Cadherin-linked molecular mechanisms governing the

CADHERIN-LINKED MOLECULAR MECHANISMS GOVERNING THE TERMINAL DIFFERENTIATION OF HUMAN TROPHOBLASTIC CELLS IN VITRO

by York Hunt Ng B.S., Oklahoma City University, USA, 2001 M.Sc., The University of British Columbia, Canada, 2004

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

in

THE FACULTY OF GRADUATE STUDIES

(Reproductiveand Developmental Sciences)

THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2010 © York Hunt Ng, 2010

Abstract Background: The formation of the multinucleated syncytial trophoblast of the human placenta is a critical step in pregnancy, which is prone to failure. In these studies, I have examined the role of TWIST, a transcription factor identified as a key repressor of E-cadexpression in normal and cancer cells of diverse origins, in the differentiation of human trophoblastic cells in vitro. The invasion of extravillous cytotrophoblasts (EVTs) into the underlying maternal tissues and vasculature is a key step in human placentation. The molecular mechanisms underlying the development of the invasive phenotype of EVTs include many of those first identified as having a rolein cancer cell metastasis. In view of these observations, I have examined the expression, regulation, and function of Twist, Runx2 and N-cad in human trophoblastic cells in vitro. Materials and Methods: Gain or loss-of-function studies were then performed to

determine the role of Twist in terminal differentiation and fusion in these cells. The presence of multinucleated syncytium was confirmedby indirect immunofluorescence. Concentration- and time-dependent studies were performed to determine whether interleukin (IL)-1β and transforming growth factor (TGF)-β1 regulate Twist and Runx2 mRNA and protein levels in EVTs. Next, a siRNA strategy was employed to determine the role of Twist, Runx2 and N-cad in HTR-8/SVneo EVT cells. Results: Exogenous expression of Twist resulted in acontinuous and progressive

decrease in E-cad expression and the subsequent formation of syncytium in BeWo cells maintained under normal culture conditions. In contrast, siRNA specific for Twist

inhibited the cAMP-mediated differentiation of these cells over time in culture. The cytokines, IL-1β and TGF-β1, respectively induced the differential up- and down-

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regulation of Twist andRunx2 expression in primary cultures of EVTs in both a concentration and time-dependent manner. Use of a siRNA strategy demonstrated that a reduction in Twist, Runx2 or N-cad in HTR-8/SVneo cells concomitantly decreased the invasiveness of these cells. Conclusions: Collectively, my findings demonstrate that TWIST is an upstream regulator of the E-CAD-mediated terminal differentiation and fusion ofhuman trophoblastic cells in vitro. TWIST, RUNX2 and N-CAD are key molecules underlying the invasive capacity of EVTs.

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TABLE OF CONTENTS
ABSTRACT ................................................................................................................................. ii TABLE OF CONTENTS............................................................................................................ iv LIST OF TABLES ...................................................................................................................... ix LIST OF FIGURES ..................................................................................................................... xi LIST OF ABBREVIATIONS.................................................................................................... xv CHAPTER 1: 1.1 1.2 1.2.1 1.2.2 1.2.2.1 1.2.2.2 1.2.2.3 1.2.2.3.1 1.2.2.3.2 1.2.2.3.3 1.2.2.3.3.1 1.2.2.3.3.2 1.3 OVERVIEW ................................................................................................. 1 Introduction ................................................................................................. 1 Human implantation and placentation...