Regulation of the Rat Hepatic NADPH-Cytochrome P450 Oxidoreductase by Glucocorticoids

Regulation of the rat hepatic NADPH-cytochrome P450 oxidoreductase by glucocorticoids

Alex Vonk

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Pharmacology and Toxicology

University of Toronto

Regulation of the rat hepatic NADPH-cytochrome P450 oxidoreductase by glucocorticoids. Master of Science, 2014. Alex Vonk, Department of Pharmacology & Toxicology, University of Toronto.

NADPH-Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal P450s. Rat hepatic POR expression is induced by dexamethasone (DEX), a synthetic glucocorticoid that activates the glucocorticoid (GR) and pregnane X receptors (PXR). This thesis addressed the roles of GR and PXR in rat hepatic POR regulation. A low GR-activating DEX dose induced POR mRNA levels by 3-fold at 6 h; a high DEX dose that activated GR and PXR induced

POR mRNA levels by 5-fold at 6 h and increased POR protein and catalytic activity at 24 h.

Selective GR or PXR agonists alone failed to induce POR protein or activity. The GR antagonist

RU486 did not inhibit the DEX induction of POR expression. Induction of POR expression by DEX was significantly attenuated in PXR-knockout rats. Although GR activation may contribute to POR mRNA induction, induction of POR expression and function by DEX is primarily PXR-mediated.

ii Acknowledgements

My time in the Department of Pharmacology and Toxicology here at the University of

Toronto has been very rewarding both professionally and socially. I feel privileged to have been given the opportunity to study and learn in an environment that continues to put forth innovative research in the field of science. I would like to formally thank my supervisor, Dr. David S. Riddick, as he has been instrumental in my completion of this program. I have been very fortunate to study under his expertise, knowledge, and character. His patience and mentorship provided an excellent atmosphere to learn and allowed me to develop as a scientist.

I would like to extend my gratitude to the other members of the Riddick lab: Chunja Lee

(Laboratory Technician), Sarah Hunter (Colleague), and Dr. Anne Mullen Grey (prior lab member). I would like to thank Chunja for her help with the optimization and fine-tuning of the assays used in this study, along with her support throughout the completion of this program. I thank Sarah for aiding me in the completion of my experiments along with complementing a memorable lab experience. In addition, I thank Dr. Anne Mullen Grey for initiating studies of POR regulation by dexamethasone that provided the foundation for my thesis work.

I thank Dr. Grant for his role as my advisor during this program, providing me with positive feedback and constructive criticism throughout my time spent at the University of Toronto. I thank the Canadian Institutes of Health Research (CIHR) and the University of Toronto for financial support of this research. To the members of the Grant, Tyndale, Ramsey/Salaphour, Koren, and

Lanctot labs, it was great to attend social events with all of you and hopefully there will be more to come.

Finally I would like to thank my family (Don, Wendy, Leslie and Amanda Vonk) and friends

(Mac Kristman, Cassandra McTaggart, and Brandon Foster) for their constant support and

iii encouragement throughout the program. To my parents, Don and Wendy, I would like to thank you both for giving me the opportunity to pursue my goals.

iv Table of Contents

ABSTRACT…………………………………………………………………………………………..ii

ACKNOWLEDGEMENTS………………………………………………………………….………iii

TABLE OF CONTENTS………………………………………………………...... ………….v

LIST OF TABLES……………………………………………………………………...……………vii

LIST OF FIGURES………………………………………………………………...... ………viii

LIST OF ABBREVIATIONS……………………………………………………………….…….…xi

LIST OF ABSTRACTS…………………………………………………………………………….xiv

SECTION 1: INTRODUCTION……………………………………………………………………1

1.1 STATEMENT OF RESEARCH PROBLEM……………………………...... ………………..1 1.2 HYPOTHALAMIC-PITUITARY-ADRENAL AXIS………………..………………………....2 1.3 GLUCOCORTICOIDS……………………………………………………………...... …………7 1.3.1 Synthetic Glucocorticoids…………………………………………………………..…...9 1.3.2 Cushing’s Syndrome……………………………..…………………………………….12 1.3.3 Addison’s Disease……………………………………….……………………………..13 1.4 NUCLEAR RECEPTORS……………………………………………………………….………14 1.4.1 Glucocorticoid Receptor………………………………………………….....…………17 1.4.2 Pregnane X Receptor………………………………………………...... ………………23 1.4.3 GR-PXR Crosstalk…………………………………………....……………………….27 1.5 CYTOCHROME P450 ENZYMES………………………………………………………...... …30 1.5.1 NADPH-cytochrome P450 oxidoreductase…………………………....………………33 1.6 RESEARCH HYPOTHESIS…………………………………………………………...... ………38 1.7 SPECIFIC OBJECTIVES……………………………………………………………….....…….39 1.8 RATIONALE FOR THE EXPERIMENTAL APPROACH……………..…………………….40

SECTION 2: MATERIALS AND METHODS………………………………...………………….43

2.1 ANIMALS AND TREATMENT PROTOCOLS………………………...... ………………….43 2.2 RNA ISOLATION…………………………………………………………………...... ……..45 2.3 RNA INTEGRITY……………………………………………………………………...... ……..46 2.4 REVERSE TRANSCRIPTION…………………………………………………………...……..46 2.5 CONVENTIONAL RT-PCR…………………………………………………………...... …..48 2.6 QUANTITATIVE REAL-TIME PCR………………………...………………………………..50 2.7 MICROSOMAL FRACTIONATION……………………………………………………….....50 2.8 LOWRY PROTEIN ASSAY………………………………………………………………….…53 2.9 IMMUNOBLOTTING…………………………………………………………………...... ….54

v 2.10 CYTOCHROME C REDUCTION AS A MEASURE OF POR ACTIVITY………………...58 2.11 STATISTICAL ANALYSIS………………………………………………………...... …58

SECTION 3: RESULTS…………………………………………………………………...... …….61

3.1 DEX DOSE-RESPONSE……………………………………………………………………..….61 3.1.1 DEX dose-response: POR, TAT, CYP3A23 and PXR mRNA………...... ………..61 3.1.2 DEX dose-response: POR protein………………………………….....……………….64 3.1.3 DEX dose-response: POR activity……………………....…………………………….64 3.2 GR- AND PXR-SELECTIVE AGONISTS……………………………..……………………....67 3.2.1 GR- and PXR-selective agonists: POR, TAT, CYP3A23 and PXR mRNA…...... 67 3.2.2 GR- and PXR-selective agonists: POR protein……………………………….……….71 3.2.3 GR- and PXR-selective agonists: POR activity………………………….....………….71 3.3 GR ANTAGONISM…………………………………………………………………………….71 3.3.1 GR antagonism: POR, TAT, CYP3A23 and PXR mRNA…………………….…...…75 3.3.2 GR antagonism: POR protein…………………………………...……………………..75 3.3.3 GR antagonism: POR activity……………………………………...…………………..80 3.4 PXR-KO……………………………………………………………………………………...... 80 3.4.1 PXR-KO: POR, TAT, CYP3A23 and PXR mRNA……………………....…………..80 3.4.2 PXR-KO: POR protein…………………………...……………………………………84 3.4.3 PXR-KO: POR activity……………………………..…………………………………84

SECTION 4: DISCUSSION…………………………………………………………………...….88

4.1 SUMMARY OF MAIN FINDINGS…………………………………………………………...88 4.1.1 DEX dose-response………………………………………………....…………………88 4.1.2 GR- and PXR-selective agonists……………………………………………………….89 4.1.3 GR antagonism………………...... …………………………………………………….90 4.1.4 PXR-KO……………………………………………………………………….....……91 4.2 MOLECULAR MECHANISMS OF POR REGULATION……………………………………92 4.2.1 Transcriptional regulation of POR expression: a potential role for GR……….....…….93 4.2.2 Transcriptional regulation of POR expression: a potential role for PXR……...... ……..95 4.2.3 Post-transcriptional regulation of POR expression…………………....……………….96 4.2.4 Translational and post-translational regulation of POR expression……………...……98 4.2.5 GR-PXR crosstalk…………………………………….……………………………….99 4.3 PHYSIOLOGICAL AND PHARMACOLOGICAL RELEVANCE………………….………101 4.4 LIMITATIONS OF THE CURRENT STUDY……………………...………………………..103 4.4.1 Doses of DEX and limited time points……………………………….………………103 4.4.2 GR antagonism with RU486……………………………………...…………………..104 4.4.3 Rat strains……………………………………………....…………………………….105 4.5 FUTURE RESEARCH DIRECTIONS……………………………...... ……………………….106 4.6 SUMMARY AND SIGNIFICANCE…………………………...... ………………………109

SECTION 5: REFERENCES…………………………………………………………...... ….111

vi List of Tables

Section 2:

Table 2.1 Sequences of primers used for conventional RT-PCR and quantitative real-time PCR...... 49

vii List of Figures

Section 1:

Figure 1.1 Hypothalamic-Pituitary-Adrenal (HPA) axis……………...... ………..……....………3

Figure 1.2 Steroid Hormone biosynthetic pathway………...... …………………....……………..6

Figure 1.3 GR ligands…...... …………………………………...... 11

Figure 1.4 NR structural domains……………………………....………………………...... …...15

Figure 1.5 Signal transduction pathway of the GR…………………...... ………...... 19

Figure 1.6 Signal transduction pathway for rodent PXR………………………...... ….……..25

Figure 1.7 A model of the two-stage CYP3A23 induction by DEX in H4IIE cells……...... …....29

Figure 1.8 Cytochrome P450 catalytic cycle…………………………...... ………….32

Section 2:

Figure 2.1 Visualization of 28S and 18S rRNA in total RNA samples isolated from rats in the DEX dose-response study………………...... ……....………………………47

Figure 2.2 Conventional RT-PCR analysis with gel-based product detection to assess the specificity of TAT primers and the TAT amplicon size………...... ….…....51

Figure 2.3 Representative efficiency curves for quantitative real-time PCR analysis of POR and β-actin mRNA levels……………………...... ……………………………...52

Figure 2.4 Representative immunoblot and standard curve analysis of POR protein levels in rat liver microsomes………………...... ……………………………………...…..55

Section 3:

Figure 3.1 Real-time PCR analysis of hepatic POR mRNA levels in rats treated with varying doses of DEX…………………………………….....………….………….....62

Figure 3.2 Real-time PCR analysis of hepatic TAT and CYP3A23 mRNA levels in rats treated with varying doses of DEX……………………....………..………………....63

Figure 3.3 Real-time PCR analysis of hepatic PXR mRNA levels in rats treated with varying doses of DEX…………………………………………………...... 65

viii Figure 3.4 Immunoblot analysis of liver microsomal POR protein levels in rats treated with varying doses of DEX……………...………………………….…….….…....66

Figure 3.5 Microsomal POR-catalyzed cytochrome c reduction activity in rats treated with varying doses of DEX…………………………………....……….……..…...68

Figure 3.6 Real-time PCR analysis of hepatic POR mRNA levels in rats treated with TA (GR agonist) or PCN (PXR agonist)…………………………………...... ……..…69

Figure 3.7 Real-time PCR analysis of hepatic TAT and CYP3A23 mRNA levels in rats treated with TA (GR agonist) or PCN (PXR agonist)…………………….....….…...70

Figure 3.8 Real-time PCR analysis of hepatic PXR mRNA levels in rats treated with TA (GR agonist) or PCN (PXR agonist)………………………..…………………..…72

Figure 3.9 Immunoblot analysis of liver microsomal POR protein levels in rats treated with TA (GR agonist) or PCN (PXR agonist)…………………...………………….…73

Figure 3.10 Microsomal POR-catalyzed cytochrome c reduction activity in rats treated with TA (GR agonist) or PCN (PXR agonist)…………………...………………….…74

Figure 3.11 Real-time PCR analysis of hepatic POR mRNA levels in rats treated with low- or high-dose DEX in the absence or presence of the GR antagonist RU486…...76

Figure 3.12 Real-time PCR analysis of hepatic TAT and CYP3A23 mRNA levels in rats treated with low- or high-dose DEX in the absence or presence of the GR antagonist RU486……………………………………………………………...... ……...77

Figure 3.13 Real-time PCR analysis of hepatic PXR mRNA levels in rats treated with low- or high-dose DEX in the absence or presence of the GR antagonist RU486…...78

Figure 3.14 Immunoblot analysis of liver microsomal POR protein levels in rats treated with low- or high-dose DEX in the absence or presence of the GR antagonist RU486...79

Figure 3.15 Microsomal POR-catalyzed cytochrome c reduction activity in rats treated with low- or high-dose DEX in the absence or presence of the GR antagonist RU486...81

Figure 3.16 Real-time PCR analysis of hepatic POR mRNA levels in wild-type and PXR- knockout rats treated with low- or high-dose DEX………………...... …..….82

Figure 3.17 Real-time PCR analysis of hepatic TAT and CYP3A23 mRNA levels in wild-type and PXR-knockout rats treated with low- or high-dose DEX...... …….83

Figure 3.18 Real-time PCR analysis of hepatic PXR mRNA levels in wild-type and PXR- knockout rats treated with low- or high-dose DEX…………….……..….….85

ix Figure 3.19 Immunoblot analysis of liver microsomal POR protein levels in wild-type and PXR- knockout rats treated with low- or high-dose DEX…………...... ………....86

Figure 3.20 Microsomal POR-catalyzed cytochrome c reduction activity in wild-type and PXR- knockout rats treated with low- or high-dose DEX……………...... ……....87

x List of Abbreviations

ΔΔCt comparative threshold cycle method 11β-HSD1 11β-hydroxysteroid dehydrogenase type 1 isozyme 11β-HSD2 11β-hydroxysteroid dehydrogenase type 2 isozyme AA arachidonic acid ABS Antley-Bixler syndrome ACTH adrenocorticotropic hormone ADX adrenalectomized AF-1 transcriptional activation domain AF-2 transcriptional activation domain AHR aryl hydrocarbon receptor ANOVA analysis of variance AP-1 activator protein-1 AR androgen receptor ARE AU-rich element ARNT aryl hydrocarbon receptor nuclear translocator AUBP A+U binding protein AVP arginine vasopressin BLAST basic local alignment search tool BSA bovine serum albumin CAH congenital adrenal hyperplasia CAR constitutive androstane receptor CBG corticosteroid-binding globulin CCRP cytoplasmic constitutive active/androstane receptor retention protein CES2 carboxylesterase-2 ChIP chromatin immunoprecipitation CORT corticosterone Cox-2 cyclooxygenase-2 CRF-R1 corticotropin releasing factor type 1 receptor CRH corticotropin-releasing hormone

Ct threshold cycle CYP cytochrome P450 DBD DNA-binding domain DDI drug-drug interaction DEX dexamethasone DHEA dehydroepiandrosterone DIO deiodinases DME drug-metabolizing enzyme DR direct repeat EGF epidermal growth factor ER estrogen receptor FAD flavin adenine dinucleotide FMN flavin mononucleotide FXR farnesoid X receptor

xi GHRH growth hormone-releasing hormone GILZ glucocorticoid leucine zipper GnRH gonadotropin-releasing hormone GR glucocorticoid receptor GRE glucocorticoid response element GST glutathione S-transferases HAT histone acetyltransferase HDAC2 histone deacetylase 2 HDL high-density lipoprotein HNF4α hepatocyte nuclear factor-4α HPA hypothalamic-pituitary-adrenal axis hPXR human PXR HSP heat shock protein HYPX hypophysectomized IGF-1 insulin-like growth factor-1 IL interleukin IRS-1 insulin response sequence-1 JAK janus kinase KO knock out LBD ligand-binding domain LXR liver X receptor MAO-A monoamine oxidase A MC2-R melanocortin receptor type 2 miRNA microRNA MMLV moloney murine leukemia virus MR mineralcorticoid receptor MRP2 multidrug resistance-associated protein 2 NR nuclear hormone receptor NADPH reduced nicotinamide adenine dinucleotide phosphate NF-κB nuclear factor-κB nGRE negative glucocorticoid response element PB phenobarbital PCN pregnenolone-16α-carbonitrile PCR polymerase chain reaction PEPCK phosphoenolpyruvate carboxykinase POMC pro-opiomelanocortin POR NADPH-cytochrome P450 oxidoreductase PORD POR deficiency PP peroxisome proliferator PPAR peroxisome proliferator-activated receptor PR progesterone receptor PTH1R parathyroid hormone receptor PXR pregnane x receptor PXRE pregnane xenobiotic-response elements PVN paraventricular nucleus

xii qPCR quantitative real-time PCR RAR retinoic acid receptor RT-PCR reverse transcriptase-PCR RXR retinoid-X-receptor SD standard deviation SHP short heterodimer partner SRC-1 steroid receptor co-activator 1 SST somatostatin STAT signal transduction and activator of transcription StAR steroidogenic acute regulatory protein T3 triiodothyronine T4 thyroxine TA triamcinolone acetonide TAT tyrosine aminotransferase TNF-α tumor necrosis factor-α TR thyroid hormone receptor TRE thyroid response element TRH thyrotropin-releasing hormone UGT uridine-5’-diphosphate glucuronosyl transferases VDR vitamin D receptor VEH vehicle

xiii List of Abstracts

A. Vonk and D.S. Riddick. (2013) Regulation of NADPH-cytochrome P450 oxidoreductase by glucocorticoids in rat liver. FASEB Journal 27: lb627. [Experimental Biology 2013; Boston, Massachusetts; April 2013]

A. Vonk and D.S. Riddick. (2013) Regulation of NADPH-cytochrome P450 oxidoreductase by glucocorticoids in rat liver. Visions in Pharmacology, p. 43. [Toronto, Ontario; June 2013]

A. Vonk and D.S. Riddick. (2013) Induction of rat hepatic NADPH-cytochrome P450 oxidoreductase by dexamethasone: are multiple receptors involved? Drug Metabolism Reviews 45(S1): 141-142. [10th International ISSX Meeting; Toronto, Ontario; September-October 2013]

Riddick DS, Hunter SR, and Vonk A. (2014) Role of glucocorticoid receptor and pregnane X receptor in dexamethasone induction of rat hepatic aryl hydrocarbon receptor nuclear translocator and NADPH-cytochrome P450 oxidoreductase. FASEB Journal 28(S1): lb597. [Experimental Biology 2014; San Diego, California; April 2014]

xiv 1. Introduction

1.1 Statement of Research Problem

NADPH-cytochrome P450 oxidoreductase (POR) is a vital flavoprotein that donates electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to monooxygenase enzymes involved in xenobiotic metabolism and other endogenous processes. Known as a focal point in the drug metabolism world, POR is often considered a rate-limiting step for the detoxification of many substances within the body. Although POR structure-function relationships have been characterized extensively (Riddick et al., 2013), less is known about the regulation of the expression and activity of this important enzyme. A key physiological regulator of POR expression is thyroid hormone (Waxman et al., 1989), acting via both transcriptional (Ram and Waxman, 1992; O’ Leary et al., 1997) and post-transcriptional (Liu and Waxman, 2002) mechanisms. This thesis is focused on the regulation of POR expression and activity by adrenal glucocorticoids. Adrenalectomized (ADX) rats show decreased hepatic POR activity that is rescued by cortisone acetate (Castro et al., 1970).

Dexamethasone (DEX) is a potent synthetic glucocorticoid with an interesting feature of activating the glucocorticoid receptor (GR, NR3C1) at low concentrations and the pregnane X receptor (PXR,

NR1I2) at higher concentrations. High-dose DEX increases rat hepatic POR mRNA levels via mRNA stabilization (Simmons et al., 1987). High-dose DEX increases hepatic POR protein levels in wild-type and Gr-null mice (Schuetz et al., 2000). In contrast, work from our lab showed that rat hepatic POR mRNA, but not protein, levels were increased by a low dose of DEX shown to activate

GR but not PXR (Mullen Grey, 2011). The goal of my thesis research was to characterize the role of

GR and PXR in the in vivo regulation of rat hepatic POR expression and activity by glucocorticoids.

This work can inform how POR-dependent processes may be modulated in conditions involving

1 altered glucocorticoid levels, such as stress, anti-inflammatory pharmacotherapy, or diseases of excess or deficiency.

1.2 Hypothalamic-Pituitary-Adrenal axis

Mammals experience various internal and external stressors on a regular basis. In an attempt to maintain homeostasis in the presence of these real or perceived stressors, multiple physiological responses may be triggered. One of the most vital responses activated involves the hypothalamic- pituitary-adrenal (HPA) axis, with the subsequent release of glucocorticoids into the bloodstream; cortisol is regarded as the main glucocorticoid in humans while corticosterone (CORT) is regarded as the main glucocorticoid in rodents (Buckingham, 2006). Glucocorticoids circulate and interact with multiple tissues to activate energy stores, induce lipolysis and proteolysis, potentiate vasoconstriction/cardiac output, and alter a number of stress related behaviours all in an attempt to maintain homeostasis (Herman et al., 2008; Papadimitriou and Priftis, 2009). The responses from these tissues allow the body to prepare for, respond to, and cope with different physical and emotional stressors (Sapolsky, 2000).

The HPA axis is depicted in Figure 1.1. This production and release of glucocorticoids into the bloodstream begins in the hypothalamus with the stimulation of neurons located in the median eminence of the paraventricular nucleus (PVN). The hypothalamus regulates multiple functions of the pituitary, master gland of the endocrine system, and is the principal integrating region for the entire autonomic nervous system. It regulates body temperature, water balance, intermediary metabolism, blood pressure, sexual and circadian cycles, secretions of the anterior pituitary, sleep, and emotion (Molinoff, 2011). The hypothalamus communicates with the periphery largely through the release of hormones such as growth hormone-releasing hormone (GHRH), gonadotropin-releasing hormone (GnRH), thyrotropin-releasing hormone (TRH), somatostatin (SST), and corticotropin-

Figure 1.1. Hypothalamic-Pituitary-Adrenal (HPA) axis. A diagrammatic representation of the HPA axis displaying the cascade of hormone release triggered by physical or emotional stress. Stimulation of the hypothalamus results in a release of corticotropin-releasing hormone (CRH), which stimulates the increased release of adrenocorticotropic hormone (ACTH). ACTH then stimulates de novo synthesis of cortisol and other steroids within the adrenal gland. These factors circulate and exert their effects on metabolic processes in target organs and provide negative feedback regulation on the anterior pituitary and hypothalamus.

3 releasing hormone (CRH). All of these releasing hormones are secreted from the hypothalamus into the hypophyseal portal system. This system of fenestrated blood vessels connecting the hypothalamus to the anterior pituitary allows for quick communication between glands (Parker and

Schimmer, 2011). Neurons in the PVN are known to be stimulated by neurotransmitters including norepinephrine, serotonin, and acetylcholine along with stressors such as injury, infection, or pain

(Parker and Schimmer, 2011).

These stimuli acting on the PVN trigger the production of the peptide hormone known as corticotropin-releasing hormone (CRH) (Dunn and Berridge, 1990). CRH was first isolated in 1981 by Vale and colleagues and is primarily involved in the adrenocroticotropic hormone (ACTH)-stress response (Vale et al., 1981). Upon release from the hypothalamus, CRH enters into the hypophyseal portal system, allowing delivery to the anterior pituitary where it binds to a G-protein coupled receptor known as CRF-type 1 (CRF-R1) located on corticotrophs of the anterior pituitary

(Aguilera et al., 2004; Guillemin, 2005).

CRH, along with other signals such as arginine vasopressin (AVP), induces pro- opiomelanocortin (POMC) gene expression within the corticotropic cells of the anterior pituitary.

This 267 amino-acid protein is then proteolytically cleaved into ACTH (Lamberts et al., 1984).

ACTH, under normal conditions, is secreted in a pulsatile manner under circadian rhythm; however, the secretion pattern can become modified due to the influence of peripheral corticosteroids or other stimuli (Papadimitriou and Priftis, 2009). ACTH is released into the peripheral circulation, where it acts upon the adrenal gland to stimulate adrenal glucocorticoid synthesis and release (Sayers, 1950).

The adrenal gland is comprised of two regions; 90% of the gland is considered the cortex and is involved in the production of steroid type hormones, while the remaining 10% is known as the medulla, which produces catecholamines. The cortex can be divided into three zones, the zona

4 glomerulosa, zona fasciculata, and zona reticularis. Each zone of the cortex requires the precursor pregnenolone to produce steroid type hormones, with pregneolone being derived from cholesterol, as seen in Figure 1.2 (Gorman, 2013). The zona glomerulosa is the outermost layer of the adrenal cortex and the primary site where the steroid hormone aldosterone is produced. Aldosterone is a mineralcorticoid and plays a key role in the kidney by regulating blood-fluid volumes through sodium retention as well as potassium release (Hungerford and Meikle, 2010). The zona reticularis is the innermost layer of the adrenal cortex and the primary site where androgens are produced, for example dehydroepiandrosterone (DHEA) (Conley and Bird, 1997). The zona fasciculata is the central layer of the adrenal cortex and is responsible for the production of cortisol in humans and corticosterone in rodents, the major glucocorticoids that influence glucose homeostasis, protein catabolism, and to some extent blood pressure (Hungerford and Meikle, 2010).

ACTH drives a de novo production of glucocorticoids in the adrenal glands by acting on the type 2 melanocortin receptor (MC2-R), a G-protein coupled receptor that is expressed on the surface of adrenal cortical cells. MC2-R is also expressed in skin and adipocytes, mediating stress- induced lipolysis (Buckingham, 2006; Papadimitriou and Priftis, 2009). Activation of MC2-R initiates signaling cascades that induce transcription of genes encoding steroidogenic acute regulatory protein (StAR) and steroidogenic cytochrome P450s (Lin et al., 1995; Stocco and Clark, 1996). Once cholesterol has entered the cortex, StAR shuttles cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane, where it undergoes a side chain cleavage catalyzed by

CYP11A1 resulting in the production of pregnenolone; the precursor for DHEA, aldosterone, and cortisol. Pregnenolone is then shuttled to the endoplasmic reticulum where it undergoes a second conversion to either progesterone (via 3β-hydroxysteroid dehydrogenase) or 17α- hydroxypregnenolone (via CYP17A1); these or subsequent metabolic products then return to the

Figure 1.2. Steroid hormone biosynthetic pathway. A simplified schematic representation of the human adrenal biosynthetic pathway displaying the conversion of cholesterol into pregnenolone with subsequent conversions for the production of sex steroids, aldosterone, and cortisol/corticosterone. In the rodent a lack of steroidogenic CYP17A1 in the zona fasciculata prevents the production of cortisol and results in corticosterone being the main glucocorticoid.

6 mitochondrion for the final steps in cortisol or corticosterone synthesis (Nussey and Whitehead,

2001).

Upon release of cortisol (human) or corticosterone (rodent) from the adrenal cortex, serum albumin or corticosteroid-binding globulin (CBG) binds these glucocorticoids and regulates their transport in the blood to various tissues within the body (Siiteri et al., 1982). Pertinent to the HPA axis, after a stress response has been triggered glucocorticoids exhibit auto-regulation exerting a negative feedback effect on the hypothalamus and pituitary gland to diminish the adrenal production of glucocorticoids (see Fig. 1.1). This is important for termination of the stress response and to maintain glucocorticoid levels within a desirable range (Handa and Weiser, 2013). The acute response of this negative feedback loop involves inhibition of the release of CRH and ACTH from the hypothalamus and pituitary, respectively (Keller-Wood and Dallman, 1984). This rapid feedback inhibition occurs independently of protein synthesis and is mediated at the cell membrane, with recent studies demonstrating that endocannabinoids may be involved in mediating this effect at the level of the PVN (Hill and Tasker, 2012). In the case of chronic feedback inhibition, glucocorticoids activate the GR located within the hypothalamus and pituitary gland to inhibit the production of

CRH and ACTH (Handa and Weiser, 2013).

1.3 Glucocorticoids

Glucocorticoids are essential steroidal hormones that are produced primarily in the zona fasciculata of the adrenal cortex. Glucocorticoids influence almost every cell, regulate roughly 10% of our genes, and are essential for the maintenance of homeostasis and the protection from pathophysiological responses to injury and inflammation (Buckingham, 2006). Basal serum levels of glucocorticoids display pulsatility due to a circadian rhythm (Buijs and Kalsbeek, 2001; Saper et al.,

2005); however, these basal serum levels increase 5- to 10- fold with the introduction of a stressor

7 such as physical or emotional trauma. Cortisol is the main glucocorticoid found in humans with corticosterone taking the role of the main glucocorticoid in rodents. This divergence exists due to a lack of 17α-hydroxylase expression (CYP17A1) within the zona fasciculata of the rodent adrenal cortex (Keegan and Hammer, 2002).

Glucocorticoids exert their biological effects primarily by binding to the cytoplasmic GR.

Glucocorticoid effects are usually identified with the term “permissive”, referring to glucocorticoids inducing changes in protein synthesis to modify the tissue’s responsiveness to other hormones. For example, in hypoadrenalism, there is a reduced response to the vasoconstrictors norepinephrine and angiotensin II, caused by a decreased expression of adrenoceptors, target genes for the GR (Sato et al., 2011).

There are a multitude of effects exerted by glucocorticoids with a central role involving modulation of carbohydrate and protein metabolism to increase blood glucose levels. Within the liver they stimulate the formation of glucose from amino acids and glycerol and the storage of glucose as liver glycogen. However, in the periphery glucose use is hindered and protein breakdown and lipolysis are increased, providing amino acids and glycerol for gluconeogenesis. This is believed to be a protective mechanism in order to prevent glucose-dependent tissues from starving. Glucocorticoids play a permissive role in lipid metabolism, facilitating the lipolytic effects of growth hormone and β- adrenergic receptor agonists, thereby increasing the concentrations of free fatty acids. Electrolyte and water balance are mainly taken care of by aldosterone; however, glucocorticoids do contribute to tubular function and glomerular filtration. Within the cardiovascular system, glucocorticoids enhance vascular reactivity to other vasoactive substances, while in the skeletal muscle permissive concentrations are required for normal function (reviewed in Schimmer and Funder, 2011).

8 In addition to these permissive effects, glucocorticoids also exert important suppressive effects, exemplified by their anti-inflammatory and immunosuppressive actions. When given at pharmacological doses, glucocorticoids reduce lymphocyte numbers within the blood, suppress macrophage activation, inhibit the release of pro-inflammatory cytokines such as interlukin-6 (IL-6) via upregulation of the glucocorticoid leucine zipper (GILZ), and inhibit tumor necrosis factor-α

(TNF-α) and nuclear factor-κB (NF-κB) through protein-protein interactions (Berrebi et al., 2003;

Schimmer and Funder, 2011; Wang et al., 2012; Huang et al., 2013).

The mineralocorticoid receptor (MR) readily binds aldosterone and is known to have a higher binding affinity for cortisol and corticosterone when compared to the GR; however the effects of glucocorticoids described above are due to GR activation. Most biological effects of glucocorticoids result from GR activation as opposed to MR activation because of the tissue distribution of these two receptors. MR’s tissue distribution pattern is restricted to tissues involved in Na+/K+ balance such as the sweat glands and the renal tubule along with certain brain regions such as the hypothalamus (Funder, 2005). The type 2 isozyme of 11 β-hydroxysteroid dehydrogenase (11β-

HSD2) is key in maintaining MR specificity for aldosterone in the face of much higher glucocorticoid levels. This enzyme metabolizes glucocorticoids such as cortisol into inactive 11-keto derivatives such as cortisone (binds neither MR or GR) to prevent hypokalemia and mineralcorticoid-related hypertension due to over-activation of the MR (Yang and Zhang, 2004). The GR is known to have a widespread distribution pattern within the body (De Kloet et al., 1998), allowing this receptor to mediate the observed biological effects of glucocorticoids in various tissues (Reul and de Kloet, 1985;

Edwards et al., 1988; Buckingham, 2006).

9 1.3.1 Synthetic Glucocorticoids

Synthetic GR agonists were derived mainly to exploit the anti-inflammatory and immunosuppressive effects seen at higher circulating concentrations of glucocorticoids. These are often used to treat inflammatory and autoimmune conditions along with hematological malignancies; from inflammatory arthritis, ulcerative colitis, asthma and skin diseases to Hodgkin’s lymphoma, acute lymphoblastic leukemia, and multiple myelomas (Dvorak and Pavek, 2010; Biddie et al., 2012).

Structures of selected GR ligands are shown in Figure 1.3. This figure illustrates chemical features of natural endogenous glucocorticoids as well as synthetic GR agonists and antagonists.

DEX, for example, has an additional 9α-fluoro group, a 1,2 unsaturated bond and a 16α- methyl group in comparison to cortisol. These structural changes make DEX approximately 25-times more potent than cortisol, possessing a half-life of 36-54 hours, with much greater specificity for GR vs. MR (McEwen et al., 1988; Burgess and Handa, 1992, Jacobs and Bijlsma, 2013). The reported range of Kd values for DEX binding to rat liver GR consists of 0.9 nM (Izawa et al., 1985) to 5.3 nM

(Isohashi et al., 1979), compared to CORT which ranges from 3 nM (Izawa et al., 1985) to 77 nM

(Allera and Wildt, 1992). These features allow DEX to be used widely as an anti-inflammatory agent with little worry of MR-mediated effects such as salt retention or hypertension. Synthetic GR agonists are administered in a variety of ways including orally, parenterally, or by various topical routes. Upon administration, they circulate the body as free steroid (up to 30%) or in a complex bound with albumin; however, they do not readily bind CBG since the synthetic steroids have a low affinity for this carrier protein (Cole, 2006). Long term and/or high dose glucocorticoid administration can result in hyperglycaemia, weight gain, hypertension, osteoporosis, depression, and decreased immunological function (Biddie et al., 2012).

Figure 1.3. GR ligands. Chemical structures of endogenous hormones (cortisol and corticosterone), synthetic agonists (dexamethasone and triamcinolone acetonide), and synthetic antagonists (mifepristone) of the glucocorticoid receptor. Differences in chemical structure result in altered potencies, half-lives, and specificity for GR vs. MR.

11 Ligand antagonists of the GR are also used clinically in cases of hypercortisolemia such as

Cushings’s syndrome, and more recently in the treatment of depression (Flores et al., 2006). A pertinent steroidal antagonist is mifepristone or RU486, shown in Figure 1.3 with its chemical structure compared to endogenous and synthetic glucocorticoids. The most notable differences are the dimethylaminophenyl group at the 11β-position of the steroid ring and the hydroxyl and propynyl substituents located at the C17 position (Baulieu, 1997). This antagonist of the GR is perhaps better known as a progesterone receptor (PR) antagonist, leading to its use as an abortion pill for the termination of early pregnancy (Cole, 2006). RU486 competes for the ligand-binding pocket where progesterone and cortisol bind in the PR and GR, respectively. In regards to the GR,

RU486 is known to bind the ligand-binding domain (LBD) of the rat hepatic cytoplasmic GR with a

Kd value of 1.5 nM (Gagne et al., 1985), this binding subsequently stabilizes the GR-heat shock protein 90 (HSP90) complex (Distelhorst and Howard, 1990; Beck et al., 1993). However, this stabilization between the GR and HSP90 fails to prevent the GR’s translocation to the nucleus (Qi et al., 1990; Rupprecht et al., 1993; Jewell et al., 1995). RU486 is believed to produce a conformational change in the GR, but one that is distinct from that achieved by agonists such as cortisol, thus leading to a decreased recruitment of transcriptional co-activators and chromatin remodelling complexes. It is hypothesized that the translocation of the GR-HSP90 complex adds another level of antagonism by allowing a competition between cortisol-activated GR and RU486-activated GR for binding sites on glucocorticoid response elements (GREs) within the regulatory regions of target genes (Peeters et al.,

2008)

1.3.2 Cushing’s syndrome

Cushing’s syndrome or hypercortisolism is described as an excess amount of cortisol within the body (Winter et al., 2012; Gorman, 2013). It can be caused by elevation of endogenous cortisol

12 levels or exogenous administration of glucocorticoids, the latter accounting for 99% of cases.

Endogenous Cushing’s syndrome can be either ACTH-independent or ACTH-dependent. ACTH- independent Cushing’s syndrome accounts for about 10-20% of adult endogenous cases and is caused by a tumor in the adrenal gland, leading to excessive amounts of cortisol and other adrenal cortical hormones being released into the circulation (Stratakis, 2008). More common is ACTH- dependent Cushing’s syndrome, accounting for about 80-90% of adult endogenous cases. In this scenario, a benign pituitary adenoma secretes excess ACTH.

Cushing’s syndrome patients typically display weight gain located centrally around the abdomen, “moon face” or rounding to their facial features, fat deposition along the back and shoulders, hypertension, immune suppression, osteoporosis, and metabolic insulin resistance

(Nussey and Whitehead, 2001). In cases of exogenous Cushing’s syndrome, the treatment involves careful tapering and eventual cessation of the glucocorticoid being given medically. However, if

Cushing’s syndrome is endogenous in nature, then a high dose DEX test is performed to differentiate between ACTH-dependent and ACTH-independent cases. If DEX administration results in suppression of ACTH, cortisol, and urine free cortisol, the syndrome may be diagnosed as ACTH- dependent and most likely involving a pituitary adenoma. If DEX administration reveals a lack of suppression of ACTH and cortisol, the syndrome may be diagnosed as ACTH-independent and most likely the result of an adrenal adenoma. Surgery will then be performed to remove the tumor or a combination of radiation and/or medication may be used to suppress the excess cortisol production

(Gorman, 2013).

1.3.3 Addison’s disease

Addison’s disease or hypocortisolism is described as chronic adrenal insufficiency or a lack of sufficient cortisol production within the body. This condition arises from chronic primary

13 adrenocortical insufficiency, thereby being distinguished from both acute primary adrenocortical insufficiency and secondary adrenal insufficiency. About 80-90% of patients with Addison’s disease have autoimmune adrenalitis and therefore cannot produce a sufficient amount of adrenal steroid hormones. This can arise as an isolated condition or as part of an autoimmune polyendocrine syndrome (Betterle et al., 2002). Secondary adrenal insufficiency is more commonly the result of impaired ACTH production by the pituitary (Kasperlik-Zaluska et al., 1998). The main symptoms portrayed by adrenal insufficiency are fatigue, weakness, loss of energy, weight loss, anorexia, and in rare cases calcification of the adrenal gland (Arlt and Allolio, 2003; Gorman, 2013). Most of the symptoms presented are not specific to Addison’s disease; hence, 50% of patients will have the disease for up to a year before it is properly diagnosed, and by this time up to 70% of the adrenal gland may have already been lost. Morning measurements of plasma cortisol and ACTH levels can identify patients with primary adrenal insufficiency while an ACTH stimulation test can help to distinguish secondary adrenal insufficiency from healthy individuals (Arlt and Allolio, 2003).

Treatment for Addison’s disease involves replacement of missing cortisol (Gorman, 2013).

1.4 Nuclear Receptors

Nuclear hormone receptors (NRs) are key players in the field of endocrinology. NRs bind a variety of small-molecule chemical messengers and relay or transduce a complex set of signals that are determined by the properties of the ligand. However, the response of the NR may vary depending on which tissue it resides in and what ligand may bind to it (Bain et al., 2007). The typical NR consists of three modules shown in Figure 1.4: an N-terminal transcriptional activation domain (AF-1), the central DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD). The LBD is responsible for the dimerization of NRs and contains a hydrophobic ligand binding pocket where the respective hormones or ligands of a specific NR bind. This, along with the important ligand-regulated

Figure 1.4. NR structural domains. At the top is a simplified diagrammatic representation of the NR structural domains: the N-terminal transcriptional activation domain (AF-1), the DNA binding domain (DBD), and the C-terminal ligand-binding domain (LBD). Below are various NRs from different categories. DBDs and LBDs across different NR families and between members of the same NR family show conserved amino acid length with the N-terminal regions varying greatly (adapted from Moore et al., 2006).

15 transcriptional activation domain (AF-2), allows for the recruitment of various co-activating proteins that promote an interaction between the NR and chromatin-remodelling complexes (Wrange et al.,

1984; Xu and Li, 2003). The DBD is the most conserved region of the NRs, responsible for docking the receptor to the hexanucleotide response elements within NR-regulated promoters. Each DBD contains two α helices and two asymmetric zinc binding modules coordinated by eight conserved cysteine residues. The first helix binds the major groove of the DNA through base-specific contacts, while the second helix is responsible for less specific interactions with the DNA backbone (Luisi et al., 1991; Roemer et al., 2006). The DBD and LBD are connected via a short amino acid sequence termed the hinge region. This is a flexible region with the ability to become phosphorylated and acetylated and both modifications have been linked to increased transcriptional activation (Knotts et al., 2001; Kino and Chrousos, 2011). The N-terminal domain contains a transcriptional activation function termed AF-1. This sequence shows weak conservation across the nuclear receptor super- family and has the ability to function as a ligand-independent transcriptional activator (Thompson and Kumar, 2003). The NRs typically function as transcription factors where they bind response elements and regulate the expression of target genes. They attract accessory proteins such as co- activators and co-repressors to regulate gene transcription. However, NRs do not influence gene transcription purely through direct binding to DNA, but can also engage in protein-protein interactions resulting in crosstalk with other important signal-transduction molecules such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) (Moore et al., 2006).

It is helpful to consider the NRs as belonging to five categories: the classic steroid hormone receptors, the retinoid-X-receptor (RXR) heterodimer receptors, the xenobiotic receptors, the orphan receptors, and the NR-like DBD-less repressors. The steroid hormone receptors include the progesterone receptor (PR, NR3C3), estrogen receptors (ER, NR3A1 and NR3A2), glucocorticoid

16 receptor (GR, NR3C1), androgen receptor (AR, NR3C4), and the mineralocorticoid receptor (MR,

NR3C2). These are typically bound to a chaperone complex involving heat shock proteins within the cytoplasm and, upon ligand activation, translocate to the nucleus where they bind as homodimers to imperfect palindromic (inverted repeats) response elements in target genes. The RXR heterodimer receptors include the liver X receptors (LXR, NR1H2 and NR1H3), farnesoid X receptor (FXR,

NR1H4), peroxisome proliferator-activated receptors (PPAR, NR1C1-NR1C3), thyroid hormone receptors (TR, NR1A1 and NR1A2), the retinoic acid receptors (RAR, NR1B1-NR1B3), and vitamin D receptor (VDR, NR1I1). These receptors associate with the RXR to form heterodimers and bind as a dimeric complex to direct repeat (DR) response elements. The xenobiotic receptors include PXR (NR1I2) and the constitutive androstane receptor (CAR, NR1I3), receptors that also dimerize with RXR and regulate expression of multiple drug-metabolizing enzymes and transporters in response to a range of endogenous and exogenous chemicals. The orphan receptor category includes those NRs with no known endogenous ligands, one example being hepatocyte nuclear factor-

4α (HNF4α, NR2A1). Short heterodimer partner (SHP) is an example of a NR-like, DBD-less repressor involved in the regulation of cholesterol catabolism.

1.4.1 Glucocorticoid Receptor

Located on human chromosome 5 and mouse/rat chromosome 18, the GR gene (NR3C1) encodes a 9-exon transcript (in humans) and a 11-exon transcript (in rodents) producing a member of the nuclear receptor super-family of ligand-dependent transcription factors (Encio and Detera-

Wadleigh, 1991). Gr-knock out (KO) mice die shortly after birth of respiratory failure due to severely impaired lung development, demonstrating that GR is a crucial nuclear receptor needed for proper development and sustained life (Cole et al., 1995). Free cortisol/corticosterone diffuses across the cellular membrane and activates the cytoplasmic GR to increase or suppress the transcription of 17 various genes. Two mechanisms contribute to the control of free cortisol levels. First, within the bloodstream, cortisol binds to CBG, as discussed earlier, along with serum albumin. This binding prevents cortisol from freely diffusing across the cell membrane and therefore limits the availability of free cortisol. Second, enzymatic transformations can regulate intracellular free cortisol levels.

After diffusion of cortisol into the cell, the type 2 form of 11β-hydroxysteroid dehydrogenase (11β-

HSD2) can metabolize cortisol into the inactive metabolite cortisone, thereby preventing the activation of the MR and GR (Walker and Andrew, 2006; Naray-Fejes-Toth and Fejes-Toth, 2007).

As discussed earlier, this mechanism is important for preventing over-activation of the high affinity

MR and this enzyme is primarily present in tissues concerned with electrolyte balance. On the other hand, the 11β-hydroxysteroid dehydrogenase type 1 isozyme (11β-HSD1) is responsible for the conversion of cortisone into cortisol, contributing to the intracellular bioavailability of cortisol in the glucocorticoid-responsive metabolic tissues such as liver, fat, lung, and central nervous system (Seckl and Walker, 2004; Walker and Andrew, 2006).

Figure 1.5 provides a simple illustration of how GR activation can alter gene expression both via the canonical pathway involving GRE binding and via protein-protein interactions with other transcription factors. The GR resides in the cytoplasm as a multi-protein heterocomplex that contains chaperone proteins such as HSP90, HSP56, HSP40, and p23, which are important for localization and conformation of the unliganded GR (Pratt et al., 2006). The cytoplasmic complex also includes the immunophilins FKBP51 and FKBP52. Free cortisol/corticosterone binds to the

LBD of the GR causing a conformational change in the receptor leading to the dissociation of chaperone proteins. The activated receptor then translocates to the nucleus and homodimerizes with another activated GR (Freedman and Yamamoto, 2004). Once inside the nucleus, the GR homodimer binds to the DNA in a sequence-specific manner; these sequences are known as GREs and are 18

Figure 1.5. Signal transduction pathway of the GR. GR resides in the cytoplasm bound to chaperone proteins such as the heat shock proteins (HSPs), p23, and immunophilins. Once activated by a glucocorticoid (G), the GR dissociates from the chaperone proteins and translocates to the nucleus where it will homodimerize to another GR and induce transcription of classic GR target genes, such as tyrosine aminotransferase (TAT). However, at elevated levels of endogenous glucocorticoids and pharmacological doses of synthetic glucocorticoids activation of the GR results in anti-inflammatory effects; this occurs through protein-protein interactions with NF-κB, preventing the transcription of inflammatory cytokines (adapted from Qi and Rodrigues, 2007).

19 characterized by the 15-bp consensus sequence 5’-GGTACAnnnTGTTCT-3’, consisting of two imperfect inverted half-sites separated by a 3-bp spacer (Lu et al., 2006). The full consensus sequence is not always utilized; it has been shown that GR is able to bind to GRE half-sites

(Schoneveld et al., 2004). Not all target genes contain just one response element, there may be multiple GREs present in their proximal promoters but numbers and location can vary (Jantzen et al., 1987; Wieland et al., 1990). Once bound, the GR recruits co-activators and co-repressors for the induction or suppression of multiple genes.

In the case of GRE-mediated transcriptional up-regulation, co-activators and other transcription factors such as histone acetyltransferases (HATs) are recruited by the DNA-bound GR to allow for the opening of chromatin, promoting easier access to target genes by the basal transcription machinery (Biddie et al., 2012). Examples of genes regulated by this mechanism are

TAT, alanine aminotransferase, and phosphoenolpyruvate carboxykinase (PEPCK), which are all involved in liver gluconeogenesis (Revollo and Cidlowski, 2009). Activated GR can also recruit co- repressors and other factors such as histone deacetylase 2 (HDAC2) to inhibit transcription; this is accomplished through the prevention of transcription factor binding via closing of the chromatin for various genes. This cis-repression can utilize the lesser known negative GREs (nGREs), which are low-affinity binding sites for the GR. These nGREs are poorly conserved and have been shown to be close to, or overlapping with, other transcription factor binding sites and, on some occasions, the

TATA-box (Dostert and Heinzel, 2004). Genes such as pro-opiomelanocortin (POMC), CRH, prolactin, and neuronal serotonin receptor have been shown to be suppressed through this cis- repression mechanism (reviewed in Revollo and Cidlowski, 2009).

20 Other regulation pathways involve protein-protein interactions between the GR and other transcription factors, resulting in either induction or repression of gene transcription. Insulin-like growth factor-1 (IGF-1) produced in the liver is important for postnatal growth. It is regulated through the GR interaction with signal transduction and activator of transcription – 5 (STAT-5).

STAT-5 is phosphorylated by Janus kinase (JAK), allowing it to dimerize to other STAT proteins leading to DNA binding; STAT-5 homodimers can recruit GR to the chromatin, where it interacts with STAT-5 without directly binding DNA. This interaction attracts other transcription factors and opens the chromatin structure allowing for easier access of the basal transcription machinery

(Tronche et al., 2004). On the other hand, trans-repression is central to the ability of glucocorticoids to exert their anti-inflammatory effects. This mechanism of signalling has been demonstrated by crosstalk with NF-κB, AP-1, and Smad3. In regards to NF-κB, GR is known to bind one of its subunits (p65) and repress NF-κB-mediated transcription in multiple ways. GR is able to prevent the formation of active NF-κB complexes by sequestering the p65 component; however, if NF-κB is already bound to the DNA, GR can bind and prevent the recruitment of other transcription factors, thereby inhibiting the transcription of cytokines, chemokines, and other inflammatory factors

(McKay and Cidlowski, 1998). Finally, GR and NF-κB share similar co-activators (CBP/p300) and is thought that GR activation can limit the availability of such factors for use by NF-κB (McKay and Cidlowski, 2000; Almawi and Melemedjian, 2002).

Non-genomic actions of glucocorticoids are less understood but seem to involve physicochemical interactions with the cellular membrane, membrane-bound GR, or cytoplasmic GR

(Song and Buttgereit, 2006; Lowenberg et al., 2008). One of the most researched mechanisms is the inhibition of the release of the pro-inflammatory molecule arachidonic acid (AA) through the inhibition of the epidermal growth factor (EGF) signaling pathway. Upon GR activation, Src kinase 21 is released from the GR chaperone complex leading to the phosphorylation of lipocortin-1 resulting in inhibition of AA release (Croxtall et al., 2000).

The GR is ubiquitously expressed in almost every tissue, although its concentration is subject to change based on certain physiological conditions. Levels of GR are modulated through auto- regulation by glucocorticoids, neural influence, and different stages of life (Sapolsky et al., 1984;

Antakly et al., 1985). A single dose of DEX caused a 60-80% decrease in rat hepatic GR mRNA levels at about 4 h with a nadir at 18-24 h; the GR protein level followed suit but failed to have a comparable decline to the mRNA (Okret et al., 1991). This robust regulation of the GR is important for the maintenance of the physiological response to changes in glucocorticoid concentrations. In addition to regulation of expression, the GR is subject to alternative splicing resulting in various isoforms. These isoforms are known as the “active” GR-α and the “inactive” GR-β. Of the 9 exons present in the human GR gene, only exons 2-9 are incorporated into the mature mRNA. Exon 1 represents the 5’-untranslated region and contains several independent translational start sites (1A-

1J), each with its own unique promoter and various transcription factor binding sites; this is thought to be the mechanism used for expressing GR in multiple tissues and allowing for a varied response upon activation by glucocorticoids (Turner et al., 2006; Preseul et al., 2007, Gross and Cidlowski,

2008). However, the two major isoforms that have been rigorously studied are known as GR-α and

GR-β, with both containing exons 2-8 and having alternative splicing of exon 9. This results in two proteins that have identical N-termini but a key difference with respect to the C-terminal LBD

(Oakley et al., 1997, Gross and Cidlowski, 2008). GR-α is the longer of the two (777 amino acids) and is the isoform responsible for the binding of glucocorticoids and modulating their effects. GR-β is shorter (742 amino acids) and is expressed at much lower levels than GR-α. GR-β is unable to bind to endogenous glucocorticoids and most synthetic glucocorticoids, with the exception of RU486 22 (Lewis-Tuffin et al., 2007); however, GR-β appears to affect gene regulation by competing with GR-

α for GREs within target genes. This has lead to the belief that GR-β acts as a dominant negative regulator of GR-α (Oakley et al., 1999, Yudt et al., 2003). Due to the dominant negative nature of

GR-β, the ratio of GR-α/GR-β is important in cells with a decreased ratio correlating with cardiovascular disease, glucocorticoid-resistant asthma, ulcerative colitis, and rheumatoid arthritis

(Lewis-Tuffin and Cidlowski, 2006). The GR-β isoform is also present in other species. Of particular relevance to this project, the GR-β isoform was identified within rat liver (DuBois et al.,

2013).

1.4.2 Pregnane X receptor

The PXR (NR1I2) belongs to the nuclear hormone receptor super-family of ligand-activated transcription factors. It is a 434-amino acid, 50 kDa protein that is primarily expressed in the liver and intestinal tissue (Lehmann et al., 1998). Although it can be categorized as a xenobiotic receptor, many view PXR as an orphan receptor since it does not have an identified endogenous ligand; it has been hypothesized that it may not possess one. Instead PXR is activated by a wide variety of structurally-diverse compounds including endogenous, synthetic and xenobiotic compounds, leading to increased transcription of a large gene battery including important players in drug metabolism and transport (Zhou et al., 2009).

This ligand promiscuity is fostered by PXR’s unusually large ligand-binding pocket, which is attributed to an expandable pore that is able to accommodate larger molecules (Timsit and Negishi,

2007). This knowledge has lead researchers to identify the PXR as an established xenosensor and a master regulator of xenobiotic responses (Gao and Xie., 2010). However, the ligand-binding pocket seems to vary between species; for example, the human PXR (hPXR) and rodent PXRs have only

23 76% amino acid similarity within their LBDs. In comparison, other orthologous NRs show a 90% amino acid similarity between human and rodent LBDs (Tolson and Wang, 2010). Due to this lack of similarity, some chemicals have been shown to selectively activate the human PXR, such as the antibiotic rifampicin, and some have been shown to selectively activate rodent PXRs, such as the anti-glucocorticoid pregnenolone-16α-carbonitrile (PCN). This has lead to the development of humanized PXR mouse models allowing for the study of human PXR in xenobiotic detoxification within a rodent model (reviewed in Wang et al., 2012). To further elucidate the role of PXR, Pxr-null mice (Xie et al., 2000) and Pxr-null rats have been created and studied. These rodents are viable and exhibit no particular phenotypic abnormality or overt differences in free and high-density lipoprotein

(HDL) cholesterol, steroid hormones (progesterone, estradiol, testosterone, and corticosterone), triglycerides, transaminases, albumin, or total bile acids (Staudinger et al., 2001) However, these

PXR-knockout animals show compromised induction of target genes when challenged with xenobiotic PXR activators.

Figure 1.6 displays the signalling cascade for rodent PXR, showing a cytoplasmic localization similar to the GR. PXR in the unactivated state is bound to chaperone proteins such as cytoplasmic constitutive active/androstane receptor retention protein (CCRP) and HSP90 in the cytoplasm (Squires et al., 2004). Upon ligand activation of the PXR, the chaperone proteins dissociate and PXR translocates to the nucleus where it heterodimerizes with RXR and binds to

DNA response elements. These response elements are known as pregnane xenobiotic-response elements (PXREs) and are generally located within the 5’-flanking region of PXR target genes.

PXREs contain two copies of the half site consensus sequence AG(G/T)TCA with various spacing, including direct repeats DR-3, DR-4, and DR-5, and everted repeats ER-6, and ER-8 (Orans et al.,

2005; Ihunnah et al., 2011). Upon heterodimer binding to the DNA, there is a recruitment of co- 24

Figure 1.6. Signal transduction pathway for rodent PXR. In the dormant state, PXR resides in the cytoplasm in a complex with chaperone proteins CCRP and HSPs. Once activated by agonists, PXR dissociates from the chaperone proteins and translocates to the nucleus. Upon entering the nucleus it heterodimerizes with RXR and induces transcription of PXR target genes, such as CYP3A23 in the rat (adapted from Ripp, 2008).

25 activators such as the p160 family and steroid receptor co-activator 1 (SRC-1) to facilitate the transcription of various target genes (McKenna et al., 1999; Li and Chiang, 2005). In addition to classical ligand-dependent PXR activation, PXR can be activated through post-translational modification. Protein kinase A modulates PXR activation through the signalling cascade triggered by forskolin while protein kinase C represses PXR signalling (Ding and Staudinger, 2005). Members of the cytochrome P450 CYP3A subfamily, the uridine-5’-diphosphate glucuronosyl transferases

(UGTs), glutathione S-transferases (GSTs), sulfotransferases, and multidrug resistance-associated protein 2 (MRP2) are all regulated by PXR (Chai et al., 2013). Each of these enzymes/transporters contributes to the detoxification and elimination of foreign/harmful chemicals in the body. Human

CYP3A4, along with its PXR-regulated relatives CYP3A23 (rat), and CYP3A11 (mouse), is highly expressed in the liver and intestine and has broad substrate specificity, with CYP3A4 being responsible for metabolism of over 50% of pharmaceuticals on the market today (Ihunnah et al.,

2011). With this information, it has become apparent that PXR regulation of drug-metabolizing enzymes can cause serious drug-drug interactions (DDI) in humans. For example, PXR activation by its prototypical agonist rifampicin leads to CYP3A4 induction, along with stimulated metabolism, decreased bioavailability, and diminished therapeutic effect of the antihypertensive/antiarrhythmic verapamil (Fuhr., 2000). PXR is also important in many endobiotic pathways involving gluconeogensis and bile acid homeostasis. In gluconeogensis, PXR activation results in inhibition of forkhead transcription factor FoxO1 binding to insulsin response sequence-1 (IRS-1), attenuating the stimulation of gluconeogenic genes such as PEPCK and thereby preventing the production of more glucose in the liver (Kodama et al., 2004). PXR protects against the hepatotoxic effects of accumulated bile acids by preventing further conversion of cholesterol into bile acid and detoxifying oxidized cholesterol (Staudinger et al., 2001; Xie et al., 2001).

26 Similar to the GR, the PXR displays multiple isoforms that result from alternative splicing.

They have been observed in human liver, breast, colon, and small intestine (Dotzlaw et al., 1999;

Fukuen et al., 2002; Lamba et al., 2004). There are multiple splice variants for PXR; however, the most abundant alternative isoform results from a 111-bp deletion leading to a 37-amino acid deletion in the LBD. This variant is referred to as PXR.2 with the original PXR known as PXR.1. PXR.2 accounts for nearly 7% of PXR expression in human liver, with ratios of PXR.1 and PXR.2 staying fairly consistent and maintaining a high correlation (Lamba et al., 2004). The 37-amino acid deletion resides in the unique strand of 50 amino acids that provides PXR with the ability to expand its ligand binding pocket; therefore, it is thought that PXR.2 is unable to bind ligands with the promiscuity and efficiency of PXR.1. However, it is possible that PXR.2 is still able to bind a select set of ligands as it contains a partial LBD that may retain some function. Further in vitro testing in HepG2 cells showed that PXR.2 acts as a dominant negative regulator (similar to GR-β) in that it is unable to bind the prototypical PXR agonists but is still able to bind PXREs located on the CYP3A4 promoter, preventing the binding of activated PXR.1 (Lin et al., 2009).

1.4.3 GR-PXR Crosstalk

The GR is known to regulate the expression of many genes, including some encoding drug- metabolizing cytochromes P450 and multiple regulators of the P450s. The GR has been shown to interact with or engage in crosstalk with the aryl hydrocarbon receptor (AHR), and nuclear receptors such as PXR, CAR, and RXRα, which all modulate transcription of a wide variety of genes (Dvorak and Pavek, 2010). This regulation of transcription factors by a nuclear receptor has therefore been referred to as the “regulation of the regulator” (Harper et al., 2006). Initial studies showed induction of a nuclear receptor protein (RXRα) through a GR-activated pathway in rat hepatoma cell lines

27 (Wan et al., 1994), leading to further studies examining this same phenomenon in rat hepatocytes

(Yamaguchi et al., 1999).

The regulation of PXR expression via GR activation is central to this thesis. PXR, as mentioned above, is known for its large hydrophobic ligand-binding pocket and its vast array of gene targets, one of which is human CYP3A4. DEX, at submicromolar concentrations (100 nM) in cultured human hepatocytes, was able to synergistically enhance the induction of CYP3A4 in response to PXR activators (Pascussi et al., 2000). In addition, there was an increase in RXRα and

PXR mRNA levels that mimicked the induction of TAT (a classic GR target gene) mRNA levels in terms of time-course and concentration-dependence (Pascussi et al., 2000; Shi et al., 2010). This finding suggested that GR activation by low DEX concentrations leads to increased transcription of the PXR gene, ultimately making more PXR protein available for activation by ligands. A follow-up study showed that at supramicromolar concentrations (10 µM), DEX binds and activates the PXR and induces CYP3A4 mRNA levels independent of GR (Pascussi et al., 2001; Shi et al., 2010). These studies were crucial in establishing interplay between the GR and PXR pathways. The rodent counterpart of CYP3A4, CYP3A23, was studied in a similar fashion in H4IIE rat hepatoma cells, leading to the mechanistic model outlined in Figure 1.7. This model displays the interplay between the GR and PXR, where at low concentrations of DEX there is an activation of the GR leading to increased synthesis of PXR and RXR, the key regulators of CYP3A23 induction by diverse agonists.

At higher DEX concentrations, GR activation can still stimulate PXR and RXR production, but in addition DEX is able to bind PXR as an agonist resulting in further induction of CYP3A23 (Huss and

Kasper, 2000). It has been shown in silico that the proximal promoter of the PXR harbours two

GREs located at 1.9- and 1.7-kb upstream of the transcriptional start site (Gibson et al., 2006), further solidifying this two-stage activation pathway for the induction of CYP3A genes by DEX. 28

Figure 1.7. A model of the two-stage CYP3A23 induction by DEX in H4IIE cells. At low DEX concentrations there is a GR-dependent increase in PXR and RXRα mRNA levels, leading to an increased induction of CYP3A23 mRNA when in the presence of a PXR agonist. In addition to this mechanism, DEX at higher concentrations can directly induce CYP3A23 expression by acting as a PXR agonist (adapted from Huss and Kasper, 2000).

29 1.5 Cytochrome P450 enzymes

Drug-metabolizing enzymes (DMEs) aid in the detoxification and excretion of drugs and other xenobiotic compounds that may pose a threat to the human body. Some water-soluble compounds are readily excreted without the need for metabolism. However, lipophilic compounds that enter the body often undergo biotransformation, rendering them less hydrophobic (more water-soluble) and allowing for easier excretion; this biotransformation is performed by Phase I and Phase II DMEs.

Phase I DMEs are primarily made up of the monooxygenases known as the cytochrome P450s, a super-family of hemoproteins that catalyze the oxidation of diverse substrates into more polar derivatives. Such products of Phase I metabolism typically have a functional group exposed or created through oxidation, providing a chemical site for a phase II conjugation enzyme (UGT, GST, sulfotransferases) to generate a highly polar drug conjugate. This facilitates excretion of the foreign chemical from the body (reviewed in Riddick, 2007).

The P450s are generally regarded as the most important DMEs. Their discovery is attributed to Klingenberg (1958), identifying them in rat liver microsomes as a carbon monoxide-binding pigment with a maximal absorbance at 450 nm. Further characterization and naming of this hemoprotein followed shortly thereafter (Omura and Sato, 1962). The human genome encodes 57 putatively functional P450s, with the corresponding figure for rat and mouse being 89 and 103, respectively (Nelson et al., 2004; Nelson, 2009). These enzymes are grouped according to their sequence similarity, resulting in 18 families and 44 subfamilies in humans (Nelson, 2009). P450s are not only involved in the metabolism of xenobiotic compounds, many are known to have physiological roles in the body with endogenous substrates. For example the CYP11, CYP17, CYP19, and CYP21 families play crucial roles in the synthesis of cortisol, testosterone, and estrogen, with

30 mutations leading to deficient production of glucocorticoids and sex steroids (Miller et al., 1998;

Nebert and Russell, 2002). Other families such as CYP27 and CYP24 have been shown to play a role in bile acid synthesis and the metabolism of vitamin D3, respectively (Andersson et al., 1989;

Wikvall, 2001). In fact, only 3 of the 18 families are heavily focused on drug metabolism: CYP1,

CYP2, and CYP3. The human enzymes CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, and

CY3A4 account for most of the P450-mediated metabolism of therapeutic agents (Guengerich, 2008).

Polymorphisms within many of these main enzymes can produce loss-of-function or gain-of- function variant enzymes, which can potentially lead to reduced or increased clearance, respectively, of therapeutic agents. Together with genetic polymorphisms, the potential for DDIs resulting from

P450 induction or inhibition gives these enzymes prominence in clinical pharmacology and drug safety. The expression and function of P450s can be regulated by non-genetic factors such as sex, age, and disease (Wolbold et al., 2003; Elbekai et al., 2004; Stevens, 2006) as well as genetic and epigenetic aspects controlling transcription or post-transcriptional events (Hukkanen, 2012).

P450s also rely on other enzymes for their catalytic function. The central focus of my thesis is the electron transfer system utilized by all microsomal P450s involving the flavoprotein POR.

Microsomal P450s are anchored on the cytoplasmic surface of the endoplasmic reticulum of the cell and catalyze the oxidation of a substrate through a cycle of steps involving cooperation with the

POR enzyme (Figure 1.8). In step one, the substrate (RH) binds in the active site of a P450, with its heme iron in the oxidized ferric form (Fe3+). In step two, POR utilizes its flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) prosthetic groups, to shuttle an electron (e-) from NADPH to the P450 heme, reducing the hemoprotein from the ferric (Fe3+) to ferrous form

2+ (Fe ). This reduction permits the binding of molecular oxygen (O2) in step three. Step four involves

- donation of a second electron (e ) via either POR or a cytochrome b5/cytochrome b5 reductase route 31

Figure 1.8. Cytochrome P450 catalytic cycle. A simplified diagram of the cytochrome P450 catalytic cycle showing the initial binding of the substrate (RH) to the ferric (Fe3+) hemoprotein, with successive electron (e-) donations from POR at two integral steps, and the subsequent release of the hydroxylated product (ROH). See text for a description of steps one through nine. (adapted from Isin and Guengerich, 2008).

32 (not shown). Following a molecular rearrangement (step five), cleavage of the oxygen-oxygen bond occurs, producing water and an activated oxygen (step six). This activated oxygen is incorporated into the substrate resulting in production of the hydroxylated product (ROH) (steps seven and eight). The product ROH is then released from the P450 with regeneration of the ferric P450 (step nine) (reviewed in Hrycay and Bandiera, 2012). Although not a focus of my thesis, it is important to point out that a secondary electron-transport system involving cytochrome b5 and cytochrome b5 reductase using NADH as a cofactor also exists in the endoplasmic reticulum. New mouse models with global (McLaughlin et al., 2010) or conditional hepatic (Finn et al., 2008) knockout of cytochrome b5 have begun to clarify the P450 isoform- and substrate-specific effects of this secondary system on P450 electron transfer and catalytic function (Henderson et al., 2013).

1.5.1 NADPH-cytochrome P450 oxidoreductase

POR is a 78-kDa microsomal flavoprotein responsible for the donation of electrons to various oxygenase enzymes. The POR gene is located on human chromosome 7q11.2 and consists of 15 coding exons and one untranslated exon (Pandey and Flück, 2013). POR is best characterized for its transfer of electrons in the catalytic cycle of the microsomal P450s; however, POR also donates electrons to heme oxygenase, squalene monooxygenase, and cytochrome b5 to aid in heme degradation, sterol biosynthesis, and fatty acid desaturation/elongation, respectively (Riddick et al.,

2013). The transfer of electrons occurs through the two flavin moieties within the POR enzyme;

NADPH donates its electrons to the flavin adenine dinucleotide (FAD) moiety of POR, followed by a transfer to the flavin mononucleotide (FMN) moiety of POR. The P450 heme iron accepts electrons in two discrete single electron transfers to support P450 catalytic function (Vermillion et al., 1981). In mice, the germ-line knockout of POR results in multiple developmental defects and

33 embryonic lethality (Shen et al., 2002; Otto et al., 2003), confirming the essential role of POR in mammalian development and physiology. Mice with conditional hepatic POR deletion have a severe decrease in all hepatic microsomal P450 activities, compensatory increased expression of several

P450 proteins, reduced circulating cholesterol and triglycerides, and hepatic lipidosis (Gu et al., 2003;

Henderson et al., 2003). These studies establish the key role of POR in hepatic microsomal P450- dependent metabolism, with POR-catalyzed electron transfer often viewed as a rate-limiting determinant of P450 catalysis. Further evidence for this hypothesis comes from the observation that human liver POR is expressed at levels 5-10 fold lower than the microsomal P450 pool, suggesting that a single POR supports reduction of multiple P450s (Gomes et al., 2009).

Genetic variation in the human POR gene has important implications for drug and steroid metabolism. POR deficiency (PORD) involves mutations in the POR gene that result in an enzyme with compromised activity that can range in severity. A severely compromised POR enzyme can produce a form of congenital adrenal hyperplasia (CAH) with ambiguous genitalia and skeletal malformations resembling Antley-Bixler syndrome (ABS), inducing craniosynostosis, midface hypoplasia, joint contractures, and bowing of the femora (Adachi et al., 2004; Flück et al., 2004).

PORD results in CAH through the reduced activity of CYP17A1, CYP19A1, and CYP21A2, key enzymes that aid in the production of steroids and require the donation of electrons from POR in order to carry out their function. There are more than 200 POR mutations and polymorphisms with

41 defined alleles listed on the CYPalleles website characterized by nucleotide changes within various regions of the POR protein such as the FAD- (eg. V492E, G504R), FMN- (eg. Y181D, Q153R), and

NADPH-binding domains (eg. C569Y, V608F) (Pandey and Flück, 2013). A common polymorphism

(A503V) appearing in 19% to 37% of individuals of all major ethnicities and resulting in 68% and

58% activity of CYP21A2 and CYP17A1, respectively, is POR*28 (Huang et al., 2005; Zanger and 34 Schwab., 2013). This particular variant maintained >50% of wild-type activity towards several P450 enzymes as well as preserving the heme oxygenase activity with 97% of wild-type activity (Riddick et al., 2013). However, this polymorphism results in 107% of the wild-type activity for CYP3A4

(as measured in vitro), leading to a 1.6-fold increase in CYP3A midazolam 1’-hydroxylase activity (in vivo phenotyping) and possible dosing alterations in patients that possess this variant to optimize drug therapy (Oneda et al., 2009; Flück and Pandey, 2011) Overall, in addition to POR mutations that cause disease, relatively common allelic variants influence P450 drug metabolism activities in

P450 isoform- and substrate-specific ways (Riddick et al., 2013).

POR expression is regulated by multiple factors including endogenous hormones and xenobiotics with many non-genetic factors including sex, age, smoking or drinking habits having minimal effect on POR levels in human liver (Zanger and Schwab, 2013). In mice, both PXR and

CAR activation induce levels of POR in the liver and intestine upon administration of PCN and phenobarbital (PB), respectively; of central importance to this thesis, the induction of POR by PCN is reduced in Pxr-null mice (Maglich et al., 2002; Ueda et al., 2002). Activation of the PPARα by peroxisome proliferators (PP) such as hypolipidemic drugs, plasticizers, and industrial solvents seems to have complex effects on POR expression. Depending on the specific PP used, POR was shown to be induced in male rat liver at the mRNA level with a subsequent down-regulation at the protein level; these effects were diminished in PPARα-null mice, suggesting a role for PPARα in POR regulation (Fan et al., 2003). Treatment of mice with 3-methylcholanthrene or 2,3,7,8- tetrachlorodibenzo-p-dioxin causes a modest induction of hepatic POR expression, with evidence of

AHR-dependence (Lee and Riddick, 2012; Lee et al., 2013).

35 A key physiological regulator of hepatic POR expression is thyroid hormone. Waxman et al.

(1989) showed that hypophysectomized (HYPX) rats have reduced levels of hepatic POR protein and diminished POR activity, suggesting that pituitary-dependent factors stimulate POR expression.

Hormone replacement in these HYPX rats showed that thyroxine (T4) was effective in restoring

POR protein, while growth hormone, ACTH, and chorionic gonadotropin were not (Waxman et al.,

1989). Some evidence suggests that thyroid hormones regulate POR expression via post- transcriptional mechanisms, involving effects on mRNA stability (Liu and Waxman, 2002) and translation (Apetalina et al., 2003). Other studies point to POR regulation by thyroid hormones via a transcriptional mechanism (Ram and Waxman, 1992; Li et al., 2001), with the response particularly pronounced in hypothyroid versus euthyroid rats. Consistent with a transcriptional mechanism, putatively functional thyroid hormone responsive elements have been identified in the promoter regions of the rat (O’Leary et al., 1997) and human (Tee et al., 2011) POR genes.

As an additional endocrine aspect of hepatic POR regulation, the central focus of my thesis is the adrenal glucocorticoids. ADX rats have reduced hepatic POR activity that is rescued with the administration of cortisone acetate over a period of eight days (Castro et al., 1970). Many studies of

POR regulation in mouse and rat models have used DEX as a potent synthetic glucocorticoid with the interesting property of activating GR at low concentrations and PXR at higher concentrations.

Most rodent studies in this field tend to utilize relatively high doses of DEX (in the range of 10 to 80 mg/kg) that are expected to activate both GR and PXR. For example, DEX (10 mg/kg/day for 2 days) increased hepatic POR activity in both sham and ADX rats (Sherratt et al., 1989) and DEX

(10 mg/kg single dose) increased hepatic POR activity in neonatal and adolescent rats (Linder and

Prough, 1993). A very high DEX dose (80 mg/kg) was shown to increase rat hepatic POR mRNA levels via a mechanism involving mRNA stabilization (Simmons et al., 1987). A role for PXR in 36 regulation of hepatic POR expression by high-dose DEX was supported by the observation that

DEX (50 mg/kg) induced hepatic POR protein levels in both wild-type and Gr-null mice (Schuetz et al., 2000).

In contrast, the focus of a recent Ph.D. graduate of our laboratory (Dr. Anne Mullen Grey) was the use of relatively low DEX doses that are expected to activate GR selectively. Two rat ADX models were used in her work: an acute model in which rats recovered for four days following ADX surgery and then received a single DEX dose (1.5 mg/kg) six hours prior to euthanasia, and a subacute model in which rats recovered for thirteen days following ADX surgery and then received a daily

DEX dose (1 mg/kg) for seven days with euthanasia the following day. In the acute study, ADX caused a decrease in hepatic POR protein and activity, with no change in POR mRNA levels (Mullen

Grey and Riddick, 2011); POR mRNA levels, but not protein levels, were increased by DEX treatment in both sham and ADX rats (Mullen Grey, 2011). The response to 3-methyl-cholanthrene in the acute ADX rats was suppressed for some, but not all, AHR-mediated responses and the decreased POR activity seen after ADX was viewed as contributing to the decreased capacity for

P450-dependent metabolism (Mullen Grey and Riddick, 2011). In the subacute study, ADX caused a decrease in hepatic POR protein and activity, with no change in POR mRNA levels; POR mRNA and protein levels were increased by DEX treatment in both sham and ADX rats (Mullen Grey,

2011).

In a time-course study, intact rats received a single DEX dose (1.5 mg/kg) with euthanasia at

3, 6, 12, and 27 hours after treatment. Hepatic POR mRNA levels were increased by DEX at 3, 6, and 12 hours, with no observed changes in POR protein levels at any time-point (Mullen Grey,

2011). This DEX dosing regimen caused significant induction of hepatic TAT mRNA levels, a classic GR-mediated response (Mullen Grey and Riddick, 2009), whereas no induction of CYP3A23

37 mRNA levels was seen, suggesting a lack of PXR activation at this low DEX dose (Mullen Grey,

2011). Although the induction of POR mRNA by low-dose DEX was consistent with GR involvement, in vivo chromatin immunoprecipitation (ChIP) assays did not detect DEX-inducible enrichment of GR recruitment to five putative GREs identified within the 5’-flanking region of the rat POR gene (Mullen Grey, 2011).

The induction of POR mRNA levels by DEX is also observed in H-4-II-E rat hepatoma cells in culture. Compared to the induction of a GR-regulated gene (TAT, EC50 ≈ 5 nM) and a PXR- regulated gene (CYP3A23, EC50 ≈ 3 µM), DEX was found to have intermediate potency for induction of POR mRNA in these cells (EC50 ≈ 40 nM). This suggests a role for both GR and PXR in the regulation of POR; however, this idea does not consider varying affinities of nuclear receptors to response elements (Mullen Grey, 2011). Taken together, the in vivo and in vitro evidence suggest that the roles of GR and PXR in the induction of hepatic POR expression and function by DEX remain unclear; this question formed the basis for the investigations pursued in this thesis.

1.6 Research Hypothesis

From the literature review discussed above, it is clear that the removal of physiological levels of glucocorticoids via ADX surgery as well as the administration of exogenous glucocorticoids can regulate the expression and activity of hepatic POR in rodent models. Many of the in vivo rodent studies have used DEX as a potent synthetic glucocorticoid with the interesting property of activating the GR selectively at low concentrations and both GR and PXR at higher concentrations

(Pascussi et al., 2001). Furthermore, many published in vivo studies in this area have used relatively high DEX doses expected to activate both GR and PXR (Simmons et al., 1987; Sherratt et al., 1989;

Linder and Prough, 1993; Schuetz et al., 2000), making it challenging to discern the relative contributions of these two nuclear receptors in the mechanism of POR regulation. Recent studies

38 from our laboratory using low DEX doses known to selectively activate GR have demonstrated pronounced POR induction at the mRNA level with minimal impact on protein levels (Mullen Grey,

2011).

An additional layer of complexity is provided by the potential for GR-PXR crosstalk in the regulation of hepatic gene expression. At low concentrations, DEX activates GR leading to increased transcription of the PXR gene, ultimately making more PXR protein available for activation by ligands. At higher concentrations, DEX can also bind PXR as an agonist resulting in further induction of target genes such as CYP3A23 (Pascussi et al., 2000; Huss and Kasper, 2000; Pascussi et al., 2001). The involvement of GR and PXR in the regulation of rat hepatic POR expression and function requires clarification. As well, it is unclear whether POR expression is regulated in a two- stage activation pathway involving both GR and PXR as has been established for the induction of

CYP3A target genes. These gaps in our current understanding provided the rationale for the following idea to be tested in this thesis research.

Hypothesis: The in vivo induction of rat hepatic POR expression and function by DEX is mediated by the GR at low doses and by both the GR and PXR at high doses.

1.7 Specific Objectives

This thesis research had four main objectives:

1. To determine whether rat hepatic POR is induced by DEX doses that activate GR (≥ 0.1

mg/kg) or PXR (≥ 10 mg/kg).

2. To determine whether TA (a selective GR agonist) or PCN (a selective PXR agonist) cause

induction of rat hepatic POR expression and activity.

39 3. To determine whether rat hepatic POR induction by DEX is altered by the GR antagonist

RU486.

4. To determine whether rat hepatic POR induction by DEX is altered in PXR-knockout rats.

1.8 Rationale for the Experimental Approach

The regulation of hepatic POR expression and function by glucocorticoids was investigated in male Fischer 344 rats as well as in PXR-knockout rats (in comparison to their wild-type Sprague-

Dawley controls). POR mRNA levels were assessed by quantitative real-time PCR, POR protein levels were assessed by immunoblot, and POR catalytic activity was assessed by the rate of cytochrome c reduction. As positive controls for GR and PXR activation, mRNA levels for TAT and CYP3A23 were assessed, respectively.

I used an in vivo approach in order to study the complex interactions between exogenous chemical administration and endogenous hormonal regulatory pathways in intact animals with functional endocrine circuits. Recent studies from our laboratory showing induction of hepatic POR mRNA levels by low-dose DEX were performed with male Fischer 344 rats (Mullen Grey, 2011) and so I continued with this rat strain as my main experimental model. Continuing with our recent focus on the regulation of rat hepatic POR expression and function, my thesis research also utilized a newly developed PXR-knockout rat strain, created by SAGE Technologies via a zinc finger nuclease approach. Since hepatic POR expression has been shown to be induced by high-dose DEX and PCN in mouse models, it would also be of interest to conduct additional studies in mice. Although there are Pxr-null mice that are viable and fertile (Xie et al., 2000), there are important health issues with both Gr-null mice (Cole et al., 1995) and hepatocyte-specific conditional Gr-null mice (Tronche et al., 2004). For these reasons, I focused on wild-type and PXR-knockout rat models, and utilized

40 pharmacological antagonism with RU486 to block GR function in the liver (Gagne et al., 1985; Honer et al., 2003).

In the DEX dose-response study, all DEX doses (0.1, 1, 10, 50 mg/kg) were anticipated to activate GR with the expectation that PXR would be activated only at the highest DEX doses (10 and 50 mg/kg). My results from this study showed an increase in hepatic POR mRNA levels in rats treated with all of these DEX doses. Based on this outcome, I chose DEX doses of 1 and 50 mg/kg for my work with PXR-knockout rats, since the low 1 mg/kg dose activates GR selectively whereas the high 50 mg/kg dose activates both GR and PXR. For the GR antagonism study, I arrived at a

RU486 dose of 50 mg/kg based on a pilot study performed in our laboratory and a previously published study (Gagne et al., 1985). In both cases, a RU486:DEX dose ratio of 100:1 was shown to evoke effective in vivo hepatic GR antagonism. Therefore, I tested the ability of RU486 (50 mg/kg) to antagonize the effects of a low 0.5 mg/kg DEX dose that activates GR selectively and a high 50 mg/kg DEX dose that activates both GR and PXR. Keeping in mind that RU486 can also activate PXR at high concentrations (Kliewer et al., 2002), this dose was chosen so as to achieve in vivo GR antagonism without causing substantial PXR activation by RU486 alone. In order to avoid complications caused by the GR-PXR crosstalk involved in the two-stage mechanism of DEX action,

I also used triamcinolone acetonide (TA) as a selective GR agonist (Runge-Morris et al., 1996) and

PCN as a selective PXR agonist (Hartley et al., 2004) in order to study activation of each receptor in isolation. Since the potency of TA is approximately one-fifth that of DEX (Schimmer and Funder,

2011) and I found effective GR activation by DEX at 1 mg/kg, I selected a TA dose of 5 mg/kg. A standard PXR-activating and CYP3A-inducing dose of PCN (50 mg/kg) was used in my study

(Schuetz et al., 2000).

41 Animals were euthanized at 6 h and 24 h following glucocorticoid treatment in all in vivo rat studies. The 6 h time-point was selected in order to capture early effects on mRNA levels as shown in a previous time-course study of DEX effects on rat hepatic POR mRNA levels (Mullen Grey,

2011). The 24-h time-point was chosen in order to observe later effects on POR protein and catalytic activity, which were anticipated to require more time for changes to occur in comparison to alterations in steady-state mRNA levels.

42 2. Materials and Methods

2.1 Animals and Treatment Protocols

All animals were cared for in accordance with the principles of the Canadian Council on

Animal Care and all animal experimentation was approved by the University of Toronto Animal

Care Committee. For the studies examining the DEX dose-response, GR- and PXR-selective agonists, and GR antagonism, male Fischer 344 rats (7 weeks old at the time of procurement) were purchased from Charles River Laboratories Canada (St. Constant, QC). Upon arrival at the Division of Comparative Medicine at the University of Toronto, rats were allowed to acclimatize for one week, during which they were subjected to regular handling, under standard housing conditions: two rats per cage, 12-h light / 12-h dark cycle with lights on at 7 am, and ad libitum access to Purina

Rodent Laboratory Chow No. 5001 and water. For the final animal study, male PXR-knockout rats

(SD-NR1i2tm1sage) and wild-type Sprague-Dawley controls (7 weeks old at the time of procurement) were obtained from SAGE Laboratories (Boyertown, PA). Upon arrival in the Division of

Comparative Medicine at the University of Toronto, these rats were placed in quarantine for one week, followed by acclimatization to standard housing for an additional period of 3 to 10 days.

Housing and diet were as described above for the Fischer 344 rats. The PXR-knockout rats are homozygous for a 20-bp deletion within exon 2 of the PXR gene created by zinc finger nuclease technology, leading to multiple premature stop codons, and non-functional PXR protein as evidenced by a lack of hepatic CYP3A induction following PCN treatment (www.sageresearchlabs. com/research-models/knockout-rats/pxr-knockout-rat). For all studies, rats were restrained during the injection procedures by an experienced animal technician to minimize stress.

43 For the DEX dose-response study, rats received a single intraperitoneal (i.p.) injection at 10 am, consisting of either corn oil vehicle or DEX (Sigma Chemical Company, St. Louis MO) at a dose of 0.1, 1, 10, or 50 mg/kg.

For the study of GR- and PXR-selective agonists, rats received a single i.p. injection at 10 am, consisting of either corn oil vehicle, TA (Sigma Chemical Company, St. Louis MO) at a dose of 5 mg/kg, or PCN (Sigma Chemical Company, St. Louis MO) at a dose of 50 mg/kg.

For the study of GR antagonism, rats received an i.p. injection of corn oil vehicle or RU486

(Sigma Chemical Company, St. Louis MO) at a dose of 50 mg/kg at 9:30 am, followed by a second i.p. injection at 10 am, consisting of corn oil vehicle or DEX at doses of 0.5 or 50 mg/kg.

In the final animal study, PXR-knockout rats and wild-type Sprague Dawley controls received a single i.p. injection at 10 am, consisting of either corn oil vehicle or DEX at doses of 1 or

50 mg/kg.

Following the injection procedures in all studies, rats were housed individually until the time of euthanasia. At 4 pm on the injection day (6 h post-injection) or 10 am the following day (24 h post-injection), rats were lightly anesthetized by isoflurane inhalation (Abbott Laboratories, North

Chicago IL), weighed, and euthanized by decapitation.

Immediately following euthanasia, each liver was perfused in situ with 30 ml of ice-cold

HEGD buffer (25 mM HEPES / 1.5 mM EDTA /10% glycerol / 1 mM dithiothreitol, pH 7.4).

Livers were then excised and weighed, and several individual pieces (each ~ 0.1 g) were frozen by immersion in liquid nitrogen and stored at -70°C for subsequent RNA isolation (see section 2.2).

The remaining liver tissue was processed by homogenization followed by subcellular fractionation, with my particular interest being the isolation of hepatic microsomes (see section 2.7).

44 2.2 RNA isolation

Total RNA was isolated from liver tissue by the acid guanidinium thiocyanate-phenol- chloroform extraction method (Chomczynski and Sacchi, 1987) using TRI-reagent from Sigma-

Aldrich (St. Louis, MO). Prior to any RNA work, all pipette tips and polypropylene tubes were autoclaved and any lab equipment involved was treated with RNase Zap from Ambion, Inc. (Austin,

TX). Each ~0.1 g piece of liver was submerged in 1 ml of TRI-reagent and homogenized using a motor-driven teflon-glass homogenizer (Caframo, Wiarton ON). Homogenized samples were transferred to polypropylene tubes and kept at room temperature for 5 min. to allow the complete dissociation of nucleoprotein complexes. Chloroform was added (0.2 ml per ml of TRI-reagent) and samples were shaken vigorously; each sample was then kept at room temperature for an additional 5 min. The resulting mixture was centrifuged at 12,000 x g for 15 min. at 4°C, separating the RNA into the upper aqueous phase. The aqueous phase was transferred to a new polypropylene tube.

Isopropanol was added (0.5 ml per ml of TRI-reagent) to the aqueous phase and samples were gently mixed and kept at room temperature for 5 min. to allow the precipitation of RNA. Samples were then subjected to centrifugation at 12,000 x g for 10 min. at 4°C, yielding a gel-like pellet (RNA precipitate) on the side/bottom of the tube. The supernatant was removed and the pellet was washed with 75% ethanol by vortexing, followed by centrifugation at 7,500 x g for 5 min. at 4°C.

RNA pellets were air-dried for 10 min. and dissolved by incubating the pellet in 100 µl of RNase-free water in a Fischer Scientific Dry Bath Incubator at 55°C for 20 min. The dissolved RNA samples were treated with 20 U DNase I (Invitrogen Life Technologies, Carlsbad, CA) for 20 min. at 37°C in a dry bath incubator in order to remove any DNA contamination. The enzyme was inactivated by incubation at 55°C for 20 min. Following the incubation RNA purity was assessed by determining

45 the A260/A280 ratio (>1.7), and the RNA yield was derived from the absorbance at 260 nm. RNA samples were stored at -70°C until subsequent use.

2.3 RNA integrity

Gel-electrophoresis was used to assess the integrity of isolated RNA samples. Each sample was run on a 1% agarose gel prepared in TAE buffer (40 mM Tris-acetate/ 1 mM EDTA, pH 8.0) with ethidium bromide (0.05 µl/ml of gel). This 1% agarose gel was then poured into a gel tray with comb in place, and allowed to polymerize for 20-30 min. RNA loading samples were prepared by adding RNA (0.5 µg/µl) from each sample to a mixture of RNase-free water (Invitrogen) and 6x dye from Fermentas Inc. (Glen Burnie, MD). A solution of 1x TAE was then poured into the Bio-Rad

Mini Sub DNA cell followed by the submersion of the polymerized gel with the wells of the gel closest to the negatively charged cathode. RNA samples (10 µl = 1 µg of RNA) were loaded, the gel apparatus was attached to a Bio-Rad PowerSupply and run at 70 V for 20-30 min. Afterwards the gel was visualized using the UVP BioDoc-It UV transilluminator, and samples were considered to have sufficient integrity if two distinct bands were present representing the 28S and 18S ribosomal

RNAs, with the 28S rRNA band showing greater intensity (Figure. 2.1).

2.4 Reverse Transcription

Reverse transcription was carried out based on the optimized conditions described in Franc et al. (2001). All cDNA synthesis reactions were done in a Perkin- Elmer DNA Thermal Cycler 480.

RNA (1 µg) was incubated with oligo d(T)18 (2 µg; Invitrogen) at 60°C for 5 min. A negative control

(C1) was introduced in this first phase by incubating RNase-free water with oligo d(T)18 in the absence of RNA. Primer-annealed samples were then incubated in a final volume of 40 µl containing

MMLV (Moloney murine leukemia virus)-reverse transcriptase (400 U; Invitrogen), RNA

Guard (60 U; Thermo Scientific, Wilmington, DE), 1mM of each dNTP (Fermentas), 10 mM

Figure 2.1. Visualization of 28S and 18S rRNA in total RNA samples isolated from rats in the DEX dose-response study. Total RNA (1 µg) from rats treated with corn oil vehicle or DEX at the indicated doses for 6 h or 24 h was run on a 1% agarose gel with ethidium bromide staining.

47 dithiothreitol, and 1X reverse transcriptase (50 mM Tris-HCl/75 mM KCl/3 mM MgCl2) buffer. A second negative control (C2) was introduced in this phase by adding the same mixture in the absence of MMLV reverse transcriptase to a primer annealed sample. Reactions continued for 60 min. at

37°C followed by incubation at 70°C for 10 min. cDNA samples were stored at -70°C until subsequent use.

2.5 Conventional RT-PCR

Conventional PCR with gel-based product detection was used qualitatively in this study to ensure successful reverse transcription, along with testing the specificity of PCR primers and the size of PCR amplicons. PCR reactions were performed based on optimized conditions for each primer set; primer specificity within the rat genome was verified by BLAST search and the thermodynamic properties of primers were assessed online with tools provided by Integrated DNA

Technologies (Coralville, IA) also the supplier of all primers. Table 2.1 lists the sequences of all primers used for both conventional PCR and quantitative real-time PCR in this thesis.

All conventional PCR reactions were done in the PTC-100 Peltier Thermal Cycler, starting with a hot start phase of 3 min. at 95°C and ending with an final extension phase of 7 min. at 72°C.

A total of 29 cycles was run with the following parameters: denaturation (30 sec at 94°C), annealing

(30 sec at 58°C), and extension (40 sec at 72°C). Each 50 µl PCR reaction contained input cDNA derived from 25 ng of RNA, Taq polymerase (2.5 U; Invitrogen), an optimized amount of each primer (0.1-0.3 µM), a 1.6 mM concentration of each dNTP, and 1X PCR buffer (20 mM Tris/50 mM KCl/3 mM MgCl2). Five types of negative controls were analyzed: the no RNA (C1) and no

MMLV reverse transcriptase (C2) controls were carried forward from the cDNA synthesis stage; a

PCR reaction lacking cDNA; PCR reactions lacking either forward or reverse primers. After cooling at 4°C, samples were diluted in 6x dye (Fermenta) and analyzed on 1% agarose gels with ethidium

Table 2.1

Sequences of primers used for conventional RT-PCR and quantitative real-time PCR

Gene Sequence Product size Primer Reference (bp) Conc. (nM) POR FP = 5’ GCC TGC CTG AGA TCG ACA AG 3’ 64 300 Muguruma et RP = 5’ GGG TCG CCC TCT CCG TAT GT 3’ al., 2006 Mullen Grey and Riddick, 2011 TAT FP = 5’ CAG CAA CGT GCT TCG AGT ACC 3’ 137 100 Mullen Grey RP = 5’ CTC CTC CTG GCT GCC TTC AG 3’ and Riddick, 2009

CYP3A23 FP = 5’ TGG GTC CTC CTG GCA GTC GT 3’ 55 200 Mullen Grey, RP = 5’ GTG TGC GGG TCC CAA ATC CGT 3’ 2011

PXR FP = 5’ CAT GTT CAA GGG CGT CAT CA 3’ 157 300 Hosoe et al., RP = 5’ GCA CTC CCA GGT TCC TGT T 3’ 2005 Gueguen et al., 2006

β-ACTIN FP = 5’ GAC CCA GAT CAT GTT TGA GAC CTT C 3’ 109 100 Tijet et al., RP = 5’ GGA GTC CAT CAC AAT GCC AGT G 3’ 2006

FP = Forward Primer RP = Reverse Primer

49 bromide staining, as described in section 2.3 and as shown in Figure. 2.2.

2.6 Quantitative Real-time PCR

For quantitative real-time PCR, the cDNA synthesis step was performed as described in

Section 2.4. POR, TAT, CYP3A23, and PXR hepatic mRNA levels were determined by the comparative threshold cycle (ΔΔCt) relative quantitation method after confirming similar efficiencies for the target genes and endogenous reference gene β-actin. Each gene underwent optimization in which the input cDNA ranged from 0.01 to 25 ng and primer concentrations ranged from 100 to 500 nM. Final optimized primer concentrations are shown in Table 2.1. An example of efficiency curves for optimization of POR and β-actin amplification is shown in Figure. 2.3.

Each PCR reaction was prepared in a final volume of 10 µl and contained an optimized amount of cDNA (derived from 25 ng of RNA), 100-300 nM final primer concentrations, and 1X

Power SYBR Green Master Mix from Applied Biosystems (Foster City, CA). Cycling conditions were: initial cycle of 2 min at 50°C and 10 min at 95°C, followed by 40 cycles at 95°C for 15 s, and

60°C for 1 min. Each reaction was run in triplicate and analyzed on the ABI Prism 7500 Sequence

Detection System. A vehicle-treated 6 h control sample was used as the calibrator on each plate within a study and β-actin was used as the endogenous reference gene for each sample measured. Ct values for each sample were normalized to the Ct value for β-actin (ΔCt) and then normalized to the corresponding value for the common calibrator sample (ΔΔCt). Relative fold-change (RQ) was calculated as 2-ΔΔCt.

2.7 Microsomal Fractionation

Approximately 8 g of each liver was homogenized in 3 volumes of HEGD buffer; using a motor-driven teflon-glass homogenizer (Caframo, Wiarton ON). Microsomal fractions were then

1 2 3 4 5 6 7

Lane 1 = 50 bp ladder Lane 2 = - ve control: cDNA absent in PCR reaction Lane 3 = - ve control: TAT reverse primer absent in PCR reaction Lane 4 = - ve control: TAT forward primer absent in PCR reaction Lane 5 = Complete RT-PCR reaction for TAT Lane 6 = RT control (C1): RNA absent in RT reaction Lane 7 = RT control (C2): MMLV reverse transcriptase absent in RT reaction

Figure 2.2. Conventional RT-PCR analysis with gel-based product detection to assess the specificity of TAT primers and the TAT amplicon size. Hepatic RNA isolated from an untreated male Fischer 344 rat underwent reverse transcription and conventional PCR amplification (29 cycles) to detect the presence of TAT transcript. PCR products were analyzed on ethidium bromide-stained 1% agarose gels. Lane 2, negative control showing lack of exogenous DNA contamination in PCR reaction; Lane 3-4, negative controls showing requirement for TAT primers for product detection; Lane 5, in a complete reaction the TAT product migrates to a position consistent with its predicted size of 137 bp; Lane 6, negative control showing lack of exogenous RNA contamination in RT reaction; Lane 7, negative control showing lack of exogenous DNA contamination in RT reaction.

Figure 2.3. Representative efficiency curves for quantitative real-time PCR analysis of POR and β-actin mRNA levels. Rat hepatic POR and β-actin mRNA levels as quantified by real-time

PCR with input cDNA derived from 0.01, 0.1, 1 or 10 ng of RNA. The Ct value from each PCR reaction run in triplicate is plotted against the input cDNA amount displayed on a log scale. The lines of best fit were generated by least-squares linear regression, with the equations and the coefficients of determination (r2) shown. The efficiency of the POR reaction is 92.9% (slope = - 3.505) and the efficiency of β-actin reaction is 87.1% (slope = -3.676). The high amplification efficiencies and the parallel nature of the efficiency curves supported the use of the ΔΔCt method.

52 prepared from the homogenized liver by differential centrifugation. Liver homogenate was first centrifuged at 9000 x g for 20 min. at 4°C using a JA-17 rotor in a J2-21M centrifuge (Beckman,

Fullerton CA). Supernatant was removed and added to pre-cooled ultracentrifuge tubes and placed in a 70Ti rotor, followed by centrifugation at 106,000 x g for 60 min at 4°C in a L-80 Ultracentrifuge

(Beckman). Following this, the supernatant (liver cytosol) was donated to lab colleagues and the microsomal pellet was re-suspended by homogenization in TGE storage buffer (10 mM Tris/20% glycerol/1 mM EDTA, pH 7.4). Microsomes were frozen by immersion in liquid nitrogen and stored at -70°C until subsequent use.

2.8 Lowry Protein Assay

Microsomal protein concentrations were determined by the method of Lowry et al. (1951).

Briefly, a protein standard was created using bovine serum albumin (BSA) at concentrations of 0,

0.08, 0.16, 0.24, 0.32, and 0.4 mg/ml. Rat liver microsomal samples and BSA standards were prepared at 0.5 ml volumes. To all standards and samples, 0.5 ml of 0.5 N NaOH was added at timed intervals; the solution was then mixed and kept at room temperature for 30 min. Following those same timed intervals, 5 ml of copper reagent (a mixture of 100 parts 2% sodium carbonate/1 part 1% cupric sulphate/1 part 2% sodium potassium tartrate) was added to the alkaline solution.

After mixing and a further 10 min. incubation, 0.5 ml of 1 N Folin phenol reagent was added with immediate mixing. After 30 min., the absorbance at 670 nm was read using the Beckman DU-

65 spectrophotometer. Blank solutions that lacked BSA were defined as displaying zero absorbance.

The Quant II Quadratic Soft-Pac module (Beckman) was used to fit the standard curve via non-linear regression. Microsomal protein concentrations were determined by interpolation from the generated standard curve.

53 2.9 Immunoblotting

Protein loading amounts and antibody dilutions were optimized using hepatic microsomes prepared from vehicle-treated male Fischer 344 rats. Standard curves relating the band intensity for the protein of interest (POR) and the reference protein loading control (β-actin) to the amount of loaded microsomal protein were generated, and an example for POR detection is shown in Figure.

2.4. Such optimization experiments suggested that loading 6 µg of microsomal protein would permit relative comparisons between different rat microsome samples.

Microsomal samples were prepared at an appropriate protein concentration in sample loading buffer to give a final composition of 0.0625 M Tris-HCl/10% glycerol/2% SDS/5% β- mercaptoethanol/0.001% bromophenol blue/pH 6.8. Samples were boiled for 4-5 min. to denature proteins. Immunoblotting samples were then cooled to room temperature and stored at -70°C until subsequent use. Along with rat hepatic microsomal samples being analyzed, all gels were also loaded with a protein molecular mass ladder (Thermo Scientific) and a constant calibrator sample to allow comparisons across multiple gels.

Microsomal proteins were resolved by SDS-PAGE procedures developed by Laemmli (1970) and transferred to a nitrocellulose membrane (Hybond Enhanced Chemiluminescence, GE Healthcare) for measurement of POR and β-actin immunoreactive protein levels. The Bio-Rad “Mini-Protean II” gel apparatus was set up with glass plates having dimensions of 7 cm x 8 cm x 0.5 mm which would contain the polyacrylamide gel. The separating gel consisted of 10% acrylamide/BIS; 0.375 M Tris-

HCl, pH 8.8; 0.1% SDS; 0.05% ammonium persulfate; and 0.05% TEMED. The stacking gel consisted of 4% acrylamide/BIS; 0.125 M Tris-HCl, pH 6.8; 0.1% SDS; 0.05% ammonium persulfate; and 0.1% TEMED. The entire gel assembly was placed in an electrophoresis tank filled with 1x running buffer (0.025 M Tris-HCl/0.192 M glycine/0.1% SDS/pH 8.3). After

Figure 2.4. Representative immunoblot and standard curve analysis of POR protein levels in rat liver microsomes. (A) Immunoblot analysis of liver microsomal protein (1-15 µg) from a vehicle-treated male Fischer 344 rat using a polyclonal POR antibody. (B) The standard curve shows a linear relationship between the amount of protein loaded and the immunoreactive POR signal intensity. The line of best fit was generated by least-squares linear regression, with the equation and the coefficient of determination (r2) shown.

55 loading prepared samples into the wells of the stacking gel, the apparatus was then attached to the

Bio-Rad model 1000/500 Power Supply and run at 180V for ~45 min.

Following completion of the SDS-PAGE run, the gel assembly was dismantled. The stacking gel was cut from the separating gel, and the separating gel was allowed to equilibrate in 1x transfer buffer (25 mM Tris-HCl/192 mM glycine/20% methanol/pH 8.3) for 15 min., as was the nitrocellulose membrane. Electrophoretic transfer of proteins (Towbin et al., 1979) required the assembly of a “sandwich” in which the separating gel is in direct contact with the nitrocellulose membrane, surrounded on both sides by filter papers and outer foam pads, locked into a cassette.

Two at a time, cassettes were placed in the Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell with the gel face closest to the cathode; the tank was then filled with 1x transfer buffer and the buffer chamber was equipped with a prefrozen Bio-Ice cooling unit and a stir bar. The entire apparatus was then placed on a magnetic stir plate, attached to a Bio-Rad Model 200/2.0 Power Supply and run at

100V for 60 min.

Following completion of the transfer step, cassettes were dismantled and the efficiency of transfer was assessed by exposing the nitrocellulose membrane to 0.2% Ponceau S stain solution

(Sigma-Aldrich) for ~1 min. After protein transfer had been confirmed, the nitrocellulose membrane was cut to separate the top (high molecular mass range including POR) and bottom (low molecular mass range including β-actin) portions of the membrane. This was followed by several rinses of the nitrocellulose with distilled H20 and three rinses with 1x TNT (20 mM Tris/137 mM NaCl/0.1%

Tween-20/pH 7.6) concluded the wash. A “Blotto” solution (5% skim milk powder in TNT buffer with a pinch of thimerasol) was prepared for blocking non-specific binding sites on the nitrocellulose.

Membranes were incubated in “Blotto” overnight on an orbital shaker at 4°C. On the following day,

“Blotto” was removed and nitrocellulose membranes were washed with 1x TNT buffer via 3 quick

56 rinses and then a change of TNT buffer every 10 min. for ~30 min. Following the wash, primary antibodies were prepared in Blotto using the optimized dilution factors determined in preliminary experiments. The rabbit polyclonal antibody raised against amino acids 1-300 of human POR (Santa

Cruz Biotechnology, Santa Cruz CA) was prepared at a dilution factor of 1:5,000, while the mouse monoclonal antibody raised against β-actin (Abcam Inc., Cambridge, MA) was prepared at a dilution factor of 1:100,000. The top portion (high molecular mass range) of the nitrocellulose was incubated in the POR primary antibody solution and the bottom portion (low molecular mass range) of the nitrocellulose was incubated in the β-actin primary antibody solution, both with shaking for 1 h at room temperature. Membranes were then washed as described above. Secondary antibody- horseradish peroxidase conjugates were prepared in Blotto using the optimized dilution factors. Blots previously incubated in POR antibody were next incubated in a 1:5000 dilution of a donkey anti- rabbit Ig-horseradish peroxidase conjugate (GE Healthcare), with shaking for 1 h at room temperature. Blots previously incubated in β-actin antibody were next incubated in a 1:5000 dilution of a sheep anti-mouse Ig-horseradish peroxidase conjugate (Novus Biologicals, Littleton CO), with shaking for 1 h at room temp. Membranes were then washed as described above. After the wash, membranes were bathed for 1 min. in a solution consisting of equal proportions of the two

Amersham ECL Detection reagents (GE Healthcare), at a volume of 0.125 ml per cm2 of membrane.

Detection reagent was drained off; membranes were wrapped in Saran Wrap and placed in an autoradiography x-ray cassette. These were then carried to the dark room where a sheet of Bioflex

Scientific Imaging film was exposed for 1 min. as well as other exposure times as required. The film was developed in a Kodak M35A X-omatic processor. Films were scanned using an Epson

Perfection V500 Scanner and band intensities were quantified using IPLabGel software (Signal

Analytics, Vienna, VA). Signal intensities for POR were first normalized to the constant calibrator

57 sample to adjust for inter-gel differences and then expressed relative to the β-actin signal intensity for the corresponding sample lane.

2.10 Cytochrome C Reduction as a measure of POR activity

Microsomal samples were analyzed in triplicate for POR activity based on the reduction of cytochrome c measured spectrophotometrically at 550 nm (Strobel and Dignam, 1978). Reaction mixtures with a final volume of 1 ml containing 300 mM potassium phosphate buffer (pH 7.7), 30

µg of microsomal protein, and 70 µM cytochrome c (Sigma C-3131) were assembled in cuvettes and reactions were initiated upon the addition of NADPH (Sigma N-1630) to a final concentration of 1 mM. The rate of cytochrome c reduction at room temperature was determined spectrophotometrically at 550 nm based on an extinction coefficient of 21 mM-1cm-1. The rate of enzyme-catalyzed reaction was determined by subtracting the rate of reaction occurring in the absence of microsomes. Normalization for microsomal protein concentration yielded the final activity in nmol/min./mg protein.

2.11 Statistical Analyses

NOTE: This section was prepared by Dr. David Riddick in consultation with Dr. José Nobrega.

The same description also appears in the M.Sc. thesis of Sarah Hunter, who studied the regulation of the aryl hydrocarbon receptor nuclear translocator (ARNT) using tissues derived from the same experimental animals.

The DEX dose-response, the GR- and PXR-selective agonists, and the GR antagonism studies utilized a sample size of four rats per treatment group. This decision was based on a formal sample size/power calculation. This sample size provided 80% power (β = 0.20), at a level of significance of α = 0.05, to detect cases where the ratio of the estimated standard deviation (SD) to the minimum effect magnitude is 0.5. A typical example from previous and current studies of the

58 regulation of constitutive rodent liver P450s performed in our lab would be a 50% increase or decrease in expression with a SD for the measured parameter equal to 25% of the mean. For the study involving PXR-knockout rats, we were limited to a sample size of three rats per treatment group due to the high cost of these genetically-modified rats. This sample size provided 80% power

(β = 0.20), at a level of significance of α = 0.05, to detect cases where the ratio of the estimated SD to the minimum effect magnitude is 0.45. In cases where a study design offers inadequate power to detect a difference, potentially interesting data patterns are described conservatively as trends that did not achieve statistical significance.

All data are expressed as mean ± SD. All statistical analyses were performed on the original raw data and not on the percent control data presented in the figures. A result was considered statistically significant if P < 0.05.

For the DEX dose-response, data were analyzed initially using a randomized-design two-way analysis of variance (ANOVA) using Graphpad Prism 4 to identify significant influences of the two independent variables and their interaction (dose, time, dose x time interaction). Post test analyses for the planned comparisons (dose effect, time effect) were performed to assess whether there were significant differences between particular groups. Post tests were Bonferroni-corrected for multiple comparisons and used the mean square residual (pooled variance) and corresponding degrees of freedom from the two-way ANOVA and were performed using the online tool provided by

Graphpad Software (www.graphpad.com).

For the study of GR- and PXR-selective agonists, data were analyzed initially using a randomized-design two-way ANOVA using Graphpad Prism 4 to identify significant influences of the two independent variables and their interaction (agonist, time, agonist x time interaction). Post test analyses for the planned comparisons (agonist effect, time effect) were performed to assess

59 whether there were significant differences between particular groups. Post tests were Bonferroni- corrected for multiple comparisons and used the mean square residual (pooled variance) and corresponding degrees of freedom from the two-way ANOVA and were performed using the online tool provided by Graphpad Software (www.graphpad.com).

For the study of GR antagonism, data were analyzed initially using a randomized-design three-way ANOVA using Statview 4.0.2 to identify significant influences of the three independent variables and their interactions (dose, time, antagonist, dose x time interaction, dose x antagonist interaction, time x antagonist interaction, dose x time x antagonist interaction). Post test analyses for the planned comparisons (dose effect, time effect, antagonist effect) were performed to assess whether there were significant differences between particular groups. Post tests were Bonferroni- corrected for multiple comparisons and used the mean square residual (pooled variance) and corresponding degrees of freedom from the three-way ANOVA and were performed using the online tool provided by Graphpad Software (www.graphpad.com).

For the study of PXR-knockout rats, data were analyzed initially using a randomized-design three-way ANOVA using Statview 4.0.2 to identify significant influences of the three independent variables and their interactions (dose, time, genotype, dose x time interaction, dose x genotype interaction, time x genotype interaction, dose x time x genotype interaction). Post test analyses for the planned comparisons (dose effect, time effect, genotype effect) were performed to assess whether there were significant differences between particular groups. Post tests were Bonferroni- corrected for multiple comparisons and used the mean square residual (pooled variance) and corresponding degrees of freedom from the three-way ANOVA and were performed using the online tool provided by Graphpad Software (www.graphpad.com).

60 3. Results

My thesis research is focused on the roles of GR and PXR in the induction of rat hepatic

POR expression and function by DEX. To explore this topic, I performed four in vivo rat studies:

(1) a DEX dose-response study, (2) a GR- and PXR-selective agonist study, (3) a GR antagonism study, and (4) a PXR-knockout rat study.

3.1 DEX dose-response

The potent synthetic glucocorticoid, DEX, activates both GR and PXR, albeit with different dose-response relationships (Pascussi, 2001; Shi et al., 2010). The GR is activated in vivo by relatively low DEX doses due to the high affinity of this interaction; thus, all DEX doses used in this study (0.1, 1, 10, 50 mg/kg) were expected to cause GR activation. Only the highest DEX doses used

(10 and 50 mg/kg) were expected to activate the low affinity xenosensor PXR. This dose-dependent differential activation of GR and PXR was used to better understand the regulation of rat hepatic

POR at the mRNA, protein, and catalytic activity levels.

3.1.1 DEX dose-response: POR, TAT, CYP3A23, and PXR mRNA

Figure 3.1 depicts the induction of POR mRNA levels in response to increasing doses of

DEX. POR mRNA levels were upregulated at the 6 h time-point by all DEX doses; the 0.1 and 1 mg/kg doses caused approximately 3-fold increases, whereas the magnitude of induction by the 10 and 50 mg/kg doses was approximately 4- and 5-fold, respectively. With only the 10 and 50 mg/kg doses, a lower magnitude induction persisted at the 24 h time-point.

In all studies, I measured TAT mRNA levels as a positive control to demonstrate conditions in which a known GR target gene was induced. Figure 3.2(A) depicts the induction of TAT mRNA levels by DEX. Hepatic TAT mRNA levels were increased at the 6 h time-point by approximately

2- to 3-fold at all DEX doses tested. No induction was detected at 24 h.

Figure 3.1. Real-time PCR analysis of hepatic POR mRNA levels in rats treated with varying doses of DEX. Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle or DEX at doses of 0.1, 1, 10, or 50 mg/kg and were euthanized at 6 or 24 h following treatment. Quantitative analysis of POR mRNA levels, relative to ß-actin. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the two-way ANOVA main effects were: P = <0.0001 (dose), P = <0.0001 (time), P = 0.0044 (dose x time interaction). Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched vehicle control; †significantly different (P < 0.05) from DEX dose- matched opposite time-point.

62 A

Figure 3.2. Real-time PCR analysis of hepatic TAT and CYP3A23 mRNA levels in rats treated with varying doses of DEX. Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle or DEX at doses of 0.1, 1, 10, or 50 mg/kg and were euthanized at 6 or 24 h following treatment. Quantitative analysis of TAT (A) and CYP3A23 (B) mRNA levels, relative to ß-actin. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the two-way ANOVA main effects were: P = <0.0001 (dose), P = <0.0001 (time), P = <0.0001 (dose x time interaction) for TAT mRNA; P = <0.0001 (dose), P = <0.0001 (time), P = <0.0001 (dose x time interaction) for CYP3A23 mRNA. Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched vehicle control; †significantly different (P < 0.05) from DEX dose-matched opposite time-point.

63 In all studies, I measured CYP3A23 mRNA levels as a positive control to demonstrate conditions in which a known PXR target gene was induced. Figure 3.2(B) depicts the induction of

CYP3A23 mRNA levels by DEX. CYP3A23 levels were induced by approximately 5-fold at the 50 mg/kg dose at the 6 h time-point, whereas the magnitude of induction by the 10 and 50 mg/kg doses was approximately 5- and 30-fold, respectively, at the 24 h time-point. No induction was seen at the lower DEX doses.

As an additional dimension to the system under investigation, GR activation is known to increase the expression of PXR via a transcriptional mechanism (Pascussi, 2000; Pascussi, 2001). I monitored PXR mRNA levels to gain insight into whether PXR expression alterations may impact

POR regulation by DEX. Figure 3.3 depicts the induction of PXR mRNA levels by DEX. The 1 and 10 mg/kg doses caused an induction of PXR mRNA levels at the 6 h time-point, with a maximal induction of approximately 3-fold at the 10 mg/kg dose. A smaller magnitude induction was seen at the 24 h time-point, but only at the 50 mg/kg dose.

3.1.2 DEX dose-response: POR protein

Microsomal POR protein levels were analyzed to determine whether the increased amount of

POR mRNA resulted in an increase in POR protein. A representative immunoblot is shown in

Figure 3.4(A). The POR protein band migrates at approximately 78 kDa, as detected using a rabbit polyclonal antibody raised against human POR protein. Regardless of the time-point, DEX doses of

0.1, 1, and 10 mg/kg failed to produce any induction of POR protein levels; however the 50 mg/kg dose produced a 2-fold induction of POR protein levels at the 24 h time-point.

3.1.3 DEX dose-response: POR activity

To determine if an increase in POR protein levels has a functional consequence, I used a spectrophotometric assay of microsomal cytochrome c reduction as an index of POR catalytic

Figure 3.3. Real-time PCR analysis of hepatic PXR mRNA levels in rats treated with varying doses of DEX. Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle or DEX at doses of 0.1, 1, 10, or 50 mg/kg and were euthanized at 6 or 24 h following treatment. Quantitative analysis of PXR mRNA levels, relative to ß-actin. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the two-way ANOVA main effects were: P = 0.0003 (dose), P = 0.1693 (time), P = 0.0003 (dose x time interaction). Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched vehicle control; †significantly different (P < 0.05) from DEX dose- matched opposite time-point.

65 A

Figure 3.4. Immunoblot analysis of liver microsomal POR protein levels in rats treated with varying doses of DEX. Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle or DEX at doses of 0.1, 1, 10, or 50 mg/kg and were euthanized at 6 or 24 h following treatment. (A) Immunoblot of microsomal protein (6 µg) using a polyclonal antibody against POR and a monoclonal antibody against ß-actin, showing results for one rat per treatment group. The POR protein band migrates at approximately 78 kDa and ß-actin migrates at approximately 42 kDa. VEH = vehicle-treated rat. (B) Quantitative analysis of POR immunoreactivity levels, relative to ß-actin. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the two-way ANOVA main effects were: P = 0.0391 (dose), P = 0.0003 (time), P = 0.1749 (dose x time interaction). Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched vehicle control; †significantly different (P < 0.05) from DEX dose-matched opposite time- point.

66 activity. Figure 3.5 shows that POR catalytic activity was increased at the 24 h time-point by approximately 1.5-fold by the 10 and 50 mg/kg doses of DEX.

3.2 GR- and PXR- selective agonists

In my next phase of experimentation, I examined whether rat hepatic POR expression and function were regulated by chemicals that selectively activate either GR or PXR. To this end, I used

TA as a GR-selective agonist at a dose of 5 mg/kg (Runge-Morris et al., 1996) and PCN as a PXR- selective agonist at a dose of 50 mg/kg (Hartley et al., 2004). Similar to the DEX dose-response, POR mRNA, protein, and catalytic activity were all assessed as well as TAT and CYP3A23 mRNA levels as positive controls for the activation of GR and PXR, respectively.

3.2.1 GR- and PXR-selective agonists: POR, TAT, CYP3A23 and PXR mRNA

Figure 3.6 shows that TA alone was unable to induce POR mRNA levels significantly at either the 6 h or 24 h time-point; however, there appears to be a trend at 6 h for an increased amount of POR mRNA corroborated by the fact that POR mRNA levels were higher at 6 h compared to the

24 h time-point. PCN induced POR mRNA levels by approximately 3- fold at the 6 h time-point.

Induction of PCN was no longer observed at 24 h.

Figure 3.7(A) shows an approximately 3.5-fold induction of TAT mRNA levels in response to TA at 6 h; this induction was no longer seen at 24 h. PCN treatment did not alter the levels of

TAT mRNA. Figure 3.7(B) shows an approximately 70- fold induction of CYP3A23 mRNA levels by PCN at 24 h; the trend for a smaller magnitude increase in CYP3A23 mRNA levels at 6 h following PCN treatment did not achieve statistical significance. TA treatment did not alter the levels of CYP3A23 mRNA.

Figure 3.5. Microsomal POR-catalyzed cytochrome c reduction activity in rats treated with varying doses of DEX. Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle or DEX at doses of 0.1, 1, 10, or 50 mg/kg and were euthanized at 6 or 24 h following treatment. Quantitative spectrophotometric analysis of microsomal POR-catalyzed cytochrome c reduction activity. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the two-way ANOVA main effects were: P = <0.0001 (dose), P = <0.0001 (time), P = <0.0001 (dose x time interaction). Outcomes of Bonferroni- corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched vehicle control; †significantly different (P < 0.05) from DEX dose-matched opposite time-point.

Figure 3.6. Real-time PCR analysis of hepatic POR mRNA levels in rats treated with TA (GR agonist) or PCN (PXR agonist). Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle (VEH), TA (5 mg/kg), or PCN (50 mg/kg) and were euthanized at 6 or 24 h following treatment. Quantitative analysis of POR mRNA levels, relative to ß-actin. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the two-way ANOVA main effects were: P = <0.0001 (agonist), P = <0.0001 (time), P = 0.0151 (agonist x time interaction). Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched vehicle control; †significantly different (P < 0.05) from agonist-matched opposite time-point.

69 A

Figure 3.7. Real-time PCR analysis of hepatic TAT and CYP3A23 mRNA levels in rats treated with TA (GR agonist) or PCN (PXR agonist). Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle (VEH), TA (5 mg/kg), or PCN (50 mg/kg) and were euthanized at 6 or 24 h following treatment. Quantitative analysis of TAT (A) and CYP3A23 (B) mRNA levels, relative to ß-actin. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the two-way ANOVA main effects were: P = <0.0001 (agonist), P = <0.0001 (time), P = <0.0001 (agonist x time interaction) for TAT mRNA; P = 0.0018 (agonist), P = 0.0284 (time), P = 0.0114 (agonist x time interaction) for CYP3A23 mRNA. Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched vehicle control; †significantly different (P < 0.05) from agonist-matched opposite time-point.

70 Figure 3.8 shows that PXR mRNA levels were not altered by either of the selective agonists at either time-point. There was a trend for elevated PXR mRNA levels at 6 h following administration of TA, but this did not achieve statistical significance.

3.2.2 GR- and PXR-selective agonists: POR protein

Figure 3.9 shows that neither TA nor PCN altered microsomal POR protein levels at the 6 h or 24 h time-points. The trend for elevated POR protein levels at 24 h following PCN treatment did not achieve statistical significance.

3.2.3 GR- and PXR-selective agonists: POR activity

Figure 3.10 shows there was no induction of POR catalytic activity by either selective agonist at either time-point. Again, the trend for increased POR activity at 24 h following PCN treatment did not achieve statistical significance.

3.3 GR antagonism:

The previous section suggested that activation of GR or PXR alone is insufficient to alter

POR protein levels or activity. For my final two studies, I therefore returned to DEX because of its ability to activate both GR and PXR. My next approach was to selectively eliminate the contributions of GR. Since a viable rat model lacking GR does not exist, I used a pharmacological strategy to test the impact of GR antagonism with RU486 (Bertagna et al., 1984) on POR induction by two doses of DEX. The low DEX dose (0.5 mg/kg) activates GR selectively, whereas the high

DEX dose (50 mg/kg) activates both the GR and the PXR. The RU486 dose (50 mg/kg) was chosen to achieve a 100:1 ratio of RU486 to DEX in rats receiving low dose DEX for effective GR antagonism without PXR activation by RU486 alone.

Figure 3.8. Real-time PCR analysis of hepatic PXR mRNA levels in rats treated with TA (GR agonist) or PCN (PXR agonist). Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle (VEH), TA (5 mg/kg), or PCN (50 mg/kg) and were euthanized at 6 or 24 h following treatment. Quantitative analysis of PXR mRNA levels, relative to ß-actin. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the two-way ANOVA main effects were: P = 0.0660 (agonist), P = 0.0861 (time), P = 0.5703 (agonist x time interaction). Bonferroni-corrected post tests for the planned comparisons yielded no statistically significant agonist or time effects.

72 A

Figure 3.9. Immunoblot analysis of liver microsomal POR protein levels in rats treated with TA (GR agonist) or PCN (PXR agonist). Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle (VEH), TA (5 mg/kg), or PCN (50 mg/kg) and were euthanized at 6 or 24 h following treatment. (A) Immunoblot of microsomal protein (6 µg) using a polyclonal antibody against POR and a monoclonal antibody against ß-actin, showing results for one rat per treatment group. The POR protein band migrates at approximately 78 kDa and ß-actin migrates at approximately 42 kDa. (B) Quantitative analysis of POR immunoreactivity levels, relative to ß-actin. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the two-way ANOVA main effects were: P = 0.2562 (agonist), P = 0.2061 (time), P = 0.4255 (agonist x time interaction). Bonferroni-corrected post tests for the planned comparisons yielded no statistically significant agonist or time effects.

Figure 3.10. Microsomal POR-catalyzed cytochrome c reduction activity in rats treated with TA (GR agonist) or PCN (PXR agonist). Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle (VEH), TA (5 mg/kg), or PCN (50 mg/kg) and were euthanized at 6 or 24 h following treatment. Quantitative spectrophotometric analysis of microsomal POR-catalyzed cytochrome c reduction activity. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the two-way ANOVA main effects were: P = 0.0243 (agonist), P = 0.1613 (time), P = 0.9062 (agonist x time interaction). Bonferroni- corrected post tests for the planned comparisons yielded no statistically significant agonist or time effects.

74 3.3.1 GR antagonism: POR, TAT, CYP3A23 and PXR mRNA

POR mRNA levels were induced approximately 4-fold at 6 h following treatment with DEX at 0.5 mg/kg (Figure 3.11) and this effect was not altered by RU486. DEX at 50 mg/kg also induced

POR mRNA levels at the 6 h time-point in the absence and presence of RU486, and this response was potentiated by RU486. At the 24 h time-point, there was a trend for increased POR mRNA levels following high-dose DEX in the absence or presence of RU486, but this did not achieve statistical significance.

Figure 3.12(A) shows that TAT mRNA levels were induced approximately 4- to 5-fold at 6 h in rats treated with either 0.5 or 50 mg/kg DEX. The induction of TAT mRNA levels at 6 h by the low DEX dose, but not the high DEX dose, was attenuated by RU486. TAT mRNA levels were not altered at 24 h by either DEX dose in the absence or presence of RU486.

Figure 3.12(B) shows that CYP3A23 mRNA levels were induced approximately 30-fold at

24 h following treatment with DEX at 50 mg/kg and this strong induction was also seen in the rats co-treated with RU486. There was no induction seen at either 6 h or 24 h in rats treated with the low

DEX dose in the absence or presence of RU486. There was a tendency for RU486 alone to increase

CYP3A23 mRNA levels, but statistical significance was not achieved.

Figure 3.13 shows that PXR mRNA levels were increased approximately 2-fold at 6 h following treatment with DEX at 50 mg/kg, but only in rats co-treated with RU486.

3.3.2 GR antagonism: POR protein

POR protein levels were induced approximately 2-fold at 24 h following treatment with DEX at 50 mg/kg (Figure 3.14), and this response was potentiated by RU486.

Figure 3.11. Real-time PCR analysis of hepatic POR mRNA levels in rats treated with low- or high-dose DEX in the absence or presence of the GR antagonist RU486. Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle or RU486 (50 mg/kg) followed 30 min later by an i.p. injection or corn oil vehicle or DEX at doses of 0.5 or 50 mg/kg, with euthanasia at 6 or 24 h following the second injection. Quantitative analysis of POR mRNA levels, relative to ß-actin. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the three-way ANOVA main effects were: P = <0.0001 (dose), P = <0.0001 (time), P = 0.0117 (antagonist), P = 0.0024 (dose x time interaction), P = 0.4304 (dose x antagonist interaction), P = 0.0373 (time x antagonist interaction), P = 0.3814 (dose x time x antagonist interaction). Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched, antagonist-matched vehicle (no DEX) control; †significantly different (P < 0.05) from DEX dose-matched, antagonist- matched opposite time-point; ‡significantly different (P < 0.05) from time-matched, DEX dose-matched vehicle (no RU486) control.

76 A

Figure 3.12. Real-time PCR analysis of hepatic TAT and CYP3A23 mRNA levels in rats treated with low- or high-dose DEX in the absence or presence of the GR antagonist RU486. Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle or RU486 (50 mg/kg) followed 30 min later by an i.p. injection or corn oil vehicle or DEX at doses of 0.5 or 50 mg/kg, with euthanasia at 6 or 24 h following the second injection. Quantitative analysis of TAT (A) and CYP3A23 (B) mRNA levels, relative to ß-actin. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the three-way ANOVA main effects were: P = <0.0001 (dose), P = <0.0001 (time), P = 0.1887 (antagonist), P = 0.0127 (dose x time interaction), P = 0.3678 (dose x antagonist interaction), P = 0.1853 (time x antagonist interaction), P = 0.1309 (dose x time x antagonist interaction) for TAT mRNA; P = <0.0001 (dose), P = <0.0001 (time), P = 0.0365 (antagonist), P = <0.0001 (dose x time interaction), P = 0.8073 (dose x antagonist interaction), P = 0.2859 (time x antagonist interaction), P = 0.8408 (dose x time x antagonist interaction) for CYP3A23 mRNA. Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched, antagonist-matched vehicle (no DEX) control; †significantly different (P < 0.05) from DEX dose-matched, antagonist-matched opposite time- point; ‡significantly different (P < 0.05) from time-matched, DEX dose-matched vehicle (no RU486) control.

Figure 3.13. Real-time PCR analysis of hepatic PXR mRNA levels in rats treated with low- or high-dose DEX in the absence or presence of the GR antagonist RU486. Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle or RU486 (50 mg/kg) followed 30 min later by an i.p. injection or corn oil vehicle or DEX at doses of 0.5 or 50 mg/kg, with euthanasia at 6 or 24 h following the second injection. Quantitative analysis of PXR mRNA levels, relative to ß-actin. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the three-way ANOVA main effects were: P = <0.0001 (dose), P = 0.4020 (time), P = 0.7225 (antagonist), P = 0.2211 (dose x time interaction), P = 0.0289 (dose x antagonist interaction), P = 0.8507 (time x antagonist interaction), P = 0.2845 (dose x time x antagonist interaction). Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched, antagonist-matched vehicle (no DEX) control.

Figure 3.14. Immunoblot analysis of liver microsomal POR protein levels in rats treated with low- or high-dose DEX in the absence or presence of the GR antagonist RU486. Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle or RU486 (50 mg/kg) followed 30 min later by an i.p. injection or corn oil vehicle or DEX at doses of 0.5 or 50 mg/kg, with euthanasia at 6 or 24 h following the second injection. (A) Immunoblot of microsomal protein (6 µg) using a polyclonal antibody against POR and a monoclonal antibody against ß-actin, showing results for one rat per treatment group. The POR protein band migrates at approximately 78 kDa and ß-actin migrates at approximately 42 kDa. VEH = vehicle-treated rat. (B) Quantitative analysis of POR immunoreactivity levels, relative to ß-actin. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the three-way ANOVA main effects were: P = <0.0001 (dose), P = <0.0001 (time), P = <0.0001 (antagonist), P = 0.0184 (dose x time interaction), P = 0.1123 (dose x antagonist interaction), P = 0.3189 (time x antagonist interaction), P = 0.9890 (dose x time x antagonist interaction). Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched, antagonist-matched vehicle (no DEX) control; †significantly different (P < 0.05) from DEX dose-matched, antagonist-matched opposite time- point; ‡significantly different (P < 0.05) from time-matched, DEX dose-matched vehicle (no RU486) control.

79 3.3.3 GR antagonism: POR activity

Due to the increases in POR protein, a cytochrome c reduction assay was carried out to determine if the increase in protein resulted in an increase in POR activity. Figure 3.15 shows an induction of POR catalytic activity by approximately 2-fold at the 24 h time-point following treatment with DEX at 50 mg/kg in both the absence and presence of RU486.

3.4 PXR-KO

In my final study, I used a genetically modified rat model lacking PXR to selectively eliminate the contributions of PXR. Thus, I studied POR regulation by two doses of DEX in PXR-knockout

(PXR-KO) rats created by SAGE laboratories via zinc finger nuclease technology, and wild-type

Sprague-Dawley controls. The low DEX dose (1 mg/kg) activates GR selectively, whereas the high

DEX dose (50 mg/kg) activates both the GR and PXR.

3.4.1 PXR-KO: POR, TAT, CYP3A23 and PXR mRNA

Figure 3.16 shows that POR mRNA levels were induced approximately 7- to 8-fold at 6 h following treatment with either 1 or 50 mg/kg DEX. This induction response was present but significantly attenuated in the PXR-KO rats. The approximately 4.5-fold induction at 24 h following treatment with DEX at 50 mg/kg was not observed in PXR-KO rats. The magnitude of POR mRNA induction by DEX in the Sprague-Dawley rat strain was greater than seen previously in the Fischer

344 rat strain (Fig. 3.1 and 3.11).

Figure 3.17(A) shows that TAT mRNA levels were induced approximately 6- to 9-fold at the 6 h time-point when dosed with DEX at 1 or 50 mg/kg. This induction response was present but partly attenuated in the PXR-KO rats. No induction of TAT mRNA levels was seen at 24 h. Again,

Sprague-Dawley rats display a greater magnitude of TAT mRNA induction by DEX compared to the response in Fischer 344 rats (Fig. 3.2 and 3.12).

Figure 3.15. Microsomal POR-catalyzed cytochrome c reduction activity in rats treated with low- or high-dose DEX in the absence or presence of the GR antagonist RU486. Adult male Fischer 344 rats received an i.p. injection of corn oil vehicle or RU486 (50 mg/kg) followed 30 min later by an i.p. injection or corn oil vehicle or DEX at doses of 0.5 or 50 mg/kg, with euthanasia at 6 or 24 h following the second injection. Quantitative spectrophotometric analysis of microsomal POR-catalyzed cytochrome c reduction activity. Data represent the mean ± SD of determinations from four rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the three-way ANOVA main effects were: P = <0.0001 (dose), P = <0.0001 (time), P = 0.0314 (antagonist), P = <0.0001 (dose x time interaction), P = 0.1571 (dose x antagonist interaction), P = 0.1409 (time x antagonist interaction), P =0.7438 (dose x time x antagonist interaction). Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched, antagonist-matched vehicle (no DEX) control; †significantly different (P < 0.05) from DEX dose-matched, antagonist-matched opposite time-point.

Figure 3.16. Real-time PCR analysis of hepatic POR mRNA levels in wild-type and PXR-knockout rats treated with low- or high-dose DEX. Adult male wild-type Sprague-Dawley rats (WT) or PXR-knockout rats (PXR-KO) received an i.p. injection of corn oil vehicle or DEX at doses of 1 or 50 mg/kg and were euthanized at 6 or 24 h following treatment. Quantitative analysis of POR mRNA levels, relative to ß-actin. Data represent the mean ± SD of determinations from three rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the three-way ANOVA main effects were: P = <0.0001 (dose), P = <0.0001 (time), P = <0.0001 (genotype), P = <0.0001 (dose x time interaction), P = 0.0051 (dose x genotype interaction), P = 0.0614 (time x genotype interaction), P = 0.0611 (dose x time x genotype interaction). Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched, genotype-matched vehicle control; †significantly different (P < 0.05) from DEX dose-matched, genotype-matched opposite time-point; ‡significantly different (P < 0.05) from time-matched, DEX dose-matched wild-type group.

82 A

Figure 3.17. Real-time PCR analysis of hepatic TAT and CYP3A23 mRNA levels in wild-type and PXR- knockout rats treated with low- or high-dose DEX. Adult male wild-type Sprague-Dawley rats (WT) or PXR- knockout rats (PXR-KO) received an i.p. injection of corn oil vehicle or DEX at doses of 1 or 50 mg/kg and were euthanized at 6 or 24 h following treatment. Quantitative analysis of TAT (A) and CYP3A23 (C) mRNA levels, relative to ß-actin. Data represent the mean ± SD of determinations from three rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the three-way ANOVA main effects were: P = <0.0001 (dose), P = <0.0001 (time), P = 0.0003 (genotype), P = <0.0001 (dose x time interaction), P = 0.0079 (dose x genotype interaction), P = 0.0017 (time x genotype interaction), P = 0.1458 (dose x time x genotype interaction) for TAT mRNA; P = <0.0001 (dose), P = <0.0001 (time), P = <0.0001 (genotype), P = <0.0001 (dose x time interaction), P = <0.0001 (dose x genotype interaction), P = <0.0001 (time x genotype interaction), P = <0.0001 (dose x time x genotype interaction) for CYP3A23 mRNA. Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched, genotype-matched vehicle control; †significantly different (P < 0.05) from DEX dose-matched, genotype-matched opposite time-point; ‡significantly different (P < 0.05) from time- matched, DEX dose-matched wild-type group.

83 Figure 3.17(B) shows that CYP3A23 mRNA levels were induced approximately 27-fold at

24 h following treatment of wild-type rats with DEX at 50 mg/kg. This induction response was completely absent in the PXR-KO rats, thus satisfying the main diagnostic criterion for successful

PXR knockout.

Figure 3.18 shows trends for induction of PXR mRNA levels by low and high DEX doses in both rat genotypes particularly at the 6 h time-point, but these were not statistically significant. The high DEX dose induced PXR mRNA levels by approximately 3-fold at the 24 h time-point in PXR-

KO rats.

3.4.2 PXR-KO: POR protein

In wild-type rats, POR protein levels were induced approximately 4.5-fold at 24 h following treatment with DEX at 50 mg/kg (Figure 3.19). This induction response was completely absent in

PXR-KO rats.

3.4.3 PXR-KO: POR activity

Figure 3.20 shows that POR activity was induced approximately 2-fold in wild-type rats at

24 h following treatment with DEX at 50 mg/kg. This induction response was completely absent in

PXR-KO rats.

Figure 3.18. Real-time PCR analysis of hepatic PXR mRNA levels in wild-type and PXR-knockout rats treated with low- or high-dose DEX. Adult male wild-type Sprague-Dawley rats (WT) or PXR-knockout rats (PXR-KO) received an i.p. injection of corn oil vehicle or DEX at doses of 1 or 50 mg/kg and were euthanized at 6 or 24 h following treatment. Quantitative analysis of PXR mRNA levels, relative to ß-actin. Data represent the mean ± SD of determinations from three rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the three-way ANOVA main effects were: P = 0.0004 (dose), P = 0.2570 (time), P = 0.1335 (genotype), P = 0.4566 (dose x time interaction), P = 0.3258 (dose x genotype interaction), P = 0.2425 (time x genotype interaction), P = 0.5141 (dose x time x genotype interaction). Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched, genotype-matched vehicle control.

85 A

Figure 3.19. Immunoblot analysis of liver microsomal POR protein levels in wild-type and PXR-knockout rats treated with low- or high-dose DEX. Adult male wild-type Sprague-Dawley rats (WT) or PXR-knockout rats (PXR- KO) received an i.p. injection of corn oil vehicle or DEX at doses of 1 or 50 mg/kg and were euthanized at 6 or 24 h following treatment. (A) Immunoblot of microsomal protein (6 µg) using a polyclonal antibody against POR and a monoclonal antibody against ß-actin, showing results for one rat per treatment group. The POR protein band migrates at approximately 78 kDa and ß-actin migrates at approximately 42 kDa. (B) Quantitative analysis of POR immunoreactivity levels, relative to ß-actin. Data represent the mean ± SD of determinations from three rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the three-way ANOVA main effects were: P = 0.1087 (dose), P = 0.0104 (time), P = 0.5606 (genotype), P = 0.4553 (dose x time interaction), P = 0.0563 (dose x genotype interaction), P = 0.2365 (time x genotype interaction), P = 0.1426 (dose x time x genotype interaction). Outcomes of Bonferroni-corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched, genotype-matched vehicle control; †significantly different (P < 0.05) from DEX dose-matched, genotype-matched opposite time-point; ‡significantly different (P < 0.05) from time-matched, DEX dose-matched wild- type group.

Figure 3.20. Microsomal POR-catalyzed cytochrome c reduction activity in wild-type and PXR-knockout rats treated with low- or high-dose DEX. Adult male wild-type Sprague-Dawley rats (WT) or PXR-knockout rats (PXR- KO) received an i.p. injection of corn oil vehicle or DEX at doses of 1 or 50 mg/kg and were euthanized at 6 or 24 h following treatment. Quantitative spectrophotometric analysis of microsomal POR-catalyzed cytochrome c reduction activity. Data represent the mean ± SD of determinations from three rats per group, expressed as a percentage of the mean for the 6 h vehicle control. The P values for the three-way ANOVA main effects were: P = 0.0012 (dose), P = 0.0068 (time), P = 0.1519 (genotype), P = 0.0541 (dose x time interaction), P = 0.1267 (dose x genotype interaction), P = 0.1075 (time x genotype interaction), P = 0.0752 (dose x time x genotype interaction). Outcomes of Bonferroni- corrected post tests for the planned comparisons were: *significantly different (P < 0.05) from time-matched, genotype- matched vehicle control; †significantly different (P < 0.05) from DEX dose-matched, genotype-matched opposite time- point; ‡significantly different (P < 0.05) from time-matched, DEX dose-matched wild-type group.

87 4. Discussion

4.1 Summary of Main Findings

Four in vivo rat studies were conducted in order to gain insight into the role of GR and PXR in the induction of hepatic POR expression and function by DEX. First, a DEX dose-response study took advantage of the ability of DEX to selectively activate the GR at relatively low doses compared to the higher DEX doses required for PXR activation. Second, a GR-selective agonist

(TA) and a PXR-selective agonist (PCN) were studied to determine if activation of either receptor alone was sufficient to induce hepatic POR expression and function. Third, pharmacological antagonism of GR using RU486 allowed me to assess contributions of GR to the induction of hepatic

POR expression and function by DEX. Finally, I used PXR-KO rats to examine the induction of hepatic POR expression and function by DEX in a rat model lacking functional PXR. In all studies, induction of TAT and CYP3A23 mRNA levels was used to monitor activation of GR and PXR, respectively.

4.1.1 DEX dose-response

Low DEX doses (0.1 and 1 mg/kg), shown to activate the GR selectively, induced POR mRNA levels by 3-fold at the 6 h time-point. High DEX doses (10 and 50 mg/kg), shown to activate both the GR and PXR, induced POR mRNA levels by 4- to 5-fold at the 6 h time-point with a smaller elevation of POR mRNA levels persisting at the 24 h time-point. A common occurrence throughout most studies in this thesis is the apparent decrease in POR mRNA levels at 24 h compared to the 6 h vehicle control. Rodent POR mRNA has been shown to display a marked circadian fluctuation with a 3.8-fold peak/trough ratio, with peaks occurring at 6:00 and 10:00 pm and troughs occurring at 6:00 and 10:00 am (Zhang et al., 2008). Circadian fluctuation is a plausible explanation for the apparent decrease in POR mRNA levels at 24 h in the vehicle control due to

88 euthanizations in these studies occurring at 4:00 pm and 10:00 am for the 6 h and 24 h time-point, respectively.

Low DEX doses failed to induce POR protein levels at either time-point. Only DEX at 50 mg/kg was able to induce POR protein levels, shown by a 2-fold elevation at the 24 h time-point.

However, elevation of protein levels may not result in a functional consequence; therefore, POR catalytic activity was monitored by the rate of cytochrome c reduction. High DEX doses (10 and 50 mg/kg) induced POR catalytic activity by approximately 1.5-fold at the 24 h time-point.

The results of this study demonstrate that low DEX doses capable of activating GR selectively can induce POR mRNA levels without affecting the protein or catalytic activity levels; on the other hand, high DEX doses that activate both GR and PXR can induce POR mRNA, protein, and catalytic activity levels. Although GR activation by DEX may contribute to the observed POR induction at the mRNA level, it seems that induction of hepatic POR protein and catalytic activity requires the in vivo administration of DEX doses capable of activating both GR and PXR.

4.1.2 GR- and PXR-selective agonists

TA, a selective GR agonist, showed a trend for increased hepatic POR mRNA levels at the 6 h time-point, although statistical significance was not achieved. TA treatment did not alter POR protein or catalytic activity levels. PCN, a selective PXR agonist, induced hepatic POR mRNA levels by 3-fold at 6 h after dosing. The trends for elevated POR protein and catalytic activity levels at 24 h following PCN treatment did not achieve statistical significance.

The results from this study suggest that both GR and PXR may contribute to induction of rat hepatic POR mRNA levels but activation of either receptor alone seems insufficient to elevate POR protein and activity.

89 4.1.3 GR antagonism

GR antagonism was achieved with the use of RU486 (50 mg/kg) and deemed successful based on the inhibition of TAT induction following the administration of the low DEX dose (0.5 mg/kg).

POR mRNA levels were induced approximately 4-fold at 6 h following administration of DEX at 0.5 mg/kg in the absence of RU486, and this effect was not altered by RU486. DEX at 50 mg/kg induced

POR mRNA levels by approximately 5-fold at the 6 h time-point in the absence of RU486, and this response was potentiated in the presence of RU486.

POR protein levels were induced approximately 2-fold at 24 h by a DEX dose of 50 mg/kg; this response was potentiated in the presence of RU486. POR catalytic activity was also induced by approximately 2-fold at the 24 h time-point following treatment with DEX at 50 mg/kg in both the absence and presence of RU486.

Although RU486 attenuated the induction of TAT mRNA levels by a low DEX dose known to selectively activate GR, RU486 did not inhibit the induction by DEX of POR expression at the mRNA, protein, and catalytic activity levels. In fact, the effects of DEX at 50 mg/kg on POR mRNA and protein levels were potentiated by RU486. These results are consistent with a limited role for GR activation in the induction of hepatic POR expression by DEX; however, this experiment is complicated by the possibility that the 50 mg/kg dose of RU486 used in order to achieve effective

GR antagonism may also be capable of some PXR activation in rodent liver (Bertilsson et al., 1998).

A previous mouse study involving PCN administration suggested an important role for PXR in the induction of POR mRNA levels (Maglich et al., 2002), and the extent to which PXR activation by

RU486 may have influenced the outcome of my study remains uncertain.

90 4.1.4 PXR-KO

PXR-KO rats were confirmed to lack functional PXR protein based on the complete absence of CYP3A23 mRNA induction by high dose DEX. PXR mRNA levels were measurable in the liver of both the wild-type and PXR-KO rats since my real-time primers amplify a region spanning exons

3 and 4 of the PXR cDNA; therefore, the amplified region was unaffected by the 20 bp deletion present in exon 2 of the PXR-KO rats.

In wild-type Sprague-Dawley rats, POR mRNA levels were induced 7- to 8-fold at 6 h following administration of DEX at 1 or 50 mg/kg, respectively. This mRNA induction response was present but significantly attenuated in PXR-KO rats. A similar effect was seen for TAT mRNA levels in PXR-KO rats compared to wild-type.

POR protein levels were induced approximately 4.5-fold at 24 h following administration of

DEX at 50 mg/kg in wild-type rats; this response was completely absent in the PXR-KO rats.

Finally, POR activity was induced approximately 2-fold at 24 h following administration of DEX at

50 mg/kg in wild-type rats, and this induction was once again completely absent in the PXR-KO rats.

The results from this study suggest that activation of the GR by low and high dose DEX contributes to the induction of POR mRNA levels both in rats expressing and lacking functional

PXR. The fact that the induction of POR mRNA levels is attenuated in PXR-KO rats suggests that activation of PXR, in addition to GR, is required for a full POR mRNA induction response. These results clearly establish that the induction of POR protein and catalytic activity seen at the high

DEX dose requires PXR.

91 Taken together, the four in vivo rat studies suggest that although GR activation may contribute to POR mRNA induction, the induction of rat hepatic POR expression and function by

DEX is primarily PXR-mediated.

4.2 Molecular mechanisms of POR regulation

The induction of POR mRNA levels following the administration of DEX may be due to a transcriptional mechanism or post-transcriptional mechanism. Since GR and PXR both function as ligand-activated transcription factors, it is important to consider transcriptional mechanisms for POR regulation involving these receptors. Depending on the dose given, DEX may activate either the GR and/or PXR, resulting in their translocation to the nucleus and subsequent binding to DNA response elements (GREs or PXREs), thus leading to the recruitment of co-activators and an increase in the rate of transcription of target genes. However, post-transcriptional mechanisms for the regulation of

POR mRNA stability have been implicated in the context of DEX (Simmons et al., 1987) and thyroid hormone (Liu and Waxman, 2002). Thus, it is important to consider the possibility that

DEX, acting via the GR, PXR and/or other mechanisms, may increase the stability of POR mRNA by preventing degradation, thereby increasing steady-state levels of POR mRNA.

In prokaryotes, an increase in mRNA transcript levels is often positively correlated with translational processes, resulting in an increase in protein levels (Pavesi, 1999); however, this is not always the case in eukaryotes. Schwanhausser et al. (2011) showed on a genome-wide scale that mammalian mRNA levels explained approximately 40% of the variability in protein levels; therefore an increase in mRNA transcript may not be paralleled by an increase in protein and catalytic activity levels. In this thesis research, I investigated POR protein and catalytic activity levels, in addition to mRNA levels, to gain a more complete understanding of the mechanisms and functional consequences of POR induction by DEX. Low doses of DEX induced POR mRNA levels with no accompanying

92 change in protein or catalytic activity levels. High doses of DEX induced POR mRNA, protein, and catalytic activity levels. Since changes in POR mRNA and protein levels do not always parallel each other, it is important to consider effects on translational efficiency or protein stability in the overall regulation of POR expression and function.

4.2.1 Transcriptional regulation of POR expression: a potential role for GR

Results from my DEX-dose response study, GR antagonism study, and PXR-KO rat study demonstrate that low doses of DEX (in the range of 0.1 to 1 mg/kg), shown to activate GR selectively, induce POR mRNA levels without a corresponding increase in POR protein or catalytic activity levels. The GR is known to regulate genes through various processes as discussed in Section

1.4.1. One common mechanism involves the direct binding of ligand- activated GR to GREs within proximal promoter regions of target genes; once bound, co-activators are recruited followed by the basal transcriptional machinery causing an induction of transcription. Another mechanism involves the tethering of the GR to other transcription factors through protein-protein interactions. A protein known to interact with the GR may first bind appropriate DNA recognition sites within the proximal promoter of target genes, followed by the recruitment of the GR along with other co- activators resulting in an increase in transcription.

Recent work from our laboratory established that a DEX dose sufficient to activate GR but not PXR caused induction of rat hepatic POR expression at the mRNA but not protein and activity level, thus stimulating my interest in a potential role for GR in this response. Mullen Grey (2011) showed a 2- to 3-fold induction of rat hepatic POR mRNA levels at 3, 6, and 12 h following the administration of DEX at 1.5 mg/kg. A bioinformatic analysis identified five putative GREs within the proximal 10 kb of the 5’-flanking region of the rat POR gene. ChIP analysis did not detect enhanced recruitment of the GR to these GREs in rat liver tissue at 3 h following administration of

93 DEX at 1.5 mg/kg. Possible explanations for this result were a lack of sensitivity of the assay, improper timing for analyzing recruitment, the GR acting through an indirect mechanism such as protein-protein interactions, and possible post-transcriptional mechanisms (Mullen Grey, 2011).

The presence of these putative GREs within the 5’ flanking region of the rat POR gene suggests that the observed induction of POR mRNA levels by glucocorticoids may be mediated via direct transcriptional activation. However, additional ChIP experiments will be required to determine whether the induction of POR mRNA levels is preceded by the recruitment of the GR to GREs.

The GR is able to interact with multiple transcription factors through protein-protein interactions including AP-1, STAT-5, and NF-κB (Jonat et al., 1990; Kerppola et al., 1993; McKay and Cidlowski, 1998; Wyszomierski et al., 1999). In the case of POR regulation, it is possible that interactions with Sp1 may allow the tethering of the GR to sites within the POR promoter. O’Leary et al. (1994) analyzed the rat POR promoter and found that it displayed many characteristics of a housekeeping gene; the promoter was found to be GC-rich (approximately 62%) with the absence of a TATA or CCAAT box, and it contained multiple binding sites for the transcription factor Sp1.

These Sp1 binding sites have been shown to be highly conserved within the rat and human POR gene.

Soneda et al. (2011) discovered a 2,487-bp deletion within the untranslated exon 1 of the POR gene, where the Sp1 sites are located, resulting in PORD in three patients. This finding suggests that these

Sp1 sites are indispensable for the regulation of POR transcription. An interesting parallel with another target gene is supported by a positive interaction between the Sp1 transcription factor and the GR in the regulation of monoamine oxidase A (MAO-A) in human neuroblastoma cells. ChIP assays revealed that the positive regulation of MAO-A through the Sp1/GR interaction was increased upon GR activation by DEX (Ou et al., 2006). Due to the importance and the multitude of

94 Sp1 sites within POR promoter region, this may be a potential GRE-independent mechanism by which GR regulates the expression of POR mRNA levels.

4.2.2 Transcriptional regulation of POR expression: a potential role for PXR

PXR is widely known for its role as a master regulator of genes involved in xenobiotic metabolism and transport. Maglich et al. (2002) demonstrated that PCN, a PXR-selective agonist, at

100 mg/kg induces POR mRNA levels in mouse liver and this induction is absent in Pxr-null mice; this strongly supports a key role for PXR in the induction of POR expression. Results from my

DEX-dose response study, GR antagonism study, and PXR-KO rat study demonstrate that high doses of DEX (in particular 50 mg/kg), shown to activate both GR and PXR, induce POR mRNA, protein and catalytic activity levels. My GR- and PXR-selective agonist study showed that activation of either receptor alone was not sufficient to induce POR protein or catalytic activity levels, suggesting that the unique ability of high dose DEX to activate both GR and PXR may be instrumental in inducing POR expression at the protein and catalytic activity levels. An essential role for PXR in the induction of POR protein and catalytic activity by high dose DEX was confirmed by the complete absence of this response in the liver of PXR-KO rats.

TREs, EREs, GREs, XREs and multiple binding sites for other transcription factors have been found in the POR proximal promoter; however, a PXRE within the POR promoter has yet to be defined (O’Leary et al., 1994; Mullen Grey, 2011; Tee et al., 2011). Based on the data presented in this thesis, it is possible that DEX induces POR mRNA levels via PXR-mediated direct transcriptional activation. The PXR and CAR activate a wide array of genes with considerable overlap in the xenobiotic-metabolizing genes controlled by each of these xenosensors. Studies have shown that PXR is able to bind and activate CAR response elements and vice versa; however, work by Faucette et al. (2006) has questioned the importance of each receptor activating opposing

95 response elements in that the induction of gene transcription may not be as efficacious in such cases compared to response elements activated by the corresponding nuclear receptor (Xie et al., 2000;

Sueyoshi and Negishi, 2001; Goodwin et al., 2002; Kast et al., 2002). In the context of POR regulation, it is well established that thyroid hormones are important physiological modulators operating at least partially via a transcriptional mechanism (Ram and Waxman, 1992; O’Leary et al.,

1997). Deiodinases (DIO) convert T4 to T3 in liver and other tissues and DIO-1 has been shown to be regulated by the TR. Interestingly, Pascussi et al. (2008) have shown that PXR/RXR heterodimers are able to bind the same TRE in the DIO-1 promoter that TR/RXR heterodimers bind, suggesting that TR and PXR may share DNA response elements and target genes. This raises the possibility that PXR activation may induce POR mRNA levels via direct transcriptional activation using the TRE half site.

4.2.3 Post-transcriptional regulation of POR expression

An additional mechanism that may be responsible for the increase in POR mRNA levels in response to DEX involves alterations of mRNA stability. The poly(A) tail is known to play a key role in nuclear mRNA processing, export to the cytoplasm, translation, and transcript stability.

Typically, a longer poly(A) tail results in more efficient translation and greater stability due to the major degradation pathway of mRNA transcripts in mammalian cells involving deadenylation followed by exonucleolytic degradation in the 3’ to 5’ direction (reviewed in Knapinska et al., 2005).

However, longer mRNA transcripts may not always be associated with increased stability. A study looking at the generation of alternative CYP3A4 mRNA transcripts in response to developmental signals and drugs revealed that the mRNA transcript with the shortest 3’-UTR was more stable and more translatable in comparison with the canonical CYP3A4 transcript (Li et al., 2012).

96 In regards to POR regulation, T3 induction of POR mRNA levels is associated with an elongation of the 3’-UTR of the POR transcript due to increasing the poly(A) tail length from approximately 100 to 205-bp. However, this alteration was not associated with any increase in POR mRNA translatability as evidenced by the absence of a T3-stimulated increase in POR protein levels

(Liu and Waxman, 2002). Simmons et al. (1987) showed that POR mRNA and protein levels were significantly induced upon the administration of the synthetic glucocorticoid DEX; however, they found no increase in the rate of POR gene transcription or levels of intranuclear pre-mRNA. They attributed the increase in mRNA and protein levels to an increase in POR mRNA stability caused by

DEX. In a general sense, the involvement of DEX as a modulator of mRNA stability is consistent with altered mRNA stability in the regulation of β-actin and β-tubulin expression by other hormonal factors (Jefferson et al., 1984). DEX has more recently been shown to display mRNA stabilizing effects towards the parathyroid hormone receptor (PTH1R) in transcriptionally-arrested cells, while in contrast displaying indirect mRNA destabilizing effects towards cyclooxygenase-2 (Cox-2) by inhibiting mitogen-activated protein kinase p38 (Lasa et al., 2001; Haramoto et al., 2007).

Another factor in the control of mRNA stability involves A+U binding proteins (AUBPs), such as the family of Hu proteins, which bind to AU-rich elements (AREs) in transcripts, preventing the recruitment of the exosome complex and therefore resulting in the stabilization of the mRNA

(Knapinska et al., 2005).

My findings suggest that both GR and PXR activation may play a role in the induction of hepatic POR mRNA levels by DEX. Additional experiments will be required to determine whether

POR mRNA levels are increased via direct transcriptional activation or via alterations in mRNA stability and the molecular details of how GR and/or PXR are involved in these processes. My data strongly implicate PXR in the regulation of POR protein and catalytic activity levels and it is this

97 regulation at the protein, as opposed to mRNA, level that ultimately may have functional consequences.

4.2.4 Translational and post-translational regulation of POR expression

The increases in POR protein and activity levels seen with PXR-activating doses of DEX could be attributed to multiple mechanisms. Taken as a whole, mRNA transcript levels do not always reliably predict how much protein is present due to factors affecting the translational efficiency, regulation of gene translation by microRNAs (miRNAs), and protein stability

(Schwanhausser et al., 2011). Altered translational efficiencies arise from the redundancy of the genetic code, in which most amino acids can be synthesized from more than one codon, allowing for the cell to fine tune the efficiency and accuracy of protein production. Translational efficiency also relies on cellular resources such as transfer RNA (tRNA) pools being adequate to ensure proper protein production (reviewed in Gingold and Pilpel, 2011). PXR activation by DEX may lead to alterations in these translational factors and result in a more efficient translation of POR mRNA, although I am not aware of a specific precedent for this.

miRNAs are highly conserved noncoding small RNAs that act at the post-transcriptional level to inhibit translation or increase degradation of the target mRNA (Bartel, 2004). Increases in miRNA-214 levels can result in down-regulated POR protein expression in rat liver, raising the possibility that this miRNA may be a key regulator of hepatic POR protein expression (Dong et al.,

2014). Nuclear receptors have been shown to regulate the expression of several miRNAs and therefore, activation of the GR or PXR by DEX may result in a down-regulation of multiple miRNAs leading to increased translation and therefore increased levels of POR protein seen in my studies after a 24-h exposure to high dose DEX (reviewed in Yang and Wang, 2011). As a specific

98 example, activation of PXR by rifampin in human hepatocytes results in the up- and down- regulation of multiple miRNAs (Ramamoorthy et al., 2013).

Finally Apletalina et al. (2003) showed that the increase in rat hepatic POR mRNA levels caused by T3 treatment was not paralleled by an increase in POR protein and catalytic activity levels. The discordance between mRNA and protein levels could not be attributed to differences in mRNA-polysome association or translational efficiency, but instead the authors suggested that POR may be regulated at least partially at the level of protein stability in the hyperthyroid rat liver.

Relating to my work, it may be possible that the POR protein is stabilized when rats are treated with

PXR-activating doses of DEX, leading to increased levels of POR protein and catalytic activity.

Regardless of the mechanism involved, my experiments indicate that PXR is vital for the induction of

POR protein and catalytic activity levels, a conclusion supported by the high DEX doses required for effects at the protein and activity levels, the lack of inhibition of this response by RU486, and the complete absence of this response in PXR-KO rats.

4.2.5 GR-PXR crosstalk

There is a well-established sequential crosstalk between the GR and the PXR in both in vitro and in vivo studies. GR activation induces PXR mRNA and protein levels in a dose-dependent manner. This induction appears to be via direct transcriptional activation through the GR, based on experiments involving actinomycin D (inhibitor of transcription), cycloheximide (inhibitor of translation), and RU486 antagonism (Pascussi et al., 2000; Pascussi et al., 2001; Shi et al., 2010).

Huss and Kasper (2000) proposed a model for the regulation of PXR through the activation of the

GR by DEX or possibly other glucocorticoids, see Fig. 1.7. This induction of PXR expression creates an opportunity for augmented regulation of PXR target genes such as rat CYP3A23, human

CYP2B6, and carboxylesterase-2 (CES2) upon the introduction of a PXR ligand (Huss and Kasper,

99 2000; Pacussi et al., 2000; Pascussi et al., 2001; Wang et al., 2003; Scheer et al., 2010; Shi et al.,

2010; Zhang et al., 2012).

I also attempted to explore this type of sequential GR-PXR crosstalk in my studies by measuring PXR mRNA levels; the assessment of PXR protein levels in rat liver continues to undergo optimization by another member of our lab group. The DEX dose-response showed increased PXR mRNA levels at DEX doses of 1 and 10 mg/kg at the 6 h time-point. The induction at 50 mg/kg failed to reach statistical significance at this time-point, although it would be interesting to assess this response at earlier times. The induction of PXR mRNA levels, particularly at the intermediate DEX doses, may be instrumental in providing additional PXR protein that can then be activated at the highest DEX doses to drive maximal induction of POR mRNA levels. Perhaps the clearest manifestation of this potential mechanism is the induction of POR protein and catalytic activity levels by high dose DEX at the later time-point, possibly due to increased amounts of PXR protein.

However, further studies need to be done to analyze PXR protein and test this hypothesis.

The results of my other studies were not entirely clear with regard to the importance of increased PXR mRNA levels in the regulation of POR expression and activity. The GR-selective agonist TA showed a trend towards increased PXR mRNA levels at 6 h but this did not achieve statistical significance, while the GR antagonism experiment only displayed increases in PXR mRNA levels following administration of both RU486 and DEX at 6 h. Since both RU486 and DEX were given at doses that can potentially activate PXR in vivo, it is tempting to speculate that PXR activation may result in auto-induction of PXR mRNA levels; however, the role of GR in the transcriptional regulation of PXR expression is more firmly established. Finally the PXR-KO rat study showed trends towards increased PXR mRNA levels in both the wild-type and PXR-KO rats at DEX doses of 1 and 50 mg/kg at 6 h, although statistical significance was not achieved. This may

100 have been due to the small sample size and the conservative nature of the statistical approach; however, there was a significant induction of PXR mRNA levels following administration of DEX at

50 mg/kg in the PXR-KO rats at 24 h. Since the PXR-KO rats have non-functional PXR protein, this might be a compensatory attempt to increase PXR expression to aid in the detoxification and elimination of the high DEX dose given.

4.3 Physiological and pharmacological relevance

Since POR is an important electron transfer partner for multiple enzymes with critical life functions, the induction of POR by DEX investigated in this thesis may have important physiological and pharmacological implications. As stated previously, the GR exhibits multiple

“permissive” effects throughout the body, producing changes in the protein makeup and therefore allowing cells to react appropriately to stressors or endogenous factors. From the data gathered within this study, I propose that DEX at low GR-activating doses acts as a permissive factor by

“priming” the drug metabolism system to be prepared for potential threats to homeostasis. At low

DEX doses, GR activation can trigger an increase in PXR expression and increases in POR mRNA levels with no subsequent change in protein or catalytic activity; therefore, the active GR may be

“priming” the drug metabolism system in order to have it ready for any future threats that may come into play. At high DEX doses, GR activation continues thereby maintaining its “priming” effect toward the drug metabolism system; however, the excess DEX within the system created by high dose administration may act as an additional stressor resulting in PXR-dependent increases in the expression and activity of POR and associated drug-metabolizing enzymes to speed the elimination of the excess drug. This sequential GR-PXR crosstalk triggered by DEX is not restricted to the POR enzyme. In fact human CYP2B6 and CYP3A4, rat CYP3A23, and CES2 all display similar induction patterns with minor variations. In many published studies, low GR-activating DEX doses are used to

101 “prime” the system and the additional stressor can be one of several PXR-activating xenobiotics

(Huss and Kasper, 2000; Pacussi et al., 2000; Pascussi et al., 2001; Wang et al., 2003; Scheer et al.,

2010; Shi et al., 2010; Zhang et al., 2012).

Because of POR’s central role in transferring electrons to all microsomal forms of P450, modulation of POR expression caused by perturbations of glucocorticoid levels within the body may have important functional consequences for pharmacotherapy. Stress is a common cause for elevation of glucocorticoid levels within the body of a wide range of organisms (Sapolsky et al.,

2000). In cases of chronic stress, exogenous glucocorticoid therapy, or diseases of glucocorticoid excess, elevated PXR expression and increased POR mRNA levels may create a “primed” state as described above. However, the main circulating glucocorticoids in humans (cortisol) and rodents

(CORT) are poor activators of the PXR; therefore, full induction of POR may only arise when glucocorticoids are given in combination with other PXR-activating drugs. Glucocorticoids are given in conjunction with chemotherapeutic drugs to reduce inflammation and other side effects caused by cancer chemotherapy (Salmon et al., 1994; Melhem et al., 2009). Some cancer chemotherapeutic agents such as paclitaxel activate the PXR and we might expect that induction of POR and P450- mediated metabolism would impact the kinetics and disposition of the drugs and potentially the balance of toxic and therapeutic effects as well (Synold et al., 2001; Mani et al., 2005). This combination of synthetic glucocorticoids with PXR activators resulting in POR induction could result in stimulated metabolism and reduced pharmacological activity of co-administered drugs that are biotransformed by P450s with increased activity caused by modulation of POR. In cases where

P450-dependent biotransformation detoxifies the parent drug in question, a DDI triggered by a combination of glucocorticoids and PXR activators would be expected to decrease pharmacological

102 activity. In cases where P450-dependent biotransformation leads to parent drug bioactivation, such

DDIs would be expected to increase therapeutic or toxic effects.

4.4 Limitations of the current study

4.4.1 Doses of DEX and limited time-points

The DEX dosing regimen used in my studies raises some limitations. The clinical relevance of the doses used in this study is questionable since they tend to be higher than what is used in humans.

For example, in a phase II multicenter study DEX was given orally at 40 mg for 4 consecutive days in conjunction with thalidomide, vincristine, and liposomal doxorubicin for the treatment of multiple myeloma. Assuming an average person of 70 kg is receiving this treatment, the dosing of DEX equates to 0.6 mg/kg/day, not accounting for bioavailability (Zervas et al., 2004). This relatively high clinical dose of DEX is in the range of what I refer to in my studies as “low dose DEX”; this helps to reinforce that the “low” (0.1 to 1 mg/kg) and “high” (10 to 50 mg/kg) DEX doses in my studies both represent high pharmacological doses, with my “low” doses being in the range of the highest doses used clinically. However, the doses of DEX used were chosen not for their clinical relevance, but in order to achieve reliable activation of GR vs. PXR in an in vivo rat model to study their influence on

POR levels. These doses were chosen in accordance with other studies showing the selective activation of the GR at low dose DEX (Pascussi et al., 2000) and the activation of both the GR and

PXR at high dose DEX (Pascussi et al., 2001). Experiments analyzing lower doses of DEX, in addition to the range that I studied, would be useful for studying impacts on POR regulation under conditions that are closer to what would be seen in clinical treatment regimens or in acute vs. chronic stress scenarios.

The use of only two time-points also presents a limitation of this study. The two time- points (6 and 24 h) were chosen to best capture potential changes at the mRNA level and the

103 protein/catalytic activity level, respectively. However when striving to establish mechanistic relationships, the inclusion of earlier and/or later time-points may have helped to establish more clearly the involvement of the GR versus the PXR in POR regulation and may have provided opportunities to evaluate minimal and maximal induced levels of POR. DuBois et al. (1995) showed that a 50 mg/kg dose of methylprednisolone induced TAT activity levels between 2 to 6 h with a subsequent dissipation, suggesting that GR activation can induce target genes earlier than 6 h and these earlier time-points could be quite relevant to POR induction mechanisms. Shi et al. (2010) showed a maximal induction of PXR mRNA levels at 24 h following treatment with a PXR-activating dose of DEX (in vitro); this suggests that 24 h may not be a long enough time to observe maximal induction of POR mRNA, protein, and catalytic activity in vivo if the process depends on initial induction of PXR expression followed by PXR activation.

4.4.2 GR antagonism with RU486

Since Gr-null mice die shortly after birth, it seems reasonable to predict that a GR-KO rat strain would not be viable. Therefore, to further investigate the role of GR in the induction of POR expression and function by DEX, RU486 was chosen to pharmacologically antagonize the GR in vivo. GR antagonism by RU486 was deemed successful if the induction of hepatic TAT mRNA levels by a low GR-activating DEX dose was attenuated significantly. A significant problem from my perspective is that RU486 is a known PXR agonist at high concentrations, and therefore it was desirable to achieve effective GR antagonism by RU486 without causing PXR activation. Studies have shown that RU486 at a dose of 10 mg/kg is able to bind the GR and prevent nuclear translocation of the DEX•GR complex, with subsequent inhibition of TAT induction (Alexandrova,

1992). I completed a pilot study examining the ability of RU486 at 10 mg/kg to block TAT induction by DEX (1 mg/kg); however, GR antagonism was not achieved under this condition. Based on the

104 report of Gagne et al. (1985), I decided to achieve a 100:1 ratio of RU486 to DEX in the rat by using a RU486 dose of 50 mg/kg against a DEX dose of 0.5 mg/kg DEX. Effective GR antagonism was achieved as evidence by attenuated TAT induction; however, RU486 at 50 mg/kg can cause some degree of PXR activation in vivo (Bertilsson et al., 1998; Kast et al., 2002; Teng et al., 2003).

Although I found that the induction of POR expression by DEX was not inhibited by RU486, the interpretation of this finding is clouded by the potential for PXR activation by RU486. Future in vivo studies could involve the use of an alternative GR antagonist, such as cyproterone, although use of this compound could be compromised by its anti-androgenic and progestin-like actions. Cell culture studies involving siRNA-mediated knockdown of GR may be the most definitive means of assessing the role of GR in POR induction by DEX.

4.4.3 Rat strains

The PXR-KO rat strain created by zinc finger nuclease technology by SAGE Laboratories was instrumental in demonstrating a key role for PXR in the induction of POR expression and function by DEX. These rats were created in a Sprague-Dawley background, whereas my three preceding studies used Fischer 344 rats. This produces a limitation in that it becomes difficult to compare completely the results from my PXR-KO rat study with the other three studies. My results showed that the magnitude of induction of POR and TAT mRNA levels by DEX was greater in

Sprague-Dawley rats compared to Fischer 344 rats. Dhabhar et al. (1993) described the differences between the Fischer 344 and the Sprague-Dawley strains in relation to the stress response, adrenal steroid levels, and CBG levels. Fischer 344 rats were found to have significantly higher diurnal and stress corticosterone profiles along with significantly higher CBG levels in plasma when compared to the Sprague-Dawley strain; however, both strains maintained similar levels of the GR in analyzed

105 tissues. These differences may contribute to the increased magnitude for both POR and TAT induction seen in the male Sprague-Dawley rats.

4.5 Future research directions

My four in vivo rat studies suggest that although GR activation may contribute to POR mRNA induction, the induction of rat hepatic POR expression and function by DEX is primarily

PXR-mediated. Future research will be essential to clarify the molecular mechanisms involved in

POR regulation and the functional consequences of POR induction by glucocorticoids.

My decision to conduct my studies in an in vivo rat model was based on the importance of studying POR regulation in an intact animal with functional endocrine circuits. However, such an approach presents limitations for molecular mechanistic investigations. Cell lines offer considerable advantages over in vivo models for the study of biological processes. Cell lines are cost effective, easy to use, they eliminate ethical concerns associated with whole animal studies, and provide consistent samples for reproducible results (Donato et al., 2008; Kaur and DuFour, 2012). A suitable cell line for future studies of POR regulation would be the rat hepatoma H-4-II-E cell line, which shows robust and reproducible induction of POR mRNA levels in response to a range of DEX concentrations (Mullen Grey, 2011). Below I outline a series of studies to be undertaken with H-4-

II-E cells in culture to elucidate more fully the roles of GR and PXR in POR induction and the molecular mechanisms involved.

Although we have already established the DEX dose-response for induction of POR mRNA levels in H-4-II-E cells, it will be important to also examine the DEX dose-response for the induction of POR protein and catalytic activity. A major advantage of using a cell line is that the effects of multiple doses (e.g. DEX at 1 nM to 100 µM) can be easily assessed at multiple time-points (e.g. 6,

12, 24 h), thereby providing insight into the minimal and maximal responses of POR induction as

106 well as GR vs. PXR involvement. Silencing experiments can be carried out by using siRNAs targeted towards the GR and PXR mRNA transcripts. siRNAs are designed to be perfect complements to their mRNA target sequences allowing for the knockdown of either GR or PXR via translational inhibition (Gong et al., 2005; Knapinska et al., 2005). By knocking down one or both receptors we should develop a greater understanding of the role of GR and PXR in POR regulation by DEX.

A transcriptional inhibitor such as actinomycin D can be used in H-4-II-E cells to help decipher whether GR and/or PXR activation by DEX modulates POR mRNA levels via a transcriptional or post-transcriptional mechanism. If the induction of POR mRNA levels by DEX is inhibited by actinomycin D, then this would suggest transcriptional regulation. In addition, following treatment of cells with actinomycin D, the half-life of POR mRNA can be assessed in the absence and presence of DEX. If this analysis suggests that DEX is inducing POR mRNA levels through a process involving increased mRNA stability, then luciferase reporter constructs containing sections of the POR 3’-UTR could be designed and tested for determinants of mRNA stability.

If the above analysis suggests that transcriptional regulation is more important, then ChIP assays along with luciferase reporter constructs could be implemented to assess the direct transcriptional influence of the GR and PXR. ChIP assays would be performed at varying time- points to determine if DEX activation causes the recruitment of the GR and/or PXR to any part of the POR 5’-flanking region. Furthermore, luciferase reporter plasmids containing the POR 5’-flank and promoter region could be constructed and transfected into the H-4-II-E cells. Key regions involved in GR- or PXR-dependent transcriptional regulation could be elucidated via deletion and site-directed mutagenesis approaches.

A translational inhibitor such as cycloheximide can be used in H-4-II-E cells to help decipher whether GR and/or PXR activation by DEX modulates POR protein levels via a translational or

107 post-translational mechanism. If the induction of POR protein levels by DEX is inhibited by cycloheximide, then this would suggest translational regulation. In addition, following treatment of cells with cycloheximide, the half-life of POR protein can be assessed in the absence and presence of

DEX. These studies taken as a whole will help elucidate the mechanisms involved in induction of

POR mRNA, protein, and catalytic activity levels by DEX and provide valuable information on the interplay of multiple receptors and enzymes.

An ultimate goal of this work is to determine if the induction of POR expression by glucocorticoids has functional consequences. In this thesis research, I assessed the rate of cytochrome c reduction as an indicator of one functional end-point. However, cytochrome c is an artifical surrogate electron acceptor and it will be important to assess whether modulation of POR levels influences the catalytic function of physiological POR partner proteins. These functional consequences should be assessed in both in vivo and in vitro H-4-II-E cell culture studies. Using a series of substrates, each of which is a selective diagnostic marker for a particular microsomal P450, it should be possible to examine how alterations of POR protein levels influence the activity of

P450s, perhaps in isozyme- and substrate-dependent fashion. Another important POR electron acceptor is heme oxygenase, and the activity of this enzyme can be assessed by monitoring the conversion of heme to biliverdin.

Overall, these proposed experiments will provide comprehensive insight into the mechanisms and functional impact of POR induction by DEX in both in vivo and in vitro contexts, suggesting new ways in which P450-dependent drug metabolism may be modulated by fluctuations in glucocorticoid levels.

108 4.6 Summary and significance

POR is a key component in the microsomal P450-dependent metabolism of xenobiotic and endogenous compounds. Microsomal P450 catalytic activity relies on the function of POR for electron transport, stressing the potential importance for regulated expression of POR in maintaining normal function of multiple P450s and other electron acceptor enzymes. Thyroid hormones are well- known regulators of the rat POR gene, with contributions to this regulation by glucocorticoids

(Castro et al., 1970; Waxman et al., 1989; Linder and Prough, 1993).

In this work I have demonstrated that low doses of DEX activate the GR and subsequently induce rat hepatic POR mRNA levels while high doses of DEX activate both the GR and PXR resulting in the induction of POR mRNA, protein, and catalytic activity levels. Activation of the GR alone by TA is unable to induce POR mRNA, protein or catalytic activity levels and activation of the PXR alone by PCN is unable to induce POR protein or catalytic activity levels. Induction of

POR protein and catalytic activity levels appears to require the administration of DEX doses sufficient to activate both GR and PXR. Taking into consideration the results from my GR antagonism and PXR-KO rat study, I conclude that although GR activation may contribute to POR mRNA induction, the induction of rat hepatic POR expression and function by DEX is primarily

PXR-mediated. Therefore, this work has important pharmacological implications in that increased glucocorticoid levels throughout the body due to stress, exogenous administration, or conditions of glucocorticoid excess along with the presence of PXR activators, endogenous or exogenous, may result in increased hepatic POR expression and function. Increased POR levels may influence microsomal P450s in isozyme- and substrate-dependent ways, with a potentially important role in drug-drug interactions.

109 Overall, this thesis contributes to the understanding of how adrenal-dependent factors and xenobiotic stressors regulate the expression of the rat hepatic POR gene, while giving insight into the interplay between the GR and PXR. Future studies should pursue the mechanisms by which the GR and PXR regulate POR expression and the functional consequences of this regulation for the various

P450 electron acceptor partners. If these responses are also demonstrated to be operative and important in human cells and tissues, then this work may help us to understand how the activity of

POR-dependent processes can be modulated by stress, steroidal therapies, and conditions of glucocorticoid excess or deficiency.

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