cadherin can simultaneously regulate these pathways and/or how the influence of E-cadherin on these signalling cues dictates epithelial cell behaviour, particularly within the tissue context. We have previously developed a culture system for normal human urothelial (NHU) cells [12]. In low calcium (0.09 mM), serum-free culture medium, NHU cells adopt a highly prolifera- tive, regenerative phenotype which is driven via the ERK, mitogen-activated protein kinase (MAPK) cascade downstream of an EGFR-activated autocrine pathway, with amphiregulin and HB-EGF identified as the key endogenous ligands [13]. A switch to physiological (2 mM) calcium induces stratification and the formation of adherens and tight junctional complexes, but does not induce urothelial cytodifferentiation [14], although the cells retain the capacity to differentiate [15] and to form a functional barrier epithelium [16]. To aid interpretation of the cellular basis of tissue regulatory mechanisms, we have previously developed the ‘‘Epitheliome’’, an agent-based computational model of epithelial cell behaviour [17,18,19]. By incorporating rule sets to describe concepts of calcium-dependent bonding and a G1 checkpoint operated by contact inhibition, we have shown good qualitative agreement between in virtuo simulations and experimentally-acquired growth kinetics for NHU cell cultures. In low (0.09 mM) exogenous calcium concentrations, both real and simulated cell cultures display higher growth rates and achieved greater final cell densities than equivalent cultures grown in physiological (2 mM) calcium, where the tendency towards colony formation results in early exit from cell cycle due to contact inhibition [17]. The single discrepancy was that low-density NHU cell cultures in 2 mM calcium conditions consistently showed a higher growth rate compared to cultures in 0.09 mM calcium, which was not replicated in the simulated system [17]. This has been examined computationally by exploring growth signals augmented through E-cadherin-dependent cell-cell contacts [20]. These observations predict a more complex interplay between signalling mechanisms for the regulation of cellular growth at different population densities. The aim of our present study was to identify the growth- promoting pathways that underpin the above observations and contribute to the regulation of epithelial tissue homeostasis and regeneration. The urothelium, which is able to switch from a mitotically quiescent to a highly-proliferative regenerative tissue in response to damage, represents an ideal model system. Using a functional inactivation approach, we demonstrate that E-cadherin interactions simultaneously regulate multiple growth-promoting signalling pathways and that the pattern of activation of these pathways accounts for the cell-growth differences observed. Our findings not only reveal a complex role for E-cadherin in regulating growth signals in normal epithelial cells, but also provide further insight into the functional consequences of E- cadherin loss in neoplastic disease. Results The stability of NHU cell-cell contacts differentially regulates the EGFR/ERK and PI3-K/AKT signalling pathways Growth curves were constructed from cell counts of NHU cell cultures maintained in low (0.09 mM) and near physiological (2 mM) calcium concentrations. In all cases, cultures were maintained in the presence of saturating concentrations of exogenous EGF in order to limit any confounding effects from autocrine/juxtacrine EGFR activation [13]. NHU cell cultures maintained in 2 mM calcium exhibited higher growth rates during E-Cadherin Growth Regulation exponential growth phase than cultures grown in 0.09 mM calcium conditions. By contrast, cultures maintained in 0.09 mM calcium grew more slowly, but attained a higher ultimate cell density (Figure 1A). As EGFR/ERK signalling is critical in NHU cell proliferation [13], we used immunofluorescence microscopy to examine the expression of EGFR and its downstream signalling effector, ERK, 24 hours following a switch from 0.09 mM to 2 mM calcium. The calcium switch resulted in a major down-regulation of nuclear phospho-ERK, despite the total amount of ERK remaining unchanged (Figure 1B). Along with an increase in the overall expression level of EGFR, we observed a change in localisation from diffuse cytoplasmic to intense, punctate labelling in cytoplasmic and perinuclear areas (Figure 1B). Immunoblotting studies, carried out to monitor EGFR and phospho-ERK over a 24-hour time-course, confirmed the increase in EGFR expression, which occurred within 12 hours of treatment, as well as the decrease in phospho-ERK as early as 3 hours following the calcium-switch (Figure 1C). The reduction in phospho-ERK activity indicated that formation of calcium-mediated, ‘stable’ intercellular contacts did not result in increased EGFR/ERK signalling and suggested that this pathway is unlikely to be responsible for the increased proliferation rate in low-density cultures. As activation of the PI3- K/AKT pathway has been previously implicated in cell contact- dependent proliferation [9,21], the possibility of an induction of AKT activity was investigated. Immunoblotting studies revealed the emergence of phosphorylated AKT on Ser-473, which occurred within 12 hours of switching to 2 mM calcium (Figure 2A); by contrast no phospho-AKT on Tyr-308 was observed (not shown). The findings were confirmed by immuno- fluorescence microscopy, which showed phospho-AKT to be nucleus-localised (Figure 2B). Only cells making direct contacts with neighbouring cells showed intense activation of AKT, whilst lone cells showed little, if any, detectable phospho-AKT (Figure 2B, right top panel, denoted by arrows). This implied that it was the formation of cell-cell contacts, rather than addition of calcium alone, which was critical for AKT activation. To corroborate direct functional involvement of the PI3-K/AKT pathway in the enhancement of growth rates, population growth was assessed for NHU cell cultures grown in 0.09 mM or 2 mM calcium in the presence or absence of the specific PI3-K inhibitor LY294002 over a 7 day time-course (Figure 2C). In confirmation of the kinetic data shown above (Figure 1A), NHU cells grown in 2 mM calcium showed higher proliferation rates during exponential growth, whereas cultures grown in 0.09 mM calcium achieved a higher density by day 7. The increased proliferation rate, characteristic of cultures grown in 2 mM calcium, was completely abolished by LY294002 treatment, whereas LY294002 had little effect on the exponential growth rate of cultures grown in low calcium medium (Figure 2C). These results indicate that stable, calcium-mediated, cell-cell interactions enhanced proliferation by activation of the PI3-K/AKT pathway in sub-confluent cultures. By contrast, the enhanced cell-cell contacts produced in physiological calcium conditions appeared to promote earlier exit from the cell cycle (as indicated by the lower growth rates at later time points), presumably due to cell contact inhibition. Expression of a dominant-negative E-cadherin mutant interferes with expression and localisation of endogenous E-cadherin and catenins To examine further the importance of cell-cell contacts in regulating proliferation, the effect of modifying adherens junctions was examined. Due to the critical role of E-cadherin in the PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13621