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Hepatic Xenobiotic Receptors in the Ubiquitin-Proteasome System

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Hepatic Xenobiotic Receptors in the Ubiquitin-Proteasome System HEPATIC XENOBIOTIC RECEPTORS IN THE UBIQUITIN-PROTEASOME SYSTEM by Jiong Yan Bachelor of Science, Xi’an Jiaotong University, 2009 Master of Science, Xi’an Jiaotong University, 2011 Submitted to the Graduate Faculty of School of Pharmacy in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2018 UNIVERSITY OF PITTSBURGH SCHOOL OF PHARMACY This dissertation was presented by Jiong Yan It was defended on Feb 27, 2018 and approved by Donald B. DeFranco, PhD, Professor, Pharmacology and Chemical Biology Paul A. Johnston, PhD, Associate Professor, Pharmaceutical Sciences Xiaochao Ma, PhD, Associate Professor, Pharmaceutical Sciences Yong Wan, PhD, Professor, Cell Biology Dissertation Advisor: Wen Xie, MD, PhD, Professor, Pharmaceutical Sciences ii Copyright © by Jiong Yan 2018 iii HEPATIC XENOBIOTIC RECEPTORS IN THE UBIQUITIN-PROTEASOME SYSTEM Jiong Yan, PhD University of Pittsburgh, 2018 Constitutive androstane receptor (CAR) and aryl hydrocarbon receptor (AhR) are liver-enriched xenobiotic receptors that are essential in the regulation of drug-metabolizing enzymes (DMEs) and drug transporters. Emerging evidence has also implicated CAR and AhR in the energy metabolism, cell proliferation and immune response, in addition to their classical function of xenobiotic detoxification. The cellular effects mediated by these xenobiotic receptors can be achieved canonically by the transcriptional modulation via direct interaction with the genomic DNA. There are also indirect mechanisms via protein-protein interactions by which CAR and AhR can alter the transcriptome. The preliminary results together with previous studies by others have suggested an interplay between the xenobiotic receptors and ubiquitin-proteasome system (UPS). In this dissertation study, I studied the E3 ubiquitin ligase activity of CAR and AhR in the context of hepatic gluconeogenesis and hepatic stellate cell (HSC) activation, respectively. My results demonstrated that (1) CAR suppresses hepatic gluconeogenic gene expression through post-translational regulation of the subcellular localization and degradation of PPAR-γ coactivator 1α (PGC1α). Activated CAR translocates into the nucleus and serves as a substrate adaptor protein recruiting PGC1α to the Cullin1 E3 ligase complex for ubiquitylation. The interaction between CAR and PGC1α also leads to their sequestration within the promyelocytic leukemia protein-nuclear bodies (PML-NBs), where PGC1α and CAR subsequently undergo proteasomal degradation, which is required for CAR-mediated inhibition of PGC1α. (2) AhR negatively regulates HSC activation by disrupting the interaction of Smad3 with β-catenin and iv impairing β-catenin-dependent stabilization of phosphorylated Smad2/3, which is independent of its E3 ubiquitin ligase activity. AhR is highly expressed in HSCs and activation of AhR prevents fibrogenesis and proliferation of HSCs. The expression of AhR in HSCs declines with the onset of HSC activation. Primary HSCs isolated from the AhR-/- mice exhibits accelerated spontaneous activation. Treatment with an AhR antagonist promotes, whereas the AhR agonists inhibit the activation of mouse and human HSCs, respectively. In vivo ablation of AhR in HSCs sensitizes mice to liver fibrosis. Overall, this dissertation elucidates a novel concept of xenobiotic receptors as the essential components in the UPS. v TABLE OF CONTENTS PREFACE .................................................................................................................................. XII ABBREVIATIONS ........................................................................................................................ I 1.0 INTRODUCTION ........................................................................................................ 1 1.1 HYPOTHESIS AND SPECIFIC AIMS............................................................. 2 1.2 DISSERTATION OUTLINE .............................................................................. 4 2.0 XENOBIOTIC RECEPTORS AND HEPATIC METABOLISM .......................... 5 2.1 HOST DEFENSE MECHANISMS .................................................................... 5 2.2 DISCOVERY OF XENOBIOTIC RECEPTORS ............................................ 7 2.3 XENOBIOTIC REGULATION BY PXR, CAR AND AHR ......................... 11 2.4 MODULATION OF XENOBIOTIC RECEPTORS: LIGAND-INDUCED NUCLEAR TRANSLOCATION ...................................................................................... 13 2.5 MODULATION OF XENOBIOTIC RECEPTORS: TRANSCRIPTIONAL REGULATION ................................................................................................................... 18 2.6 MODULATION OF XENOBIOTIC RECEPTORS: POST- TRANSLATIONAL MODIFICATIONS (PTMS) .......................................................... 20 2.7 ROLE OF UBIQUITIN PROTEOLYTIC SYSTEM IN XENOBIOTIC RECEPTORS ...................................................................................................................... 23 3.0 CAR AND HEPATIC GLUCONEOGENESIS ...................................................... 25 vi 3.1 RESEARCH BACKGROUND ......................................................................... 25 3.2 MATERIALS AND METHODS ...................................................................... 27 3.3 EXPERIMENTAL RESULTS ......................................................................... 32 3.4 DISCUSSION AND CONCLUSION ............................................................... 53 4.0 AHR AND LIVER FIBROSIS .................................................................................. 58 4.1 RESEARCH BACKGROUND ......................................................................... 58 4.2 MATERIALS AND METHODS ...................................................................... 60 4.3 EXPERIMENTAL RESULTS ......................................................................... 67 4.4 DISCUSSION AND CONCLUSION ............................................................... 86 5.0 SUMMARY ................................................................................................................ 90 5.1 NON-GENOMIC (NON-CANONICAL) FUNCTION OF XENOBIOTIC RECEPTORS ...................................................................................................................... 90 5.2 CAR IN THE REGULATION OF ENERGY METABOLISM .................... 92 5.3 AHR IN THE HEPATIC TOXICITY AND HOMEOSTASIS ..................... 95 APPENDIX A .............................................................................................................................. 99 APPENDIX B ............................................................................................................................ 100 BIBLIOGRAPHY ..................................................................................................................... 102 vii LIST OF TABLES Table 1. Antibody information ..................................................................................................... 99 Table 2. Real-time PCR primers sequences ................................................................................ 100 viii LIST OF FIGURES Figure 1. Historical landmarks in the discovery of PXR, CAR and AhR .................................... 11 Figure 2. Paradigm of xenobiotic response by mouse PXR, CAR and AhR ................................ 13 Figure 3. Nuclear translocation of xenobiotic receptors ............................................................... 17 Figure 4. PGC1α is a central transcriptional coactivator in hepatic gluconeogenesis .................. 26 Figure 5. CAR suppresses gluconeogenic gene expression in primary mouse and human hepatocytes .................................................................................................................................... 34 Figure 6. CAR suppresses gluconeogenesis through inhibiting PGC1α activity ......................... 35 Figure 7. CAR and PGC1α are co-regulated during fasting and in diet-induced obesity ............. 36 Figure 8. CAR decreases the DNA-binding of PGC1α on the gluconeogenic genes ................... 38 Figure 9. Activation of CAR leads to the redistribution of PGC1α to PML-NBs ........................ 39 Figure 10. CAR promotes ubiquitylation-proteasomal degradation of PGC1α ............................ 41 Figure 11. CAR’s interaction with PGC1α is essential for the ubiquitylation and degradation of PGC1α ........................................................................................................................................... 41 Figure 12. PML-NBs are required for CAR to induce PGC1α degradation and suppress gluconeogenic gene expression in vitro ........................................................................................ 43 Figure 13. PML-NBs are required for CAR to induce PGC1α degradation and suppress gluconeogenic gene expression in vivo ........................................................................................ 44 ix Figure 14. CAR interacts with the CUL1 E3 ligase components and forms a unique complex ... 46 Figure 15. CAR recruits the CUL1 E3 ligase to promote the ubiquitylation of PGC1α .............. 47 Figure 16. CAR-mediated inhibition of PGC1α requires active AF2 domain.............................. 50 Figure 17. CAR-mediated inhibition of PGC1α is independent of DNA-binding ....................... 51 Figure 18. N-terminal PGC1α fragment is resistant to the inhibition by CAR ...........................
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