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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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Related in: MedlinePlus

Loss of E-cadherin function enhances NHU cell proliferation and activates the EGFR/ERK and β-catenin/TCF signalling pathways.(A) NHU-Con and NHU-ECmut cells were seeded in 96-well plates and cultured as above. [3H]-thymidine (TdR) precursor was added 24 hours later and 16 hours post-pulsing, cells were harvested and TdR uptake measured by scintillation spectrometry. Data represent mean of cpm counts (±S.E.M.) for 12 replicate wells. ns, non-significant; *, P<0.05; **, P<0.01. Of note, NHU-Con cells showed higher proliferation levels in low calcium compared to physiologic calcium conditions, hence there was no apparent calcium-mediated enhancement in growth described above. This is because [3H]-thymidine incorporation studies assessed proliferation in NHU cultures that were at >50% confluence, thus missing the initial phase where cells exhibit calcium-mediated increased growth at low-density. (B) NHU-Con and NHU-ECmut cells were cultured in medium containing 0.09 mM (low Ca) or 2.0 mM (phys Ca) [Ca2+] and proliferation assessed by cell counting as in Figure 1A. Each data point represents the mean (±S.E.M.) of 3 replicates and results are representative of at least two independent experiments. Results for days 3, 5 and 7 are also presented in the form of bar graphs (lower panel) for the purpose of statistical analysis and clarity. ns, non-significant; *, P<0.05; **, P<0.01; ***, P<0.001. (C) NHU-Con and NHU-ECmut cells were cultured in medium containing low (-Ca2+) and physiological (+Ca2+) calcium levels for 24 hours and protein lysates were prepared. Expression of phospho-ERK (p-ERK) and -AKT (p-AKT) as well as total ERK and AKT was determined using primary and secondary antibodies described in Figures 1C and 2A. Densitometry was performed to determine fold induction of p-ERK and p-AKT expression following normalisation against total ERK and total AKT, respectively. Bar graphs represent results relative to the expression level for NHU-Con in low calcium (-Ca2+). (D) NHU-Con and NHU-ECmut cells were transfected with the TCF/LEF firefly luciferase reporter TOPflash or the FOPflash control plasmid, alongside the Renilla luciferase expression vector pRL-tk. Four hours post-transfection, culture supernatants were adjusted for the desired level of calcium concentration, i.e. 0.09 mM (Low Ca2+) or 2.0 mM (Phys Ca2+). Cell lysates were prepared and reporter activity assessed in a 96-well format using the Dual-Luciferase Reporter Assay on a microplate reader for pair-wise detection of luminescence activity for each reporter. Firefly luciferase values were normalised against those of Renilla luciferase and were then expressed as fold activity with respect to that obtained for FOPflash in NHU-Con cells cultured in low or physiological calcium. Bars represent mean fold Firefly luciferase activity (±S.E.M.) for 6 replicate samples.
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pone-0013621-g004: Loss of E-cadherin function enhances NHU cell proliferation and activates the EGFR/ERK and β-catenin/TCF signalling pathways.(A) NHU-Con and NHU-ECmut cells were seeded in 96-well plates and cultured as above. [3H]-thymidine (TdR) precursor was added 24 hours later and 16 hours post-pulsing, cells were harvested and TdR uptake measured by scintillation spectrometry. Data represent mean of cpm counts (±S.E.M.) for 12 replicate wells. ns, non-significant; *, P<0.05; **, P<0.01. Of note, NHU-Con cells showed higher proliferation levels in low calcium compared to physiologic calcium conditions, hence there was no apparent calcium-mediated enhancement in growth described above. This is because [3H]-thymidine incorporation studies assessed proliferation in NHU cultures that were at >50% confluence, thus missing the initial phase where cells exhibit calcium-mediated increased growth at low-density. (B) NHU-Con and NHU-ECmut cells were cultured in medium containing 0.09 mM (low Ca) or 2.0 mM (phys Ca) [Ca2+] and proliferation assessed by cell counting as in Figure 1A. Each data point represents the mean (±S.E.M.) of 3 replicates and results are representative of at least two independent experiments. Results for days 3, 5 and 7 are also presented in the form of bar graphs (lower panel) for the purpose of statistical analysis and clarity. ns, non-significant; *, P<0.05; **, P<0.01; ***, P<0.001. (C) NHU-Con and NHU-ECmut cells were cultured in medium containing low (-Ca2+) and physiological (+Ca2+) calcium levels for 24 hours and protein lysates were prepared. Expression of phospho-ERK (p-ERK) and -AKT (p-AKT) as well as total ERK and AKT was determined using primary and secondary antibodies described in Figures 1C and 2A. Densitometry was performed to determine fold induction of p-ERK and p-AKT expression following normalisation against total ERK and total AKT, respectively. Bar graphs represent results relative to the expression level for NHU-Con in low calcium (-Ca2+). (D) NHU-Con and NHU-ECmut cells were transfected with the TCF/LEF firefly luciferase reporter TOPflash or the FOPflash control plasmid, alongside the Renilla luciferase expression vector pRL-tk. Four hours post-transfection, culture supernatants were adjusted for the desired level of calcium concentration, i.e. 0.09 mM (Low Ca2+) or 2.0 mM (Phys Ca2+). Cell lysates were prepared and reporter activity assessed in a 96-well format using the Dual-Luciferase Reporter Assay on a microplate reader for pair-wise detection of luminescence activity for each reporter. Firefly luciferase values were normalised against those of Renilla luciferase and were then expressed as fold activity with respect to that obtained for FOPflash in NHU-Con cells cultured in low or physiological calcium. Bars represent mean fold Firefly luciferase activity (±S.E.M.) for 6 replicate samples.

Mentions: 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 [3H]-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.


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

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

Loss of E-cadherin function enhances NHU cell proliferation and activates the EGFR/ERK and β-catenin/TCF signalling pathways.(A) NHU-Con and NHU-ECmut cells were seeded in 96-well plates and cultured as above. [3H]-thymidine (TdR) precursor was added 24 hours later and 16 hours post-pulsing, cells were harvested and TdR uptake measured by scintillation spectrometry. Data represent mean of cpm counts (±S.E.M.) for 12 replicate wells. ns, non-significant; *, P<0.05; **, P<0.01. Of note, NHU-Con cells showed higher proliferation levels in low calcium compared to physiologic calcium conditions, hence there was no apparent calcium-mediated enhancement in growth described above. This is because [3H]-thymidine incorporation studies assessed proliferation in NHU cultures that were at >50% confluence, thus missing the initial phase where cells exhibit calcium-mediated increased growth at low-density. (B) NHU-Con and NHU-ECmut cells were cultured in medium containing 0.09 mM (low Ca) or 2.0 mM (phys Ca) [Ca2+] and proliferation assessed by cell counting as in Figure 1A. Each data point represents the mean (±S.E.M.) of 3 replicates and results are representative of at least two independent experiments. Results for days 3, 5 and 7 are also presented in the form of bar graphs (lower panel) for the purpose of statistical analysis and clarity. ns, non-significant; *, P<0.05; **, P<0.01; ***, P<0.001. (C) NHU-Con and NHU-ECmut cells were cultured in medium containing low (-Ca2+) and physiological (+Ca2+) calcium levels for 24 hours and protein lysates were prepared. Expression of phospho-ERK (p-ERK) and -AKT (p-AKT) as well as total ERK and AKT was determined using primary and secondary antibodies described in Figures 1C and 2A. Densitometry was performed to determine fold induction of p-ERK and p-AKT expression following normalisation against total ERK and total AKT, respectively. Bar graphs represent results relative to the expression level for NHU-Con in low calcium (-Ca2+). (D) NHU-Con and NHU-ECmut cells were transfected with the TCF/LEF firefly luciferase reporter TOPflash or the FOPflash control plasmid, alongside the Renilla luciferase expression vector pRL-tk. Four hours post-transfection, culture supernatants were adjusted for the desired level of calcium concentration, i.e. 0.09 mM (Low Ca2+) or 2.0 mM (Phys Ca2+). Cell lysates were prepared and reporter activity assessed in a 96-well format using the Dual-Luciferase Reporter Assay on a microplate reader for pair-wise detection of luminescence activity for each reporter. Firefly luciferase values were normalised against those of Renilla luciferase and were then expressed as fold activity with respect to that obtained for FOPflash in NHU-Con cells cultured in low or physiological calcium. Bars represent mean fold Firefly luciferase activity (±S.E.M.) for 6 replicate samples.
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pone-0013621-g004: Loss of E-cadherin function enhances NHU cell proliferation and activates the EGFR/ERK and β-catenin/TCF signalling pathways.(A) NHU-Con and NHU-ECmut cells were seeded in 96-well plates and cultured as above. [3H]-thymidine (TdR) precursor was added 24 hours later and 16 hours post-pulsing, cells were harvested and TdR uptake measured by scintillation spectrometry. Data represent mean of cpm counts (±S.E.M.) for 12 replicate wells. ns, non-significant; *, P<0.05; **, P<0.01. Of note, NHU-Con cells showed higher proliferation levels in low calcium compared to physiologic calcium conditions, hence there was no apparent calcium-mediated enhancement in growth described above. This is because [3H]-thymidine incorporation studies assessed proliferation in NHU cultures that were at >50% confluence, thus missing the initial phase where cells exhibit calcium-mediated increased growth at low-density. (B) NHU-Con and NHU-ECmut cells were cultured in medium containing 0.09 mM (low Ca) or 2.0 mM (phys Ca) [Ca2+] and proliferation assessed by cell counting as in Figure 1A. Each data point represents the mean (±S.E.M.) of 3 replicates and results are representative of at least two independent experiments. Results for days 3, 5 and 7 are also presented in the form of bar graphs (lower panel) for the purpose of statistical analysis and clarity. ns, non-significant; *, P<0.05; **, P<0.01; ***, P<0.001. (C) NHU-Con and NHU-ECmut cells were cultured in medium containing low (-Ca2+) and physiological (+Ca2+) calcium levels for 24 hours and protein lysates were prepared. Expression of phospho-ERK (p-ERK) and -AKT (p-AKT) as well as total ERK and AKT was determined using primary and secondary antibodies described in Figures 1C and 2A. Densitometry was performed to determine fold induction of p-ERK and p-AKT expression following normalisation against total ERK and total AKT, respectively. Bar graphs represent results relative to the expression level for NHU-Con in low calcium (-Ca2+). (D) NHU-Con and NHU-ECmut cells were transfected with the TCF/LEF firefly luciferase reporter TOPflash or the FOPflash control plasmid, alongside the Renilla luciferase expression vector pRL-tk. Four hours post-transfection, culture supernatants were adjusted for the desired level of calcium concentration, i.e. 0.09 mM (Low Ca2+) or 2.0 mM (Phys Ca2+). Cell lysates were prepared and reporter activity assessed in a 96-well format using the Dual-Luciferase Reporter Assay on a microplate reader for pair-wise detection of luminescence activity for each reporter. Firefly luciferase values were normalised against those of Renilla luciferase and were then expressed as fold activity with respect to that obtained for FOPflash in NHU-Con cells cultured in low or physiological calcium. Bars represent mean fold Firefly luciferase activity (±S.E.M.) for 6 replicate samples.
Mentions: 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 [3H]-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.

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