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The Pennsylvania State University The Graduate School Department of Veterinary and Biomedical Science IDENTIFICATION OF ENDOGENOUS MODULATORS FOR THE ARYL HYDROCARBON RECEPTOR A Thesis in Genetics by Christopher R. Chiaro © 2007 Christopher R. Chiaro Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December, 2007 The thesis of Christopher R. Chiaro was reviewed and approved* by the following: Gary H. Perdew John T. and Paige S. Smith Professor in Agricultural Sciences Thesis Advisor Chair of Committee C. Channa Reddy Distinguished Professor of Veterinary Science A. Daniel Jones Senior Scientist Department of Chemistry John P. Vanden Heuvel Professor of Veterinary Science Richard Ordway Associate Professor of Biology Chair of Genetics Graduate Program *Signatures are on file in the Graduate School iii ABSTRACT The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor capable of being regulated by a structurally diverse array of chemicals ranging from environmental carcinogens to dietary metabolites. A member of the basic helix-loop- helix/ Per-Arnt-Sim (bHLH-PAS) super-family of DNA binding regulatory proteins, the AhR is an important developmental regulator that can be detected in nearly all mammalian tissues. Prior to ligand activation, the AhR resides in the cytosol as part of an inactive oligomeric protein complex comprised of the AhR ligand-binding subunit, a dimer of the 90 kDa heat shock protein, and a single molecule each of the immunophilin like X-associated protein 2 (XAP2) and p23 proteins. Functioning as chemosensor, the AhR responds to both endobiotic and xenobiotic derived chemical ligands by ultimately directing the expression of metabolically important target genes. Primarily responsible for mediating the toxicological and biological effects of dioxin and other environmentally persistent carcinogens, the AhR was originally characterized for its role in orchestrating the adaptive metabolic response to xenobiotic compounds. Recently, however, the AhR has been identified as performing a critical role in a number of physiologically important life functions, including proper embryonic and liver development, immune system homeostasis, resolution of fetal vasculature, and maintenance of normal cardiac physiology. Currently, the most potent AhR agonists to be identified are of synthetic origin, yet an increasing number of natural compounds have been shown to activate the receptor. Although essential roles for the AhR in normal cellular biology have already been established and continue to evolve, no high iv affinity physiologically relevant endogenous ligand has been identified. Therefore, the ultimate goal of this research project was to identify such ligands. The initial data presented in this thesis confirms the presence of a putative endogenous ligand(s) for the AhR in the CV-1 cell line, while demonstrating the existence of an AhR regulated feedback mechanism functioning to control putative endogenous ligand levels. Derived from the kidney epithelium of the African green monkey, the CV-1 cell line is an immortalized cell culture line exhibiting minimal AhR expression. Consequentially, the level of AhR-regulated cytochrome P450 metabolism is also compromised allowing for subsequent accumulation of cellular metabolites, including potential intracellular endogenous ligands for the AhR. However, the ectopic expression of AhR-regulated cytochrome P450s from the 1A or 1B families effectively reduced the high level of constitutive AhR activity observed in CV-1 cells. Meanwhile cytochrome P450 2E1, an isoform not regulated by AhR, exhibited no significant effect. Furthermore, extracts of lung tissue prepared from Ahr-null mice clearly revealed, by the increased AhR activation potential compared to “wild-type” mice, the accumulation of an endogenous ligand for the AhR. Coupled with the high level of AhR-dependent CYP1A1 constitutive activity normally seen in lung, these observations support the existence, at least in mouse lung tissue, of an auto-regulatory loop between the AhR and CYP1A1 functioning to modulate endogenous ligand levels. Presented in subsequent chapters are results identifying and characterizing the ability of several eicosanoid molecules to activate AhR signaling. For instance, 12(R)-hydroxy- 5(Z),8(Z),10(E),14(Z)- eicosatetraenoic acid (12(R)-HETE) and 5(S), 12(R)-dihydroxy-6(E),8(E),10(E),14(Z)- eicosatetraenoic acid (6-trans-LTB4), two potent pro-inflammatory metabolites of v arachidonic acid, were discovered as indirect activators of the AhR, capable of activating AhR signaling, but failing to bind of the receptor. Surprisingly, other structurally similar isomers, such as 12(S)-HETE and LTB4 respectively, did not activate AhR signaling. In addition, the 5,6- and 14,15- positional isomers of leukotriene A4 (LTA4) along with various dihydroxyeicosatetraenoic acid (DiHETE) metabolites were also identified as potent activators of the AhR. Furthermore, conclusive evidence is presented demonstrating that 5,6-DiHETE isomers, cellular metabolites of 5,6-LTA4, can serve as endogenous ligands for the AhR, capable of directly binding and transforming the receptor to its DNA binding form. Although several of these eicosanoid molecules function as direct ligands for the receptor, others appear to activate the AhR through an indirect mechanism. Nevertheless, lipoxygenase metabolites are physiologically important bioactive lipid mediators that now comprise an exciting new class of endogenous modulators for the AhR. vi TABLE OF CONTENTS LIST OF FIGURES .....................................................................................................ix LIST OF TABLES.......................................................................................................xii ABBREVIATIONS .....................................................................................................xiii ACKNOWLEDGEMENTS.........................................................................................xv CHAPTER 1 ................................................................................................................1 INTRODUCTION .......................................................................................................2 1.1 THE ARYL HYDROCARBON (AH) RECEPTOR......................................2 1.1.1 Structural features of the AhR..............................................................3 1.1.1.1 The basic and helix-loop-helix domains ....................................3 1.1.1.2 The Per-Arnt-Sim (PAS) domain...............................................5 1.1.1.3 The hsp90 and ligand binding domain .......................................6 1.1.1.4 The transactivation domain ........................................................6 1.2 THE AH RECEPTOR SIGNAL TRANSDUCTION PATHWAY................7 1.2.1 The un-liganded AhR core complex.....................................................10 1.2.2 Transformation of the Ah receptor.......................................................10 1.2.2.1 Ligand binding ...........................................................................11 1.2.2.2 Translocation of the AhR core complex ....................................11 1.2.2.3 AhR and ARNT Heterodimerization and DNA Binding ...........12 1.3 LIGANDS FOR THE AH RECEPTOR.........................................................13 1.3.1 Halogenated aromatic hydrocarbons ....................................................13 1.3.2 Polycyclic aromatic hydrocarbons .......................................................17 1.3.3 Dietary ligands......................................................................................21 1.3.4 Endogenous ligands..............................................................................25 1.4 AHR MEDIATED BIOLOGICAL RESPONSES .........................................30 1.4.1 The AhR regulates xenobiotic metabolism ..........................................30 1.4.1.1 AhR regulation of phase I enzymes ...........................................31 1.4.1.2 AhR regulation of phase II enzymes ..........................................37 1.4.2 The AhR mediates the carcinogenic and toxicological response to environmental pollutants........................................................................42 vii 1.5 PHYSIOLOGICAL ROLES FOR THE AHR................................................45 1.5.1 AhR influences normal vascular development.....................................45 1.5.2 AhR influences cardiac development...................................................48 1.6 EICOSANOIDS..............................................................................................49 1.6.1 Overview ..............................................................................................49 1.6.2 Arachidonic acid cascade .....................................................................51 1.6.3 Cyclooxygenase Metabolites................................................................54 1.6.4 Lipoxygenase Metabolites....................................................................55 1.6.4.1 Arachidonate 5-LOX metabolites ..............................................57 1.6.4.2 Arachidonate 8-LOX metabolites ..............................................66 1.6.4.3 Arachidonate 12-LOX metabolites ...........................................67 1.6.4.4 Arachidonate 15-LOX metabolites ...........................................76 1.6.5 Cytochrome P450 monooxygenase metabolites...................................83