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

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Regulation of the Rat Hepatic NADPH-Cytochrome P450 Oxidoreductase by Glucocorticoids Regulation of the rat hepatic NADPH-cytochrome P450 oxidoreductase by glucocorticoids by 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 © Copyright by Alex Vonk (2014) Abstract 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
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