DIFFERENTIAL IMPACT OF VEGF AND FGF2 SIGNALING MECHANISMS ON FLT1 PRE-MRNA SPLICING Laura Beth Payne Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biomedical and Veterinary Sciences William R. Huckle Carla Finkielstein Ian Herring William Eyestone April 27, 2016 Blacksburg, Virginia Keywords: Akt, Alternative splicing, FGF2, Flt1, ERK, Preeclampsia, pre-mRNA, Signal transduction, soluble Flt1, SR proteins, VEGF DIFFERENTIAL IMPACT OF VEGF AND FGF2 SIGNALING MECHANISMS ON FLT1 PRE-MRNA SPLICING Laura Beth Payne ABSTRACT, Academic The human proteome is exponentially derived from a limited number of genes via alternative splicing, where one gene gives rise to multiple proteins. Alternatively spliced gene products, although crucial for normal physiology, are also linked to an increasing number of pathologies. Consequently, a growing focus is currently being placed on elucidating the extrinsic cues and ensuing signaling mechanisms which direct changes in gene splicing to yield functionally distinct proteins. Of note is the dysregulation of the vascular endothelial growth factor (VEGF) receptor, Flt1 and its soluble splice variants, sFlt1_v1 and sFlt1_v2, in the pregnancy-related disorder, preeclampsia. Preeclampsia is characterized by proteinuria and hypertension and is responsible for almost 600,000 maternal and fetal yearly deaths, worldwide. Here, we examined the impact of endothelial mitogens VEGF and FGF2 (fibroblast growth factor 2), both of which are upregulated in preeclampsia, on Flt1 transcript variants in umbilical vein endothelial cells. We tested the hypothesis that VEGF modulates the expression of Flt1 variants via the signaling kinase Akt and its impact on SR proteins. VEGF was observed to induce expression of overall Flt1 mRNA, principally as variants Flt1 and sFlt1_v1. Conversely, FGF2 induced a shift in splicing toward sFlt1_v2 without significant increase in overall Flt1. Based on inhibitor studies, the VEGF and FGF2 signals were transduced via ERK, but with the involvement of different upstream components. We mapped predicted SR protein binding to Flt1 pre-mRNA and identified two candidate proteins, SRSF2 and SRSF3, that may be involved in VEGF- or FGF2-induced Flt1 pre-mRNA splicing. Examination of SRSF2 and SRSF3 relative mRNA expression levels, following inhibition of VEGF- and FGF2-activated kinases, indicates that FGF2 significantly downregulates SRSF3 mRNA levels via PKC- independent activation of ERK. Additionally, our data suggest that FGF2 may impact Flt1 and sFlt1_v1 via SR protein kinases Akt and SRPK, while conversely regulating sFlt1_v2 levels via Clk. We did not find evidence of VEGF-induced Flt1 variant splicing via SR protein kinase activation or SRSF2 and SRSF3 mRNA levels. Thus, VEGF and FGF2 signals were tranduced via related but distinct mechanisms to differentially influence Flt1 pre-mRNA splicing. These findings implicate VEGF and FGF2 and their related intracellular signaling mechanisms in soluble Flt1 regulation. ABSTRACT, Public About 95% of the genes in the human body are each processed in different ways to ultimately yield multiple different proteins – complex molecules that make up structural and functional features of cells and tissues of the body. In pathology and disease, cues from outside the cell may direct a particular gene to produce an incorrect protein or balance of proteins. One such case is preeclampsia - a pregnancy-related disorder affecting as many as 1 out of every 20 pregnancies in the US alone. Preeclampsia is characterized by hypertension and protein in the urine and can lead to organ failure and death of both mother and baby. Currently there is no treatment to effectively control the disorder during pregnancy, the only cure being delivery. The originating cause of preeclampsia remains unknown. What is known, however, is some of the cells in the placenta incorrectly express proteins from the gene called Flt1. Flt1 is protein that crosses through the outer layer of the cell (a cell-surface receptor) and binds vascular endothelial growth factor (VEGF), which controls growth of blood vessels. VEGF binds to two main receptors on the surface of the cell, which then relay the “signal” inside the cell, leading to the appropriate changes in cell function. The responses to VEGF signaling include blood vessel growth or branching. In the placenta, the VEGF signal, along with other signaling molecules such as fibroblast growth factor 2 (FGF2), directs blood vessels to grow properly in order to deliver nutrients and oxygen to the growing fetus. In preeclampsia, production (expression) of three protein receptors that come from the gene Flt1, called Flt1, sFlt1_v1 and sFlt1_v2, is imbalanced. In turn, the development of the blood vessels in the placenta is inadequate, leading to pregnancy complications. We investigated the influence of VEGF and FGF2 on the expression of the different forms (variants) of Flt1. We found that VEGF and FGF2 cause the different variants of Flt1 to be expressed differently. We investigated proteins inside the cells, by which they send (transduce) their signals, and found that VEGF and FGF2 use different but related proteins to influence Flt1 variant expression. We also examined SR proteins, which are directed by cues outside the cell (such as VEGF or FGF2) to directly interact with and influence which variants are expressed from a particular gene. We used prediction tools to identify specific SR proteins that may be directed by VEGF or FGF2 to bind to Flt1 before it is processed into different variants and, in turn, influence which variants are expressed. We identified two candidate SR proteins and found that FGF2 appears to affect the expression of one of them, SRSF3. Overall, VEGF and FGF2, which are imperative for proper blood vessel growth in the placenta, differently affect the amounts of Ftl1 variants, likely by sending their signals through related but different proteins inside the cell. Additionally, our results indicate that FGF2 may influence Flt1 variant expression by activating SR proteins and, in the case of SRSF3, by affecting how much is expressed. Our findings provide new evidence for the role of FGF2 in preeclampsia and sheds light on how FGF2 and VEGF influence Flt1 variant expression. Ultimately, understanding how and why imbalanced Flt1 variant expression occurs in preeclampsia may lead to life-saving therapies where none currently exist. Our multi-level investigation of proteins that influence Flt1 variant expression, leading from outside the cell and into the nucleus, reveals different levels for further investigation and eventual targeted therapy pursuit. DEDICATION This manuscript is dedicated to my loving family for their unwavering support. To my parents, Bill and Bonnie Payne, thank you for sowing and nurturing the seeds of personal growth and optimism. You have made all my accomplishments possible. Thank you, from the bottom of my heart. I love you. To my sisters, thank you for believing in me and seeing my true heart. Such gifts have seen me through the days of doubt. You have my undying gratitude and love. To my parents-in-law, I am indebted to you for the incredibly gracious support you have given me during this journey. I offer my love and gratitude. Finally, this manuscript is dedicated to my wonderful husband, John Eustis, and my magical daughter, Graelyn Eustis. To John, my thanks is unending – your support and encouragement have been monumental and foundational to my success. Thank you for being so solid and sharing your love with me. I love you very much. To Graelyn, words fall short – your very presence in my life has inspired me in so many ways. You truly can achieve whatever you set your sights on…but the most important achievement is remembering that life’s true meaning is found in the present, regardless of where you have been or where you are headed. My love for you is, and will always be, immeasurable and unconditional. vi ACKNOWLEDGMENTS Funding support was provided by an assistantship provided by the Virginia Maryland Regional College of Veterinary Medicine. I thank my fellow BMVS members for allowing me to pick their brains and occasionally borrow the random reagent. I would also like to thank my fellow lab-mate, Erwin Kristobal Gudenschwager Basso, for allowing me to bump shoulders while we pursued our respective projects. I thank my committee members for their support, encouragement, and expertise during the course of my graduate program. I am grateful for the generous time an effort you put forth for me. Finally, I thank my advisor Dr. Huckle. Thank you so much for your calm and encouraging guidance as I learned the ropes throughout this process called graduate school. Thank you for allowing me to be an individual and a mom, while also wearing the hat of student. Most of all, thank you for believing in me and for sharing experiential words of direction… gifts that will live far beyond the technical skills defining my degree. Thank you. vii TABLE OF CONTENTS CHAPTER 1: INTRODUCTION………………………………………………………… 1 CHAPTER 2: LITERATURE REVIEW………………………………………………… 3 2.1 Placental Biology………………………………………………………………… 3 2.2 VEGFs and VEGFRs…………………………………………………………….. 3 2.3 FGFs and FGFRs………………………………………………………………… 5 2.4 VEGF and FGF Signaling – Akt and ERK……………………………………… 6 2.5 Alternative Splicing……………………………………………………………… 7 2.6 SR Proteins………………………………………………………………………. 8 2.7 SR Protein Kinases……………………………………………………………….