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The Role of the Aryl Hydrocarbon Receptor in 2,3,7,8- Tetrachlorodibenzo-Ρ-Dioxin-Induced Mitochondrial Dysfunction in Mouse Hepatoma Cells

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The Role of the Aryl Hydrocarbon Receptor in 2,3,7,8- Tetrachlorodibenzo-Ρ-Dioxin-Induced Mitochondrial Dysfunction in Mouse Hepatoma Cells THE ROLE OF THE ARYL HYDROCARBON RECEPTOR IN 2,3,7,8- TETRACHLORODIBENZO-ρ-DIOXIN-INDUCED MITOCHONDRIAL DYSFUNCTION IN MOUSE HEPATOMA CELLS By Hye Jin Hwang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry and Molecular Biology - Doctor of Philosophy 2015 ABSTRACT THE ROLE OF THE ARYL HYDROCARBON RECEPTOR IN 2,3,7,8- TETRACHLORODIBENZO-ρ-DIOXIN-INDUCED MITOCHONDRIAL DYSFUNCTION IN MOUSE HEPATOMA CELLS By Hye Jin Hwang The aryl hydrocarbon receptor (AHR) is a Per-Arnt-Sim (PAS) domain family protein and ligand-activated transcription factor. The AHR directly regulates the expression of a large number of genes, including many that encode Phase I and Phase II drug metabolizing enzymes. Environmental contaminants such as polycyclic aromatic hydrocarbon or halogenated aryl hydrocarbon act as AHR ligands. The most potent AHR ligand, 2,3,7,8-tetrachlorodibenzo-ρ- dioxin (TCDD), causes various deleterious physiological effects, such as tumor promotion, chloracne, wasting syndrome, and hepatic steatosis. Over the last several decades, the AHR has been the focus of extensive research. Even with this depth of inquiry, there is a lack of understanding to the mechanism of action for AHR-mediated toxicity. Several possibilities have been proposed. For example, since the AHR is a transcription factor, it has been proposed that one, or more, of the genes that is regulated by the AHR leads to toxicity. The cytochrome P450 monooxygenases (CYPs) and TCDD-inducible poly (ADP-ribose) polymerase (TiPARP) are primary targets of AHR-regulated expression and are capable of promoting oxidative stress. In addition, the AHR can participate in crosstalk with other signaling pathways related to proliferation, development, differentiation, immune suppression, lipid metabolism and apoptosis. Several of these pathways are also involved in mediating the cellular response to oxidative stress. Given that oxidative stress is central to multiple pathways involved in AHR-mediated TCDD-induced toxicity and that the mitochondria, which consume most of cellular oxygen, are the main site generating oxidative stress, the role of the mitochondria in AHR signaling and toxicity was explored. Using the protease protection assay with digitonin extraction, the AHR was localized to the inter-membrane space (IMS) of the mitochondria. TCDD exposure induced a degradation of mitochondrial AHR similar to that of cytosolic AHR. Using siRNA in mouse hepatoma hepa1c1c7 cells, TOM20 and AHR-interacting protein (AIP) knockdown revealed that these two proteins are involved in the import of AHR into the mitochondria. The inhibition of heat shock protein-90kDa (HSP90) activity using an ATPase inhibitor, geldanamycin, also suggested that activity of HSP90 decided the level of mitochondrial AHR. TCDD altered the metabolic rate of cells in an AHR-dependent manner as measured by oxygen consumption rate (OCR) using the XF24 Extracellular Flux Analyzer. 30 nM TCDD exposure in hepa1c1c7 cells caused a significant AHR-dependent decrease in the OCR, but no change in the respiratory control ratio and the enzyme activities in ETC and ATP synthase. On the other hand, 30 nM TCDD exposure in AHR-deficient hepac12 cells caused no significant changes but the increased the respiratory control ratio and the activity of complex I. The mitochondria proteome was investigated using stable isotope labeling by amino acids in cell culture (SILAC). SILAC identified a battery of proteins that were influenced by TCDD in an AHR-dependent manner. The proteomic data suggested TCDD-induced AHR-mediated regulation in heme metabolism, lipid metabolism, redox potential, and mitochondrial biogenesis. Collectively, the data suggest the model 1) mitochondrial AHR in the IMS binds to components in the inner-membrane (IM), 2) TCDD induces the loss of interaction of AHR between a component in the IM, possibly causing an alteration in respiratory machinery, 3) AHR might help maintain homeostasis in respiratory efficiency against TCDD via regulating complex I or complex IV activity, and 4) AHR regulates the expression of genes that encode proteins that are important for various mitochondrial metabolic pathway and this regulation can be a result of mitochondrial retrograde signaling. This model supports a role for the mitochondria and possibly mitochondrial localized AHR in TCDD-induced toxicity, including metabolic dysfunction, wasting syndrome and hepatic steatosis. ACKNOWLEDGEMENTS The graduate school life in the MSU brought me wonderful memories within my life. Those couldn’t have been made without people whom I have had known, and I would never have achieved a PhD without them. First, I would like to thank Dr. John LaPres, my PI, for his consistent support, encouragement, and patience. He is a good model of scientist. He has taught me how and what a scientist must think and consider. He was always accessible and provided valuable advice. His guidance made me get through tough challenges from experiments and endless learning. Next, I would like to thank my committee members, for their precious time and supportive advice, Drs. Shelagh Ferguson-Miller, Kathy Gallo, Bill Henry, and Jeff MacKeigan. In every committee meeting, I got valuable guidance and insightful comments. I would especially like to thank Dr. Ferguson-Miller for providing a wealth of knowledge and collaboration regarding mitochondria and experimental techniques. Also, I had luck to work with wonderful people, the members of LaPres lab. I thank Drs. Scott Lynn, Dorothy Tappenden, and Ajith Vengellur for being exceptional colleagues, scientific mentors, and friends. I also thank Dr. Steve Proper for help and friendship. Finally, I want to thank the current members, Peter Dornbos and Michelle Steidemann, and the past graduate rotation students and undergraduate students, Taylor, Sarah, Mike, Larissa, Kai, for their huge efforts and help in the project. I would like to thank the Biochemistry & Molecular Biology program, and RTSF facility for their assistance. In addition, I also want to thank Dr. Laurie Kaguni for giving me the opportunity to attend the mitochondria summer schools. Lastly, I would like to thank my family for their love, supports and being there. Without you, I could not have done all of mine here. I love you and thank you. iv TABLE OF CONTENTS LIST OF TABLES ........................................................................................................................ vii! LIST OF FIGURES ..................................................................................................................... viii! KEY TO SYMBOLS AND ABBREVIATIONS ................................................................................ x! CHAPTER 1. LITERATURE REVIEW .......................................................................................... 1! PER-ARNT-SIM (PAS) DOMAIN PROTEINS ........................................................................... 2! ARYL HYDROCARBON RECEPTOR ...................................................................................... 3! Domains of AHR ................................................................................................................... 4! Transactivation of AHR ......................................................................................................... 8! Deactivation of AHR .............................................................................................................. 9! AHR Chaperones ................................................................................................................ 10! Ligands of AHR ................................................................................................................... 11! Endogenous ligands ....................................................................................................... 11! Xenobiotics ..................................................................................................................... 14! AHR Target Genes ............................................................................................................. 17! Physiological Roles of AHR ................................................................................................ 20! MITOCHONDRIA .................................................................................................................... 23! Structure of Mitochondria .................................................................................................... 23! Mitochondrial DNA (mtDNA) ............................................................................................... 24! Synthesis of Mitochondrial Proteins .................................................................................... 25! Synthesis of mitochondrial gene encoded proteins ........................................................ 25! Synthesis of nuclear gene encoded mitochondrial proteins ............................................ 26! Nuclear-encoded Mitochondrial Protein Translocation ....................................................... 26! Nuclear Transcription Factors for Mitochondrial Biogenesis ............................................... 28! Mitochondrial Respiration and Reactive Oxygen Species .................................................. 29! TCA Cycle ........................................................................................................................... 33! Fatty Acid β-oxidation and Ketogenesis ............................................................................
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