NUMB and Syncytiotrophoblast Development and Function: Investigation Using BeWo Choriocarcinoma Cells By Julie Carey This thesis is submitted as a partial fulfillment of the M.Sc. program in Cellular and Molecular Medicine Faculty of Medicine, University of Ottawa May, 2012 © Julie Carey, Ottawa, Canada, 2012 ABSTRACT The role of NUMB, a protein important for cellular differentiation and endocytosis in non-placental cells, was investigated in syncytiotrophoblast development and function in the human placenta. The BeWo choriocarcinoma cell line was used as a model for villous cytotrophoblast cells and syncytiotrophoblast to investigate NUMB’s involvement in differentiation and epidermal growth factor receptor (EGFR) endocytosis. NUMB isoforms 1 and 3 were found to be the predominant isoforms and were upregulated following forskolin-induced differentiation. Overexpression of NUMB isoforms 1 and 3 did not mediate differentiation or EGFR signaling. Immunofluorescence analysis revealed that NUMB colocalized with EGFR at perinuclear late endosomes and lysosomes following EGF stimulation. We have demonstrated for the first time that NUMB isoforms 1 and 3 are expressed in BeWo cells, are upregulated in forskolin- differentiated BeWo cells and are involved in ligand-dependent EGFR endocytosis in BeWo cells. ii TABLE OF CONTENTS ABSTRACT …………………………………………………………………………….. ii LIST OF TABLES ……………………………………………………………………... v LIST OF FIGURES ………………………………………………………………...…. vi LIST OF ABBREVIATIONS ………………………………………………...……… vii ACKNOWLEDGEMENTS ……………………………………………...…..……….. ix CHAPTER 1: INTRODUCTION 1.1 Formation and development of the human placenta ........................................ 1 1.2. Functions of the human placenta ........................................................................ 4 1.3 Pregnancy complications associated with placental aberrations ..................... 8 1.4 NUMB i. NUMB structure and function ..................................................................... 13 ii. NUMB and placental development ............................................................ 16 1.5 Rationale for investigation i. NUMB and villous cytotrophoblast differentiation .................................. 19 ii. NUMB and EGFR endocytosis in syncytiotrophoblast ............................ 21 iii. BeWo choriocarcinoma cells as an in vitro model for human villous trophoblast cells ............................................................................... 23 iv. Summary ....................................................................................................... 25 1.6 Hypothesis, Overall Objective and Specific Objectives .................................. 26 CHAPTER 2: MATERIALS AND METHODS 2.1 Cell culture ......................................................................................................... 27 2.2 Immunoblotting (Western blot) ........................................................................ 27 2.3 Forskolin-induced differentiation of BeWo cells and assessment of NUMB isoform expression ................................................................................ 31 2.4 BeWo transfection efficiency ............................................................................ 31 i. GFP fluorescence ............................................................................................ 32 ii. V5 immunodetection ...................................................................................... 33 iii. lacZ staining .................................................................................................. 34 2.5 Overexpression of NUMB isoforms .................................................................. 35 iii 2.6 Cell morphology ................................................................................................. 36 2.7 Cell viability ........................................................................................................ 36 2.8 Overexpression of NUMB to assess effects on BeWo differentiation ............ 37 2.9 Overexpression of NUMB to assess effects on EGFR signaling .................... 38 2.10 Streptavidin pull-down assay ............................................................................ 38 2.11 Immunofluorescence (immunocytochemistry) ................................................ 39 2.12 Statistical analysis .............................................................................................. 41 CHAPTER 3: RESULTS 3.1 NUMB protein expression in undifferentiated and forskolin-treated BeWo cells ........................................................................................................... 42 3.2 BeWo cell transfection efficiency assessment .................................................. 47 3.3 Effects of NUMB1 and NUMB3 overexpression on BeWo cell morphology and viability .................................................................................. 51 3.4 Effects of NUMB1 and NUMB3 overexpression on BeWo differentiation ..................................................................................................... 54 3.5 Effects of NUMB1 and NUMB3 overexpression on EGFR signaling in BeWo cells ........................................................................................................... 56 3.6 Membrane EGFR levels in EGF-treated BeWo cells ..................................... 58 3.7 Effects of EGF on the subcellular localization of NUMB, EGFR and LAMP-2 in BeWo cells .............................................................................. 60 CHAPTER 4: DISCUSSION & CONCLUSIONS ...................................................... 64 4.1 NUMB isoform expression in the placenta ...................................................... 64 4.2 Regulation of NUMB1 and NUMB3 in BeWo cells ......................................... 67 4.3 Investigating the roles of NUMB1 and NUMB3 in BeWo cells .................... 69 4.4 Postulated role of NUMB in EGFR endocytosis ............................................. 72 4.5 Study limitations ................................................................................................ 75 4.6 Future directions ................................................................................................ 75 4.7 Conclusions ......................................................................................................... 77 REFERENCES ………………………………………………...……………………… 78 iv LIST OF TABLES Table 1: Human placental cells and tissues .............................................................. 7 Table 2: Antibodies used for Western blotting (WB) and immunocytochemistry (ICC) ................................................................... 29 Table 3: NUMB expression in the placenta: Summary of current data ................. 66 v LIST OF FIGURES Figure 1: Schematics of first and third trimester chorionic villi cross-sections ........ 3 Figure 2: Comparison of trophoblast invasion during healthy and pre-eclamptic pregnancies ................................................................. 12 Figure 3: Schematic representation of NUMB isoforms 1 to 9 .............................. 18 Figure 4: NUMB protein expression in human placental villi at term ................... 20 Figure 5: NUMB protein expression in BeWo and HTR-8/SVneo cells ................ 44 Figure 6: NUMB protein expression in forskolin-treated BeWo cells .................... 45 Figure 7: Resolution of NUMB isoforms in forskolin-treated BeWo cells ............. 46 Figure 8: BeWo transfection efficiency assessment using GFP fluorescence ......... 48 Figure 9: BeWo transfection efficiency assessment using V5 immunodetection ... 49 Figure 10: BeWo transfection efficiency assessment using lacZ staining................. 50 Figure 11: Assessment of BeWo cell morphology following NUMB1 and NUMB3 overexpression .................................................................... 52 Figure 12: Assessment of BeWo cell viability following NUMB1 and NUMB3 overexpression .................................................................... 53 Figure 13: Influence of NUMB1 and NUMB3 overexpression on BeWo differentiation ........................................................................................... 55 Figure 14: Influence of NUMB1 and NUMB3 overexpression on EGFR signaling in BeWo cells ................................................................ 57 Figure 15: Membrane EGFR levels in EGF-treated BeWo cells .............................. 59 Figure 16: Colocalization of EGFR and NUMB in EGF-treated BeWo cells .......... 61 Figure 17: Colocalization of LAMP-2 and NUMB in EGF-treated BeWo cells ...... 62 Figure 18: Colocalization of LAMP-2 and EGFR in EGF-treated BeWo cells ........ 63 Figure 19: Current model for EGFR endosomal trafficking ..................................... 74 vi LIST OF ABBREVIATIONS aa Amino acid AEC 3-amino-9 ethylcarbazole aPKC Atypical protein kinase C APP Amyloid precursor protein ANOVA Analysis of variance ARF6 ADP-ribosylation factor 6 BSA Bovine serum albumin CaMKI Ca2+/calmodulin-dependent protein kinase I cAMP Cyclic adenosine monophosphate CT Cytotrophoblast DMSO Dimethyl sulfoxide dNUMB Drosophila NUMB E-cadherin Epithelial cadherin ECM Extracellular cell matrix EGF Epidermal growth factor EGFR Epidermal growth factor receptor EGTA Ethylene glycol tetraacetic acid EH Eps15 homology EPS15 EGFR pathway substrate clone 15 EVT Extravillous trophoblast FBS Fetal bovine serum FV Floating villus GAPDH Glyceraldehyde 3’ phosphate dehydrogenase