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Differential regulation of growth-promoting signalling pathways by E-cadherin.

Georgopoulos NT, Kirkwood LA, Walker DC, Southgate J - PLoS ONE (2010)

Bottom Line: The potential for E-cadherin to co-ordinate different proliferation-associated signalling pathways has yet to be fully explored.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 β-catenin-TCF signalling.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.

View Article: PubMed Central - PubMed

Affiliation: Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York, 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 β-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.

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Calcium-induced increase in proliferation in low-density NHU cultures occurs via activation of the PI3-K/AKT pathway.(A) NHU cells were seeded in medium containing low [Ca2+] (0.09 mM) and left to attach overnight. Calcium concentration was then increased to 2 mM and protein lysates were prepared at the indicated time-points for immunoblotting. Expression of phospho-AKT (green) and total AKT (red) was determined using rabbit and mouse antibodies, respectively, followed by fluorochrome-conjugated secondary antisera as in Figure 1C. Immunolabelling was visualised and densitometry to determine fold induction of p-AKT expression with respect to total AKT was performed as in Figure 1C. (B) NHU cells were cultured in medium containing 0.09 mM (Low Ca2+) or 2.0 mM (Phys Ca2+) [Ca2+]. Expression of phospho-AKT (p-AKT) was determined by immunofluorescence microscopy using anti-p-AKT rabbit antibody followed by goat anti-rabbit antibody conjugated with Alexa Fluor 488 (green). Cell nuclei were visualised using Hoechst 33258 (blue). (C) NHU cells were seeded into 96-well plates and cultured for a period of 7 days in medium containing either low (-Ca) or physiological (+Ca) calcium levels, in the presence or absence of 5 µM of the PI3-K inhibitor LY294002. Proliferation was determined on the basis of cell biomass using the MTT assay. Data points represent mean absorbance values for 6 replicate wells (±S.E.M.). Results for all data points are also presented in the form of bar graphs (lower panel) for the purpose of statistical analysis. ns, non-significant; **, P<0.01; ***, P<0.001.
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pone-0013621-g002: Calcium-induced increase in proliferation in low-density NHU cultures occurs via activation of the PI3-K/AKT pathway.(A) NHU cells were seeded in medium containing low [Ca2+] (0.09 mM) and left to attach overnight. Calcium concentration was then increased to 2 mM and protein lysates were prepared at the indicated time-points for immunoblotting. Expression of phospho-AKT (green) and total AKT (red) was determined using rabbit and mouse antibodies, respectively, followed by fluorochrome-conjugated secondary antisera as in Figure 1C. Immunolabelling was visualised and densitometry to determine fold induction of p-AKT expression with respect to total AKT was performed as in Figure 1C. (B) NHU cells were cultured in medium containing 0.09 mM (Low Ca2+) or 2.0 mM (Phys Ca2+) [Ca2+]. Expression of phospho-AKT (p-AKT) was determined by immunofluorescence microscopy using anti-p-AKT rabbit antibody followed by goat anti-rabbit antibody conjugated with Alexa Fluor 488 (green). Cell nuclei were visualised using Hoechst 33258 (blue). (C) NHU cells were seeded into 96-well plates and cultured for a period of 7 days in medium containing either low (-Ca) or physiological (+Ca) calcium levels, in the presence or absence of 5 µM of the PI3-K inhibitor LY294002. Proliferation was determined on the basis of cell biomass using the MTT assay. Data points represent mean absorbance values for 6 replicate wells (±S.E.M.). Results for all data points are also presented in the form of bar graphs (lower panel) for the purpose of statistical analysis. ns, non-significant; **, P<0.01; ***, P<0.001.

Mentions: 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 immunofluorescence 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.


Differential regulation of growth-promoting signalling pathways by E-cadherin.

Georgopoulos NT, Kirkwood LA, Walker DC, Southgate J - PLoS ONE (2010)

Calcium-induced increase in proliferation in low-density NHU cultures occurs via activation of the PI3-K/AKT pathway.(A) NHU cells were seeded in medium containing low [Ca2+] (0.09 mM) and left to attach overnight. Calcium concentration was then increased to 2 mM and protein lysates were prepared at the indicated time-points for immunoblotting. Expression of phospho-AKT (green) and total AKT (red) was determined using rabbit and mouse antibodies, respectively, followed by fluorochrome-conjugated secondary antisera as in Figure 1C. Immunolabelling was visualised and densitometry to determine fold induction of p-AKT expression with respect to total AKT was performed as in Figure 1C. (B) NHU cells were cultured in medium containing 0.09 mM (Low Ca2+) or 2.0 mM (Phys Ca2+) [Ca2+]. Expression of phospho-AKT (p-AKT) was determined by immunofluorescence microscopy using anti-p-AKT rabbit antibody followed by goat anti-rabbit antibody conjugated with Alexa Fluor 488 (green). Cell nuclei were visualised using Hoechst 33258 (blue). (C) NHU cells were seeded into 96-well plates and cultured for a period of 7 days in medium containing either low (-Ca) or physiological (+Ca) calcium levels, in the presence or absence of 5 µM of the PI3-K inhibitor LY294002. Proliferation was determined on the basis of cell biomass using the MTT assay. Data points represent mean absorbance values for 6 replicate wells (±S.E.M.). Results for all data points are also presented in the form of bar graphs (lower panel) for the purpose of statistical analysis. ns, non-significant; **, P<0.01; ***, P<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2964323&req=5

pone-0013621-g002: Calcium-induced increase in proliferation in low-density NHU cultures occurs via activation of the PI3-K/AKT pathway.(A) NHU cells were seeded in medium containing low [Ca2+] (0.09 mM) and left to attach overnight. Calcium concentration was then increased to 2 mM and protein lysates were prepared at the indicated time-points for immunoblotting. Expression of phospho-AKT (green) and total AKT (red) was determined using rabbit and mouse antibodies, respectively, followed by fluorochrome-conjugated secondary antisera as in Figure 1C. Immunolabelling was visualised and densitometry to determine fold induction of p-AKT expression with respect to total AKT was performed as in Figure 1C. (B) NHU cells were cultured in medium containing 0.09 mM (Low Ca2+) or 2.0 mM (Phys Ca2+) [Ca2+]. Expression of phospho-AKT (p-AKT) was determined by immunofluorescence microscopy using anti-p-AKT rabbit antibody followed by goat anti-rabbit antibody conjugated with Alexa Fluor 488 (green). Cell nuclei were visualised using Hoechst 33258 (blue). (C) NHU cells were seeded into 96-well plates and cultured for a period of 7 days in medium containing either low (-Ca) or physiological (+Ca) calcium levels, in the presence or absence of 5 µM of the PI3-K inhibitor LY294002. Proliferation was determined on the basis of cell biomass using the MTT assay. Data points represent mean absorbance values for 6 replicate wells (±S.E.M.). Results for all data points are also presented in the form of bar graphs (lower panel) for the purpose of statistical analysis. ns, non-significant; **, P<0.01; ***, P<0.001.
Mentions: 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 immunofluorescence 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.

Bottom Line: The potential for E-cadherin to co-ordinate different proliferation-associated signalling pathways has yet to be fully explored.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 β-catenin-TCF signalling.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.

View Article: PubMed Central - PubMed

Affiliation: Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York, 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 β-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.

Show MeSH
Related in: MedlinePlus