From the Developmental Signalling and ¶ Signal
Transduction Laboratories, Cancer Research UK London Research
Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United
Kingdom
Received for publication, September 4, 2002, and in revised form, November 7, 2002
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ABSTRACT |
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In normal epithelial cells, transforming growth
factor- Transforming growth factor- We previously described a model tissue culture system that exhibits
certain parallels to the process of tumorigenesis with respect to the
role of TGF- The signaling pathways downstream of the TGF- Here we have investigated how the TGF- Plasmids--
pBabe-puro EGFP-tagged Cell Culture and Retroviral Infection--
MDCK cell lines
stably expressing Whole Cell and Nuclear Extracts--
Nuclear extracts were
prepared as described previously (11). Whole cell extracts were
prepared in buffer containing 1% Triton X-100 as described previously
(12) with the exception of the experiment shown in Fig. 4A
where cells were lysed directly into SDS-PAGE gel sample buffer and the
experiment shown in Fig. 4B where whole cell extracts were
prepared in RIPA buffer.
Antibodies, Western blotting, Electrophoretic Mobility Shift
Assay (EMSA), and Indirect Immunofluorescence--
The following
antibodies were used: ERK2/p42 MAPK (raised against the peptide
CEETARFQPGYRS); cyclin A (H432, Santa Cruz Biotechnology); Smad4 (B8,
Santa Cruz Biotechnology); Smad2 cross-reacting with Smad3
(Transduction Laboratories); Smad3 (Zymed Laboratories
Inc.); E-cadherin (Transduction Laboratories);
p21WAF1/Cip1 (Santa Cruz Biotechnology); pRb (BD
Biosciences); and GRB2 (Transduction Laboratories). The
anti-PCNA antibody (PC10) was obtained from the Imperial Cancer
Research Fund hybridoma unit. For Western blotting, proteins
were resolved by SDS-PAGE and transferred onto polyvinylidene
difluoride membrane (Millipore), and immunoreactive proteins were
visualized by ECL (Amersham Biosciences). EMSAs and indirect
immunofluorescence microscopy were performed as described previously
(4).
Cell Cycle Analysis--
To examine cell cycle distribution,
cells were fixed in 70% ethanol, treated with ribonuclease (100 µg/ml) for 5 min at room temperature, stained with 50 µg/ml
propidium iodide (BD Biosciences) for 5 min, and analyzed by flow
cytometry using excitation at 488 nm .
RNase Protection Assays--
All of the probes with the
exception of that for E-cadherin used for RNase protection were
designed against the human sequences but cross-reacted with the dog
sequences. The Smad2 probe recognized the region encoding amino acids
9-86 of the human sequence; Smad3, the region encoding amino acids
107-208; Smad4, the region encoding amino acids 94-227; and GAPDH,
the region encoding amino acids 18-77. The Long Term Activation of Raf Leads to Down-regulation of Smad3
Expression--
To understand how MDCK cells alter their response to
exogenous TGF-
Overall, the expression of Smad proteins in these cells was analyzed in
whole cell extracts (Fig. 1B). Short or long term activation
of Raf did not affect expression levels of Smad2 and Smad4. However,
the activation of Raf for long periods caused a marked decrease in the
level of Smad3. After 14 days of Raf activation, Smad3 expression was
markedly reduced, and moreover, in cells grown continuously in 4HT
(RafT cells), Smad3 could not be detected. Both of these conditions
caused EMT as demonstrated by the loss of the epithelial marker
E-cadherin. To determine whether the expression of Smad3 was altered at
the transcriptional level, the mRNA levels of Smad2, Smad3, and
Smad4 were directly measured by RNase protection assay (Fig.
1C). As a control for EMT, the levels of E-cadherin mRNA
were also monitored. The level of Smad3 mRNA was very strongly
reduced in RafT cells and also in MDCK
To address the question of whether long term activation of Raf is
sufficient to induce loss of Smad3 in the absence of EMT, the function
of the type I TGF-
A related issue is whether Smad3 down-regulation could occur in
response to prolonged TGF- Re-expression of Smad3 in MDCK RafT Cells--
To determine
whether the loss of Smad3 expression seen during EMT was responsible
for the altered responsiveness to TGF-
The ability of exogenous Smad3 to form DNA-binding complexes with Smad4
on the Smad-binding element of c-Jun (11) was also tested by an EMSA.
In MDCK
The EMT process requires continued synergism between Raf and TGF- Re-expression of Smad3 in MDCK RafT Cells Restores Sensitivity to
the Growth Inhibitory Effects of TGF-
When epithelial cells are stimulated with TGF-
Another event implicated in cell cycle arrest by TGF-
Cyclin A expression is essential for the progression of cell cycle
(20). MDCK Loss of Smad3 during EMT Leads to Resistance to the
Anti-proliferative Effects of TGF-
Previous studies on keratinocytes and mouse embryo fibroblasts
in which Smad3 had been deleted have shown that this also leads to a
loss of TGF-
An analysis of Smad3 expression levels in tumor cell lines shows that
its loss is not uncommon. In a panel of five colorectal tumor cell
lines (Colo741, Colo205, HCT116, CaCo2, and
HT29,2 only one cell
line, HT29, expresses appreciable levels of Smad3. However, this cell
line also lacks Smad4, and as a result, both Smad2 and Smad3 function
will be compromised. Colo741, Colo205, and HT29 all have activating
mutations in B-Raf (24). HCT116 has an activating mutation in K-Ras and
CaCo2 has an inactivating mutation in Smad4 (25). Only one of these
lines, Colo741, is obviously mesenchymal; therefore, it is certainly
not the case that low Smad3 expression is sufficient for EMT. Instead,
it is likely that the loss of Smad3 function by decrease in its
expression (or that of Smad4) is a requirement for a tumor cell to be
able to proliferate and/or survive in the presence of prolonged TGF- (TGF-
) typically causes growth arrest in the
G1 phase of the cell cycle and may eventually lead to
apoptosis. However, transformed cells lose these inhibitory
responses and often instead show an increase in malignant character
following TGF-
treatment. In the canine kidney-derived epithelial
cell line, MDCK, synergism between activation of the Raf/MAPK pathway
and the resulting autocrine production of TGF-
triggers transition
from an epithelial to a mesenchymal phenotype. During this process,
these cells become refractive to TGF-
-induced cell cycle arrest and
apoptosis. TGF-
signals are primarily transduced to the nucleus
through complexes of receptor-regulated Smads, Smad2 and Smad3 with the
common mediator Smad, Smad4. Here we show that the transition from an
epithelial to mesenchymal phenotype is accompanied by gradual
down-regulation of expression of Smad3. Restoration of Smad3 to
previous levels of expression restores the cell cycle arrest induced by
TGF-
without reverting the cells to an epithelial phenotype or
impacting on the MAPK pathway. Regulation of apoptosis is not affected
by Smad3 levels. These data attribute to Smad3 a critical role in the control of cell proliferation by TGF-
, which is lost following an epithelial to mesenchymal transition.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(TGF-
)1 plays two rather
paradoxical roles in cancer (1-3). At early stages, it acts as a tumor suppressor primarily through its ability to induce growth arrest and
apoptosis in epithelial cells from which the majority of human tumors
are derived. However, as tumors progress, they frequently become
resistant to the growth inhibitory and pro-apoptotic effects of
TGF-
, and at late stages, TGF-
acts as a tumor promoter. It acts
directly on tumor cells to enhance epithelial to mesenchymal transition
(EMT), increase motility, invasiveness, and metastasis and acts
indirectly on the surrounding stroma to enhance angiogenesis and
decrease immune surveillance (1-3).
signaling (4). Madin-Darby canine kidney (MDCK) cells
are an untransformed immortalized dog kidney epithelial cell line that
are characterized by the formation of a simple monolayer of cells with
high electrical resistance across it and the expression of epithelial
markers such as E-cadherin and ZO-1, which are components of adherens
and tight junctions (5). The activation in these cells of the Raf/ERK
MAPK pathway by an inducible Raf construct,
Raf-ER, causes autocrine
expression of TGF-
(4). This synergizes with ERK MAPK to induce a
gradual change in phenotype from epithelial to mesenchymal, a process during which epithelial markers are lost, adherens and tight junctions degrade, and the cells express mesenchymal markers such as vimentin and
become more motile (4). The activation of Raf in MDCK cells leads to
rapid induction of protection from apoptosis induced by exogenous
TGF-
and other death stimuli (within 1 day) followed by much slower
protection from the specific growth inhibitory effects of TGF-
.
Cells remain responsive to TGF-
even after prolonged Raf activation,
showing increased invasive behavior.
receptors are now
known in some detail. The major signal transducers are the Smads.
Receptor activation leads to phosphorylation and activation of the
receptor-regulated Smads, Smad2 and Smad3. They form complexes with the
common mediator Smad, Smad4, that accumulate in the nucleus and are
directly involved in transcriptional activation of target genes,
usually in conjunction with other transcription factors (6).
signaling pathway is
perturbed in these Raf-expressing MDCK cells to allow them to become
specifically resistant to the anti-proliferative effects of TGF-
while maintaining other TGF-
responses. We demonstrate that a
gradual down-regulation of Smad3 during the process of EMT is
sufficient for the cells to lose their ability to undergo growth arrest
in response to TGF-
. Responsiveness is restored upon re-expression
of Smad3 to previous levels of expression. These data suggest that
Smad3 plays a critical role in the control of cell proliferation by
TGF-
, which is lost following EMT.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
Raf-1:hbER DD (referred
to as
Raf-ER) was kindly provided by M. McMahon (7). Human Smad3
cDNA was subcloned into the EcoRI/SalI
cloning sites of the pBabe-hygromycin and pBabe-bleomycin vectors (8).
The CAGA12-luciferase reporter was as described previously
(9).
Raf-ER and their derivative RafT cells, which had
been grown continuously in 100 nM 4-OH tamoxifen (4HT),
have been described previously (4). They express the ecotropic
retrovirus receptor. MDCK RafT cells stably expressing human Smad3 were
generated by retroviral infection. GP+E-packaging cells were
transfected using LipofectAMINE (Invitrogen) with either Smad3-pBabe-bleomycin, Smad3-pBabe-hygromycin, or empty vector and then
selected with the appropriate antibiotic (100 µg/ml hygromycin or 25 µg/ml bleomycin) for 8 days. MDCK RafT cells were then incubated with
retrovirus-containing supernatants and then cultured in medium containing the appropriate antibiotic as described above to select for
virus-infected cells. TGF-
1 (PeproTech) was used at the
concentrations indicated in the figure legends. The TGF-
type I
receptor inhibitor SB-431542 was used as described previously (10).
-actin probe was as
described previously (13). For the E-cadherin probe, an E-cadherin
fragment was prepared by RT-PCR from total RNA from MDCK cells using
the primers (forward 5'-TGACAGAGCCTCTGGATAGAG-3' and reverse
5'-CTCGTTCTCAGGCACCTGAC-3') and subcloned into pGEM-T vector (Promega)
for probe preparation. All of the probe preparation and RNase
protection assays were as described previously (13, 14).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
during the process of Raf-induced EMT, we examined
the functions of the Smads in cells expressing
Raf-ER but in its inactive form and in the same cells treated with 4HT for different times to activate the
Raf-ER. Initially, the translocation of the
three TGF-
-regulated Smads (the receptor-regulated Smads, Smad2 and
Smad3, and the common mediator Smad, Smad4) to the nucleus upon TGF-
treatment was studied (Fig.
1A). TGF-
-induced
translocation of Smad4 to the nucleus was observed in MDCK cells
expressing
Raf-ER that were untreated or cells that had been induced
with 4HT for a short (24 h) or long (14 days) period. Cells growing continuously in 4HT (RafT) also showed this translocation. Similar results were seen for the translocation of Smad2. However, we noted
that in cells in which Raf had been activated for longer periods and in
which EMT had occurred (see below), Smad3 failed to translocate to the
nucleus in response to TGF-
treatment. As expected, in all cases,
4HT treatment induced the phosphorylation of ERK2 as visualized by a
shift in mobility in an ERK2 Western blot.
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Fig. 1.
Long term activation of Raf leads to
down-regulation of Smad3 expression. A, nuclear extracts
were analyzed for Smad2, Smad3, and Smad4 and for phosphorylation of
ERK2 by Western blotting. They were free from cytoplasmic contamination
as demonstrated by the lack of GRB2. Total lysate from untreated MDCK
Raf-ER cells was used as a control (Total). PCNA was used
as a loading control. MDCK
Raf-ER cells untreated or treated with
100 nM 4-OH tamoxifen (4HT) for 24 h or 14 days and
MDCK RafT cells were treated with 2 ng/ml TGF-
for 1 h as
indicated. MDCK RafT cells are MDCK
Raf-ER cells grown continuously
in 100 nM 4HT. B, total lysates were analyzed
for expression of Smad2, Smad3, Smad4, E-cadherin, PCNA, GRB2, and for
phosphorylation of ERK2 by Western blotting. C, RNase
protection analysis was performed with RNA from MDCK
Raf-ER cells
either untreated or treated with 100 nM 4HT as indicated
and RafT cells using probes against E-cadherin, Smad4, Smad3, Smad2,
and GAPDH. D, MDCK
Raf-ER cells were grown either
untreated or with 100 nM 4HT or with 100 nM 4HT
and 5 µM SB-431542, a type I TGF-
receptor inhibitor
for 18 days. Whole cell lysates were then immunoblotted for E-cadherin,
Smad2/3, and GRB2. Data are representative of at least three
independent experiments.
Raf-ER cells treated with 4HT
for 14 days. The E-cadherin probe detected no E-cadherin mRNA in
RafT cells and minimal amounts in MDCK
Raf-ER cells treated with 4HT
for 12 or 14 days. Smad2 and Smad4 mRNA levels were not altered
during the process of EMT. Taken together, these results demonstrate
that Smad3 expression is down-regulated in MDCK cells that have
undergone EMT. The time course of loss of Smad3 expression may lag
marginally behind that of E-cadherin loss. Because the reduction in
expression of E-cadherin during EMT is thought to involve methylation
of the regulatory region of its gene (15), it is possible that
methylation may also be involved in the loss of Smad3 expression.
receptor ALK5 was inhibited using the
potent and specific drug SB-431542 (10). This blocks the operation of
the Raf-induced TGF-
autocrine loop that is essential for EMT (4)
and allows us to assay the effects of Raf signaling in the absence of
TGF-
signaling. In the presence of SB-431542, prolonged Raf
activation failed to induce EMT as assessed by E-cadherin expression
levels in immunoblot (Fig. 1D) and immunofluorescence (data
not shown). As well as preventing Raf-induced E-cadherin loss,
SB-431542 also blocked down-regulation of Smad3 expression (Fig.
1D), indicating that prolonged Raf activation is not
sufficient to cause loss of Smad3 expression in the absence of EMT.
signaling in the absence of Raf stimulation. However, in the absence of Raf activation by 4HT treatment, TGF-
induces apoptosis in MDCK
Raf-ER cells
(data not shown) (4), so this cannot be addressed directly. It cannot be ruled out that prolonged TGF-
signaling under conditions where apoptosis or growth arrest are blocked by means other than Raf activation would lead to reduction in Smad3 expression.
, we restored the levels of
Smad3 expression in RafT cells by introducing human Smad3 cDNA in
retroviral expression vectors. Two different vectors for Smad3 were
used, one being selectable in hygromycin and the other in bleomycin.
After drug selection, individual cell clones were grown up from pools
of resistant cells. Three clones were chosen for further study, two of
which showed fairly similar levels of expression of Smad3 to wild type
MDCK and uninduced MDCK
Raf-ER cells (H2, H3) while a third clone,
B4b, had higher levels (Fig.
2A). These clones along with
parental MDCK RafT cells were maintained in 4HT to maintain the
activation of
Raf-ER. To test the functionality of Smad3, its
translocation to the nucleus in response to TGF-
was assessed (Fig.
2A). As noted previously, Smad3 translocates to the nucleus
in response to TGF-
in MDCK
Raf-ER cells not treated with 4HT but
is absent from RafT cells. The stimulation of RafT clones H2 and H3,
which re-express Smad3, with TGF-
induced its translocation into the
nucleus at similar levels as in
Raf-ER cells. In the case of clone
B4b, high levels of Smad3 protein were found in the nucleus before
stimulation with TGF-
, most probably because of the high
overexpression of Smad3 in this clone. In all of the cases with the
exception of B4b, the amount of nuclear phosphorylated Smad2 upon
TGF-
induction was close to normal levels. In the case of B4b, very
low levels of phosphorylated Smad2 were detected, suggesting that the
high levels of expression of Smad3 may interfere with Smad2
phosphorylation.
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Fig. 2.
Re-expression of Smad3 in MDCK RafT cells
without reversal of EMT. A, nuclear accumulation of Smad3
and phosphorylated Smad2 following treatment with 2 ng/ml TGF- 1 for
1 h. Parental MDCK RafT cells or clones re-expressing Smad3 (H2,
H3, or B4b) were grown in the presence of 4HT (100 nM).
MDCK
Raf-ER cells were grown without 4HT. 30 µg of nuclear
extracts were analyzed by Western blotting. B, formation of
Smad3·Smad4 complexes. The 32P-labeled c-Jun Smad-binding
element probe was incubated for 15 min with 10 µg of protein from the
same extracts used in A and also extracts from MDCK
Raf-ER cells treated with 4HT for 24 h and analyzed by EMSA.
The Smad3·Smad4 DNA-binding complex is indicated. C, ERK2
MAPK activation. Cells as in B were grown sparsely and were
left untreated or treated with 7.5 ng/ml TGF-
1 for 24 h. Whole
cell extracts (40 µg) were resolved by SDS-PAGE, blotted, and probed
with an antibody against ERK2, which recognizes both unphosphorylated
and phosphorylated forms. D, restoration of Smad3 expression
does not reverse EMT or affect
Raf-ER expression. MDCK cells
expressing
Raf-ER not induced with 4HT, MDCK RafT cells
re-expressing Smad3, or not were grown on nitrocellulose filters and
methanol:acetone-fixed. Cells were stained with an antibody recognizing
E-cadherin and examined by confocal laser scanning microscopy for
E-cadherin and EGFP-
Raf-ER expression. In the uninduced
Raf-ER
cells, basal levels of EGFP:
Raf-ER were too low to be detected (4).
E, expression of Smad3 and E-cadherin was detected by RNase
protection. 20 µg of total RNA extracted from MDCK
Raf-ER cells,
RafT cells, and RafT cells where Smad3 had been re-expressed (clone H3)
were analyzed by RNase protection with probes for canine E-cadherin or
human Smad3. Pretreatment with 4HT was for 24 h in the case of
MDCK
Raf-ER cells as indicated and continuous for RafT and H3 cells.
The protected fragments (E-cadherin or Smad3) are indicated.
-Actin
probe was used as a loading control. Data are representative of at
least three independent experiments.
Raf-ER cells untreated or treated with 4HT for only 24 h, TGF-
induced the formation of a nuclear Smad3/Smad4 DNA-binding
complex (Fig. 2B). This complex is greatly decreased in the
MDCK RafT cells because of the very low expression of Smad3. In all of
the MDCK RafT Smad3 re-expressing clones, a DNA-binding complex was
formed exclusively upon induction with TGF-
. Even in the case of B4b
cells where the high levels of expression of Smad3 caused its
localization to the nucleus without TGF-
treatment, virtually no
DNA-binding complex was detected in unstimulated cells. In all of the
cases, supershift analysis showed that the TGF-
-induced complex
contained Smad3 and Smad4 (data not shown). Note that induction of the
PAI-1-derived reporter gene CAGA12-luciferase (9)
was dependent on TGF-
in all of the Smad3 re-expressing clones,
suggesting that high nuclear levels of Smad3 alone were insufficient
for the induction of TGF-
-dependent transcriptional
responses (data not shown).
(Fig. 1D) (4, 16). To confirm that the clones used here had not lost Raf expression, the level of phosphorylation of MAPK
in cultures containing 4HT was determined. All of the cells treated
with 4HT showed high levels of activation of ERK2 (Fig. 2C).
In addition, we checked the clones for expression of the
Raf-ER
construct, which is fused to EGFP (7). The clones all continue to
express
Raf-ER (Fig. 2D). Moreover, staining for
E-cadherin showed that the re-expression of Smad3 had not lead to a
reversal of EMT and that the cells still showed a mesenchymal phenotype. An RNase protection assay on RNA extracted from unstimulated and 4HT-stimulated MDCK
Raf-ER cells, RafT, and RafT clone H3 cells
also showed that re-expression of Smad3 did not restore E-cadherin
expression (Fig. 2E). The expression of Smad3 mRNA was
strongly suppressed in RafT cells but restored to at least wild type
levels in H3 cells.
--
RafT cells in which
continuous activation of
Raf-ER has led to acquisition of a
mesenchymal phenotype are resistant to cell cycle inhibition in
response to TGF-
(4). To analyze whether re-expression of Smad3 in
RafT cells restores sensitivity to the growth inhibitory effects of
TGF-
, cell cycle distribution was examined in MDCK RafT cells and
MDCK RafT cells re-expressing Smad3. Cells were analyzed 24 h
after treatment with TGF-
(Fig. 3). In
MDCK
Raf-ER cells not treated with 4HT, stimulation with TGF-
led
to an increased percentage of cells in G1 and a reduced percentage of cells in S phase. This effect was the same whether or not
Raf was activated by 4HT treatment over a period of up to 2 days (4).
In contrast, in RafT cells where Raf has been continuously activated,
no significant changes in cell cycle distribution in response to
TGF-
were observed. However, the re-expression of Smad3 in RafT
cells rendered these cells sensitive to TGF-
-induced cell cycle
arrest, even in the continued presence of 4HT. RafT clones and pooled
populations expressing Smad3 showed a reduction in the number of cells
in S phase and an increase in the number of cells in G1.
Thus, there is a positive correlation between Smad3 expression and the
TGF-
-induced cell cycle regulation. Re-expression of Smad3 did not
resensitize RafT cells to the pro-apoptotic effects of TGF-
(data
not shown).
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Fig. 3.
Re-expression of Smad3 in MDCK RafT cells
restores sensitivity to TGF- -induced growth
arrest. MDCK
Raf-ER cells, MDCK RafT cells, and either pooled
(hygromycin (Hygro); bleomycin (Bleo)) or clonal
(H2, H3, B4b) populations of MDCK RafT cells stably re-expressing Smad3
were stimulated with TGF-
1 (7.5 ng/ml) for 24 h. Cell cycle
distribution was assayed by flow cytometry after propidium iodide
staining. The data for the hygromycin- and bleomycin-resistant cells
are from two separate experiments with appropriate controls. Data are
representative of at least three independent experiments.
, the retinoblastoma
susceptibility product (pRb) is dephosphorylated, leading to inhibition
of E2F, a crucial event for cell cycle arrest (17). To understand the
molecular mechanisms involved in the rescue of TGF-
-induced cell
cycle arrest upon Smad3 re-expression in RafT cells, we looked at the
levels of phosphorylation of pRb. MDCK
Raf-ER, RafT, and RafT-Smad3
cells growing in serum were treated with TGF-
for 8 h. MDCK
Raf-ER cells not induced with 4HT or those that had been induced for
24 h with 4HT showed a shift in mobility of pRb protein toward the
dephosphorylated state upon TGF-
stimulation independently of the
induction of the Ras/MAPK pathway (Fig.
4A). In contrast, in MDCK RafT
cells, very little pRb was dephosphorylated in response to TGF-
.
When the MDCK RafT-Smad3 clones were examined, all of them showed clear
dephosphorylation of pRb in response to TGF-
. Thus, Smad3 was
responsible for mediating TGF-
-induced pRb dephosphorylation.
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Fig. 4.
Smad3 re-expression in MDCK RafT cells
restores molecular events associated with
TGF- -induced cell cycle arrest. A,
growing MDCK cells were treated with TGF-
for 8 h as indicated.
Whole cell extracts were resolved by SDS-PAGE and Western blotted with
an antibody recognizing both hyperphosphorylated (slower migrating,
pRbphos) and hypophosphorylated (faster migrating,
pRb) forms of retinoblastoma protein. In the case of MDCK
Raf-ER, the treatment with 100 nM 4HT was for 24 h
before stimulation with 7.5 ng/ml TGF-
1 as indicated. The RafT, H2,
H3, and B4b cells were maintained in 4HT. B, total RIPA
buffer lysates (50 µg) from cells left unstimulated or stimulated
with 2 ng/ml TGF-
1 for the given times were Western blotted using
antibodies against p21WAF1/Cip1, GRB2 (loading control),
Smad3, and E-cadherin. C, total lysates from cells left
unstimulated or stimulated with 7.5 ng/ml TGF-
1 for 24 h were
Western blotted using an antibody against cyclin A. Data are
representative of at least three independent experiments.
is the
induction of expression of p21WAF1/Cip1 (hereafter referred
to as p21), an inhibitor of activated cyclin-dependent kinases (18, 19). Its expression is thought to contribute to the
inhibition of CDK4/6-cyclin D and CDK2/cyclin E complexes, leading to
hypophosphorylation of pRb and thus preventing the progression of the cell cycle (18). MDCK
Raf-ER cells responded to
TGF-
by increasing the levels of p21 mRNA (data not shown) and
protein (Fig. 4B). However, in MDCK RafT cells, TGF-
did not induce expression of p21 (Fig. 4B). By contrast, MDCK
RafT-Smad3 cells showed low p21 levels, which increased when stimulated
with TGF-
(Fig. 4B). Thus, the re-expression of Smad3 in
these cells restores TGF-
-induced expression of p21.
Raf-ER cells not induced with 4HT or those that had been
induced for 24 h with 4HT down-regulate cyclin A expression in
response to TGF-
, whereas MDCK RafT cells are resistant to the
TGF-
-induced down-regulation of cyclin A (Fig. 4C) (4). In the MDCK RafT-Smad3 clones, this function was restored, because all
of them showed a clear down-regulation of cyclin A in response to
TGF-
(Fig. 4C).
--
The data presented here
provide an explanation of how cells can become refractory to the growth
inhibitory effects of TGF-
during the process of epithelial to
mesenchymal transition driven by constitutive activation of the
Raf/MAPK pathway that is accompanied by autocrine TGF-
production.
Smad3 expression is lost with a time course similar to the loss of
E-cadherin expression, while Smad2 and Smad4 are maintained and appear
to function normally. Exogenous re-expression of Smad3 restores
TGF-
-induced growth arrest.
-induced growth arrest (21-23). Mouse embryo fibroblasts deleted in Smad2 were likewise resistant to the
antiproliferative effects of TGF-
(22), suggesting that Smad2 is
also required although not sufficient for TGF
-induced growth arrest.
The loss of Smad3 during EMT driven by oncogenic Ras together with
TGF-
in cells that normally express a high Smad3 to Smad2 ratio
could be critical in releasing them from the growth inhibitory effects of TGF-
. Other TGF-
responses that may provide a competitive advantage for the tumor cells could then be maintained via Smad2 (21),
whereas effects on host cells, especially angiogenesis and evasion of
immune response, may also make TGF-
production advantageous to the tumor.
stimulation either resulting from autocrine production or from the
stroma. Smad3 down-regulation may be able to occur by a number of
mechanisms, one of which is linked to the process of EMT as seen in the
MDCK cells.
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ACKNOWLEDGEMENTS |
---|
We thank Richard Treisman for GAPDH cDNA,
Martin McMahon for pBabe-puro EGFP Raf-1:hbER DD, Peter ten Dijke
for Smad3 cDNA and the anti-phosphorylated Smad2 antibody, and
Alastair Reith and Nicholas Laping at GlaxoSmithKline Pharmaceuticals
for SB-431542. We are grateful to the ICRF FACS laboratory for
fluorescence-activated cell sorter analysis, Gordon Peters for comments
on the paper, and Richard Marais for sharing data prior to publication.
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FOOTNOTES |
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* This work was supported by ICRF (now Cancer Research UK) and an MRC postdoctoral training fellowship (to F. J. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
Present address: metaGen GmbH, Oudenarder Strasse 16, D-13347
Berlin, Germany.
** To whom correspondence may be addressed. E-mail: caroline.hill@cancer.org.uk or julian.downward{at}cancer.org.uk.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M209019200
2 F. J. Nicolás, P. H. Warne, J. Downward, and C. S. Hill, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
MDCK, Madin-Darby canine kidney;
ERK, extracellular signal-regulated kinase;
MAPK, mitogen-activated protein
kinase;
ER, estrogen receptor;
EGFP, enhanced green fluorescent
protein;
EMT, epithelial to mesenchymal transition;
PCNA, proliferating
cell nuclear antigen;
4HT, 4-OH tamoxifen;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
RIPA, radioimmune
precipitation assay.
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