From the Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM) and Departamento de Bioquímica (UAM), Arturo Duperier 4, 28029 Madrid, Spain
Received for publication, November 5, 2002
, and in revised form, March 20, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The molecular mechanisms underlying down-regulation of E-cadherin during EMTs and tumor progression are starting to be uncovered. Genetic alterations of the E-cadherin loci have been found in a scarce number of tumors, particularly in lobular breast carcinomas and diffuse gastric carcinomas (3, 11, 12), whereas the majority of carcinomas with down-regulated E-cadherin maintain an intact E-cadherin locus. Hypermethylation of the E-cadherin promoter and transcriptional alterations have emerged as the main mechanisms responsible for E-cadherin down-regulation in most carcinomas (5, 13). Several transcriptional repressors of E-cadherin have been isolated recently, including the zinc finger factors Snail (14, 15) and Slug (16, 17), the two-handed zinc factors ZEB-1 and SIP-1 (18, 19), and the bHLH factor E12/E47 (20). Snail family factors are in fact involved in EMTs when overexpressed in epithelial cell lines (14, 15, 17), as well as in embryonic development (reviewed in Ref. 21), and are proposed to act as inducers of the invasion process (14, 22). Generation of Snail knockout mice has further established the role of this factor in EMT and as the E-cadherin gene repressor. The null Snail embryos die at gastrulation as they fail to undergo a complete EMT process, forming an altered mesodermal layer that maintains the expression of E-cadherin (23). Nevertheless, the mechanisms that regulate the expression of Snail factors are still poorly understood (6, 21).
Different growth factors and cytokines have also been implicated in the
process of EMTs in both epithelial cell systems and in embryonic development.
Studies on development have indicated the participation of several members of
the transforming growth factor (TGF)/bone morphogenetic family of growth
factors in specific EMT processes in different species
(24,
25), whereas fibroblast growth
factor (FGF) signaling has been reported recently
(26) as a determinant for
mesoderm cell fate specification in the mouse embryo. Several studies have
also indicated that a multiple cross-talk among TGF
/bone morphogenetics,
FGF, and Wnt signals could be required for some EMTs in development
(26,
27,
28). In epithelial cell
systems, several growth factors have been widely studied and reported to
induce a scattering phenotype or a complete EMT depending on the specific cell
system analyzed (reviewed in Refs.
6 and
29). Among them, TGF
has
been identified as an important molecular player of EMT both in vitro
and in vivo (30,
31,
32,
33,
34). In some cell systems, a
synergistic cooperation between H-Ras activation and TGF
signaling
appears to be required for induction of a complete EMT
(33,
35,
36). Recently, TGF
has
been reported to induce the expression of Snail in fetal and in
immortalized murine hepatocytes and in human mesothelial cells
(37,
38,
39), but whether this is a
direct or indirect effect has not yet been established.
The participation of specific signaling pathways activated by TGF
and/or H-Ras activation in EMTs has been analyzed previously with somewhat
contradictory results as regard to the specific implication of Smad,
mitogen-activated protein kinase (MAPK) and/or phosphatidylinositol 3-kinase
(PI3K) pathways (36,
40,
41,
42,
43). The issue has been
unraveled recently (36) in the
EpRas model with the implication of MAPK in TGF
-induced EMT,
tumorigenesis, and metastasis, whereas PI3K is involved in cell scattering and
resistance to TGF-
induced apoptosis. It remains to be established,
however, if the same situation applies to other systems and, more importantly,
the identification of the target genes involved in the specific growth factor
signaling leading to EMTs.
We have used the prototypic epithelial MDCK cells to further analyze the
process of EMT induced by TGF and FGF. We have previously used this cell
system to show that Snail overexpression leads to the full repression
of E-cadherin expression and induction of a complete EMT
(14). In the present work we
have investigated the ability of TGF
1 and FGF2 to induce an EMT in MDCK
cells and ask whether Snail is a target gene of this process. We
present evidence that TGF
1 treatment induces an EMT process linked to
Snail induction in MDCK cells. Analysis of the mouse Snail
promoter indicates that it is directly induced by TGF
1 and that FGF2 and
activated H-Ras cooperate with TGF
1 in induction of the Snail
promoter. Our results also indicate that the MAPK and PI3K pathways are
involved in the TGF
1- and H-Ras-mediated induction of Snail
promoter. These results strongly support that Snail is a direct
target of TGF
1 and oncogenic H-Ras and open the way for future studies
on the molecular mechanisms and targets of EMTs and the invasion process.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RT-PCR AnalysesTotal RNA was isolated from the different cell lines, and RT-PCR analyses were carried out as described previously (14, 17, 20). Canine PCR products were obtained after 3035 cycles of amplification with an annealing temperature of 6065 °C. Primer sequences were as follows: for canine E-cadherin (sequence kindly provided by Y. Chen, Harvard Medical School), forward: 5'-GGAATCCTTGGAGGGATCCTC-3'; reverse: 5'-GTCGTCCTCGC-CACCGCCGTACAT-3' (amplifies a fragment of 560 bp); for canine Snail, forward: 5'-CCCAAGCCCAGCCGATGAG-3'; reverse: 5'-CTTGGCCACGGAGAGCCC-3' (amplifies a fragment of 200 bp); and for canine glyceralde-hyde-3-phosphate dehydrogenase, forward: 5'-TGAAGGTCGGT-GTGAACGGATTTGGC-3'; reverse: 5'-CATGTAGGCCATGAGGTCCACCAC-3' (amplifies a fragment of 900 bp).
3TP-Lux, E-cadherin, and Snail Promoter AnalysesFor 3TP-Lux
assays a reporter construct containing the
12-O-tetradecanoylphorbol-13-acetate and TGF response elements
fused to the Luciferase reporter gene
(44) was used. The generation
of mouse E-cadherin promoter constructs containing -178/+92 sequences
in its wild-type or mutant Epal fused to Luciferase has been reported
previously (17). Generation of
full-length mouse Snail promoter construct (-900 bp) has also been
described recently (17).
Deletion constructs of the Snail promoter mutants were obtained by
PCR amplification from the full-length -900 bp promoter using appropriate
primers containing BamHI and KpnI restriction sites and the
corresponding PCR products cloned into the same restriction sites in the
pXP1-Luciferase vector.
To determine the activity of 3TP-Lux and the Snail promoter 2 x 105 cells grown in 24-well plates were transiently transfected with 200 to 500 ng of the indicated reporter constructs and 20 ng of TK-Renilla construct (Promega) as a control of transfection efficiency. Luciferase and renilla activities were measured using a dual-luciferase reporter assay kit (Promega), and after normalization the results were referred to the wild-type promoter activity detected in mock-transfected cells. Results represent the mean ± S.D. of at least two independent experiments performed in duplicate samples.
For the cotransfection experiments 500 ng of the following plasmids were
used: pSmad4 DN (1514) in pCMV5 vector (provided by J. Massagué,
Sloan-Kettering Memorial Cancer Center)
(44); pLXSNHRasV12,
pLXSNHRasN17, and the different mutants of HRasV12 (pLXSNHRasV12S35,
pLXSNHRasV12C40, and pHras-LXSNRasV12G37) in the pLXSN vector (a gift of P.
Rodriguez-Viciana, University of California Cancer Research Institute)
(45); -catenin S33Y
(provided by A. Ben-Ze'ev, Weizmann Institute) and Lef-1 (provided by H.
Clevers, Utrecht University Hospital) cloned in pcDNA3. The corresponding
empty vectors, pLXSN, pCMV5, or pcDNA3 were used in control transfections and
for normalization of the total amount of DNA.
Immunofluorescence and Western Blot AnalysesFor
immunofluorescence staining cells grown on coverslips were fixed in methanol
(-20 °C, 30 s) and stained for E-cadherin, vimentin, cytokeratin-8, and
fibronectin as described previously
(14,
17,
20). For F-actin stain, cells
were fixed in 3.7% formaldehyde, 0.5% Triton X-100 for 30 min at room
temperature, stained with tetramethyl rhodamine isothiocyanate-conjugated
phalloidin (Sigma) and washed four times in phosphate-buffered saline. The
cells were mounted on Mowiol, and the preparations were visualized using a
Leica confocal TCS SP2 microscope. For Western blot, whole cell extracts of
control and treated cells were obtained in radioimmune precipitation assay
buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet
P-40, 5% deoxycholate, 0.1% SDS) and analyzed for the indicated molecules by
Western blot and enhanced chemiluminescence detection as described previously
(14,
17,
20). Primary anti-bodies
included rat monoclonal anti-E-cadherin ECCD-2 (1:100) (provided by M.
Takeichi, Kyoto University), mouse monoclonal antivimentin (1:200) (Dako),
mouse monoclonal anti-cytokeratin 8 (1:200) (Progen), rabbit polyclonal
anti-fibronectin (1:100), and mouse monoclonal anti--tubulin (1:1000)
(Sigma). For cell signaling analysis, Western blots were carried out on cell
extracts obtained by lysis in Buffer A (20 mM Hepes, pH 7.5, 10
mM EGTA, 40 mM
-glycerophosphate, 2.5
mM MgCl2, 1% Nonidet P-40, 1 mM
dithiothreitol), containing the appropriate protease and phosphatase
inhibitors, during 30 min. at 4 °C. Primary antisera included goat
anti-AKT (1:1000) (Santa Cruz Biotechnology, Inc.), rabbit anti-phospho
(Ser-473)-AKT (1:500), rabbit anti-ERK1/2 and anti-phospho
(Thr-202/Tyr-204)-ERK1/2 (1:1000) (Cell Signaling Technology), and rabbit
anti-Smad2/3 and rabbit anti-phospho (Ser-465/Ser-467)-Smad2/3 (1:500)
(Upstate Biotechnology). Secondary antibodies were BODIPY-conjugated goat
anti-rat, anti-mouse and anti-rabbit IgG (Molecular Probes), and horseradish
peroxidase-conjugated sheep anti-mouse (1:1000) (Amersham Biosciences), donkey
anti-goat (1:1000) (Santa Cruz Biotechnology, Inc.), goat anti-rat (1:10,000)
(Pierce), and goat anti-rabbit (1:4000) (Nordic) IgG.
Cell Proliferation AssaysThe indicated number of cells (2.5
x 105 or 5 x 105) were seeded in triplicate
samples in 92 plates and grown in complete medium for 3 h. After washing in
phosphate-buffered saline, TGF1 (10 ng/ml) in FBS-free medium was added,
and the cells were grown for an additional 24 h. [3H]Thymidine was
added during the last 5 h of treatment. The cells were collected using a cell
harvester device, and [3H]thymidine incorporation was determined in
a scintillation counter. The values, representing the mean ± S.D., were
normalized to those obtained in control untreated cells.
Migration AssaysThe migratory/motility behavior of MDCK
cells was analyzed in in vitro wound healing assays as described
previously (14,
17). Monolayers of confluent
cultures were lightly scratched with a Gilson pipette tip and, after washing
to remove detached cells, treated with TGF1 (10 ng/ml) and/or PD98059
(10 µM), as indicated. Cultures were observed at timely
intervals for up to 36 h post-incision.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
We then analyzed the implication of MAPK and PI3K pathways in the
phenotypic effects induced by TGF1 in MDCK cells, because they have been
implicated previously (33,
35,
36,
40,
42,
43) in epithelial cell
scattering induced in MDCK and in other cell systems by several growth
factors. A 1-h pretreatment with the specific MEK1/2 inhibitor PD98059 (10
µM) abolished the cell dissociation and scattering induced by
TGF
1 (Fig. 1A,
e) or by the combination of TGF
1 and FGF2
(Fig. 1A, f)
treatments. No significant effect of PD98059 on the cell phenotype was
observed in control untreated (Fig. 1A,
d) or FGF2-treated cells (data not shown), although increased
intracellular vacuolization was observed in all PD98059-treated samples. In
agreement with those observations, PD98059 pretreatment also blocked the
TGF
1-induced migration of MDCK cells
(Fig. 1B, h) but did
not have any effect on the migration of untreated cells
(Fig. 1B, f).
Pretreatment with the PI3K inhibitor LY294002 (30 µM) followed
by TGF
1 treatment caused cell disintegration (data not shown), thus
precluding further studies on the implication of PI3K in the phenotypic or
migratory effects of TGF
1.
Activation of the MAPK and PI3K pathways following TGF1 treatment of
MDCK cells was confirmed by Western blot analyses of phosphorylated ERK1/2 and
AKT, respectively, using phosphospecific antibodies to both effectors. As
shown in Fig. 3, increased
levels of phospho-ERK2 (Pp-42) were detected after 30 min of TGF
1
treatment, peaking after 1 h and slowly decreasing thereafter
(Fig. 3, upper
panels). A similar kinetics was observed in the levels of phospho-ERK1
(Pp-44), although to a lesser extent. Interestingly, increased levels of
phospho-ERK1/2 were detected even after 6 h of TGF
1 treatment,
indicating a sustained response of the MAPK pathway. Activation of PI3K
followed a slower kinetics in response to TGF
1, increased levels of
P-AKT were first detected after 1 h of TGF
1 treatment, peaked by 3 h,
and decreased thereafter (Fig.
3, middle panels). TGF
1 treatment also induced a
fast and sustained activation of the Smad pathway in MDCK cells, because
P-Smad2 was detected after 15 min and was maintained up to 3 h of TGF
1
treatment (data not shown).
|
These results indicate that TGF1 induces a scattering and motile
phenotype in MDCK cells, apparently depending on MAPK signaling, and suggest
that activation of the PI3K pathway might be required for survival in the
presence of TGF
1. FGF2 by itself does not have a significant effect on
the MDCK phenotype, although it can potentially collaborate with
TGF
1.
TGF1 Treatment Induces EMT Associated with Snail
Induction and E-cadherin Repression in MDCK CellsThe phenotypic
changes and increased motility observed in MDCK cells after TGF
1
treatment were reminiscent of those observed after stable transfection of MDCK
cells with the Snail repressor
(14) and suggested that
Snail might be induced by TGF
1. To analyze this hypothesis, the
endogenous levels of Snail transcripts in MDCK cells following
TGF
1 treatment were analyzed by RT-PCR
(Fig. 4A). Twenty-four
h of treatment with TGF
1 led to a 2- to 3-fold induction of
Snail mRNA over the basal levels; the level of Snail
transcripts decreased thereafter but remained above the basal levels, at least
up to 72 h of treatment. Analysis of E-cadherin mRNA levels showed no
significant changes after 24 h of TGF
1 treatment, but a marked decrease
was detected after 48 and 72 h of TGF
1 treatment, when
E-cadherin mRNA was almost undetectable
(Fig. 4A).
Densitometric analyses showed that by 72 h of TGF
1 treatment the
Snail and E-cadherin transcripts were present at levels
representing 150 and 20%, respectively, of those detected in control untreated
cells. In agreement with those data, analysis of an exogenous mouse
E-cadherin promoter
(17) by transient transfection
showed a 5060% inhibition after 48 to 72 h of TGF
1 treatment
(Fig. 4B, left
panel). Furthermore, E-cadherin promoter inhibition by
TGF
1 depends on the presence of Snail-binding site, the E-pal element
(14,
17), because its mutation
fully abolished the TGF
1 effect (Fig.
4B, right panel). These data support that the
TGF
1-induced repression of E-cadherin can be mediated by Snail
expression. Western blot analyses showed a moderate decrease (around 35% of
control cells) in the total level of E-cadherin but strong reduction of other
epithelial markers, such as cytokeratin 8 (more than 50% of control levels)
after 72 h of TGF
1 treatment (Fig.
4C). The inhibition of E-cadherin promoter
activity and decreased mRNA levels detected between 48 and 72hofTGF
1
treatment contrast with the levels of E-cadherin protein detected at this time
point. This apparent discrepancy has also been observed in other cell systems
(47) and can be explained by
the long half-live of the E-cadherin protein, estimated in more than 40 h in
other cell systems (49). The
above described results suggest that Snail induction, even at
moderate levels, could be required to trigger the repression of
E-cadherin and, potentially, of other epithelial genes that
eventually lead to the EMT induced by TGF
1 treatment in MDCK cells.
|
To further investigate whether TGF1 indeed induces a full EMT in MDCK
cells, we analyzed the expression and localization pattern of E-cadherin,
cytokeratin 8, as well as that of vimentin and fibronectin as prototypic
markers of epithelial and mesenchymal cells, respectively. Confocal
immunofluorescence analysis showed that 24 to 48 h of treatment with
TGF
1 led to a redistribution of E-cadherin from the cell-cell contacts
to the cytoplasm (data not shown). By 72 h of TGF
1 treatment almost
complete disappearance of E-cadherin at cell-cell interactions was observed
(Fig 5A, b), as
compared with control untreated cells (Fig.
5A, a). Cotreatment with TGF
1 and FGF2 induced a
similar redistribution of E-cadherin (Fig.
5A, d). In agreement with the lack of phenotypic effects,
FGF2 treatment alone did not produce redistribution of the E-cadherin
molecules (Fig. 5A,
c). The TGF
1-induced redistribution of E-cadherin was
fully abolished by pretreatment of MDCK cells with PD98059
(Fig 5A, e), which
showed a similar E-cadherin stain as control cells pretreated with PD98059
(Fig. 5A, f).
Forty-eight h of treatment of MDCK cells with TGF
1 also induced a marked
decrease and disorganization of cytokeratin 8 stain
(Fig. 5B, b), also
confirmed by Western blot (Fig.
4C), and increased staining of vimentin
(Fig. 5B, e) and
fibronectin (Fig. 5B,
h), which were organized in clear intermediate filaments and
apparently secreted matrix, respectively, although no changes in total
vimentin levels were detected (Fig.
4C). Staining for F-actin also showed a marked
reorganization of the microfilament network with appearance of stress fibers
and membrane protrusions, resembling lamellipodia and filopodia, in
TGF
1-treated MDCK cells (Fig. 5B,
k, arrows), in contrast to untreated control cells
that showed a more defined cortical actin filaments
(Fig. 5B, j). These
results, together with those shown in Fig.
1, indicate that TGF
1 induces a full EMT in MDCK cells.
Furthermore, the multiple changes detected in the different markers and in
cytoskeleton organization after TGF
1 treatment were fully abolished by
pre-treatment of MDCK cells with PD98059
(Fig. 5B, c, f, i, and
l), as well as E-cadherin redistribution
(Fig. 5A, e),
indicating that the MAPK activity is necessary for TGF
1-induced EMT in
this cell line.
|
TGF1 and FGF2 Signaling Pathways Collaborate in Snail
Promoter Induction and Depend on MAPK ActivityTo investigate
whether the observed induction of Snail expression is a direct effect of
TGF
1, we analyzed the effect of the growth factor on the mouse
Snail promoter (17)
by transient transfection assays. As shown in
Fig. 6A, TGF
1
treatment of MDCK cells induced the Snail promoter activity in a
dose-dependent manner. TGF
1 at 10 ng/ml was able to induce the promoter
activity by 3-fold, whereas treatments with lower concentrations of 1 and 5
ng/ml induced Snail promoter activity by 1.3- and 2-fold,
respectively. To determine whether this effect was restricted to MDCK cells,
we analyzed two other epithelial cell lines, the mouse epidermal keratinocyte
MCA3D and PDV cells, representing immortalized and transformed stages of the
mouse skin carcinogenesis model, respectively
(49,
50). TGF
1 (10 ng/ml)
treatment induced the Snail promoter activity about 2-fold in both
MCA3D and PDV cell lines (Fig.
6B). These results indicate that Snail promoter
could in fact be controlled by signals downstream of TGF
1 in epithelial
cell lines. Although the level of Snail promoter induction by
TGF
1 in the analyzed cell lines is only moderate, it is consistent with
the induction of Snail mRNA level detected in MDCK cells (see
Fig. 4A).
|
As indicated previously
(26), FGF signaling has been
implicated recently in the regulation of Snail expression during
embryonic development, and previous work
(51) in epithelial NBT-II
cells also suggested its involvement in the regulation of Slug (a
closely related homolog of Snail). We, therefore, analyzed the effect
of FGF2, alone or in combination with TGF1, on the Snail
promoter activity in MDCK cells (Fig.
6C). FGF2 (100 ng/ml) treatment induced a slight
activation of the Snail promoter (1.5-fold), lower than that induced
by TGF
1 at 10 ng/ml (3-fold activation), but an additive effect on the
Snail promoter activity (4.5-fold induction) was observed by the
combination of both FGF2 and TGF
1
(Fig. 6C). The
collaboration between both factors has also been observed in other contexts,
such as in embryonic development where this synergism is necessary for the
subsequent correct development of the EMTs areas, together with others
signals, such as Wnt (28).
However, the canonical Wnt signaling pathway seems not to play a significant
role in the regulation of Snail expression in MDCK cells, as no
effect on the Snail promoter activity was observed by the treatment
with TGF
1 in the presence of activated
-catenin and Lef-1 factor
(data not shown). These latter results are also in agreement with a previous
report (52) showing that
integrin linked kinase-induced activation of the human Snail promoter
in colon cancer cells is independent of the
-catenin/Tcf complex.
We next investigated the TGF1 and FGF2 signaling pathways involved in
the regulation of Snail promoter. Cotransfection of a dominant
negative version of Smad4 (1514) that blocks the classical
TGF
-Smad signaling pathway
(44), as confirmed here by its
action on the responsive 3TP-lux promoter
(Fig. 6E), did not
significantly change the TGF
1-mediated induction of the Snail
promoter activity and even increased the combined effect of TGF
1 and
FGF2 on the promoter activity (Fig.
6D). These results indicated that Smad4 signaling is not
involved directly in the regulation of Snail promoter activity by
TGF
1. We then analyzed the participation of the MAPK pathway in the
regulation of Snail promoter by TGF
1 and FGF2, because it has
been implicated recently (36,
53) in TGF
signaling in
other contexts. To that end, the activity of the Snail promoter was
analyzed in MDCK cells pretreated with the MEK1/2 inhibitor PD98059 (10
µM) before treatment with TGF
1 and/or FGF2. Pre-treatment
with PD98059 decreased the basal activity of Snail promoter to about
60% (see Fig. 6C and
Fig. 7A). More
significantly, PD98059 pretreatment fully blocked the Snail promoter
induction observed by treatment with TGF
1, FGF2, or the combination of
both factors (Fig.
6C). These results strongly suggest that MAPK signaling
is one of the pathways implicated in the TGF
1-mediated regulation of
Snail expression in MDCK cells.
|
TGF1 and Ras Pathways Collaborate in Snail
Induction Several recent works have shown the requirement of Ras
downstream signaling in the process of EMT in different epithelial cell
systems, in some cases in cooperation with TGF
(33,
35,
36). It was, therefore,
important to determine the potential contribution of Ras, either by itself or
in cooperation with TGF
1, to the regulation of the Snail
promoter. Cotransfection of a dominant active version of Ras (HRasV12) induced
a 3- to 4-fold activation of the Snail promoter activity
(Fig. 7, A and
C), similar to that observed in the presence of
TGF
1 (Fig. 7, B and
D), whereas a dominant negative version of Ras (HRasN17)
did not have any significant effect on the Snail promoter
(Fig. 7A).
Pretreatment with the MEK1/2 inhibitor PD98059 or the PI3K inhibitor LY294002
resulted in the total blockade of Snail promoter induction after
HRasV12 cotransfection (Fig.
7A), indicating that both MAPK and PI3K signaling
pathways are involved in Snail promoter induction by activated H-Ras.
In contrast to PD98059, the LY294002 inhibitor did not have a significant
effect on the basal non-induced Snail promoter
(Fig. 7A).
Interestingly, activated H-Ras seems to be required for, and synergistically
cooperates with, TGF
1-mediated Snail induction. Cotransfection
with HRasV12 and TGF
1 treatment induced a much stronger activation of
the Snail promoter (about 8- to 12-fold) than that induced separately
by the growth factor or HRasV12 (Fig. 7,
B and D). Furthermore, cotransfection with the
dominant negative HRasN17 resulted in a 60% reduction of the
TGF
1-mediated induction of Snail promoter
(Fig. 7B).
The above results indicated the participation of activated H-Ras and its
cooperation with TGF1 in the regulation of Snail induction,
with the involvement of both MAPK and PI3K signaling pathways. To confirm
these results we used different mutants of activated HRasV12 that are able to
transduce signals by specific pathways
(45). We cotransfected the
mutants RasV12C40 (activated PI3K pathway), RasV12S35 (activated MAPK
pathway), and RasV12G37 (activated Ral-GDS) and analyzed the induction of the
Snail promoter in the absence or presence of TGF
1 treatment.
Results indicate that both V12S35 and V12C40 mutants maintain high levels of
Snail promoter activity both in the absence
(Fig. 7C) and presence
of TGF
1 (Fig.
7D), accounting for about 70% of the level obtained by
HRasV12 in both situations. In contrast, the V12G37 mutant had a lower
activity, accounting for only about 50% of the level obtained with HRasV12
mutant. Of note, under TGF
1 treatment, the V12G37 mutant did not show
any significant Snail promoter induction as compared with the
TGF
1 treatment alone (Fig.
7D). Taken together, these results indicate that both
MAPK and PI3K pathways are required for the H-Ras and TGF
1/H-Ras
mediated induction of Snail promoter, whereas the Ral-GDS pathway
might play a more modest role in Snail induction by activated H-Ras
alone.
Characterization of the Snail Promoter Regulatory
ElementsFinally, to get further insights into the regulation of
Snail promoter activity by TGF1 and H-Ras signals, we have
performed initial studies on the putative regulatory elements implicated.
In silico analysis of the cloned mouse Snail promoter region
(-900 bp) indicated the presence of several putative interaction sites for
different transcriptional regulators, including AP-4, AP-1, STAT, MZF-1, or
MyoD consensus sites (Fig.
8A). The organization of this promoter led us to generate
several deletion mutants containing the different control elements, as
indicated in the schematics of Fig.
8B. Particularly, we were interested in the AP-1 site
located at the -23/-33 position (from the ATG start codon), because AP-1 sites
are highly sensitive to downstream signals generated in response to TGF
and RasV12 pathways (54).
|
Transfection of MDCK cells with the different Snail promoter
mutants showed that the -900 bp construct exhibited the highest activity, and
decreased activities (4025%) were detected in most of the other
constructs (results not shown). The -100-bp construction has not significant
activity as compared with the other mutants or the full-length -900-bp
construct and could, therefore, be considered as a minimal basal promoter
region (data not shown). The effect of TGF1 and HRasV12 was analyzed on
the different Snail promoter constructs, and the activities were
normalized to that of the basal activity of each promoter construct
(Fig. 8B).
Surprisingly, the -100-bp promoter region was enough to respond to HRasV12
cotransfection, which induced a 3.6-fold activation of this basic
Snail promoter (Fig.
8B). The other Snail promoter constructions
showed a similar sensitivity to HRasV12 cotransfection, with exception of the
-575-bp construct that exhibited a stronger activation (5.8-fold)
(Fig. 8B). These
results suggested that the proximal AP-1 site could be the main regulatory
element implicated in H-Ras induction of Snail promoter and point to
the potential involvement of negative regulatory elements for H-Ras signals
located between -900 and -575 position of the Snail promoter.
Analyses of the various Snail promoter constructs in response to
TGF1 treatment showed that the -900-bp construct exhibited the stronger
induction (3-fold activation over basal non-stimulated control)
(Fig. 8B). Deletion of
sequences from the -575-bp position greatly reduced TGF
1 activation, and
no response to TGF
1 was achieved with the -100-bp construct
(Fig. 8B). Two
additional constructs containing -675- and -200-bp sequences showed the same
induction by TGF
1 as the -575- and -300-bp constructs, respectively
(data not shown). These results suggest that the TGF
1 response elements
are located between the -675- and -900-bp positions, correlating with all the
experiments done with the full-length construction. Several putative binding
regions for different regulatory transcription factors (AP-4, MZF-1) are
present between the -675- and -900-bp region that could be responsible of the
TGF
1 activation of the Snail promoter.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TGF1 induces a scattering phenotype in MDCK cells characterized by
the quick internalization and further loss of E-cadherin from the cell
surface, decreased expression of cytokeratins, induction/reorganization of
mesenchymal markers, reorganization of the actin cytoskeleton, and increased
cell motility. The phenotypic and differentiation markers changes observed
here are consistent with some of the operational criteria proposed recently
(36) for the definition of a
complete EMT process. Nevertheless, it should be kept in mind that the EMT
process induced by TGF
1 in MDCK cells occurs concomitantly to a growth
inhibitory response, in agreement with previous reports
(30,
35,
50,
53) on MDCK cells and other
non-transformed epithelial cell types. The phenotypic changes induced by
TGF
1 in MDCK cells are similar to those observed in other epithelial
cell systems (37,
40,
56) but differ in the extent
of E-cadherin repression observed in the different systems. They also differ
from those observed in the mammary EpH4 cells in which TGF
is not able
to induce by itself an EMT process
(33), indicating that the
sensitivity and phenotypic response to TFG
can be modulated by the
specific epithelial cell type.
Our present results provide the following evidence supporting that Snail is
mediating the EMT triggered by TGF1 in MDCK cells. (a) 24-h
treatment of MDCK cells with TGF
1 leads to a 2- to 3-fold induction of
Snail mRNA level, and the Snail transcripts are maintained
above the basal levels after 72 h of treatment. (b) The
Snail promoter activity is induced to a similar level after 24 h of
TGF
1 treatment. (c) The increase and maintenance of
Snail transcripts after TGF
-1 treatment is correlated with the
reduction of E-cadherin mRNA levels and promoter activity detected
between 48 and 72 h of treatment and with the overall changes in the cell
phenotype. The partial repression of the exogenous mouse E-cadherin
promoter and the fact that total E-cadherin protein levels are only slightly
decreased after 72 h of TGF
1 treatment might argue against a direct
repression of E-cadherin by Snail. However, the repression of the
exogenous E-cadherin promoter activity after TGF
-1 treatment is
dependent on the Snail-interacting promoter sequences
(Fig. 4B) located in
the E-pal element (14,
17). Furthermore, a strong
repression of E-cadherin mRNA is indeed observed after 72 h of
TGF
-1 treatment. The partial repression of the E-cadherin
promoter observed here might reflect intrinsic differences between endogenous
and exogenous E-cadherin promoter regulation in MDCK cells or be
explained by the moderate levels of Snail induction under those conditions. On
the other hand, the slow turnover of E-cadherin protein
(49) might well explain the
moderate decrease in E-cadherin protein levels observed after 72 h of
TGF
1 treatment. Despite this fact, the strong redistribution of
E-cadherin induced by TGF
1 in MDCK cells suggests that perturbation of
the functional localization of E-cadherin at cell-cell contacts should be
enough to initiate the EMT, which could be later sustained by effective
repression of E-cadherin mRNA following TGF
1 treatment.
Furthermore, Snail might regulate other genes required, in conjunction with
E-cadherin downregulation, for the EMT process. Indeed,
Snail-mediated repression of cytokeratin 18 has been reported recently in
colon carcinoma HT29 cells
(57), and our ongoing studies
on Snail target genes indicate that besides E-cadherin, expression of
genes coding for several cytokeratins, desmogleins, and desmoplakins are
strongly repressed in Snail-expressing MDCK
cells.2
A direct effect of TGF1 signaling in Snail expression is
supported by our analysis of the mouse Snail promoter, because the
growth factor consistently induced the promoter activity by 35-fold
over the basal levels in MDCK cells and also induced the Snail
promoter in other epithelial cell lines. In contrast, FGF2 had a milder effect
on the Snail promoter activity, but a cooperation between FGF2 and
TGF
1 was clearly detected (Fig.
6C). These results are also in agreement with the
phenotypic effects observed in MDCK cells in the presence of these two
factors, because FGF2 alone was unable to induce significant phenotypic
changes or decreased E-cadherin organization at the cell-cell contacts (see
Figs. 1 and
5). Interestingly, activated
H-Ras is also able to induce the Snail promoter activity and, more
significantly, synergistically cooperates with TGF
1
(Fig. 7). These results might
explain the apparently increased induction of Snail mRNA levels
observed by TGF
treatment in murine hepatocytes after H-Ras
transformation (35). The
cooperation between TGF
and activated H-Ras has been reported previously
(33,
36) to be required for a
complete EMT in some cell systems, such as in EpRas cells, where indeed both
signals participate into the invasive and metastatic phenotype.
The specific signaling pathways involved in EMT mediated by TGF and
activated H-Ras have been also addressed in the present study. Our results do
not support a direct involvement of the Smad pathway in Snail
promoter regulation, although an indirect involvement cannot be presently
discarded, because the Smad pathway is activated by TGF
1 in MDCK cells
(data not shown). In fact, the cooperation between FGF2 and TGF
1 in
Snail promoter induction is magnified by a dominant negative version
of Smad4 (Fig. 6D),
suggesting a potential cross-talk between Smad and growth factor signals, as
reported in other cell systems
(50). In contrast, the MAPK
pathway appears to be directly involved in the EMT process driven by
TGF
1 in MDCK cells. This conclusion is supported by the strong and
sustained activation of ERK1/2 after TGF
1 treatment, the blockade of the
phenotypic effects of the growth factor by the MEK1/2 inhibitor PD98059, and,
more significantly, from the studies on the Snail promoter. Even the
basal activity of the Snail promoter is inhibited by PD98059,
suggesting the requirement of active MAPK for expression of Snail
promoter in MDCK cells. An active MAPK pathway is also required for the
induction of Snail promoter by activated H-Ras, as deduced from the
studies with PD98059 and specific RasV12 mutants (see Figs.
6 and
7). The PI3K pathway, although
apparently not required for the activity of the basal Snail promoter,
is needed for Snail promoter activation by oncogenic H-Ras, alone or
in cooperation with TGF
1 (see Figs.
6 and
7). These results are in
agreement with the observed activation of the PI3K pathway after TGF
1
treatment (Fig. 3) and with
recent findings indicating that PI3K activity is necessary for cell scattering
and survival after TGF
1 treatment in other cell systems
(36,
38) and for the maintenance of
the fibroblastic phenotype in H-Ras transformed murine hepatocytes
(35). Taken together, our
results support a major role for the MAPK pathway in TGF
1-mediated
induction of Snail promoter and the cooperation between MAPK and PI3K
pathways in the synergistic induction of Snail mediated by TGF
1
and activated H-Ras. The participation of MAPK pathway into the EMT and
invasive phenotype of MDCK cells has been reported previously either in stable
transfectants with an activated MEK1 version
(42) or by using an inducible
form of c-Raf (Raf-ER), which also led to the autocrine production of
TGF
(53). This latter
report established a strong link and synergism between TGF
and the
Raf-MAPK pathway in the promotion of invasiveness and in vivo
malignancy. The requirement of TGF
signaling for invasiveness and
metastasis has also been established previously
(58) in the EpRas cell
system.
A large body of evidence strongly supports that TGF acts as
stimulator of malignant progression in late stages of carcinogenesis (reviewed
in Refs. 59 and
60). The results presented
here provide the first evidence to link TGF
1 signaling to Snail
repressor and EMTs, further reinforcing the important role of this growth
factor into the malignant progression. Furthermore, the cooperation between
H-Ras and TGF
1 in Snail promoter induction reported here can be
of biological significance, because activating mutations of H-Ras are present
in a high number of tumors and can eventually contribute, together with
acquired resistance to the anti-proliferative effects of TGF
, to the
malignant conversion. Interestingly, H-Ras activation can lead to the
autocrine production of TGF
in various cell systems
(35,
53). These findings, together
with the over-production of TGF
observed in a high percentage of human
tumors (61) and the fact that
most tumors maintain a functional TGF
signaling system
(59,
60), further reinforce the
cooperation between H-Ras and TGF
signals in malignancy. Our present
results add a further step into the mechanisms of tumor progression, linking
TGF
signaling and oncogenic Ras activation to induction of the promoter
of invasion Snail.
The promoter region of Snail transcription factor contains several
potential control elements for H-Ras and TGF downstream signals. The
signals that are highly induced by H-Ras seem to activate the minimal promoter
region of Snail near the initiation site. In contrast, this proximal
region is not sensitive for TGF
1 signals, indicating that transduction
of the different signals could require the coordination of several response
elements in the Snail promoter. On other hand, the central region of
Snail promoter (from -900 to -575 bp) appears to negatively regulate
its basal expression and the signal-mediated induction, suggesting the
presence of negative regulators in this region. It is tempting to speculate
that those putative control elements can be involved in the fine regulation of
Snail expression in normal biological process. Although further
studies are clearly required to characterize the specific control elements and
transcription factors responsible of Snail expression in different
biological situations, the results reported here can contribute to a better
understanding of the molecular mechanisms of malignant progression, involving
some relevant regulators, such as H-Ras, TGF
, and Snail. They also open
the way to future studies in which positive regulators of EMT should be
considered as promising targets for new anti-tumor therapies.
![]() |
FOOTNOTES |
---|
To whom correspondence should be addressed. Tel.: 34-91-585-4411; Fax:
34-91-585-4401; E-mail:
acano{at}iib.uam.es.
1 The abbreviations used are: EMTs, epithelial-mesenchymal transitions; AP,
activator protein; ERK, extracellular signal-regulated kinase; FGF, fibroblast
growth factor; MAPK, mitogen-activated protein kinase; MDCK, Madin-Darby
canine kidney; MEK, MAPK/ERK kinase; PI3K, phosphatidylinositol 3-kinase; RT,
reverse transcription; TGF, transforming growth factor
; FBS,
fetal bovine serum.
2 H. Peinado, A. Fabra, J. Palacios, and A. Cano, manuscript in
preparation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|