QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 11 December 2002
doi: 10.1242/jcs.00224

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bolós, V. Articles by Cano, A. Search for Related Content PubMed PubMed Citation Articles by Bolós, V. Articles by Cano, A. Social Bookmarking                         What's this?

The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors

Victoria Bolós¹, Hector Peinado¹, Mirna A. Pérez-Moreno^1,*, Mario F. Fraga², Manel Esteller² and Amparo Cano^1,

¹ Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM), Arturo Duperier, 4, 28029 Madrid, Spain
² Centro Nacional de Investigaciones Oncológicas, Melchor Fernández Almagro, 4, 28029 Madrid, Spain
^* Present address: Laboratory of Mammalian Cell Biology and Development, The Rockefeller University. 1230 York Avenue, Box 300. New York, NY 10021, USA

View larger version (65K): [in a new window]   Fig. 1. The Slug transcription factor binds to the E-pal element of E-cadherin promoter through the E-boxes. (A) Recombinant GST-Slug protein (1 µg) was incubated with the 32P-labeled E-pal probe in the absence or presence of 500-fold molar excess of wild-type or mutant cold oligonucleotides or in the presence of 5 µg of anti-Slug monoclonal antibody or control mouse IgG. The retarded complexes are indicated by arrows and the supershifted complex by an arrowhead. (B). Recombinant GST, GST-Snail, GST-Slug and GST-E47 proteins were incubated at the indicated combinations with the 32P-labeled wild-type E-pal probe. The different retarded complexes detected are indicated by arrows. The complete sequence of the E-pal probe is indicated at the bottom of the figure with the two E-boxes showed in black letters. Asterisks indicate the position of the mutated nucleotides in the mEpal oligonucleotide.

View larger version (55K): [in a new window]   Fig. 2. Differential binding of Snail and Slug to the E-pal element. Recombinant GST-Slug (left panel) and GST-Snail (right panel) proteins were incubated at the indicated concentrations (50-500 ng) with the 32P-labeled wild-type E-pal probe in the presence or absence of 1,000-fold molar excess of the wild-type or mutant cold oligonucleotides or in the presence of 5 µg of anti-Slug monoclonal antibody (left panel) or anti-Snail polyclonal antibody (right panel). The different retarded complexes are indicated by arrows and the supershifted complexes by arrowheads. Wild-type E-pal and mE-pal oligonucleotides are as shown in Fig. 1.

View larger version (21K): [in a new window]   Fig. 3. Binding affinity of Snail, E47 and Slug recombinant proteins to E-pal synthetic oligonucleotide estimated by capillary electrophoretic mobility shift assays (CEMSA). (A,B) Electrophoregrams for mixtures of 6-FAM-labeled wild-type E-pal oligonucleotide (24 nM) and (A) 1 nM of control GST, GST-Snail, GST-E47 and GST-Slug or (B) 1 µM of GST-Snail, GST-E47 and GST-Slug, subjected to CEMSA analysis as described in Materials and Methods. RFU, relative fluorescence units; min, elution time in minutes. The elution of the main E-pal oligo—GST-Snail complex is indicated by arrows (E-pal/protein) in both panels. (C) Concentration-dependent binding of GST-Snail (white circles), GST-E47 (black circles) and GST-Slug (white squares) to the 6-FAM wild-type E-pal probe. The indicated concentrations of the different recombinant proteins were analyzed by CEMSA, in duplicated samples, in three independent experiments. The single-site ligand-binding fit was performed using GraFit 3.1 software. R, saturation ([complex]/[complex]+[DNA]). [protein], protein concentration of intact GST-factors. Results are expressed as mean±s.d. (D) Western blot analysis of purified recombinant proteins. 50 µg of the purified recombinant proteins analyzed with anti-GST or anti-E47 polyclonal antibodies, as indicated. Migration of the molecular weight markers (in kDa) and of the different intact recombinant proteins is indicated at the side of the panel.

View larger version (16K): [in a new window]   Fig. 4. Slug represses the activity of the mouse E-cadherin promoter both in transient and stable MDCK transfectants. (A) MDCK cells were transiently cotransfected with 200 ng of the -178 wild-type E-cadherin promoter construct fused to the luciferase reporter gene in the presence of the indicated amounts of pcDNA3 (Mock), pcDNA3-Slug or pcDNA3-Snail vectors. Luciferase and renilla activities were determined 24 hours after transfection. The activity of the promoter is expressed relative to that obtained in the mock-transfected cells. Results represent the mean±s.d. of at least two independent results. (B) The activity of the -178 wild-type E-cadherin promoter is strongly reduced or completely silenced in Slug- and Snail-expressing cells. MDCK-mock, two independent MDCK-Slug clones and MDCK-Snail cells were transiently transfected with the -178 wild-type (white bars) or mE-pal (grey bars) E-cadherin constructs fused to the CAT reporter gene. Luciferase and CAT activities were determined 24 hours after transfection. The activity of the promoter constructs is represented relative to that of the -178 wt construct detected in the mock-transfected clone. Results represent the mean±s.d. of two independent experiments. The relative levels of activity for both constructs in each cell line are also indicated at the bottom. Slu1I represents one subclone isolated from an original Slu1 clone; Slu3 represents an independently isolated clone.

View larger version (100K): [in a new window]   Fig. 5. Stable transfection of Slug into MDCK cells induces epithelial to mesenchymal conversion concomitantly with the loss of epithelial markers and the gain of mesenchymal markers. (a-f) Phase-contrast images of living, subconfluent cultures of a mock-transfected clone (a), four Slug-transfected clones (b,c,d,e) and Snail-transfected cells (f). (g-x) Immunofluorescent images of the indicated cell lines showing the localization and organization of E-cadherin (g-l), fibronectin (m-r) and vimentin (s-x). See the loss of E-cadherin stain and the increased expression and fibrous organization of fibronectin and vimentin in the Slug and Snail transfectants. Slu2 and Slu3 represent independent clones; Slu1I and Slu1 III represent two subclones isolated from an original Slu1 clone. Bars, 40 µm (a-f); 20 µm (g-x).

View larger version (59K): [in a new window]   Fig. 6. The phenotypic effects induced by ectopic Slug expression in MDCK cells are associated with a full repression of E-cadherin expression and are independent of endogenous Snail expression. (A) Western blot analysis of whole cell extracts of the indicated proteins in mock- and Slug-transfected clones. E-cad, E-cadherin; Plako, plakoglobin; ß-cat, ß-catenin; VN, vimentin. Detection of -tubulin (-tub) levels was used as a loading control. (B) The presence of canine E-cadherin, mouse Slug and canine Snail transcripts in mock- and Slug-transfected clones was analyzed by RT-PCR. The expression of GAPDH was analyzed in the same samples as a control for the amount of cDNA present in each sample. The —RT lane shows the results of amplification in the absence of template. (C) Inmunofluorescence analysis for Slug expression in mock (a) and Slu 3 clone (b,c). Ectopic expression of the Slug protein was observed in the nuclei of the Slug-transfected cells (arrows in b and c). Bar, 20 µm. (D) The activity of the mSnail promoter in the Slug-transfectants corresponds to the endogenous Snail mRNA levels. The indicated cell lines were transiently transfected with the wild-type mouse Snail promoter construct (white bars) or with the mutant E-box (at -221) construct (grey bars) fused to a luciferase reporter gene. Luciferase and renilla activities were determined 24 hours after transfection. The activity of the promoter is expressed relative to that obtained in the mock-transfected cells with the wild-type construct. Results represent the mean±s.d. of at least two independent experiments.

View larger version (141K): [in a new window]   Fig. 7. Slug expression in epithelial cells induces a migratory and motile phenotype. (A) The motility/migratory behaviour of mock- (a-c) and Slug-transfected (d-i) cells was analyzed in an in vitro wound model. Confluent cultures of the mock and Slug-transfected clones were gently scratched with a pipette tip to produce a wound. Photographs of the cultures were taken immediately after the incision (a,d,g) and after 6 hours (b,e,h) and 9 hours (c,f,i) in culture. Bar, 40 µm. (B). The organization of F-actin was analyzed in fixed and permeabilized cells by incubation with FITC-phalloidin. Fluorescence images of mock (a), two Slug-transfected cell lines (b,c), and Snail-transfected cells (d). F-actin is organized in abundant stress fibres and lamellipodia-like structures in Slug-expressing cells. Bar, 20 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter