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Gene Regulation:
The Anti-proliferative Function of the
TGF- β1 Signalling Pathway Involves the
Repression of the Oncogenic TBX2 by its
Homologue TBX3
Jarod Li, Deeya Ballim, Mercedes Rodríguez,
Rutao Cui, Colin R. Goding, Huajian Teng
and Sharon Prince
J. Biol. Chem. published online November 4, 2014
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TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
The anti-proliferative function of the TGF-β1 signalling pathway involves the repression of the
oncogenic TBX2 by its homologue TBX3
1
1
Jarod Li , Deeya Ballim , Mercedes Rodriguez2, Rutao Cui3, Colin R. Goding2, Huajian Teng1,
Sharon Prince1
1
Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Observatory, 7925,
Cape Town, South Africa. 2Ludwig Institute for Cancer Research, Oxford University, Old Road Campus,
Headington, Oxford, OX3 7DQ, United Kingdom. 3Department of Dermatology, Boston University
School of Medicine, Boston, MA, USA
Running Title: TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
To whom correspondence should be addressed: A/Prof Sharon Prince, Department of Human
Biology, Faculty of Health Sciences, University of Cape Town, Observatory, 7925, South Africa, Tel:
27-21-406-6240, Fax: 27-21-448-7226, E-mail: [email protected]
CAPSULE
Background: The transcription factor TBX3
regulates the anti-proliferative function of the
TGF-β1 pathway but its downstream target(s)
responsible for this is unknown.
Results: TBX3 downstream of TGF-β1 signalling
represses TBX2 to inhibit proliferation.
Conclusion: The downregulation of TBX2 is
required for the anti-proliferative function of the
TGF-β1-TBX3 axis.
Significance: This study sheds light on the
mechanisms involved in the anti-proliferative role
of TGF-β1.
ABSTRACT
A growing body of work has shown that the highly
homologous T-box transcription factors TBX2 and
TBX3 play critical but distinct roles in embryonic
development and cancer progression. For example,
TBX2 and TBX3 are upregulated in several
cancers and recent evidence suggests that whereas
TBX2 functions as a pro-proliferative factor,
TBX3 inhibits cell proliferation but promotes
cancer cell migration and invasion. While the
molecular mechanisms regulating these functions
of TBX2 and TBX3 are poorly understood we
recently reported that the TGF-β1 signalling
pathway upregulates TBX3 expression to mediate,
in part, its well described anti-proliferative and
pro-migratory roles. The TBX3 targets responsible
for these functions were however not identified.
Here we reveal for the first time that the TGF-β1
signalling
pathway
represses
TBX2
transcriptionally and we provide a detailed
mechanism to show that this is mediated by TBX3.
Furthermore, we implicate the downregulation of
TBX2 in the anti-proliferative function of the
TGF-β1-TBX3 axis. These findings have
important implications for our understanding of the
regulation of TBX2 and TBX3 and shed light on
the mechanisms involved in the anti-proliferative
and pro-migratory roles of TGF-β1.
The transforming growth factor β1 (TGF-β1)
pathway plays critical roles in a wide range of
cellular processes including cell proliferation,
apoptosis, differentiation and the immune response.
This multi-functional cytokine generally exerts its
effects by binding and activating the TGFβ
receptors I and II, which initiate a cascade of
signalling events frequently involving Smad
proteins and co-factors but which can also be
Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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Keywords: TBX2, TBX3, TGF-β1, proliferation, MCF-12A, B16
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
has no significant effects on this process (19–21).
Furthermore, we and others have also shown that
TBX2 and TBX3 play distinct roles in cancer
progression, with TBX2 functioning as a powerful
pro-proliferative factor and TBX3 playing a role in
migration and invasion (22, 23). Together with our
previously published data showing that TBX2 and
TBX3 exhibit mutually exclusive expression in
some melanoma cell lines (24), we therefore
speculated that the anti-proliferative function of
TGF-β1 may be due to its ability to downregulate
TBX2 expression. Indeed, here we show that in
human breast epithelial and mouse melanoma cells,
TGF-β1 negatively regulates TBX2 protein and
mRNA levels at a transcriptional level and
importantly, we demonstrate that this regulation is
dependent on increased TBX3 levels. Using both
in vitro and in vivo assays we reveal that TBX3
binds and regulates the TBX2 promoter in response
to TGF-β1 through a T-element at -186 bp.
Furthermore, we show that the downregulation of
TBX2 expression is responsible for the decrease in
cell proliferation induced by TGF-β1 and that this
is possibly mediated by increased p21 levels.
The T-box family of transcription factors are
highly conserved in evolution and play critical
roles in embryonic development. TBX3 is a
member of the TBX2 subfamily and along with its
closely related family member, TBX2, it plays an
important role in the development of the limbs,
heart and mammary glands (8). TBX2 and TBX3
are overexpressed in a number of cancers,
including breast, pancreatic, ovarian, liver, cervical
and melanoma (9–13). While there is evidence to
suggest that they both repress the cell cycle
regulators p19ARF and p21WAF1/CIP1/SDII, they have
distinct functions in embryonic development and
cancer (8–10, 14–19). For example, mouse studies
examining the effects of loss of function mutations
have shown that Tbx3 plays an important role
during mammary placode induction while Tbx2
EXPERIMENTAL PROCEDURES
Cell Lines and Culture Conditions—The normal
human breast epithelial MCF-12A cells were
maintained in complete media consisting of
DMEM/Ham’s F12 supplemented with 10% FBS,
100 U/ml penicillin, 0.1 μg/ml cholera toxin
(Sigma, MO, USA), 0.5 μg/ml hydrocortisone
(CALBIOCHEM, MA, USA), 10 μg/ml insulin
(Novorapid, Denmark), 20 ng/ml EGF (GIBCO,
CA, USA) and 5% horse serum (Highveld
Biological, Lyndhurst, UK). B16 mouse melanoma
cells were maintained in RPMI 1640 (Highveld
Biological, Lyndhurst, UK) supplemented with 10%
FBS, 100 U/mL penicillin, and 100 µg/mL
streptomycin. All cell lines were maintained in a 5%
CO2 humidified incubator at 37oC. For TGF-β1
2
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Smad-independent (1). One of the well-known
roles of TGF-β1 is its ability to inhibit the
proliferation of epithelial cells but promote their
migration (2). TGF-β1 has been shown to inhibit
proliferation primarily by inducing a G1 cell cycle
arrest through its ability to transcriptionally
upregulate expression of the cyclin dependent
kinase inhibitors p15Ink4b and p21Cip1 (3). Its
pro-migratory effects, however, appear to be
mainly due to its ability to regulate key players of
epithelial-mesenchymal transition (EMT) (4).
These anti-proliferative and pro-migratory roles of
TGF-β1 are important in embryonic development,
for example during branching morphogenesis of
the mammary gland, as well as during oncogenesis
(5, 6). We recently reported that TGF-β1
upregulates expression of the T-box transcription
factor, TBX3, through a mechanism involving the
co-operation of Smad proteins and JunB. This was
shown to be a critical downstream event for
mediating TGF-β1-induced anti-proliferation and
pro-migration of breast epithelial cells and
keratinocytes but the TBX3 target genes
responsible for this were not described (7).
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
treatment, we used 5 ng/ml rhTGF-β1 (R&D
systems, MN, USA), diluted in 4mM HCl, 1mg/ml
BSA. For inhibition of de novo protein synthesis
experiments, cells were pre-treated with 30 µg/ml
cycloheximide (Sigma-Aldrich) for 1 hr and then
treated with TGF-β1 for 8 hrs.
Generation of stable cell lines expressing
TBX2—To generate stably transfected cell lines,
MCF-12A cells were transfected with either the
empty expression vector pcDNA3.1 (+) or with
this vector containing the full-length human TBX2
cDNA using X-tremeGENE HD DNA transfection
reagent (Roche Diagnostics, Basel, Switzerland).
Stable transfected cells were selected by 400 μg/ml
G-418.
Generation of MCF-12A cell line over-expressing
TBX3 - The MCF-12A cells were transiently
transduced using adenovirus with human TBX3
cDNA cloned into the vector component of an
Adeno-X Tet-off expression system 1 (Clontech,
CA, USA). The virus was purified using the
Adeno-X Maxi purification kit (Clontech, USA).
All steps were performed according to the
manufacturer’s instructions.
Transfection and luciferase assays—Transient
transfections were performed using X-tremeGENE
HD DNA transfection reagent (Roche Diagnostics,
Basel,
Switzerland)
according
to
the
manufacturer’s instructions. MCF-12A cells were
plated at 1 x 105 cells per well of a 12-well plate,
24 hrs before transfection. Cells were
co-transfected with 400 ng of a TBX2-luciferase
reporter plasmid plus 100 ng of the TBX3
expression plasmids or corresponding amounts of
an empty-vector plasmid. Luciferase activities
were measured using the Luminoskan Ascent
luminometer (Thermo Labsystems, Franklin, MA,
USA). All transfections were performed in
duplicate and at least three independent
experiments were done to confirm reproducibility.
Firefly luciferase values were expressed relative to
empty-vector control.
The TBX3 N-terminal construct was constructed
by excising the C-terminal portion of the
pCMV-TBX3 construct with BglII and SpeI and
re-ligated.
Western Blot Analysis—Cells were harvested and
protein prepared as described previously (27).
Primary antibodies used were as follows: goat
polyclonal anti-TBX2 antibody (sc-17880, Santa
Cruz Biotechnology, CA, USA), rabbit polyclonal
anti-TBX3 (42-4800, Zymed, Invitrogen, USA),
rabbit polyclonal anti-p21 (sc-397, Santa Cruz
Biotechnology, CA, USA) and rabbit polyclonal
anti-p38 (M0800, Sigma, Germany).
Quantitative Real-Time PCR (qRT-PCR) —Cells
were harvested and mRNA prepared as described
previously (28). Primers used to amplify the
human
TBX3
(QT00022484),
TBX2
(QT00091266) and GUSB (QT00046046) were
purchased from Qiagen, USA.
Site-directed
mutagenesis
—Site-directed
mutagenesis was performed on the −218/+33 bp
TBX2
promoter-luciferase
reporter
or
3
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Plasmid constructs—The human TBX2 promoter
luciferase reporter constructs have been described
previously (25). The pcDNA 3.1(+) containing the
full-length human TBX2 cDNA was supplied by
Prof. van Lohuizen (Antoni van Leeuwenhoek
hospital, Amsterdam, The Netherlands). The TBX2
cDNA was then excised and the vector re-ligated
to generate the pcDNA3.1 (+) empty vector, into
which the human TBX3 cDNA was cloned using
the NheI and BamHI sites. The p21-pRc/CMV
plasmid, which was prepared in the laboratory of
Prof William Kaelin, was obtained from Addgene
(Addgene plasmid 20814) (26).
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
is the ChIP of interest and ∆Ct2 is the IgG.
pCMV-TBX3 expression construct as a template
using PFU polymerase reagents (Promega, WI,
USA). Mutations were introduced as follows (only
sense-strand presented and mutations indicated
with lowercase letters): T-element (−186): 5′GCTGAGGCTTCCaAaAaCTTCTCCCAGGCC
-3′; TBX3 DNA binding domain mutant (R133G):
5’
CATTACCAAGTCGGGAgGGgGAATGTTTCCT
CCATTTAAAG 3’. The integrity of each mutant
construct was verified using agarose gel
electrophoresis and sequencing.
siRNA sequences and transfection—Transient
knockdown of TBX2 and TBX3 was achieved by
siTBX2#1 (SI02656563), siTBX2#2 (SI03238802),
siTBX3#1
(SI00083503)
and
siTBX3#2
(SI03100426) purchased from Qiagen (MD, USA).
Knockdown of p21 was achieved using
ON-TARGETplus SMARTpool Human CDKN1A
siRNA (sip21#1) (Thermo Scientific) and sip21#2
(SI00299810) from Qiagen (MD, USA). The cells
were transfected with siRNAs to TBX2, TBX3,
p21 or a control (non-silencing) siRNA (1027310;
Qiagen, USA) using HiPerFect (Qiagen, USA)
according to manufacturer's instructions.
Growth Curves—Short-term growth of cells was
compared over a 3-day period, as described
previously (29). 0.5 x 104 cells were seeded in
triplicate in 24 well plates on day zero and treated
with vehicle or 5 ng/ml TGF-β1 for 2-3 days.
Chromatin immunoprecipitation assays (ChIP)
—ChIP assays were carried out as previously
described (10). Briefly, MCF-12A cells treated
with TGF-β1 for 24 hrs were fixed in 1%
formaldehyde and the chromatin extracted,
sonicated and immunoprecipitated using antibodies
against TBX3 (sc-17871) or IgG (negative control,
Santa Cruz Biotechnology, USA). DNA
precipitated was analyzed by qRT-PCR using
human TBX2-specific primer pairs or a
non-specific promoter region (GUSB, Qiagen,
USA). Crossing values (Ct) of TBX3 precipitated
DNA were normalized against the Ct values of IgG.
Fold enrichment was determined using the ∆∆Ct
method: Fold enrichment= 2-(∆Ct1-∆Ct2), where ∆Ct1
Statistical Analysis—Statistical analysis was
performed by using the 2-sample t-test (Excel).
RESULTS
TGF-β1
transcriptionally
represses
TBX2
expression in MCF-12A breast epithelial and B16
melanoma cells. TBX2 and TBX3 are highly
homologous T-box factors with distinct oncogenic
roles. We recently reported that TBX3 is
transcriptionally activated by the TGF-β1
signalling pathway (7) and we therefore examined
whether the expression of TBX2 is also responsive
to this pathway. To this end we exposed MCF-12A
4
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DNA affinity immunoblot assay—Biotinylated
DNA
oligos
were:
WT:
5′-GCTGAGGCTTCCGACACCTTCTCCCAGG
CC-3′,
MT:
5′-GCTGAGGCTTCCaAaAaCTTCTCCCAGGC
C-3′,
Consensus:
5′-CTTAGGGAATTTCACACCTAGGTGTGAAA
TTCCCT-3′. For each DNA-binding reaction,
nuclear extract was incubated with biotinylated
DNA probe in binding buffer (20 mM Tris-HCl pH
7.6, 50 mM NaCl, 1 mM MgCl2, 0.2 mM EDTA,
0.5 mM dithiothreitol, 5% glycerol, and 10 ng/µl
poly(dI-dC)). The beads were washed with binding
buffer and boiled in protein loading buffer (125
mM Tris-HCl, pH 6.5, 0.4% SDS, 10%
β-mercaptoethanol, and 20% glycerol). Proteins
bound to the biotinylated probes were analysed by
SDS–PAGE, followed by immunoblotting using
goat polyclonal TBX3 (sc-17871) antibody (Santa
Cruz Biotechnology, Santa Cruz, CA).
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
little effect on TBX2 promoter activity suggesting
that the regulation of TBX2 by TGF-β1 either
requires a Smad co-factor(s) or involves a
mechanism that does not directly require the
Smads. We therefore repeated these experiments in
the presence or absence of the well-known Smad
co-factors, JunB or Sp1, but no repression was
observed (data not shown). Based on our previous
findings we speculated that TBX2 may be
downstream of TBX3 in the TGF-β1 pathway. To
address this we screened the TBX2 promoter for
T-elements and a T-element which is highly
conserved between Tbx2 promoters from several
species was identified at -186 bp (Fig. 3C). Indeed,
depleting TBX3 levels by siRNA in MCF-12A
cells diminished the ability of TGF-β1 to repress
TBX2 protein and mRNA levels (Fig. 3D, E). It is
worth noting that while the fold repression of
TBX2 by TGF-β1 in the siTBX3 cells was still
significant, it was lower than that of the control
cells and may be due to incomplete knockdown of
TBX3. Furthermore, knocking down TBX3 in
untreated cells resulted in an increase in TBX2
mRNA and protein levels suggesting that TBX3
represses basal and TGF-β1 regulated TBX2
levels.
The downregulation of TBX2 by TGF-β1 is
mediated by TBX3 in breast epithelial cells. To
narrow down the region of the TBX2 promoter
mediating its repression by TGF-β1, 5’ deletion
constructs of the human TBX2 promoter driving a
firefly luciferase reporter were tested in luciferase
assays. The results show that the repression of
TBX2 by TGF-β1 treatment is sustained even in
the -218 bp TBX2 promoter construct (Fig. 3A).
To investigate whether Smad3 and Smad4
(Smad3/4) are involved in this regulation,
MCF-12A cells were co-transfected with
increasing amounts of Smad3/4 expression vectors
and the -218 bp TBX2 promoter luciferase
construct. Figure 3B shows that Smad3/4 had very
TBX3 binds to the TBX2 promoter at -186 bp in
response to TGF-β1 treatment. We next wanted to
confirm that TBX3 can directly repress TBX2 in
vivo and to this end we performed a chromatin
immunoprecipitation (ChIP) assay. Cross-linked
chromatin was prepared from TGF-β1-treated and
control
MCF-12A
cells
and
DNA
immunoprecipitated with an antibody to TBX3
was subjected to qRT-PCR with primers spanning
the -186 bp putative T-element in the TBX2
promoter. The results obtained show that compared
to the untreated control, the binding of TBX3 to
the TBX2 promoter was enhanced by 8.5 fold in
the presence of TGF-β1 (Fig. 4A). To determine
whether TBX3 binds specifically to the -186 bp
5
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breast epithelial and B16 mouse melanoma cells to
TGF-β1 or vehicle over a time course spanning 1 –
36 hours. Western blot analyses show that TBX2
levels decrease substantially in response to
TGF-β1 treatment in both cell lines from 8 hours
in MCF-12A cells and 4 hours in B16 cells (Fig.
1A, B). This robust repression was also observed
in WI-38 normal human fibroblasts (data not
shown). Quantitative real-time PCR (qRT-PCR)
experiments performed on cells treated as above
demonstrate a corresponding decrease in TBX2
mRNA levels that precedes the decrease in protein
levels (Fig. 1C, D). To investigate if this regulation
is transcriptional, we tested whether the -1604 bp
TBX2 promoter is responsive to TGF-β1 treatment
using a luciferase assay and the results showed a
time-dependent decrease in reporter activity in
response to TGF-β1 (Fig. 2A). To distinguish the
need for de novo protein synthesis, cells were
pre-treated with cycloheximide (CHX), a protein
synthesis inhibitor, which abolished the repression
of TBX2 mRNA and protein levels by TGF-β1
(Fig. 2B, C). Together these results suggest that
TGF-β1 mediated repression of TBX2 is
transcriptional and that it requires the production
of nascent protein.
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
Ectopic expression of TBX2 is able to rescue
TGF-β1 inhibited cell proliferation. We next
investigated whether overexpression of TBX2 in
MCF-12A cells could rescue the inhibition of cell
proliferation by TGF-β1. Briefly, a pcDNA-TBX2
expression vector or the empty control vector was
transfected into MCF-12A cells and G418-resistant
clones were pooled for subsequent analysis.
Overexpression of TBX2 was confirmed by
western blotting (Fig. 5A) and growth curves show
that while cell proliferation was significantly
reduced by TGF-β1 in the control cells,
overexpressing TBX2 was sufficient to rescue this
phenotype (Fig. 5B). This was confirmed by flow
cytometry which showed that TGF-β1 treatment
induced a significant G1 cell cycle arrest in the
control cells but had no effect on the cell cycle
profile of the TBX2-overexpressing cells (Fig. 5C).
These results correlated with changes in the
protein and mRNA levels of the cyclin dependent
kinase inhibitor p21 (Fig. 5A and E). Importantly,
the increase in p21 mRNA and protein levels in
TGF-β1 treated cells are abrogated when TBX2 is
overexpressed suggesting that TBX2 may be the
upstream regulator of p21 in the TGF-β1 pathway.
Indeed, when TBX2-overexpressing cells were
transfected with a p21 expression vector they had a
decreased proliferative ability which could not be
further reduced by TGF-β1 treatment (Fig. 5D).
Interestingly, TBX2 overexpression failed to have
any significant effect on the TGF-β1-induced
activation of p15 mRNA levels (Fig. 5F), another
cell cycle inhibitor which has been shown to
mediate a G1 arrest in response to TGF-β1 (30).
Together,
these
data
imply
that
the
anti-proliferative effect of TGF-β1 on MCF-12A
cells occurs primarily through upregulation of p21,
since the continued repression of p21 in TBX2
overexpressing cells allowed the cells to proliferate
even in the presence of TGF-β1 and despite the
upregulation of p15. To confirm this, p21
To test whether TBX3 can repress the TBX2
promoter and whether the T-element at -186 bp
mediates this repression, MCF-12A cells were
co-transfected with a TBX3 expression vector and
either a wild type -218 bp TBX2 promoter
luciferase construct or a construct in which the
T-element was mutated by site-directed
mutagenesis. The results show that TBX3
repressed the TBX2 promoter by approximately 18
fold and that the ability of TBX3 to repress TBX2
was significantly reduced when the -186 bp
T-element was mutated (Fig. 4C). To identify the
functional domains of the TBX3 protein that are
responsible for this regulation, the wild type -218
bp TBX2 promoter luciferase construct was
co-transfected with vectors expressing either a WT
TBX3 protein or a TBX3 protein in which the
DNA binding site was mutated or only a
N-terminal TBX3 protein lacking the dominant
repression domain. The data obtained indicate that
the TBX3 DNA binding domain and C-terminus
containing a repression domain are important for
its ability to repress the TBX2 promoter (Fig. 4D).
All TBX3 constructs were expressed at similar
levels (Fig. 4C and D, right).
6
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T-element in the TBX2 promoter, we performed a
DNA affinity immunoblot (DAI) using nuclear
extract isolated from TGF-β1-treated MCF-12A
cells and a biotin-labelled probe containing the
wild-type or mutated T-element (Fig. 4B). As an
additional control a probe containing a full
T-element consensus site was included in these
experiments and protein-bound biotinylated DNA
was isolated and analysed by western blotting
using an anti-TBX3 antibody. The results showed
that while TBX3 bound strongly to both the
consensus and wild-type probes, it displayed a
decreased affinity for the probe with the mutated
T-element (Fig. 4B). Together, these results
suggest that TBX3 binds the TBX2 promoter
specifically at the T-element at -186 bp.
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
promote invasiveness by repressing E-cadherin
expression (24), is a downstream target of the
TGF-β1 signalling pathway and plays an important
role in exerting its anti-proliferative and
pro-invasive effects (7). However, how TBX3
might exert its anti-proliferative function, and what
suppresses TBX2 expression was not known. In
this study we show that when the TGF-β1 pathway
is stimulated in breast epithelial and melanoma
cells, TBX2 expression is repressed through the
direct binding of TBX3 to a half T-element in the
TBX2 promoter. Our results are also consistent
with TBX2 being a powerful pro-proliferative
factor (22, 23), and indicate that the
downregulation of TBX2 blocks proliferation
through the de-repression of the TBX2 target gene,
p21, a cdki that initiates the TGF-β1-induced G1
arrest (32). Together these data provide an
explanation for how the TGF-β1 pathway exerts its
anti-proliferative effect and reveal an upstream
mechanism involved in regulating TBX2 and
TBX3 in contexts where they are differentially
expressed. Moreover, since overexpression of
TBX2 can override the anti-proliferative effects of
TGF-β1 without affecting p15 levels, up-regulation
of p15 is unlikely to be required for the effect of
TGF-β1 on the proliferative ability of these cells,
though it may be involved in other
TGF-β1-mediated functions.
TBX2 and TBX3 are highly homologous T-box
transcription factors which have distinct roles
during embryonic development and in breast
cancer and melanoma cell lines where they are
both overexpressed (7, 19–22). Importantly,
whereas TBX2 is essential for promoting cell
proliferation, TBX3 inhibits cell proliferation but
is required for cell migration. These results
together with data from the current study suggest
an interesting interplay between TBX2, TBX3 and
TGF-β1 signalling in normal epithelial cells
(Figure 7). Briefly, in this context TGF-β1
signalling activates TBX3 expression in a
DISCUSSION
The TGF-β1 signalling pathway controls a wide
spectrum of cellular functions and deregulated
TGF-β signalling has been linked to severe human
diseases, including cardiovascular disease, skeletal
and muscular disorders and cancer (31). TGF-β1 is
a potent inhibitor of cell proliferation, which is
thought to result from its ability to induce a G1 cell
cycle arrest through the up-regulation of the cyclin
dependent kinase inhibitors (cdki), p15 and p21
(30). However, it can also stimulate invasiveness
by promoting an ‘epithelial to mesenchyme
transition’.
Understanding
how
TGF-β1
coordinates its effects on the cell cycle and
invasion in different cell types is a key issue, both
for development and disease.
Here we have dissected the interrelated roles of
the TBX2 and TBX3 transcription factors, which
are widely implicated in cancer progression, in the
response to TGF-β1. Previously we demonstrated
that the TGF-β1-mediated growth arrest observed
in melanoma could be bypassed by ectopic TBX2
expression (36), and that TBX3, which can
7
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expression was knocked down using a siRNA
approach and growth curve analyses performed in
the presence and absence of TGF-β1. As expected,
cells in which p21 were effectively knocked down
proliferated faster than the control cells in the
absence of TGF-β1 (Fig. 6A). Importantly, the
p21-depleted cells also failed to show a significant
decrease in cell proliferation in response to
TGF-β1. In addition, when the MCF-12A cells
were depleted of TBX2 the untreated cells had a
significant decrease in cell proliferation which
could not be significantly further decreased in
response to TGF-ß1 treatment depleted TBX2 in
(Fig. 6B). These data together with western blot
results from TBX3 overexpressing cells (Fig. 6C)
suggest that the down-regulation of TBX2 by
TBX3 mediates the anti-proliferative effect of
TGF-β1 through allowing upregulation of p21.
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
consensus MYC binding site, raising the
possibility that MYC will impact the TBX3-TBX2
axis identified here. The expression of MYC is
usually down-regulated by TGF-β1 treatment, and
ectopic expression of MYC in mouse keratinocytes
desensitized the cells to TGF-β1 mediated growth
inhibition (33–35). Furthermore, sustained MYC
expression in ovarian cancers coincides with
resistance to the anti-proliferative effects of
TGF-β1 (36). Although, the precise relationship
between MYC and TBX2 and TBX3 expression
remains to be determined, there is no doubt that
TBX2 and TBX3 are likely to represent key
effectors of TGF-β1 signalling in multiple cell
types.
8
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Smad/JunB dependent manner and TBX3 then
represses TBX2 transcription which allows for the
de-repression of p21 and a G1 cell cycle arrest.
Interestingly,
malignant
cells
acquire
immortalising mutations that enable them to evade
the anti-proliferative effects of TGF-β1 signalling.
For example, one of the mechanisms by which
TGF-β1 exerts its anti-proliferative effects is
through inhibiting expression of MYC that leads to
the de-repression of p21. Many malignant cells
however have mutations that result in the
constitutive expression of MYC and hence these
cells are resistant to the anti-proliferative effects of
TGF-β1. It is therefore possible that cancers in
which TBX2 is overexpressed will similarly
disregard the TGF-β1 anti-proliferative signals.
Intriguingly, the TBX3 promoter has a full
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
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TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
Acknowledgments–This work was supported by grants from the SA Medical Research Council, the National Research
Foundation (NRF), Cancer Association of South Africa (CANSA), Cancer Research Initiative of South Africa
(CARISA) and the University of Cape Town. The content is solely the responsibility of the authors and does not
necessarily represent the official views of the funding agencies.
FIGURE LEGENDS
FIGURE 1. TGF-β1 represses TBX2 protein and mRNA expression. TBX2 protein from TGF-β1 (5ng/ml) treated
MCF-12A cells (A) or B16 cells (B) were prepared after indicated times and was examined by western blot analysis.
p38 was used as a loading control. Total RNA extracted from MCF-12A cells (C) or B16 cells (D) after indicated
times with TGF-β1 treatment were reverse-transcribed and subjected to qRT-PCR using primers specific to TBX2.
mRNA levels were normalized to GUSB. Bars, SD. ** P<0.001
FIGURE 2. TBX2 is transcriptionally regulated by TGF-β1. (A) Luciferase assay using MCF-12A cells which were
transfected with full length human TBX2 promoter (400 ng) constructs and were treated with 5 ng/ml TGF-β1. Mean
values (± SD) are presented as fold activity over that of an empty firefly luciferase reporter and are representative of at
least three independent experiments. MCF-12A (B) and B16 (C) cells were pre-treated with vehicle (control) or 30
µg/ml cycloheximide (CHX) for 1 hr and treated with TGF-β1 for 8 hrs for MCF-12A cells and 4 hrs for B16 cells.
RNA and protein were harvested for use in qRT-PCR and western blotting analysis. The expression of TBX2 was
quantified using UN-SCAN-IT gel 6.1 software and the Densitometric values normalised to p38 levels. Bars, SD. *
P<0.05, ** P<0.001
FIGURE 3. The downregulation of TBX2 by TGF-β1 is mediated by TBX3 (A) Luciferase assay of MCF-12A cells
transfected with 400 ng of human TBX2 5’- deletion promoter constructs and treated with 5 ng/ml TGF-β1 or vehicle.
(B) Luciferase assay of MCF-12A cells co-transfected with increasing amounts of Smad3/4 expression vectors and
400ng of -218 bp TBX2 promoter reporter construct. (C) Alignment of the T-element on -186 bp of TBX2 promoter of
human, chimpanzee, mouse and zebrafish. (D, E) Upper panels: siTBX3#1 lower panels: siTBX3#2. Serum starved
MCF-12A cells were transfected with either 50 nM siControl or siTBX3 for 48 hrs, followed by 2 days of TGF-β1
treatment and subjected to western blotting (D) or qRT-PCR analysis (E). Bars, SD. * P<0.05, ** P<0.001
FIGURE 4. TGF-β1 repression of the TBX2 promoter is mediated by a T-element at −186 bp. (A) MCF-12A cells
were treated with 5 ng/ml TGF-β1 for 24 hrs and chromatin immunoprecipitation assays performed with antibodies
against TBX3 or IgG (negative control). Immunoprecipitated DNA was assayed by qRT-PCR with primers against the
TBX2 proximal promoter. (B) Biotinylated DNA probes of the TBX2 promoter containing the consensus T-element or
homologous WT or MT T-element were immobilized on streptavidin beads, and incubated with nuclear extracts from
MCF-12A cells treated with 5 ng/ml TGF-β1 for 24 hrs. The DNA-bound protein complexes were isolated and
analysed by western blotting using antibodies to TBX3. (C) Luciferase assay of MCF-12A cells co-transfected with
100ng of TBX3 expression vector or empty control vector and 400ng of either TBX2 WT or T-element mutated (MT)
promoter luciferase reporter. (D) Luciferase assay of MCF-12A cells co-transfected with 400 ng of TBX2 promoter
luciferase reporter and 100 ng of an empty control vector or WT TBX3 or DNA binding domain mutant (DBM) or
TBX3 N-terminal truncated expression vector. Western blots (C, D, right) show equal expression of TBX3 protein.
Bars, SD. *p < 0.05, ** P<0.001
FIGURE 5. Ectopic TBX2 expression rescues TGF-β1 induced growth inhibition through downregulating p21 in
breast epithelial cells. (A) Western blot analyses with indicated antibodies and (B) Growth curve assays of MCF-12A
14
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
control or TBX2 overexpressing cells treated with vehicle or 5 ng/ml TGF-β1 for 3 days. (C) Flow cytometry assays
show relative fold change of the percentage of TGF-β1-treated MCF-12A TBX2 overexpressing and control cells in
G1, S and G2/M phases compared to their corresponding vehicle-treated cells. (D) MCF-12A TBX2 overexpressing
cells were transiently transfected with either pRc/CMV (TBX2 Control) or pRc/CMV-p21 (TBX2 p21) and treated
with vehicle or 5 ng/ml TGF-β1 for 3 days. Replicate wells from growth curve analyses (right) were pooled and
protein harvested for western blot analyses (left). (E, F) MCF-12A control or TBX2 cell lines were treated with
vehicle or TGF-β1 for 24 hrs and RNA was harvested for use in qRT-PCR analyses to examine the expression of p21
(E) or p15 (F). Bars, SD. *p < 0.05, ** P< 0.001
FIGURE 6. The down-regulation of TBX2 by TBX3 mediates the TGF-β1 anti-proliferative effect through
upregulation of p21. (A) MCF-12A cells were transiently transfected with either 25 nM sip21 or siControl for 12 hrs
followed by 3 days of 5 ng/ml TGF-β1 treatment. Replicate wells from growth curve analyses (right) were pooled and
protein harvested for western blot analyses (left). Upper panel: sip21#1, lower panel sip21#2. (B) Growth curve
analyses of MCF-12A cells transiently transfected with either 10 nM siTBX2 or siControl for 12 hrs followed by 3
days of 5 ng/ml TGF-β1 treatment. Left: siTBX2#1, right: siTBX2#2. (C) Western blot analyses of MCF-12A cells
transduced with adeno-TBX3 tet-off virus which allows TBX3 expression to be regulated by 10 µg/ml doxycycline
(Dox). Cells were treated as indicated and protein harvested was subjected to western blot analyses using antibodies
specific to TBX3, TBX2 and p21 and p38 was used as a loading control. Bars, SD. * P<0.05, ** P<0.001
FIGURE 7. Proposed model linking TBX2 and TBX3 to the TGF-β1 signalling pathway.
15
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
Figure 1
16
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
Figure 2.
17
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
Figure 3.
18
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
Figure 4.
19
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
Figure 5.
20
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
Figure 6.
21
TBX2 is repressed by TBX3 in the TGF-β1 signalling pathway
Figure 7.
22