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First published online June 18, 2008
doi: 10.1242/10.1242/jcs.021667

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E-cadherin controls β-catenin and NF-B transcriptional activity in mesenchymal gene expression

Guiomar Solanas^1,*, Montserrat Porta-de-la-Riva^2,*, Cristina Agustí², David Casagolda¹, Francisco Sánchez-Aguilera², María Jesús Larriba³, Ferran Pons², Sandra Peiró², Maria Escrivà², Alberto Muñoz³, Mireia Duñach^1,, Antonio García de Herreros^2,4, and Josep Baulida^2,

¹ Unitat de Biofísica-CEB, Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain
² Programa de Recerca en Càncer, IMIM-Hospital del Mar, E-8003, Barcelona, Spain
³ Instituto de Investigaciones Biomédicas `Alberto Sols', Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain
⁴ Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain

View larger version (43K): [in this window] [in a new window]   Fig. 1. E-cadherin represses fibronectin and LEF1 gene expression. (A) Levels of E-cadherin and Snail1 proteins in SW-480 transfectants. SDS-protein extracts obtained from SW-480 cells stably transfected with the indicated genes and grown until 50-60% confluence were analysed by western blot (WB) with the indicated antibodies. (B) E-cadherin expression downregulates fibronectin and LEF1 RNA levels. Fibronectin and LEF1 RNAs were determined by qRT-PCR in SW-480 cell lines. Values are relative to that obtained in control SW-480 cells. Graphics show the average ± s.d. of the three values obtained for every sample. (C) E-cadherin expression downregulates fibronectin and LEF1 promoter activity. Activities of –341/+265 fibronectin promoter and –735/+1077 LEF1 promoter were determined after transfection of these promoters, which were inserted into pGL3 plasmid as described, into subconfluent SW-480 stable transfectants. The values show the average ± s.d. of two experiments performed in triplicate samples, and are relative to the value obtained in control SW-480 cells. (D) Cell-culture confluence regulates SW-480 E-cadherin levels. SW480 ADH cells were grown in standard conditions until 50-60% confluence (Sub-Conf) or 3 days after 100% confluence (Conf). 1% SDS total protein extracts were obtained and analysed by western blot with anti-E-cadherin or anti-pyruvate-kinase mAbs. (E) Expression of fibronectin and LEF1 decrease in confluent SW-480 cells. Fibronectin and LEF1 RNAs were determined by qRT-PCR and values (average ± s.d.) referred to the value obtained in the sub-confluent cells. (F,G) Interference of E-cadherin expression upregulates fibronectin and LEF1 RNA levels. Cells expressing an siRNA specific to E-cadherin or a scrambled control (Irr) were cultured until confluence, and E-cadherin and actin levels were determined by western blot (F). In parallel, fibronectin, LEF1 or HPRT RNA content were determined in these cells by semi-quantitative RT-PCR (G).

View larger version (38K): [in this window] [in a new window]   Fig. 2. β-catenin depletion downregulates fibronectin and LEF1 transcript levels. (A,B) Snai1l increases fibronectin and LEF1 gene expression in LS174T cells. RNAs were extracted from LS174T control cells transfected with Snail1 in a eukaryotic expression vector and analysed by semi-quantitative PCR (A) or qRT-PCR (B) with specific oligonucleotides for the indicated genes. Representative clones are shown in A; the average of the results obtained with three different clones are shown in B. (C) Inducible repression of β-catenin in LS-174T clones. Total-cell protein extracts or RNAs were obtained from clones expressing Snail1 (clones S) or controls (clones C). Doxycycline (1 µg/ml) was added for 6 days prior to the preparation of the extracts as indicated. β-catenin and Snail1 expression were analysed by western blot with specific mAbs. Anti--tubulin was used as a loading control. Endogenous full-length TCF4 mRNA and exogenous TCF4 plus endogenous TCF4 mRNA were also analysed by RT-PCR. As a control, HPRT levels were determined. (D) β-catenin siRNA decreases fibronectin and LEF1 transcript levels. RNA was obtained from the above-mentioned cell clones and levels of fibronectin, LEF1 and Myc were determined by qRT-PCR. As a control, HPRT RNA levels were determined. The figure shows the values of fibronectin, LEF1 and Myc RNA levels determined in the presence of doxycycline and referred to the level of the corresponding RNA in the absence of this drug. The average ± s.d. of two independent experiments performed in duplicate with two representative clones is shown.

View larger version (13K): [in this window] [in a new window]   Fig. 3. E-cadherin controls the transcriptional activity of β-catenin on the fibronectin promoter. (A) Snail1 and E-cadherin modulate β-catenin and TCF4 transcriptional activity. The activity of a β-catenin–TCF4-dependent promoter (TOP) was determined in SW-480 cells stably transfected with Snail1-HA, E-cadherin or both. The results are the average ± s.d. of three experiments. (B) Binding of β-catenin to the fibronectin promoter is sensitive to E-cadherin. ChIP assays were carried out as described in the Materials and Methods, immunoprecipitating crosslinked nuclear extracts from SW-480 cells stably transfected with the indicated genes. The average ± s.d. of two experiments is shown.

View larger version (32K): [in this window] [in a new window]   Fig. 4. E-cadherin inhibits NF-B transcriptional activity on the fibronectin promoter. (A) Binding of NF-B to the fibronectin promoter is sensitive to E-cadherin. ChIP assays were carried out as described in the Materials and Methods, immunoprecipitating crosslinked nuclear extracts from SW-480 cells stably transfected with the indicated genes. Semi-quantitative analysis from one experiment of the three performed (right) or the average ± s.d. of quantitative analysis of three experiments (left) is shown. (B) E-cadherin controls p65 association to DNA. Gel shift assays were performed as described in the Materials and Methods, with an oligonucleotide containing the NF-B-binding element present in the human fibronectin promoter. Nuclear extracts form SW-480 cells transfected with Snail1 alone or both Snail1 and E-cadherin were used. In the experiment shown in the left panel, binding of the radioactive probe was competed with a 50- or 100-fold excess of non-radioactive probe containing a consensus binding element for NF-B, either wild-type (WT) or mutated (MUT). When indicated (right panel), binding was carried out in the presence of an irrelevant IgG or a mAb specific for p65, as indicated in the Materials and Methods. The arrows show the specific band detected with this assay; the arrowhead shows the migration of the free probe. Note that the upper band was not competed either by the NF-B consensus oligonucleotide or by the p65 antibody; therefore, we considered that it did not correspond to a p65 complex. The results of a representative experiment of the four (right) or five (left) that were performed are shown. (C) Snail1 and E-cadherin modulate NF-B transcriptional activity. The activity of an NF-B-dependent promoter (NF3) was determined in SW-480 cells stably transfected with Snail1-HA, E-cadherin or both. The same experiment was performed in MiaPaca-2 cells transiently transfected with these two cDNAs. The results correspond to the average ± s.d. of three experiments.

View larger version (51K): [in this window] [in a new window]   Fig. 5. E-cadherin prevents β-catenin and NF-B nuclear localisation. (A) Cell fractionation of p65 and β-catenin. Subcellular fractions were prepared from SW-480 cells and analysed by western blot. Lamin B1 and TBP expression were used as nuclear markers; pyruvate kinase and E-cadherin were used as markers for the cytosolic-plus-membrane fraction. (B) Immunolocalisation of p65 and β-catenin is affected by E-cadherin expression. Analysis of β-catenin (green) and NF-B (red) subcellular localisation was carried out in SW-480 Snail1-transfected and SW-480 Snail1- and E-cadherin-transfected cells. E-cadherin-positive cells (right) were grown at equal cell density to E-cadherin-negative cells (left), or to a lower density (middle) in order to better visualize cell colonies. The analysis was performed by immunofluorescence using mAbs against β-catenin and NF-B. No signal was obtained when the same analysis was performed in the absence of primary antibody. (C) Subcellular co-distribution of p65 and E-cadherin. The subcellular distribution of NF-B p65 subunit (green) and E-cadherin (red) was determined by immunofluorescence in SW-480 E-cadherin-transfected cells as mentioned above, using specific mAbs against these two proteins. The upper row shows an amplified area selected from the panels shown below (boxed). An xz section is shown in the bottom row.

View larger version (48K): [in this window] [in a new window]   Fig. 6. NF-B associates with E-cadherin and other components of the junctional complex. (A) NF-B co-immunoprecipitates with E-cadherin and β-catenin. The p65 subunit of NF-B was immunoprecipitated from whole-cell extracts of SW-480 cells stably transfected with Snail1-HA and E-cadherin. The associated proteins were analysed with specific mAbs against E-cadherin, and β-, - and p120-catenin. (B)NF-B associates with E-cadherin and E-cadherin-associated proteins. Pull-down assays were performed by incubating 5 pmol of the different GST-fused proteins with 500 µg of cell extracts from confluent SW-480 cells prepared in RIPA buffer. Protein complexes were affinity-purified with glutathione-Sepharose and analysed by western blotting with anti-p65 mAb. Blots were re-analysed with anti-GST antibodies to ensure equal loading of samples. 3% of the total-cell extracts used for the assay was loaded in the input lane. When indicated, 25 pmols of cytoE-cadherin recombinant protein were added to the binding assays. (C) Recombinant purified p65 [1 (+) or 2 (++) pmol] was incubated with 5 pmol of either cytosolic fragment of E-cadherin fused to GST (GST–cytoE-cad) or GST as a control in the absence or presence of 500 µg of SW-480 cell extracts. Protein complexes were affinity-purified with glutathione-Sepharose and analysed by western blot as above. Known amounts of extract (3 µg) and recombinant p65 (0.05 pmol, 3 ng) were included in the same blot as controls (Input). (D) NF-B interacts only with E-cadherin-associated β-catenin. Extracts, prepared as in A, from IEC-18 cells expressing E-cadherin were immunoprecipitated with a mAb specific to E-cadherin in two successive rounds. The presence of p65, E-cadherin and β-catenin was analysed before and after E-cadherin immunodepletion by western blot (input). Extracts before or after immunodepletion were immunoprecipitated with mAbs against E-cadherin, β-catenin or an irrelevant IgG as control and presence of E-cadherin, β-catenin or p65 in the immunocomplex was determined by western blot. (E) IB prevents association of NF-B with E-cadherin. NIH3T3 fibroblasts were transfected with 7.5 µg of pcDNA3 control, pcDNA3–E-cadherin or pCdna3-IB S32,S36 mutant (a kind gift from A. Bigas, IDIBELL, Barcelona, Spain). After 48 hours, cell extracts were prepared and the p65 subunit of NF-B was immunoprecipitated. The presence of E-cadherin, catenins and IB in the complexes was analysed by western blot with specific mAbs. The results shown correspond to a representative experiment out of the three performed.

View larger version (39K): [in this window] [in a new window]   Fig. 7. Disruption of E-cadherin-mediated contacts activates NF-B transcriptional activity. (A) K-ras expression downregulates E-cadherin interaction with p65. The p65 subunit of NF-B was immunoprecipitated from total-cell extracts prepared from IEC or IEC K-ras-transfected cells. Immunocomplexes were analysed by western blot with mAbs against E-cadherin, and β-, - and p120-catenin. The results given correspond to a representative experiment out of the three performed. (B) E-cadherin siRNA affects the association of p65 to junctional-complex components. IEC cells were transfected with an siRNA specific to E-cadherin or with an irrelevant siRNA as control. Total extracts were prepared, the p65 subunit of NF-B was immunoprecipitated and immunocomplexes were analysed by western blot with mAbs against E-cadherin, and β-, - and p120-catenin. The results given correspond to a representative experiment out of the three performed. (C) E-cadherin siRNA increases p65 nuclear levels. Presence of p65 in the nucleus was determined by western blot analysis of cell fractions prepared from cells transfected with E-cadherin siRNAs or irrelevant siRNAs (Irr), as indicated above. Lamin B1 and TBP or pyruvate kinase were used as nuclear and cytosolic markers, respectively. (D,E) E-cadherin siRNA increases NF-B transcriptional activity. IEC cells were transfected with irrelevant or E-cadherin siRNAs and NF3 reporter plasmid. Luciferase activity was determined after 48 hours and is shown as the average ± s.d. of three experiments performed in triplicate. In E, cells were deprived of serum for 16 hours and incubated with TNF (20 ng/ml) for 6 hours before analysis of NF3 promoter activity.

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