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The effect and underlying mechanisms of bone morphogenetic protein 2 in regulating human trophoblast… Zhao, Hongjin 2019

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THE EFFECT AND UNDERLYING MECHANISMS OF BONE MORPHOGENETIC PROTEIN 2 IN REGULATING HUMAN TROPHOBLAST CELL INVASION  by  Hongjin Zhao Bachelor of Medicine, Shandong University, 2008 Master of Medicine, Shandong University, 2010  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Reproductive and Developmental Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2019  © Hongjin Zhao, 2019  ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: THE EFFECT AND UNDERLYING MECHANISMS OF BONE MORPHOGENETIC PROTEIN 2 IN REGULATING HUMAN TROPHOBLAST CELLS INVASION  submitted by Hongjin Zhao in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Reproductive and Developmental Sciences  Examining Committee: Dr. Peter C.K. Leung Supervisor  Dr. Xuesen Dong Supervisory Committee Member  Dr. Dan Rurak Supervisory Committee Member Dr. Alexander Guillermo Beristain University Examiner Dr. Ivan Robert Nabi University Examiner  Additional Supervisory Committee Members: Dr. Christian Klausen Supervisory Committee Member Dr. Evica Separovic Supervisory Committee Member  iii  Abstract  During placentation, extravillous cytotrophoblasts (EVTs) derived from villous cytotrophoblasts invade into the uterine wall for proper placentation and successful establishment of human pregnancy. Insufficient trophoblast invasion contributes to several pregnancy complications including preeclampsia, which is a leading cause of maternal mortality and affects 2-8% of pregnancies worldwide. As an important member of the transforming growth factor β (TGF-β) superfamily, bone morphogenetic protein 2 (BMP2) is abundantly produced at the maternal-fetal interface and its expression is spatiotemporally correlated with embryo placentation. BMP2 is crucial for endometrial decidualization in humans and normal fertility in mice. In addition, BMP2 exerts pro-invasive effects in a variety of cancer cells. However, whether BMP2 can promote trophoblast cell invasion during placentation remains unknown. BMPs increase mesenchymal adhesion molecule N-cadherin expression, activin A production, an inhibitor of DNA-binding protein 1 (ID1) expression, and WNT/β-catenin signaling in different cell types. All of the above mentioned molecules and signals have been shown to positively regulate human trophoblast or cancer cell invasion, thus we hypothesized that BMP2 could promote human trophoblast cell invasion by regulating the expression of N-cadherin, activin A and ID1 as well as the activation of canonical WNT/β-catenin signaling. Primary and immortalized (HTR8/SVneo) cultures of human EVT cells were used as study models. Activin receptor-like kinase 2/3 (ALK2/3) inhibitor DMH1 and ALK4/5/7 inhibitor SB431542 were used to block receptor-mediated signaling. Small interfering RNA (siRNA) was used to study the involvement of key signaling molecules. Cell invasiveness was examined using the Matrigel-coated transwell invasion assay. Overall, our results demonstrate that BMP2 promotes trophoblast cell invasion iv  via the following mechanisms: 1) Up-regulating N-cadherin via non-canonical ALK2/3/4-SMAD2/3-SMAD4 signaling; 2) Up-regulating inhibin βA and activin A production via ALK3-BMPR2/ACVR2A-SMAD1/5/8-SMAD4 signaling; 3) Inducing ID1-mediated up-regulation of insulin-like growth factor binding protein 3 (IGFBP3); and 4) Inducing WNT/β-catenin signaling activation mediated by bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI). These findings deepen our understanding of the roles of BMP2 in placentation and provide insights into the molecular mechanisms of human trophoblast invasion.  v  Lay summary  During pregnancy, cells of the placenta must invade into the uterine wall for proper placental formation. Insufficient invasion of these cells contributes to several pregnancy complications including preeclampsia, which is a leading cause of maternal mortality and affects 2-8% of pregnancies worldwide. Thus, a better understanding the regulation of placental cell invasion will benefit the development of diagnostic and therapeutic approaches for this and other adverse pregnancy outcomes. Bone morphogenetic protein 2 (BMP2) is an essential factor in maintaining normal fertility in mice and promotes invasive behaviour of several human cancers. Here we aimed to investigate whether BMP2 could promote placental cell invasion. By exposing these cells to BMP2 and subsequently analyzing cell behaviour, we demonstrated that BMP2 promotes human placental cell invasion through four underlying mechanisms. These findings broaden our knowledge on the roles of BMP2 in placenta formation and provide insights into the molecular mechanisms involved. vi  Preface   This study was approved by the University of British Columbia Research Ethics Board. Certificate Number: H07-01149.  A version of Chapter 3 has been published Hong-Jin Zhao, Christian Klausen, Yan Li, Hua Zhu, Yan-Ling Wang and Peter C.K. Leung. (2018) Bone morphogenetic protein 2 promotes human trophoblast cell invasion by upregulating N-cadherin via non-canonical SMAD2/3 signaling. Cell Death & Disease 9(2):174 I designed and carried out all the experiments as well as statistical analysis with the help and supervision of my supervisor Dr. Peter C.K. Leung, Dr. Christian Klausen and Dr. Yan-Ling Wang. I wrote the manuscript, which was further revised by Dr. Christian Klausen and Dr. Peter C.K. Leung. Dr. Yan Li and Dr. Hua Zhu helped me a lot in culturing primary human EVT cells.  A version of Chapter 4 has been published Hong-Jin Zhao, Hsun-Ming Chang, Hua Zhu, Christian Klausen, Yan Li and Peter C.K. Leung. (2018) Bone Morphogenetic Protein 2 Promotes Human Trophoblast Cell Invasion by Inducing Activin A Production. Endocrinology 159:2815-2825 I designed and performed all the experiments and data analysis with the help and supervision of my supervisor Dr. Peter C.K. Leung and Dr. Hua Zhu. I wrote the manuscript which was further refined by Hsun-Ming Chang, Dr. Christian Klausen and Dr. Peter C.K. Leung. Dr. Yan Li and Dr. Hua Zhu helped me in isolating and culturing primary human EVT cells.  vii  A version of Chapter 5 is in preparation for submission Hong-Jin Zhao, Christian Klausen, Yan Li, Hua Zhu, Peter C.K. Leung. Bone morphogenetic protein 2 promotes human trophoblast cell invasion through ID1-mediated up-regulation of IGF binding protein-3. I was responsible for study design, execution and statistical analysis with the help and supervision of my supervisor Dr. Peter C.K. Leung and Dr. Christian Klausen. I wrote the manuscript, which was revised by Dr. Christian Klausen and Dr. Peter C.K. Leung. Dr. Yan Li and Dr. Hua Zhu helped me in getting and culturing primary human EVT cells.  A version of Chapter 6 is in preparation for submission Hong-Jin Zhao, Christian Klausen, Hua Zhu, Yan Li, Peter C.K. Leung. Bone morphogenetic protein 2 promotes human trophoblast invasion through BAMBI-mediated activation of WNT/β-catenin signaling. I was responsible for study design, execution and analysis with the help and supervision of my supervisor Dr. Peter C.K. Leung and Dr. Christian Klausen. I wrote the manuscript, which was revised by Dr. Christian Klausen and Dr. Peter C.K. Leung. Dr. Yan Li and Dr. Hua Zhu helped me in getting and culturing primary human EVT cells.  viii  Table of contents  Abstract ......................................................................................................................................... iii  Lay summary ..................................................................................................................................v  Preface ........................................................................................................................................... vi  Table of contents ........................................................................................................................ viii  List of figures ............................................................................................................................... xii  List of abbreviations .................................................................................................................. xvi  Acknowledgements .................................................................................................................... xix  Chapter 1: Introduction ................................................................................................................1  1.1 Placenta ........................................................................................................................... 1 1.2 Embryo implantation and placentation ........................................................................... 2 1.2.1 Endometrial decidualization ....................................................................................... 2 1.2.2 Trophoblast differentiation ......................................................................................... 3  1.3 Study models for placentation ........................................................................................ 6 1.3.1.1 Immortalized trophoblastic cell lines .................................................................. 6 1.3.1.2 Primary human trophoblastic cells...................................................................... 7 1.3.1.3 3D culture models ............................................................................................... 7 1.3.1.4 Animal models .................................................................................................... 8  1.4 Major signaling pathways responsible for the regulation of EVT invasion ................... 9 1.4.1 TGF-β signaling ........................................................................................................ 10 1.4.1.1 TGF-β receptors and signal transduction pathway ........................................... 10 1.4.1.2 BMP2 ................................................................................................................ 11 ix  1.4.1.3 Activins ............................................................................................................. 13 1.4.2 IGF signaling ............................................................................................................ 13 1.4.2.1 IGFs in placenta ................................................................................................ 13 1.4.2.2 IGF binding proteins (IGFBPs) ........................................................................ 14 1.4.3 WNT/β-catenin signaling .......................................................................................... 15 1.5 Key molecules involved in EVT or cancer cell invasion .............................................. 17 1.5.1 Cadherins .................................................................................................................. 18 1.5.1.1 The classification of cadherins .......................................................................... 18 1.5.1.2 E-cadherin and N-cadherin in cancer and EVT invasion .................................. 18 1.5.2 The inhibitor of DNA-binding proteins (IDs) ........................................................... 19 1.5.3 Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) ..... 20 Chapter 2: Rationale, hypothesis and objectives ......................................................................22  2.1 Rationale ....................................................................................................................... 22  2.2 Overall hypothesis ........................................................................................................ 26 2.3 Study models ................................................................................................................. 26  2.4 The objectives of the study ........................................................................................... 27 Chapter 3: Bone morphogenetic protein 2 promotes human trophoblast cell invasion by up-regulating N-cadherin via non-canonical SMAD2/3 signaling...........................................29 3.1 Introduction ................................................................................................................... 29  3.2 Materials and Methods .................................................................................................. 31  3.3 Results ........................................................................................................................... 36 3.4 Discussion ..................................................................................................................... 39  x  Chapter 4: Bone morphogenetic protein 2 promotes human trophoblast cell invasion by inducing activin A production ....................................................................................................60  4.1 Introduction ................................................................................................................... 60  4.2 Materials and Methods .................................................................................................. 62  4.3 Results ........................................................................................................................... 65 4.4 Discussion ..................................................................................................................... 68  Chapter 5: Bone morphogenetic protein 2 promotes human trophoblast cell invasion through ID1-mediated up-regulation of IGF binding protein-3..............................................81 5.1 Introduction ................................................................................................................... 81  5.2 Materials and Methods .................................................................................................. 83  5.3 Results ........................................................................................................................... 86 5.4 Discussion ..................................................................................................................... 88  Chapter 6: Bone morphogenetic protein 2 promotes human trophoblast cell invasion through BAMBI-mediated activation of WNT/β-catenin signaling ......................................101 6.1 Introduction ................................................................................................................. 101  6.2 Materials and Methods ................................................................................................ 103  6.3 Results ......................................................................................................................... 105 6.4 Discussion ................................................................................................................... 107  Chapter 7: Conclusion ...............................................................................................................120  7.1 Conclusion .................................................................................................................. 120 7.2 General discussion of this study ................................................................................. 122 7.3 Limitations of this study ............................................................................................. 126 7.4 Significance and translational potential ...................................................................... 127  xi  7.5 Future directions ......................................................................................................... 128 Bibliography ...............................................................................................................................136  xii  List of figures  Figure 1.1 Schematic diagram showing the differentiation of cytotrophoblasts and localization of trophoblast subpopulations in chorionic villi .................................................................................. 5  Figure 3.1 BMP2 increases HTR8/SVneo cell invasion ............................................................... 44 Figure 3.2 BMP2 increases primary human EVT cell invasion ................................................... 45  Figure 3.3 BMP2 increases N-cadherin mRNA in HTR8/SVneo and primary human EVT cells 46 Figure 3.4 BMP2 increases N-cadherin protein levels in HTR8/SVneo and primary human EVT cells ............................................................................................................................................... 47  Figure 3.5 Knockdown of N-cadherin attenuates basal and BMP2-induced HTR8/SVneo cell invasion ......................................................................................................................................... 48  Figure 3.6 Knockdown of N-cadherin attenuates basal and BMP2-induced primary human EVT cell invasion .................................................................................................................................. 49  Figure 3.7 BMP2 activates canonical SMAD1/5/8 and noncanonical SMAD2/3 signaling ........ 50 Figure 3.8 Noncanonical SMAD2/3 signaling mediates the up-regulation of N-cadherin by BMP2 ............................................................................................................................................ 51  Figure 3.9 BMP and activin/TGF-β type I receptors contribute to BMP2-induced phosphorylation of SMAD2/3....................................................................................................... 52  Figure 3.10 BMP and activin/TGF-β type I receptors contribute to BMP2-induced up-regulation of N-cadherin mRNA levels ......................................................................................................... 53  Figure 3.11 BMP and activin/TGF-β type I receptors contribute to BMP2-induced up-regulation of N-cadherin protein levels.......................................................................................................... 54  xiii  Figure 3.12 BMP (ALK2/3) and activin (ALK4) type I receptors mediate the up-regulation of N-cadherin by BMP2 ........................................................................................................................ 55  Figure 3.13 BMP2 increases the expression of several EMT-associated genes in immortalized human trophoblast cells ................................................................................................................ 56  Figure 3.14 Treatment with BMP2 (< 2 hours) has no effects on AKT or ERK1/2 phosphorylation/activation ............................................................................................................ 57  Figure 3.15 Canonical SMAD1/5/8 signaling mediates the up-regulation of furin by BMP2 ..... 58 Figure 3.16 Proposed model of the signaling pathway mediating BMP2-induced N-cadherin up-regulation and increased human trophoblast cell invasion ........................................................... 59  Figure 4.1 BMP2 up-regulates inhibin βA mRNA levels and increases activin A accumulation in primary human EVT cells ............................................................................................................. 73  Figure 4.2 Knockdown of inhibin βA abolishes the BMP2-induced trophoblast cell invasion in primary human EVTs .................................................................................................................... 74  Figure 4.3 DMH1 abolishes the BMP2-induced up-regulation of inhibin βA in primary human EVTs ............................................................................................................................................. 75  Figure 4.4 ALK3 type I receptor mediates the BMP2-induced up-regulation of inhibin βA in primary human EVTs .................................................................................................................... 76  Figure 4.5 BMPR2 and ACVR2A but not ACVR2B type II receptors mediate the BMP2-induced up-regulation of inhibin βA in primary human EVTs .................................................................. 77  Figure 4.6 BMP2 activates canonical SMAD1/5/8 and uncanonical SMAD2/3 signaling in primary human EVTs .................................................................................................................... 78  Figure 4.7 Non-canonical SMAD2/3 signaling is not involved in the BMP2-induced up-regulation of inhibin βA in primary human EVTs ........................................................................ 79  xiv  Figure 4.8 Schematic diagram of the proposed molecular mechanisms for BMP2-induced increases in inhibin βA expression and activin A production in human trophoblast cells ........... 80 Figure 5.1 BMP2 increases ID1 mRNA levels in HTR8/SVneo and primary human EVT cells. 91 Figure 5.2 BMP2 increases ID1 protein levels in HTR8/SVneo and primary human EVT cells. 92 Figure 5.3 BMP2 increases IGFBP3 mRNA levels in HTR8/SVneo and primary human EVT cells ............................................................................................................................................... 93  Figure 5.4 BMP2 increases IGFBP3 protein levels in HTR8/SVneo and primary human EVT cells ............................................................................................................................................... 94  Figure 5.5 BMP2 increases IGFBP3 production in HTR8/SVneo and primary human EVT cells....................................................................................................................................................... 95  Figure 5.6 ID1 mediates BMP2-induced IGFBP3 up-regulation in HTR8/SVneo cells .............. 96 Figure 5.7 ID1 mediates BMP2-induced IGFBP3 up-regulation in human primary EVT cells ... 97 Figure 5.8 Knockdown of ID1 or IGFBP3 abolishes BMP2-induced trophoblast cell invasion in HTR8/SVneo cells ........................................................................................................................ 98  Figure 5.9 Knockdown of ID1 or IGFBP3 abolishes BMP2-induced cell invasion in primary human EVTs ................................................................................................................................. 99  Figure 5.10 ID1 and IGFBP3 are involved in BMP2-induced up-regulation of Slug in HTR8/SVneo cell line ................................................................................................................. 100  Figure 6.1 BMP2 increases BAMBI mRNA levels through ALK2 and/or ALK3 type I receptor in both HTR8/SVneo and primary human EVT cells ................................................................. 112 Figure 6.2 BMP2 increases active β-catenin protein levels in HTR8/SVneo and primary human EVT cells .................................................................................................................................... 113  xv  Figure 6.3 BMP2 increases cyclin D1 protein levels in HTR8/SVneo and primary human EVT cells ............................................................................................................................................. 114  Figure 6.4 BAMBI mediates the up-regulation of active β-catenin and cyclin D1 by BMP2 in HTR8/SVneo cells ...................................................................................................................... 115  Figure 6.5 BAMBI mediates the up-regulation of active β-catenin and cyclin D1 by BMP2 in primary human EVT cells ........................................................................................................... 116  Figure 6.6 BAMBI knockdown attenuates BMP2-induced human trophoblast cell invasion .... 117 Figure 6.7 TGF-β1 reduces BAMBI mRNA levels in primary human EVT cells ..................... 118 Figure 6.8 Proposed model of the signaling pathway mediating BMP2-induced BAMBI up-regulation, canonical WNT/β-catenin signaling activation and increased human trophoblast cell invasion ....................................................................................................................................... 119  Figure 7.1 Schematic presentation of the proposed mechanisms underlying BMP2-promoted human trophoblast invasion ........................................................................................................ 130  Figure 7.2 Activin A contributes to BMP2-induced up-regulation of N-cadherin and IGFBP3 in primary human EVT cells ........................................................................................................... 132  Figure 7.3 Non-canonical SMAD2/3 accounts for N-cadherin up-regulation while canonical SMAD1/5/8 signaling mediates the up-regulation of furin and ID1 induced by BMP2 ............ 134 Figure 7.4 ID1 mediates BMP2-induced cyclin D1 up-regulation in HTR8/SVneo and primary human EVT cells......................................................................................................................... 135   xvi  List of abbreviations  ALK                                       Activin receptor-like kinase ANOVA                                 Analysis of variance BAMBI                                  Bone morphogenetic protein and activin membrane-bound inhibitor BMP                                       Bone morphogenetic protein Bmpr2                                     BMP type II receptor BSA                                        Bovine serum albumin °C                                            Degrees Celsius cDNA                                      Complementary DNA DMEM                                    Dulbeco’s modified eagle’s medium DMSO                                     Dimethyl sulfoxide DNA                                        Deoxyribonucleic acid dNTP                                       Deoxynucleoside triphosphate ECL                                         Enhanced chemiluminescence ECM                                        Extracellular matrix ELISA                                     Enzyme-linked immunosorbent assay EMT                                        Epithelial mesenchymal transition ERK                                         Extracellular signal-regulated kinase EVT                                         Extravillous cytotrophoblast g                                               Gram GAPDH                                   Glyceraldehyde-3-phosphate dehydrogenase GSK-3β                                    Glycogen synthase kinase 3β  xvii  H                                                 Hour hCG                                            Human chorionic gonadotropin  HIF1                                           Hypoxia-inducible factor 1 HLA-G                                       Human leukocyte antigen G   ID                                               The inhibitor of DNA-binding protein IGF                                             Insulin-like growth factor                                          IUGR                                          Intrauterine growth restriction kDa                                             Kilodalton min                                              Minutes ml                                                Milliliter mRNA                                         Messenger RNA MTT                                            3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide L                                               Microliter nM                                               Nanomolar PAGE                                          Polyacrylamide gel electrophoresis PBS                                             Phosphate buffered saline RNA                                            Ribonucleic acid R-SMAD                                     Receptor-regulated SMAD RT-qPCR                                    Reverse transcription quantitative polymerase chain reaction SA                                               Spiral artery SD                                               Standard deviation SDS                                             Sodium dodecyl sulfate SEM                                            Standard error of the mean xviii  siRNA                                         Small interfering RNA SMAD                                         Sma- and Mad-related protein SV40                                            Simian vacuolating virus-40 TCF/LEF                                     T cell factor/ lymphoid enhancer factor TGF-                                          Transforming growth factor-beta TBS                                             Tris-buffered saline Tris-HCl                                      Tris (hydroxylmethyl)-aminomethane-hydrochloric acid  xix  Acknowledgements  Firstly, I would like to express my deepest gratitude to my supervisor, Dr. Peter C.K. Leung for his valuable guidance and continuous support during my PhD study. His optimism and enthusiasm encouraged me to conduct this research and will benefit me a lot in my future research career.   I would like to sincerely thank my supervisory committee members, Dr. Xuesen Dong, Dr. Dan Rurak, Dr. Christian Klausen, and Dr. Evica Separovic, for their scientific guidance and suggestions on my research. Their precious comments enlarged my scientific vision and enhanced my knowledge in research. Particular appreciation to Dr. Catherine Pallen, who mentored and inspired me at the beginning of my Ph. D study. I am deeply grateful for all their help and support.  I offer my enduring gratitude to Dr. Christian Klausen for his scientific training of me and detailed revision of my manuscripts and thesis. I am really grateful to Dr. Hua Zhu and Dr. Hsun-Ming Chang for their great help, technic advice, and assistance with manuscript preparation. I would like to thank all my lab mates for our efficient collaboration in Dr. Leung’s lab.  Special thanks are owed to my parents and my parents in-law for their consistent support and understanding during my education. I would also like to thank my wife Yan for her great xx  contribution to my family and being supportive beside me all the time. My children Emily and Leo, I love you both very much and we will always be living together in the future. 1  Chapter 1: Introduction  1.1 Placenta     The placenta is a temporary organ connecting the growing fetus through the umbilical cord to maternal uterus wall during pregnancy.  It is crucial for successful pregnancy establishment and maintenance as well as the health of both the fetus and mother (1). In humans, the placenta appearance is discoid due to the arrangement of chorionic villi in a circular plate. During placental development, human trophoblast cells from anchoring villi invade into the endometrium and inner one third of the myometrium, and remodel maternal uterine tissues and spiral arteries (2) (Figure 1.1). Because of the direct contact of fetal trophoblast cells with maternal blood, the highly invasive placenta in human is classified as haemochorial (3). Functionally, the human placenta physically links the mother and fetus, providing the growing fetus with sufficient oxygen, nutrition while eliminating fetal wastes across the maternal-fetal interface (4). In addition, as an endocrine organ, the human placenta supplies hormones, cytokines and growth factors essential for fetal growth and pregnancy maintenance (5). Moreover, the placenta protects the allogeneic fetus from attack by the maternal immune system (6). Besides its essential function for fetal growth, the placental size at birth is associated with the incidence of specific diseases in adults (7).     Abnormal placentation accounts for several pregnancy disorders, including recurrent pregnancy loss, preeclampsia, intrauterine growth restriction (IUGR), preterm birth and stillbirth (8-11). For instance, abnormal implantation and poor placental development are responsible for miscarriages early in gestation (12). Insufficient placental development characterized by shallow trophoblast invasion and inadequate spiral artery remodeling is associated with preeclampsia, 2  characterized by hypertension, proteinuria and systemic chronic inflammatory disorders after 20 weeks gestation (13, 14). Therefore, further studies on placentation regulation are needed to improve the clinical therapy for those pregnancy complications.  1.2 Embryo implantation and placentation     Embryonic development starts from an ovum fertilized by a sperm, usually in the ampullary region of the Fallopian tube. At about day 5 after fertilization, the zygote becomes a blastocyst which is characterized by a large inner cavity. The outer layer of the blastocyst is comprised of trophectodermal cells which will differentiate into trophoblast cells, whereas the inner cell mass will develop into the embryo (15). Embryo implantation begins when the trophectodermal cells of the blastocyst attach to the luminal epithelium of the uterus at day 6-7 after fertilization (16). Both implantation and subsequent placentation are highly coordinated processes requiring the interaction and cooperation of endometrium and trophoblast cells.  1.2.1 Endometrial decidualization      Endometrial decidualization is of great importance for blastocyst implantation and placentation. Impaired implantation due to insufficient endometrial decidualization causes sterility in mice (17, 18). Endometrial decidualization is a hormone-dependent process starting from the late secretory phase of the menstrual cycle, and is often referred to as the "uterine receptivity window" since it is a period conducive to embryo implantation and trophoblast invasion (19, 20). Typically, endometrial decidualization comprises several changes, including morphological changes of stromal cells into polyhedral or circular shape, increased glandular structures and vascularization, infiltration of uterine natural killer cells marked with CD56, 3  abundant production of prolactin, insulin-like growth factor binding protein-1 (IGFBP1) and enhanced accumulation of glycogen and lipid (21-23). In this way, the human uterus adapts to be permissive enough for pregnancy establishment.  1.2.2 Trophoblast differentiation      During implantation, trophectodermal cells and their derived syncytiotrophoblasts located in the outermost layer of the pre-implantation blastocyst attach and finally penetrate through the uterine epithelium and basement membrane to implant in the endometrium (24). In addition, trophectodermal cells differentiate into cytotrophoblast cells which further proliferate, differentiate and remodel the uterine environment to form chorionic villi including floating villi and anchoring villi (25, 26).      Mitotically active cytotrophoblasts are progenitor cells of other trophoblast lineages. Through asymmetric cell division, one villous cytotrophoblast cell gives rise to two daughter cells, one of which maintains this proliferative progenitor pool and the other differentiates into more specialized trophoblast subpopulations, including syncytiotrophoblasts and extravillous cytotrophoblasts (EVTs) (27). In humans, the differentiation route taken by cytotrophoblast depends on its location in the placenta (28). In floating villi, mononucleated cytotrophoblasts fuse to become multinucleated syncytiotrophoblasts, as demonstrated by observation of remnants of cytotrophoblastic junctional complexes within syncytiotrophoblasts (29, 30). Syncytiotrophoblast is terminally differentiated and maintained by the fusion of underlying cytotrophoblasts (31, 32). Compared with cytotrophoblasts, the syncytiotrophoblast has more developed organelles, such as rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi apparatus and secretary vesicles. The syncytiotrophoblast is characterized by its secretion 4  of hormones essential for pregnancy maintenance, including human chorionic gonadotropin (hCG), human placental lactogen and progesterone (33-35). Structurally, mature floating villi are composed of a mesenchymal core containing the fetal vasculature, a layer of cytotrophoblast cells sitting on basement membrane, and the outer syncytiotrophoblast bathed in maternal blood. Via this haemochorial floating villi, fetal circulation performs gas and nutrient exchange with maternal blood but is anatomically insulated from the maternal circulation throughout pregnancy (28).     Anchoring villi are in direct contact with the uterine wall and anchor the embryo to the uterus. Here, cytotrophoblasts differentiate into EVTs which can be further sub-classified into column EVTs, interstitial EVTs and endovascular EVTs according to their proliferative ability, molecular markers and location (36). Once detached from the basement membrane, polarized cytotrophoblasts differentiate gradually into column EVTs which are still mitotically active and proliferate extensively to form cell columns (36, 37). After leaving the tips of the cell columns, column EVTs further differentiate into interstitial EVTs that invade into the endometrium and inner one third of the myometrium to remodel decidual tissues and regulate maternal immune function (38). Endovascular EVTs move into maternal spiral arteries and cooperate with the uterine natural killer cells to remodel these vessels to have lower resistance in order to ensure sufficient supply of maternal blood at a relatively low pressure to the placenta and growing fetus (36, 39, 40) (Figure 1.1). Like cytotrophoblasts, EVTs express cytokeratin, indicating their epithelial nature. In addition, EVTs uniquely express human leukocyte antigen G (HLA-G) as a cell marker to differentiate them from villous cytotrophoblasts (41-43).  5   (Adapted from Zhou et al., 1997. J Clin Invest. 99(9):2139-51)    Figure 1.1 Schematic diagram showing the differentiation of cytotrophoblasts and localization of trophoblast subpopulations in chorionic villi In floating villi, villous cytotrophoblasts fuse to form syncytiotrophoblasts. In anchoring villi, once detached from basement membrane, polarized cytotrophoblasts differentiate into column EVTs, which further give rise to interstitial EVTs invading the uterine wall, and endovascular EVTs for spiral artery remodeling. BM, basement membrane; CT, cytotrophoblast; EVT, extravillous cytotrophoblast; PV, placental vessel; SA, spiral artery; SCT, syncytiotrophoblast.  6  1.3 Study models for placentation 1.3.1.1 Immortalized trophoblastic cell lines     Early knowledge on placentation was mostly from histological analysis of pre-mature abortion or term placenta samples because of the ethical restrictions on in vivo placenta research in the human (44, 45). To explore the mechanisms of human trophoblast differentiation and invasion, several immortalized trophoblastic cell lines have been developed.     The HTR8/SVneo cell line was derived from first trimester human EVTs transfected with simian virus 40 large T antigen (46). Compared with primary EVTs, the HTR8/SVneo cell line has a much longer lifespan, high invasive capacity, and similar gene expression, all of which make it an excellent tool used in human trophoblast biological research (46, 47). In particular, HTR8/SVneo cells have been reported to form endothelial-like tubes when cultured on certain concentrations of Matrigel, displaying the phenotype of endovascular EVTs (48, 49). To date, the HTR8/SVneo cell line has been used extensively to investigate human trophoblast features, including differentiation, migration and invasion (50-53). However, cytokeratin 7 down-regulation and vimentin up-regulation in HTR8/SVneo cells suggests these cells have more mesenchymal features than primary EVTs (54).     SGHPL-5 cells are another immortalized trophoblastic cell line established by transfection of human EVTs with simian virus 40 large T antigen (55). In addition to retaining invasive capacity, SGHPL-5 cells express human chorionic gonadotrophin (hCG) and cytokeratin 7, indicative of its epithelial origin (55, 56). Though not as popular as the HTR8/SVneo cell line, SGHPL-5 cells have also been widely utilized for human trophoblast research. For example, the positive role that canonical WNT signaling plays in human EVT invasion was initially reported using SGHPL-5 cells (56). 7     1.3.1.2 Primary human trophoblastic cells     Primary human trophoblast cells are ideal for in vitro studies of trophoblast biology because they mostly maintain the phenotype and gene expression profile of trophoblast cells in situ and lack artificial modification compared with immortalized cell lines. The enzyme digestion and gradient centrifugation method is broadly used to isolate human trophoblastic cells from first or third trimester placental villi, which are further purified through immunoselection including flow cytometry or immunomagnetic microspheres (31, 57-59).     An alternative method for isolation of human first trimester EVTs has also been used to study the biological properties of EVTs. In this method, villi tips from first trimester placenta are finely minced and cultured for 3-4 days in flasks. Non-attached pieces are removed and attached villous tissue fragments are further cultured to allow for outgrowth of EVTs, which are then  separated from the fragments by trypsinization (60). Primary cells obtained with this method stain positive for cytokeratin-7 (epithelial marker) and HLA-G (EVT specific marker), both of which are features of human EVTs in situ (60, 61). In addition, these EVTs express matrix metalloproteinase (MMP) 2 and 9, which are crucial for extracellular matrix (ECM) degradation during EVT invasion (62, 63).  1.3.1.3 3D culture models     To examine interactions between trophoblast cells and other cell types, some 3D co-cultures of trophoblasts have been developed with the help of the ECM-like compound Matrigel. For example, 3D co-cultures with immune cells such as macrophages and natural killer cells were used to determine the interactions between trophoblast cells and immune cells (64-66). Along 8  with the establishment of the culture conditions for human trophoblast stem cells (67), trophoblast organoids have been developed (68, 69), which will novel methods of studying  human trophoblast differentiation.  1.3.1.4 Animal models     Besides in vitro research, in vivo studies are also of great importance to correlate trophoblast differentiation and spiral artery remodeling with placental function and fetal growth in a physiological context. Due to ethical restrictions in human pregnancy, animals with haemochorial placentas have been used as in vivo models in placental research (70).     Non-human primates are evolutionarily close to humans. With similarities to humans in spiral artery remodeling and the pattern of circulation in the intervillous space, monkeys are deemed as good models for research on trophoblast modulation of the maternal vasculature (71, 72). In particular, patas monkeys display preeclampsia-like symptoms and could be of value in studies of this condition (73). However in contrast to humans, endovascular trophoblast invasion in these monkeys is largely limited to the decidua and thus does not fully recapitulate trophoblast invasion in human placentation. Chimpanzees and gorillas display deep trophoblast invasion comparable to that of humans, but their endangered status precludes their use in laboratory research (70).     The placentas of most rodent animals are classified as haemochorial as well, suggesting a more affordable and appropriate candidate for human placenta research (74). From an evolutionary perspective, mice are fairly closely related to primates and this, combined with their relative ease of use, has supported a considerable amount of mouse-based research on human physiology and disease. In placental research, mice are a good model due to their short 9  pregnancy and small placenta which can be easily manipulated in experiments. In addition, advances in transgenic mouse models has improved our understanding of the functions of many genes in mouse placentation, which provides clues to their possible roles in human placental development (75). However, one disadvantage is that trophoblast invasion in mice is restricted to the decidual basalis of the uterus (76). Alternatively, placentas from rat and guinea pig have deeper trophoblast invasion comparable to humans, though rodent labyrinthine placentas are different from villous hemochorial placentas in primates (77-80). In particular, preeclampsia-associated toxemia was discovered in natural pregnancy in guinea pigs (81). Subsequently, a guinea pig model with preeclampsia-like manifestations was created by inducing uteroplacental ischemia by constricting the uterine and ovarian arteries (82). Together, these studies suggest that guinea pigs serve as an adequate model for the study of mechanisms underlying trophoblast invasion and vasculature remodeling in humans (78).  1.4   Major signaling pathways responsible for the regulation of EVT invasion     Trophoblast differentiation and EVT invasion are tightly controlled by diverse hormones, cytokines and growth factors (54). Dysregulation of trophoblast invasion leads to pregnancy-associated complications. For example, superficial trophoblast invasion and inadequate vascular remodeling are associated with pregnancy complications including preeclampsia and IUGR (83-85). In contrast, uncontrolled trophoblast invasion leads to hydatidiform moles or even choriocarcinoma (86). Such pregnancy disorders are harmful to the health of the growing fetus as well as the mother. Therefore, it is of great importance to broaden our knowledge on the regulation of EVT invasion and underlying signaling pathways in order to improve the diagnosis and therapeutic management of these pregnancy disorders. To date, a number of signaling 10  pathways are known to be involved in human EVT invasion, including transforming growth factor-beta (TGF-β) signaling, insulin-like growth factor (IGF) signaling and WNT/β-catenin signaling.   1.4.1 TGF-β signaling     The TGF-β superfamily comprises over 40 members which are further classified into different subfamilies including TGF-1-3, activins and inhibins, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), anti-Müllerian hormone (AMH), nodal and others (87). Many of them are abundantly expressed at the maternal-fetal interface and are closely associated with uterine decidualization as well as human trophoblastic differentiation and invasion (88). Different TGF-β members have synergistic or antagonistic functions on certain activities during pregnancy. For example, both activin A and BMP2 play positive roles in uterine decidualization (89) whereas activin A and TGF-β exert totally opposite effects on human trophoblast invasion; activin A promotes whereas TGF-β inhibits EVT invasion (51, 90). It is recognized that endometrial decidualization serves to create a local microenvironment beneficial for human trophoblast invasion. Therefore, the precise spatiotemporal regulation of TGF-β superfamily members with similar or differing functional effects is essential for pregnancy establishment and maintenance.  1.4.1.1 TGF-β receptors and signal transduction pathway     Bioactive TGF-β superfamily members are usually homodimers or heterodimers linked covalently via a disulfide bond; notable exceptions include BMP3, GDF9 and BMP15 (91, 92). They function through binding and activating heterotetrameric complexes of TGF-β type I and 11  type II serine/threonine kinase receptors. These receptors consist of an N-terminal extracellular domain, a transmembrane domain, and a C-terminal serine/threonine kinase domain. Mammals, have five type II receptors (TGFBR2, ACTR2A, ACTR2B, BMPR2 and AMHR2) and seven type I receptors (ACTRL1, ACTR1, BMPR1A, ACTR1B, TGFβRI, BMPR1B and ACTR1C); the latter are also called activin receptor-like kinases (ALKs) 1-7 (93). The activation of receptors induced by TGF-β ligands leads to phosphorylation and activation of downstream molecules referred to as receptor-regulated SMADs (R-SMADs), including SMAD1, SMAD2, SMAD3, SMAD5 and SMAD8. Different TGF-β ligands activate different R-SMADs; TGF-βs, activins and GDFs activate SMAD2/3 while BMPs and AMH activate SMAD1/5/8. Activated R-SMADs then complex with common SMAD4 and translocate into the nucleus for canonical TGF-β signaling-associated gene transcription (94). In addition, there are also inhibitory SMADs including SMAD6 and SMAD7, whose functions are to antagonize R-SMAD-dependent TGF-β signaling (95). Besides SMAD-mediated canonical signaling, TGF-β superfamily members are also reported to activate non-canonical signaling such as phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) signaling (96).  1.4.1.2 BMP2     Since BMPs were first shown to induce ectopic bone formation in the 1960’s, over 20 BMP members have been identified in humans (97). As the biggest subfamily of the TGF-β superfamily, BMPs exert diverse effects in developmental processes and tissue homeostasis beyond those initially identified in bone, leading some scholars to suggest their name be changed to Body Morphogenetic Proteins (98). According to similarities in sequence and functions, BMPs are further sub-grouped into BMP2/4, BMP5/6/7/8a/8b, BMP9/10 and BMP12/13/14 (92). 12  Classically, BMPs bind to TGF-β type I receptors ALK2, ALK3 and/or ALK6 in combination with type II receptors BMPR2, ACTR2A and/or ACTR2B, and activate SMAD1/5/8-mediated canonical signaling (92). In addition to canonical SMAD1/5/8 signaling, BMP2 has also been shown to activate SMAD2/3 signaling, especially in embryonic and transformed cells (99). Moreover, BMP2 exerts varying effects on cancer progression by regulating PI3K/AKT or MAPK signaling (100, 101). Functionally, there is considerable evidence that BMP2 is involved in cell motility and EMT-like processes in both physiological and pathological conditions. For instance, BMP2 is essential for cardiac cushion formation due to its ability to induce endocardial EMT (102). As well, BMP2 promotes endothelial cell migration during angiogenesis (103, 104). In cancers, BMP2 also induces EMT processes for cancer cell invasion (105, 106). BMP2 is expressed at the maternal-fetal interface and EMT is involved in trophoblast invasive differentiation; however, there are no reports of the effects of BMP2 on human trophoblast invasion. Interestingly, the expression of Bmp2 is spatiotemporally correlated with embryo implantation, suggesting potential roles for BMP2 in implantation and early placentation (107). In addition, conditional knockout and in vitro studies have revealed that Bmp2 is crucial for endometrial decidualization and fertility in mice and humans (17, 18). Furthermore, treatment of bovine trophoblast cells with BMP2 alleviates the expression of interferon-tau, which is rapidly down-regulated when natural implantation starts in vivo (108), suggesting BMP2 contributes to bovine trophoblast differentiation along the invasive pathway.   13  1.4.1.3 Activins      Activins are disulfide-linked homodimers or heterodimers of inhibin β subunits including inhibin A and B. Different combinations of inhibin β subunits gives rise to three main isoforms of activin; activin A (A-A), activin AB (A-B) and activin B (B-B) (109, 110). Inhibin A and B subunits are expressed abundantly at the human maternal-fetal interface, suggesting their potential functions during pregnancy (111, 112). Indeed, activin A has been shown to positively regulate endometrial decidualization (111), trophoblast hormone production (e.g. hCG, progesterone and estradiol) (113, 114), and trophoblast invasion (51, 52). Moreover, activin A facilitates endothelial-like tube formation in HTR8/SVneo cells, suggesting it may contribute to trophoblast endovascular differentiation (49). These results suggest that activin A may regulate important aspects of human placentation and maternal vasculature remodeling. Moreover, elevated levels of activin A in placentas from women with severe preeclampsia (compared with normal pregnancies) suggests that it might contribute to this pregnancy complication (115), A possible explanation for this apparent discrepancy is that increased production of activin A is a compensatory response to severe preeclampsia.  1.4.2 IGF signaling 1.4.2.1 IGFs in placenta     IGFs including IGF1 and IGF2 are polypeptides with homologous structure to proinsulin (116). Both of them are expressed at the maternal-fetal interface in humans, however the production of IGF2 is higher than that of IGF1 throughout gestation (117). IGFs play essential roles in placental development by stimulating cytotrophoblast proliferation, syncytiotrophoblast formation and EVT migration (118-121). The effects of IGFs in placenta are mainly mediated by 14  IGF type I receptor though sometimes IGF type 2 receptor is also involved (119-122). After binding to their receptors, IGFs induce the phosphorylation of insulin receptor substrate-1, which then activates PI3K/AKT signaling for cell migration and RAS/MAPK signaling for cell proliferation and differentiation (123-127).  1.4.2.2 IGF binding proteins (IGFBPs)     The bioavailability of IGFs is modulated by IGFBPs. IGFBPs regulate the functions of IGFs in two opposite ways; first, IGFBPs sequester IGFs from binding to their receptors to block their bioactivity, second, IGFBPs increase the half-life and localized levels of IGFs to potentiate the functions of IGFs (128-131). In addition to the above IGF-dependent actions, IGFBPs have IGF-independent effects via direct association with cell surface proteins (128, 130, 132). To date, six IGFBPs (IGFBP1-6) have been described in human placenta, of which IGFBP1 and IGFBP3 are expressed most abundantly at the fetal-maternal interface (117). IGFBP1 has been shown to stimulate human EVT invasion in an IGF-independent manner (133), whereas IGFBP5 has been shown to counteract IGF2-induced HTR8/SVneo cell migration (134, 135). These examples highlight the complex effects of IGFBPs on human placentation.  In addition to modulating the interaction between IGFs and IGF receptors, IGFBP3 has been shown to up-regulate IGF1 and directly activate IGF type I receptor in human umbilical vein endothelial cells (136). IGF1 exerts positive effects on trophoblast invasion and its expression is reduced in placentas from women with preeclampsia compared to normal controls (69, 137). However, whether or not IGFBP3 regulates human EVT invasion is unknown. In guinea pigs, maternal circulating IGFBP3 is positively correlated with placental growth late in gestation (138). In human placenta, in situ hybridization was used to show that IGFBP3 is primarily 15  expressed in first trimester column EVTs (117), implying that it may be up-regulated during trophoblast invasive differentiation. Moreover, IGFBP3 is strongly down-regulated in preeclamptic placentas whereas microRNA-210, which targets IGFBP3, is elevated (139-141), suggesting IGFBP3 may play a role in this disease. Pro-invasive effects of IGFBP3 have been reported in melanoma (142), esophageal cancer (143), nasopharyngeal carcinoma (144) and so on, though IGFBP3 is generally considered to be a tumor suppressor in human cancers. In angiogenesis, IGFBP3 promotes the differentiation of endothelial precursor cells to mature endothelial cells (145) and up-regulates MMP2 and MMP9 for ECM remodeling (146). Furthermore, IGFBP3 enhances endothelial cell migration and capillary-like network formation to facilitate angiogenesis (136, 146).  1.4.3 WNT/β-catenin signaling     WNT signaling is conserved from Drosophila and plays essential roles in embryogenesis, organogenesis and homeostasis. Aberrant WNT signaling has been described in pregnancy complications, various cancers, diabetes, and neurodegenerative diseases (147-149). WNT ligands are cysteine-rich glycoproteins with low solubility, thus they are transported in lipoprotein-complexes in the circulation (148). Since the first discovered WNT ligand WNT1, the human homologue of Drosophila Wingless, there have been a total of 19 WNT ligands described in humans (150, 151). They can activate canonical WNT/β-catenin signaling, non-canonical WNT/Ca2+ signaling and WNT/planar cell polarity signaling, of which canonical WNT/β-catenin signaling is reported to be associated with cell motility (56, 152, 153). In static cells, β-catenin mainly binds to cadherin for normal adhesion junction formation. Cytosolic levels of β-catenin are very low because almost all of it is phosphorylated by glycogen synthase 16  kinase 3β (GSK-3β) which leads to ubiquitination and proteasomal degradation (154). Phosphorylation of β-catenin by GSK-3β is blocked when canonical WNT ligands bind to the seven-transmembrane frizzled receptor which then interacts and heterodimerizes with the single-transmembrane co-receptor low density lipoprotein receptor-related protein 5/6 (LRP5/6). The increase in cytosolic β-catenin results in its translocation into the nucleus to cooperate with T cell factor/ lymphoid enhancer factor (TCF/LEF) for target gene expression, including cyclin D1, c-myc and MMPs (149, 155, 156).      In the human placenta, 14 WNT ligands are detectable, of which WNT1, WNT2b and WNT11 are most abundantly expressed in human EVTs (150). Canonical WNT/β-catenin signaling has been widely displayed to function in the regulation of human trophoblast invasion (56, 157, 158). As human cytotrophoblasts differentiate along the invasive pathway, the nuclear accumulation of β-catenin and the up-regulation of TCF3/4 strongly suggest that canonical WNT signaling is activated (56). In vitro studies with human trophoblast cell lines and/or primary EVT cells have shown that trophoblast cell migration is increased by WNT3A-induced  canonical WNT/β-catenin signaling, but is decreased by reduced cyclin D1 expression (56, 159, 160). In severe preeclamptic placentas, the levels of WNT1, β-catenin and cyclin D1 are decreased whereas those of Dickkopf-related protein 1 (DKK1), an antagonist of canonical WNT/β-catenin signaling, are increased compared to those in normal placentas (161). These findings suggest an association between deficiencies in WNT/β-catenin signaling and preeclampsia. In contrast, abundant nuclear accumulation of β-catenin, which is a sign of hyper-activated WNT signaling, has been described in invasive EVTs from complete hydatidiform moles (56, 162). In addition to its effects on EVT invasion, WNT/β-catenin signaling plays critical roles in progesterone or BMP2-mediated endometrial decidualization in mice or human endometrial stromal cells, 17  respectively (163, 164). Moreover, WNT/β-catenin signaling has also been linked to glial cells missing 1 (GCM1)-dependent cytotrophoblast fusion (165), demonstrating the broad effects of canonical WNT signaling during placental development.  1.5 Key molecules involved in EVT or cancer cell invasion     During placentation, invasion of EVTs into the uterine wall is spatiotemporally controlled which is in contrast to the dysregulated invasion typical of cancer cells. For example, human trophoblasts exhibit no signs of tumor formation in immunodeficient mice (46), which is totally different from malignant cancer cells. However, the differentiation of villous cytotrophoblasts to EVTs involves EMT, which is often associated with malignancy (166, 167). Epithelial cells are normally held tightly together by cell adhesions and junctions to form a polarized epithelial sheet. In this way, individual epithelial cells are prevented from moving away from this epithelial monolayer. On the other hand, adhesions among mesenchymal cells are much weaker compared with epithelial cells, conferring them with elevated motility. In addition to these cadherin-dependent changes in adhesion, EMT is also associated with changes in integrin expression, increases in MMP secretion, and enhanced WNT/β-catenin signaling, all of which cooperate to convert immotile polarized epithelial cells to invasive mesenchymal cells (168). In addition to EMT, trophoblast invasive differentiation and cancer cell invasion share other molecular mechanisms (169). Therefore, human EVTs are often considered as pseudo-malignant cells and key molecules involved in cancer cell invasion may also regulate human trophoblast differentiation and invasion.   18  1.5.1 Cadherins 1.5.1.1 The classification of cadherins     Cadherins are transmembrane proteins mediating cell-cell adhesion in specialized cell junctions. Based on structural similarity, vertebrate cadherins can be divided into 5 groups: classical cadherins (type I and type II), desmosome cadherins, protocadherins, Flamingo cadherins and FAT-like cadherins, of which classical cadherins are the most studied (170, 171). Classical cadherins participate in the assembly of adherens junctions which mediate cell adhesion, cell polarity, cell sorting, and cell migration/invasion (172). Structurally, classical cadherins contain 5 N-terminal extracellular cadherin repeats (or EC domains) responsible for calcium binding, and a C-terminal cytoplasmic domain which interacts with catenins and elements of the actin cytoskeleton (170, 173). Type I classical cadherins such as epithelial-cadherin (E-cadherin, CDH1) and neural-cadherin (N-cadherin, CDH2) have a conserved HAV (histidine-alanine-valine) motif in the most N-terminal EC domain (EC1) that is responsible for homodimer formation, whereas type II classical cadherins do not (e.g. vascular endothelial-cadherin (VE-cadherin, CDH5) and osteoblast-cadherin (OB-cadherin, CDH11)) (170, 171).  1.5.1.2 E-cadherin and N-cadherin in cancer and EVT invasion     During cancer progression, cancer cells of epithelial origin undergo a switch from E-cadherin to N-cadherin to weaken their adhesion to neighbouring cells so as to acquire a higher invasive capacity (168). E-cadherin is often employed as a marker of epithelial cells because of its crucial effects in strong cell-cell adhesion among epithelial cells (174). Down-regulation or impaired function of E-cadherin resulting from mutations, aberrant protein processing, epigenetic modifications and transcriptional repression is involved in the initiation or progression of certain 19  cancers (173, 174). In contrast, N-cadherin is often used as a mesenchymal marker since it is highly expressed among invasive mesenchymal cells. N-cadherin is responsible for F-actin reassembly as well as cell motility during neural tube formation in embryogenesis (175, 176). N-cadherin up-regulation has also been shown to correlate with the invasive properties of cancer cells (177). Indeed, experimental overexpression of N-cadherin promotes cell motility and metastasis in cancer models (178, 179). Mechanistically, N-cadherin-mediated cell motility is thought to involve fibroblast growth factor (FGF) signaling since it can be abrogated by an inhibitor of the FGF receptor (180, 181). Based on its positive effects on cancer cell invasion, N-cadherin has been suggested as a possible target molecule for clinical anticancer therapy (177).     Studies suggest that trophoblast invasion shares several features with tumor cell invasion, though the latter lacks strict physiological control. In human placenta, E-cadherin is expressed by villous cytotrophoblasts and its expression decreases as cytotrophoblasts differentiate to invasive EVTs (182). In contrast, N-cadherin levels are high in primary human EVTs as well as HTR8/SVneo cells whereas they are much lower in choriocarcinoma cell lines with reduced invasiveness (61, 183). Moreover, N-cadherin up-regulation is involved in activin-induced human EVT invasion (51, 52). Switching expression from E-cadherin to N-cadherin is a key step during trophoblast invasive differentiation and failure to switch is associated with insufficient trophoblast invasion and abnormal placentation (166, 167).   1.5.2 The inhibitor of DNA-binding proteins (IDs)     IDs including ID1-4 are a family of small proteins containing a helix-loop-helix (HLH) motif which they use to dimerize with other basic HLH proteins, mainly E-box binding proteins (E proteins), that function as transcription factors (184). Since IDs have no DNA binding domain 20  close to the HLH motif, they sequester homodimers of E proteins or heterodimers of E protein and other tissue-specific basic HLH proteins from binding to DNA, resulting in the inhibition of associated target gene transcription (185, 186). In addition to E protein-dependent effects, E-protein-independent actions of IDs have also been implicated in a number of cellular activities (186). For example, ID1 binds to the integral membrane protein caveolin 1, which lacks an HLH motif, to induce EMT and promote cell invasion in prostate cancer cells (187). Generally, IDs function to keep the stem features of progenitor cells, including inhibiting cell differentiation and senescence while enhancing cell-cycle progression and cell motility. Due to their essential regulatory roles in physiological processes, IDs are evolutionarily conserved from Drosophila to humans (186).      ID1 is the best studied of the four ID members. It is considered to be an oncogene because it is up-regulated in most cancers and its expression is positively correlated with poor clinical prognosis and chemotherapy resistance (187-189). In addition to its effects on the malignancy of cancer cells, ID1 is crucial for normal angiogenesis. For example, ID1 up-regulation mediates BMP6-induced bovine aortic endothelial cell migration and capillary-like tube formation (190). In human placenta, ID1 is repressed during the differentiation of cytotrophoblasts into syncytiotrophoblasts (191). In contrast,  increased ID1 expression is observed in hydatidiform moles compared to normal placentas (192), suggesting the roles of ID1 in both normal placentation and the occurrence of pregnancy complications.  1.5.3 Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI)     BAMBI is a transmembrane protein and evolutionarily conserved in vertebrates (193). It is structurally similar to the TGF-β type I receptor but has no intracellular serine/threonine kinase 21  for signal transduction. As a pseudoreceptor of the TGF-β family members, BAMBI negatively regulates TGF-β, activin and BMP signaling by influencing the formation of functional ligand-receptor complexes (193). However, in human preadipocytes BAMBI knockdown attenuates SMAD1/5/8 phosphorylation and adipogenic effects induced by BMP4, which belongs to the same BMP subfamily as BMP2. These findings suggest positive roles for BAMBI in BMPs signaling, at least in some cell types (194). In addition, BAMBI positively regulates canonical WNT signaling. For example, BAMBI facilitates WNT/β-catenin signaling during rodent myoblast cell differentiation, and reduced BAMBI expression blocks the nuclear translocation of β-catenin in gastric cancer cells and preadipocytes (194-196).     In certain carcinomas, BAMBI exerts oncogenic functions to promote malignancy (197). BAMBI is over-expressed in several cancers including osteosarcoma (198), ovarian (197), and colorectal cancers (199). In particular, BAMBI mediates the loss of epithelial polarity (200), which is typically included in EMT during human trophoblast differentiation along the invasive pathway (167). To date, there are no reports about the effects of BAMBI on human trophoblast differentiation and invasion. However, the mRNA level of BAMBI in human EVTs is over 2 fold higher than those in villous cytotrophoblasts (47, 162), suggesting it may play a role in EVT invasion. As well, BAMBI may regulate human trophoblast differentiation and invasion by modulating the activation of WNT/β-catenin signaling, which is essential for trophoblast invasion. 22  Chapter 2: Rationale, hypothesis and objectives  2.1 Rationale     Human trophoblast differentiation and EVT invasion into maternal endometrium and myometrium are tightly controlled by a variety of hormones, growth factors and cytokines. Appropriate EVT invasive differentiation is essential for normal placentation and the maintenance of pregnancy (54). Transforming growth factor-β (TGF-β) superfamily members exert divergent effects on trophoblast invasion during placentation. TGF-β1 suppresses EVT invasiveness by down-regulating matrix metalloproteinase 9 (MMP9) and vascular endothelial-cadherin (VE-cadherin) (90, 201, 202), whereas activin A promotes EVT invasion by up-regulating N-cadherin and matrix metalloproteinase 2 (MMP2) (51, 52). However, the roles of bone morphogenetic proteins (BMPs), which are also expressed at the maternal-fetal interface, in regulating EVT invasion have not been investigated.  As the biggest subfamily of the TGF-β superfamily, BMPs consist of over 20 isoforms whose roles in organogenesis and embryogenesis are conserved from insects to human (203, 204). BMP2 and BMP4 share the most similarities in structure and function and are grouped in the BMP2/BMP4 subgroup (92). Indeed, BMP2 and BMP4 are of great importance during embryo development and placentation.  In vitro studies have revealed that BMP4 induces the differentiation of human embryonic stem cells into cytokeratin 7/HLA-G-positive trophoblast cells with enhanced invasive ability to penetrate Matrigel (205). However, compared with Bmp4, Bmp2 expression is spatiotemporally correlated with embryo implantation, emphasizing the crucial roles of Bmp2 in embryo implantation and early placentation (107). In addition, Bmp2 is crucial for endometrial decidualization and fertility in mice, and the effect of BMP2 on 23  endometrial decidualization has been confirmed in human stromal cells (17, 18). Similarly, conditional Bmp type II receptor knockout (Bmpr2 cKO) in mouse uterus, which disturbs the Bmpr2-dependent function of Bmp2 and other growth factors, leads to significant impairments in both endometrial decidualization and trophoblast invasion (206). Importantly, treatment of bovine trophoblast cells with BMP2 alleviates the expression of interferon-tau, which is rapidly down-regulated when natural implantation starts in vivo (108), suggesting BMP2 may specifically contribute to bovine trophoblast differentiation along the invasive pathway. Of note, pro-invasive effects of BMP2 have been reported in breast, colon, gastric, and pancreatic cancer cell lines (99, 105, 106, 207-209). Thus, it is likely that BMP2 is involved in regulating EVT invasion. N-cadherin is a mesenchymal adhesion molecule and its up-regulation is correlated with invasiveness in cancer cells (177). Studies suggest that trophoblast invasion shares several common characteristics with tumor cell invasion, though the latter lacks strict physiological control. For example, the switch in expression from E-cadherin (epithelial marker) to N-cadherin (mesenchymal marker) during cancer initiation and progression is also involved in trophoblast differentiation along the invasive pathway. Failure to adequately switch cadherin expression is associated with insufficient invasion and abnormal placentation (166, 167). In addition, N-cadherin is involved in activin A-mediated human EVT invasion (51). Interestingly, BMP2 exerts positive effects on gastric cancer cell invasion and N-cadherin up-regulation (208). To date, it has not been studied whether BMP2 has the same effects on N-cadherin up-regulation and human trophoblast invasion.     Activin A is a homodimer of inhibin βA subunits and a member of the TGF-β superfamily. Similar as BMP2, activin A is abundantly expressed at the fetal-maternal interface and plays 24  essential roles in regulating endometrial decidualization (210, 211). Previous studies have shown that activin A acts as a local regulator to stimulate the differentiation and invasion of human trophoblast cells during the first trimester of pregnancy (212, 213). Our recent studies have also demonstrated that activin A promotes human EVT invasion by up-regulating N-cadherin expression (51) and SNAIL-mediated MMP2 expression (195). Additionally, we have previously shown that BMP4 and BMP7 are strong stimulators of inhibin βA expression and mature activin A production in human granulosa cells (214). However, whether BMP2 regulates inhibin βA expression in human trophoblast cells is unclear. As a classical downstream signaling molecule of BMP2 (191), ID1 is also expressed in human trophoblast cells. In addition, the expression level of ID1 in column EVTs of human first trimester placenta is significantly higher compared to cytotrophoblasts (192). Moreover, increased ID1 expression is observed in hydatidiform moles compared to normal placentas (192). An in vitro study has shown that leukemia inhibitory factor increases HTR8/SVneo invasion and ID1 expression (215), indicating a positive correlation between ID1 expression and HTR8/SVneo invasion. During human organ fibrosis or cancer progression, ID1 promotes EMT including the down-regulation of E-cadherin and zonula occludens-1 (216) as well as the up-regulation of MMP2 (217) and MMP9 (218). Especially, ID1 is deemed as a pro-oncogene owing to its ability to promote tumor invasion and malignancy (186). However, whether ID1 is involved in BMP2-induced human trophoblast invasive differentiation is still unknown.  IGFBP3, whose expression can be regulated by ID1 (219, 220), is one of the major IGFBP proteins present at the maternal-fetal interface in human placenta (117). In situ hybridization studies demonstrated that IGFBP3 is mainly expressed in column EVTs compared with cytotrophoblasts and syncytiotrophoblasts in first trimester human placenta (117). Likewise, 25  whole genome microarray analysis showed that IGFBP3 mRNA levels in EVTs are significantly higher than in cytotrophoblasts (221). In addition, IGFBP3 up-regulation is concomitant with increased HTR8/SVneo cell invasion under hypoxic conditions (222). Together, these findings suggest that IGFBP3 up-regulation may facilitate invasive trophoblast differentiation. Indeed, IGFBP3 is involved in TGF-β1-induced EMT such as the switch of E-cadherin to N-cadherin in transformed human esophageal cells (223). Additionally, BMP2, ID1 and IGFBP3 have each been shown to promote human endothelial cell migration (104, 136, 145, 146, 190). However, whether IGFBP3 is involved in ID1-mediated BMP2 signaling for human EVT invasion needs to be elucidated.  BMP2 was first identified for its essential effects on bone formation; however, the cooperation of canonical WNT/β-catenin signaling is required for this process (103, 224). In addition, WNT/β-catenin signaling plays critical roles in BMP2-mediated endometrial decidualization and pulmonary angiogenesis (103, 163, 164, 224). Importantly, canonical WNT/β-catenin signaling has been broadly reported to positively regulate human trophoblast invasion (56, 148, 157). Indeed, levels of WNT1, β-catenin and cyclin D1 are decreased whereas those of DKK1, a canonical WNT/β-catenin antagonist, are increased in placentas from women with severe preeclampsia compared to normal controls (161). Together, these results suggest that BMP2 signaling may cooperate with WNT/β-catenin signaling to regulate human trophoblast invasion. Moreover, it has been reported that BAMBI, a target gene of BMP2 in granulosa cells (225), facilitates canonical WNT signaling in different cell types (194-196). Importantly, BAMBI is overexpressed in several cancers and increases cancer cell migration (197). In human trophoblasts, the mRNA levels of BAMBI in human EVTs is over 2-fold higher than those in villous cytotrophoblasts (47, 162), suggesting its potential role in trophoblast invasive 26  differentiation. Moreover, BAMBI is up-regulated and mediates the loss of epithelial polarity induced by hypoxia-inducible factor 1 (HIF1) in canine kidney epithelial cells (200). Intriguingly, in addition to the effect on BAMBI up-regulation, HIF1 is also a positive regulator for HTR8/SVneo invasion (222). However, whether BMP2 can promote human trophoblast invasion through BAMBI-mediated activation of canonical WNT/β-catenin signaling is not known.  2.2  Overall hypothesis We hypothesize that BMP2 promotes human EVT invasion by increasing the expression of invasion-associated genes N-cadherin, activin A, ID1, IGFBP3 and activating WNT/β-catenin signaling pathway involved in EVT invasive differentiation.  2.3 Study models     The HTR8/SVneo cell line (immortalized human EVTs) and primary human first trimester EVTs were employed as study models. HTR8/SVneo cells were kindly provided by Dr. P. K. Lala (The Western University, Canada). Compared with primary EVTs, HTR8/SVneo cells have longer lifespan and reduced variability between biological replicates. However, the primary EVTs isolated from first trimester (6-10 gestation weeks) human chorionic villous explants have characteristics closer to the in vivo condition. We employed both study models together in order to balance potential disadvantages of each system. Studies using primary human EVTs were approved by the Research Ethics Board of the University of British Columbia and all patients provided informed written consent.  27  2.4 The objectives of the study The general objective is to determine the role of BMP2 in regulating human EVT invasion and the underlying molecular mechanisms. Objective 1: To investigate the effect of BMP2 on human trophoblast invasion and the involvement of N-cadherin and SMAD signaling. (Presented in Chapter 3) (1) To study the effect of BMP2 on human trophoblast invasion. (2) To examine the effect of BMP2 on N-cadherin expression (3) To determine the involvement of N-cadherin in BMP2-induced trophoblast invasion. (4) To investigate the effects of BMP2 on SMAD signaling pathway including canonical SMAD1/5/8 signaling and non-canonical SMAD2/3 signaling. (5) To determine the involvement of non-canonical SMAD2/3 signaling in BMP2-induced up-regulation of N-cadherin. (6) To investigate the involvement of TGFβ type I receptors (ALKs) in BMP2-induced SMAD phosphorylation and N-cadherin up-regulation.  Objective 2: to determine the effect of BMP2 on activin A production and its role in human trophoblast invasion. (Presented in Chapter 4) (1) To determine the effects of BMP2 on inhibin βA transcription and activin A accumulation. (2) To study the involvement of inhibin βA in BMP2-induced trophoblast invasion. (3) To investigate the involvement of the type I receptors ALKs in BMP2-induced up-regulation of inhibin βA. (4) To investigate the involvement of TGFβ type II receptors in BMP2-induced inhibin βA up-regulation. 28  (5) To determine the role of non-canonical SMAD2/3 signaling in BMP2-induced up-regulation of inhibin βA.  Objective 3: to determine the effects of BMP2 on ID1 and IGFBP3 expression, the association between ID1 and IGFBP3, and their roles in human trophoblast invasion. (Presented in Chapter 5) (1) To determine the effects of BMP2 on ID1 expression.  (2) To determine the effects of BMP2 on IGFBP3 expression. (3) To study the role of ID1 in BMP2-induced IGFBP3 up-regulation. (4) To investigate the involvement of ID1 and IGFBP3 in BMP2-induced human trophoblast invasion.  Objective 4: To study the effect of BAMBI on BMP2-induced WNT/β-catenin signaling activation and human trophoblast invasion. (Presented in Chapter 6) (1) To study the effect of BMP2 on BAMBI mRNA levels. (2) To determine the effects of BMP2 on the activation of WNT/β-catenin signaling as demonstrated by the increased expression of active (non-phospho) β-catenin and cyclin D1. (3) To determine the involvement of BAMBI in BMP2-induced WNT/β-catenin signaling activation. (4) To investigate the role of BAMBI in BMP2-induced human trophoblast invasion.  29  Chapter 3: Bone morphogenetic protein 2 promotes human trophoblast cell invasion by up-regulating N-cadherin via non-canonical SMAD2/3 signaling  3.1 Introduction Extravillous cytotrophoblasts (EVTs) derived from villous cell columns invade into the maternal uterine wall for proper placentation and successful establishment of human pregnancy (54). Insufficient trophoblast invasion is thought to contribute to several pregnancy complications, such as preeclampsia which affects 2-8% of pregnancies worldwide and is a leading cause of maternal mortality (8, 83). Therefore, it is essential to better understand the regulation of trophoblast invasion and identify key signaling molecules underlying this process in order to improve the diagnosis and treatment of these conditions. Transforming growth factor-β (TGF-β) superfamily members exert a variety of regulatory effects on trophoblast invasion during embryo implantation. TGF-β1 suppresses EVT invasiveness by down-regulating matrix metalloproteinase 9 and vascular endothelial-cadherin (201, 202), whereas activin A promotes invasion by up-regulating N-cadherin and matrix metalloproteinase 2 (51, 52). However, there have been no reports about the effects of bone morphogenetic proteins (BMPs) on trophoblast cell invasion. BMPs are the biggest subfamily of the TGF-β superfamily and consist of over 20 isoforms. Their roles in organogenesis are conserved from insects to humans and they may also play key roles in placentation (203, 204). Classically, BMPs function by activating heterotetrameric complexes of type I ALK (activin receptor-like kinases) and type II transmembrane serine-threonine kinase receptors, which subsequently phosphorylate and activate receptor-regulated SMAD1/5/8. Phosphorylated 30  SMAD1/5/8 then bind to common SMAD4 and translocate into the nucleus to mediate BMP-regulated gene expression (92, 226, 227).  In situ hybridization studies in mice have demonstrated that, unlike Bmp 4, 5, 6, 7, 8a and 8b, uterine expression of Bmp2 was spatiotemporally correlated with embryo implantation, suggesting important functions for Bmp2 during implantation and early placentation (107).  Conditional knockout and in vitro studies revealed that Bmp2 was crucial for endometrial decidualization and fertility in mice and humans (17, 18). Although the decidua produces BMP2, it is not known whether BMP2 regulates trophoblast cell invasiveness. However, pro-invasive effects of BMP2 have been reported in breast, colon, gastric, and pancreatic cancer cell lines, and likely involve aspects of EMT including up-regulation of N-cadherin (99, 105, 106, 207-209). Cadherins are transmembrane proteins mediating calcium-dependent cell-cell adhesion with the cytoplasmic domain interacting with catenin and elements of the actin cytoskeleton (173). N-cadherin is a mesenchymal adhesion molecule and its up-regulation has been shown to correlate with invasive properties of cancer cells (177). Studies suggest that trophoblast invasion shares several features with tumor cell invasion, though the latter lacks strict physiological control. Interestingly, switching expression from E-cadherin (epithelial marker) to N-cadherin (mesenchymal marker) is involved in trophoblast differentiation along the invasive pathway and failure to switch is associated with insufficient invasion and abnormal placentation (166, 167). However, it is not known whether BMP2 can promote human trophoblast cell invasion or if such an effect involves the up-regulation of N-cadherin. In the present study, we have examined the effects of BMP2 on human trophoblast cell invasion and the regulation and involvement of N-cadherin in these effects. Our results show that BMP2 treatment enhances trophoblast cell invasion and N-cadherin expression. Furthermore, the 31  pro-invasive effects of BMP2 on trophoblast invasion are mediated by up-regulating N-cadherin via non-canonical SMAD2/3-SMAD4-dependent signaling.  3.2 Materials and Methods Culture of HTR8/SVneo human EVT cell line     The HTR8/SVneo simian virus 40 large T antigen immortalized first trimester human EVT cell line was kindly provided by Dr. P. K. Lala (Western University, Canada) (46). Cells were cultured in DMEM (Life Technologies) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies). Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2 in air.  Primary human EVT isolation and culture     This study was approved by the Research Ethics Board of the University of British Columbia and all women provided informed written consent. 17 first-trimester human placentas (6–10 weeks gestation) were collected from women undergoing elective termination of pregnancy. Primary human EVT cells were isolated from chorionic villous explants as previously described and cultured at 37°C in a humidified 5% CO2/air atmosphere (51, 60). Briefly, the placental villi tips were finely minced and cultured for 3-4 days in flasks with DMEM (Life Technology) supplemented with 10% (vol/vol) FBS, 100 U/mL penicillin, and 100μg/mL streptomycin. Non-attached pieces were removed and attached villous tissue fragments were cultured for another 10-14 days to allow for EVT outgrowth.  EVT cells were subsequently separated from villous explants by trypsinization. The purity of EVT cell cultures was verified by immunocytochemical staining for cytokeratin-7 and human leukocyte antigen G (HLA-G). Only cultures showing 32  more than 99% positive staining for cytokeratin-7 and HLA-G were used in this study. Each experiment performed with primary EVT cells was replicated with cells from 5 different placentas.  Reagents and antibodies     Recombinant human BMP2 and DMH1 (ALK2/3 inhibitor) were obtained from R&D Systems. SB431542 (ALK4/5/7 inhibitor) (catalog no. S4317) was purchased from Sigma-Aldrich. Mouse monoclonal anti-cytokeratin 7 (OV-TL 12/30) and anti-HLA-G (4H84) were purchased from Millipore and Exbio, respectively. Mouse monoclonal anti-N-cadherin antibody (catalog no. 610920) was obtained from BD Biosciences. Rabbit monoclonal anti-phospho-SMAD2 (Ser465/467; 138D4), mouse monoclonal anti- SMAD2 (L16D3), rabbit monoclonal anti-phospho-SMAD3 (Ser423/425; C25A9), rabbit monoclonal SMAD3 (C67H9), rabbit polyclonal anti-SMAD4 (catalog no. 9515) and anti-phospho-SMAD1 (Ser463/465)/SMAD5 (Ser 463/465)/SMAD8 (Ser426/428; catalog no. 9511) were purchased from Cell Signaling Technology. Rabbit polyclonal anti-SMAD1/5/8 (N-18; catalog no. sc-6031-R) and mouse monoclonal anti-α-Tubulin (B-5-1-2; catalog no. sc-23948) were obtained from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories.  Matrigel-coated transwell invasion assay     Trophoblast cell invasiveness was examined using Corning Biocoat Growth Factor Reduced Matrigel Invasion Chamber (pore size, 8 μm; catalog no. 354483) as per the guidelines for use.  Briefly, cells were pre-treated with vehicle or BMP2 (25 or 50 ng/mL) for 20 minutes. Then each 33  insert was seeded with 5×104 cells suspended in 250 μL vehicle/BMP2-containing DMEM medium supplemented with 0.1% (vol/vol) FBS and 750 μL DMEM medium with 10% (vol/vol) FBS was added to the lower chamber. After 24 hours incubation, cells in each insert were retreated with vehicle or BMP2 (25 or 50 ng/mL) and incubated for a further 12 hours (36 hours in total). At the end of the experiment, non-invading cells were removed from the upper side of the membrane and cells on the lower side were fixed with cold methanol (-20°C) and air dried. Cell nuclei were stained with Hoechst 33258 (Sigma-Aldrich) and imaged with a fluorescent microscope followed by analysis with Image-J software. Duplicate inserts were used for each individual experiment, and five random microscopic fields were counted per insert.  Reverse transcription quantitative real-time PCR (RT-qPCR)     Total RNA was extracted with TRIzol Reagent (Life Technologies) as per the manufacturer’s instructions. Reverse transcription was carried out with 2 μg RNA, random primers, and Moloney murine leukemia virus reverse transcriptase (Promega) in a final volume of 20 μL. SYBR Green or TaqMan RT-qPCR was performed on an Applied Biosystems 7300 Real-Time PCR System equipped with 96-well optical reaction plates. Each 20 μL SYBR Green RT-qPCR reaction contained 1×SYBR Green PCR Master Mix (Applied Biosystems), 20 ng cDNA, and 250 nM of each specific primer. The primers used were:  N-cadherin (CDH2), 5'-GGACAGTTCCTGAGGGATCA-3' (forward) and 5'-GGATTGCCTTCCATGTCTGT-3' (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GAGTCAACGGATTTGGTCGT-3' (forward) and 5'- GACAAGCTTCCCGTTCTCAG-3' (reverse). The specificity of each assay was validated by dissociation curve analysis and agarose gel electrophoresis of PCR products. TaqMan gene expression assays for BMP2 (catalog no. 34  Hs00154192_m1), ALK2 (catalog no. Hs00153836_m1), ALK3 (catalog no. Hs01034913_g1), ALK4 (catalog no. Hs00244715_m1), ALK5 (catalog no. Hs00610320_m1) and GAPDH (catalog no. Hs02758991_g1) were purchased from Applied Biosystems. Each 20 μL TaqMan RT-qPCR reaction contained 1×TaqMan Gene Expression Master Mix (Applied Biosystems), 20 ng cDNA, and 1×specific TaqMan assay containing primers and probe. Each sample was assayed in triplicate and a mean value from at least three independent experiments was used for relative quantification of mRNA levels by the comparative Cq method with GAPDH as the reference gene and using the formula 2-ΔΔCq.  Western blot analysis     Cells were lysed in ice-cold lysis buffer (Cell Signaling Technology) with added protease inhibitor cocktail (Sigma-Aldrich). Extracts were centrifuged at 13 000 rpm for 15 minutes at 4°C and supernatant protein concentrations were determined using the DC Protein Assay (Bio-Rad Laboratories) with BSA as the standard. Equal amounts of protein were separated by standard Tris-glycine SDS-PAGE and electrotransferred to PVDF membranes. Membranes were blocked with Tris-buffered saline containing 5% (wt/vol) nonfat dry milk for 1 hour and then immunoblotted overnight at 4°C with specific primary antibodies diluted in Tris-buffered saline with 5% (wt/vol) non-fat dried milk and 0.1% (vol/vol) Tween-20. After incubation with appropriate horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature, signals were detected with enhanced chemiluminescent or SuperSignal West Femto chemiluminescent substrates (Thermo Fisher) and CL-XPosure film (Thermo Fisher). Membranes were stripped with stripping buffer (62.5mM Tris-HCl [pH 6.8], 100 mM β-mercaptoethanol, and 2% (wt/vol) SDS) at 50°C for 30 minutes and reprobed as described above 35  with antibodies against α-tubulin, SMAD2, SMAD3, or SMAD1/5/8. Densitometric quantification was performed using Image-Pro Plus software with α-tubulin, SMAD2, SMAD3 or SMAD1/5/8 for normalization.  Small interfering RNA (siRNA) transfection     Cells at approximately fifty percent confluency were transfected for 48 hours with 25 nM ON-TARGETplus NON-TARGETINGpool siRNA or ON-TARGETplus SMARTpool siRNA  targeting human N-cadherin (L-011605-00-0005), SMAD2 (L-003561-00-0005), SMAD3 (L-020067-00-0005), SMAD4 (L-003902-00-0005), ALK2 (L-004924-00-0005), ALK3 (L-004933-00-0005), ALK4 (L-004925-00-0005) or ALK5 (L-003929-00-0005; Dharmacon) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. Knockdown efficiency was assessed by RT-qPCR or Western blot analysis.  MTT assay     MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma) assay was used to assess cell viability. HTR8/SVneo cells were plated in 24-well plates (1.2×104 cells/well in 1 mL 10% FBS medium) and incubated for 12 hours before starvation with 0.1% FBS medium for another 24 hours. Cells were then treated every 24 hours with or without BMP2 in medium containing 0.1% FBS for total 72 hours. Cells were incubated with 0.5 mg/mL MTT for 4 hours after which the medium was replaced with 1 mL dimethylsulfoxide (DMSO) and absorbances were measured at 490 nm using a microplate reader.  Statistical analysis 36      Results are presented as the mean ± SEM of at least three independent experiments. Multiple group comparisons were analyzed by one-way ANOVA followed by Newman-Keuls test using PRISM software (GraphPad Software, Inc). Means were considered significantly different if P < 0.05 and columns without letters in common are significantly different.  3.3 Results BMP2 enhances human trophoblast cell invasion     To determine the effects of BMP2 on trophoblast cell invasion, Matrigel-coated transwell invasion assays were carried out following treatment of HTR8/SVneo cells with 25 or 50 ng/mL recombinant human BMP2. The results showed BMP2 significantly increased HTR8/SVneo cell invasion in a concentration-dependent manner (Figure 3.1, A and B). To examine whether the enhanced trophoblast invasion induced by BMP2 might be influenced by any effect of BMP2 on cell viability, MTT assays were performed and the results confirmed that treatment with 25 ng/mL or 50 ng/mL BMP2 for as long as 72 hours had no significant effect on HTR8/SVneo cell viability (Figure 3.1, C). Similar to HTR8/SVneo cells, treatment with 25 ng/mL BMP2 significantly enhanced cell invasion in primary human EVT cells (Figure 3.2). Since 25ng/mL BMP2 could promote human trophoblast cell invasion significantly, this concentration of BMP2 was used to treat HTR8/SVneo cell line as well as primary trophoblast cells in the following experiments.  BMP2 up-regulates trophoblast N-cadherin levels     To examine the effects of BMP2 on N-cadherin expression, HTR8/SVneo and primary EVT cells were treated with 25 ng/mL BMP2 for 3, 6, 12, 24 or 48 hours. RT-qPCR results showed 37  that BMP2 treatment increased N-cadherin mRNA levels in both HTR8/SVneo and primary EVT cells between 6 and 24 hours (Figure 3.3). Similarly, Western blot analysis revealed up-regulation of N-cadherin protein levels following treatment of HTR8/SVneo and primary EVT cells with BMP2 for 24 and 48 hours (Figure 3.4).   N-cadherin up-regulation contributes to BMP2-induced trophoblast cell invasion      To determine whether N-cadherin up-regulation is involved in BMP2-induced trophoblast cell invasion, we performed siRNA-mediated knockdown of N-cadherin. Pre-treatment of HTR8/SVneo cells with siRNA targeting N-cadherin for 48 hours suppressed both basal and BMP2-induced N-cadherin protein levels (Figure 3.5, A). Matrigel-coated transwell invasion assays revealed that depletion of N-cadherin attenuated both basal and BMP2-induced HTR8/SVneo cell invasion (Figure 3.5, B). Importantly, the contribution of N-cadherin to basal and BMP2-induced cell invasion was also confirmed in first-trimester primary human EVT cells (Figure 3.6).   Noncanonical SMAD2/3 signaling is required for BMP2-induced N-cadherin up-regulation     BMPs exert their biological effects primarily via canonical SMAD1/5/8-SMAD4 signaling, however several reports have demonstrated the involvement of noncanonical SMAD2/3-SMAD4 and/or SMAD-independent signaling (99, 227-230). Thus, we used Western blot to measure changes in the phosphorylation/activation of SMAD1/5/8 or SMAD2/3 following treatment of HTR8/SVneo or primary EVT cells with BMP2. BMP2 treatment increased not only the phosphorylation of canonical SMAD1/5/8 as expected, but also induced the phosphorylation of noncanonical SMAD2/3 in HTR8/SVneo cells (Figure 3.7). Confirmatory experiments 38  performed with primary EVT cells likewise showed that BMP2 treatment increased the phosphorylation of both SMAD1/5/8 and SMAD2/3 (Figure 3.7). To investigate the involvement of SMAD signaling in BMP2-induced N-cadherin production, we first performed knockdown of common SMAD4. As shown in Figure 3.8, the stimulatory effect of BMP2 on N-cadherin protein level was abolished by knockdown of common SMAD4 in HTR8/SVneo cells. Next, we performed combined knockdown of SMAD2/3 since we have previously shown that SMAD2/3 mediates N-cadherin up-regulation in response to activin A in human trophoblast cells (51). Interestingly, depletion of SMAD2/3 completely abolished the up-regulation of N-cadherin by BMP2 in HTR8/SVneo cells (Figure 3.8), suggesting that noncanonical SMAD2/3 signaling is essential for BMP2-induced N-cadherin production.  BMP2 increases N-cadherin production via both BMP (ALK2/3) and activin (ALK4) type I receptors     BMPs generally activate SMAD1/5/8 signaling via BMP type I receptors ALK2, ALK3 and/or ALK6, whereas activins/TGF-βs induce the activation of SMAD2/3 via ALK4, ALK5 or ALK7. To determine which ALKs are involved in the activation of noncanonical SMAD2/3 and up-regulation of N-cadherin by BMP2, we first used a pharmacological approach with selective inhibitors of BMP or activin/TGF-β type I receptors. Western blot was used to measure BMP2-induced SMAD phosphorylation in HTR8/SVneo cells following pre-treatment with the ALK2/3 inhibitor DMH1 (231) or the ALK4/5/7 inhibitor SB431542 (232). HTR8/SVneo cells were also treated with activin A as a positive control since it has previously been shown to up-regulate N-cadherin and induce SMAD2/3 signaling, both of which can be totally blocked by pre-treatment with SB431542 (51). As expected, BMP2-induced phosphorylation of SMAD1/5/8 was 39  abolished by pre-treatment with DMH1 but was not influenced by SB431542 pre-treatment (Figure 3.9). Interestingly, DMH1 also abolished BMP2-induced phosphorylation of SMAD2 and SMAD3, whereas pre-treatment with SB431542 was partially inhibitory (Figure 3.9). In contrast, activin A-induced SMAD2/3 phosphorylation was totally blocked by pre-treatment with SB431542 (Figure 3.9) as previously reported (51). In both HTR8/SVneo and primary EVT cells, the up-regulation of N-cadherin mRNA (Figure 3.10) and protein (Figure 3.11) levels by BMP2 was completely inhibited by treatment with DMH1 and partially inhibited by treatment with SB431542. Next, we used siRNA-mediated knockdown approach to determine which ALKs are involved in the up-regulation of N-cadherin by BMP2. HTR8/SVneo cells were pre-treated for 48 hours with siRNA targeting ALK2, ALK3, ALK4 or ALK5 prior to treatment with BMP2 for 24 hours. BMP2-induced increases in N-cadherin mRNA levels were abolished by knockdown of ALK3 and partially inhibited by depletion of ALK2 or ALK4 (Figure 3.12). In contrast, down-regulation of ALK5 did not affect the up-regulation of N-cadherin by BMP2 (Figure 3.12). Together, these results suggest that BMP2 increases N-cadherin production via ALK2, ALK3 and ALK4 in human trophoblast cells.   3.4 Discussion     We propose that as well as its role in endometrial decidualization (17, 18), BMP2 may contribute to embryo implantation and early placental development by promoting trophoblast invasion. In particular, we have shown that BMP2 promotes primary and immortalized human EVT cell invasion by up-regulating N-cadherin. Moreover, we demonstrate that whereas BMP2 activates both canonical SMAD1/5/8 and non-canonical SMAD2/3 signaling, only SMAD2/3 40  signaling seems to be required for its up-regulation of N-cadherin. In vivo studies of the putative functions of BMP2 in endometrial decidulization have been performed in mice using a conditional knockout approach because total knockout of Bmp2 or Bmp receptors in mice leads to embryonic lethality or severe defects in cardiac development (206, 233). Progesterone receptor locus-guided conditional knockout of BMP type II receptor (Bmpr2 cKO) in the uterus revealed impairments in both endometrial decidualization and trophoblast invasion (206). At the time it was suggested that the defects in trophoblast invasion were simply due to impaired decidualization. However, it should be noted that mouse trophoblast cells also express progesterone receptor (234) and that some of this impairment could reflect direct effects in trophoblast cells due to Bmpr2 haploinsufficiency in a majority of the embryos produced by Bmpr2 cKO mice. BMPR2 binds BMPs as well as some growth differentiation factors so it remains to be determined exactly what the in vivo functions of BMP2 are in early placental development. However, we have detected high levels of BMP2 mRNA in primary human EVT cells (data not shown) which suggests that the effects of BMP2 on trophoblast invasion could be mediated in an autocrine as well as paracrine manner. During differentiation to invasive EVTs, trophoblasts of epithelial lineage must undergo epithelial-mesenchymal transition (EMT) to acquire the motility and invasive potential necessary for remodeling of the decidua and spiral arteries (166, 167). Defects in this process are associated with pre-eclampsia, a common pregnancy complication characterized, in part, by shallow trophoblast invasion (83). EMT is a fundamental process that is important for embryonic development as well as cancer progression; however its involvement in invasive trophoblast differentiation has yet to be fully delineated. BMP2 has been implicated in EMT-like processes in both physiological and pathological contexts. For example, BMP2 was shown to be critical for 41  cardiac cushion formation by inducing endocardial EMT (102). In cancer cells, BMP2 increases cell invasion via PI3K/AKT signaling and induces EMT-like changes in gene expression, such as down-regulation of E-cadherin and up-regulation of SNAIL or SLUG  (105, 106). In addition to increasing N-cadherin and cell invasion, treatment of trophoblastic HTR8/SVneo cells with BMP2 increased the expression of several EMT-associated genes, including matrix metalloproteinase 2, SNAIL and SLUG (Figure 3.13). Induction of these EMT-associated genes could act in parallel with N-cadherin to increase HTR8/SVneo cell invasion, and might account for inability of N-cadherin knockdown to completely inhibit BMP2-induced HTR8/SVneo cell invasion. However, BMP2 has also been reported to suppress colorectal cancer cell growth by inhibiting proliferation and inducing apoptosis (235). As well, BMP2 has been shown to inhibit liver cancer cell migration and growth by down-regulating PI3K/AKT signaling (100). Thus, BMP2 can exert varying, and even opposing, effects on cancer cells depending on type and context. In EVT cells the effects of BMP2 appear to be largely restricted to the promotion of invasiveness since we did not observe any significant changes in cell viability. Nevertheless, future studies are required to investigate the effects of BMP2 on other trophoblast cell functions, especially in cytotrophoblast or syncytiotrophoblast cell populations. BMP2-induced activation or inactivation of SMAD-independent signaling, especially PI3K/AKT and MAPK pathways, has been implicated in the promotion or suppression of cancer progression (100, 101). However, our study revealed that short term treatment with BMP2 (< 2 hours) had no effects on AKT or ERK1/2 phosphorylation/activation (Figure 3.14). Rather, BMP2 treatment induced the activation of both canonical SMAD1/5/8 and noncanonical SMAD2/3 signaling, although only the latter mediated N-cadherin up-regulation in human trophoblast cells. In a study of 46 normal and cancer cell lines, BMP2 was shown to induce 42  SMAD2/3 signaling preferentially in embryonic and transformed cells (99). Our findings of BMP2-induced SMAD2/3 phosphorylation in human EVT cells are in agreement since trophoblast cells are derived from the blastocyst trophectoderm. Interestingly, Holtzhausen et al. also noted greater co-expression of genes responsive to SMAD1/5/8 and SMAD2/3 in breast and liver cancer samples compared to normal tissues, suggesting cooperation between these two SMAD signaling pathways in cancer (99). Our findings do not exclude a role for SMAD1/5/8 signaling in BMP2-induced trophoblast invasion. Indeed, BMP2-induced up-regulation of furin, which is a pro-protein convertase that can directly activate membrane type 1 MMP (MT1-MMP) (236), is blocked by SMAD4 knockdown but not by combined knockdown of SMAD2/3, suggesting regulation by SMAD1/5/8-SMAD4 signaling (Figure 3.15). Trophoblast cells are often thought of as pseudomalignant because they share a number of features with cancer cells. Future studies investigating functional cooperation between canonical SMAD1/5/8 and non-canonical SMAD2/3 signaling in human trophoblast cells will be of great interest. TGF-β superfamily members signal in a SMAD-dependent manner to regulate trophoblast invasion during embryo implantation. However, in contrast to the classical notion of BMP signaling, we have shown that BMP2 enhances EVT cell invasion by up-regulating N-cadherin via non-canonical SMAD2/3 signaling. Generally, BMPs are thought to bind ALK2/3/6 type I receptors and activate SMAD1/5/8, whereas TGF-βs/activins usually bind ALK4/5/7 to induce SMAD2/3. However, recent studies in cancer, pituitary and ovarian cells have demonstrated BMP-induced activation of SMAD2/3 signaling via several mechanisms. Holtzhausen et al. used a variety of techniques to show that BMP2 could induce heterodimeric type I receptor complexes composed of ALK5/7 and ALK3/6 which were capable of phosphorylating SMAD2/3 in a variety of cancer cell lines (99). Direct activation of SMAD2 and SMAD3 by ALK3 has been 43  demonstrated in BMP2-treated melanoma and pituitary gonadotrope cells, respectively (229, 230). In human ovarian granulosa cells, BMP4 was shown to activate SMAD2 via ALK3 and ALK4/5, whereas it activated SMAD3 via ALK3 and ALK4 (228). Together our inhibitor and knockdown results suggest that BMP2-induced increases in SMAD2/3 phosphorylation and N-cadherin production are mediated by both ALK2 and ALK3. In contrast to previous studies in cancer and pituitary gonadotrope cells (99, 230, 237), our study in trophoblast cells is the first to implicate ALK2 in BMP2-induced non-canonical SMAD2/3 signaling. Our inhibitor and knockdown results also show that ALK4, but not ALK5, is partially involved in the SMAD2/3-dependent up-regulation of N-cadherin by BMP2. These results are unique compared to previous studies in cancer cells implicating only ALK5 or both ALK4/5 in BMP2-induced SMAD2/3 signaling (99, 230). In agreement with the classical notion of BMP signaling, our SB431542 results show that only ALK2/3 are involved in the activation of canonical SMAD1/5/8 signaling. Future studies are required to investigate in greater detail the precise roles of SMAD2/3 vs. SMAD1/5/8 signaling in the effects of BMP2 on human trophoblast invasion as well as other biological responses. Overall, our study demonstrates for the first time that BMP2 promotes human trophoblast invasion in addition to its well-established roles in endometrial decidualization. The pro-invasive effects of BMP2 are mediated, in part, by up-regulation of N-cadherin via noncanonical SMAD2/3-SMAD4 signaling. BMP2 induces SMAD2/3 signaling via ALK2, ALK3 and ALK4, whereas it activates canonical SMAD1/5/8 signaling via ALK2/3 (Figure 3.16). These findings deepen our understanding of the roles of BMP2 in placentation and provide insight into the molecular mechanisms of human trophoblast invasion.   44     Figure 3.1 BMP2 increases HTR8/SVneo cell invasion A and B, HTR8/SVneo cells were treated with vehicle (Ctrl) or BMP2 (25 or 50 ng/mL) and cell invasiveness was examined by Matrigel-coated transwell assay. Representative images of the invasion assay (A; scale bar 100 μm) and summarized quantitative results (B) are shown separately. C, HTR8/SVneo cells were seeded in 24-well plates and treated every 24 hours with vehicle or BMP2 (25 or 50 ng/mL) for a total of 72 hours. Cell viability was determined by MTT assay at 24, 48, and 72 hours after BMP2 treatment. Results are displayed as the mean ± SEM of at least three independent experiments and in Figure 3.1B columns without letters in common are significantly different (P < 0.05). 45       Figure 3.2 BMP2 increases primary human EVT cell invasion  Primary human EVT cells were treated with or without 25 ng/mL BMP2 and cell invasiveness was examined by Matrigel-coated transwell assay. Representative images of the invasion assay (left panel; scale bar 100 μm) and combined quantitative results (right panel) are shown separately (n=5). Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).      46       Figure 3.3 BMP2 increases N-cadherin mRNA in HTR8/SVneo and primary human EVT cells  HTR8/SVneo cells (left panel) or primary EVT cells (right panel) were treated with vehicle (Ctrl) or 25 ng/mL BMP2 for different lengths of time (3, 6, 12 or 24 h), and N-cadherin mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).       47       Figure 3.4 BMP2 increases N-cadherin protein levels in HTR8/SVneo and primary human EVT cells  HTR8/SVneo cells (left panel) or primary EVT cells (right panel) were treated with or without 25 ng/mL BMP2 every 24 hours for 48 hours, and N-cadherin protein levels were analyzed by Western blot and normalized to α-tubulin. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).     48     Figure 3.5 Knockdown of N-cadherin attenuates basal and BMP2-induced HTR8/SVneo cell invasion A. HTR8/SVneo cells were transfected for 48 hours with 25 nM non-targeting control siRNA (si-Ctrl) or 25 nM siRNA targeting N-cadherin (si-N-cad) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2. Western blot was used to measure N-cadherin protein levels in HTR8/SVneo 24 hours after treatment with BMP2. B, Matrigel-coated transwell assays were used to examine the effects of N-cadherin knockdown on BMP2-induced invasion in HTR8/SVneo cells. Summarized quantitative results are expressed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).    49     Figure 3.6 Knockdown of N-cadherin attenuates basal and BMP2-induced primary human EVT cell invasion A, Primary human EVT cells were transfected for 48 hours with 25 nM non-targeting control siRNA (si-Ctrl) or 25 nM siRNA targeting N-cadherin (si-N-cad) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2. Western blot was used to measure N-cadherin protein levels in primary EVT cells 24 hours after treatment with BMP2. B, Matrigel-coated transwell assays were used to examine the effects of N-cadherin knockdown on BMP2-induced invasion in primary EVT cells. Summarized quantitative results are expressed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).   50      Figure 3.7 BMP2 activates canonical SMAD1/5/8 and noncanonical SMAD2/3 signaling  HTR8/SVneo (left panel) and primary EVT (right panel) cells were treated with vehicle (Ctrl) or 25 ng/mL BMP2 for different lengths of time (10, 30, 60 or 120 min). Levels of phosphorylated SMAD1/5/8 (P-SMAD1/5/8), SMAD2 (P-SMAD2) and SMAD3 (P-SMAD3) were examined by Western blot with corresponding phospho-specific antibodies. Membranes were stripped and re-probed with antibodies for total SMAD1/5/8 (T-SMAD1/5/8), SMAD2 (T-SMAD2) and SMAD3 (T-SMAD3).     51      Figure 3.8 Noncanonical SMAD2/3 signaling mediates the up-regulation of N-cadherin by BMP2  HTR8/SVneo cells were transfected for 48 h with 25 nM non-targeting control siRNA (si-Ctrl), 25 nM siRNA targeting SMAD2+SMAD3 (si-S2+3) or 25 nM siRNA targeting SMAD4 (si-S4) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for 24 hours. Protein levels of N-cadherin, T-SMAD2, T-SMAD3 and T-SMAD4 were examined by Western blot. Summarized quantitative results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).    52      Figure 3.9 BMP and activin/TGF-β type I receptors contribute to BMP2-induced phosphorylation of SMAD2/3 HTR8/SVneo cells were pre-treated for 1 hour with vehicle control (DMSO), 1 μM DMH1 (ALK2/3 inhibitor) or 10 μM SB431542 (ALK4/5/7 inhibitor) prior to treatment for 30 minutes with vehicle (Ctrl), 25 ng/mL BMP2 or 50 ng/mL activin A. Protein levels of phosphorylated SMAD1/5/8 (P-SMAD1/5/8), SMAD2 (P-SMAD2) and SMAD3 (P-SMAD3) were examined by Western blot with corresponding phospho-specific antibodies. Membranes were stripped and re-probed with antibodies for total SMAD1/5/8 (T-SMAD1/5/8), SMAD2 (T-SMAD2) and SMAD3 (T-SMAD3). Results are expressed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).   53        Figure 3.10 BMP and activin/TGF-β type I receptors contribute to BMP2-induced up-regulation of N-cadherin mRNA levels HTR8/SVneo and primary EVT cells were pre-treated for 1 hour with DMH1 (1 μM) or SB431542 (10 μM ) prior to treatment for 12 hour with or without 25 ng/mL BMP2, and N-cadherin mRNA levels were measured by RT-qPCR. Results are expressed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).      54      Figure 3.11 BMP and activin/TGF-β type I receptors contribute to BMP2-induced up-regulation of N-cadherin protein levels  HTR8/SVneo and primary EVT cells were pre-treated for 1 hour with DMH1 (1 μM) or SB431542 (10 μM) prior to treatment for 24 hours with BMP2 (25 ng/mL) or activin A (50 ng/mL), and N-cadherin protein levels were analyzed by Western blot. Results are expressed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).      55   Figure 3.12 BMP (ALK2/3) and activin (ALK4) type I receptors mediate the up-regulation of N-cadherin by BMP2 A and B, HTR8/SVneo cells were transfected for 48 h with 25 nM non-targeting control siRNA (si-Ctrl) or 25 nM siRNA targeting ALK2 (si-ALK2), ALK3 (si-ALK3), ALK4 (si-ALK4) or ALK5 (si-ALK5). Cells were treated for a further 24 hours with vehicle (Ctrl) or 25 ng/mL BMP2 and RT-qPCR was used to measure the mRNA levels of N-cadherin (A) and ALKs (B). Summarized quantitative results are displayed as the mean ± SEM of three independent experiments and columns without letters in common are significantly different (P < 0.05). 56   Figure 3.13 BMP2 increases the expression of several EMT-associated genes in immortalized human trophoblast cells  HTR8/SVneo cells were treated with vehicle (Ctrl) or with 25 ng/mL BMP2 for different lengths of time (3, 6, 12 or 24 hours), and MMP2, SNAIL and SLUG mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. Summarized quantitative results are displayed as the mean ± SEM of three independent experiments and columns without letters in common are significantly different (P < 0.05). 57     Figure 3.14 Treatment with BMP2 (< 2 hours) has no effects on AKT or ERK1/2 phosphorylation/activation  HTR8/SVneo cells were treated with vehicle (Ctrl) or 25 ng/mL BMP2 for different lengths of time (10, 30, 60 or 120 min). Levels of phosphorylated ERK (P-ERK) and AKT (P-AKT) were examined by Western blot with corresponding phospho-specific antibodies. Membranes were stripped and re-probed with antibodies for total ERK (T-ERK) and AKT (T-AKT).  58     Figure 3.15 Canonical SMAD1/5/8 signaling mediates the up-regulation of furin by BMP2  HTR8/SVneo cells were transfected for 48 h with 25 nM non-targeting control siRNA (si-Ctrl), 25 nM siRNA targeting SMAD2+SMAD3 (si-S2+3) or 25 nM siRNA targeting SMAD4 (si-S4) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for 24 hours. Protein levels of furin, N-cadherin, T-SMAD2, T-SMAD3 and T-SMAD4 were examined by Western blot.   59    Figure 3.16 Proposed model of the signaling pathway mediating BMP2-induced N-cadherin up-regulation and increased human trophoblast cell invasion BMP2 binds to a complex of type I and II receptors leading to the activation of both canonical SMAD1/5/8 and noncanonical SMAD2/3 signaling. Activation of receptor complexes containing ALK2, ALK3 and ALK4 lead to the phosphorylation of SMAD2/3 which complexes with common SMAD4 and translocates into the nucleus to increase the transcription of N-cadherin, which promotes human trophoblast cell invasion. BMP2 activates canonical SMAD1/5/8 signaling via ALK2/3 however the functional consequences of this signaling require further investigation.  60  Chapter 4: Bone morphogenetic protein 2 promotes human trophoblast cell invasion by inducing activin A production  4.1 Introduction     Embryo implantation is a highly regulated process controlled by a complex interplay between the placental trophoblasts and endometrium. After attachment of trophoblast cells to the epithelial surface of the endometrium, extravillous cytotrophoblasts (EVTs) invade the uterine stroma and remodel the  maternal vasculature, a process that is critical for the establishment and maintenance of early pregnancy (2). Studies have shown that at the fetal-maternal interface, the invasion of human EVTs is stringently modulated by various factors, such as extracellular matrix components, growth factors, and several adhesion molecules in an autocrine/paracrine manner (90, 238). Failure or dysregulation of trophoblast invasion and inadequate vasculature transformation may result in preeclampsia and fetal growth restriction (8, 239). First identified as inducers of bone formation, bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily that mediate various physiological and developmental activities, including placental development (240, 241). BMP2 belongs to the BMP subfamily that is especially critical in placental development (108). BMP2 is expressed in mouse and human stromal/decidual cells as well as primary human EVT cells (89, 242). In particular, the expression of BMP2 is spatiotemporally correlated with implantation sites in mice (107). In knockout mice, conceptuses lacking BMP2 display developmental arrest at early stage without the formation of mesoderm and placental vasculature (243). Conditional ablation of Bmp2 in the uterus using the (PR-cre) mice shows that their uterine stroma is incapable of undergoing decidualization to support further placental development, which causes 61  sterility (17). Similarly, mice with conditional depletion of Bmpr2 (type II receptor for BMP2) in uterus demonstrate fetal growth retardation and severe hemorrhage at the implantation sites, which leads to fetal demise and placental abruption (206). We have recently shown that BMP2 treatment can promote human EVT cell invasion by up-regulating N-cadherin (242). Collectively, these results suggest a critical role for BMP2 in the process of embryo implantation and any abnormality in uterine BMP2-mediated signaling can have adverse consequences in maintaining pregnancy. However, the molecular mechanisms underlying BMP2-induced trophoblast cell invasion are yet to be elucidated. Activin A is a homodimer of inhibin βA that belongs to the TGF-β superfamily. Activin A and its functional receptors and binding proteins are highly expressed in human preimplantation embryos, endometrium, and placenta (210, 211). Previous studies have shown that activin A acts as a local regulator to stimulate the differentiation, outgrowth, and invasion of human cytotrophoblast cells during the first trimester of pregnancy (212, 213). Our recent studies also demonstrated that activin A promotes human trophoblast cell invasion by up-regulating N-cadherin expression (51) and SNAIL-mediated MMP2 expression (195). Additionally, activin A facilitates human trophoblast cell endothelial-like tube formation by inducing the expression of vascular endothelial growth factor-A (49). Although the functional roles of activin A have been extensively studied in human trophoblast cells, much less is known regarding its intracellular regulation. We have previously shown that BMP4 and BMP7 are strong stimulators for inhibin βA expression and mature activin A production in human granulosa cells (214). At present, whether BMP2 can up-regulate inhibin βA expression in human trophoblast cells is unclear. In addition, if inhibin βA expression and activin A levels can be up-regulated by BMP2, whether activin A mediates BMP2–induced human trophoblast cell invasion remains unknown. In the 62  present study, we examined the effect of BMP2 on activin A production and the underlying molecular mechanisms in primary human EVTs. We also investigated the role of activin A in BMP2-induced increases in human trophoblast cell invasion.  4.2 Materials and Methods Primary human EVT cell isolation and culture This study was approved by the Research Ethics Board of the University of British Columbia and all patients provided informed written consent. First-trimester human placental samples (6–8 weeks) were collected from women undergoing elective termination of pregnancy. Primary human EVT cells were isolated and cultured as described in Chapter 3.   Reagents and antibodies     Recombinant human BMP2 and DMH-1 were obtained from R&D Systems. Mouse monoclonal anti-cytokeratin 7 (catalog no. MAB3554) and anti-HLA-G (catalog no. 11-499-C100) were obtained from Millipore and EXBIO Praha, respectively. Rabbit monoclonal anti-phospho-SMAD2 (catalog no. 3108), mouse monoclonal anti-SMAD2 (catalog no. 3103), rabbit monoclonal anti-phospho-SMAD3 (catalog no. 9520), rabbit monoclonal SMAD3 (catalog no. 9523) and anti-phospho-SMAD1/5/8 (catalog no. 13820) were purchased from Cell Signaling Technology. Rabbit polyclonal anti-SMAD1/5/8 (catalog no. sc-6031-R), and mouse monoclonal anti-α-Tubulin (catalog no. sc-23948) were obtained from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories.  63  Matrigel invasion assay     Trophoblast cell invasiveness was examined using Corning Biocoat Growth Factor Reduced Matrigel Invasion Chamber (8 μm pore size; catalog no. 354483) as described in Chapter 3.  Reverse transcription quantitative real-time PCR (RT-qPCR)     Total RNA was extracted with TRIzol Reagent (Life Technologies) as per the manufacturer’s instructions. RT-qPCR was carried out as delineated in Chapter 3. The primers used were:  human inhibin α subunit (INHA), 5'-GTCTCCCAAGCCATCCTTTT-3' (forward) and 5'-TGGCAGCTGACTTGTCCTC-3' (reverse); human inhibin βA subunit (INHBA), 5'-CTCGGAGATCATCACGTTTG-3' (forward) and 5'-CCTTGGAAATCTCGAAGTGC-3' (reverse); human inhibin βB subunit (INHBB), 5'- ATCAGCTTCGCCGAGACA-3' (forward) and 5'-GCCTTCGTTGGAGATGAAGA-3' (reverse); human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GAGTCAACGGATTTGGTCGT-3' (forward) and 5'- GACAAGCTTCCCGTTCTCAG-3' (reverse). TaqMan gene expression assays for ALK2 (Hs00153836_m1), ALK3 (Hs01034913_g1), ALK6 (Hs01010965_m1), BMPR2 (Hs00176148_m1), ACVR2A (Hs00155658_m1), ACVR2B (Hs00609603_m1) and GAPDH (Hs02758991_g1) were purchased from Applied Biosystems and were performed as shown in Chapter 3.  Western blot analysis Cells were lysed in ice-cold lysis buffer (Cell Signaling Technology) containing protease inhibitor cocktail (Sigma-Aldrich). Western blot analysis was performed as described in Chapter 3. 64   Small interfering RNA (siRNA) transfection     Cells at approximately fifty percent confluency were transfected for 48 h with 25 nM ON-TARGETplus Non-targeting control pool or separate ON-TARGETplus SMART pools targeting ALK2 (ACVR1), ALK3 (BMPR1A), ALK6 (BMPR1B), BMPR2, ACVR2A, ACVR2B, SMAD2, SMAD3, or SMAD4 (Dharmacon) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. Knockdown efficiency was assessed by RT-qPCR.  Measurement of activin A      Culture medium was collected and assayed immediately or stored at -80°C until assayed. Activin A accumulation in conditioned medium was measured using a human activin A Quantikine ELISA kit (R&D Systems, DAC00B) according to the manufacturer’s instructions. Inter- and intra-assay coefficients of variation for this assay were less than 10% and the detection limit was 7.85 pg/mL. Each sample was assayed in triplicate and activin A levels were normalized to total cellular protein content.  Statistical analysis     Results are presented as the mean ± SEM of at least three independent experiments. Results were analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison test using PRISM software (GraphPad Software). Means were considered significantly different if P < 0.05 and columns without letters in common are significantly different.  65  4.3 Results BMP2 increases inhibin βA mRNA levels and activin A accumulation in primary human EVT cells     To investigate the effects of BMP2 on the transcription of inhibin subunits, primary human EVT cells were starved in DMEM with 0.1% FBS for 18 hours prior to treatment with or without 25 ng/mL BMP2 for 3, 6, 12, or 24 hours in DMEM with 0.1% FBS, and the mRNA levels of inhibin α, βB, and βA were examined using RT-qPCR. Treatment with 25 ng/mL BMP2 did not affect the mRNA levels of inhibin α and inhibin βB (Figure 4.1, A and B), whereas it increased inhibin βA mRNA levels starting at 12 hour, and the stimulatory effect persisted until 24 hour (Figure 4.1, C). Next, we measured the accumulation of activin A in conditioned medium to determine whether BMP2 enhances the production of activin A in primary human EVT cells. Basal activin A accumulation in 24 h controls was 940.41±234.39 pg/mL. Cells were treated for 24 or 48 hours with 25 ng/mL BMP2 in 0.1% FBS medium and activin A accumulation was measured using an enzyme immunoassay. As shown in Figure 4.1D, treatment with BMP2 for 24 or 48 hours increased the accumulation of activin A.   BMP2 promotes primary EVT cell invasion by up-regulating the expression of inhibin βA      It has been shown that activin A can promote human trophoblast cell invasion in primary EVT cells and EVT cell lines (51, 195). Small interfering RNA (siRNA; 25 nM) targeting inhibin βA subunit (INHBA) was used to investigate its involvement in BMP2-induced human trophoblast cell invasion. As shown in Figure 4.2A, transient transfection with siRNA targeting INHBA for 48 hours effectively suppressed the mRNA levels of inhibin βA. Treatment with BMP2 (25 ng/mL) for 36 hours significantly enhanced primary human EVT cell invasion and, interestingly, 66  this effect was inhibited by INHBA knockdown for 48 hours prior to BMP2 treatment (Figure 4.2, B). These data suggest that BMP2 promotes human trophoblast invasion by up-regulating the expression of inhibin βA.   ALK3 type I receptor mediates BMP2-induced up-regulation of inhibin βA in primary human EVT cells     BMPs function mainly through three TGF-β type I receptors: ALK2, ALK3, or ALK6 (244). To determine which ALK type I receptor mediates the BMP2-induced up-regulation of inhibin βA, a specific ALK2/ALK3 inhibitor DMH1 (231) was used in this study. Following starvation for 18 h in DMEM with 0.1% FBS, primary human EVTs in DMEM with 0.1% FBS were pretreated with dimethylsulfoxide (DMSO) or DMH1 (1 μM) for 1 hour and then treated with 25 ng/mL BMP2 for a further 12 hours (inhibin βA mRNA levels) or 24 hours (activin A accumulation). As shown in Figure 4.3A, pre-treatment with DMH-1 for 1 h completely reversed the BMP2-induced increase in inhibin βA mRNA levels in primary human EVT cells. Similarly, pre-treatment with DMH1 for 1 hour totally blocked the ability of BMP2 to increase activin A accumulation in conditioned medium (Figure 4.3, B; basal activin A in 24 hour controls was 618.54±367.02 pg/mL). To further confirm which ALK mediates BMP2-induced activin A production in primary human EVT cells, an siRNA-based inhibition approach was used to specifically knockdown each type I receptor (ALK2, ALK3, or ALK6). Analysis of knockdown efficiency by RT-qPCR showed that transfection of cells with 25 nM siRNA targeting ALK2, ALK3, or ALK6 for 48 hours specifically suppressed the mRNA levels of the targeted ALK (Figure 4.4, A). Most importantly, only knockdown of ALK3 completely abolished the BMP2-induced increase in inhibin βA mRNA levels (Figure 4.4, B), indicating that ALK3 type I 67  receptor is required for the effects of BMP2 on inhibin βA in primary human EVT cells. Unexpectedly, ALK2 knockdown up-regulated basal inhibin βA mRNA level, however further increases were observed after BMP2 treatment (Figure 4.4, B).   BMPR2 and ACVR2A type II receptors are required for the up-regulation of inhibin βA by BMP2 in primary human EVT cells     Three TGF-β type II receptors (BMPR2, ACVR2A and ACVR2B) have been shown to mediate the effects of BMPs in human cells (245). A similar siRNA-based inhibition approach was used to determine which TGF-β type II receptors are involved in the BMP2-induced up-regulation of inhibin βA. RT-qPCR analysis confirmed efficient and specific knockdown of targeted type II receptors (Figure 4.5, A). Interestingly, knockdown of BMPR2, ACVR2A, or ACVR2B alone did not affect the BMP2-induced up-regulation of inhibin βA (Figure 4.5, B), indicating at least two different TGF-β type II receptors could be involved. Indeed, only combined knockdown of BMPR2 and ACVR2A totally abolished the increase in inhibin βA mRNA levels induced by BMP2 (Figure 4.5, C). In contrast, neither combined knockdown of BMPR2 and ACVR2B nor combined knockdown of ACVR2A and ACVR2B affected the BMP2-induced inhibin βA mRNA levels. These results suggest that both BMPR2 and ACVR2A are required for the up-regulation of inhibin βA by BMP2 in primary human EVT cells.  Non-canonical SMAD2/3 signaling is not involved in BMP2-induced up-regulation of inhibin βA in primary human EVT cells     Western blot was used to investigate whether BMP2 activates canonical SMAD1/5/8 and non-canonical SMAD2/3 signaling in primary human EVTs. Our results show that treatment with 25 68  ng/mL BMP2 increased the levels of phosphorylated SMAD1/5/8 at all time-points examined (10, 30, 60, and 120 min; Figure 4.6, A). Similarly, treatment with BMP2 for 30, 60, or 120 min increased the levels of phosphorylated SMAD2 (Figure 4.6, B) and SMAD3 (Figure 4.6, C). Interestingly, combined knockdown of SMAD2 and SMAD3 did not affect BMP2-induced up-regulation of inhibin βA (Figure 4.7, B). However, knockdown of common SMAD4 (required for both SMAD1/5/8 and SMAD2/3 signaling) completely abolished the up-regulation of inhibin βA induced by BMP2 treatment (Figure 4.7, B). These results suggest that canonical SMAD1/5/8, rather than non-canonical SMAD2/3, signaling mediates the up-regulation of inhibin βA by BMP2 in primary human EVT cells (Figure 4.8).  4.4 Discussion     During early embryo implantation, both trophoblast and decidual cells secrete a variety of growth factors and cytokines to regulate trophoblast differentiation and invasion at the maternal-fetal interface. In the present study, we demonstrate for the first time that BMP2 increases inhibin βA expression and activin A production to promote human trophoblast cell invasion, revealing a coordinated regulation of trophoblast cell invasion by divergent growth factors. This result is consistent with our previous study showing both BMP4 and BMP7 induced the expression of inhibin βA in human granulosa cells (214). Similar results were reported in murine gonadotrope LβT2 cells, as BMP2 and activin A synergistically stimulate the synthesis of FSH (246). Interestingly, an activin A/BMP2 chimera promotes murine osteogenesis and bone healing more potently than recombinant BMP2 (247). Though both are members of the same superfamily, BMP2 and activin A have more often been shown to exert effects that are antagonistic to one another. These two growth factors can stimulate different target gene 69  expression and suppress each other’s actions through competing for SMAD4, a common regulator of the downstream signaling for BMPs (via SMAD1/5/8) and activins (via SMAD2/3). For instance, activin A induces dorsalization of Xenopus mesoderm, whereas BMPs induce its ventralization and can inhibit the effects induced by activin A (248, 249). Activin A maintains the pluripotency of human embryonic stem cells and can inhibit BMP-induced differentiation (250). Collectively, these observations suggest that two ligands, activin A and BMP2, may generate synergistic or antagonistic effects through employing different, nonoverlapping mechanisms in different cell types. Our results clarify the mechanisms by which BMP2 regulates activin A production and shed light on the cooperative effects of these two ligands. From a clinical perspective, understanding the cellular and molecular mechanisms underlying BMP actions will improve our knowledge of early implantation and could lead to the development of pharmaceutical approaches for the treatment of pregnancy complications related to trophoblast-dysfunction. BMP2 initiates downstream signaling through binding to ALK type I and type II transmembrane serine/threonine kinase receptors. The kinase domains of the type II receptors are constitutively active, whereas the kinase (GS) domains of type I receptors are phosphorylated upon ligand-receptor binding (251). Thus, the specificity of the downstream signaling is principally determined by type I receptors (251). The present study used a specific BMP type I receptor inhibitor DMH1 to investigate which receptors are required for the biological effects of BMP2 in human primary EVT cells. Our result showed that pre-treatment with DMH1 completely abolished the stimulatory effect of BMP2 on inhibin βA expression. Considering the limitations and off-target effect of the inhibitor, we therefore used an siRNA-based approach to precisely determine which ALK mediates the actions of BMP2. Our results strongly suggest that ALK3 is the major physiological receptor for BMP2 in primary human 70  EVT cells. This result differs from our recent studies demonstrating that BMP2 functions through ALK2 and ALK3 to down-regulate connexin 43 and pentraxin 3 in human granulosa cells (252). These differences suggest that the molecular mechanisms underlying BMP2-mediated target gene regulation are cell type-specific (253). Unexpectedly, we found that knockdown of ALK2 increased basal and BMP2-induced inhibin βA mRNA levels, suggesting a negative role for ALK2 in regulating basal inhibin βA expression. At present, it is unclear exactly which TGF-β superfamily member acts through ALK2 to suppress inhibin βA expression in human trophoblast cells. Future studies focusing on how ALK2 contributes to the regulation of activin A production in human trophoblast cells will be of interest. It has been a general assumption that the TGF-β ligands all bind to and signal via their receptors in a similar manner. Instead, the limited number of receptors suggest complex and promiscuous interactions with different TGF-β ligands (254). Moreover, multiple TGF-β ligands produced in complex tissues may signal through differential and overlapping subsets of receptors (231). Studies have shown that BMPs bind to three distinct type II receptors, BMPR2, ACVR2A, and ACVR2B (251, 255). Currently, which type II receptors are used to mediate the physiological action of BMP2 in human trophoblast cells remains unclear. Our results showed that individual knockdown of BMPR2, ACVR2A, or ACVR2B did not abolish the increases in inhibin βA expression induced by BMP2. Rather, combined knockdown of both BMPR2 and ACVR2A was required to totally abolish the stimulatory effects of BMP2 on inhibin βA expression. Similarly, our most recent study reported that BMP2 could act via both BMPR2 and ACVR2A to exert cellular function in human granulosa cells (256). Compared to ACVR2B type II receptor, ACVR2A has a broader specificity and is able to bind to both BMPs and activins (257). A study using recombinant receptor extracellular domain demonstrated that ACVR2A 71  could bind to BMP2, BMP7 and activin with different affinities (257). The high degree of sequence identity of the binding domains in these ligands may explain why ACVR2A is commonly used as the functional type II receptor for different ligands. Aside from canonical SMAD1/5/8 signaling, recent studies have shown that non-canonical SMAD2/3 signaling (activated by activins and TGF-βs) can be induced by BMPs to modulate hormone production and cancer progression (99, 229). Indeed, our previous study showed that both BMP4 and BMP15 induce non-canonical SMAD2/3 signaling to induce hyaluronan production in human granulosa cells (228). We have also shown that BMP2 enhances N-cadherin expression via non-canonical SMAD2/3 signaling in HTR8/SVneo cells (242). In this study, we showed that BMP2 could induce the phosphorylation of both SMAD1/5/8 and SMAD2/3, however knockdown of SMAD2/3 did not block the effects of BMP2 on inhibin βA. Since BMP2-induced inhibin βA expression was completely blocked by knockdown of common SMAD4, our results suggest that these effects are mediated by canonical SMAD1/5/8 signaling. Previous studies have demonstrated that the expression levels of type I and II receptors in cells are the main contributing factors for the differential activation of non-canonical SMAD2/3 signaling induced by BMP2. Specifically, cell types that have higher expression levels of receptors (and hence higher receptor binding properties) tend to display non-canonical signaling (230). Therefore, it is of great importance to determine to what extent the expression levels of receptors might contribute to the BMP2-induced activation of non-canonical SMAD2/3 signaling in human trophoblast cells. Nonetheless, our results clearly show that non-canonical SMAD2/3 signaling is not required for the induction of activin A production by BMP2 in primary human EVT cells. 72  In summary, the present study demonstrates that BMP2 up-regulates inhibin βA expression and activin A production, which contributes to the trophoblast cell invasion (Figure 4.8). In addition, our results show that non-canonical SMAD2/3 signaling is not required for BMP2-induced up-regulation of inhibin βA expression. Our inhibitor and knockdown studies demonstrate that ALK3 type I and BMPR2/ACVR2A type II receptors are the functional receptors that mediate BMP2-induced up-regulation of inhibin βA expression in human trophoblast cells (Figure 4.8). This study provides important insights into the molecular mechanisms that mediate BMP2-driven early pregnancy establishment.             73   Figure 4.1 BMP2 up-regulates inhibin βA mRNA levels and increases activin A accumulation in primary human EVT cells  A-C, Primary EVTs were treated with vehicle control or 25 ng/mL BMP2 for 3, 6, 12, or 24 h, and the mRNA levels of inhibin α (A), Inhibin βB (B), or Inhibin βA (C) were examined using RT-qPCR. D, Primary EVTs were treated with vehicle control or 25 ng/mL BMP2 for 24 or 48 h, and the activin A accumulation levels in the conditioned medium were measured using an enzyme immunoassay. The results are expressed as the mean ± SEM of at least three independent experiments. Columns without letters in common indicate significant differences (P <0.05). 74   Figure 4.2 Knockdown of inhibin βA abolishes the BMP2-induced trophoblast cell invasion in primary human EVTs  A and B, Primary human EVTs were transfected for 48 h with 25 nM non-targeting control siRNA (siCtrl) or 25 nM siRNA targeting inhibin βA (siINHBA) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for additional 36 h. The knockdown efficiency of inhibin βA was evaluated using RT-qPCR (A). Trophoblast cell invasiveness was examined using a Matrigel invasion assay (B; scale bar 100 μm) and combined quantitative results are shown graphically underneath. The results are expressed as the mean ± SEM of at least three independent experiments. Columns without letters in common are significantly different (P <0.05). 75      Figure 4.3 DMH1 abolishes the BMP2-induced up-regulation of inhibin βA in primary human EVTs A and B, Primary human EVTs were pretreated with dimethylsulfoxide (DMSO) or the specific receptor inhibitor, DMH1 (1 μM) for 1 h, and then treated with 25 ng/mL BMP2 for additional 12 h (for inhibin βA mRNA level) or 24 h (for activin A accumulation). The mRNA levels of inhibin βA (A) were examined using RT-qPCR and activin A accumulation levels (B) in the conditioned medium were determined with an enzyme immunoassay, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Columns without letters in common are significantly different (P <0.05). 76     Figure 4.4 ALK3 type I receptor mediates the BMP2-induced up-regulation of inhibin βA in primary human EVTs  Primary EVT cells were transfected with siCtrl, siALK2, siALK3, or siALK6 for 48 h before 25 ng/mL BMP2 treatment for an additional 12 h. The knockdown efficiency and specificity of each siRNA related ALKs (A) and the inhibin βA mRNA levels (B) were detected using RT-qPCR, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Columns without letters in common are significantly different (P <0.05).    77   Figure 4.5 BMPR2 and ACVR2A but not ACVR2B type II receptors mediate the BMP2-induced up-regulation of inhibin βA in primary human EVTs A, The knockdown efficiency and specificity of BMPR2, ACVR2A and ACVR2B siRNAs (transfected for 48 h) were evaluated using RT-qPCR. B, Primary human EVTs were transfected with siCtrl, siBMPR2, siACVR2A, or siACVR2B for 48 h before treatment with 25 ng/ml BMP2 for an additional 12 h, and the inhibin βA mRNA levels were examined using RT-qPCR. C, Primary human EVTs cells were transfected with siCtrl, combined siBMPR2 with siACVR2A (siBMPR2 + siACVR2A), combined siBMPR2 with siACVR2B (siBMPR2 + siACVR2B), or combined siACVR2A with siACVR2B (siACVR2A + siACVR2B) for 48 h before treatment with 25 ng/mL of BMP2 for an additional 12 h, and the inhibin βA mRNA levels were examined using RT-qPCR. The results are expressed as the mean ± SEM of at least three independent experiments. Columns without letters in common are significantly different (P <0.05). 78   Figure 4.6 BMP2 activates canonical SMAD1/5/8 and uncanonical SMAD2/3 signaling in primary human EVTs  Primary human EVTs were treated vehicle control or 25 ng/mL BMP2 for 10, 30, 60, and 120 min. The phosphorylated protein levels of SMAD1/5/8 (A), SMAD2 (B), and SMAD3 (C) were examined using Western blot analysis. The results are expressed as the mean ± SEM of at least three independent experiments. Columns without letters in common are significantly different (P <0.05). 79    Figure 4.7 Non-canonical SMAD2/3 signaling is not involved in the BMP2-induced up-regulation of inhibin βA in primary human EVTs  Primary human EVTs were transfected with 25 nM siCtrl, combined siSMAD2 and siSMAD3 (siSMAD2 + siSMAD3), or siSMAD4 for 48 h, and then they were treated with 25 ng/mL BMP2 for an additional 12 h. The knockdown efficiency of each siRNA related SMADs (A) and the inhibin βA mRNA levels (B) were examined using RT-qPCR. The results are expressed as the mean ± SEM of at least three independent experiments. Columns without letters in common are significantly different (P <0.05).   80     Figure 4.8 Schematic diagram of the proposed molecular mechanisms for BMP2-induced increases in inhibin βA expression and activin A production in human trophoblast cells  BMP2 signals through binding to ALK3 type I receptor and BMPR2 or ACVR2A type II receptors. The ligand-receptor binding activates the phosphorylation of SMAD1/5/8 and SMAD2/3. Phosphorylated SMAD1/5/8 but not SMAD2/3 then associates with common SMAD (SMAD4), which translocate into the nucleus and up-regulate inhibin βA expression. This process promotes the production of mature activin A, which stimulates human trophoblast cell invasion. 81  Chapter 5: Bone morphogenetic protein 2 promotes human trophoblast cell invasion through ID1-mediated up-regulation of IGF binding protein-3  5.1 Introduction During placentation, human extravillous trophoblasts (EVTs) invade into the uterine wall and differentiate into interstitial and endovascular EVTs, both of which participate in spiral artery remodeling. In particular, endovascular EVTs facilitate spiral artery remodeling by penetrating the spiral arteries and replacing the residing endothelial cells lining the arterial lumen (70, 258). Inadequate vascular remodeling caused by insufficient EVT invasion in the first trimester of pregnancy is one of the pathological characteristics of preeclampsia (8, 83). To date, many cytokines and growth factors have been reported to regulate human trophoblast invasion, including transforming growth factor-β (TGF-β) superfamily members (54). Different TGF-β superfamily members play divergent roles in human trophoblast invasion. TGF-β1 inhibits trophoblast invasion by down-regulating matrix metaloproteinase 9 (MMP9) whereas activin A promotes trophoblast invasion by up-regulating N-cadherin and MMP2 (51, 90, 195). As the biggest subfamily of the TGF-β superfamily, bone morphogenetic proteins (BMPs) are essential for placental development (241). In particular, the expression of Bmp2 is tightly correlated with embryo implantation and is essential for endometrial decidualization and fertility in mice (17, 89, 107, 242). In humans, BMP2 is expressed in both endometrial tissue and trophoblast cells, and plays essential roles in endometrial decidualization and EVT invasion (242, 259).  As a classical downstream signaling molecule of BMP2, inhibitor of DNA-binding 1 (ID1) is a member of the helix-loop-helix (HLH) family of proteins (184). Since ID1 has no DNA 82  binding domain, it dimerizes with other basic HLH proteins (primarily E-box binding proteins) to sequester them from binding DNA, resulting in inhibition of associated target gene transcription (185). ID1 is repressed during the differentiation of cytotrophoblasts into syncytiotrophoblasts (191). In contrast, the expression level of ID1 in column EVTs of human first trimester placenta is significantly higher compared to cytotrophoblasts (192), suggesting that ID1 up-regulation may contribute to the invasive differentiation of cytotrophoblasts. Moreover, increased ID1 expression is observed in hydatidiform moles compared to normal placentas (192). An in vitro study has shown that leukemia inhibitory factor increases HTR8/SVneo invasion and ID1 expression (215), indicating a positive correlation between ID1 expression and HTR8/SVneo invasion. Since ID1 up-regulation has been shown to mediate BMP6-induced bovine aortic endothelial cell migration (190), we hypothesized that ID1 is involved in BMP2-induced human EVT cell invasion. Insulin-like growth factor binding protein-3 (IGFBP-3) is one of the major IGFBP proteins present at the maternal-fetal interface (117). IGFBP3 exerts its effects by regulating insulin-like growth factor (IGF) bioavailability, though IGF-independent effects of IGFBP3 have also been reported (260). In situ hybridization studies demonstrated that IGFBP3 is mainly expressed in column trophoblasts compared with cytotrophoblasts and syncytiotrophoblasts in  first trimester human placenta (117). Likewise, whole genome microarray analysis showed that IGFBP3 mRNA levels in EVTs are significantly higher than in cytotrophoblasts (221). In addition, IGFBP3 up-regulation is concomitant with increased HTR8/SVneo cell invasion under hypoxic conditions (222). Together, these findings suggest that IGFBP3 up-regulation may facilitate invasive trophoblast differentiation. In preeclamptic placentas, IGFBP3 is strongly down-regulated whereas microRNA-210, which targets IGFBP3, is elevated compared with normal 83  placentas (139-141). IGFBP3 shows the same localization and possible function on HTR8/SVneo cell invasion as ID1. In addition, BMP2, ID1 and IGFBP3 have each been shown to promote human endothelial cell migration (104, 136, 145, 146, 190). Zhao et al. reported that ID1 could regulate IGFBP3 expression in mouse endothelial cells (219). Based on this evidence, we hypothesized that IGFBP3 is involved in ID1-mediated BMP2-induced human EVT cell invasion.  In the present study, we determined the effects of BMP2 on the expression of ID1 and IGFBP3 as well as their involvement in BMP2-induced human trophoblast invasion. Our results indicate that BMP2 induces the up-regulation of ID1, which subsequently mediates the increased expression of IGFBP3. Moreover, both ID1 and IGFBP3 play essential roles in BMP-induced trophoblast invasion.  5.2 Materials and Methods Culture of HTR8/SVneo human EVT cell line     The HTR8/SVneo simian virus 40 large T antigen immortalized first trimester human EVT cell line was kindly provided by Dr. P. K. Lala (Western University, Canada) (46). Cells were cultured as described in Chapter 3.  Primary human EVT isolation and culture     This study was approved by the Research Ethics Board of the University of British Columbia and all women provided informed written consent. First-trimester human placentas (6–8 weeks gestation) were collected from women undergoing elective termination of pregnancy. Primary human EVT cells were isolated from chorionic villous explants as described in Chapter 3. 84  Reagents and antibodies     Recombinant human BMP2 and DMH1 were obtained from R&D Systems. Mouse monoclonal anti-cytokeratin 7 (catalog no. MAB3554) and anti-HLA-G (catalog no. 11-499-C100) were obtained from Millipore and EXBIO Praha, respectively. Rabbit polyclonal anti-ID1 antibody (catalog no. sc-488) was obtained from Santa Cruz Biotechnology. Mouse monoclonal anti-IGFBP3 (catalog no. MAB305) was purchased from R&D Systems. Rabbit monoclonal anti-Slug (catalog no. 9585) were purchased from Cell Signaling Technology. Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories.  Matrigel-coated transwell invasion assay Trophoblast cell invasiveness was examined using Corning Biocoat Growth Factor Reduced Matrigel Invasion Chamber (pore size, 8 μm; catalog no. 354483) as described in Chapter 3.  Reverse transcription quantitative real-time PCR (RT-qPCR) Total RNA was extracted with TRIzol Reagent (Life Technologies) as per the manufacturer’s instructions. RT-qPCR was carried out as delineated in Chapter 3. The primers used were:  ID1, 5'- CTCTACGACATGAACGGCTGT-3' (forward) and 5'-TGCTCACCTTGCGGTTCTG-3' (reverse); IGFBP3, 5'-CAGAGCACAGATACCCAGAACTTC-3' (forward) and 5'- TTCTCTACGGCAGGGACCAT-3' (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GAGTCAACGGATTTGGTCGT-3' (forward) and 5'- GACAAGCTTCCCGTTCTCAG-3' (reverse).   85  Western blot analysis     Cells were lysed in ice-cold lysis buffer (Cell Signaling Technology) with added protease inhibitor cocktail (Sigma-Aldrich). Western blot analysis was performed as shown in Chapter 3.  Small interfering RNA (siRNA) transfection     Cells at approximately fifty percent confluency were transfected for 48 hours with 25 nM ON-TARGETplus NON-TARGETINGpool siRNA or ON-TARGETplus SMARTpool siRNA targeting human ID1 (L-005051-00-0005; Dharmacon), IGFBP3 (L-004777-00-0005; Dharmacon) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. Knockdown efficiency was assessed by RT-qPCR or Western blot analysis.  Measurement of IGFBP3 accumulation       Culture medium was collected and assayed immediately or stored at -80°C until assayed. IGFBP3 accumulation in conditioned medium was measured using a Human IGFBP3 Quantikine ELISA kit (R&D Systems, DGB300) according to the manufacturer’s instructions. Inter- and intra-assay coefficients of variation for this assay were less than 10% and the detection limit was 0.14 ng/mL. Each sample was assayed in triplicate and IGFBP3 levels were normalized to total cellular protein content.  Statistical analysis     Results are presented as the mean ± SEM of at least three independent experiments. Multiple group comparisons were analyzed by one-way ANOVA followed by Newman-Keuls test using 86  PRISM software (GraphPad Software, Inc). Means were considered significantly different if P < 0.05 and columns without letters in common are significantly different.  5.3 Results BMP2 induces the up-regulation of ID1 in human trophoblast cells     To test whether BMP2 can induce ID1 expression in human trophoblast cells, HTR8/SVneo and primary EVT cells were treated with 25 ng/mL BMP2 for 3, 6, 12 and 24 hours. RT-qPCR and Western blot analyses showed that BMP2 significantly increased the levels of ID1 mRNA (Figure 5.1) and protein (Figure 5.2) in both HTR8/SVneo and primary EVT cells.  BMP2 increases IGFBP3 production in human trophoblast cells To determine the effects of BMP2 on IGFBP3 expression in human trophoblast cells, HTR8/SVneo and primary EVT cells were treated with 25 ng/mL BMP2 for 3, 6, 12 and 24 hours. RT-qPCR and Western blot analyses showed that BMP2 treatment significantly increased IGFBP3 mRNA (Figure 5.3) and protein (Figure 5.4) levels at 12 and 24 hours in both HTR8/SVneo and primary EVT cells.  IGFBP3 is a secreted protein that functions by adjusting the bioavailability of IGF or exerting its effects via receptor-mediated endocytosis (260). IGFBP3 accumulation in conditioned medium was measured to determine whether BMP2 enhances IGFBP3 secretion in human trophoblast cells. HTR8/SVneo and primary EVT cells were treated with 25 ng/mL BMP2 in 0.1% FBS DMEM medium for 12 or 24 hours and IGFBP3 accumulation was measured using an enzyme immunoassay. As shown in Figure 5.5, BMP2 treatment significantly increased IGFBP3 accumulation in both HTR8/SVneo and primary EVT cells. 87   ID1 mediates BMP2-induced IGFBP3 up-regulation      Next, we used siRNA-mediated ID1 knockdown to determine if ID1 up-regulation is essential for BMP2-induced increases in IGFBP3 production. HTR8/SVneo cells were pre-treated with siRNA targeting ID1 for 48 hours followed by BMP2 treatment for another 12 hours (RNA levels) or 24 hours (protein levels). RT-qPCR results showed that the up-regulation of IGFBP3 by BMP2 was attenuated by ID1 knockdown (Figure 5.6, A). This result was confirmed at the protein level by Western blot in HTR8/SVneo cells (Figure 5.6, B). Importantly, the essential role of ID1 in BMP2-induced IGFBP3 up-regulation was also demonstrated in primary human EVTs by Western blot (Figure 5.7).   Up-regulation of ID1 and IGFBP3 contributes to BMP2-induced trophoblast cell invasion     To determine whether the up-regulation of ID1 and IGFBP3 is involved in BMP2-induced trophoblast cell invasion, siRNA-mediated knockdown of ID1 or IGFBP3 was performed prior to BMP2 treatment and transwell Matrigel invasion assays. Western blot analysis showed that pre-treatment of HTR8/SVneo cells with siRNA targeting ID1 or IGFBP3 for 48 hours suppressed the protein levels of ID1 and IGFBP3. Moreover, ID1 knockdown attenuated the up-regulation of IGFBP3 by BMP2 (Figure 5.8, A-C). Importantly, knockdown of ID1 or IGFBP3 attenuated BMP2-induced increases in HTR8/SVneo cell invasion (Figure 5.8, D and E). Likewise, the contribution of ID1 and IGFBP3 to BMP2-induced trophoblast invasion was also confirmed in human primary EVT cells (Figure 5.9).  88  5.4 Discussion During placentation, a host of molecules are recruited to cooperate with each other for the regulation of human trophoblast invasion. Here we have demonstrated for the first time that BMP2 increases the expression of ID1 and IGFBP3, both of which contribute to BMP2-induced invasiveness in primary and immortalized human EVTs. Moreover, BMP2-induced IGFBP3 up-regulation is mediated by ID1 in human trophoblast cells. Therefore, ID1 and IGFBP3 are integrated in BMP2 signaling in a coordinated manner to regulate human trophoblast invasion.  We have already shown that BMP2 promotes human trophoblast invasion by up-regulating N-cadherin expression and activin A production (242, 259). Here we further demonstrate that ID1 is involved in BMP2-induced trophoblast invasion. However, it has been reported that Id1 is down-regulated during the differentiation of rat Rcho-1 choriocarcinoma cells into trophoblast giant cells, which are thought to be somewhat equivalent to human EVTs. Moreover, Id1 overexpression in Rcho-1 cells inhibits the differentiation to rat trophoblast giant cells (261). Compared to rat Rcho-1 cells, differentiated rat trophoblast giant cells in vitro show an immotile epithelial phenotype characterized by reduced membrane protrusive activity and well-organized actin stress fibers (262, 263). Conversely, human EVTs differentiated from epithelial cytotrophoblasts are mesenchymal-like cells with enhanced invasive ability. In humans, ID1 expression is significantly higher in column EVTs than in cytotrophoblast cells (192), suggesting ID1 up-regulation facilitates cytotrophoblast differentiation to motile EVTs. Indeed, ID1 up-regulation is associated with increased invasion of HTR8/SVneo cells induced by leukemia inhibitory factor (215).  According to our in vitro research, ID1 is essential for BMP2-induced IGFBP3 up-regulation in human EVTs. In contrast, Igfbp3 was shown to be up-regulated in the endothelial cells of Id3 89  knockout mice with conditional knockout of Id1 (219), suggesting that Id1 can repress Igfbp3 expression in the absence of Id3 in vivo. This discrepancy may be due to the impact of Id3 knockout because ID3 is also up-regulated when human EVTs are treated with BMP2 (data not shown). Several clinical studies suggest that both ID1 and IGFBP3 are up-regulated in pathological processes such as Alzhermer’s and psoriatic diseases (264, 265). Moreover, BMP2, ID1 and IGFBP3 have each been implicated in the promotion of endothelial cell migration (104, 145, 146, 190), but whether they function together in normal angiogenesis is unknown. Similarly, hypoxic conditions have been shown to increase ID1 expression in human trophoblasts (266) as well as IGFBP3 expression and cell invasion in HTR8/SVneo cells (222). ID1 is generally considered to be an inhibitor of gene expression due to its ability to antagonize the binding of basic HLH transcription factors to promoter regions (184). However, there is evidence that ID1 can increase gene expression in several biological processes, including the up-regulation of matrix metalloproteinase 9 (MMP9) (184, 218). Interestingly, Li et al. described a novel mechanism whereby ID1 mediates IGF2 up-regulation by stabilizing the transcription factor E2F1 in esophageal cancer cells (267). Although our study demonstrates that ID1 is critical for IGFBP3 up-regulation by BMP2, future studies are required to determine if this mechanism involves E2F1 stabilization or interaction with other transcription factors.      In our study, BMP2 enhances IGFBP3 production to increase trophoblast transwell Matrigel invasion, which is in agreement with the up-regulation of IGFBP3 during invasive differentiation of cytotrophoblasts (117, 221). In transformed human esophageal cells, IGFBP3 plays a positive role in EMT, a process important to invasive differentiation of human trophoblasts (223). Indeed, IGFBP3 is also involved in BMP2-ID1-induced up-regulation of the EMT-associated transcription factor Slug (168) (Figure 5.10), suggesting the possible downstream signaling for 90  enhanced trophoblast invasion. However, others have shown that treatment with recombinant human IGFBP3 has no effect on migration of human EVTs through fibronectin-coated transwell inserts (268), suggesting its effects may be context dependent. Indeed, IGFBP3 has been shown to exert both positive and negative effects on malignancy (260), and has even been shown to exert opposing functions on malignant vs. non-malignant breast epithelial cells (269). Different post-translational modifications of IGFBP3, such as glycosylation and phosphorylation, may at least partially account for the divergent functions of IGFBP3 (270). In addition, IGF-dependent and IGF-independent effects of IGFBP3 suggest that cytokines, growth factors, extracellular matrix (ECM) components in the microenvironment might influence how IGFBP3 functions (260).     In guinea pigs, maternal circulating IGFBP3 is closely correlated with placental growth in late gestation (138). In human preeclampsia placentas, IGFBP3 is downregulated while microRNA 210, one target gene of which is IGFBP3, is significantly upregulated in contrast to normal placenta controls (140, 141). During cytotrophoblasts differentiation, epithelial to endothelial transformation is also required for endovascular EVT differentiation, and column EVTs acquire endothelial-like features including the expression of endothelial markers (271, 272). IGFBP3 has been shown to facilitate the differentiation of endothelial precursor cells into endothelial cells (145). Thus it is tempting to suggest that IGFBP3 may be involved in endovascular EVT differentiation, and future studies investigating this will be of great interest.      In summary, our study reveals that BMP2 up-regulates ID1 and IGFBP3 expression, both of which promote human trophoblast cell invasion. In addition, we show for the first time that ID1 mediates BMP2-induced up-regulation of IGFBP3. These findings broaden our knowledge on the molecular mechanisms underlying the pro-invasive effects of BMP2 on human trophoblasts.  91         Figure 5.1 BMP2 increases ID1 mRNA levels in HTR8/SVneo and primary human EVT cells HTR8/SVneo cells (left panel) or primary EVT cells (right panel) were treated with vehicle (Ctrl) or 25 ng/mL BMP2 for different lengths of time (3, 6, 12 or 24 h). ID1 mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).      92      Figure 5.2 BMP2 increases ID1 protein levels in HTR8/SVneo and primary human EVT cells HTR8/SVneo cells (left panel) or primary EVT cells (right panel) were treated with vehicle (Ctrl) or 25 ng/mL BMP2 for different lengths of time (3, 6, 12 or 24 h). ID1 protein levels were examined by Western blot and normalized to α-tubulin. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).  93         Figure 5.3 BMP2 increases IGFBP3 mRNA levels in HTR8/SVneo and primary human EVT cells  HTR8/SVneo cells (left panel) and primary EVT cells (right panel) were treated with or without 25 ng/mL BMP2 for different lengths of time (3, 6, 12 or 24 h). IGFBP3 mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).      94      Figure 5.4 BMP2 increases IGFBP3 protein levels in HTR8/SVneo and primary human EVT cells  HTR8/SVneo cells (left panel) and primary EVT cells (right panel) were treated with or without 25 ng/mL BMP2 for different lengths of time (3, 6, 12 or 24 h). IGFBP3 protein levels were examined by Western blot and normalized to α-tubulin. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).      95      Figure 5.5 BMP2 increases IGFBP3 production in HTR8/SVneo and primary human EVT cells  HTR8/SVneo cells (left panel) or primary EVT cells (right panel) were treated with or without 25 ng/mL BMP2 for 12 or 24 h. IGFBP3 accumulation in conditioned medium was measured using an enzyme immunoassay. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).         96      Figure 5.6 ID1 mediates BMP2-induced IGFBP3 up-regulation in HTR8/SVneo cells  A and B, HTR8/SVneo cells were transfected for 48 hours with 25 nM non-targeting control siRNA (siCtrl) or 25 nM siRNA targeting ID1 (siID1) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2. A, RT-qPCR was used to measure IGFBP3 and ID1 mRNA levels 12 hours after treatment with BMP2. B, Western blot was used to measure IGFBP3 and ID1 protein levels 24 hours after BMP2 treatment. Summarized quantitative results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).  97      Figure 5.7 ID1 mediates BMP2-induced IGFBP3 up-regulation in human primary EVT cells  Primary EVT cells were transfected for 48 hours with 25 nM non-targeting control siRNA (siCtrl) or 25 nM siRNA targeting ID1 (siID1) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for another 24 hours. Western blot was used to measure IGFBP3 and ID1 protein levels. Summarized quantitative results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).   98   Figure 5.8 Knockdown of ID1 or IGFBP3 abolishes BMP2-induced trophoblast cell invasion in HTR8/SVneo cells  A-C, HTR8/SVneo cells were transfected for 48 h with 25 nM siCtrl or siID1 or siIGFBP3 prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for additional 36 h. Knockdown efficiency of ID1 and IGFBP3 were evaluated by Western blot. D and E, HTR8/SVneo cell invasiveness was examined by Matrigel-coated transwell invasion assay and summarized quantitative results are shown in E. Results are presented as the mean ± SEM of at least three independent experiments. Columns without letters in common are significantly different (P <0.05). 99   Figure 5.9 Knockdown of ID1 or IGFBP3 abolishes BMP2-induced cell invasion in primary human EVTs  A-C, Primary human EVTs were transfected for 48 h with 25 nM siCtrl, siID1 or siIGFBP3 prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for a further 36 h. Knockdown efficiency of ID1 and IGFBP3 were evaluated by Western blot. D and E, Primary human EVT cell invasiveness was examined by Matrigel-coated transwell invasion assay and summarized quantitative results are shown in E. The results are presented as the mean ± SEM of at least three independent experiments. Columns without letters in common are significantly different (P <0.05). 100    Figure 5.10 ID1 and IGFBP3 are involved in BMP2-induced up-regulation of Slug in HTR8/SVneo cell line A, HTR8/SVneo cells were treated with vehicle (Ctrl) or 25 ng/mL BMP2 for different lengths of time (3, 6, 12 or 24 h). Slug protein levels were examined by Western blot. B, HTR8/SVneo cells were transfected for 48 h with 25 nM siCtrl or siID1 or siIGFBP3 prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for additional 24 h. Slug, IGFBP3 and ID1 protein levels were examined by Western blot. 101  Chapter 6: Bone morphogenetic protein 2 promotes human trophoblast cell invasion through BAMBI-mediated activation of WNT/β-catenin signaling  6.1 Introduction Appropriate extravillous cytotrophoblast (EVT) invasion into the uterine wall is a prerequisite for normal pregnancy. Shallow EVT invasion leads to several pregnancy complications, such as preeclampsia and intrauterine growth restriction, both of which are harmful to the health of both mothers and fetuses (273). Thus, research on the control of EVT invasion is of great importance. To date, a variety of signaling pathways have been linked to EVT invasion, including transforming growth factor-β (TGF-β) and WNT/β-catenin signaling pathways (274, 275). As an important TGF-β superfamily member first discovered for its pivotal role in bone formation (276), BMP2 has since been shown to be expressed in many tissues including human decidual cells and primary human EVT cells (89, 242). Moreover, BMP2 is expressed at embryonic implantation sites in mice (107). Conditional knockout studies show that Bmp2 is essential for endometrial decidualization and fertility (17). In vitro research has demonstrated that BMP2 promotes human trophoblast invasion through SMAD-mediated signaling (242, 259). Like BMP2, canonical WNT/β-catenin signaling has also been reported to function in regulating bone formation and human trophoblast invasion (56, 277). Canonical WNT proteins, including WNT1 and WNT3A, bind to seven-transmembrane frizzled receptor and a single-transmembrane co-receptor known as low density lipoprotein receptor-related protein 5/6 (LRP5/6). Receptor binding results in the accumulation of active (non-phosphorylated) β-catenin in the cytoplasm and its translocation into the nucleus to cooperate with T cell factor/ lymphoid enhancer factor (TCF/LEF) in the regulation of gene expression (e.g. cyclin D1) (155, 156). During the 102  differentiation of rat trophoblast cells to giant cells, which are similar to human EVTs, cyclin D1 is up-regulated to allow for transition from normal mitotic cell cycle to endoreduplication cycle (278). Similar changes in cell cycle occur during the acquisition of invasive capacity in human EVTs (279, 280), suggesting potential effects of cyclin D1 on invasive differentiation of human trophoblasts. In vitro studies with human trophoblast cell lines and/or primary EVTs show that trophoblast cell invasion is enhanced by WNT3A-induced canonical WNT signaling, but is impaired by reduced expression of cyclin D1 (56, 159, 160). Therefore, BMP2 and WNT/β-catenin signaling play complementary roles in human trophoblast invasion.  Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is a transmembrane protein that is highly conserved among vertebrates (193). It shares structural similarities with TGF-β type I receptors but lacks an intracellular serine/threonine kinase domain for signal transduction. Therefore, BAMBI often functions as an antagonist of TGF-β signaling by blocking the normal interaction between TGF-β type I and type II receptors (193). In addition, BAMBI facilitates canonical WNT signaling during rodent myoblast cell differentiation (195). Indeed, decreased BAMBI expression blocks the nuclear translocation of β-catenin and inhibits WNT/ β-catenin signaling in gastric cancer cells and preadipocytes (194, 196). Notably, BAMBI is overexpressed and involved in the malignancy of several cancers including osteosarcoma (198), ovarian (197) and colorectal (199). In canine kidney epithelial cells, BAMBI is up-regulated to mediate the loss of epithelial polarity induced by hypoxic-inducible factor 1 (HIF1) (200). Intriguingly, HIF1 is also a positive regulator of HTR8/SVneo cell invasion (222), suggesting a possible link between BAMBI and HIF1-mediated human EVT invasion. BAMBI levels in human EVTs are over 2-fold higher than those in villous cytotrophoblast (47, 162), suggesting that BAMBI may be associated with trophoblast invasive differentiation. BAMBI is a 103  target molecule of BMP2 in human granulosa cells (225), but it is unknown whether BAMBI is involved in BMP2 and/or WNT/β-catenin signaling in human trophoblast cells.  In the present study, we have examined the effect of BAMBI on the activation of canonical WNT/β-catenin signaling and human EVT cell invasion by BMP2. Our results show that BMP2 up-regulates BAMBI and activates WNT/β-catenin signaling as demonstrated by increased active β-catenin accumulation and cyclin D1 expression. Moreover, BAMBI is essential for BMP2-induced activation of WNT/β-catenin signaling as well as human trophoblast invasion.  6.2 Materials and Methods Culture of HTR8/SVneo cells The HTR8/SVneo simian virus 40 large T antigen immortalized first trimester human EVT cell line was kindly provided by Dr. P. K. Lala (Western University, Canada) (46). Cells were cultured as described in Chapter 3.  Primary human EVT cell isolation and culture This study was approved by the Research Ethics Board of the University of British Columbia and all patients provided informed written consent. First-trimester human placental samples (6–8 weeks) were collected from women undergoing elective termination of pregnancy. Primary human EVT cells were isolated and cultured as described in Chapter 3.  Reagents and antibodies Recombinant human BMP2 and DMH1 were obtained from R&D Systems. Mouse monoclonal anti-cytokeratin 7 (catalog no. MAB3554) and anti-HLA-G (catalog no. 11-499-104  C100) were obtained from Millipore and EXBIO Praha, respectively. Rabbit monoclonal anti-non-phosphorylated-(Active)-β-catenin Ser45 (catalog no. 19807), and mouse monoclonal anti-cyclin D1 (catalog no. 2926) were purchased from Cell Signaling Technology. Mouse monoclonal anti-β-catenin (catalog no. 610153) was purchased from BD Biosciences. Mouse monoclonal anti-α-Tubulin (catalog no. sc-23948) was obtained from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories.  Matrigel-coated transwell invasion assay Trophoblast cell invasiveness was examined using Corning Biocoat Growth Factor Reduced Matrigel Invasion Chambers (pore size, 8 μm; catalog no. 354483) as described in Chapter 3.  Reverse transcription quantitative real-time PCR (RT-qPCR) Total RNA was extracted with TRIzol Reagent (Life Technologies) as per the manufacturer’s instructions. RT-qPCR was carried out as described in Chapter 3. The primers used were:  BAMBI, 5'-GGCCTCAGGACAAGGAAACAG-3' (forward) and 5'- CGGAACCACAACTCTTTGGAAG-3' (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GAGTCAACGGATTTGGTCGT-3' (forward) and 5'- GACAAGCTTCCCGTTCTCAG-3' (reverse).   Western blot analysis 105  Cells were lysed in ice-cold lysis buffer (Cell Signaling Technology) with added protease inhibitor cocktail (Sigma-Aldrich). Western blot analysis and densitometric quantification was performed as described in Chapter 3.  Small interfering RNA (siRNA) transfection Cells at approximately fifty percent confluency were transfected for 48 hours with 25 nM ON-TARGETplus NON-TARGETINGpool siRNA or ON-TARGETplus SMARTpool siRNA targeting human BAMBI (L-019596-00-0005; Dharmacon) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. Knockdown efficiency was assessed by RT-qPCR.  Statistical analysis Results are presented as the mean ± SEM of at least three independent experiments. Multiple group comparisons were analyzed by one-way ANOVA followed by Newman-Keuls test using PRISM software (GraphPad Software, Inc). Means were considered significantly different if P < 0.05 and columns without letters in common are significantly different.  6.3 Results BMP2 increases BAMBI mRNA levels in human trophoblast cells     BMP2 has recently been reported to up-regulate BAMBI in human granulosa cells (225). To examine the effects of BMP2 on BAMBI mRNA levels in human trophoblast cells, HTR8/SVneo and primary EVT cells were treated with 25 ng/mL BMP2 for different lengths of time (3, 6, 12 and 24 hours). RT-qPCR results showed BMP2 increased BAMBI mRNA levels in 106  both cell models from 3 to 24 hours (Figure 6.1, A and B). According to previous experiments, two TGF-β type I receptors, activin receptor-like kinases (ALK) 2 and/or ALK3, were generally used by BMP2 to exert its effects on human trophoblast cells (242, 259). In this study, HTR8/SVneo and primary EVT cells were pre-treated with the ALK2/ALK3 inhibitor DMH1 for 1 hour prior to treatment with BMP2 for a further 12 hours. As shown in Figure 6.1 C and D, pre-treatment with DMH1 totally blocked BMP2-induced BAMBI up-regulation in both HTR8/SVneo and primary human EVT cells, demonstrating that BMP2 increases BAMBI mRNA levels via ALK2 and/or ALK3.  BMP2 increases accumulation of active β-catenin and its downstream target cyclin D1 in human trophoblast cells     BMP2 has been shown to activate and cooperate with canonical WNT/β-catenin signaling for bone formation and pulmonary angiogenesis (103, 224). Accordingly, we investigated the effects of BMP2 on WNT/β-catenin signaling in both HTR8/SVneo and primary human EVT cells. Western blot analysis showed that BMP2 treatment increased the levels of active (non-phosphorylated) β-catenin from 12 to 24 hours in HTR8/SVneo cells and from 6 to 24 hours in primary EVT cells (Figure 6.2). In addition, BMP2 treatment also increased cyclin D1 protein levels at 24 and 48 hours in both cell models (Figure 6.3). These results suggest that BMP2 enhances canonical WNT/β-catenin signaling in human trophoblast cells.  BAMBI is essential for the enhancement of WNT/β-catenin signaling and trophoblast invasion by BMP2 107      BAMBI is a positive regulator of WNT/β-catenin signaling (195). Thus, we used siRNA targeting BAMBI to examine its contribution to BMP2-induced WNT/β-catenin signaling. Transient transfection of HTR8/SVneo cells with siRNA targeting BAMBI for 48 hours significantly suppressed BAMBI mRNA levels (Figure 6.4, A). Western blot results showed that treatment of HTR8/SVneo cells with BMP2 significantly increased the levels of active β-catenin and cyclin D1, and these effects were totally abolished by BAMBI knockdown (Figure 6.4, B-D). Similar experiments carried out in primary EVT cells confirmed that BAMBI was essential for BMP2-induced activation of WNT/β-catenin signaling (Figure 6.5).      Next, we examined the effects of BAMBI knockdown on BMP2-induced trophoblast invasion in both cell models. Matrigel-coated transwell invasion assays showed that BMP2 treatment for 36 hours significantly increased HTR8/SVneo cell invasion as previously reported. Importantly, this effect was abolished by BAMBI knockdown (Figure 6.6, A and B). Likewise, the involvement of BAMBI in BMP2-induced trophoblast invasion was further confirmed in primary human first trimester EVT cells (Figure 6.6, C and D).   6.4 Discussion During placentation, a diversity of growth factors and cytokines secreted by trophoblast cells or decidual tissues cooperate with each other to tightly control human trophoblast invasion (274, 275). Here, we show that BMP2 induces the up-regulation of BAMBI and the activation of canonical WNT/β-catenin signaling as demonstrated by accumulation of active β-catenin and increased cyclin D1 expression.  Moreover, we demonstrate that BAMBI is essential for BMP2-induced canonical WNT/ β-catenin signaling and trophoblast invasion.  108  Consistent with higher mRNA levels of BAMBI in human EVTs compared with villous cytotrophoblasts (47, 162), our research shows that BAMBI facilitates BMP2-activated WNT/β-catenin signaling to promote human trophoblast invasion. As a pseudo-receptor of TGF-β superfamily members, BAMBI has been shown to negatively regulate TGF-β, activin and BMP signaling by interfering with the formation of functional ligand-receptor complexes (193). However, despite belonging to the same superfamily, BMP2 and TGF-β can exert opposing functions under certain conditions. For example, BMP2 inhibits TGF-β1-induced differentiation of murine epicardial cells to smooth muscle cells (281). Opposing effects of BMP2 and TGF-β1 also occurs in human trophoblasts; whereby BMP2 promotes and TGF-β1 inhibits human EVT cell invasion (90, 202, 242, 259). Even for the regulation of BAMBI expression in primary EVTs, BMP2 up-regulates whereas TGF-β1 down-regulates BAMBI mRNA levels (Figure 6.1 B and Figure 6.7). Our recent work has shown that BMP2 up-regulates BAMBI to inhibit TGF-β1 signaling and function in human granulosa cells (225). Such inhibitory effects of BAMBI on TGF-β1 signaling may facilitate BMP2-induced human trophoblast invasion because human EVTs are known to secrete TGF-β1 (88, 282). Possible mechanisms whereby BAMBI could inhibit TGF-β1 signaling include complexing with SMAD7, SMAD3 and TGF-β1 type I receptor (ALK5) to sequester the phosphorylation of SMAD3 and thereby disrupt TGF-β1 signaling (283). In addition, BAMBI co-translocates with SMAD2/3 to the nucleus and alter TGF-β1-responsive gene expression in ovarian cancer cells (197). On the other hand, knockdown of BAMBI in human preadipocytes attenuates SMAD1/5/8 phosphorylation and adipogenic effects induced by BMP4, suggesting positive roles for BAMBI in BMP signaling in this cell type (194). Indeed, our results demonstrate that BAMBI knockdown abolishes BMP2-induced activation of WNT/β-catenin signaling and EVT cell invasion. 109  Previous studies have shown that BMP2 can enhance canonical WNT/ β-catenin signaling by increasing total β-catenin levels (103, 224). In human trophoblast cells, BMP2 had no effects on total β-catenin but increased the accumulation of active (non-phosphorylated) β-catenin as well as the expression of its downstream target cyclin D1. Similarly, treatment of human granulosa cells with lithium chloride (LiCl), a well-known pharmacological activator of WNT/β-catenin signaling, also leads to increased levels of active β-catenin without altering total β-catenin levels (284). Thus, the activation of the WNT/β-catenin signaling depends more on active β-catenin accumulation compared with total β-catenin expression. In our study, BMP2-induced increases in BAMBI mRNA levels (3 hours) occurred prior to the increases in active β-catenin (12 hours in HTR8/SVneo and 6 hours in primary EVT cells), suggesting BAMBI might contribute to BMP2-induced WNT/β-catenin signaling. This was confirmed by BAMBI knockdown, which attenuated BMP2-induced accumulation of active β-catenin and cyclin D1, although the precise mechanisms involved are not clear. In WNT1 ligand-induced β-catenin signaling, BAMBI has been shown to strengthen the interaction between frizzled receptor and disheveled protein to inhibit β-catenin phosphorylation and subsequent degradation (285). Intriguingly, BMP2 up-regulates several WNT ligands and their receptors (18, 286). Moreover, the WNT pathway inhibitor Dickkopf-related protein 1 (DKK1), which blocks WNT ligands from binding to their co-receptors (287, 288), inhibits both BMP2-induced WNT/β-catenin signaling and osteoblast differentiation (224). Thus, it is possible that BMP2 induces WNT/β-catenin signaling via canonical WNT ligand-dependent mechanisms, during which BAMBI exerts its effects as previously reported (285). However, Vinicio et al. demonstrated that BMP2 directly induces β-catenin accumulation through BMP type II receptor-mediated GSK3β inhibition in pulmonary artery endothelial cells, in a WNT ligand-independent manner (103). Thus, future studies 110  examining exactly how BAMBI contributes to BMP2-induced WNT/β-catenin signaling will be of great interest.      Like BMP2, WNT/β-catenin signaling enhances human trophoblast invasion (56). In placentas from women with severe preeclampsia, levels of WNT1, β-catenin and cyclin D1 are decreased whereas those DKK1 are increased compared to normal placentas (161), suggesting deficiencies in WNT/β-catenin signaling may contribute to preeclampsia. In contrast, abundant nuclear recruitment of β-catenin, which is a marker of hyper-activated WNT signaling, has been demonstrated in invasive EVTs from complete hydatidiform moles (56, 162). Integration of BMP2 and WNT/β-catenin signaling has already been reported in osteoblast differentiation as well as osteogenesis, because β-catenin conditional knockout mice exhibit impaired BMP2-induced bone formation (224). In agreement, our study shows that BMP2-induced human EVT cell invasion is abolished when active β-catenin and cyclin D1 levels return to the basal following BAMBI knockdown. Notably, canonical WNT/β-catenin signaling usually leads to increased cell proliferation, especially since cyclin D1 plays key roles in the transition from G1 to S phase of the cell cycle (160).  However, despite increasing canonical WNT/β-catenin signaling and cyclin D1, we have previously shown that BMP2 has no significant effects on HTR8/SVneo cell viability as assessed by MTT assay (242). Similarly, others have shown that canonical WNT signaling has no effect on cell proliferation but promotes migration of human immortalized SGHPL-5 and primary EVT cells (158). Importantly, cyclin D1 is involved in cell cycle transition to endoreduplication and the acquisition of polyploidy during rat trophoblast differentiation to giant cells (278), both of which occur during human trophoblast invasive differentiation (279). Thus, in this context cyclin D1 may exert effects on trophoblast differentiation rather than proliferation.  111  In summary, our study demonstrates for the first time that BMP2 induces the up-regulation of BAMBI and the activation of canonical WNT/β-catenin signaling, as demonstrated by increased active β-catenin accumulation and cyclin D1 expression in human trophoblast cells. Furthermore, BAMBI up-regulation is responsible for BMP2-induced canonical WNT/β-catenin signaling activation and human EVT cell invasion (Figure 6.8). These findings strengthen our knowledge on placentation by highlight a new molecular mechanism underlying the integration of BMP2 and WNT/β-catenin signaling in the regulation of human trophoblast invasion.  112    Figure 6.1 BMP2 increases BAMBI mRNA levels through ALK2 and/or ALK3 type I receptor in both HTR8/SVneo and primary human EVT cells  A-B, HTR8/SVneo cells (A) or primary EVT cells (B) were treated with vehicle (Ctrl) or 25 ng/mL BMP2 for different lengths of time (3, 6, 12 or 24 h), and BAMBI mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. C-D, HTR8/SVneo cells (C) or primary EVT cells (D) were pre-treated with vehicle control (dimethylsulfoxide, DMSO) or 1 μM DMH1 (ALK2/3 inhibitor, 1 μM) for 1 hour prior to treatment for 12 hours with or without 25 ng/mL BMP2, and BAMBI mRNA levels were measured by RT-qPCR. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05). 113       Figure 6.2 BMP2 increases active β-catenin protein levels in HTR8/SVneo and primary human EVT cells  HTR8/SVneo cells (left panel) and primary EVT cells (right panel) were treated with vehicle (Ctrl) or 25 ng/mL BMP2 for different lengths of time (3, 6, 12 or 24 h), and active (non-phosphorylated) β-catenin and total β-catenin protein levels were examined by Western blot and normalized to α-tubulin. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).  114       Figure 6.3 BMP2 increases cyclin D1 protein levels in HTR8/SVneo and primary human EVT cells HTR8/SVneo cells (left panel) or primary EVT cells (right panel) were treated with or without 25 ng/mL BMP2 every 24 hours for 48 hours, and cyclin D1 protein levels were analyzed by Western blot and normalized to α-tubulin. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).     115    Figure 6.4 BAMBI mediates the up-regulation of active β-catenin and cyclin D1 by BMP2 in HTR8/SVneo cells HTR8/SVneo cells were transfected for 48 h with 25 nM non-targeting control siRNA (siCtrl) or 25 nM siRNA targeting BAMBI (siBAMBI) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for 12 hours (mRNA levels) or 24 hours (protein levels). A, RT-PCR was used to evaluate the knockdown efficiency of BAMBI mRNA levels. B-D, Representative images of Western blots for active β-catenin, total β-catenin, cyclin D1 and α-tubulin. Combined quantitative results for active β-catenin (C, normalized to total β-catenin) and cyclin D1 (D, normalized to α-tubulin) are shown. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).  116    Figure 6.5 BAMBI mediates the up-regulation of active β-catenin and cyclin D1 by BMP2 in primary human EVT cells  Primary EVT cells were transfected for 48 h with 25 nM non-targeting control siRNA (siCtrl) or 25 nM siRNA targeting BAMBI (siBAMBI) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for 12 hours (mRNA levels) or 24 hours (protein levels). A, RT-PCR was used to evaluate the knockdown efficiency of BAMBI mRNA levels. B-D, Representative images of Western blots for active β-catenin, total β-catenin, cyclin D1 and α-tubulin. Combined quantitative results for active β-catenin (C, normalized to total β-catenin) and cyclin D1 (D, normalized to α-tubulin) are shown. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05).   117    Figure 6.6 BAMBI knockdown attenuates BMP2-induced human trophoblast cell invasion HTR8/SVneo or primary human EVT cells were transfected for 48 hours with 25 nM non-targeting control siRNA (siCtrl) or 25 nM siRNA targeting BAMBI (siBAMBI) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for a further 36 h. Matrigel-coated transwell invasion assays were used to examine the effects of BAMBI knockdown on BMP2-induced invasion in HTR8/SVneo (A and B; scale bar 100 μm) and primary EVT cells (C and D). Summarized quantitative results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05). 118          Figure 6.7 TGF-β1 reduces BAMBI mRNA levels in primary human EVT cells  Primary EVT cells were treated with vehicle (Ctrl) or 5 ng/mL TGF-β1 for different lengths of time (3, 6, 12 or 24 h), and BAMBI mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. Results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05). 119     Figure 6.8 Proposed model of the signaling pathway mediating BMP2-induced BAMBI up-regulation, canonical WNT/β-catenin signaling activation and increased human trophoblast cell invasion  BMP2 binding to type I receptors ALK2 and/or ALK3 leads to up-regulation of BAMBI, which in turn activates WNT/β-catenin signaling, as indicated by the increase in the accumulation of active (non-phosphorylated) β-catenin and the up-regulation of the downstream target gene cyclin D1, to promote human EVT cell invasion. 120  Chapter 7: Conclusion  7.1 Conclusion     The objective of this thesis was to study the role of BMP2 expressed at the fetal-maternal interface in regulating human trophoblast invasion and the underlying molecular mechanisms. Our results demonstrate that BMP2 exerts positive effects on trophoblast invasion in addition to its well-known role in promoting endometrial decidualization. Our findings broaden current knowledge on the roles of BMP2 in placentation and provide insights into the molecular mechanisms underlying human trophoblast invasion.     As shown in Chapter 3, the regulatory role of BMP2 on human EVT invasion and the involvement of SMAD signaling as well as N-cadherin were studied. Treatment with recombinant human BMP2 increased HTR8/SVneo cell invasion as well as N-cadherin mRNA and protein levels, but had no significant effect on cell proliferation. Likewise, BMP2 treatment enhanced primary human EVT cell invasion and N-cadherin production. Basal and BMP2-induced invasion were attenuated by siRNA-mediated down-regulation of N-cadherin in both HTR8/SVneo and primary EVT cells. Intriguingly, BMP2 induced the phosphorylation/activation of both canonical SMAD1/5/8 and non-canonical SMAD2/3 signaling in HTR8/SVneo and primary EVTs. Knockdown of SMAD2/3 or common SMAD4 totally abolished the effects of BMP2 on N-cadherin up-regulation in HTR/SVneo cells. Up-regulation of SMAD2/3 phosphorylation and N-cadherin were totally abolished by ALK2/3 inhibitor DMH1, and knockdown of ALK2 or ALK3 inhibited N-cadherin up-regulation. Interestingly, activation of SMAD2/3 and up-regulation of N-cadherin were partially attenuated by ALK4/5/7 inhibitor SB431542 or knockdown of ALK4, but not ALK5. In summary, BMP2 121  promotes trophoblast cell invasion by up-regulating N-cadherin via non-canonical ALK2/3/4-SMAD2/3-SMAD4 signaling.     As presented in Chapter 4, the effect of BMP2 on activin A production and its role in human trophoblast invasion were investigated. BMP2 treatment significantly increased inhibin βA mRNA levels and activin A production without altering inhibin α and βB levels. BMP2-induced EVT invasion was attenuated by knockdown of inhibin βA. The increased inhibin βA transcription and activin A production by BMP2 were blocked by type I receptor activin receptor-like kinases 2/3 (ALK2/3) inhibitor DMH1. BMP2-induced inhibin βA up-regulation was also inhibited by knockdown of type I receptor ALK3 or combined knockdown of type II receptors BMPR2 and ACTR2A. Whereas BMP2 initiated both canonical SMAD1/5/8 and noncanonical SMAD2/3 signaling, only knockdown of SMAD4, but not SMAD2 and SMAD3, abolished the effects of BMP2 on inhibin βA. Our results show that BMP2 increases human trophoblast invasion by up-regulating inhibin βA and activin A production via ALK3-BMPR2/ACVR2A-SMAD1/5/8-SMAD4 signaling.     In Chapter 5, the roles of BMP2 on ID1 and IGFBP3 expression and their roles on human trophoblast invasion were examined. BMP2 treatment increased ID1 and IGFBP3 mRNA and protein levels in HTR8/SVneo cell lines and primary human EVTs. Intriguingly, ID1 is essential for BMP2-induced IGFBP3 up-regulation in both study models. Furthermore, BMP2-induced trophoblast invasion was attenuated by down-regulation of ID1 or IGFBP3 with corresponding small interfering RNA in both HTR8/SVneo and primary EVT cells. Our results reveal that BMP2 promotes trophoblast cell invasion by ID1-mediated IGFBP3 up-regulation.     In Chapter 6, the effects of BAMBI on BMP2-induced activation of WNT/β-catenin signaling and human trophoblast invasion were determined. BMP2 treatment increased BAMBI mRNA 122  levels in HTR8/SVneo and primary human EVT cells, and these effects were totally blocked by the activin receptor-like kinases (ALK) 2/3 inhibitor DMH1. In addition, BMP2 increased the levels of active β-catenin as well as its downstream target gene cyclin D1 in both cell models, suggesting the activation of canonical WNT/β-catenin signaling. Importantly, BMP2-induced active β-catenin and cyclin D1 expression as well as human trophoblast invasion were totally blocked by siRNA-mediated knockdown of BAMBI in HTR8/SVneo and primary human EVT cells. Collectively, BMP2 promotes trophoblast cell invasion via BAMBI-mediated activation of WNT/β-catenin signaling. In summary, our experimental results reveal the positive regulatory role of BMP2 on human trophoblast invasion and the underlying mechanisms. BMP2 up-regulated N-cadherin via non-canonical SMAD2/3 signaling while increased activin A production through canonical SMAD1/5/8 signaling, showing the cooperation of SMAD2/3 and SMAD1/5/8 signaling in human trophoblast invasion. Moreover, we showed for the first time ID1-mediated IGFBP3 up-regulation by BMP2 and BAMBI-mediated the crosstalk between BMP2 signaling and canonical WNT signaling in human trophoblast invasion. Together, these mechanisms emphasize the essential roles of BMP2 in regulating human trophoblast invasion (Figure 7.1), which could lead to advances in the development of clinical, diagnostic and therapeutic approaches for pregnancy disorders.  7.2 General discussion of this study      In HTR8/SVneo and primary human EVT cells, BMP2 treatment induced the activation of both canonical SMAD1/5/8 and noncanonical SMAD2/3 signaling. In a study of 46 normal and cancer cell lines, BMP2 was shown to induce SMAD2/3 signaling preferentially in embryonic 123  and transformed cells (99). Our findings of BMP2-induced SMAD2/3 phosphorylation in human EVT cells are in agreement since trophoblast cells are derived from the blastocyst trophectoderm. Interestingly, Holtzhausen et al. also noted greater co-expression of genes responsive to SMAD1/5/8 and SMAD2/3 in breast and liver cancer samples compared to normal tissues, suggesting cooperation between these two SMAD signaling pathways in cancer (99). Consistently, both SMAD2/3-mediated N-cadherin up-regulation (Chapter 3) and SMAD1/5/8-mediated activin A accumulation (Chapter 4) are involved in pro-invasive effects of BMP2 on human EVT cells, which share a lot of similarities with cancer cell invasion. Intriguingly, Chapter 5 shows that BMP2-induced IGFBP3 up-regulation is partially but not totally mediated through ID1 in primary human EVTs (Figure 5.7, 5.9A and 5.10). In addition, inhibin βA knockdown attenuates the up-regulation of IGFBP3 whereas it has no effects on ID1 up-regulation induced by BMP2 (Figure 7.2B). Given that ID1 up-regulation is mediated by SMAD1/5/8 (Figure 7.3) and activin A signals via SMAD2/3, these findings suggest that IGFBP3 is likely to be up-regulated through both SMAD2/3 and SMAD1/5/8 signaling in primary human EVT cells. Thus, our studies have revealed that collaboration between canonical SMAD1/5/8 and non-canonical SMAD2/3 signaling is important for human trophoblast invasion.     BMP2 and activin A are both expressed at the maternal-fetal interface and exert pro-invasive effects on human trophoblast cells as well as promoting endometrium decidualization (17, 18, 210-213, 242). The stimulatory effects of BMP2 on activin A production identified in our studies may at least partially account for some of these similarities. Though BMP2 is indispensable for human endometrial decidualization and trophoblast invasion in vitro, to date there is no clinical data showing a correlation between BMP2 levels in maternal serum or amniotic fluid and the occurrence of abnormal placentation-associated pregnancy complications. In Chapter 4, BMP2 is 124  shown to promote human EVT invasion by enhancing the production of activin A in an ACVR2A/BMPR2-dependent manner (259). However, women with preeclampsia were shown to have increased activin A protein levels in placenta and maternal serum (115) as well as increased placental ACVR2A levels (functional receptors for both activin A and BMP2) (289). This would seem to contradict the positive effects of activin A and BMP2 on trophoblast invasion as demonstrated by in vitro studies. However, the functions of activin A on human trophoblasts differ at different stages of gestation. For example, activin A promotes cytotrophoblast invasion at 6-10 weeks, but not at 10-12 weeks (213), suggesting the pro-invasive effects of activin A on human trophoblast in early gestational age may contribute to maternal spiral artery remodeling initiating at 10 weeks of human pregnancy (45). Our studies show that BMP2 increases the production of activin A by human EVT cells isolated from 6-8 week placental villi (259), which is in agreement with the pro-invasive effects of activin A at this gestational age. In addition, plasma activin A levels in preeclampsia patients are not significant increased until 25-30 weeks when preeclampsia-associated manifestations may already be obvious (115). The up-regulation of activin A and ACVR2A levels manifested in preeclampsia patients may be due to the pro-apoptotic effects activin exerts in later gestational stages or may constitute a compensatory response.     Besides its critical roles in BMP2-induced human EVT invasion early in gestation, activin A is crucial for the up-regulation of N-cadherin protein levels at 24 hours following BMP2 treatment (Figure 7.2). However, BMP2 calso increases N-cadherin in an activin A-independent manner because knockdown of inhibin βA only partially blocks increases in N-cadherin mRNA levels at 12 hours (Figure 7.2). In addition to N-cadherin, activin A also contributes to BMP2-induced 125  IGFBP3 up-regulation (Figure 7.2, B), suggesting IGFBP3 may also be involved in activin A-induced human trophoblast invasion.     In prostate cancer, increased expression of ID1 promotes cancer cell invasion and is associated with poor prognosis of patients (290-293). At the same time, IGFBP3 is considered a suppressive factor for cancer progression since its expression is positively correlated with patient survival (294, 295). Correspondingly, ectopic overexpression of ID1 down-regulates IGFBP3 while immortalizing rat normal prostate epithelial cells (220). In human trophoblast cells, ID1 and IGFBP3 are both up-regulated during villous cytotrophoblast differentiation to column EVTs (117, 192, 221). Under different conditions, increased expression of ID1 or IGFBP3 are respectively reported to be associated with enhanced HTR8/SVneo cell invasion (215, 222). In addition, IGFBP3 expression is down-regulated in preeclampsia compared with normal controls (140). Our study confirms the positive effects of ID1 and IGFBP3 on human trophoblast invasion, further showing that ID1 mediates BMP2-induced IGFBP3 up-regulation (Chapter 5). Therefore, correlations between ID1 and IGFBP3 expression are cell type- and/or context-specific and the underlying mechanism would be of great interest for future investigations.     ID1 and cyclin D1 are both up-regulated by BMP2 in human EVT cells (Chapter 5 and 6). However, Nicholas et al. showed that cyclin D1 occupied the ID1 promotor region and repressed ID1 expression in MDA-MB-231 breast cancer cells. Furthermore, cyclin D1low/ID1high breast cancers showed higher expression of EMT markers, correlating with decreased recurrence free survival (160). In contrast, we found in HTR8/SVneo cells that basal ID1 levels were reduced following knockdown of cyclin D1 (Data not shown). Furthermore, ID1 mediates BMP2-induced cyclin D1 up-regulation in both HTR8/SVneo and primary EVT cells (Figure 7.4). These findings suggest that interactions between ID1 and cyclin D1 are context-dependent. As a 126  classical downstream gene of WNT/β-catenin signaling, cyclin D1 expression is reduced in placentas from women with preeclampsia (161) and has been shown to promote HTR8/SVneo cell migration (160). Therefore, in human trophoblast cells cyclin D1 may be involved in ID1-mediated human trophoblast differentiation into motile mesenchymal-like EVT cells. In addition, cyclin D1 is involved in cell cycle transition to endoreduplication and the acquisition of polyploidy during rat trophoblast differentiation to giant cells (278), both of which occur during human trophoblast invasive differentiation (279). Thus, in this context cyclin D1 may exert effects on invasive trophoblast differentiation.     Altogether, our studies show that BMP2 promotes human trophoblast cell invasion by up-regulating N-cadherin, activin A, ID1, IGFBP3 and BAMBI, all of which are reported to be either EMT markers or inductive factors of EMT during trophoblast invasive differentiation or cancer progression. In addition, we show cooperation between BMP2 and activin A, SMAD2/3 and SMAD1/5/8 signaling, ID1 and IGFBP3, and BMP2 and WNT/β-catenin signaling, all of which are integrated in BMP2-induced human EVT cell invasion. Our research improves our understanding of molecule interactions underlying human trophoblast differentiation and invasion.  7.3 Limitations of this study      We performed this study with both an immortalized human EVT cell line (HTR8/SVneo) and primary EVTs from first trimester placentas. The HTR8/SVneo cell line was derived from primary EVTs transfected with simian virus 40 large T antigen. Due to the longer lifespan compared with primary EVTs, HTR8/SVneo cells have been widely used to explore trophoblast-associated features including invasion and endothelial-like phenotypes. However, the 127  immortalization process may result in some different phenotypes compared to primary EVTs. For example, HTR8/SVneo cells have been reported to express more vimentin and less cytokeratin 7 than primary human EVTs. Moreover, the expression of HLA genes in HTR8/SVneo cells is also different from that in primary EVTs. To mitigate against some of the limitations of HTR8/SVneo cells, primary EVTs isolated from first trimester placentas were also used to confirm the results obtained using HTR8/SVneo cells.      We are aware that our in vitro system allows us to study the function of BMP2 on trophoblast invasion, but that it does not address the mutual effects among different types of cells or complex interactions among other biological factors characteristic of in vivo conditions. Indeed, trophoblast cells in the human placenta have extensive interactions with other kinds of cells including maternal stromal cells, natural killer cells and macrophages. Trophoblast phenotypes reflect the total effects of numerous factors acting on these cells. Of note, the medium in which trophoblast cells are cultured in vitro cannot totally mimic the real microenvironment in vivo. Therefore, in vivo studies or at least co-culture systems are required to confirm the effects of BMP2 on trophoblast invasion.   7.4 Significance and translational potential Our studies show for the first time the positive role of BMP2 in regulating human trophoblast invasion and the underlying molecular mechanisms. BMP2 increases activin A production which further contributes to the upregulation of N-cadherin and IGFBP3 induced by BMP2, suggesting the collaboration between BMP2 and activin A in human EVT invasion. Our research also demonstrates the cooperation between canonical SMAD1/5/8 (for the up-regulation of inhibin βA, ID1) and non-canonical SMAD2/3 (for N-cadherin up-regulation) signaling in BMP2-128  induced human EVT cells invasion. To the best of our knowledge, our study is the first to show both ID1-mediated IGFBP3 up-regulation and BAMBI-mediated BMP2-WNT/β-catenin signaling. In particular, our study challenges the current belief about the antagonistic effects of BAMBI on BMP2 signaling. Our findings implicate BMP2 in the regulation of human trophoblast cell invasion and confer new insights into the molecular mechanisms underlying normal placentation, which could inform advances in clinical diagnostic and therapeutic approaches for pregnancy complications.   7.5 Future directions     Previous studies by other research groups showed the crucial function of BMP2 on promoting endometrial decidualization and mice fertility. Currently we show that BMP2 also promotes human trophoblast invasion through regulating several key signaling pathways. Therefore, BMP2 expressed at the fetal-maternal interface is of great importance for placental development. However, there are still some unknown aspects that require further investigation. 1. Though BMP2 is currently shown to positively regulate trophoblast invasion in vitro, whether BMP2 functions on trophoblast invasion in the similar way in vivo needs to be examined. 2. BMP2 activates canonical WNT/β-catenin signaling in human EVTs through up-regulating BAMBI. However, whether canonical WNT ligands or frizzle receptors or their inhibitors are involved in BMP2-induced canonical WNT signaling is unknown. Furthermore, non-canonical signaling has been implicated in the antagonism of canonical signaling in cancers as well as in trophoblast invasion. Whether BMP2 acts via non-canonical signaling should be investigated. 129  3. BMP2, ID1 and IGFBP3 have each been reported to promote endothelial tube-like formation in endothelial precursor or endothelial cells. Whether BMP2-ID1-IGFBP3 signaling is involved in the differentiation of human trophoblasts to endovascular EVTs remains to be elucidated. 4. Though ID1 mediates BMP2-induced IGFBP3 in human trophoblast cells, the detailed mechanisms involved are unknown. It has been reported that ID1 mediates IGF2 up-regulation by stabilizing E2F1, a transcription factor of IGF2, in esophageal cancer cells.  Whether E2F1 is also involved in ID1-mediated IGFBP3 up-regulation in response to BMP2 treatment in human trophoblasts would be an interesting area for future study. 5. BMP2 was reported to participate in progesterone-induced signaling during endometrial decidualization, but whether BMP2 plays essential roles in other progesterone-mediated functions during pregnancy could be further explored.         130     Figure 7.1 Schematic presentation of the proposed mechanisms underlying BMP2-promoted human trophoblast invasion  BMP2 binds to type I and II receptors leading to the activation of both canonical SMAD1/5/8 and noncanonical SMAD2/3 signaling. Activation of receptor complexes containing ALK2, ALK3 and ALK4 lead to the phosphorylation of SMAD2/3 which subsequently combines with common SMAD4 and translocates into the nucleus to increase the transcription of N-cadherin for human trophoblast cell invasion. BMP2 activates canonical SMAD1/5/8 signaling to increase 131  activin A production which also contributes to human EVT invasion. Increased production of activin A by BMP2 also contributes to the up-regulation of N-cadherin and IGFBP3. In addition, BMP2 induces ID1-mediated IGFBP3 up-regulation which is associated with human trophoblast invasion and up-regulation of the EMT-associated transcription factor Slug. BAMBI-mediated activation of canonical WNT/β-catenin signaling, as displayed by the up-regulation of active β-catenin and cyclin D1, is involved in BMP2-induced human trophoblast invasion during placentation. ID1 also functions to up-regulate cyclin D1 which has already been shown to be essential for the migration of HTR8/SVneo cells.   132   Figure 7.2 Activin A contributes to BMP2-induced up-regulation of N-cadherin and IGFBP3 in primary human EVT cells  Primary EVT cells were transfected for 48 hours with 25 nM non-targeting control siRNA (siCtrl) or 25 nM siRNA targeting Inhibin βA (siInhibin βA) prior to treatment with vehicle (Ctrl) or 25 133  ng/mL BMP2. A, RT-qPCR was used to measure Inhibin βA and N-cadherin mRNA levels 12 hours after treatment with BMP2. B, Western blot was used to measure N-cadherin, IGFBP3, and α-Tubulin protein levels 24 hours after BMP2 treatment. Summarized quantitative results are displayed as the mean ± SEM of at least three independent experiments and columns without letters in common are significantly different (P < 0.05). 134     Figure 7.3 Non-canonical SMAD2/3 accounts for N-cadherin up-regulation while canonical SMAD1/5/8 signaling mediates the up-regulation of furin and ID1 induced by BMP2  HTR8/SVneo cells were transfected for 48 h with 25 nM non-targeting control siRNA (siCtrl), 25 nM siRNA targeting SMAD2+SMAD3 (siSMAD2+3) or 25 nM siRNA targeting SMAD4 (siSMAD4) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for 24 hours. Protein levels of N-cadherin, Furin, ID1, T-SMAD2, T-SMAD3 and T-SMAD4 were examined by Western blot.  135    Figure 7.4 ID1 mediates BMP2-induced cyclin D1 up-regulation in HTR8/SVneo and primary human EVT cells HTR8/SVneo (left panel) and primary EVT (right panel) cells were transfected for 48 h with 25 nM non-targeting control siRNA (siCtrl) or 25 nM siRNA targeting ID1 (siID1) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for 24 hours. Protein levels of cyclin D1, ID1 and α-tubulin were examined by Western blot.  136  Bibliography  1. Gude NM, Roberts CT, Kalionis B, King RG 2004 Growth and function of the normal human placenta. Thromb Res 114:397-407 2. Lyall F, Bulmer JN, Duffie E, Cousins F, Theriault A, Robson SC 2001 Human trophoblast invasion and spiral artery transformation: the role of PECAM-1 in normal pregnancy, preeclampsia, and fetal growth restriction. Am J Pathol 158:1713-1721 3. 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