* Centre National de la Recherche Scientifique-Institut Curie, 75231 Paris Cedex 05, France; and Craniofacial Developmental
Biology and Regeneration Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland
20892-4370
Epithelial-mesenchymal transition (EMT) is an essential morphogenetic process during embryonic development. It can be induced in vitro by hepatocyte growth factor/scatter factor (HGF/SF), or by FGF-1 in our NBT-II cell model for EMT. We tested for a central role in EMT of a zinc-finger protein called Slug. Slug mRNA and protein levels were increased transiently in FGF-1-treated NBT-II cells. Transient or stable transfection of Slug cDNA in NBT-II cells resulted in a striking disappearance of the desmosomal markers desmoplakin and desmoglein from cell-cell contact areas, mimicking the initial steps of FGF-1 or HGF/SF- induced EMT. Stable transfectant cells expressed Slug protein and were less epithelial, with increased cell spreading and cell-cell separation in subconfluent cultures. Interestingly, NBT-II cells transfected with antisense Slug cDNA were able to resist EMT induction by FGF-1 or even HGF/SF. This antisense effect was suppressed by retransfection with Slug sense cDNA. Our results indicate that Slug induces the first phase of growth factor-induced EMT, including desmosome dissociation, cell spreading, and initiation of cell separation. Moreover, the antisense inhibition experiments suggest that Slug is also necessary for EMT.
Epithelial cells adhere to each other through specialized structures essential for the maintenance of
epithelial organization and differentiation. Among
these, structures linked to the cytokeratin intermediate filament network appear to provide the strongest and most
resilient adhesion (12, 15). The core unit of such structures
is the desmosome, which appears early during epithelial differentiation (13, 24, 30, 37). Desmoglein and desmocollins are desmosome-specific cadherins that mediate cell-
cell binding (15, 34). They are part of a molecular complex
involving Individualization of cells emerging and dissociating from
an epithelial sheet is one of the basic mechanisms involved
in embryonic development. In early postimplantation mouse
embryos, it appears that all cells that contribute to embryonic tissues are epithelial cells expressing desmosomes
(30). Therefore, cellular dissociation involves the disintegration of these desmosomes and other cell-cell adhesion
systems. Depending on the species, this necessary process of epithelial-mesenchymal transition (EMT)1 occurs at several critical stages during development, such as gastrulation, neural crest cell emigration, and organogenesis (for
reviews see references 19, 27, 28, 38, 52).
Several inducers, including extracellular molecules (5,
32, 33, 50, 54, 67, 68) and growth-factors from the transforming growth factor (TGF) Epithelial cells can be induced to dissociate by treatment with hepatocyte growth factor/scatter factor (HGF/
SF), and in some cases by other growth factor members of
the FGF (65), EGF, and TGF families (3, 16, 43). The EMT
process is initiated by cell-cell dissociation, which is preceded by the internalization of desmosomal components
and progressive disappearance from cell-cell contact areas
(7). As studied in Madin-Darby canine kidney (MDCK)
cells, these scatter factors can also act as growth factors and morphogenetic factors, using specific transduction
pathways in each case (1, 17, 25, 51, 58).
We have previously characterized a rat bladder carcinoma cell line, NBT-II, that is induced by FGF-1 to undergo an EMT characterized by a switch to a fibroblastoid
phenotype and by cell migration (7, 65). Desmosomal
components, including desmoplakin and desmoglein, were
found to be internalized and disappear from the surface
starting 4 h after initiation of FGF-1 treatment. By 6 h,
>50% of the cells no longer express desmosomes. By 12 h,
cells undergo active migration on the substrate (7). In contrast, adherens junction components such as E-cadherin
and catenins were not altered quantitatively during this
epithelial-mesenchymal transition (8). However, E-cadherin
was redistributed from cell-cell contact areas to a diffuse
distribution on the cell surface. FGF-1 activation was mediated through the receptor FGFR2c/KGFR, which underwent alternative splicing during the EMT process (53). The FGFR2c/KGFR tyrosine kinase domain was found to
be involved in transducing the EMT process through phosphorylation (6). In addition, an EMT-specific activation of
pp60c-src was demonstrated (49), and overexpression of normal c-src in NBT-II cells was found to oversensitize them
to EMT induced by FGF-1. Transcriptional and translational events were also found to be required for EMT
since actinomycin D inhibited EMT (Savagner, P., unpublished observation), as did cycloheximide (8).
Prompted by the developmental studies cited above, we
investigated the role and cell biological effects of the zincfinger protein Slug in FGF-1-induced EMT in NBT-II
cells, extended our studies to HGF/SF, and tested for a potential direct role for Slug in inducing EMT.
Reagents
Human recombinant FGF-1 was kindly provided by Dr. M. Jaye (Rhône
Poulenc Rorer Central Research Inc., King of Prussia, PA). HGF/SF was
purchased from PeproTech (Rocky Hill, NJ). Mouse monoclonal antibodies against bovine desmoplakins 1 and 2 (clone DP 2.15, 2.17, and 2.20)
and bovine desmoglein ("band 3": Clone DG 3.10) were purchased from
American Research Products (Solon, OH). Monoclonal antibodies against
E-cadherin (Clone 34) were purchased from Transduction Laboratories
(Lexington, KY). Monoclonal antibodies against human vimentin (clone
V 9) and bovine cytokeratins (pan-cytokeratin) were purchased from
Zymed Labs (S. San Francisco, CA). Mouse antibodies against chicken
Slug (39) were a gift from Dr. T. Jessell (Columbia University, New York).
Cell Culture
The rat bladder carcinoma NBT-II cell line was initially obtained from
Prof. Marc Mareel (University Hospital, Ghent, Belgium). Cells were cultured in DME supplemented with glutamine, antibiotics, and 10% heatinactivated FCS as previously described (7). Human keratinocyte primary
culture YF29 (newborn foreskin, fourth passage) was grown by A. Rochat
and Y. Barrandon (Ecole Normale Supérieure, Paris, France) in DME
supplemented with glutamine, antibiotics, and 10% heat-inactivated FCS.
RNA Preparation and PCR
Total RNA was extracted from human keratinocytes and NBT-II cells
that were growth-arrested by serum deprivation and incubated in serumcontaining medium for 6 h by the acid guanidinium thiocyanate-phenol method (11). Synthesis of cDNA was performed using AMV reverse transcriptase as specified by the supplier (Stratagene, La Jolla, CA). The total
volume for each reaction was 100 µl for 4 µg of RNA. PCR reactions included 5 µl of cDNA as template, 0.4 µg of each specific primer, 0.2 mM
dNTPs, and 2 U of Taq polymerase in a buffer supplied with the enzyme
(Perkin-Elmer Corp., Norwalk, CT). Annealing temperature was 46°C.
Primers
Specific primers were designed based on a published chicken Slug sequence (46) and mouse Snail sequence (45, 55). The primer sequences
were P41: CTTCGGATGTGCATCTTCAGAC (Mouse Snail bp 744-
764), P57: AT(ACT)GA(AG)GC(ACGT)GA(AG)AA(AG)TT(TC)CA
(GA)TG (chicken Slug, amino acids 125-131), P64: AAGCCCAACTATAGCGAGCTG (mouse Snail bp 46-66), P85: CTTGTAGTCGGATCCGTGTGCCACACAGCAGCCAGA (Mouse Slug 3 Cloning and Sequencing
The PCR fragments P57-P41 and P64-P41 obtained from human keratinocyte cDNA, NBT-II cell cDNA, and a mouse cDNA library derived from the putative neural crest cell line NC15 (a gift from Dr. Karen
Brown, National Institute of Dental Research) were cloned in PCR II (Invitrogen, San Diego, CA) and sequenced using an automated DNA sequencer. The PCR fragment S64-41, obtained from a mouse cDNA library, was cloned in the PCR II vector and used as a probe for screening a
mouse cDNA library (kindly provided by Dr. Karen Brown) that was
based on Transfection and Cell Selection
Expression vectors containing full-length mouse slug sequence were transfected into NBT-II cells plated in 16-well slide chambers (Nunc) with 0.25 µl
lipofectamine (Life Technologies, Grand Island, NY). For transient transfections in NBT-II cells, 0.1 µg of IL-2R construct was added during the
transfection to serve as a marker for transfection (20). Wells were washed
several times after 6 h of incubation. NBT-II cells were cultured for another 48 h before fixation. For stable transfections, NBT-II cells were incubated with DNA/lipofectamine for 15 h, washed repeatedly, and then
treated with 400 µg/ml active G418 (Life Technologies) for 7 d. Surviving cells were cloned individually by limiting dilution.
Immunofluorescence Microscopy
Cells cultured on 16-well multiwell glass slides (Nunc) were treated with
FGF-1 for 2 d after cell plating. Heparin (Choay Laboratories, Paris,
France) at 10 µg/ml was added to the culture medium to stabilize the biological activity of FGF-1 (22). Growth factors were renewed every other
day for long term activation. NBT-II cells were processed for immunocytochemistry as described previously (8).
Electron Microscopy
Cells were cultured for 72 h before fixation in 2% glutaraldehyde, 2%
paraformaldehyde in cacodylate buffer. After postfixation in 1% osmium
tetroxyde, they were progressively dehydrated in ethanol and then lifted
from the culture dishes before embedding in Epon resin. Ultrathin sections were stained with 2% uranyl acetate and 1% lead citrate before visualization with a transmission electron microscope (JEOL; Tokyo, Japan).
Nucleus-to-Nucleus Distance
High-magnification photographs were measured with a ruler to find the
distance from the geometrical center of nuclei from adjacent cells. More
than 50 measurements were done in each case. Adjacent cells were chosen
randomly, and cells located at the edges of aggregates were excluded.
Motility Assays
Motility assays were performed using cells seeded on plastic tissue culture
dishes and cultured for 2 d in standard medium. Dishes were covered with
a glass slide, and time-lapse video cinematography was performed over 15 h
after addition of growth factor. The average speed of locomotion was calculated from more than 30 distinct cell tracks chosen randomly. For each
cell, total cell migration distance during the time of migration (5-6 h) was
determined.
Northern Analysis
After separation on a 1.2% formaldehyde-containing agarose gel, RNAs
were transferred overnight to a Nylon membrane (Nytran; Schleicher and
Schuell, Keene, NH). The P64-P41 slug probe was excised from PCR II
constructs and a ribosomal human 28S probe was excised from HHCD07
(American Type Culture Collection, Rockville, MD). Both probes were
[32P]dCTP-labeled using a Primer II kit (Stratagene) and then incubated overnight at 42°C with filters in hybridization solution (5× SSC, 5× Denhardt's, 50% formamide, 10% dextran sulfate, 50 µg/ml salmon sperm
DNA, and 0.4% SDS). After washing twice for 10 min at 42°C in 0.25×
SSC and 0.1% SDS, filters were dried and exposed to Hyperfilm-MP (Amersham Corp., Arlington Heights, IL) for 2 d. Quantitation was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Cloning and Sequence Analysis of Mammalian
Slug cDNA
We cloned by PCR several Slug cDNA fragments using
primers derived from the chick sequence. We used the
fragment P64-P41, amplified with primer set 64/41 to screen
a mouse cDNA library. We obtained a full-length mouse
Slug cDNA clone; its deduced amino acid sequence is
shown in Fig. 1. This clone has 92% amino-acid identity
with the chicken Slug sequence. It includes the "trademark" MPRSFLV K/R K amino-terminal sequence characteristic
of the Snail family (42, 46, 61). As in chicken Slug, it contains four classic CXXC (12 X) HXXXH zinc-fingers and
a fifth one representing a structural variant apparently specific for the Snail family: CXXXC (12 X) HXXXXC (61). As
noted for Xenopus, mouse Slug is very similar (90% identity) to the mouse Snail protein in the carboxy-terminal
half of the molecule after amino acid 160. This region includes the four zinc-finger domains expressed by the mouse
Snail gene.
Amplification products derived from the 57/41 or 64/41
primer sets were also cloned and sequenced from human
keratinocytes and rat bladder carcinoma cells (NBT-II).
Interestingly, the mouse amino acid sequence in this region, covering the first two zinc-finger domains, was 100%
identical to the sequence of the human Slug clone and
98% identical to the sequence of the rat clone (Fig. 1).
Slug mRNA Expression Rises Early after
FGF-1 Treatment
RNA was extracted from NBT-II cells at various times after EMT-triggered FGF-1 treatment. The slug fragment
S64-41 was labeled with 32P and used for Northern blot analysis. As shown in Fig. 1, Slug RNA was induced as early as
3 h after FGF-1 treatment. Expression reached a peak at 6 h
and then decreased after 15 h. Under these conditions,
NBT-II cells cultured with FGF-1 for more than 6 d no
longer expressed Slug mRNA.
Slug-transfected Cells Display a Modified Phenotype
The full-length cDNA encoding mouse slug was inserted
in both orientations in the expression vector PCR 3 under
the control of a cytomegalovirus promoter. Constructs were
cotransfected transiently into NBT-II cells using lipofectamine together with an expression vector encoding a truncated IL-2R described as a marker for DNA uptake and
expression (20, 36). Desmoplakin immunolabeling was
used to localize desmosomes. As described previously (65), untreated parental NBT-II cells have a localization of desmoplakin at punctate sites corresponding to desmosomes,
which is restricted to cell-cell contact areas. However, this
pattern disappears when cells are induced to undergo EMT
with FGF-1, and cells treated for >12 h with FGF-1 are devoid of desmosomes. To examine for similar modulation in
the transiently transfected cells, we counted 100-200 cells
displaying IL-2R labeling for each condition. We then
calculated the proportion of desmoplakin-positive cells
among the cells expressing the IL-2R. Cells cotransfected
with Slug cDNA were compared to cells cotransfected
with the control vector PCR 3 (Fig. 2). The proportion of
cells displaying desmosomes after slug transfection was
less than half that of control-transfected cells, and this difference was highly significant (P < 0.004).
Since transient transfections indicated a Slug-mediated
modulation of cell phenotype, we established clones of stable transfectants with Slug cDNA in either orientation together with a Neomycin resistance gene to produce sense
and antisense transfectants, as well as transfecting with expression vector alone. Cells were selected in the presence
of Geneticin (G 418) and cloned individually by limiting
dilution. A total of 37 independent transfectant clones including 9 controls, 13 antisense clones, and 15 sense clones were established and analyzed. slug (sense and antisense)-
transfected cells, as well as control-transfected cells, displayed cell-cell associations classified as tight, intermediate, or loose according to their morphology (Fig. 3 A).
"Tight" cell contacts were defined in this system as sites
visualized by phase-contrast microscopy as simple phase
refractile (bright) lines, outlining flat apical areas of epithelioid cells. "Loose" cell contacts showed a much more
complex pattern at cell-cell contact areas, with irregular cell-cell boundaries that often showed phase-refractile
zones at flattening surfaces of cells that were internal to
phase-dark zones at flat sites of variable cell-cell contact.
When the cell population phenotype was heterogeneous,
cells were considered to display an intermediate morphology. In subconfluent cultures, the normal pattern of NBT-II
cell distribution, in which clusters of cells organize themselves into closely packed epithelial islands surrounded by
empty spaces, was replaced by a modified phenotype in
slug-transfected cells, in which cell-cell associations appeared much looser. Two sense clones, S4 and S6, are
shown in Fig. 3 B. They show a modification of cell-cell
contact areas that is particularly apparent at higher magnification (Fig. 4 A), with a wider cell-cell junction area or
separations at cell junctions that disrupted the more cuboidal epithelial pattern of parental NBT-II cells. The proportion of the cell periphery involved in contact with another cell was substantially decreased. Cells located within
the interior of groups of cells had >90% of their peripheral membrane in contact with an adjacent cell, whereas
cells transfected with the sense slug construct had only
46% of their membrane in contact, as estimated from photographs. As a consequence, there were spaces of varying
size in the monolayer even when adjacent cells were in
contact (Fig. 4 A).
To study in detail the nature of the cell-cell contact areas, S6 and untransfected NBT-II cells were examined in
electron microscopy. NBT-II cells were found to exhibit
characteristic morphological features of epithelial cells
such as desmosomes (Fig. 4 B) and putative adherens junctions (data not shown). They displayed numerous interdigitating processes, similar to those reported in bladder epithelial cells in vivo (9). In contrast, the Slug sense clone
cDNA S6 showed flattened and spread cells devoid of desmosomes, still in contact with each other through much
more limited lateral contact areas (Fig. 4 B). Interdigitating processes similar to those in untransfected cells were
found, as well as putative cell adherens junctions (data not
shown).
Phenotype modulation was also characterized by an increased spreading apart of the transfected cells on the substrate. To quantify this alteration, we calculated the average nucleus-to-nucleus distance in the stable transfectants.
We found that sense clone S6 displayed twice the nucleusto-nucleus distance as compared to the parental NBT-II
cells or to the antisense-transfected cells (Fig. 4 C). On the
other hand, antisense-transfected clones such as AS2.8
and AS2.10 displayed the typical cobblestone epithelial phenotype expressed by parental NBT-II cells (Fig. 3 B).
To check mRNA expression in stable transfectants, Northern analysis was performed using RNA isolated from seven
different transfected clones including S3, S4, AS2.8, and
AS2.10 clones (Fig. 5). As quantified with a PhosphorImager, transfected clones expressed 10 to 50 times more
Slug RNA than parental NBT-II cells (data not shown).
Expression of Slug Protein in Parental and Transfected
NBT-II Cells
We compared the levels of Slug protein in untransfected
and transfected NBT-II cells by immunofluorescence using anti-chicken Slug antibodies (Fig. 6). The level of Slug
protein expression was very low in parental epithelial
NBT-II cells. It was restricted to rare cells that were usually located at the periphery of cell aggregates. After 2 h of
FGF-1 treatment, a substantial subpopulation of cells that
was also mostly located at the edge of cell aggregates was
clearly positive. This population became predominant after 6 h of FGF-1 treatment (Fig. 6). However, after 48 h of
FGF-1 treatment and full conversion to a mesenchymal
phenotype, Slug protein expression was downregulated.
In contrast, sense-transfected cells expressed Slug before any FGF-1 treatment and maintained it after 6 h of
treatment. Antisense-transfected cells did not express Slug
before the FGF-1 treatment, and only a few cells located
at the periphery of cell aggregates expressed Slug even after 6 h of FGF-1 treatment (Fig. 6).
Immunofluorescence Shows Disruption of
Desmosomes in slug-transfected Cells
To analyze potential modulation of desmosomal cell-cell
adhesion structures, we localized desmoplakin by immunofluorescence (Fig. 7). To quantitate this modulation, we
calculated the percentage of cells displaying desmoplakin
localization characteristic of the epithelial phenotype (desmosome-positive) compared to the total number of cells.
This ratio was calculated for untreated cells or at different
times after FGF-1 treatment for six independent slug-
transfected clones, and the results are reported in Fig. 7 B. Four of these clones, including clones S4 and S6 (Fig. 7 A),
had a low to null percentage of desmosome-positive cells
before any FGF-1 treatment. Cytoplasmic immunoreactivity was found with antidesmoplakin antibodies, suggesting
a cytoplasmic desmoplakin pool in S4 and S6 cells. This
immunoreactivity was no longer found after 48 h of FGF-1
treatment (Fig. 7 A for clone S4; data not shown for S6), as
published previously for the parental NBT-II cells (7). In
comparison, control clones (C1, C2, and C3) had >95%
desmosome-positive cells before treatment, a ratio similar to that for the parental NBT-II cells. In all cases, immunoreactivity was no longer present after prolonged FGF-1
treatment, as published previously for the parental NBT-II
cells (7).
Expression of Antisense Slug Inhibits EMT Induced by
FGF-1 or HGF/SF
Cells stably transfected with the antisense Slug construct
were examined for altered sensitivity to inducers of Slug
and EMT. Phase-contrast microscopy showed no significant morphological effects of FGF-1 in these cells (data
not shown). Moreover, desmoplakin in desmosomes continued to be localized normally at cell-cell contact areas in
antisense-transfected AS2.8 and AS2.10 cells treated with
5 ng/ml FGF-1 (Fig. 7 C). The percentage of desmosomepositive cells was determined for seven independent antisense-transfected clones. Five of them showed significant
inhibition of FGF-1-induced internalization of desmosomes. In a time-course study, we determined the percentage of desmosome-positive cells at several times after initiation of the FGF-1 treatment for three antisense clones, AS2.1, AS2.8, and AS2.10 (Fig. 7 D). Even after 48 h of
FGF-1 treatment, >70% of the cells remained clearly epithelial and desmosome-positive in AS2.8 and AS2.10 cells.
This high retention of desmosomes should be compared to
the very low level of 0.3% desmosome-positive cells observed in control transfectants (Fig. 7 B). These results demonstrate inhibition of the entire process of EMT by antisense slug transfection.
To confirm that the suppression of EMT was due only to
the antisense Slug, the antisense mouse Slug stable-transfectant AS2.8 cell line was cotransfected with mouse Slug
cDNA and marker-truncated IL-2R in a "rescue transfection" attempt. After this transient transfection and 48 h of
culture, cells expressing IL-2R (>100) were examined for
desmosome expression as previously described. 85 ± 6%
of cells cotransfected with the PCR 3 vector alone expressed desmosomes, a proportion similar to that in control AS2.8 cells, whereas only 26 ± 14% of the AS2.8 cells
cotransfected with sense Slug cDNA expressed desmosomes, showing a highly significant (P < 0.001) decrease in
the number of cells expressing desmosomes (Fig. 8).
Finally, we examined for the ability of antisense Slug to
block EMT induced by a potent scatter factor, HGF/SF,
which was previously described to induce EMT in NBT-II
cells as well as in a variety of other epithelial cells (2, 18,
51, 58). Interestingly, the two clones tested, AS2.8 and
AS2.10, also resisted HGF/SF action. Double immunofluorescence using antibodies against desmoplakin and cytokeratin clearly indicated a persistence of desmosomes in
antisense-transfected cells with retention of cytokeratin filament insertion into points of cell-cell contact rich in
desmoplakin (Fig. 9 A). Quantitation of desmosome-positive cells showed a pattern of resistance to dissociation
similar to that obtained with FGF-1 (Fig. 9 B). At higher
concentrations of HGF/SF or of FGF-1, more cell dissociation was observed, suggesting a competitive mechanism.
Modulation of Desmosomal Cadherins in
slug-transfected Cells
We examined the distribution of two members of the cadherin family involved in most cell-cell adhesion structures.
We chose desmoglein, which is a cadherin found specifically in desmosomes, and E-cadherin, a ubiquitous cadherin also present in cell-cell adherens junctions. Desmoglein was found to disappear from cell-cell contact areas
very similarly to desmoplakin in slug-transfected cells, as
reported previously for NBT-II cells treated with FGF-1 to induce EMT (8). In contrast, desmoglein localization
persisted in antisense-transfected clone AS2.8, even after
FGF-1 treatment (Fig. 10 A). E-cadherin immunostaining
was previously described to persist in NBT-II cells undergoing EMT, but it became relocalized diffusely over the cell
surface (8). We found a similar distribution in untreated
slug-transfected S4 cells, which showed a partial relocalization of E-cadherin to regions not involved in cell-cell contacts (Fig. 10 B, arrowhead). FGF-1 treatment did not
significantly change this localization, although E-cadherin
immunoreactivity appeared significantly decreased overall
in association with a decrease in total cell surface area involved in cell-cell contacts. On the other hand, antisense
stable transfectant AS2.8 showed little change in total
staining compared with untreated NBT-II cells. However,
some E-cadherin expression could also be detected in regions not involved in cell-cell contact areas (Fig. 10 B).
Slug-transfected Clones Continue to Express
Cytokeratin, but Its Organization Is Altered
Cytokeratin protein levels are progressively downregulated in NBT-II cells treated with FGF-1 for several days
(8). Since desmosomes are linked to a cytokeratin network
in epithelial cells, we studied cytokeratin localization in
slug transfectants. Both sense and antisense S4 and AS2.8
cells continued to express a dense cytokeratin mesh, indicating that this later step in EMT is not triggered by Slug.
However, in accord with the absence of desmoplakin and
desmoglein in S4 cells, these slug-transfected cells failed to
display the typical pattern of cell-cell junctional cytokeratin filaments anchored tightly in desmosomes as was observed in parental NBT-II cells as well as in AS2.8 cells
(Figs. 9 and 11). Epithelial NBT-II cells do not express vimentin intermediate filaments until they are treated for
several days with FGF-1 (7). Similarly, <5% of the slugtransfected S4 cells were found to express vimentin in absence of FGF-1 treatment. This number increased progressively after FGF-1 treatment and exceeded 95% after 3-4 d
in culture, similarly to the alteration observed with parental NBT-II cells. In contrast, very few (<1%) of the antisense slug-transfected AS2.8 cells expressed vimentin,
even after FGF-1 treatment. Two of the latter rare cells
are shown by double staining for cytokeratin and vimentin
in Fig. 11 A and were found to express simultaneously cytokeratin and vimentin filaments. In addition, they clearly express typical cell-cell connecting cytokeratin filaments
indicative of functional desmosomes, even though these cells
are also synthesizing vimentin.
Video Time-Lapse Analysis of Transfectant
Cell Migration
An important characteristic of NBT-II cells expressing vimentin filaments associated with the complete mesenchymal phenotype achieved after FGF-1 treatment is the appearance of a motile phenotype that can be quantified by
time-lapse video microscopy (65). We quantified the motility of slug transfectants plated on plastic substrates by
analyzing video recordings with or without FGF treatment (Fig. 11 B). Interestingly, even in the absence of desmosomes, S4 cells did not express a motile phenotype until
treated with FGF-1. Only after treatment did they begin
migrating similarly to the parental NBT-II cells treated
with FGF-1. On the other hand, with or without prior
FGF-1 treatment, antisense slug-transfected AS2.8 or 2.10 cells did not express any significant motility, in accord with
the persistence of desmosomes and cell-cell contacts.
In this study, we tested directly whether the newly described zinc-finger protein Slug plays a central role in regulating one or more steps in the process of EMT, using
both gain-of-function and loss-of-function approaches. We
cloned and sequenced a mouse full-length cDNA encoding slug, which has known homologues in chicken and Xenopus. We also cloned fragments of human and rat slug;
sequences from all species were found to be highly conserved. We transfected full-length slug cDNA into NBT-II
cells to generate transient as well as stable transfectants for
testing its function. Slug induced a striking total dissociation
of desmosomes in NBT-II epithelial cells incorporating
the DNA. It also induced increased spreading and separation of these cells, with a doubled average nucleus-to-nucleus
distance and a marked decrease in the proportion of
membrane involved in cell-cell contact. Conversely, we
found that NBT-II cells transfected with antisense Slug
cDNA resisted the actions of both FGF-1 or HGF/SF for
inducing desmosomal dispersal and for cell scattering in
the EMT process, i.e., antisense slug interferes with a necessary Slug expression step in EMT. The specificity of the
antisense slug activity was supported by a "rescue" slug transfection experiment, in which transient overexpression
of a slug sense construct permitted dissociation of stable
antisense transfectants. We conclude from these and other
experiments that Slug expression is necessary and sufficient for the first key steps of EMT involving desmosomal
dissociation and cell separation, as determined for FGFinduced EMT in NBT-II cells. The resistance of antisensetransfected cells to EMT mediated by FGF-1 as well as
HGF/SF suggests that it is a necessary step on which several pathways leading to cell-cell dissociation may converge,
initiated by distinct factors such as FGFs or HGF/SF.
Sequence and Expression Homologies between Chicken,
Human, Rat, and Mouse Slug
The full-length sequence of mouse Slug shows 92% amino
acid sequence identity to the chicken Slug sequence and
89% identity to Xenopus Slug. Interestingly, this homology is much stronger than the homology between mouse
and Xenopus Snail proteins (initial members of the Snail
transcription factor family to which Slug belongs), partially reflecting the disappearance of a zinc-finger motif in
the mouse Snail protein. In the DNA segments we cloned and sequenced from other species, mouse Slug showed
100% identity to human Slug in the deduced amino acid
sequence and 98% identity to rat Slug. These data, added
to the similarity of distribution patterns between chicken
and Xenopus Slug protein in embryos (42, 46), suggest a
conserved function for Slug in vertebrates. Snail is known
to be a transcriptional repressor in Drosophila, probably acting by direct competition with transcriptional activators
(23, 31). A DNA-binding site motif has been defined for
the Drosophila Snail (41), apparently shared by the related escargot gene (14). Escargot was recently described
to play a key role during Drosophila trachea morphogenesis (60).
It is likely that Slug is also a transcription factor that
may recognize specific sites in gene regulatory regions.
Snail probably downregulates its own expression through
a negative regulatory loop mechanism (41). This could also
be the case for Slug, which could explain the transient pattern of expression we observed in NBT-II cells. This transient expression pattern also suggests that Slug initiates a
genetic program subsequently dependent on a distinct
FGF-1-induced activator. Direct involvement of distinct transcription factors like Ets-1 (66), E1a (21), and c-fos (48)
in the induction of cell phenotype modulation, including EMT phases, has been suggested in several cases. However, these factors do not show functional specificity in
vivo comparable to that which we describe in the present
study of Slug with NBT-II cells.
Slug Targets Desmosomal Proteins
Transient and stable slug transfectants showed a characteristic morphological separation of normally tightly apposed plasma membranes at cell-cell boundaries, suggesting that Slug targets some critical cell-cell adhesion
system. This separation was accompanied by an increased
spreading of the cells well characterized by electron microscopy, resulting in a doubled nucleus-to-nucleus distance. We therefore compared its effects on desmosomal
and cadherin-based adhesion systems. Transient and stable transfectants with slug showed a disappearance of the
desmosomes characteristic of epithelial NBT-II cells. This
effect was also found by transient transfections of MDCK
cells, a different epithelial cell line normally expressing numerous desmosomes at the cell-cell junctions; although
MDCK cells undergo an EMT-like response to HGF but not
FGF, Slug can also lead to losses of desmosomes in this
cell line (Savagner, P., unpublished data). Desmosome dissociation was observed in most stable transfectant NBT-II
clones using three markers. First, desmoplakin and desmoglein, two essential desmosome components, disappeared
from cell-cell contact areas. The third desmosomal marker
was the focal insertion of cytokeratin filaments, which anchored the desmosomes to the cytokeratin meshwork. The
stable transfectant line S4 did express a cytokeratin filament
network comparable to parental cells as observed by immunofluorescence, but they did not express any membraneanchoring cytokeratin filaments. This altered cytokeratin
localization pattern was comparable to that observed in
epithelial cells transfected with a dominant-negative desmoplakin polypeptide (4). In accord with the immunofluorescence results, Western blot analysis confirmed the presence of desmoglein in untreated Slug transfectants, at a
level similar to the untransfected NBT-II cells (Savagner,
P., unpublished observation). The three markers were
downregulated after several days of FGF-1 treatment, as for
the untransfected NBT-II cells (Results and Savagner, P., unpublished observation). Conversely, the three desmosomal markers were positive in slug antisense-transfected
cells, even after FGF-1 or HGF/SF treatment, demonstrating the continued presence of desmosomes in these cells.
Other cell adhesion structures were also investigated. Adherens junction components including E-cadherin and
Interestingly, this relocalization was not described in
epithelial cells transfected with a dominant-negative desmoplakin polypeptide (4), further suggesting that the desmosomes are not the only target for Slug.
Slug Induces the First Phase in the Process of EMT
Our observations suggest that Slug induction triggers the
steps of desmosomal disruption, cell spreading, and partial
separation at cell-cell borders, which comprise the first
and necessary phase of the EMT process in our NBT-II
cell model, but that Slug cannot trigger the second phase,
which includes the induction of cell motility, repression of
the cytokeratin expression, and activation of vimentin expression (Fig. 12). Our studies therefore provide the first
demonstration of discrete mechanistic phases in EMT. It is
conceivable that the overexpression and the persistence of
Slug expression in transfected clones might interfere with a Slug-triggered induction of this second phase. However,
additional FGF treatment of such transfectants readily induces this second phase, demonstrating the absence of a
block. Therefore, we propose that a distinct mechanism is
responsible for the induction of motility in NBT-II cells
treated with FGF-1. Such a mechanism is in accord with
previous observations analyzing phases in endothelial cell
conversion to mesenchymal cells that differentiate into the valves and membranous portion of the atrial and ventricular septum during heart morphogenesis (44, 47). Consequently, EMT appears to be a sequential set of events that
involves at least two necessary and distinct phases that can
be dissected by gain-of-function and loss-of-function
transfection approaches. Our studies provide a mechanistic explanation of the results of previous in vivo experiments in which antisense oligonucleotides targeting Slug
were found to interfere with embryonic processes involving EMT as a prelude to subsequent differentiation (46).
Slug is essential for initiating growth factor-induced EMT,
and its actions include induction of desmosome dissociation and disruption of cell-cell adhesive junction.
-catenin/plakoglobin, Band 6/plakophilin, and
desmoplakin, among other components (26, 29, 40, 56, 57,
62). Desmoplakin can bind directly to cytokeratin filaments in vitro and appears to enhance desmosome stability
(35, 56, 57). Studies with dominant-negative variants indicate that desmoplakin is required in vivo for attaching intermediate filaments to the desmosome (4). Little is known,
however, about how desmosomal assembly is regulated.
and FGF families (10, 47), have been suggested to play a role during these embryonic
phenotype modulations. Transcription factors are likely to
be involved at some point during the process. For example, the zinc-finger protein Slug was found to be expressed
in chicken neural crest cells just before they emerge from
the neural tube and later during their migration phase
(46). Interestingly, the same report described Slug expression by epiblast cells lining the primitive streak during gastrulation, just before the emergence of mesenchymal cells.
Treatment of developing embryos with antisense oligonucleotides from slug was found to interfere with these two
processes, suggesting a potential causal role for Slug in the
EMT process in vivo. Slug and closely related members of
the Snail family were also found to express similar patterns of localization in Xenopus and zebrafish embryos,
providing an early marker for neural crest cells.
Materials and Methods
end), and P86:
TCGAATTCGCGGTCGCTGTC.
ZAP II (Stratagene). A clone was excised in pBluescript and
sequenced in both directions using an automated DNA sequencer and
various primers derived from successive sequencing. Slug expression vectors were prepared by cloning the mouse PCR product P86-P85 containing the full-length mouse slug sequence in a PCR 3 vector (Invitrogen).
The interleukin-2 receptor (IL-2R) expression vector was described previously (36). Sequences were analyzed using the Wisconsin package (Genetics Computer Group, Madison, WI) and Mac Vector (Oxford Molecular
Group, Campbell, CA) software.
Results
Fig. 1.
(A) Comparison of mouse, rat, and human sequences
of Slug cDNA. The mouse Slug (M slug) cDNA was isolated from
a mouse cDNA library. Rat (R slug) and human (H slug) PCR P64P41 and P57-P41 fragments were amplified by PCR using primers
P57, P64, and P41. Sequences were aligned with chick (C slug)
and Xenopus (X slug) Slug sequences, highlighting the five zincfinger domains. These sequence data are available from GenBank/EMBL/DDBJ under accession numbers U97059, U97060,
and U97061. (B) Northern analysis of Slug expression in NBT-II
cells. NBT-II cells were treated with FGF-1 (20 ng/ml) for the
time periods indicated. RNA was separated on a formaldehydeagarose gel, subsequently transferred to a Nylon membrane and
hybridized with the slug fragment P64-P41 probe, and then, after
washing, was reprobed with a ribosomal 28S probe. Slug relative
expression was normalized in both cases to the basal level of Slug
expression (t = 0) and the corresponding level of 28S RNA to define the time course shown on the graph. Insert displays a different Northern analysis experiment showing a similar transient expression pattern. Northern analysis was repeated in five independent experiments and gave similar results.
[View Larger Version of this Image (31K GIF file)]
Fig. 2.
Slug transient transfections. NBT-II cells were cotransfected with vectors containing mouse full-length cDNA for Slug and a
truncated (IL-2R) cDNA used as a transfection marker. Alternatively, a control vector pCR 3 (Control) was cotransfected with IL-2R cDNA. After 48 h, cells were fixed and processed for double immunofluorescence using antibodies against IL-2R and antidesmoplakin
(A); note that cells expressing the IL-2R transfection marker are positive for desmoplakin in control transfectants and negative in slugtransfected cells. Cells expressing desmoplakin at cell-cell boundaries were counted as desmosome-positive (DP+), i.e., fully epithelial.
More than 100 cells expressing IL-2R were analyzed at the same time point for desmosome expression. The number of desmosome-positive cells displaying IL-2R labeling was normalized to the total number of cells expressing IL-2R (B). Bar, 9 µm.
[View Larger Versions of these Images (108 + 12K GIF file)]
Fig. 3.
slug-transfected cells display a modified morphological
phenotype. After selection and cloning, stable transfectant clones were cultured under standard conditions. (A) Phenotype expression of stable slug transfectants, comparing sense, antisense, and control clones. 37 clones were analyzed by phase-contrast microscopy for their phenotype and classified according to the appearance of cell-cell junctions as tight, intermediate, and loose. (B) Phase-contrast micrographs of sense (S6 and S4) and antisense (AS2.8 and AS2.10) slug transfectants were taken after 48-72 h of cell culture. The morphology of the cell junctions in S4 and S6
were classified as loose, whereas the other four were classified as
tight for quantification for A. Bar, 10 µm.
[View Larger Versions of these Images (20 + 81K GIF file)]
Fig. 4.
Slug transfectant clone exhibits looser cell contact phenotype. (A) NBT-II and S6 cells were observed at higher magnification. Arrowheads indicate cell edges, which are often free for
S6 cells compared to involvement in tight cell-cell contacts for
control NBT-II cells. (B) Electron micrographs of NBT-II cells
and S6 cells. NBT-II cells (a, c, and d) or S6 cells (b) were plated for 48 h before fixation and processing for electron microscopy. NBT-II cells displayed an epithelial phenotype characterized by numerous desmosomes (arrow in a, higher magnifications of
other examples in c and d). Conversely, S6 cells appeared flatter,
more spread, and less epithelial. No desmosomes could be found.
(C) slug-transfected S6 cells are more widely spaced in subconfluent cultures than parental NBT-II cells. Cell-to-cell spacing was evaluated by the average distance between the geometrical centers of nuclei of adjacent cells. Only cells in contact with each
other were used for the calculation. More than 50 distances were
measured in each case to calculate the average nucleus-to-
nucleus distance. Bars: (A) 10 µm; (B, a and b ) 2 µm; (B, c) 1 µm; (B, d) 0.2 µm.
[View Larger Versions of these Images (143 + 163 + 113 + 28K GIF file)]
Fig. 5.
Northern analysis of Slug-mRNA expression by stable
transfectants. slug-transfected cells expressed large amounts of
Slug RNA. Northern analysis using slug transfectant clones
(sense and antisense) were probed as described for Fig. 2. The
double-stranded DNA probe allowed detection of both sense and
antisense RNA in the transfectants; Ct 3 was transfected with
vector alone.
[View Larger Version of this Image (105K GIF file)]
Fig. 6.
Immunofluorescence analysis of Slug protein expression by transfected and untransfected NBT-II cells. Cells were plated for 24 h and then treated with FGF-1 as indicated. After fixation, cells were immunostained with anti-Slug antibodies. A stable slug transfectant clone (S6) and an antisense stable slug transfectant (AS2.8) were compared to the parental NBT-II cells. Arrowheads indicate
Slug-positive cells. Bar, 35 µm.
[View Larger Version of this Image (73K GIF file)]
Fig. 7.
Desmosome expression by slug-transfected
clones. (A) slug-transfected
NBT-II clones do not express desmosomal structures. Cells were plated for 24 h
and then treated with FGF-1
(5 ng/ml) for 48 h in the case
of NBT-II cells and S4 cells,
and 6 h for S6 cells. After fixation, cells were immunostained with antidesmoplakin antibodies. Two clones of
stable slug transfectants, S4
and S6, were compared to
the parental NBT-II cells.
(B) Proportion of epithelial
cells among FGF-1-treated
and -untreated slug-transfected NBT-II clones. Cells
expressing desmoplakin at
cell-cell boundaries (DP+) were counted after 0 (nt), 3, 6, or 48 h of treatment with FGF-1. slug-transfected clones (S9, S6, S3, and S4)
were compared to control clones C1, C2, and C3 transfected with vector alone. (C) Antisense slug-transfected NBT-II clones resist
FGF-1-induced desmosome dissociation. Cells were plated for 24 h and then treated with FGF-1 (5 ng/ml for 24 h) as indicated. After
fixation, cells were incubated with antidesmoplakin antibodies. Two clones of stable antisense slug transfectants, AS2.8 and AS2.10,
were processed for desmoplakin immunolocalization and photographed at two different magnifications, with or without prior FGF-1
treatment, as indicated. (D) Proportion of epithelial cells among FGF-1-treated and -untreated antisense slug-transfected NBT-II
clones. Cells expressing desmoplakin at cell-cell boundaries (DP+) were counted after 0 (nt), 3, 6, or 48 h of treatment with FGF-1, as
indicated. Antisense slug-transfected clones (AS 2.1, AS2.8, and AS2.10) should be compared to control clones C1, C2, and C3 transfected with vector alone in B. Bars, 10 µm.
[View Larger Versions of these Images (100 + 22 + 124 + 26K GIF file)]
Fig. 8.
Antisense slug stable
transfectants can be reverted by
transient rescue transfection.
Antisense slug stable transfectant AS2.8 cells were cotransfected with Slug cDNA and
truncated IL-2R. After 48 h culture, >100 cells expressing IL2R were scored for the presence of desmosomes, as determined
by desmoplakin immunolocalization. Cells cotransfected with
PCR 3 alone (Control) were
compared with cells cotransfected with Slug cDNA.
[View Larger Version of this Image (12K GIF file)]
Fig. 9.
(A) Antisense slug-
transfected cells also resist the
dissociating activity of HGF/SF.
Control cells (Ct 3) and antisense slug-transfected AS2.8
cells were plated for 24 h and
then treated with HGF/SF at 2 ng/ml for 24 h before fixation
and double labeling with antibodies against desmoplakin or
cytokeratin as indicated. (B)
Proportion of desmosome-positive (DP+) cells among the
AS2.8 cells after HGF/SF treatment. Cells were processed for
desmoplakin immunofluorescence as described above. Desmosome-positive (DP+) cells
were counted after 3, 6 or 48 h of
treatment with HGF/SF. Antisense slug-transfected clones
(AS2.8 and AS2.10) can be compared to control clone Ct 3, which was transfected with the
pCR 3 vector.
[View Larger Versions of these Images (132 + 23K GIF file)]
Fig. 10.
Cadherin expression by slug transfectants. Stable slug transfectants S4 (sense), AS2.8 (antisense), and parental NBT-II cells were prepared for immunofluorescence using antibodies against desmoglein (A) or E-cadherin (B). Cells were treated with FGF-1 (5 ng/ml) for 24 h as indicated. Bars, 10 µm.
[View Larger Versions of these Images (121 + 110K GIF file)]
Fig. 11.
Other processes involved in EMT. (A) slug-transfected cells display a modified cytokeratin network and require
FGF-1 treatment to undergo migration. Slug transfectant clone
S4 and antisense clone AS2.8 were processed for immunofluorescence as described above after 24 h of culture. In addition, AS2.8
was treated for 48 h with HGF/SF. Cells were labeled with antibodies against cytokeratin or against vimentin as indicated. The
AS2.8 cells were double-labeled, and they generally showed little
or no reactivity with vimentin antibody, even after HGF/SF treatment. However, shown here is an unusual example of simultaneous expression by two cells of cytokeratin intermediate filaments (including desmosome-connecting filaments) and vimentin
intermediate filaments. (B) Because initiation of motility often
accompanies EMT, NBT-II cell migration after slug transfection
was evaluated by video time-lapse analysis. Cells were plated for
24 h on culture dishes before a random field was followed for 5 h
by time-lapse microscopy. More than 30 cells were individually
tracked in each case, and the average of the distances traversed
by the cells over 5 h was plotted as the motility. Bars, 10 µm.
[View Larger Versions of these Images (27 + 100K GIF file)]
Discussion
-catenin (Savagner, P., unpublished observation) were
found to be modestly relocalized but still present in S4
cells. They were present as normal in cell-cell contact areas, but they were also present sometimes in membrane
regions not involved in cell-cell contacts. This relocalization was mostly present in cells treated with FGF-1. Interestingly, a slug antisense stable transfectant exhibited the
same relocalization after FGF-1 treatment in spite of the
persistence of the desmosomes. This relocalization was very similar to that previously described for E-cadherin on
FGF-1-treated NBT-II cells (8). The mechanism of this relocalization of cadherin membrane and intracytoplasmic
adhesion molecules and its functional significance are not
clear. NBT-II cells were found previously (8) to aggregate
similarly via Ca2+- and E-cadherin-dependent mechanisms
with or without FGF-1 treatment, suggesting these complexes were indeed functional in spite of the motility of the
mesenchymal NBT-II cells. However, the strength of cell-
cell adhesion might be reduced after such relocalization as
in v-src-transfected MDCK cells (59), which would account for the dominant phenotype of Slug-induced cell
shape change and cell separation even though components
of the E-cadherin system are still present.
Fig. 12.
EMT phases. Slug transfection induced the transition
from epithelial cells to an intermediate phenotype stage characterized by a drastic modulation of the cell-cell adhesion system,
including the dissociation of desmosomes. A second stage is postulated to occur when cells are treated with FGF-1, involving the
replacement of cytokeratin intermediate filaments by vimentin
intermediate filaments and the appearance of cell motility. Both
phases are blocked if the first phase is blocked by antisense slug
transfection (¢).
[View Larger Version of this Image (29K GIF file)]
Received for publication 13 September 1996 and in revised form 18 April 1997.
1. Abbreviations used in this paper: EMT, epithelial-mesenchymal transition; HGF/SF, hepatocyte growth factor/scatter factor; IL-2R, interleukin-2 receptor; MDCK, Madin-Darby canine kidney.This work was supported by the Centre National de la Recherche Scientifique, the Association pour la Recherche contre le Cancer (ARC 6465), the Ligue Francaise contre le Cancer (National Committee and Committee of Paris), the National Cancer Institute of the National Institutes of Health (2R01 CA 49417-06), the Philippe Foundation and the Human Frontier Science Program Organization, and the National Institute of Dental Research intramural program.
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