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Phosphatidylinositol-3 Kinase/Akt and Fos Like Protein 1

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Phosphatidylinositol-3 Kinase/Akt and Fos Like Protein 1 PHOSPHATIDYLINOSITOL-3 KINASE/AKT AND FOS LIKE PROTEIN 1 REGULATION OF TROPHOBLAST DIFFERENTIATION BY Lindsey N. Kent Submitted to the graduate degree program in Pathology and Laboratory Medicine and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Graduate committee: ______________________________ Chair: Michael J. Soares, Ph.D. ______________________________ Dr. Linheng Li, Ph.D. ______________________________ Dr. Margaret G. Petroff, Ph.D. ______________________________ Dr. Jay L. Vivian, Ph.D. ______________________________ Dr. Michael W. Wolfe, Ph.D. Date defended: ________________ PHOSPHATIDYLINOSITOL-3 KINASE/AKT AND FOS LIKE PROTEIN 1 REGULATION OF TROPHOBLAST DIFFERENTIATION BY Lindsey N. Kent Submitted to the graduate degree program in Pathology and Laboratory Medicine and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ______________________________ Chair: Michael J. Soares, Ph.D. Date approved: ________________ 2 ACKNOWLEDGEMENTS I would like to thank my family for all of their support, encouragement and enthusiasm. I would also like to acknowledge all members (past and present) of the Soares Laboratory, all of which taught me important lesions and that have made me a better scientist. I would like to thank Dr. Toshiro Konno for his instruction and advice with all of the histology and statistical analysis; Dr. Dong- Soo Lee for his help with the embryo transfer experiments and Dr. Mohammad Rumi for his help with the lentiviral and shRNA experiments as well as his advice that has greatly improved my molecular biology knowledge and technique. I would also like to thank Mrs. Stacy McClure for her advice and support. I would also like to thank my committee members for their help and advice in guiding my research project over the years. Most especially I would like to thank my mentor Dr. Michael Soares. I would never have considered graduate school without his support and a large part of my success is due directly to his guidance and encouragement. 3 ABSTRACT Hemochorial placentation is an effective strategy facilitating nutrient delivery and development of the fetus within the female reproductive tract. Remodeling of the uterine vasculature by trophoblast cells is one of the key processes necessary for a successful pregnancy. Several pathways have been implicated in the regulation of trophoblast invasion including the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. In this research, we identified genes characteristic of trophoblast stem cell and differentiated states, including those involved in trophoblast invasion and vasculature remodeling. We also evaluated the role of the PI3K/AKT signaling pathway in the regulation of trophoblast cell differentiation. This investigation utilized the Rcho-1 trophoblast stem cells as a model system for trophoblast differentiation. These cells can be maintained in a trophoblast stem cell state or induced to differentiate into trophoblast giant cells, which have the capability to invade. mRNA expression profiling using microarrays provided insights about trophoblast stem cell renewal and differentiation. Genes linked to trophoblast invasion and/or vasculature remodeling were identified. These putative ‘invasion related genes’ were upregulated during differentiation, corresponding to an increase in PI3K/AKT signaling during differentiation. Using pharmacological inhibition of PI3K or AKT we determined that expression of several ‘invasion related genes’ was dependent on the PI3K/AKT signaling pathway. We also showed that the invasive ability of trophoblast cells was decreased when the PI3K/AKT signaling pathway was inhibited. Further analysis using lentiviral delivered shRNAs to 4 knockdown Akt1, Akt2 or Akt3 further validated the involvement of the PI3K/AKT pathway in the regulation of these events. Fos like antigen 1 (FOSL1 or FRA1) was upregulated during trophoblast cell differentiation and shown to be a candidate downstream mediator of the PI3K/AKT signaling pathway. Modulation of the PI3K/AKT signaling pathway resulted in a decrease in FRA1 protein level. Knockdown of FRA1 using lentiviral delivered shRNA resulted in a decrease in a subset of the PI3K/AKT sensitive ‘invasion related genes’ and decreased trophoblast invasion. In conclusion, the PI3K/AKT signaling pathway regulates trophoblast differentiation by regulating FRA1 and the expression of key genes involved in the invasion process. In this research, we have identified key factors contributing to the regulation of rodent hemochorial placentation. The conservation of these regulatory factors in the process of human placentation and their potential involvement in pregnancy-related diseases remain to be determined. 5 TABLE OF CONTENTS Abstract……………………………………………………………………………….…4 Table of Contents………………………………………………………………….…..6 List of Figures and Tables…………………………………………..…………..….10 CHAPTER 1: GENERAL INTRODUCTION………………………………………..13 General Introduction…………………………………………………………….…..14 Rodent Trophoblast Models…………………………………………….…....17 Early lineage decisions in the mammalian embryo………………….……..17 Placentation in the Rodent………………………………..…………..………17 Trophoblast Invasion and Vascular Remodeling…………………..……….23 Rcho-1 Trophoblast Stem Cells……………………………………..……….24 Overview of the Phosphatidylinositol-3 Kinase (PI3K)/AKT Signaling Pathway……………………………………………………..………….27 PI3K/AKT Pathway in Placental and Trophoblast Cell Development and Invasion…………………………………………………………………30 FOS Like Protein 1 (FOSL1 or FRA1)……………………………………….32 PI3K/AKT Signaling Pathway and FRA1…………………………...……....35 FRA1 in Placental and Trophoblast Cell Development……………………35 Objectives of our Research………………………………………………………...37 CHAPTER 2: PHOSPHATIDYLINOSITOL 3-KINASE MODULATION OF TROPHOBLAST CELL DIFFERENTIATION………………………………..…….41 Abstract………………………………………………………………………………..42 Introduction…………………………………………………………………….……..43 6 Materials and Methods………………………………………………………………46 Results…………………………………………………………………………..……..56 Identification of genes associated with trophoblast differentiation……………….56 Trophoblast stem-associated genes………………………………………...56 Trophoblast differentiation-associated genes……………………………....63 PI3K signaling and trophoblast differentiation………………………………………71 PI3K signaling: negatively regulated genes……………………………..…76 PI3K signaling: positively regulated genes…………………………………78 PI3K regulation of trophoblast Steroidogenesis…………………………....79 Discussion…………………………………………………………………………….84 Trophoblast stem cell-associated genes…………………………………………....84 Trophoblast differentiation-associated genes………………………………………88 PI3K signaling-sensitive genes……………………………………………………....88 CHAPTER 3: PHOSPHATIDYLINOSITOL 3-KINASE/AKT SIGNALING PATHWAY AND FOS LIKE ANTIGEN 1 IN THE REGULATION OF TROPHOBLAST INVASION AND VASCULAR REMODELING……………..…92 Abstract………………………………………………………………………………..93 Introduction……………………………………………………………………………95 Materials and Methods………………………………………………………………99 Results………………………………………………………………………………..109 The PI3K/AKT pathway and trophoblast cell invasion……………………………109 Trophoblast AKT is expressed, active, and sensitive to small molecule inhibition……………………………………………………………….109 7 Small molecule inhibition of PI3K and AKT in trophoblast cells decreases expression of “invasion-vascular remodeling related” genes……112 Small molecule inhibition of PI3K or AKT decreases MMP9 activity and invasive ability of trophoblast cells………………………………....112 AKT isoform regulation of the expression of ‘invasion-vascular remodeling related’ genes and the invasive ability of trophoblast…………….118 FRA1 and PI3K/AKT signaling……………………………………………………...123 FRA1 is expressed in trophoblast giant cells and invasive trophoblast cells…………………………………………………………………….123 PI3K/AKT signaling regulates FRA1……………………………………….128 FRA1 and trophoblast invasion……………………………………………………..133 FRA1 regulates expression of PI3K/AKT-sensitive ‘invasion-vascular remodeling related’ genes………………………………………..…133 FRA1 regulates in vitro trophoblast cell invasion…………………………133 FRA1 regulates in vivo trophoblast cell invasion…………………………140 Discussion…………………………………………………………………………...145 PI3K/AKT regulation of ‘invasion-vascular remodeling related’ genes…………145 AKT isoforms and trophoblast cell biology………………………………………...146 PI3K/AKT regulation of FRA1……………………………………………………….148 FRA1 and placentation………………………………………………………………149 CHAPTER 4: GENERAL DISCUSSION…………………………………………..151 I. Model……………………………………………………………………………….152 II. Regulation of trophoblast differentiation and invasion by the PI3K/AKT signaling pathway…………………………………………………………….....152 Activation of the PI3K/AKT signaling pathway in trophoblast cells……………..152 Isoform specific roles for AKT in trophoblast cells…………………………….….155 Akt isoforms in placental and embryonic development………..………...155 8 Akt isoforms in invasion……………………………………………………..157 PI3K/AKT mediators of trophoblast invasion……………………………………...158 Regulation of Fos like 1 by the PI3K/Akt signaling pathway…………….158 Crosstalk between PI3K/AKT/FRA1 and other signaling pathways and trophoblast invasion………………………………………………….160 The role of FRA1 in trophoblast invasion……………………………………….…161 Identifying targets of FRA1……………………………………………….…161 Potential binding partners of FRA1…………………………………………161 The function of FRA1 target genes in trophoblast invasion……………..162 Significance of the invasion and vascular remodeling related genes……….….163 III. Human pregnancy related disorders…………………………………………..164
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