobiotic receptors, CAR is constitutively active in immortalized cell lines, making the identification of activators rather difficult

(Baes et al., 1994; Choi et al., 1997; Yoshinari et al., 2001). To address this issue, most studies have either used a CAR mutant with low constitutive activity or antagonists, like

PK11195, to reduce CAR basal activity (Chen et al., 2010; Imai et al., 2013; Lynch et al.,

2015). High-throughput screens using these assay methods have done well identifying

CAR modulators in immortalized cells, which does not always translate to physiologically-relevant systems. There are many possible reasons for this discrepancy, but one of the major differences between immortalized cell lines and physiologically- relevant liver cells, such as HPH, is their capacity for drug metabolism. As most compounds are metabolized in the body, this is a major translational issue with high- throughput screening assays due to the change in chemical structure of a compound altering its modulation of a xenobiotic receptor. In fact, a metabolite of the CAR antagonist PK11195, ND-PK, was identified to be a CAR activator despite only one methyl group being different between the two compounds (Mackowiak et al., 2017).

Therefore, a metabolism-competent screening system in physiologically-relevant cells would be beneficial in identifying CAR modulators that exhibit clinical effects.

One metabolism-competent experiment that has been used to characterize and validate

CAR activators is visualization of CAR nuclear translocation. While CAR is

98 constitutively active and localized to the nucleus in immortalized cell lines, it is sequestered in the cytoplasm of differentiated cells, such as hepatocytes, and translocates to the nucleus upon activation (Li and Wang, 2010). The development of adenovirus- delivered EYFP-tagged hCAR allowed for fluorescent hCAR to be expressed in HPH and easily visualized with microscopy (Li et al., 2009). Over multiple studies, the CAR translocation assay in HPH has been able to accurately predict whether a compound would activate CAR and induce CYP2B6 expression in HPH. Therefore, we sought to expand this assay into a high-throughput format and determine whether FDA-approved drugs would activate CAR and induce CYP2B6 expression in HPH. With the advances in high-throughput microscopy and analysis, we were able to expand this assay to a 96-well plate format, validate an analysis method to characterize CAR translocation, and identify

FDA-approved drugs that modulate CAR. By comparing our CAR translocation results with the data from the HepG2-CYP2B6-hCAR screen of the Tox21 10K compound library by the National Center for Advancing Translational Sciences (NCATS), we were able to better understand the utility of both types of assays (Lynch et al., 2018). While we can be relatively certain that a compound alters CAR activity in some way if it can alter

CAR localization, it is harder to validate CAR activity on a functional level. To validate our dataset, we undertook a literature search for modulation of CAR target genes

CYP2B6 and CYP3A4 in HPH by compounds in our combined dataset. While induction of CYP2B6 and CYP3A4 is not a perfect, specific indicator of CAR activation, as both genes can be influenced by other factors such as PXR activation, CYP2B6 and CYP3A4 are the most widely studied target genes of CAR. This enables the collection of data for a greater number of compounds since performing CAR functional studies for each

99 compound would be costly. Combining our literature search with our own CYP2B6 induction data (Table 3.2) showed that almost all compounds which translocated CAR (% translocation >50% of PB) were able to either induce or repress coCYP2B6 or CYP3A4 expression or function (>1.5 fold) with only one compound identified as a false positive.

The CAR luciferase assay also identified many compounds that exhibit CAR target gene modulation, but additionally identified five compounds as false positives (Fig. 3.2C,

Table 3.2). Correlation of CAR translocation with target gene induction (rpb = 0.46) was superior to that of the luciferase agonist assay with target gene induction (rpb = -0.16).

Interestingly, the CAR translocation assay exhibited good correlation with CAR target gene modulation when there was at least 20% of PB translocation displayed, showing that the predictive ability of this assay may be increased by screening a range of concentrations for each drug. Clearly, combining the data from two separate CAR activity assays can improve their predictive power and increase confidence in the physiological-relevance of each compound tested.

On the other side of our dataset, we found that four out of the 32 compounds that translocated CAR were also antagonists in the CAR luciferase assay. While two of the four overlapping compounds were found to induce CAR target genes in HPH (Table 3.3), rimonabant exhibited repression of CYP2B6 expression in HPH (Table 3.1). In addition, sorafenib, a compound that was inconclusive in the luciferase assay but translocated

CAR, also repressed CYP2B6 expression (Fig. 3.3C, Table 3.1).Many studies over the years have posited that CAR translocation, from the cytoplasm to the nucleus of hepatocytes, is the first, essential step of its activation; given that CAR must be in the nucleus to enhance the transcription of its target genes, this theory is convincing. While

100

CYP2B6 repression may not be the best indicator of reduced CAR activity, several lines of evidence suggest that CAR can be translocated to the nucleus not only by agonists, but direct antagonists as well. Two potent direct antagonists of CAR, PK11195 and CINPA1, both mediate CAR nuclear translocation (Li et al., 2008) (Fig. 3.3E). While PK11195 is known to be metabolized into a CAR activator in HPH, potentially explaining its translocation, CINPA1 and its metabolites do not activate CAR but mediate CAR nuclear translocation nonetheless (Cherian et al., 2015b; Cherian et al., 2016; Mackowiak et al.,

2017). Taken together, this data suggests that CAR translocation is not limited to CAR agonists and may also occur upon stimulation with direct antagonists, which is important to note when drawing conclusions from translocation data.

One CAR activator in our validation dataset that piqued our interest was mosapride citrate, a 5-HT4 receptor agonist used to treat gastrointestinal ailments such as dyspepsia, gastritis, and irritable bowel syndromeby increasing gastric emptying and gastrointestinal motility (Curran and Robinson, 2008). Widely used to treat different forms of gastropathy in Asian countries such as Japan and Korea, mosapride has also shown potential to improve glycemic control in subjects who had impaired glucose tolerances or

Type 2 diabetes. In several clinical studies, mosapride was able to lower fasting glucose levels in as little as 10 hours after a single dose, with repeated dosing over 1-2 weeks having similar effects (Aoki et al., 2013; Koshiyama et al., 2000; Nam et al., 2010; Ueno et al., 2002; Ueno and Satoh, 2009). Multiple mechanisms for this decrease in glucose levels have been proposed, including improvement of the timing between insulin release and glucose levels after a meal due to increases in gastrointestinal motility, improvement of insulin sensitivity (patients with impaired glucose tolerance treated with mosapride

101 exhibit lower fasting insulin levels), and higher levels of glucagon-like-peptide 1 (GLP-1) occur with mosapride treatment, which increases insulin release in response to stimulation by glucose (Aoki et al., 2013; Asakawa et al., 2003; Koshiyama et al., 2000;

Nonogaki and Kaji, 2015; Ueno et al., 2002). While all these mechanisms may be a factor, the role of the liver in the glucose-lowering capability of mosapride has not been investigated. As CAR activation in mice also leads to a reduction in fasting blood glucose levels by reducing gluconeogenesis, we sought to further characterize the activation of

CAR by mosapride citrate in HPH and investigate whether mosapride can reduce gluconeogenic gene expression and glucose output in HPH.

Mosapride citrate has been previously shown to induce CYP2B6 activity and mRNA expression in HPH (Young-Hoon et al., 2015). Combining this information with our studies exhibiting that mosapride activates CAR while also translocating it to the nucleus of HPH, leads to the induction of CYP2B6 mRNA and protein expression over a range of concentrations (Fig. 3.4) (Young-Hoon et al., 2015). In addition, mosapride was able to repress the mRNA expression of G6Pase and PEPCK, two major proteins involved in gluconeogenesis (Fig. 3.5). After stimulation of gluconeogenesis with cAMP, mosapride was able to reduce glucose output from HPH over 24h (Fig. 3.6). These experiments demonstrated that mosapride can repress gluconeogenesis in HPH, providing another possible liver-specific mechanism for the reduction in fasting blood glucose levels by mosapride (Fig. 3.7). If this liver-specific mechanism for glucose lowering can be reproduced in vivo and show dependence upon CAR, it would provide a rationale for

102 clinical use of hCAR activators for metabolic disorders.

Figure 3.7 - Proposed mechanisms for mosapride-dependent improvement in glycemic control. While mosapride is known to function by increasing gastric motility in the gut and increasing insulin secretion from the pancreas, our study demonstrates that mosapride citrate represses gluconeogenesis, providing an additional, liver-specific mechanism by which mosapride alters glucose homeostasis.

While the high-throughput hCAR nuclear translocation assay was robust and able to identify both new and known CAR modulators, there were several limitations to our study. Due to the expense associated with using HPH, we were only able to screen the

FDA-approved compound library at a single concentration (10 μM) with one replicate in two separate livers. Therefore, compounds with higher EC50 values may have been missed in the translocation assay while being correctly identified in the luciferase assay, as the maximum compound concentration used in the luciferase screen was 46 - 92 μM.

While translocation data for each compound was relatively consistent between two liver donors, there were some discrepancies. Future studies may be able to utilize pooled hepatocytes from multiple donors to provide more relevant data. We also decided to consider translocation on a per cell basis and limit translocation to a yes/no;, this method

103 provided a robust analysis for detecting true translocators but also may have missed compounds which weakly translocate CAR. Our assay also relied on fixed cells, but optimization of live-cell imaging in combination with high-throughput mRNA extraction/PCR analysis or fluorescent CYP substrate probes would be more optimal ways to identify CAR modulators. However, the current assay employed here, can be adapted to the 384-well format which would reduce costs associated with HPH screening, in the future. In the current study, we were able to optimize the Ad/EYFP-hCAR nuclear translocation assay into a high-throughput format, validate the nuclear translocation assay in comparison to the HepG2-CYP2B6-hCAR luciferase assay, and identify mosapride citrate as a CAR activator that represses gluconeogenesis in HPH, providing a liver- specific mechanism for mosapride-dependent decreases in fasting blood glucose.

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Chapter 4: Examination of downstream effects of constitutive androstane receptor activation

4.1 Introduction

The xenobiotic receptors CAR and PXR are highly expressed in the liver and intestine, where they protect the body from exogenous chemicals by upregulating a set of drug- metabolizing enzymes and transporters. This “traditional” role of CAR and PXR in xenobiotic detoxification has been extensively studied in vitro and in vivo, in animals and even in translational human clinical studies, due to the ability of these receptors to mediate severe drug-drug interactions. However, accumulating evidence reveals that XRs also have significant endogenous roles functioning as signaling molecules that modulate physiological and pathophysiological functions including energy homeostasis, insulin signaling, inflammation, immune response, cell proliferation, apoptosis, autophagy, and cancer development (Gao and Xie, 2012; Yan et al., 2014; Banerjee et al., 2015; De

Mattia et al., 2016; Kazantseva et al., 2016; Roman et al., 2017; Gutiérrez-Vázquez and

Quintana, 2018).

The importance of CAR in xenobiotic metabolism began with exploration into the enzyme-inducing effects of powerful antiepileptic drug phenobarbital (PB) which potently induced the expression of CYP2B genes, and CAR was established as the key nuclear receptor that regulates the inductive expression of CYP2B by numerous PB-like chemicals (Trottier et al., 1995; Park et al., 1996; Honkakoski and Negishi, 1997;

Honkakoski et al., 1998; Sueyoshi et al., 1999; Wei et al., 2000; Staudinger et al., 2013).

Although it has long been known that PB, a prototypical CAR activator, improves insulin

105 sensitivity and decrease blood glucose levels in patients with type 2 diabetes, the connection of CAR with regulation of endogenous stimuli was not made until much more recently (Lahtela et al., 1985). Many studies in rodents have established that activation of

CAR reducing gluconeogenesis by repressing the expression of gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) through multiple different mechanisms, and this effect holds true in human primary hepatocytes as well (Miao et al., 2006) (Kodama et al., 2004) (Gao et al., 2015)(Lynch et al). While this glucose-lowering effect is consistent between rodent and human CAR, activation of PXR leads to opposite effects on gluconeogenesis between rodents ad humans. Activation of PXR in mice represses gluconeogenic gene expression, lowering circulating blood glucose levels. However, clinical studies have shown that treatment with human PXR activator rifampicin increases blood glucose levels in both tuberculosis patients and healthy volunteers (Takasu et al., 1982; Rysa et al., 2013). Several in vitro studies have reinforced this finding, demonstrating that activation of human PXR induces gluconeogenic genes in HPH and HepG2 cells ( ). These significant differences in downstream responses, paired with differences in ligand specificity between preclinical species and humans demonstrates the challenges associated with translating preclinical xenobiotic receptor data to the clinic.

In addition to the species differences seen with PXR, there are significant species differences between rodent CAR (rCAR) and human CAR (hCAR) as well. One area where the divergence in downstream effects between rCAR and hCAR is most apparent is the role of rCAR in the development of liver tumors. When activated by a range of

106 different chemicals in rodents, CAR increases DNA replication, suppresses apoptosis, and promotes liver tumor formation and progression. While the exact mechanism of rCAR-mediated tumor promotion remains elusive, the involvement of CAR in liver cancer promotion has been extensively validated. In contrast, the involvement of activated CAR in human liver cancer is much more controversial. For instance, PB- mediated activation of CAR in rodents promotes liver tumor formation but has been used safely in humans for decades without increasing the incidence of liver cancer (Braeuning,

2014; La Vecchia and Negri, 2014). PB also does not increase hepatocellular proliferation in HPH or in chimeric mice with humanized livers, illustrating the differences in the downstream effects between human and rodent CAR.

Due to the interest around the endogenous effects of CAR and PXR, multiple studies have attempted “omics”-based approaches to determine how CAR and PXR activation affect downstream biologic processes.

4.2 Materials and Methods

4.2.1 Chemicals and Biological Reagents

Phenobarbital (PB) was obtained from Sigma-Aldrich (St. Louis, MO). 6-(4-

Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime

(CITCO) was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA).

PCR primers were synthesized by Integrated DNA Technologies (Coralville, IA).

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4.2.2 HPH Culture.

HPH were obtained from Bioreclamation In Vitro Technologies (Baltimore, MD).

Hepatocytes with viability over 90% were seeded at 1.5×106 cells/well in 6-well collagen-coated plates as described previously (1). After attachment, hepatocytes were cultured in serum-free William’s E Medium supplemented with insulin, transferrin, and selenium, 0.1 μM dexamethasone, 100 U/ml penicillin, and 100 μg/ml streptomycin, and overlaid with Matrigel (0.25 mg/ml). Thirty-six hours after seeding, HPH were treated with vehicle control (0.1% DMSO), PB (1 mM), or CITCO (1 μM) for 24h before harvesting for proteomics or RNA, or 72h for protein.

4.2.3 Proteomics.

Cells were lysed by 4% sodium deoxycholate after washing in phosphate-buffered saline.

Lysates were washed, reduced, alkylated and trypsinolyzed on filter as described by

Wisniewski et al. (2009) and Erde et al. (2014) (2,3). Tryptic peptides were separated on a nanoACQUITY UPLC analytical column (BEH130 C18, 1.7 μm, 75 μm x 200 mm,

Waters) over a 180 min linear acetonitrile gradient (3 – 43%) with 0.1 % formic acid on a

Waters nano-ACQUITY UPLC system, and analyzed on a coupled Waters Synapt G2S

HDMS mass spectrometric system. Spectra were acquired using an ion mobility linked parallel mass spectrometry (UDMSe) and analyzed as described by Distler et al. (2014)

(4). Peaks were resolved using Apex3D and Peptide3D algorithms (5). Tandem mass spectra were searched against a UniProt human reference proteome and its corresponding decoy sequences using an ion accounting algorithm (6). Resulting hits were validated at a maximum false discovery rate of 0.04. Protein abundance ratios between the PB or

CITCO treated cell line and the untreated cell line were measured by comparing the MS1

108 peak volumes of peptide ions at the low collision energy cycle, whose identities were confirmed by MS2 sequencing at the elevated collision energy cycle as described above.

Label-free quantifications were performed using an aligned AMRT (Accurate Mass and

Retention Time) cluster quantification algorithm developed by Qi et al. (2012) (7).

Pathway and gene ontology analysis were performed with Qiagen Ingenuity and Panther

GO databases, as described by Krämer et al. (2014) and Mi et al. (2017), respectively

(8,9).

4.2.4 Real-time RT-PCR

Total RNA was isolated from cells using TRIzol reagent (ThermoFisher, Rockford, IL) and reverse transcribed using a High Capacity cDNA Archive kit (Applied Biosystems,

Foster, CA) according to the manufacturer’s instructions. Real-time PCR assay was performed using SYBR Green PCR Mastermix (Qiagen) on an ABI StepOnePlus Real-

Time PCR system (Applied Biosystems). Primer sequences are listed in Table 1.

4.2.5 Western Blot Analysis

Protein samples extracted from treated cells were electrophoretically separated on SDS-

PAGE gels (4-12%), and transferred to polyvinylidine fluoride membranes.

Subsequently, membranes were incubated with primary antibodies against CYP2B6

(1:200, Santa Cruz), CYP3A4 (1:5000, Sigma-Aldrich), or β-actin (1:50,000, Sigma-

Aldrich) at 4 °C overnight. Blots were developed with West Pico chemiluminescent

109 substrate (ThermoFisher) after incubation with horse-radish peroxidase secondary antibodies.

4.2.6 Statistical Analysis

0.001.

4.3 Results

4.3.1 Differential protein expression in HPH after PB and CITCO treatment

After treating HPH123 with DMSO (0.1%), PB (1 mM), or CITCO (1 μM) for 72h, HPH were harvested and subjected to the proteomics workflow detailed in Materials and

Methods. As the goal of this experiment was to identify major pathways and proteins affected by either dual activators of CAR and PXR (PB) or specific activation of CAR

(CITCO), we determined differential expression based on a significance cutoff (p < 0.05) for this discovery proteomics experiment. In total there were 306 and 560 proteins differentially expressed (p < 0.05) after PB and CITCO treatment, respectively. The top

25 upregulated and downregulated proteins for each group (PB alone, PB and CITCO, or

CITCO alone) are detailed in Figure 4.1. Several known CAR target genes are represented in the overlapping induced dataset, including CYP3A4, CYP1A2, and

ALAS1.

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A. Upregulated PB CITCO

PB PB CITCO CITCO 1 2 3 1 2 3 1 2 3 1 2 3 SQSTM1 CYP3A4 VPS8 ATP5C1 CYP2A13 RETSAT GLYCTK HSPA8 VCP TRAK1 VIM FLII ACSL1 ECT2 ORM2 SQRDL KRT23 ADH1C LRRC37A2 HSP90AB1 LONP1 ACSS3 CYP1A2 BHMT SLC39A14 FHAD1 ANXA7 SNRPB2 RAB11FIP2 LOC105371242 UGT1A1 EPB41L1 ZNF112 UGT2B28 RPL19 SEL1L RHOBTB3 RPS5 HNRNPA3 LETM1 SNAP47 FAM213A PPP2R1A ALAS1 CA2 CPNE7 EEF1A1P5 UBAP2L CISD1 PSMD6 HEATR6 AOX1 MTHFD1 ECM29 RBBP6 RLTPR CKB SAMHD1 CDH2 AKR1C3 Relative Abundance RABGEF1 PSMA5 SELENBP1 >2 ATP2B1 UGT2A3 EML5 1.6 CES1 MYZAP SOD1 1.3 SMARCB1 ZEB2 PRDX6 1.1 CYB5A TAF1 PTGR1 1 (no change vs. control)

B. Downregulated PB CITCO

PB PB CITCO CITCO 1 2 3 1 2 3 1 2 3 1 2 3 ALB CDT1 TM9SF2 EIF3L MRPL39 LAD1 TBC1D30 CERS2 SLC25A24 RUVBL1 ANPEP ALDH4A1 BCAR3 MTCH1 CCDC82 SSX2IP NPEPPS BPHL ABLIM2 UQCRC2 KIAA1033 KIF2C CYFIP2 NOL4 MAPK9 CCDC110 TTC23 CAD KRT1 TP53BP1 GPHN COX6A1 ERN1 TRA2B NIPSNAP3A ATP5O SPTAN1 HNRNPH2 RAPGEF2 RDH11 MCF2L2 ARHGAP23 BRSK2 ACOT7 NDUFB3 ATP1B1 MECOM TRIP11 ACTB FER SYCP2 CCDC144B APOC3 FGG TF PMPCA MYO18A PGM3 ATP5E RIC1 Relative Abundance MYCBPAP PPP1R3B POLD1 1 (no change vs. control) GALNT2 COX7C RGL3 0.91 CAV1 AGT GOLGA4 0.77 CNTROB PROCA1 SDHA 0.625 DHRS7 RASGEF1A GLDC <0.5 111

Figure 4.1 - Differential protein expression in HPH after PB and CITCO treatment. Proteomic analysis of differentially-expressed proteins (p<0.05) revealed that PB and CITCO downregulate a similar number of proteins while CITCO upregulates many more proteins than PB in HPH. The top 25 most significant upregulated (A) or downregulated (B) proteins in each category are shown for each biological sample in each heatmap.

4.3.2 Upstream regulators and pathways perturbed by PB and CITCO treatment

After differentially-expressed proteins were identified, pathway and upstream regulator analyses were performed. While PB and CITCO both regulated a significant amount of overlapping pathways, there were some significant differences. CITCO treatment led to the upregulation of PI3K/AKT, p70S6K, and 14-3-3-mediated signaling, while PB was most associated with downregulation of the LXR/RXR activation. The majority of the upstream regulators identified for PB and CITCO treatments overlapped, with both CAR and PXR identified as upstream regulators, as expected. Many proteins involved in cancer development and progression, such as p53, MYC, and RICTOR, were also identified as upstream regulators.

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Figure 4.2 - Upstream regulators and pathways perturbed by PB and CITCO treatment. Bioinformatic analysis was performed to determine which pathways were perturbed in PB-treated HPH (A) and CITCO-treated HPH (B), and determined the overlapping pathways between the two treatment groups (C). As expected, CAR and PXR were identified as upstream regulators for both treatment groups (D).

One novel pathway that both PB and CITCO upregulated in our analysis was the EIF2 pathway. One of the proteins integral to the pathway, EIF5, was found to be upregulated by both treatments in the proteomics experiment and validated via western blot in two separate livers (Figure 4.3).

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Figure 4.3 - PB and CITCO alter the EIF2 pathway via EIF5 upregulation. EIF2 was a novel pathway found to be upregulated by both PB and CITCO in our bioinformatic analysis. In the EIF2 pathway (A), eIF5 is a key protein that acts as a GTPase activating protein (GAP) for the eIF2 complex, allowing it to be recycled after translation initiation. In our dataset, EIF5 was upregulated by both PB and CITCO (B), which could be mediating the increase in EIF2 signaling activity. Through Western Blot, we were able to validate that EIF5 is induced by both PB and CITCO.

4.3.3 Functional consequences of PB and CITCO treatment

After identifying pathways altered by PB and CITCO treatment and upstream regulators involved, we performed biofunctional and toxicogenomic analyses to determine what the overall functional consequences of PB and CITCO treatment are, with a specific focus on dual PXR-CAR activation or selective CAR activation. PB treatment was predicted to be associated with increases in cell proliferation and xenobiotic metabolism while decreasing cell death and necrosis. Contrary to these findings, CITCO was predicted to repress cancer functions in general, particularly liver cancer.

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Figure 4.4 - Functional consequences of PB and CITCO treatment. As we were able to find a possible mechanism for the increase in signaling activity for a novel pathway in our bioinformatic analysis, we wanted to further investigate the functional changes that will be caused by these treatments. Our biofunctional analysis showed that PB may downregulate cell death and necrosis while upregulating fatty acid metabolism and cell proliferation. On the other hand, CITCO had a major impact on cancer, including hepatobiliary system cancer, liver cancer, and liver carcinoma. This is in contrast to studies in mice where activation of mCAR with PB or TCPOBOP can directly induce liver cancer. The difference in the predicted reduction in tumorigenicity with hCAR activation and increase in tumorigenicity with mCAR activation is interesting and needs further investigation.

4.4 Conclusion and Discussion

In the current study, the proteomes of HPH treated with PB, and CITCO were analyzed to determine how CAR activation affects signaling pathways and functional outcomes in the liver. PB is an anti-epileptic drug that activates CAR indirectly through antagonism of the

115 epidermal growth factor (EGF) signaling pathway, and also activates PXR. The selective, direct CAR activator CITCO was first identified in 2003 and is still the only potent and selective CAR activator that has been identified. While PB has been widely studied as a

CYP2B inducer, CITCO has been used in most recent studies of human CAR to activate

CAR specifically(Niu et al., 2018). A recent study found that the genome binding patterns in the livers of mice for hCAR activated either directly by CITCO or indirectly with PB exhibited almost identical genomic profiles, which means that the major differences between PB and CITCO treatment are most likely due to activation of PXR by PB instead of activation differences. In contrast to a previous RNA-Seq study in

HepaRG cells, CITCO treatment led to the differential expression of many more proteins than PB (Li et al., 2015a). This finding was puzzling, as PB alters many pathways other than CAR, including EGFR and PXR activation, Interestingly, CITCO upregulated many more proteins than it downregulated in our proteomics study, but a previous transcriptomics study treating HPH with CITCO found that CITCO downregulated ~5- fold more genes than it upregulated (Kandel et al., 2016). This could be due to the overall treatment time of the HPH (24h vs. 72h) or CAR activation affects the proteome outside of traditional transcriptional pathways. Both CAR and PXR induced known target proteins, such as CYP3A4, CYP1A2, and ALAS1; however, the prototypical target gene of CAR, CYP2B6, was not identified as upregulated in our proteomics analysis. This could be due to any number of factors, but enough CAR-regulated genes outside of

CYP2B6 were differentially expressed after both treatments that we can be confident

CAR was activated.

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After identifying differentially-expressed proteins, we performed pathway and upstream regulator analysis on the proteomic dataset. The pathway analysis identified many pathways that were co-regulated by both PB and CITCO, including PXR/RXR activation, xenobiotic metabolism signaling, and Nrf2 activation. Increases in PXR downstream targets and xenobiotic receptor signaling are both well-established outcomes of treatment with these two compounds, but only recently has the connection between CAR activation and Nrf2 activation been established (Corton et al., 2018). This study identified a significant correlation between CAR activation and Nrf2 activation, with 90% of chemicals that activate CAR also activating Nrf2. In addition, CAR activation was found to precede Nrf2 activation and Nrf2 was not activated by these chemicals in CAR-null mice (Corton et al., 2018). This study helps to validate our pathway analysis and our data reinforces the hypothesis that CAR activation leads to Nrf2 activation in the liver of both mice and humans.

Another pathway found to be upregulated by PB and CITCO is the EIF2 pathway. The eukaryotic initiation factor 2 (EIF2) pathway plays a vital role in the initiation of mRNA translation to protein (Kimball, 1999). To initiate translation, the 40s ribosomal unit containing EIF2 bound to Met-tRNA and GTP combines with EIF1, 1A, 3, and 5 to form the 43s pre-initiation complex (Hinnebusch, 2011). After the pre-initiation complex recognizes a start codon, EIF5 functions as a GTPase accelerating protein (GAP) to help initiate translation while also helping to recycle EIF2 by inhibiting the dissociation of

GDP (Jennings and Pavitt, 2010). Our proteomics data showed that EIF5 was upregulated by both PB and CITCO, and we confirmed this via western blotting in two separate livers

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(Figure 4.3). Interestingly, in vivo binding site analysis with WT or humanized CAR mice identified a binding site for hCAR in the promoter of Eif5, but not for mCAR (Niu et al.,

2018). This discrepancy is rather interesting, as only ~6% of all binding interactions between mCAR and hCAR exhibited differences and therefore, EIF5 should undergo further study as a potential mediator of distinct species-differences between mCAR and hCAR.

Upstream regulator analysis of our dataset revealed that the majority of upstream regulators for the proteins altered upon PB and CITCO treatment were similar to both treatments. As expected, CAR and PXR were identified as upstream regulators for both treatments, as well as Nrf2, which correlates well with our pathway analysis. Intriguingly, many of the other top overlapping upstream regulators are known to be master regulators of oncogenesis, including MYC and TP53. MYC is a known downstream target of mCAR and p53 activity is known to be regulated by Mdm2, which is induced by mCAR

(Blanco-Bose et al., 2008; Huang et al., 2005). However, the regulation of these pathways by hCAR is more controversial, as Myc induction by hCAR in mouse liver was only 2-fold as opposed to ~10-fold by mCAR while MYC induction in HPH exhibited low induction as well (Niu et al., 2018). HNF4α was also a common upstream regulator for PB and CITCO treatment, which is interesting as CAR and PXR are known to exhibit interplay with HNF4α in the regulation of their target genes (Tirona et al., 2003).

While many overlapping proteins and pathways were altered by both treatments, there functional consequences of those alterations were vastly different, with PB predicted to

118 enhance oncogenic cellular functions such as cell proliferation and inhibition of cell death, while CITCO was predicted to inhibit liver oncogenesis. In rodents, CAR acts as a tumor initiator, driving oncogenesis through multiple pathways upon activation(Wei et al., 2000). However, activation of CAR in humans is unlikely to act in a similar fashion as several important clinically-used drugs activate CAR but do not have any association with higher chances of liver cancer (Braeuning, 2014; La Vecchia and Negri, 2014).

While these studies show that CAR activation does not initiate liver cancer in humans, several other studies have found that hCAR drives cellular differentiation and reduces cell proliferation. Separate studies found that knocking-out CAR in HepaRG cells leads to the upregulation of cellular proliferation markers while CAR overexpression leads to improved differentiation of HepaRG cultures without DMSO (Li et al., 2015a; van der

Mark et al., 2017). In addition, activation of hCAR in brain tumor stem cells by CITCO in vitro and in vivo led to cell cycle arrest and enhanced apoptosis, providing evidence that hCAR may also function as a tumor suppressor (Chakraborty et al., 2011). The divergence between PB and CITCO is interesting, and may be due to the dual activation of CAR and PXR by PB compared with the selective activation of CAR by CITCO. The role of PXR in cancer development and progression is controversial and may be cell-type specific (Mackowiak et al., 2018). Further studies are warranted to determine the mechanisms behind functional changes due to PB and CITCO treatments and further validate these outcomes in physiologically-relevant liver models and humans.

Overall, a comprehensive proteomics approach was utilized to determine the functional outcomes associated CAR activation by treating HPH with PB and CITCO. We identified

119 novel proteins and pathways regulated by PB and CITCO in HPH, and provided evidence that specific CAR activation may be beneficial to prevent or treat liver cancer in humans.

Further studies are warranted to better understand how hCAR activation affects oncogenic pathways in the human liver.

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Chapter 5: Conclusion and future directions

Overall, the comprehensive studies presented above provide a model for metabolism- based changes in xenobiotic receptor activity, identified clinically-used drugs that modulate CAR activity, and determined the clinically-relevant downstream effects of

CAR activation. Metabolism-based alterations of xenobiotic receptor activity are clearly important not just for DDIs but also for downstream endogenous effects mediated by receptors such as CAR and PXR. Studies with CAR antagonist PK11195 found that

PK11195 is metabolized to a CAR activator in metabolically-competent systems, demonstrating the importance of validation in physiologically-relevant cell models. The importance of this principle extends not only to drug development, but to the thousands of potentially toxic chemicals already in use around the world. Metabolically-competent screening systems are being recognized as the way forward for toxicity screening by the

Tox21 consortium, who have begun the development of systems that will allow for metabolically-competent screening assays.

While several large-scale screening assays for CAR activation have been used to identify

CAR modulators, all of these screens were in immortalized cell lines that lack metabolic competence. This has resulted in datasets of CAR modulation that exhibit limited translation to physiologically-relevant systems. Here, we developed and validated a physiologically-relevant high-throughput method to accurately identify CAR translocation, which is a marker of CAR activation, by utilizing Ad/EYFP-hCAR and high content imaging. Hits in the CAR translocation assay were more highly correlated with CYP2B6 induction than activation in the HepG2-hCAR-CYP2B6 luciferase assay

121 and led to the identification of mosapride citrate as a CAR agonist that represses gluconeogenesis in HPH. While this assay was successful at identifying CAR activators, adding additional concentrations and moving to a 384-well system would vastly iprove the predictive ability of this assay while decreasing the cost. In addition, this assay can be used to identify modulation of other cellular receptors which exhibit any type of phenotypic localization in response to modulation. For liver-expressed proteins, screening in HPH combines metabolic-competence and physiological-relevance which is the gold standard for any assay. In the future, pooled HPH should be tested to determine whether they can reduce the interindividual variability between liver donors as this may make results more consistent.

Our proteomic analysis of HPH treated with CAR activators PB and CITCO demonstrated the importance of knowing the effects of compounds and their metabolites on CAR. We were able to confirm via pathway analysis that Nrf2 activation by CAR, only demonstrated in mice thus far, is also upregulated in HPH. In addition, a novel modulator of the EIF2 pathway, EIF5, was found to be upregulated by both PB and

CITCO in HPH, and genome binding studies of CAR suggest that this effect may be specific in humans. Additionally, functional analysis of CITCO treatment predicted that

CITCO represses liver cancer development, which is in contrast to the known initiation of cancer by mCAR but agrees with several studies that found hCAR to repress proliferation and drive differentiation in different cell types. Further studies are needed to determine exactly which pathways are affected by CAR activation and how those pathways contribute to the predicted repression of liver cancer development.

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These studies have demonstrated that metabolites of xenobiotic receptor ligands can exhibit much different effects on the xenobiotic receptors than their parent compound.

This metabolism-mediated alteration is important beyond prediction of DDIs as xenobiotic receptors are now known to affect many endogenous processes outside of xenobiotic detoxification. Thus, use of metabolically-competent systems to evaluate the potential effects of a compound are essential to determine the functional outcomes of not only exposure to the parent drug, but its metabolites as well.

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REFERENCES

Andersin T, Väisänen S and Carlberg C (2003) The Critical Role of Carboxy-Terminal Amino Acids in Ligand-Dependent and -Independent Transactivation of the Constitutive Androstane Receptor. Molecular Endocrinology 17(2): 234-246. Anderson LE, Dring AM, Hamel LD and Stoner MA (2011) Modulation of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) by 6-arylpyrrolo[2,1- d][1,5]benzothiazepine derivatives, ligands of peripheral benzodiazepine receptor (PBR). Toxicology Letters 202(2): 148-154. Andersson TB, Kanebratt KP and Kenna JG (2012) The HepaRG cell line: a unique in vitro tool for understanding drug metabolism and toxicology in human. Expert Opin Drug Metab Toxicol 8(7): 909-920. Aoki K, Kamiyama H, Masuda K, Togashi Y and Terauchi Y (2013) Mosapride citrate, a 5-HT₄ receptor agonist, increased the plasma active and total glucagon-like peptide-1 levels in non-diabetic men. Endocrine Journal 60(4): 493-499. Arnold KA, Eichelbaum M and Burk O (2004) Alternative splicing affects the function and tissue-specific expression of the human constitutive androstane receptor. Nuclear Receptor 2: 1-16. Asakawa H, Hayashi I, Fukui T and Tokunaga K (2003) Effect of mosapride on glycemic control and gastric emptying in type 2 diabetes mellitus patients with gastropathy. Diabetes Research and Clinical Practice 61(3): 175-182. Auerbach SS, Ramsden R, Stoner MA, Verlinde C, Hassett C and Omiecinski CJ (2003) Alternatively spliced isoforms of the human constitutive androstane receptor. Nucleic Acids Research 31(12): 3194-3207. Baes M, Gulick T, Choi HS, Martinoli MG, Simha D and Moore DD (1994) A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements. Molecular and Cellular Biology 14(3): 1544-1552. Bailly-Maitre B, de Sousa G, Boulukos K, Gugenheim J and Rahmani R (2001) Dexamethasone inhibits spontaneous apoptosis in primary cultures of human and rat hepatocytes via Bcl-2 and Bcl-xL induction. Cell Death And Differentiation 8: 279. Banerjee M, Robbins D and Chen T (2015) Targeting xenobiotic receptors PXR and CAR in human diseases. Drug Discovery Today 20(5): 618-628. Bauer D, Wolfram N, Kahl GF and Hirsch-Ernst KI (2004) Transcriptional Regulation of CYP2B1 Induction in Primary Rat Hepatocyte Cultures: Repression by Epidermal Growth Factor Is Mediated via a Distal Enhancer Region. Molecular Pharmacology 65(1): 172-180. Bertilsson G, Heidrich J, Svensson K, Åsman M, Jendeberg L, Sydow-Bäckman M, Ohlsson R, Postlind H, Blomquist P and Berkenstam A (1998) Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proceedings of the National Academy of Sciences 95(21): 12208-12213. Bhalla S, Ozalp C, Fang S, Xiang L and Kemper JK (2004) Ligand-activated Pregnane X Receptor Interferes with HNF-4 Signaling by Targeting a Common Coactivator PGC-1α: FUNCTIONAL IMPLICATIONS IN HEPATIC CHOLESTEROL

124

AND GLUCOSE METABOLISM. Journal of Biological Chemistry 279(43): 45139-45147. Bitter A, Rümmele P, Klein K, Kandel BA, Rieger JK, Nüssler AK, Zanger UM, Trauner M, Schwab M and Burk O (2015) Pregnane X receptor activation and silencing promote steatosis of human hepatic cells by distinct lipogenic mechanisms. Archives of Toxicology 89(11): 2089-2103. Blanco-Bose WE, Murphy MJ, Ehninger A, Offner S, Dubey C, Huang W, Moore DD and Trumpp A (2008) C-Myc and its target FoxM1 are critical downstream effectors of constitutive androstane receptor (CAR) mediated direct liver hyperplasia. Hepatology 48(4): 1302-1311. Blumberg B, Sabbagh W, Jr., Juguilon H, Bolado J, Jr., van Meter CM, Ong ES and Evans RM (1998) SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes & development 12(20): 3195-3205. Boussadia B, Gangarossa G, Mselli-Lakhal L, Rousset M-C, de Bock F, Lassere F, Ghosh C, Pascussi J-M, Janigro D and Marchi N (2016) Lack of CAR impacts neuronal function and cerebrovascular integrity in vivo. Experimental Neurology 283(Part A): 39-48. Braeuning A (2014) Liver cell proliferation and tumor promotion by phenobarbital: relevance for humans? Archives of Toxicology 88(10): 1771-1772. Braeuning A, Gavrilov A, Geissler M, Wenz C, Colnot S, Templin MF, Metzger U, Römer M, Zell A and Schwarz M (2016) Tumor promotion and inhibition by phenobarbital in livers of conditional Apc-deficient mice. Archives of Toxicology 90(6): 1481-1494. Burbach KM, Poland A and Bradfield CA (1992) Cloning of the Ah-Receptor cDNA Reveals a Distinctive Ligand-Activated Transcription Factor, p 8185, National Academy of Sciences of the United States of America. Burk O, Arnold KA, Nussler AK, Schaeffeler E, Efimova E, Avery BA, Avery MA, Fromm MF and Eichelbaum M (2005) Antimalarial Artemisinin Drugs Induce Cytochrome P450 and MDR1 Expression by Activation of Xenosensors Pregnane X Receptor and Constitutive Androstane Receptor. Molecular Pharmacology 67(6): 1954. Burk O, Piedade R, Ghebreghiorghis L, Fait JT, Nussler AK, Gil JP, Windshügel B and Schwab M (2012) Differential effects of clinically used derivatives and metabolites of artemisinin in the activation of constitutive androstane receptor isoforms. British Journal of Pharmacology 167(3): 666-681. Carazo A and Pavek P (2015) The Use of the LanthaScreen TR-FRET CAR Coactivator Assay in the Characterization of Constitutive Androstane Receptor (CAR) Inverse Agonists. Sensors (Basel) 15(4): 9265-9276. Carazo Fernández A, Smutny T, Hyrsová L, Berka K and Pavek P (2015) Chrysin, baicalein and galangin are indirect activators of the human constitutive androstane receptor (CAR). Toxicology Letters 233(2): 68-77. Chakraborty S, Kanakasabai S and Bright JJ (2011) Constitutive androstane receptor agonist CITCO inhibits growth and expansion of brain tumour stem cells. British Journal of Cancer 104(3): 448-459.

125

Chang TKH and Waxman D (2006) Synthetic Drugs and Natural Products as Modulators of Constitutive Androstane Receptor (Car) and Pregnane X Receptor (PXR). Drug Metabolism Reviews 38(1/2): 51-73. Chen T, Laurenzana Elizabeth M, Coslo Denise M, Chen F and Omiecinski Curtis J (2014) Proteasomal interaction as a critical activity modulator of the human constitutive androstane receptor. Biochemical Journal 458(1): 95-107. Chen T, Tompkins LM, Li L, Li H, Kim G, Zheng Y and Wang H (2010) A Single Amino Acid Controls the Functional Switch of Human Constitutive Androstane Receptor (CAR) 1 to the Xenobiotic-Sensitive Splicing Variant CAR3. Journal of Pharmacology and Experimental Therapeutics 332(1): 106. Chen Y, Tang Y, Chen S and Nie D (2009) Regulation of drug resistance by human pregnane X receptor in breast cancer. Cancer Biol Ther 8(13): 1265-1272. Chen Y, Tang Y, Guo C, Wang J, Boral D and Nie D (2012) Nuclear receptors in the multidrug resistance through the regulation of drug-metabolizing enzymes and drug transporters. Biochemical Pharmacology 83(8): 1112-1126. Chen Y, Tang Y, Wang MT, Zeng S and Nie D (2007) Human pregnane X receptor and resistance to chemotherapy in prostate cancer. Cancer Res 67(21): 10361-10367. Cheng J, Fang Z-Z, Nagaoka K, Okamoto M, Qu A, Tanaka N, Kimura S and Gonzalez FJ (2014) Activation of Intestinal Human Pregnane X Receptor Protects against Azoxymethane/Dextran Sulfate Sodium–Induced Colon Cancer. Journal of Pharmacology and Experimental Therapeutics 351(3): 559-567. Cheng S, Zou M, Liu Q, Kuang J, Shen J, Pu S, Chen L, Li H, Wu T, Li R, Li Y, Jiang W, Zhang Z and He J (2017) Activation of Constitutive Androstane Receptor Prevents Cholesterol Gallstone Formation. Am J Pathol 187(4): 808-818. Cherian MT, Lin W, Wu J and Chen T (2015a) CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor. Molecular Pharmacology 87(5): 878-889. Cherian MT, Lin W, Wu J and Chen T (2015b) CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor. Molecular Pharmacology 87(5): 878. Cherian MT, Yang L, Chai SC, Lin W and Chen T (2016) Identification and Characterization of CINPA1 Metabolites Facilitates Structure-Activity Studies of the Constitutive Androstane Receptor. Drug Metabolism and Disposition 44(11): 1759. Choi H-S, Chung M, Tzameli I, Simha D, Lee Y-K, Seol W and Moore DD (1997) Differential Transactivation by Two Isoforms of the Orphan Nuclear Hormone Receptor CAR. Journal of Biological Chemistry 272(38): 23565-23571. Choi Y-J, Zhou D, Barbosa ACS, Niu Y, Guan X, Xu M, Ren S, Nolin TD, Liu Y and Xie W (2018) Activation of Constitutive Androstane Receptor Ameliorates Renal Ischemia-Reperfusion–Induced Kidney and Liver Injury. Molecular Pharmacology 93(3): 239-250. Chrencik JE, Orans J, Moore LB, Xue Y, Peng L, Collins JL, Wisely GB, Lambert MH, Kliewer SA and Redinbo MR (2005) Structural Disorder in the Complex of Human Pregnane X Receptor and the Macrolide Antibiotic Rifampicin. Molecular Endocrinology 19(5): 1125-1134.

126

Clavien P-A, Lesurtel M, Bossuyt PMM, Gores GJ, Langer B and Perrier A (2010) Recommendations for liver transplantation for hepatocellular carcinoma: an international consensus conference report. The Lancet Oncology 13(1): e11-e22. Corton JC, Rooney JP, Oshida K, Kumar R and Baldwin WS (2018) Chemical Activation of the Constitutive Androstane Receptor Leads to Activation of Oxidant-Induced Nrf2. Toxicological Sciences 167(1): 172-189. Coulouarn C, Factor VM, Andersen JB, Durkin ME and Thorgeirsson SS (2009) Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. Oncogene 28(40): 3526-3536. Curran MP and Robinson DM (2008) Mosapride. Drugs 68(7): 981-991. Dai G, He L, Bu P and Wan YJ (2008) Pregnane X receptor is essential for normal progression of liver regeneration. Hepatology 47(4): 1277-1287. de Vries EM, Oosterman JE, Eggink HM, de Goede P, Sen S, Foppen E, Boudzovitch- Surovtseva O, Boelen A, Romijn JA, laFleur SE and Kalsbeek A (2017) Effects of meal composition and meal timing on the expression of genes involved in hepatic drug metabolism in rats. PLoS One 12(10): e0185520. DeKeyser JG, Laurenzana EM, Peterson EC, Chen T and Omiecinski CJ (2011) Selective Phthalate Activation of Naturally Occurring Human Constitutive Androstane Receptor Splice Variants and the Pregnane X Receptor. Toxicological Sciences 120(2): 381-391. DeKeyser JG, Stagliano MC, Auerbach SS, Prabhu KS, Jones AD and Omiecinski CJ (2009) Di(2-ethylhexyl) phthalate Is a Highly Potent Agonist for the Human Constitutive Androstane Receptor Splice Variant CAR2. Molecular Pharmacology 75(5): 1005-1013. di Masi A, Marinis ED, Ascenzi P and Marino M (2009) Nuclear receptors CAR and PXR: Molecular, functional, and biomedical aspects. Molecular Aspects of Medicine 30(5): 297-343. Ding X and Staudinger JL (2005a) Induction of Drug Metabolism by Forskolin: The Role of the Pregnane X Receptor and the Protein Kinase A Signal Transduction Pathway. Journal of Pharmacology and Experimental Therapeutics 312(2): 849- 856. Ding X and Staudinger JL (2005b) Repression of PXR-mediated induction of hepatic CYP3A gene expression by protein kinase C. Biochemical Pharmacology 69(5): 867-873. Dong B, Lee J-S, Park Y-Y, Yang F, Xu G, Huang W, Finegold MJ and Moore DD (2015) Activating CAR and β-catenin induces uncontrolled liver growth and tumorigenesis. Nature Communications 6: 5944. Dong B, Saha PK, Huang W, Chen W, Abu-Elheiga LA, Wakil SJ, Stevens RD, Ilkayeva O, Newgard CB, Chan L and Moore DD (2009) Activation of nuclear receptor CAR ameliorates diabetes and fatty liver disease. Proceedings of the National Academy of Sciences 106(44): 18831-18836. Dong H, Lin W, Wu J and Chen T (2010) Flavonoids activate pregnane × receptor- mediated CYP3A4 gene expression by inhibiting cyclin-dependent kinases in HepG2 liver carcinoma cells. BMC Biochemistry 11(1): 1-9.

127

Doricakova A, Novotna A, Vrzal R, Pavek P and Dvorak Z (2013) The role of residues T248, Y249 and T422 in the function of human pregnane X receptor. Archives of Toxicology 87(2): 291-301. Dubrac S, Elentner A, Ebner S, Horejs-Hoeck J and Schmuth M (2010) Modulation of T lymphocyte function by the pregnane X receptor. J Immunol 184(6): 2949-2957. Dussault I, Lin M, Hollister K, Fan M, Termini J, Sherman MA and Forman BM (2002) A Structural Model of the Constitutive Androstane Receptor Defines Novel Interactions That Mediate Ligand-Independent Activity. Molecular and Cellular Biology 22(15): 5270-5280. Ekins S, Kortagere S, Iyer M, Reschly EJ, Lill MA, Redinbo MR and Krasowski MD (2009) Challenges predicting ligand-receptor interactions of promiscuous proteins: the nuclear receptor PXR. PLoS Comput Biol 5(12): e1000594. Elcombe CR, Elcombe BM, Foster JR, Chang SC, Ehresman DJ and Butenhoff JL (2012) Hepatocellular hypertrophy and cell proliferation in Sprague-Dawley rats from dietary exposure to potassium perfluorooctanesulfonate results from increased expression of xenosensor nuclear receptors PPARalpha and CAR/PXR. Toxicology 293(1-3): 16-29. Elentner A, Ortner D, Clausen B, Gonzalez FJ, Fernandez-Salguero PM, Schmuth M and Dubrac S (2015) Skin response to a carcinogen involves the xenobiotic receptor pregnane X receptor. Exp Dermatol 24(11): 835-840. Elentner A, Schmuth M, Yannoutsos N, Eichmann TO, Gruber R, Radner FPW, Hermann M, Del Frari B and Dubrac S (2018) Epidermal Overexpression of Xenobiotic Receptor PXR Impairs the Epidermal Barrier and Triggers Th2 Immune Response. J Invest Dermatol 138(1): 109-120. Elias A, High AA, Mishra A, Ong SS, Wu J, Peng J and Chen T (2014) Identification and characterization of phosphorylation sites within the pregnane X receptor protein. Biochemical Pharmacology 87(2): 360-370. Elias A, Wu J and Chen T (2013) Tumor Suppressor Protein p53 Negatively Regulates Human Pregnane X Receptor Activity. Molecular Pharmacology 83(6): 1229- 1236. Faucette SR, Sueyoshi T, Smith CM, Negishi M, LeCluyse EL and Wang H (2006a) Differential Regulation of Hepatic CYP2B6 and CYP3A4 Genes by Constitutive Androstane Receptor but Not Pregnane X Receptor. Journal of Pharmacology and Experimental Therapeutics 317(3): 1200-1209. Faucette SR, Sueyoshi T, Smith CM, Negishi M, Lecluyse EL and Wang H (2006b) Differential regulation of hepatic CYP2B6 and CYP3A4 genes by constitutive androstane receptor but not pregnane X receptor. J Pharmacol Exp Ther 317(3): 1200-1209. Feng F, Jiang Q, Cao S, Cao Y, Li R, Shen L, Zhu H, Wang T, Sun L, Liang E, Sun H, Chai Y, Li X, Liu G, Yang R, Yang Z, Yang Y, Xin S and Li BA (2018) Pregnane X receptor mediates sorafenib resistance in advanced hepatocellular carcinoma. Biochim Biophys Acta 1862(4): 1017-1030. Forman BM and Evans RM (1995) Nuclear hormone receptors activate direct, inverted, and everted repeats. Ann N Y Acad Sci 761: 29-37.

128

Forman BM, Tzameli I, Choi H-S, Chen J, Simha D, Seol W, Evans RM and Moore DD (1998) Androstane metabolites bind to and deactivate the nuclear receptor CAR- [beta]. Nature 395(6702): 612-615. Gachon F, Olela FF, Schaad O, Descombes P and Schibler U (2006) The circadian PAR- domain basic leucine zipper transcription factors DBP, TEF, and HLF modulate basal and inducible xenobiotic detoxification. Cell Metab 4(1): 25-36. Gao J, He J, Zhai Y, Wada T and Xie W (2009) The Constitutive Androstane Receptor Is an Anti-obesity Nuclear Receptor That Improves Insulin Sensitivity. Journal of Biological Chemistry 284(38): 25984-25992. Gao J and Xie W (2012) Targeting xenobiotic receptors PXR and CAR for metabolic diseases. Trends in Pharmacological Sciences 33(10): 552-558. Gao J, Yan J, Xu M, Ren S and Xie W (2015) CAR Suppresses Hepatic Gluconeogenesis by Facilitating the Ubiquitination and Degradation of PGC1α. Molecular Endocrinology 29(11): 1558-1570. Gerbal-Chaloin S, Pichard-Garcia L, Fabre J-M, Sa-Cunha A, Poellinger L, Maurel P and Daujat-Chavanieu M (2006) Role of CYP3A4 in the regulation of the aryl hydrocarbon receptor by omeprazole sulphide. Cellular Signalling 18(5): 740- 750. Gotoh S, Miyauchi Y, Moore R and Negishi M (2017) Glucose elicits serine/threonine kinase VRK1 to phosphorylate nuclear pregnane X receptor as a novel hepatic gluconeogenic signal. Cellular Signalling 40: 200-209. Gotoh S and Negishi M (2014) Serum- and Glucocorticoid-Regulated Kinase 2 Determines Drug-Activated Pregnane X Receptor to Induce Gluconeogenesis in Human Liver Cells. Journal of Pharmacology and Experimental Therapeutics 348(1): 131-140. Gotoh S and Negishi M (2015) Statin-activated nuclear receptor PXR promotes SGK2 dephosphorylation by scaffolding PP2C to induce hepatic gluconeogenesis. Sci Rep 5: 14076. Gupta D, Venkatesh M, Wang H, Kim S, Sinz M, Goldberg GL, Whitney K, Longley C and Mani S (2008) Expanding the roles for pregnane X receptor in cancer: proliferation and drug resistance in ovarian cancer. Clin Cancer Res 14(17): 5332- 5340. Haines C, Elcombe BM, Chatham LR, Vardy A, Higgins LG, Elcombe CR and Lake BG (2018) Comparison of the effects of sodium phenobarbital in wild type and humanized constitutive androstane receptor (CAR)/pregnane X receptor (PXR) mice and in cultured mouse, rat and human hepatocytes. Toxicology. Hakkola J, Rysä J and Hukkanen J (2016) Regulation of hepatic energy metabolism by the nuclear receptor PXR. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1859(9): 1072-1082. Hao R, Su S, Wan Y, Shen F, Niu B, Coslo DM, Albert I, Han X and Omiecinski CJ (2016) Bioinformatic analysis of microRNA networks following the activation of the constitutive androstane receptor (CAR) in mouse liver. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1859(9): 1228-1237. Haslam IS, Pitre A, Schuetz JD and Paus R (2013) Protection against chemotherapy- induced alopecia: targeting ATP-binding cassette transporters in the hair follicle? Trends Pharmacol Sci 34(11): 599-604.

129

Hassani-Nezhad-Gashti F, Rysä J, Kummu O, Näpänkangas J, Buler M, Karpale M, Hukkanen J and Hakkola J (2018) Activation of nuclear receptor PXR impairs glucose tolerance and dysregulates GLUT2 expression and subcellular localization in liver. Biochemical Pharmacology 148: 253-264. He J, Gao J, Xu M, Ren S, Stefanovic-Racic M, O'Doherty RM and Xie W (2013) PXR Ablation Alleviates Diet-Induced and Genetic Obesity and Insulin Resistance in Mice. Diabetes 62(6): 1876-1887. Hedrich WD, Xiao J, Heyward S, Zhang Y, Zhang J, Baer MR, Hassan HE and Wang H (2016) Activation of the Constitutive Androstane Receptor Increases the Therapeutic Index of CHOP in Lymphoma Treatment. Molecular Cancer Therapeutics 15(3): 392. Hernandez JP, Mota LC and Baldwin WS (2009) Activation of CAR and PXR by Dietary, Environmental and Occupational Chemicals Alters Drug Metabolism, Intermediary Metabolism, and Cell Proliferation. Curr Pharmacogenomics Person Med 7(2): 81-105. Herzig S, Long F, Jhala US, Hedrick S, Quinn R, Bauer A, Rudolph D, Schutz G, Yoon C, Puigserver P, Spiegelman B and Montminy M (2001) CREB regulates hepatic gluconeogenesis through the coactivator PGC-1. Nature 413: 179. Hinnebusch AG (2011) Molecular Mechanism of Scanning and Start Codon Selection in Eukaryotes. Microbiology and Molecular Biology Reviews 75(3): 434. Honkakoski P, Zelko I, Sueyoshi T and 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(10): 5652-5658. Hosseinpour F, Moore R, Negishi M and Sueyoshi T (2006) Serine 202 Regulates the Nuclear Translocation of Constitutive Active/Androstane Receptor. Molecular Pharmacology 69(4): 1095-1102. Huang W, Zhang J, Chua SS, Qatanani M, Han Y, Granata R and Moore DD (2003) Induction of bilirubin clearance by the constitutive androstane receptor (CAR). Proceedings of the National Academy of Sciences 100(7): 4156-4161. Huang W, Zhang J, Washington M, Liu J, Parant JM, Lozano G and Moore DD (2005) Xenobiotic Stress Induces Hepatomegaly and Liver Tumors via the Nuclear Receptor Constitutive Androstane Receptor. Molecular Endocrinology 19(6): 1646-1653. Hudson GM, Flannigan KL, Erickson SL, Vicentini FA, Zamponi A, Hirota CL, Alston L, Altier C, Ghosh S, Rioux KP, Mani S, Chang TK and Hirota SA (2017) Constitutive androstane receptor regulates the intestinal mucosal response to injury. British Journal of Pharmacology 174(12): 1857-1871. Imai J, Yamazoe Y and Yoshinari K (2013) Novel Cell-based Reporter Assay System Using Epitope-tagged Protein for the Identification of Agonistic Ligands of Constitutive Androstane Receptor (CAR). Drug Metabolism and Pharmacokinetics 28(4): 290-298. Jackson JP, Li L, Chamberlain ED, Wang H and Ferguson SS (2016) Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures. Drug Metabolism and Disposition 44(9): 1463. Jennings MD and Pavitt GD (2010) eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation. Nature 465: 378.

130

Joannard F, Rissel M, Gilot D, Anderson A, Orfila-Lefeuvre L, Guillouzo A, Atfi A and Lagadic-Gossmann D (2006) Role for mitogen-activated protein kinases in phenobarbital-induced expression of cytochrome P450 2B in primary cultures of rat hepatocytes. Toxicology Letters 161(1): 61-72. Josse R, Aninat C, Glaise D, Dumont J, Fessard V, Morel F, Poul JM, Guguen-Guillouzo C and Guillouzo A (2008) Long-term functional stability of human HepaRG hepatocytes and use for chronic toxicity and genotoxicity studies. Drug Metab Dispos 36(6): 1111-1118. Jyrkkärinne J, Windshügel B, Mäkinen J, Ylisirniö M, Peräkylä M, Poso A, Sippl W and Honkakoski P (2005) Amino Acids Important for Ligand Specificity of the Human Constitutive Androstane Receptor. Journal of Biological Chemistry 280(7): 5960-5971. Jyrkkärinne J, Windshügel B, Rönkkö T, Tervo AJ, Küblbeck J, Lahtela-Kakkonen M, Sippl W, Poso A and Honkakoski P (2008) Insights into Ligand-Elicited Activation of Human Constitutive Androstane Receptor Based on Novel Agonists and Three-Dimensional Quantitative Structure−Activity Relationship. Journal of Medicinal Chemistry 51(22): 7181-7192. Kamino H, Moore R and Dai G (2011a) Role of a novel CAR-induced gene, TUBA8, in hepatocellular carcinoma cell lines. Cancer Genetics 204(7): 382-391. Kamino H, Yamazaki Y, Saito K, Takizawa D, Kakizaki S, Moore R and Negishi M (2011b) Nuclear receptor CAR-regulated expression of the FAM84A gene during the development of mouse liver tumors. Int J Oncol 38(6): 1511-1520. Kandel BA, Thomas M, Winter S, Damm G, Seehofer D, Burk O, Schwab M and Zanger UM (2016) Genomewide comparison of the inducible transcriptomes of nuclear receptors CAR, PXR and PPARα in primary human hepatocytes. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1859(9): 1218-1227. Kanno Y and Inouye Y (2010) A consecutive three alanine residue insertion mutant of human CAR: a novel CAR ligand screening system in HepG2 cells. The Journal of Toxicological Sciences 35(4): 515-525. Kanno Y, Suzuki M, Nakahama T and Inouye Y (2005) Characterization of nuclear localization signals and cytoplasmic retention region in the nuclear receptor CAR. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1745(2): 215- 222. Kawamoto T, Sueyoshi T, Zelko I, Moore R, Washburn K and Negishi M (1999a) Phenobarbital-Responsive Nuclear Translocation of the Receptor CAR in Induction of the <em>CYP2B</em> Gene. Molecular and Cellular Biology 19(9): 6318. Kawamoto T, Sueyoshi T, Zelko I, Moore R, Washburn K and Negishi M (1999b) Phenobarbital-Responsive Nuclear Translocation of the Receptor CAR in Induction of the CYP2B Gene. Molecular and Cellular Biology 19(9): 6318-6322. Kawana K, Ikuta T, Kobayashi Y, Gotoh O, Takeda K and Kawajiri K (2003) Molecular Mechanism of Nuclear Translocation of an Orphan Nuclear Receptor, SXR. Molecular Pharmacology 63(3): 524-531. Kazantseva YA, Yarushkin AA, Mostovich LA, Pustylnyak YA and Pustylnyak VO (2015) Xenosensor CAR mediates down-regulation of miR-122 and up-regulation

131

of miR-122 targets in the liver. Toxicology and Applied Pharmacology 288(1): 26-32. Kettner NM, Voicu H, Finegold MJ, Coarfa C, Sreekumar A, Putluri N, Katchy CA, Lee C, Moore DD and Fu L (2016) Circadian Homeostasis of Liver Metabolism Suppresses Hepatocarcinogenesis. Cancer Cell 30(6): 909-924. Kimball SR (1999) Eukaryotic initiation factor eIF2. The International Journal of Biochemistry & Cell Biology 31(1): 25-29. Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, McKee DD, Oliver BB, Willson TM, Zetterström RH, Perlmann T and Lehmann JM (1998) An Orphan Nuclear Receptor Activated by Pregnanes Defines a Novel Steroid Signaling Pathway. Cell 92(1): 73-82. Kobayashi K, Hashimoto M, Honkakoski P and Negishi M (2015) Regulation of gene expression by CAR: an update. Arch Toxicol 89(7): 1045-1055. Kobayashi K, Sueyoshi T, Inoue K, Moore R and Negishi M (2003) Cytoplasmic Accumulation of the Nuclear Receptor CAR by a Tetratricopeptide Repeat Protein in HepG2 Cells. Molecular Pharmacology 64(5): 1069-1075. Kodama S, Koike C, Negishi M and Yamamoto Y (2004) Nuclear Receptors CAR and PXR Cross Talk with FOXO1 To Regulate Genes That Encode Drug- Metabolizing and Gluconeogenic Enzymes. Molecular and Cellular Biology 24(18): 7931-7940. Kodama S, Moore R, Yamamoto Y and Negishi M (2007) Human nuclear pregnane X receptor cross-talk with CREB to repress cAMP activation of the glucose-6- phosphatase gene. Biochem J 407(3): 373-381. Koike C, Moore R and Negishi M (2007) Extracellular Signal-Regulated Kinase Is an Endogenous Signal Retaining the Nuclear Constitutive Active/Androstane Receptor (CAR) in the Cytoplasm of Mouse Primary Hepatocytes. Molecular Pharmacology 71(5): 1217-1221. Koshiyama H, Shimono D, Wada Y and Nakamura Y (2000) Improvement of glycemic control after treatment with mosapride for diabetic gastropathy. Diabetes Care 23(8): 1198. Krasowski MD, Ni A, Hagey LR and Ekins S (2011) Evolution of promiscuous nuclear hormone receptors: LXR, FXR, VDR, PXR, and CAR. Molecular and Cellular Endocrinology 334(1–2): 39-48. Küblbeck J, Laitinen T, Jyrkkärinne J, Rousu T, Tolonen A, Abel T, Kortelainen T, Uusitalo J, Korjamo T, Honkakoski P and Molnár F (2011) Use of comprehensive screening methods to detect selective human CAR activators. Biochemical Pharmacology 82(12): 1994-2007. La Vecchia C and Negri E (2014) A review of epidemiological data on epilepsy, phenobarbital, and risk of liver cancer. Eur J Cancer Prev 23(1): 1-7. Lamba JK, Lamba V, Yasuda K, Lin YS, Assem M, Thompson E, Strom S and Schuetz E (2004) Expression of Constitutive Androstane Receptor Splice Variants in Human Tissues and Their Functional Consequences. Journal of Pharmacology and Experimental Therapeutics 311(2): 811-821. LeCluyse EL, Alexandre E, Hamilton GA, Viollon-Abadie C, Coon DJ, Jolley S and Richert L (2005) Isolation and culture of primary human hepatocytes. Methods Mol Biol 290: 207-229.

132

Li D, Mackowiak B, Brayman TG, Mitchell M, Zhang L, Huang S-M and Wang H (2015a) Genome-wide analysis of human constitutive androstane receptor (CAR) transcriptome in wild-type and CAR-knockout HepaRG cells. Biochemical Pharmacology 98(1): 190-202. Li H, Chen T, Cottrell J and 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 Metabolism and Disposition 37(5): 1098-1106. Li H and Wang H (2010) Activation of xenobiotic receptors: driving into the nucleus. Expert Opinion on Drug Metabolism & Toxicology 6(4): 409-426. Li L, Chen T, Stanton JD, Sueyoshi T, Negishi M and Wang H (2008) The Peripheral Benzodiazepine Receptor Ligand 1-(2-Chlorophenyl-methylpropyl)-3- isoquinoline-carboxamide Is a Novel Antagonist of Human Constitutive Androstane Receptor. Molecular Pharmacology 74(2): 443. Li L, Hassan HE, Tolson AH, Ferguson SS, Eddington ND and Wang H (2010) Differential Activation of Pregnane X Receptor and Constitutive Androstane Receptor by Buprenorphine in Primary Human Hepatocytes and HepG2 Cells. Journal of Pharmacology and Experimental Therapeutics 335(3): 562-571. Li L, Li H, Garzel B, Yang H, Sueyoshi T, Li Q, Shu Y, Zhang J, Hu B, Heyward S, Moeller T, Xie W, Negishi M and Wang H (2015b) SLC13A5 Is a Novel Transcriptional Target of the Pregnane X Receptor and Sensitizes Drug-Induced Steatosis in Human Liver. Molecular Pharmacology 87(4): 674-682. Lichti-Kaiser K, Brobst D, Xu C and Staudinger JL (2009) A Systematic Analysis of Predicted Phosphorylation Sites within the Human Pregnane X Receptor Protein. Journal of Pharmacology and Experimental Therapeutics 331(1): 65-76. Lin W, Wu J, Dong H, Bouck D, Zeng F-Y and Chen T (2008) Cyclin-dependent Kinase 2 Negatively Regulates Human Pregnane X Receptor-mediated CYP3A4 Gene Expression in HepG2 Liver Carcinoma Cells. Journal of Biological Chemistry 283(45): 30650-30657. Ling Z, Shu N, Xu P, Wang F, Zhong Z, Sun B, Li F, Zhang M, Zhao K, Tang X, Wang Z, Zhu L, Liu L and Liu X (2016) Involvement of pregnane X receptor in the impaired glucose utilization induced by atorvastatin in hepatocytes. Biochemical Pharmacology 100: 98-111. Lynch C, Li H and Wang H (2014a) Characterization of Constitutive Androstane Receptor (CAR) Activation, in Optimization in Drug Discovery: In Vitro Methods (Caldwell GW and Yan Z eds) pp 195-205, Humana Press, Totowa, NJ. Lynch C, Mackowiak B, Huang R, Li L, Heyward S, Sakamuru S, Wang H and Xia M (2018) Identification of Modulators That Activate the Constitutive Androstane Receptor From the Tox21 10K Compound Library. Toxicological Sciences: kfy242-kfy242. Lynch C, Pan Y, Li L, Ferguson SS, Xia M, Swaan PW and Wang H (2013) Identification of Novel Activators of Constitutive Androstane Receptor from FDA-Approved Drugs by Integrated Computational and Biological Approaches. Pharmaceutical Research 30(2): 489-501. Lynch C, Pan Y, Li L, Heyward S, Moeller T, Swaan PW and Wang H (2014b) Activation of the constitutive androstane receptor inhibits gluconeogenesis

133

without affecting lipogenesis or fatty acid synthesis in human hepatocytes. Toxicology and Applied Pharmacology 279(1): 33-42. Lynch C, Zhao J, Huang R, Xiao J, Li L, Heyward S, Xia M and Wang H (2015) Quantitative High-Throughput Identification of Drugs as Modulators of Human Constitutive Androstane Receptor. Scientific Reports 5: 10405. Ma Y and Liu D (2012) Activation of Pregnane X Receptor by Pregnenolone 16 α- carbonitrile Prevents High-Fat Diet-Induced Obesity in AKR/J Mice. PLOS ONE 7(6): e38734. Mackowiak B, Hodge J, Stern S and Wang H (2018) The Roles of Xenobiotic Receptors: Beyond Chemical Disposition. Drug Metabolism and Disposition. Mackowiak B, Li L, Welch MA, Li D, Jones JW, Heyward S, Kane MA, Swaan PW and Wang H (2017) Molecular Basis of Metabolism-Mediated Conversion of PK11195 from an Antagonist to an Agonist of the Constitutive Androstane Receptor. Molecular Pharmacology 92(1): 75. Mackowiak B and Wang H (2016) Mechanisms of xenobiotic receptor activation: Direct vs. indirect. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1859(9): 1130-1140. Maeda J, Inoue K, Ichimura R, Takahashi M, Kodama Y, Saito N and Yoshida M (2015) Essential role of constitutive androstane receptor in Ginkgo biloba extract induced liver hypertrophy and hepatocarcinogenesis. Food and Chemical Toxicology 83: 201-209. Maglich JM, Parks DJ, Moore LB, Collins JL, Goodwin B, Billin AN, Stoltz CA, Kliewer SA, Lambert MH, Willson TM and Moore JT (2003) Identification of a Novel Human Constitutive Androstane Receptor (CAR) Agonist and Its Use in the Identification of CAR Target Genes. Journal of Biological Chemistry 278(19): 17277-17283. Maglich JM, Watson J, McMillen PJ, Goodwin B, Willson TM and Moore JT (2004) The Nuclear Receptor CAR Is a Regulator of Thyroid Hormone Metabolism during Caloric Restriction. Journal of Biological Chemistry 279(19): 19832-19838. Mäkinen J, Frank C, Jyrkkärinne J, Gynther J, Carlberg C and Honkakoski P (2002) Modulation of Mouse and Human Phenobarbital-Responsive Enhancer Module by Nuclear Receptors. Molecular Pharmacology 62(2): 366-378. MÄKINEN J, REINISALO M, NIEMI K, VIITALA P, JYRKKÄRINNE J, CHUNG H, PELKONEN O and HONKAKOSKI P (2003) Dual action of oestrogens on the mouse constitutive androstane receptor. Biochemical Journal 376(2): 465-472. Mani S, Huang H, Sundarababu S, Liu W, Kalpana G, Smith AB and Horwitz SB (2005) Activation of the steroid and xenobiotic receptor (human pregnane X receptor) by nontaxane microtubule-stabilizing agents. Clin Cancer Res 11(17): 6359-6369. Marija P, Elsa P and Judy LR (2017) Transactivation Assays that Identify Indirect and Direct Activators of Human Pregnane X Receptor (PXR, NR1I2) and Constitutive Androstane Receptor (CAR, NR1I3). Drug Metabolism Letters 11(2): 128-137. Marmugi A, Lukowicz C, Lasserre F, Montagner A, Polizzi A, Ducheix S, Goron A, Gamet-Payrastre L, Gerbal-Chaloin S, Pascussi JM, Moldes M, Pineau T, Guillou H and Mselli-Lakhal L (2016) Activation of the Constitutive Androstane Receptor induces hepatic lipogenesis and regulates Pnpla3 gene expression in a LXR-

134

independent way. Toxicology and Applied Pharmacology 303(Supplement C): 90- 100. Martini T, Stojan J, Rozman D and Prosenc Zmrzljak U (2017) Interaction of PER2 with the Constitutive Androstane Receptor Possibly Links Circadian Rhythms to Metabolism. Acta Chim Slov 64(3): 571-576. Masuyama H and Hiramatsu Y (2012) Treatment with a constitutive androstane receptor ligand ameliorates the signs of preeclampsia in high-fat diet-induced obese pregnant mice. Molecular and Cellular Endocrinology 348(1): 120-127. Masuyama H, Nakatsukasa H, Takamoto N and Hiramatsu Y (2007) Down-regulation of pregnane X receptor contributes to cell growth inhibition and apoptosis by anticancer agents in endometrial cancer cells. Mol Pharmacol 72(4): 1045-1053. McKenna NJ, Lanz RB and O’Malley BW (1999) Nuclear Receptor Coregulators: Cellular and Molecular Biology. Endocrine Reviews 20(3): 321-344. Mencarelli A, Migliorati M, Barbanti M, Cipriani S, Palladino G, Distrutti E, Renga B and Fiorucci S (2010) Pregnane-X-receptor mediates the anti-inflammatory activities of rifaximin on detoxification pathways in intestinal epithelial cells. Biochem Pharmacol 80(11): 1700-1707. Miao J, Fang S, Bae Y and Kemper JK (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-1α. Journal of Biological Chemistry 281(21): 14537-14546. Miki Y, Suzuki T, Kitada K, Yabuki N, Shibuya R, Moriya T, Ishida T, Ohuchi N, Blumberg B and Sasano H (2006) Expression of the steroid and xenobiotic receptor and its possible target gene, organic anion transporting polypeptide-A, in human breast carcinoma. Cancer Res 66(1): 535-542. Min G, Kemper JK and Kemper B (2002) Glucocorticoid Receptor-interacting Protein 1 Mediates Ligand-independent Nuclear Translocation and Activation of Constitutive Androstane Receptor in Vivo. Journal of Biological Chemistry 277(29): 26356-26363. Mo L, Shen J, Liu Q, Zhang Y, Kuang J, Pu S, Cheng S, Zou M, Jiang W, Jiang C, Qu A and He J (2016) Irisin Is Regulated by CAR in Liver and Is a Mediator of Hepatic Glucose and Lipid Metabolism. Molecular Endocrinology 30(5): 533-542. Moore LB, Parks DJ, Jones SA, Bledsoe RK, Consler TG, Stimmel JB, Goodwin B, Liddle C, Blanchard SG, Willson TM, Collins JL and Kliewer SA (2000) Orphan Nuclear Receptors Constitutive Androstane Receptor and Pregnane X Receptor Share Xenobiotic and Steroid Ligands. Journal of Biological Chemistry 275(20): 15122-15127. Moreau A, Téruel C, Beylot M, Albalea V, Tamasi V, Umbdenstock T, Parmentier Y, Sa-Cunha A, Suc B, Fabre J-M, Navarro F, Ramos J, Meyer U, Maurel P, Vilarem M-J and Pascussi J-M (2009) A novel pregnane X receptor and S14- mediated lipogenic pathway in human hepatocyte. Hepatology 49(6): 2068-2079. Muangmoonchai R, Smirlis D, Wong S-C, Edwards M, Phillips IR and Shephard EA (2001) Xenobiotic induction of cytochrome P450 2B1 (CYP2B1) is mediated by the orphan nuclear receptor constitutive androstane receptor (CAR) and requires

135

steroid co-activator 1 (SRC-1) and the transcription factor Sp1. Biochemical Journal 355(1): 71. Mutoh S, Osabe M, Inoue K, Moore R, Pedersen L, Perera L, Rebolloso Y, Sueyoshi T and Negishi M (2009) Dephosphorylation of Threonine 38 Is Required for Nuclear Translocation and Activation of Human Xenobiotic Receptor CAR (NR1I3). Journal of Biological Chemistry 284(50): 34785-34792. Mutoh S, Sobhany M, Moore R, Perera L, Pedersen L, Sueyoshi T and Negishi M (2013) Phenobarbital Indirectly Activates the Constitutive Active Androstane Receptor (CAR) by Inhibition of Epidermal Growth Factor Receptor Signaling. Science Signaling 6(274): ra31-ra31. Nagy L and Schwabe JWR (2004) Mechanism of the nuclear receptor molecular switch. Trends in Biochemical Sciences 29(6): 317-324. Nakamura K, Moore R, Negishi M and Sueyoshi T (2007) Nuclear Pregnane X Receptor Cross-talk with FoxA2 to Mediate Drug-induced Regulation of Lipid Metabolism in Fasting Mouse Liver. Journal of Biological Chemistry 282(13): 9768-9776. Nam JS, Nam JY, Yoo JS, Cho M, Park JS, Ahn CW, Cha BS, Lee EJ, Lim SK, Kim KR and Lee HC (2010) The effect of mosapride (5HT-4 receptor agonist) on insulin sensitivity and GLUT4 translocation. Diabetes Research and Clinical Practice 87(3): 329-334. Ngan C-H, Beglov D, Rudnitskaya AN, Kozakov D, Waxman DJ and Vajda S (2009) The Structural Basis of Pregnane X Receptor Binding Promiscuity. Biochemistry 48(48): 11572-11581. Niu B, Coslo DM, Omiecinski CJ, Bataille AR, Pugh BF and Albert I (2018) In vivo genome-wide binding interactions of mouse and human constitutive androstane receptors reveal novel gene targets. Nucleic Acids Research 46(16): 8385-8403. Nonogaki K and Kaji T (2015) Mosapride, a selective serotonin 5-HT4 receptor agonist, and alogliptin, a selective dipeptidyl peptidase-4 inhibitor, exert synergic effects on plasma active GLP-1 levels and glucose tolerance in mice. Diabetes Research and Clinical Practice 110(3): e18-e21. Oetjen LK, Trier AM and Kim BS (2018) PXR: A New Player in Atopic Dermatitis. J Invest Dermatol 138(1): 8-10. Okuda Y, Kushida M, Sumida K, Nagahori H, Nakamura Y, Higuchi H, Kawamura S, Lake BG, Cohen SM and Yamada T (2017) Editor’s Highlight: Mode of Action Analysis for Rat Hepatocellular Tumors Produced by the Synthetic Pyrethroid Momfluorothrin: Evidence for Activation of the Constitutive Androstane Receptor and Mitogenicity in Rat Hepatocytes. Toxicological Sciences 158(2): 412-430. Oladimeji PO and Chen T (2018) PXR: More Than Just a Master Xenobiotic Receptor. Mol Pharmacol 93(2): 119-127. Oladimeji PO, Lin W, Brewer CT and Chen T (2017) Glucose-dependent regulation of pregnane X receptor is modulated by AMP-activated protein kinase. Scientific Reports 7: 46751. Omiecinski CJ, Coslo DM, Chen T, Laurenzana EM and Peffer RC (2011) Multi-species Analyses of Direct Activators of the Constitutive Androstane Receptor. Toxicological Sciences 123(2): 550-562.

136

Orans J, Teotico DG and Redinbo MR (2005) The Nuclear Xenobiotic Receptor Pregnane X Receptor: Recent Insights and New Challenges. Molecular Endocrinology 19(12): 2891-2900. Osabe M and 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. Journal of Biological Chemistry 286(41): 35763-35769. Ostberg T, Bertilsson G, Jendeberg L, Berkenstam A and Uppenberg J (2002) Identification of residues in the PXR ligand binding domain critical for species specific and constitutive activation. Eur J Biochem 269(19): 4896-4904. Ouyang N, Ke S, Eagleton N, Xie Y, Chen G, Laffins B, Yao H, Zhou B and Tian Y (2010) Pregnane X receptor suppresses proliferation and tumourigenicity of colon cancer cells. British Journal Of Cancer 102: 1753. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, Stimmel JB, Willson TM, Zavacki AM, Moore DD and Lehmann JM (1999) Bile acids: natural ligands for an orphan nuclear receptor. Science 284(5418): 1365-1368. Phillips JM, Yamamoto Y, Negishi M, Maronpot RR and Goodman JI (2007) Orphan Nuclear Receptor Constitutive Active/Androstane Receptor–Mediated Alterations in DNA Methylation during Phenobarbital Promotion of Liver Tumorigenesis. Toxicological Sciences 96(1): 72-82. Polyzos SA, Kountouras J, Anastasilakis AD, Geladari EV and Mantzoros CS (2014) Irisin in patients with nonalcoholic fatty liver disease. Metabolism 63(2): 207-217. Pondugula SR, Flannery PC, Apte U, Babu JR, Geetha T, Rege SD, Chen T and Abbott KL (2015) Mg2+/Mn2+-Dependent Phosphatase 1A Is Involved in Regulating Pregnane X Receptor–Mediated Cytochrome p450 3A4 Gene Expression. Drug Metabolism and Disposition 43(3): 385-391. Pondugula SR, Pavek P and Mani S (2016) Pregnane X Receptor and Cancer: Context- Specificity is Key. Nucl Receptor Res 3. Qatanani M and Moore DD (2005) CAR, the continuously advancing receptor, in drug metabolism and disease. Current Drug Metabolism 6(4): 329-339. Ratajewski M, Grzelak I, Wiśniewska K, Ryba K, Gorzkiewicz M, Walczak-Drzewiecka A, Hoffmann M and Dastych J (2015) Screening of a chemical library reveals novel PXR-activating pharmacologic compounds. Toxicology Letters 232(1): 193- 202. Raucy JL and Lasker JM (2013) Cell-based systems to assess nuclear receptor activation and their use in drug development. Drug Metabolism Reviews 45(1): 101-109. Robbins D, Cherian M, Wu J and Chen T (2016) Human pregnane X receptor compromises the function of p53 and promotes malignant transformation. Cell death discovery 2: 16023. Roivainen A, Någren K, Hirvonen J, Oikonen V, Virsu P, Tolvanen T and Rinne JO (2008) Whole-body distribution and metabolism of [N-methyl-11C](R)-1-(2- chlorophenyl)-N-(1-methylpropyl)-3-isoquinolinecarboxamide in humans; an imaging agent for in vivo assessment of peripheral benzodiazepine receptor activity with positron emission tomography. European Journal of Nuclear Medicine and Molecular Imaging 36(4): 671-682.

137

Romeo S, Kozlitina J, Xing C, Pertsemlidis A, Cox D, Pennacchio LA, Boerwinkle E, Cohen JC and Hobbs HH (2008) Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nature Genetics 40: 1461. Rysa J, Buler M, Savolainen MJ, Ruskoaho H, Hakkola J and Hukkanen J (2013) Pregnane X receptor agonists impair postprandial glucose tolerance. Clin Pharmacol Ther 93(6): 556-563. Sakashita M, Yamaguchi T, Miyazaki H, Sekine Y, Nomiyama T, Tanaka S, Miwa T and Harasawa S (1993) Pharmacokinetics of the gastrokinetic agent mosapride citrate after single and multiple oral administrations in healthy subjects. Arzneimittelforschung 43(8): 867-872. Saradhi M, Sengupta A, Mukhopadhyay G and Tyagi RK (2005) Pregnane and Xenobiotic Receptor (PXR/SXR) resides predominantly in the nuclear compartment of the interphase cell and associates with the condensed chromosomes during mitosis. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1746(2): 85-94. Savas Ü, Wester MR, Griffin KJ and Johnson EF (2000) Rabbit Pregnane X Receptor Is Activated by Rifampicin. Drug Metabolism and Disposition 28(5): 529-537. Shizu R, Benoki S, Numakura Y, Kodama S, Miyata M, Yamazoe Y and Yoshinari K (2013) Xenobiotic-Induced Hepatocyte Proliferation Associated with Constitutive Active/Androstane Receptor (CAR) or Peroxisome Proliferator-Activated Receptor α (PPARα) Is Enhanced by Pregnane X Receptor (PXR) Activation in Mice. PLOS ONE 8(4): e61802. Shizu R, Shindo S, Yoshida T and Numazawa S (2012) MicroRNA-122 down-regulation is involved in phenobarbital-mediated activation of the constitutive androstane receptor. PLoS One 7(7): e41291. Shukla SJ, Nguyen D-T, MacArthur R, Simeonov A, Frazee WJ, Hallis TM, Marks BD, Singh U, Eliason HC, Printen J, Austin CP, Inglese J and Auld DS (2009) Identification of Pregnane X Receptor Ligands Using Time-Resolved Fluorescence Resonance Energy Transfer and Quantitative High-Throughput Screening. Assay and Drug Development Technologies 7(2): 143-169. Smagris E, BasuRay S, Li J, Huang Y, Lai K-mV, Gromada J, Cohen JC and Hobbs HH (2015) Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis. Hepatology 61(1): 108-118. Smith CM, Graham RA, Krol WL, Silver IS, Negishi M, Wang H and Lecluyse EL (2005) Differential UGT1A1 Induction by Chrysin in Primary Human Hepatocytes and HepG2 Cells. Journal of Pharmacology and Experimental Therapeutics 315(3): 1256. Soldatow V, Peffer RC, Trask OJ, Cowie DE, Andersen ME, LeCluyse E and Deisenroth C (2016) Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation. Toxicology in Vitro 36: 224-237. Sonoda J, Pei L and Evans RM (2008) Nuclear receptors: Decoding metabolic disease. FEBS Letters 582(1): 2-9. Squires EJ, Sueyoshi T and Negishi M (2004) Cytoplasmic Localization of Pregnane X Receptor and Ligand-dependent Nuclear Translocation in Mouse Liver. Journal of Biological Chemistry 279(47): 49307-49314.

138

Stanley L, Horsburgh B, Ross J, Scheer N and Roland Wolf C (2006) PXR and CAR: Nuclear Receptors which Play a Pivotal Role in Drug Disposition and Chemical Toxicity. Drug Metabolism Reviews 38(3): 515-597. Sugatani J, Hattori Y, Noguchi Y, Yamaguchi M, Yamazaki Y and Ikari A (2014) Threonine-290 Regulates Nuclear Translocation of the Human Pregnane X Receptor through Its Phosphorylation/Dephosphorylation by Ca2+/Calmodulin- Dependent Protein Kinase II and Protein Phosphatase 1. Drug Metabolism and Disposition 42(10): 1708-1718. Suino K, Peng L, Reynolds R, Li Y, Cha J-Y, Repa JJ, Kliewer SA and Xu HE (2004) The Nuclear Xenobiotic Receptor CAR: Structural Determinants of Constitutive Activation and Heterodimerization. Molecular Cell 16(6): 893-905. Swales K, Kakizaki S, Yamamoto Y, Inoue K, Kobayashi K and Negishi M (2005) Novel CAR-mediated Mechanism for Synergistic Activation of Two Distinct Elements within the Human Cytochrome P450 2B6 Gene in HepG2 Cells. Journal of Biological Chemistry 280(5): 3458-3466. Takasu N, Yamada T, Miura H, Sakamoto S, Korenaga M, Nakajima K and Kanayama M (1982) Rifampicin-induced Early Phase Hyperglycemia in Humans. American Review of Respiratory Disease 125(1): 23-27. Takwi AA, Wang YM, Wu J, Michaelis M, Cinatl J and Chen T (2013) miR-137 regulates the constitutive androstane receptor and modulates doxorubicin sensitivity in parental and doxorubicin-resistant neuroblastoma cells. Oncogene 33: 3717. Tamura K, Inoue K, Takahashi M, Matsuo S, Irie K, Kodama Y, Gamo T, Ozawa S and Yoshida M (2015) Involvement of constitutive androstane receptor in liver hypertrophy and liver tumor development induced by triazole fungicides. Food and Chemical Toxicology 78: 86-95. Tamura K, Inoue K, Takahashi M, Matsuo S, Kodama Y and Yoshida M (2016) A crucial role of constitutive androstane receptor (CAR) in liver tumor development by imazalil in mice. The Journal of Toxicological Sciences 41(6): 801-811. Terc J, Hansen A, Alston L and Hirota SA (2014) Pregnane X receptor agonists enhance intestinal epithelial wound healing and repair of the intestinal barrier following the induction of experimental colitis. Eur J Pharm Sci 55: 12-19. Timsit YE and Negishi M (2007) CAR and PXR: the xenobiotic-sensing receptors. Steroids 72(3): 231-246. Timsit YE and Negishi M (2014) Coordinated regulation of nuclear receptor CAR by CCRP/DNAJC7, HSP70 and the ubiquitin-proteasome system. PLoS One 9(5): e96092. Tirona RG, Lee W, Leake BF, Lan L-B, Cline CB, Lamba V, Parviz F, Duncan SA, Inoue Y, Gonzalez FJ, Schuetz EG and Kim RB (2003) The orphan nuclear receptor HNF4α determines PXR- and CAR-mediated xenobiotic induction of CYP3A4. Nature Medicine 9: 220. Tolson AH and Wang H (2010) Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR. Advanced Drug Delivery Reviews 62(13): 1238-1249. Tschuor C, Kachaylo E, Limani P, Raptis DA, Linecker M, Tian Y, Herrmann U, Grabliauskaite K, Weber A, Columbano A, Graf R, Humar B and Clavien P-A

139

(2016) Constitutive androstane receptor (Car)-driven regeneration protects liver from failure following tissue loss. Journal of Hepatology 65(1): 66-74. Tzameli I, Pissios P, Schuetz EG and Moore DD (2000) The Xenobiotic Compound 1,4- Bis[2-(3,5-Dichloropyridyloxy)]Benzene Is an Agonist Ligand for the Nuclear Receptor CAR. Molecular and Cellular Biology 20(9): 2951-2958. Ueda A, Hamadeh HK, Webb HK, Yamamoto Y, Sueyoshi T, Afshari CA, Lehmann JM and Negishi M (2002) Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital. Mol Pharmacol 61(1): 1-6. Ueno N, Inui A, Asakawa A, Takao F, Komatsu Y, Kotani K, Nishimura R and Kasuga M (2002) Mosapride, a 5HT-4 receptor agonist, improves insulin sensitivity and glycaemic control in patients with Type II diabetes mellitus. Diabetologia 45(6): 792-797. Ueno N and Satoh Y (2009) Rapid effect of mosapride citrate, 5-HT4 receptor agonist, on fasting blood glucose in Type 2 diabetes patients. Journal of Diabetes and its Complications 23(4): 255-256. van der Mark VA, Rudi de Waart D, Shevchenko V, Elferink RPJO, Chamuleau RAFM and Hoekstra R (2017) Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells. Drug Metabolism and Disposition 45(1): 56. Wagner M, Halilbasic E, Marschall H-U, Zollner G, Fickert P, Langner C, Zatloukal K, Denk H and Trauner M (2005) CAR and PXR agonists stimulate hepatic bile acid and bilirubin detoxification and elimination pathways in mice. Hepatology 42(2): 420-430. Wang D, Li L, Yang H, Ferguson SS, Baer MR, Gartenhaus RB and Wang H (2013) The constitutive androstane receptor is a novel therapeutic target facilitating cyclophosphamide-based treatment of hematopoietic malignancies. Blood 121(2): 329-338. Wang H, Faucette S, Moore R, Sueyoshi T, Negishi M and LeCluyse E (2004) Human Constitutive Androstane Receptor Mediates Induction of CYP2B6 Gene Expression by Phenytoin. Journal of Biological Chemistry 279(28): 29295-29301. Wang H and LeCluyse E (2003) Role of Orphan Nuclear Receptors in the Regulation of Drug-Metabolising Enzymes. Clin Pharmacokinet 42(15): 1331-1357. Wang H, Venkatesh M, Li H, Goetz R, Mukherjee S, Biswas A, Zhu L, Kaubisch A, Wang L, Pullman J, Whitney K, Kuro-o M, Roig AI, Shay JW, Mohammadi M and Mani S (2011) Pregnane X receptor activation induces FGF19-dependent tumor aggressiveness in humans and mice. J Clin Invest 121(8): 3220-3232. Wang YM, Ong SS, Chai SC and Chen T (2012) Role of CAR and PXR in xenobiotic sensing and metabolism. Expert Opin Drug Metab Toxicol 8(7): 803-817. Wang Z, Li X, Wu Q, Lamb JC and Klaunig JE (2017) Toxaphene-induced mouse liver tumorigenesis is mediated by the constitutive androstane receptor. Journal of Applied Toxicology 37(8): 967-975. Watkins RE, Davis-Searles PR, Lambert MH and Redinbo MR (2003a) Coactivator Binding Promotes the Specific Interaction Between Ligand and the Pregnane X Receptor. Journal of Molecular Biology 331(4): 815-828. Watkins RE, Maglich JM, Moore LB, Wisely GB, Noble SM, Davis-Searles PR, Lambert MH, Kliewer SA and Redinbo MR (2003b) 2.1 Å Crystal Structure of Human

140

PXR in Complex with the St. John's Wort Compound Hyperforin†,‡. Biochemistry 42(6): 1430-1438. Watkins RE, Wisely GB, Moore LB, Collins JL, Lambert MH, Williams SP, Willson TM, Kliewer SA and Redinbo MR (2001) The Human Nuclear Xenobiotic Receptor PXR: Structural Determinants of Directed Promiscuity. Science 292(5525): 2329-2333. Wei P, Zhang J, Egan-Hafley M, Liang S and Moore DD (2000) The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature 407: 920. Williamson B, Lorbeer M, Mitchell MD, Brayman TG and Riley RJ (2016) Evaluation of a novel PXR‐knockout in HepaRG(™) cells. Pharmacology Research & Perspectives 4(5): e00264. Windshügel B, Jyrkkärinne J, Poso A, Honkakoski P and Sippl W (2005) Molecular dynamics simulations of the human CAR ligand-binding domain: deciphering the molecular basis for constitutive activity. Journal of Molecular Modeling 11(1): 69-79. Wu G, Robertson DH, Brooks CL, 3rd and Vieth M (2003) Detailed analysis of grid- based molecular docking: A case study of CDOCKER-A CHARMm-based MD docking algorithm. J Comput Chem 24(13): 1549-1562. Xiao L, Nickbarg E, Wang W, Thomas A, Ziebell M, Prosise WW, Lesburg CA, Taremi SS, Gerlach VL, Le HV and Cheng KC (2011) Evaluation of in vitro PXR-based assays and in silico modeling approaches for understanding the binding of a structurally diverse set of drugs to PXR. Biochemical Pharmacology 81(5): 669- 679. Xu RX, Lambert MH, Wisely BB, Warren EN, Weinert EE, Waitt GM, Williams JD, Collins JL, Moore LB, Willson TM and Moore JT (2004) A Structural Basis for Constitutive Activity in the Human CAR/RXRα Heterodimer. Molecular Cell 16(6): 919-928. Yamada T, Okuda Y, Kushida M, Sumida K, Takeuchi H, Nagahori H, Fukuda T, Lake BG, Cohen SM and Kawamura S (2014) Human Hepatocytes Support the Hypertrophic but not the Hyperplastic Response to the Murine Nongenotoxic Hepatocarcinogen Sodium Phenobarbital in an In Vivo Study Using a Chimeric Mouse with Humanized Liver. Toxicological Sciences 142(1): 137-157. Yamamoto Y, Kawamoto T and Negishi M (2003) The role of the nuclear receptor CAR as a coordinate regulator of hepatic gene expression in defense against chemical toxicity. Arch Biochem Biophys 409(1): 207-211. Yamamoto Y, Moore R, Flavell RA, Lu B and Negishi M (2010) Nuclear Receptor CAR Represses TNFα-Induced Cell Death by Interacting with the Anti-Apoptotic GADD45B. PLOS ONE 5(4): e10121. Yamamoto Y, Moore R, Goldsworthy TL, Negishi M and Maronpot RR (2004) The Orphan Nuclear Receptor Constitutive Active/Androstane Receptor Is Essential for Liver Tumor Promotion by Phenobarbital in Mice. Cancer Research 64(20): 7197-7200. Yoshinari K, Kobayashi K, Moore R, Kawamoto T and Negishi M (2003) Identification of the nuclear receptor CAR:HSP90 complex in mouse liver and recruitment of protein phosphatase 2A in response to phenobarbital. FEBS Letters 548(1): 17-20.

141

Yoshinari K, Sueyoshi T, Moore R and Negishi M (2001) Nuclear Receptor CAR as a Regulatory Factor for the Sexually Dimorphic Induction of CYP2B1 Gene by Phenobarbital in Rat Livers. Molecular Pharmacology 59(2): 278-284. Young-Hoon K, Young-Ji B, Hyung Soo K, Hey-Jin C, Jae-Suk Y, Ji-Soon S, Won-Keun S, Yong-Moon L and and Kyoung-Moon H (2015) Measurement of Human Cytochrome P450 Enzyme Induction Based on Mesalazine and Mosapride Citrate Treatments Using a Luminescent Assay. Biomol Ther (Seoul) 23(5): 486-492. Yu L, Wang Z, Huang M, Li Y, Zeng K, Lei J, Hu H, Chen B, Lu J, Xie W and Zeng S (2016) Evodia alkaloids suppress gluconeogenesis and lipogenesis by activating the constitutive androstane receptor. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1859(9): 1100-1111. Zelko I and Negishi M (2000) Phenobarbital-Elicited Activation of Nuclear Receptor CAR in Induction of Cytochrome P450 Genes. Biochemical and Biophysical Research Communications 277(1): 1-6. Zelko I, Sueyoshi T, Kawamoto T, Moore R and Negishi M (2001a) The Peptide Near the C Terminus Regulates Receptor CAR Nuclear Translocation Induced by Xenochemicals in Mouse Liver. Molecular and Cellular Biology 21(8): 2838. Zelko I, Sueyoshi T, Kawamoto T, Moore R and Negishi M (2001b) The peptide near the C terminus regulates receptor CAR nuclear translocation induced by xenochemicals in mouse liver. Mol Cell Biol 21(8): 2838-2846. Zhang H-J, Zhang X-F, Ma Z-M, Pan L-L, Chen Z, Han H-W, Han C-K, Zhuang X-J, Lu Y, Li X-J, Yang S-Y and Li X-Y (2013) Irisin is inversely associated with intrahepatic triglyceride contents in obese adults. Journal of Hepatology 59(3): 557-562. Zhang J-H, Chung TDY and Oldenburg KR (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. Journal of Biomolecular Screening 4(2): 67-73. Zhou J, Liu M, Zhai Y and Xie W (2008) The antiapoptotic role of pregnane X receptor in human colon cancer cells. Mol Endocrinol 22(4): 868-880. Zhou J, Zhai Y, Mu Y, Gong H, Uppal H, Toma D, Ren S, Evans RM and Xie W (2006) A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent Lipogenic Pathway. Journal of Biological Chemistry 281(21): 15013-15020. Zhuo W, Hu L, Lv J, Wang H, Zhou H and Fan L (2014) Role of pregnane X receptor in chemotherapeutic treatment. Cancer Chemother Pharmacol 74(2): 217-227. Zucchini N, de Sousa G, Bailly-Maitre B, Gugenheim J, Bars R, Lemaire G and Rahmani R (2005) Regulation of Bcl-2 and Bcl-xL anti-apoptotic protein expression by nuclear receptor PXR in primary cultures of human and rat hepatocytes. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1745(1): 48-58.

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