APOCAROTENOIDS MODULATE RETINOID RECEPTORS Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Abdulkerim EROĞLU The Ohio State Biochemistry Program The Ohio State University 2012 Dissertation Committee: Earl H. HARRISON, Ph.D., Advisor Robert W. CURLEY, Ph.D. Ouliana ZIOUZENKOVA, Ph.D. Jeanette W. MARKETON, Ph.D. Copyright by Abdulkerim EROĞLU 2012 Abstract β-carotene (BC) is the major dietary source of provitamin A. Central cleavage of BC catalyzed by β-carotene oxygenase 1 yields two molecules of retinaldehyde. Subsequent oxidation produces all-trans-retinoic acid (ATRA) which functions as a ligand for a family of nuclear transcription factors, the retinoic acid receptors (RARs). Eccentric cleavage of BC at non-central double bonds is catalyzed by other enzymes and can also occur non-enzymatically. The products of these reactions are β-apocarotenals and β- apocarotenones, whose biological functions in mammals are unknown. We used reporter gene assays to show that none of the β-apocarotenoids significantly activated RARs and RXRα. β-Apo-13-carotenone was found to antagonize the activation of RXRα by 9-cis- retinoic acid and was effective at concentrations as low as 1 nM. Molecular modeling studies revealed that β-apo-13-carotenone makes molecular interactions like an antagonist of RXRα. The results suggest a possible function of BACs on RXRα signaling. Moreover, β-apo-14’-carotenal, β-apo-14’-carotenoic acid, and β-apo-13- carotenone antagonized ATRA-induced transactivation of RARs. Competitive radioligand binding assays demonstrated that these putative RAR antagonists compete directly with retinoic acid for high affinity binding to purified receptors. Molecular modeling studies confirmed that β-apo-13-carotenone can directly interact with the ligand binding site of the retinoid receptors. β-Apo-13-carotenone and the β-apo-14’- carotenoids inhibited ATRA-induced expression of retinoid responsive genes in Hep G2 ii cells. Finally, we developed an LC/MS method and found 3-5 nM β-apo-13-carotenone was present in human plasma. These findings suggest that β-apocarotenoids function as naturally-occurring retinoid antagonists. We have also tested apo-lycopenoids that have a structural resemblance to β-apo-13-carotenone to see if they exert a similar action as β- apo-13-carotenone in modulating retinoid receptor activation. We found that apo-13- lycopenone was able to block ATRA induced expression of RARβ and CYP26A1 like the action of β-apo-13-carotenone. This suggests that the ionone ring may not be prerequisite for β-apo-13-carotenone’s binding to RARs. The antagonism of retinoid signaling by these metabolites may have implications for the activities of dietary β- carotene as a provitamin A and as a modulator of risk for cardiovascular disease and cancer. iii Dedication This document is dedicated to my parents and my wife. iv Acknowledgements I am greatly indebted to people who have helped me throughout my graduate studies. It is almost impossible to individually thank everyone who provided assistance or encouragement during my studies. I want to give my sincere thanks to all the people who are not mentioned here. I am eternally grateful for my advisor, Dr. Earl Howard Harrison, who generously gave me the opportunity to study in his laboratory and supported me through out my Ph.D. I appreciate his patience and guidance in teaching me directly en route to becoming an independent scientist and in correcting my poor English writing. Dr. Robert W. Curley, Jr. helped me a lot to learn analytical chemistry techniques and forge my ideas and to test them in his laboratory. I especially enjoyed many hours I spent working with him and his crew. Both Harrison lab and Curley lab are such great places to work and I am indebted to all the members of these labs, past and present. I have benefited from the advice of many of them. I thank Dr. Jian Sun, Mrs. Shiva Raghuvanshi, Dr. Matt Feshman, Mr. Carlo Sena and other members of our lab for their assistance. I also thank Dr. Curley lab members, Dr. Suresh Narayanasamy and Damian Hruszkewycz who contributed significantly to the analytical experiments in synthesis of β-apocarotenoids and apo-lycopenoids. Without their work, the studies that presented here would not be complete. I also thank Dr. Steven J. Schwartz and his lab members for their contribution to chapter 3. They analyzed the existence of β-apo-13-carotenone in human plasma samples. I am sincerely grateful to Dr. Ouliana Ziouzenkova and Dr. Jeanette Webster Marketon for their invaluable suggestions and guidance. v I am grateful to this country for opening its doors to me as a visiting research scholar back in 2005. I am eternally grateful to my parents Kadir and Kezzi Eroğlu for whom none of this would be possible and to my sister Yasemin Eroğlu and my brother Suleyman Eroğlu who are friends as well as siblings. I am also so grateful for my wife Arife Eroğlu for cheering me up and for her devoted support, love, inspiration and wise counsel. vi Vita 2005……………B.S. Molecular Biology and Genetics, Istanbul Halic University 2009……………Virginia Vivian Research Award 2010……………American Society for Biochemistry and Molecular Biology (ASBMB) Graduate/Postdoctoral Travel Award to Attend Experimental Biology Meeting 2010 in Conjunction with ASBMB Annual Meeting 2011……………American Society for Nutrition (ASN) Travel Stipend to Attend New Developments in Carotenoid Research an International Conference 2011……………Poster Competition Winner (1st place) at New Developments in Carotenoid Research an International Conference 2011…………....Outstanding Poster Award at the 2011 Carotenoids Research Interaction Group (CARIG) Meeting 2007-2012...........Graduate Research Associate, Department of Human Nutrition, The Ohio State University Publications 1. Eroglu, A., Hruszkewycz, D.P., Curley, R.W. & Harrison, E.H. The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα. Archives of Biochemistry and Biophysics 504, 11-16 (2010). 2. Eroglu, A. et al. Naturally-occurring eccentric cleavage products of provitamin A carotene β-carotene function as antagonists of retinoic acid receptors. Journal of Biological Chemistry 287, 15886-15995 (2012). Fields of Study Major Field: Biochemistry vii Table of Contents Abstract............................................................................................................................... ii Dedication.......................................................................................................................... iv Acknowledgement ...............................................................................................................v Vita.................................................................................................................................... vii Table of Contents………………………………………………………………………..viii List of Abbreviations ......................................................................................................... xi List of Tables ................................................................................................................... xiv List of Figures....................................................................................................................xv Chapter 1: Literature review 1.1 Introduction....................................................................................................................1 1.2 Absorption, Metabolism, and Transport of β-carotene and Vitamin A…………….....2 1.3 Cleavage and Oxidation of Carotenoids to Apocarotenoids…………………………..8 1.4 Occurrence and Functions of Apo-lycopenoids and β-Apocarotenoids, in Foods, and in Mammalian Tissues and Plasma………………………………………………………12 1.5 Retinoic Acid Receptors and Retinoid X Receptors....................................................24 1.6 Figures..........................................................................................................................30 viii Chapter 2: The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα Abstract..............................................................................................................................37 2.1 Introduction .................................................................................................................37 2.2 Materials and Methods.................................................................................................39 2.3 Results and Discussion ................................................................................................44 2.4 Implications..................................................................................................................48 2.5 Figures and Tables ...................................................................................................... 49 Chapter 3: Naturally-occurring eccentric cleavage products of provitamin A carotene β- carotene function as antagonists of retinoic acid receptors Abstract..............................................................................................................................56 3.1 Introduction .................................................................................................................57 3.2 Experimental Procedures ............................................................................................58 3.3 Results and Discussion ...............................................................................................66