NATURAL KILLER CELLS, HYPOXIA, AND EPIGENETIC REGULATION OF HEMOCHORIAL PLACENTATION BY Damayanti Chakraborty Submitted to the graduate degree program in Pathology and Laboratory Medicine and the Graduate Faculty of the University of Kansas in partial fulfillment ofthe requirements for the degree of Doctor of Philosophy. ________________________________ Chair: Michael J. Soares, Ph.D. ________________________________ Jay Vivian, Ph.D. ________________________________ Patrick Fields, Ph.D. ________________________________ Soumen Paul, Ph.D. ________________________________ Michael Wolfe, Ph.D. ________________________________ Adam J. Krieg, Ph.D. Date Defended: 04/01/2013 The Dissertation Committee for Damayanti Chakraborty certifies that this is the approved version of the following dissertation: NATURAL KILLER CELLS, HYPOXIA, AND EPIGENETIC REGULATION OF HEMOCHORIAL PLACENTATION ________________________________ Chair: Michael J. Soares, Ph.D. Date approved: 04/01/2013 ii ABSTRACT During the establishment of pregnancy, uterine stromal cells differentiate into decidual cells and recruit natural killer (NK) cells. These NK cells are characterized by low cytotoxicity and distinct cytokine production. In rodent as well as in human pregnancy, the uterine NK cells peak in number around mid-gestation after which they decline. NK cells associate with uterine spiral arteries and are implicated in pregnancy associated vascular remodeling processes and potentially in modulating trophoblast invasion. Failure of trophoblast invasion and vascular remodeling has been shown to be associated with pathological conditions like preeclampsia syndrome, hypertension in mother and/or fetal growth restriction. We hypothesize that NK cells fundamentally contribute to the organization of the placentation site. In order to study the in vivo role of NK cells during pregnancy, gestation stage- specific NK cell depletion was performed in rats using anti asialo GM1 antibodies. Robust endovascular trophoblast invasion and vascular remodeling were observed in NK cell depleted animals. This depletion affected mesometrial vasculature, lowered relative oxygen concentrations at the placentation site and altered trophoblast lineage commitment at gestation d9.5. There was also a significant change in the organization of the chorioallantoic placenta on d13.5. Delivery of oxygen appears to be a key signal influencing both trophoblast cell differentiation and organization of the placentation site. Hypoxia promotes development of the invasive trophoblast lineage. We next evaluated the impact of hypoxia on the trophoblast stem (TS) cell transcriptome. DNA microarray analysis was performed on rat TS cells exposed to atmospheric and low oxygen (0.5%). Upregulation of genes characteristic of an invasive phenotype and a marked downregulation of stem state- associated genes were observed. Matrix metalloproteinase 12 (Mmp12) and lysine demethylase iii 3A (Kdm3a) were markedly upregulated in response to low oxygen, while E-cadherin (Cdh1) expression was dramatically decreased. These responses were dependent upon hypoxia inducible factor (HIF) signaling. Several of the HIF targets were identified to be Kdm3a targets. KDM3A acts on dimethyl and monomethyl histone H3K9 substrates, which prompted an examination of global histone H3K9 methylation status of trophoblast cells developing in vitro and in vivo. Hypoxia significantly impacted histone H3K9 methylation. Knockdown of KDM3A in TS cells inhibited hypoxia-induced Mmp12 gene expression and disrupted histone H3K9 methylation status at the Mmp12 locus. Ectopic expression of KDM3A stimulated MMP12 expression. KDM3A knockdown also significantly decreased hypoxia-activated TS cell invasion through Matrigel chambers. Finally, blastocysts transduced with Kdm3a shRNA expressing lentiviral particles showed reduced outgrowth when exposed to low oxygen tensions. In summary, NK cells impact oxygen delivery and uteroplacental adaptive responses, influencing critical lineage decisions affecting trophoblast differentiation and the ultimate structure and function of the chorioallantoic placenta. Hypoxia/HIF-directed epigenetic remodeling via KDM3A contributes to the control of TS cell adaptations during placentation modulating trophoblast lineage commitment and invasion. iv ACKNOWLEDGEMENTS I would like to thank my family for the unrestrained support, guidance, and encouragement that I have received from them over all these years. My father Mr. Janak Chakraborty has always instilled in me a love for science, and encouraged me to pursue my dreams of becoming a scientist wholeheartedly. My mother, Ms. Swati Chakraborty has been instrumental in imparting strong values, both moral and ethical, and an unflinching sense of commitment and integrity. I shall be forever grateful to the both of them. Special thanks to my brother Mr. Arnab Chakraborty (Tito), for helping me realize the spirit of sharing and teamwork, which was extremely valuable during my tenure as a graduate student in the laboratory. I would like to thank my best friend and collaborator, Dr. Aritra Bhattacherjee, for some very stimulating scientific discussions and brainstorming sessions, which contributed significantly towards designing experiments and interpreting experimental outcomes. I would like to thank my mentor Dr. Michael J. Soares, for supporting me, and nurturing and shaping my scientific endeavors. Without his support, this work would scarcely have been possible. His scientific spirit and enthusiasm has been quite infectious, and I hope to carry on my future research activities based on his foundation of strong scientific ethic and commitment. I would also like to thank Dr. Mohammad Rumi for his help and advice with Molecular Biology and his superior guidance in helping me delve into unchartered scientific territories fearlessly. My heartfelt thanks to Dr. Toshihiro Konno, for his advice on histological techniques, and on developing a camaraderie amongst fellow lab members. I would like to thank my committee members, for their continuous help and advice in guiding my research project. I would also like to thank Ms. Stacy McClure, Ms. Jackie Jorland and Ms. Lesley Shriver for their v support and administrative help. Special thanks to Ms. Jorland for helping me acquire the tools and reagents for my experiments, without which all of the research work would not have been possible. I would like to acknowledge all members of the Soares Laboratory (past and present), who helped me with their scientific and non-scientific advice, making me a better scientist in the process. I would like to thank Dr. Sumedha Gunewardena and Clark Bloomer in the Bioinformatics core (University of Kansas Medical Center) for helping me with generation and analysis of microarray data. I would like to thank my collaborators Dr. Kunio Shiota from University of Tokyo, Japan and Dr. Christopher Mack from University of North Carolina, Chapel Hill for kindly providing me with reagents that were valuable for my research. Last but not the least, I would like to acknowledge American Heart Association and Biomedical Research Training Grant Program (University of Kansas Medical Center) for providing me with fellowship which has partially supported my research activities. vi TABLE OF CONTENTS Abstract………………………………………………………………………………………….iii Table of Contents………………………………………………………………………………...v List of Figures and Tables……………………………………………………………………...xi Chapter 1: GENERAL INTRODUCTION………………………………………………….....1 Maternal Fetal interface…………………………………………………………………...1 The rat as a model for studying biology at the maternal-fetal interface…………………..2 i. Placentation in Rat………………………………………………………...3 ii. Blastocyst derived rat trophoblast stem (TS) cell………………………..5 NK cells and uterine vasculature……………………………………………………….....8 Uterine NK cells and invasive trophoblast cell interaction……………………………...12 Hypoxia and HIF signaling………………………………………………………………16 Hypoxia signaling and trophoblast cell development……………………………………17 Oxygen concentration and trophoblast cell invasion…………………………………….18 Hypoxia activation of epigenetic regulators……………………………………………..21 Epigenetics, Hypoxia and Cell invasion…………………………………………………22 Epigenetic regulation of trophoblast cell invasion……………………………………….23 Research Objectives…………………………………………………………………………….24 CHAPTER 2: NATURAL KILLER CELL REGULATION OF HEMOCHORIAL PLACENTATION……………………………………………………………………………...28 Abstract………………………………………………………………………………………….29 vii Introduction…………………………………………………………………………………….30 Materials and Methods…………………………………………………………………………33 Results…………………………………………………………………………………………...42 i. Depletion of uterine NK cells………………………………………………………………42 ii. NK cells, endovascular trophoblast invasion, and uterine spiral artery remodeling………43 iii. NK cells modulate uterine spiral arteries and oxygen delivery…………………………...61 iv. NK cells, hypoxia, and trophoblast lineage decisions within the placenta………………..69 v. Hypoxia signaling regulates trophoblast cell lineage decisions…………………………..77 Discussion…………………………………………………………………………………….....89 CHAPTER 3: EPIGENETIC INVOLVEMENT OF THE HISTONE H3K9 DEMETHYLASE KDM3A IN TROPHOBLAST STEM CELL ADAPTATIONS TO HYPOXIA ………………………………………………………………………………………93 Abstract………………………………………………………………………………………....94 Introduction…………………………………………………………………………………….95 Materials and Methods…………………………………………………………………………97 Results………………………………………………………………………………………….106 TS cell transcriptome responses to low oxygen…………………………………………..........106 HIF dependence of TS cell responses to low oxygen