perinuclear, Golgi/late-endosome compartments (compare left and right panels, Figure 3C), synonymous with the perinuclear localisation of the H-2Kd-E-cad chimera (lower panels, Figure 3A). Associated with the loss of surface E-cadherin in NHU-ECmut cells, there was down-regulation of a-catenin and increased expression of nuclear b-catenin (Figure 3C). These experiments confirmed that the H-2Kd-E-cad mutant expressed in NHU cells was functional, as it efficiently incapacitated endogenous E- cadherin by blocking localisation to sites of cell-cell contact and preventing recruitment of a- and b-catenin. Functional inactivation of E-cadherin enhances NHU cell proliferation and reveals a pattern of E-cadherin- mediated differential regulation of EGFR/ERK, PI3-K/AKT and b-catenin/TCF signalling Following retrovirus transduction and antibiotic selection, NHU-ECmut cells exhibited a consistently higher proliferation rate than the isogenic controls. This was noticeable during routine maintenance of the cultures and was confirmed quantitatively by 3 [ H]-thymidine incorporation assays. These experiments showed that NHU-ECmut cells displayed consistently higher proliferation rates than NHU-Con cells in both low and physiological calcium conditions (Figure 4A). Population growth curves were also constructed over a period of 9 days. NHU-Con showed similar growth characteristics to non-transduced NHU cells, with a higher growth rate at lower density when cultured in physiological calcium (Figure 4B). In comparison to the control cells, the growth rate of NHU-ECmut was elevated in low calcium conditions and NHU-ECmut cells maintained in physiological calcium showed virtually identical growth rates to NHU-Con cells cultured in low calcium (Figure 4B); this was in agreement with the results from [3H]-thymidine incorporation assays (Figure 4A). Therefore, expression of mutant E-cadherin effectively overcame the inhibition of growth seen in 2 mM calcium in NHU and NHU- Con cell cultures. We then examined the effect of abrogation of E-cadherin function on the activity of the EGFR/ERK and PI3-K/AKT pathways. The addition of physiological calcium to NHU-Con cell cultures resulted in reduced phospho-ERK levels by almost 3-fold (Figure 4C) as expected. By contrast, in NHU-ECmut cells there was little (if any) reduction in ERK phosphorylation observed (Figure 4C). At the same time, despite a substantial increase in phosphor-AKT (on Ser-473) following calcium-switching in NHU- Con cell cultures, there was no such response in NHU-ECmut cultures (Figure 4C). These findings account for the observed effect of calcium-mediated contacts in regulating growth-promoting pathways and provide direct evidence for E-cadherin-mediated modulation of EGFR/ERK and PI3-K/AKT signalling. Our observation of EGFR/ERK de-repression following E- cadherin loss-of-function implies that elevated phospho-ERK activity may account, at least in part, for the increased proliferation rate of E-cadherin-disabled cells. However, the ability of NHU-ECmut cells to display consistently higher growth rates irrespective of extracellular calcium levels (Figure 4B) was suggestive of potential involvement of additional signalling pathways in driving cell proliferation. Another signalling candidate for promoting an increased growth rate was b-catenin, as immunofluorescence microscopy experiments revealed nuclear b-catenin localisation both in NHU cells maintained in low calcium (Figure 3B) and in NHU-ECmut cells maintained in physiological calcium (Figure 3C). b-catenin is a multifunctional protein that, upon activation, can act as a cofactor for the TCF/ LEF transcription factor complex to induce or enhance cell E-Cadherin Growth Regulation proliferation [4]. We used a reporter assay to determine whether nuclear b-catenin was active and able to trigger TCF transcrip- tional activity in NHU-Con and NHU-ECmut cells. Like NHU cells (not shown), NHU-Con cells showed some b-catenin/TCF activity, which was diminished in the presence of physiological calcium (Figure 4D). By contrast, NHU-ECmut cells showed high levels of b-catenin/TCF activity, irrespective of the extracellular calcium concentration (Figure 4D). Therefore, collectively, our studies show that functional inactivation of E-cadherin in NHU cells de-repressed EGFR/ERK signalling and enhanced b- catenin/TCF transcriptional activity by several-fold. Down-regulation of b-catenin by RNA interference mimics E-cadherin engagement and influences PI3-K/AKT activity and NHU cell proliferation As shown above, whilst inducing PI3-K/AKT activity (Figure 2), E-cadherin recruits b-catenin to the points of cell contact (Figure 3B) and down-regulates b-catenin/TCF activity (Figure 4D), whereas loss of E-cadherin function abolishes PI3- K/AKT induction (Figure 4C) and confers constitutive activation of b-catenin (Figure 4B). Collectively, these observations suggest that E-cadherin-mediated sequestration of active b-catenin to the cell surface coincides with induction of PI3-K/AKT activity. We therefore next addressed the possibility that engineered loss of active b-catenin may mimic the effects of E-cadherin engagement. We produced NHU cell derivatives that were transduced with a retrovirus expressing a b-catenin-specific short-hairpin RNA (shRNA) adapted from a reported siRNA sequence [25]. Expression of active b-catenin was significantly decreased in b- catenin shRNA-expressing (NHU-b-cat-KD) cells when compared to control (NHU-Con) cells expressing control shRNA, as shown by immunoblotting experiments (Figure 5A). The shRNA- mediated b-catenin knock-down resulted in the induction of phospho-AKT and this was accompanied by an almost three-fold increase in E-cadherin expression (Figure 5A). This suggested that forced down-regulation of active b-catenin allowed induction of PI3-K/AKT activity. To confirm the functional involvement of the induced phospho-AKT, we assessed population growth for NHU-Con and NHU-b-cat-KD cell cultures in 0.09 mM or 2 mM calcium in the presence of the LY294002 inhibitor over a 6 day time-course (Figure 5B). As in NHU cells, the increased proliferation rate characteristic of cultures grown in physiological calcium was attenuated by LY294002 treatment in NHU-Con cells, whereas the inhibitor had little effect on the exponential growth rate in low calcium conditions. However, NHU-b-cat-KD cell growth in physiological calcium was significantly reduced in comparison to cultures of NHU-Con cells (Figure 5B), indicating that b-catenin knock-down rendered NHU- b-cat-KD cells very sensitive to the LY294002 inhibitor and confirming the impor- tance of PI3-K/AKT signalling in conditions that mimic E- cadherin engagement. Discussion Our study indicates a critical role for E-cadherin in monitoring the ‘‘quality’’ of cell:cell interactions and regulating density- dependent cell growth via the modulation of different growth promoting pathways. Thus, E-cadherin has the capacity to simultaneously regulate signalling through EGFR/ERK, PI3-K/ AKT and b-catenin/TCF pathways, with direct, context-specific consequences for individual cell proliferation within a population. In addition to demonstrating a central role for E-cadherin in regulating epithelial tissue homeostasis and regeneration, it also PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13621