Differential Regulation of Growth-Promoting Signalling Pathways by E-Cadherin Nikolaos T. Georgopoulos1¤, Lisa A. Kirkwood 1 , Dawn C. Walker2, Jennifer Southgate1* 1 Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York, United Kingdom, 2 Department of Computer Science, Kroto Research Institute, University of Sheffield, Sheffield, United Kingdom Abstract Background: Despite the well-documented association between loss of E-cadherin and carcinogenesis, as well as the link between restoration of its expression and suppression of proliferation in carcinoma cells, the ability of E-cadherin to modulate growth-promoting cell signalling in normal epithelial cells is less well understood and frequently contradictory. The potential for E-cadherin to co-ordinate different proliferation-associated signalling pathways has yet to be fully explored. Methodology/Principal Findings: Using a normal human urothelial (NHU) cell culture system and following a calcium- switch approach, we demonstrate that the stability of NHU cell-cell contacts differentially regulates the Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-Regulated Kinase (ERK) and Phosphatidylinositol 3-Kinase (PI3-K)/AKT pathways. We show that stable cell contacts down-modulate the EGFR/ERK pathway, whilst inducing PI3-K/AKT activity, which transiently enhances cell growth at low density. Functional inactivation of E-cadherin interferes with the capacity of NHU cells to form stable calcium-mediated contacts, attenuates E-cadherin-mediated PI3-K/AKT induction and enhances NHU cell proliferation by allowing de-repression of the EGFR/ERK pathway and constitutive activation of b-catenin-TCF signalling. Conclusions/Significance: Our findings provide evidence that E-cadherin can differentially and concurrently regulate specific growth-related signalling pathways in a context-specific fashion, with direct, functional consequences for cell proliferation and population growth. Our observations not only reveal a novel, complex role for E-cadherin in normal epithelial cell homeostasis and tissue regeneration, but also provide the basis for a more complete understanding of the consequences of E-cadherin loss on malignant transformation. Citation: Georgopoulos NT, Kirkwood LA, Walker DC, Southgate J (2010) Differential Regulation of Growth-Promoting Signalling Pathways by E-Cadherin. PLoS ONE 5(10): e13621. doi:10.1371/journal.pone.0013621 Editor: Kurt Anderson, The Beatson Institute for Cancer Research, United Kingdom Received April 16, 2010; Accepted July 13, 2010; Published October 26, 2010 Copyright: 2010 Georgopoulos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was funded by the Engineering and Physical Sciences Research Council (EPSRC; grant reference GR/S62338/01), on which NTG was employed. DCW is supported by an RCUK fellowship. LAC is a postgraduate student and JS holds a research chair supported by York Against Cancer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: js35@york.ac.uk ¤ Current address: Department of Chemical and Biological Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom Introduction E-cadherin is a member of the cadherin family of transmem- brane glycoproteins that mediates formation of adherens junctions in epithelial cells [1]. Formation of calcium-dependent ‘‘zipper- like’’ homophilic interactions between the extracellular domains of E-cadherin on adjacent cells facilitates epithelial cell-cell contact and adhesion [2]. These interactions are stabilised by the association of the cytoplasmic tail of E-cadherin with the a, b and c catenins, which anchor E-cadherin to the cytoskeleton [3]. In epithelial tissues, E-cadherin functions not only to stabilise homotypic cell interactions, but also to modulate extracellular growth-associated signals, thus providing a homeostatic mecha- nism to link cell-cell contact/adhesion with cellular proliferation and tissue growth. For instance, E-cadherin interacts with b- catenin, a multifunctional protein that regulates cell growth by acting as a transcription factor in Wnt signalling [4]. The association between E-cadherin and b-catenin not only stabilises the adherens complex itself, but also provides a mechanism by which E-cadherin can sequester b-catenin to the cell membrane and impede b- catenin/TCF-mediated transcription [5,6]. Al- though the cytoplasmic tail of E-cadherin lacks enzymatic activity, it can regulate growth-promoting cell signalling, such as the Phosphatidylinositol 3-Kinase (PI3-K)/AKT and Extracellular Signal-Regulated Kinase (ERK) pathways, by influencing the activation of receptor tyrosine kinases (RTKs). Some studies have reported positive regulation of Epidermal Growth Factor Receptor (EGFR) downstream signalling through ERK [7,8] and PI3-K/ AKT [9] following calcium-mediated formation of E-cadherin intercellular contacts in monocultures of normal keratinocytes and cancer cell lines, respectively. Yet, using the same calcium-switch approach, others have demonstrated that E-cadherin-mediated cell-cell adhesion can inhibit the ligand-dependent activation of diverse RTKs, including EGFR [10,11]. Although these studies imply a critical, context-specific role for E-cadherin-dependent cell-cell contacts in modulating growth-promoting intracellular signalling pathways, many of the described findings are contra- dictory. Equally importantly, it remains unknown whether E- PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13621