A ROLE for ADIPONECTIN in TROPHOBLAST FUNCTION by Emily A. Mcdonald Submitted to the Graduate Degree Program in Molecular and In
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A ROLE FOR ADIPONECTIN IN TROPHOBLAST FUNCTION BY Emily A. McDonald Submitted to the graduate degree program in Molecular and Integrative Physiology and the Graduate Faculty of the University of Kansas Medical Center in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ______________________________ Michael W. Wolfe, Ph.D. Chairperson Committee Members ______________________________ Lane K. Christenson, Ph.D. ______________________________ Leslie L. Heckert, Ph.D. ______________________________ Margaret G. Petroff, Ph.D. ______________________________ Michael J. Soares, Ph.D. Date Defended: July 12, 2010 The Dissertation Committee for Emily A. McDonald certifies that this is the approved version of the following dissertation: A ROLE FOR ADIPONECTIN IN TROPHOBLAST FUNCTION Committee: _______________________________ Michael W. Wolfe, Ph.D. Chairperson _______________________________ Lane K. Christenson, Ph.D. _______________________________ Leslie L. Heckert, Ph.D. _______________________________ Margaret G. Petroff, Ph.D. _______________________________ Michael J. Soares, Ph.D. Date Approved: July 29, 2010 2 Table of Contents I. Acceptance Page…………….……………………………………………2 II. Acknowledgements………………………………………………………..5 III. List of Tables and Figures…..…………………………………………......7 IV. Abstract…………………………………………………………………..10 V. Chapter One: Introduction…………………………………………….....13 1. The Human Placenta……………………………………………..14 2. Diseases of Pregnancy…………………………………………...28 3. The Impact of Other Physiological Systems on the Placenta……40 4. Adiponectin………………………………………………………52 5. Study Significance……………………………………………….66 VI. Chapter Two: Adiponectin Attenuation of Endocrine Function within Human Term Trophoblast Cells………………………………………….71 1. Abstract…………………………………………………………..72 2. Introduction………………………………………………………74 3. Materials and Methods…………………………………………...77 4. Results……………………………………………………………83 5. Discussion………………………………………………………..98 6. Acknowledgements……………………………………………..101 VII. Chapter Three: The Pro-Inflammatory Role of Adiponectin at the Maternal-Fetal Interface………………………………………………...102 1. Abstract…………………………………………………………103 2. Introduction……………………………………………………..104 3. Materials and Methods………………………………………….107 4. Results…………………………………………………………..112 5. Discussion……………………………………………………....122 VIII. Chapter Four: Signaling Cascades Initiated by Adiponectin in Trophoblast Cells…………………………………………………….………………125 3 1. Abstract…………………………………………………………126 2. Introduction……………………………………………………..128 3. Materials and Methods………………………………………….131 4. Results…………………………………………………………..135 5. Discussion………………………………………………………150 IX. Chapter Five: Microarray Analysis of Adiponectin Treatment on Trophoblast Cells……………………………………………………….156 1. Abstract…………………………………………………………157 2. Introduction……………………………………………………..159 3. Materials and Methods………………………………………….161 4. Results…………………………………………………………..165 5. Discussion………………………………………………………186 6. Acknowledgements……………………………………………..190 X. Chapter Six: Concluding Remarks….…………………………………..191 XI. Chapter Seven: References……………………………………………..198 4 II. Acknowledgements 5 Firstly, I would like to thank my advisor, Dr. Michael Wolfe, for allowing me the independence and latitude to develop a project I feel so passionately about. His patience and willingness to allow me to search for answers has made me a better scientist. I would like to thank all of my committee members: Drs. Lane Christenson, Leslie Heckert, Peggy Petroff, and Mike Soares. Each one of you has supported me and not only helped mold me into a scientist throughout my degree training, but also helped to make my future career a success. You have all shown that you truly do have my best interests at heart, and for that I thank you. I would also like to thank the members of the Center for Reproductive Sciences. The collaborative spirit and willingness to share reagents is strong among the reproductive biologists on the third floor of the KLSIC building, and has made many aspects of my research a great deal easier. I would especially like to thank members of the Heckert and Petroff labs, not only for their generosity, but also for their friendship. You have made my time at KU exciting and enjoyable. Thanks to the department of Molecular and Integrative Physiology, and its chair, Dr. Paul Cheney, for being supportive of my research and my training throughout my degree. There have also been a number of people who, though separated by distance, have demonstrated an untiring belief in me and encouragement that has gotten me through many of the most difficult times during this degree. For that I thank you. I would also like to thank those friends in Kansas City who have lent balance to my life 6 and shared in my joys and frustrations. A special thanks to Beth Dille and Lacey Luense, who am I lucky to count as great friends. Beth, you have seen me through much in the past few years, and I will be forever grateful. You have shown me strength I didn’t know I had, and provided me with someone to lean on and laugh with. Although our time of living within walking distance of each other may be ending, our friendship is one that I know will continue through the years. Lacey, not only are you a great sports and music buddy, but you have also become a great friend. You have demonstrated a belief in me like no one else, and it has encouraged me when I have been most frustrated. Thanks for all the laughs, good food, beer, games, and shows we’ve shared, but most of all thanks for helping me be comfortable in my own skin. Finally, I would like to thank my family. You have never doubted my abilities, and your love and support is what has gotten me here. A very special thanks to my mother, who, whether entirely intended or not, has shaped me into the woman I am today. It is through you that I learned my sense of drive, ambition, and a belief that I can do anything. You remain one of the strongest people I know, and I have done much in my life to emulate the strength of character and determination that I see in you. 7 III. List of Tables and Figures 8 Tables Table VI-1. Primer Sequences for Quantitative RT-PCR………………………...79 Table VII-1. Primer Sequences for Quantitative RT-PCR……………………….108 Table VIII-1. Primer Sequences for Quantitative RT-PCR……………………….134 Table IX-1. Primer Sequences for Quantitative RT-PCR……………………….164 Table IX-2. Genes down-regulated following adiponectin treatment…………...166 Table IX-3. Genes up-regulated following adiponectin treatment………………181 Table IX-4. Adiponectin-regulated genes identified by microarray and validated by quantitative RT-PCR……………………………………………………………185 Figures Figure V-1. Schematic representation of the human placenta………………..…...21 Figure VI-1. Expression of adiponectin and its receptors in the placenta…………84 Figure VI-2. Adiponectin inhibits endocrine gene expression…………………….88 Figure VI-3. Adiponectin regulates gene expression of steroidogenic enzymes in an enzyme-specific manner…………………………………………………………90 Figure VI-4. Adiponectin decreases progesterone production by trophoblast cells in vitro………………………………………………………………………………92 Figure VI-5. Adiponectin does not inhibit differentiation in trophoblast cells in vitro………………………………………………………………………………96 9 Figure VII-1. Adiponectin up-regulates expression of the pro-inflammatory cytokines, IL-1β and IL-8 in term trophoblast cells……………………………114 Figure VII-2. Both CD24 and Siglec10 are expressed by human trophoblast cells at term…………………………………………………………………………….117 Figure VII-3. Adiponectin treatment increases Siglec10 expression in term trophoblast cells………………………………………………………………...120 Figure VIII-1. Adiponectin does not stimulate AMPK in human term trophoblast cells……………………………………………………………………………..137 Figure VIII-2. Adiponectin activates ERK1/2 and JNK signaling cascades in term trophoblast cells………………………………………………………………...140 Figure VIII-3. Adiponectin activates EGFR in syncytialized trophoblast cells……143 Figure VIII-4. Adiponectin signaling in trophoblast cells requires APPL1………..146 Figure VIII-5. Adiponectin-induced activation of EGFR is blocked by leptin production in trophoblast cells...…………………………………………….….149 Figure VIII-6. Model of adiponectin signaling in the syncytiotrophoblast at term...155 10 IV. Abstract 11 As the ultimate mediator of fetal growth and well-being, the placenta is inundated with a number of molecular cues from both the maternal and fetal systems. Successful integration of these signals is critical to the ongoing health of the pregnancy, and may have important implications for the future health of both mother and child. Maternal adipose tissue releases a variety of adipokines, whose primary aim is to regulate energy metabolism. Given this, and the importance of this process in the placenta, it follows that adipokines may have profound effects on the trophoblast cells of the placenta. However, the influence of most of these adipokines on the placenta remains poorly understood. We examined the role the adipokine, adiponectin, has on the human placenta using isolated trophoblasts collected from healthy term pregnancies. Treatment of these cells with adiponectin in culture inhibited the production of human chorionic gonadotropin (hCG), placental lactogen and progesterone, all hormones produced by the placenta that play a key role in the continued health and viability of the pregnancy. In addition, adiponectin induced a pro-inflammatory environment in trophoblast cells, with increases in production of both interleukin (IL)-1β and IL-8. We have gone on to show that adiponectin can mediate its actions