TRB3 interacts with SMAD3 promoting tumor cellmigration and invasion
Fang Hua*, Rong Mu*, Jinwen Liu, Jianfei Xue, Ziyan Wang, Heng Lin, Hongzhen Yang, Xiaoguang Chenand Zhuowei Hu`
Molecular Immunology and Pharmacology Laboratory, State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute ofMateria Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Beijing, 100050, P R China
*These authors contributed equally to this work`Author for correspondence ([email protected])
Accepted 27 May 2011Journal of Cell Science 124, 3235–3246� 2011. Published by The Company of Biologists Ltddoi: 10.1242/jcs.082875
SummaryTribbles homolog 3 (TRB3, also known as TRIB3, NIPK and SKIP3), a human homolog of Drosophila Tribbles, has been found to
interact with a variety of signaling molecules to regulate diverse cellular functions. Here, we report that TRB3 is a novel SMAD3-interacting protein. Expression of exogenous TRB3 enhanced the transcriptional activity of SMAD3, whereas knocking downendogenous TRB3 reduced the transcriptional activity of SMAD3. The kinase-like domain (KD) of TRB3 was responsible for the
interaction with SMAD3 and the regulation of SMAD3-mediated transcriptional activity. In addition, TGF-b1 stimulation oroverexpression of SMAD3 enhanced the TRB3 promoter activity and expression, suggesting that there is a positive feedback loopbetween TRB3 and TGF-b–SMAD3 signaling. Mechanistically, TRB3 was found to trigger the degradation of SMAD ubiquitin
regulatory factor 2 (Smurf2), which resulted in a decrease in the degradation of SMAD2 and phosphorylated SMAD3. Moreover, TRB3–SMAD3 interaction promoted the nuclear localization of SMAD3 because of the interaction of TRB3 with the MH2 domain of SMAD3.These effects of TRB3 were responsible for potentiating the SMAD3-mediated activity. Furthermore, knockdown of endogenous TRB3expression inhibited the migration and invasion of tumor cells in vitro, which were associated with an increase in the expression of E-
cadherin and a decrease in the expression of Twist-1 and Snail, two master regulators of epithelial-to-mesenchymal transition,suggesting a crucial role for TRB3 in maintaining the mesenchymal status of tumor cells. These results demonstrate that TRB3 acts as anovel SMAD3-interacting protein to participate in the positive regulation of TGF-b–SMAD-mediated cellular biological functions.
Key words: TRB3, SMAD3, Protein interaction
IntroductionThe transcription factor SMAD3 is a downstream modulator of TGF-
b signaling, and is involved in regulating a variety of physiological
and pathological processes, such as development, differentiation,
apoptosis, immunomodulation, fibrogenesis and carcinogenesis
(Kim et al., 2005). Following the activation and phosphorylation
by TGF-b type I receptor, SMAD3 forms a heterodimer with
SMAD4. The activated heteromeric SMAD complex is translocated
into the nucleus and regulates the transcription of target genes. The
specificity for target genes and biological effects of SMAD3-
mediated signaling is under the control of many SMAD-interacting
proteins including cytoplasmic partners (Conery et al., 2004; Xue et
al., 2010; Seong et al., 2007) and transcriptional cofactors (Ding et
al., 2009; Feng et al., 2000). The epithelial-to-mesenchymal
transition (EMT) is important in development, as well as in
fibrogenesis and tumor metastasis. SMAD3 is a key signaling
molecule for the induction of EMT by TGF-b and overexpression of
SMAD3 enhanced metastases of mice injected with MCF10CA1a
tumor cells but SMAD3DC, the dominant-negative mutant,
suppressed the metastases (Tian et al., 2003).
Tribbles was first identified in Drosophila as an inhibitor of
mitosis that regulates cell proliferation, migration and
morphogenesis during development (Grosshans and Wieschaus,
2000; Mata et al., 2000; Seher and Leptin, 2000). In mammals,
there are three Tribbles homologs – TRB1, TRB2 and TRB3. They
are considered to be members of the pseudokinase family, which
contain a Ser/Thr protein kinase-like domain but lack the ATP
binding pocket and catalytic residues. Despite lacking kinase
activity, tribbles has a scaffold-like function and participates in
protein complex assembly. TRB3 is the best-studied member of
the mammalian tribbles family. The interacting partners of TRB3
range from transcription factors, ubiquitin ligase, BMP type II
receptor to members of the MAPK and PI3K signaling pathways.
Through interacting with these proteins, it coordinates crucial
cellular processes, including glucose and lipid metabolism,
apoptosis, adipocyte differentiation, cell stress and regulation of
collagen expression (Bezy et al., 2007; Chan et al., 2007; Du et al.,
2003; Ohoka et al., 2005; Qi et al., 2006; Tang et al., 2008).
Recently, emerging evidence suggests that the three mammalian
tribbles homologs are crucial modulators of tumorogenesis. TRB1
and TRB2 are both involved in myeloid leukemogenesis (Jin et al.,
2007; Keeshan et al., 2006), whereas, TRB3 is highly expressed in
many human tumor cell lines and in several human tumor tissues
(Bowers et al., 2003; Xu et al., 2007). However, the function and
mechanism of interaction of TRB3 in cancer remains unknown.
In an effort to discover SMAD3-interacting proteins using a
yeast two-hybrid screening system, we identified a number of
molecules, including TRB3, that physically interact with SMAD3
Research Article 3235
Jour
nal o
f Cel
l Sci
ence
Fig. 1. TRB3 interacts with SMAD3. (A) SMAD3–Myc was
co-transfected with TRB3–HA into HEK293T cells. Whole cell
extracts were immunoprecipitated with an anti-Myc antibody
and blotted with an anti-HA. (B) Mapping of TRB3 domains
involved in SMAD3 binding. Top: deletion mutants of TRB3.
Below: HEK293T cells were transiently transfected with the
indicated constructs of TRB3. Whole cell extracts were
immunoprecipitated (IP) with anti-SMAD3 antibody. (C) TGF-
b1 promotes the endogenous interaction between SMAD3 and
TRB3. HepG2 cells were quiescent overnight and treated with
TGF-b1 (10 ng/ml) for 2 hours, or left untreated. Whole cell
extracts were immunoprecipitated with anti-SMAD3 antibody or
equal amount of rabbit IgG and blotted with an anti-TRB3
antibody. (D) Phosphorylation of SMAD3 increases the
SMAD3–TRB3 interaction. SMAD3–Myc or its two mutants
were co-transfected with TRB3–HA into HEK293T cells. Whole
cell extracts were immunoprecipitated with an anti-Myc
antibody and blotted with an anti-HA antibody. (E) In vitro
binding between TRB3 and SMAD3. Cell lysates from
HEK293T cells overexpressing TRB3–HA were incubated with
equal amounts of GST or GST–SMAD3 and analyzed by
western blotting using the anti-HA antibody. The presence of the
GST fusion proteins was confirmed by staining gels with
Coomassie Blue.
Fig. 2. Overexpression of TRB3 augments SMAD3-mediated
transcriptional activity and targeted gene expression. (A) HEK293T
cells were transiently transfected in 12-well plates with 500 ng of SBE4–
luc, 10 ng of TK-Renilla, with (+) or without (–) 1.0 mg of SMAD3–Myc
and 1.0 mg of HA-tagged TRB3 or its truncated mutants, as indicated.
After 24 hours of transfection, cells were incubated in the presence (gray
bars) or absence (black bars) of TGF-b1 for another 24 hours and then the
luciferase activity was measured. (B) HepG2 cells were transfected with
500 ng of 3TP–lux, 10 ng of TK-Renilla, and with (+) or without (–)
1.0 mg of SMAD3–Myc and 1.0 mg of TRB3 for 24 hours. The cells were
incubated with or without TGF-b1 (10 ng/ml) for another 24 hours and
luciferase activity was measured. (C) The effect of TRB3 on TGF-b
signaling is SMAD3 dependent. HEK293T cells were transiently
transfected in 12-well plates with 500 ng of SBE4–luc, 10 ng of TK-
Renilla and with (+) or without (–) 2.0 mg of HA-tagged TRB3, and 50 nM
of universal scrambled negative control siRNA duplexes or siRNA
duplexes targeting SMAD3. After 24 hours of transfection, cells were
incubated in the presence (gray bars) or absence (black bars) of TGF-b1
for another 24 hours and then the luciferase activity was measured.
(D) HepG2 cells were transiently transfected with TRB3–HA plasmid.
After 24 hours of transfection, cells were incubated in the presence (+) or
absence (–) of TGF-b1 for another 24 hours. Whole cell extracts were
prepared and the expression of p21, PAI-1, SMAD2/3, and the
phosphorylation of Ser465 and Ser467 of SMAD2, and Ser423 and Ser425
of SMAD3 (pSmad2 and 3, respectively) were analyzed by western
blotting. Values are means ¡ s.e.m. of three independent assays.
*P,0.05, **P,0.01, ***P,0.001.
Journal of Cell Science 124 (19)3236
Jour
nal o
f Cel
l Sci
ence
(Xue et al., 2010). In this study, the functional significance and
regulatory mechanism of the TRB3–SMAD3 interaction wasfurther explored. We found that the TRB3–SMAD3 interactionplays an important role in the regulation of SMAD3-mediated
transcriptional activity and in the reciprocally positive regulationof TRB3 expression. Knockdown of endogenous TRB3expression inhibited the migration and invasion of HepG2 cellsin vitro, through inhibition of EMT. Our studies suggest a pivotal
role of TRB3 in tumor progression and metastasis.
ResultsIdentification of TRB3 as an interacting protein of SMAD3
In a previous study, we identified a number of proteins, includingTRB3, as partners of SMAD3 in a yeast two-hybrid screening
system (Xue et al., 2010). To confirm the TRB3–SMAD3interaction in mammalian cells, co-immunoprecipitation assaywas carried out using HEK293T cells co-transfected with Myc–
SMAD3 and HA–TRB3. TRB3 could be precipitated with
SMAD3 (Fig. 1A) and the kinase-like domain (KD) of TRB3
mediated the interaction (Fig. 1B). To determine whether TRB3
specifically interacted with SMAD3 or generally interacted with
other members of the SMAD family, we also examined the
possible interaction of HA–TRB3 with Myc–SMAD2 and Myc–
SMAD4. We found that SMAD2 and SMAD4 could also be co-
immunoprecipitated with HA-tagged TRB3, similar to SMAD3
(supplementary material Fig. S1A). SMAD3 is a downstream
molecule of TGF-b1 signaling, so we therefore examined whether
TGF-b1 regulated the endogenous TRB3–SMAD3 interaction. In
the absence of TGF-b1, endogenous TRB3 was weakly associated
with SMAD3. However, TGF-b1 stimulation enhanced this
interaction (Fig. 1C). Because phosphorylation of the C-terminal
serine residues in SMAD3 is a crucial step in TGF-b1 signaling
(Massague et al., 2005), we tested whether the phosphorylation
status of SMAD3 might regulate the SMAD3–TRB3 interaction.
Fig. 3. Silencing endogenous TRB3 inhibits SMAD3-
mediated transcription and the expression of TGF-b1
target genes. (A) Silencing TRB3 inhibits SBE4–luc
reporter activity. HepG2 cells stably expressing control
shRNA or TRB3 shRNA were transfected with the SBE4–
luc reporter construct for 24 hours. The cells were incubated
with (gray bars) or without (black bars) TGF-b1 (10 ng/ml)
for another 24 hours. The cells were harvested and
luciferase activity was measured. (B) Silencing of TRB3
inhibits 3TP–lux reporter activity. The assay was conducted
as described in A, except for transfecting with the 3TP–lux
reporter construct instead of SBE4–luc reporter construct.
(C) Silencing TRB3 inhibits the expression of TGF-b
signaling target genes. HepG2 cells stably expressing
control shRNA or TRB3 shRNA were incubated with (+) or
without (–) TGF-b1 (10 ng/ml) for 24 hours. Whole cell
extracts were prepared and the expression of p21, PAI-1,
SMAD2/3, and the phosphorylation of Ser465 and Ser467 of
SMAD2 and Ser423 and Ser425 of SMAD3 (p-Smad2 and
3, respectively) were analyzed by western blotting. Values
are means ¡ s.e.m. of three independent assays. **P,0.01.
Fig. 4. Activation of TGF-b1–SMAD3
increases the expression of TRB3.
(A) TGF-b1 stimulates TRB3 expression.
HepG2 cells were treated with TGF-b1
(10 ng/ml) for the indicated time. Whole
cell extracts were prepared and the
expression of TRB3, p21, PAI-1,
phosphorylated SMAD3 (pSmad3),
SMAD3 and actin were analyzed by
western blotting. (B) A diagram of the
TRB3 promoter reporter gene. Position +1
is the initiation site for TRB3 transcription.
(C) Overexpression of SMAD3 causes
TRB3 induction. HEK293T cells were
transiently transfected with the indicated
amounts of SMAD3-expressing plasmid
plus the TRB3 promoter luciferase
(pTRB3-luc) plasmid. After 24 hours of
transfection, the cells were incubated with
(grey bars) or without (black bars) TGF-b1
(10 ng/ml) for another 24 hours and
luciferase activity was measured. Values
are means ¡ s.e.m. of three independent
assays. ***P,0.001.
TRB3 interacts with SMAD3 3237
Jour
nal o
f Cel
l Sci
ence
We found that the SMAD3D mutant, which has three serine
residues replaced by aspartate to mimic phosphorylation, bound to
TRB3 as well as the wild-type SMAD3 did. By contrast, the 3A
mutant, lacking the phosphorylation sites, showed no interaction
(Fig. 1D). Using a GST pull-down assay, TRB3 was found to bind
to a GST–SMAD3 fusion protein in vitro but not GST protein
alone (Fig. 1E). These results not only provide evidence
demonstrating an interaction between SMAD3 and TRB3 but
also show that the KD of TRB3 is crucial for the interaction.
However, the phosphorylation status of SMAD3 is important, but
not absolutely required, for this interaction because the non-
phosphorylated GST–SMAD3 fusion protein expressed in
Escherichia coli can also interact with TRB3 (Fig. 1E).
TRB3 regulates SMAD3-mediated transcription and target
gene expression
To determine the effect of the TRB3–SMAD3 interaction on
SMAD3-mediated transcription, HEK293T cells were co-
transfected with Myc–SMAD3, HA-tagged TRB3 or its truncated
mutants, and the SBE4–luciferase (luc) reporter, which contains
four copies of a SMAD consensus-binding element. Together with
SMAD3, TRB3 overexpression induced an increase in SBE4–luc
reporter activity and the KD fragment but not the N- or C-terminus
of TRB3 that induced the SBE4–luc activity. Stimulation with TGF-
b1 further strengthened the TRB3-induced activity (Fig. 2A).
Additionally, we found that TRB3 induced SMAD2-mediated
transcriptional activity (supplementary material Fig. S1B).
Next, we assessed the effect of TRB3 on TGF-b1-responsive
gene transcription. Overexpression of TRB3 promoted an
increase in 3TP–lux reporter activity in the presence or absence
of TGF-b1 (Fig. 2B). This effect of TRB3 is SMAD3 dependent
because SMAD3 knockdown led to a significant reduction in the
SBE4–luc activity induced by TRB3 expression and TGF-b1
stimulation (Fig. 2C).
We then examined the regulatory effect of TRB3 on TGF-b1–
SMAD3 signaling and target gene expression. We found that
overexpression of TRB3 enhanced the expression of PAI-1 and p21
even in the absence of TGF-b1. Interestingly, TRB3 overexpression
promoted the phosphorylation of SMAD3 and upregulated the
expression of SMAD2 but had little effect on the phosphorylation
of SMAD2 (Fig. 2D), whereas knock down of TRB3 with sequence
specific shRNA (Du et al., 2003) reduced the SBE4–luc and 3TP–
lux reporter activity to 50% of the control level even in the presence
of TGF-b1 (Fig. 3A,B). In addition, knock down of TRB3 reduced
Fig. 5. TRB3 promotes cell migration and invasion.
(A) Cell migration was measured using transwell assays for
parental, control-shRNA and TRB3-shRNA HepG2 cells.
The cells were plated in the upper chamber of the filters
that had been coated with fibronectin on the underside, and
stimulated with TGF-b1 (10 ng/ml) for 24 hours or left
unstimulated. Cells migrating to the underside of the
transwell insert were measured. (B) A transwell invasion
assay was performed as described in A, except that the
chambers were coated with basement membrane Matrigel
(30 mg/well) and cells migrating to the underside of the
insert were measured 48 hours after the start of the
experiment. (C) Cell invasion was measured using
transwell assays for parental, control-shRNA and TRB3-
shRNA HCT-8 cells. 24 hours after plating, the cells
migrating to the underside of the transwell insert were
measured. The expression levels of TRB3 in these cells
were detected by western blotting. (D) Transwell migration
assays were performed using NIH3T3 cells transfected
with or without TRB3–HA for 24 hours. The cells
migrating to the underside of the transwell insert were
measured. The expression levels of TRB3 in these cells
were detected by western blot. The value from parental
cells was arbitrarily set at 100%. Values are means ¡
s.e.m. of three independent assays. **P,0.01. The original
magnification of all images was 2006.
Journal of Cell Science 124 (19)3238
Jour
nal o
f Cel
l Sci
ence
Fig. 6. TRB3 positively regulates EMT process. (A) Parental HepG2 cells and cells expressing either control shRNA or TRB3 shRNA were stimulated with
TGF-b1 (10 ng/ml) for 24 hours or left unstimulated, and then immunofluorescence staining of E-cadherin and a-SMA was examined. Scale bars: 20 mm. (B)
Parental HepG2 cells and cells expressing either control shRNA or TRB3 shRNA were treated as described in A; whole cell extracts were prepared and the
expression of E-cadherin and a-SMA was examined by immunoblotting. (C) NIH3T3 cells were transiently transfected with TRB3–HA for 24 hours. Whole cell
extracts were prepared, and the expression of E-cadherin, a-SMA, TRB3 (anti-HA antibody) and actin were analyzed by western blotting. (D) Semi-quantitative
RT-PCR analysis of the E-cadherin repressors. Cells were grown to confluence in six-well plates. Total RNA was extracted from parental, control-shRNA and
TRB3-shRNA HepG2 cells. The expression of mRNA encoding for Twist-1, Twist-2, Zeb-2, Snail, Slug, TRB3 and b-actin was determined by RT-PCR using
specific primer sets. The value from parental cells was arbitrarily set at 1.0. (E) Protein expression level of the E-cadherin repressors. Cells were grown to
confluency in six-well plates. Whole cell extracts were prepared from control-shRNA and TRB3-shRNA HepG2 cells. The expression of Twist-1, Snail, Slug,
TRB3 and actin were analyzed by western blotting. The value from control-shRNA cells was arbitrarily set at 1.0. Values are means ¡ s.e.m. of three independent
assays. **P,0.01.
TRB3 interacts with SMAD3 3239
Jour
nal o
f Cel
l Sci
ence
the expression of PAI-1, p21, SMAD2 and phosphorylated SMAD3
(Fig. 3C), which was consistent with the result of Fig. 2D. Taken
together, these results indicated that TRB3 is a positive modulator
of TGF-b–SMAD3 signaling.
TGF-b1–SMAD3 stimulates TRB3 expression and activates
TRB3 promoter activity
Because TRB3 promoted SMAD3-mediated transcriptional
activity, we wondered if the expression of TRB3 could be
regulated by TGF-b1–SMAD3 signaling. We found that TGF-b1
induced a time-dependent expression of TRB3 as well as the
SMAD3-targeted proteins, such as p21 and PAI-1 in HepG2 cells
(Fig. 4A). The SMAD3-targeted proteins were induced much
more quickly than TRB3. The expression of p21 and PAI-1 was
induced 12 hours after TGF-b1 stimulation, whereas the
phosphorylation of SMAD3 was observed 1 hour after
stimulation and a high level of phosphorylation occurred at
6 hours after stimulation. TGF-b1 stimulation also induced a time-
Fig. 7. See next page for legend.
Journal of Cell Science 124 (19)3240
Jour
nal o
f Cel
l Sci
ence
dependent expression of these proteins in HCT-8 cells, a human
colorectal carcinoma cell line (supplementary material Fig. S2).
Using the promoter region of human TRB3 (–1038 , +847) as a
luciferase reporter system (TRB3–luc; Fig. 4B), we found that
overexpression of SMAD3 induced a concentration-dependent
transactivation of TRB3 and that stimulation with TGF-b1 further
induced its activation (Fig. 4C). Taken together with the data in
Figs 2 and 3, our study suggests that TRB3 can be regulated by
TGF-b1–SMAD3 signaling, and there exists a positive feedback
loop between TRB3 and TGF-b1–SMAD3 signaling.
TRB3 is essential for the migration and invasion of
tumor cells
TRB3 mRNA is highly expressed in several human tumor tissues
and multiple human tumor cell lines (Bowers et al., 2003; Xu et
al., 2007), indicating a potential role of TRB3 in tumorigenesis
and tumor progression. A large number of studies have provided
strong evidence for the role of TGF-b1–SMAD3 in tumor
invasion and metastasis (Micalizzi et al., 2009; Wesolowska et
al., 2007). Therefore, we examined the regulatory effect of TRB3
on migratory and invasive ability in HepG2 cells using transwell
migration and invasion assays. We found that TGF-b1 promoted
the migration and invasion of HepG2 cells, whereas knocking
down of TRB3 blocked cell migration and invasion in the
absence or presence of TGF-b1 (Fig. 5A,B). HCT-8 is a human
colorectal carcinoma cell line, with a high level of expression of
TRB3. Knockdown of TRB3 inhibited cell invasion as observed
in HepG2 cells (Fig. 5C). By contrast, overexpression of TRB3in NIH3T3 cells, which express low levels of endogenous TRB3,
promoted migration of the cells (Fig. 5D). Taken together, thesedata suggest that TRB3 participates in the regulation of tumorcells migration and invasion.
TRB3 is required for maintaining the mesenchymal statusof HepG2 cells
Epithelial-mesenchymal transition (EMT) plays an important rolein the conversion of early stage tumors into invasive malignancies(Kang and Massague, 2004). Epithelial cells undergoing EMT
typically lose the expression of epithelial markers such as E-cadherin and acquire the expression of mesenchymal markersincluding a-SMA and vimentin. Because we found that silencing
endogenous TRB3 blocked the migration and invasion of HepG2cells, we reasoned that TRB3 might have a role in the regulationof EMT. We examined EMT markers in HepG2 cells. As a
hepatoma cell line, parental and control short hairpin (shRNA)-expressing HepG2 cells showed modest a-SMA staining and lostthe staining of E-cadherin (Fig. 6A). It is generally accepted thatTGF-b can promote an invasive phenotype of tumor cells in later
stages of carcinoma by inducing EMT. We found that TGF-b1stimulation enhanced the expression of a-SMA but reduced theexpression of E-cadherin in HepG2 cells. Silencing TRB3
significantly reduced the expression of a-SMA but markedlyenhanced the expression of epithelial marker E-cadherin, even inthe presence of TGF-b1 (Fig. 6A). Western blot analysis of
cellular extracts from TRB3-silenced or TRB3-overexpressingcells confirmed the expression changes of a-SMA and E-cadherin(Fig. 6B,C).
We next examined the effect of TRB3 on the expression of the E-cadherin repressors known to regulate EMT. RT-PCR analysisshowed that Zeb-2 and slug (snail2) were constitutively expressed
in parental HepG2 cells and in the control-shRNA and TRB3-shRNA transfectants. However, expression of Twist-2 mRNA wasundetectable in all the three cell types. The expression of Twist-1
and Snail, two transcription factors contributing to EMT and
metastasis, was detectable in parental HepG2 cells and the control-shRNA transfectants but was significantly decreased in TRB3-shRNA transfectants (Fig. 6D). Western blotting confirmed the
expression change of Twist-1 and Snail (Fig. 6E). These dataindicate that TRB3 is crucial in maintaining the mesenchymalstatus of HepG2 cells and in the regulation of TGF-b1-induced
EMT.
TRB3–SMAD3 interaction maintains the nuclearlocalization of SMAD3
Previous work indicated that SMAD3 is distributed predominantlyin the cytoplasm (Xu et al., 2002) but TRB3 localizes in the nucleus
(Xu et al., 2007) in quiescent cells. When SMAD3 and TRB3 werecoexpressed, SMAD3 was concentrated and colocalized withTRB3 in the nucleus (Fig. 7A). To confirm those observations in
intact cells, nuclear and cytosolic fractions were extracted fromHepG2 cells transfected with TRB3–HA-expressing plasmid andthe level of SMAD3 in each fraction was determined. The cytosolic
and nuclear fractions were immunoblotted for histone H3 as anuclear marker and GAPDH as a cytosolic marker. We found thatno histone H3 was detected in the cytosolic fractions and no
GAPDH was detected in the nuclear fractions, verifying an efficientisolation of these fractions (Fig. 7B). Consistent withimmunofluorescence results, SMAD3 localized predominantly in
Fig. 7. TRB3 interacts with the MH2 domain of SMAD3 to maintain the
nuclear localization of SMAD3. (A) HepG2 cells were transiently
transfected with plasmid expressing SMAD3–Myc, TRB3–HA or co-
transfected with the both plasmids for 24 hours. The cells were
immunostained with an anti-Myc or anti-HA antibody, followed by FITC-
conjugated anti-mouse secondary antibody (green, for TRB3) or Alexa Fluor
594 anti-rabbit secondary antibody (red, for SMAD3). Scale bar: 25 mm. The
number of cells showing a predominant nuclear distribution of SMAD3 was
divided by the total number of cells to obtain the percentage of cells with
nuclear localization. Values are means ¡ s.e.m. of three independent
experiments. (B) HepG2 cells were transfected with TRB3–HA-expressing
plasmid or left untransfected. After 24 hours of transfection, the nuclear and
cytosolic fractions were extracted and assessed by western blot analysis. To
verify separation of nuclear and cytosolic fractions, histone H3 (nuclear
marker) and GAPDH (cytosolic marker) were immunoblotted. C.E. and N.E.,
cytosolic extract and nuclear extract, respectively. Values are means ¡ s.e.m.
of three independent assays. The value from untransfected HepG2 cells was
arbitrarily set at 1.0. (C) HepG2 cells expressing either control shRNA or TRB3
shRNA were stimulated with TGF-b1 (10 ng/ml) for 2 hours or left
untransfected. The cells were immunostained with an anti-SMAD3 antibody,
followed by FITC-conjugated anti-rabbit secondary antibody. DAPI was used
to stain the nuclei. Scale bars: 30 mm. The number of cells showing a
predominant nuclear distribution of SMAD3 was divided by the total number of
cells to obtain the percentage of cells with nuclear localization. Values are
means ¡ s.e.m. of three independent experiments. (D) HepG2 cells expressing
either control shRNA or TRB3 shRNA were stimulated with TGF-b1 (10 ng/
ml) for 2 hours or left untransfected. The nuclear and cytosolic fractions were
extracted and assessed as described in B. Values are means ¡ s.e.m. of three
independent assays. Cells expressing control shRNA without TGF-b1
stimulation were used as a control and the value from these cells was arbitrarily
set at 1.0. (E) Cell lysates from HEK293T overexpressing TRB3–HA were
incubated with equal amounts of GST–MH1, GST–MH2 or GST–linker and
analyzed by western blotting using the anti-HA antibody. The presence of the
GST fusion proteins (arrows) was confirmed by staining gels with Coomassie
Blue. Data is representative of two assays with identical results.
TRB3 interacts with SMAD3 3241
Jour
nal o
f Cel
l Sci
ence
the cytoplasm under basal condition. Transient transfection ofTRB3–HA resulted in accumulation of SMAD3 in the nucleus
(Fig. 7B). The effect of endogenous TRB3 on SMAD3 nuclear
localization was also examined. As shown in Fig. 7C,D, knockingdown TRB3 decreased the nuclear accumulation of SMAD3 and
inhibited TGF-b1-induced nuclear translocation of SMAD3 to acertain extent. Because SMAD proteins are continuously shuttling
between the nucleus and the cytoplasm regardless of the presenceof a TGF-b signal (Hill, 2009), we further examined how TRB3
regulates the localization of SMAD3. SMAD3 consists of twoglobular domains, namely the N-terminal MH1 domain and the C-
terminal MH2 domain, coupled by a linker region (Fig. 7E, upperpanel). The MH2 domain of SMAD3 is responsible for nuclear
export of SMAD3 through interaction with exportin 4 (Kurisaki etal., 2006). Using a GST pull-down assay we detected which domain
of SMAD3 interacted with TRB3. As illustrated in the lower panelof Fig. 7E, there was clear interaction between TRB3 and GST–
MH2, but not TRB3 and GST–linker. A weak interaction betweenGST–MH1 and TRB3 was also observed. These results indicatethat TRB3 binds directly to the MH2 domain of SMAD3, which
interferes with the nuclear export of SMAD3 (Kurisaki et al., 2006).
TRB3 interacts with Smurf2 and promotes its degradation
Overexpression of TRB3 increased the phosphorylation of SMAD3,whereas silencing of TRB3 caused the opposite effect. Because
TRB3 is a pseudokinase and lacks the kinase activity, it mightregulate the phosphorylation of SMAD3 by changing the stability ofphosphorylated SMAD3. Wu et al. reported recently that
phosphorylated SMAD3 is a target for SMAD ubiquitin regulatoryfactor 2 (Smurf2) ubiquitylation (Wu et al., 2008). TRB3 has been
Fig. 8. TRB3 interacts with Smurf2 and promotes its ubiquitylation. (A) Semi-quantitative RT-PCR analysis of Smurf2. Cells were grown to confluence in six-
well plate. Total RNA was extracted from parental, control-shRNA and TRB3-shRNA HepG2 cells. The expression of mRNA encoding for Smurf2, TBR3 and b-
actin was determined by RT-PCR using specific primer sets. The value from parental cells was arbitrarily set at 1.0. Values are means ¡ s.e.m. of three independent
assays. (B) The whole cell extracts of parental HepG2 cells and HepG2 cells stably expressing control shRNA or TRB3 shRNA were prepared, and the expression of
Smurf2, TRB3 and actin were analyzed by western blotting. The value from parental cells was arbitrarily set at 1.0. Values are means ¡ s.e.m. of three independent
assays. **P,0.01. (C) HEK293T cells were transiently transfected with increasing amounts of TRB3–HA-expressing plasmid together with Smurf2-DDK plasmid
for 24 hours. The cells were also transfected with an identical amount of pEGFP-N1 plasmid to monitor the transfection efficiency. The expression of Smurf2,
TRB3 or GFP was examined by western blotting with an anti-DDK antibody (for Smurf2), an anti-HA antibody (for TRB3), or an anti-GFP antibody, respectively.
(D) The interaction between TRB3 and Smurf2 was examined by immunoprecipitation assay. Smurf2-DDK was co-transfected with TRB3–HA into HEK293T
cells. Whole cell extracts were immunoprecipitated with an anti-DDK antibody and blotted with the anti-HA antibody. (E) Overexpression of TRB3 mediates the
ubiquitylation of Smurf2. HEK293T cells were transfected with ubiquitin, Smurf2–Myc, and TRB3–HA expression constructs. Whole cell extracts were prepared
and immunoprecipitated with the anti-Myc antibody. The precipitates were then blotted with an anti-ubiquitin antibody. (F) Overexpression of Smurf2 reverses the
regulatory effect of TRB3 on SMAD3-mediated transcriptional activity. HEK293T cells or HepG2 cells were transfected with different plasmids as indicated for
24 hours and luciferase activity was measured. Values are means ¡ s.e.m. of three independent assays. **P,0.01.
Journal of Cell Science 124 (19)3242
Jour
nal o
f Cel
l Sci
ence
reported to interact with Smurf1, which shows a high homology withSmurf2 (Lin et al., 2000), and to promote its degradation through the
ubiquitin–proteasome pathway (Chan et al., 2007). We thusexamined the regulatory effect of TRB3 on the expression ofSmurf2 in HepG2 cells. We found that silencing TRB3 increased theprotein but not mRNA expression of Smurf2 (Fig. 8A,B), whereas
increasing TRB3–HA expression decreased the levels of Smurf2(Fig. 8C). Moreover, the immunoprecipitation assay revealed astrong interaction between TRB3 and Smurf2 (Fig. 8D). We further
determined whether the ubiquitylation of Smruf2 was TRB3dependent. Compared with the control cells, many moreubiquitylated proteins (molecular mass .86 kDa) were
immunoprecipitated with anti-Myc (Smurf2–Myc) from TRB3-transfected cells (Fig. 8E), suggesting that TRB3 is a negativeregulator of Smurf2 by promoting Smurf2 ubiquitylation. Indeed,knockdown of SMURF2 using SMURF2-targeting siRNA duplexes
caused the accumulation of SMAD2 and phosphorylated SMAD3 inthe cells (supplementary material Fig. S3). Similar results had beenreported previously (Lin et al., 2000; Zhang et al., 2001; Wu et al.,
2008). Therefore we investigated whether TRB3 regulation ofSMAD3-mediated transcriptional activity was Smurf2 dependent.We found that expression of TRB3 enhanced SBE4–luc reporter
activity but this effect was significantly attenuated in the cellscoexpressing TRB3 and Smurf2 (Fig. 8F). Taken together, ourstudies indicate that TRB3 augments TGF-b1–SMAD3 signaling by
promoting ubiquitylation and degradation of Smurf2, whichattenuates the degradation of SMAD2 and phosphorylatedSMAD3 (Fig. 9).
DiscussionThe major finding of this study is that TRB3, an intracellularadaptor molecule, augments TGF-b1–SMAD3-mediated
transcriptional activity and cellular functions by physicallyinteracting with SMAD2/3. Knockdown of TRB3 expression intumor cells significantly inhibits the invasive and metastatic
ability of the cells by promoting mesenchymal-epithelialtransition. Moreover, TRB3 interacts with SMAD2 andSMAD4, two members of TGF-b–SMAD signaling family. Thehighly conserved MH2 domain of the SMAD proteins, as
indicated previously (Massague et al., 2005) might mediatetheir interaction with TRB3. However, the highly conservedtribbles kinase-like domain in TRB3 is also necessary for TRB3–
SMAD3 interaction and SMAD3-mediated transcription activity,suggesting an important role for the biological function of thisregion. Although only receptors and SMAD proteins are essential
components of the canonical TGF-b1–SMAD pathway, thispathway can be modulated by, and have a crosstalk with, othersignaling molecules through interaction with SMAD2/3 (Chen et
al., 2007). Thus, our studies indicate that TRB3 is a novel partnerand modulator of TGF-b–SMAD3 signaling, which might play animportant role in tumor progression and metastasis.
There is evidence that the activation and nuclear translocation
of SMAD3 results from TGF-b-induced phosphorylation at theC-terminal serine residues of SMAD3 (Massague, 1998;Moustakas et al., 2001). The observations that the positive
regulation of the endogenous TRB3–SMAD3 interaction byTGF-b1 and that the SMAD3D mutant can interact with TRB3confirm that the phosphorylation of SMAD3 is important for the
formation of the TRB3–SMAD3 complex. However, we foundthat GST–SMAD3 can also interact with TRB3 in pull-downassay while GST–SMAD3 fusion protein cannot be
phosphorylated when it is expressed in E. coli. These results
suggest that the subcellular localization rather than the
phosphorylation status is essential for the interaction between
TRB3 and SMAD3. In particular, the enhancement of the TRB3–
SMAD3 interaction when SMAD3 is phosphorylated might occur
because phosphorylated SMAD3 moves into the nucleus more
readily (Massague, 1998; Moustakas et al., 2001) and is therefore
more accessible for interaction. Moreover, previous work
indicates that SMAD proteins are continuously shuttling
between the nucleus and the cytoplasm and the MH2 domain
of SMAD3 is responsible for nuclear export of SMAD3 (Kurisaki
et al., 2006). We found in the current study that TRB3 interacts
with the MH2 domain of SMAD3, and overexpression of TRB3
increases the nuclear localization of SMAD3. On the basis of
these observations, we propose that the interaction between
TRB3 and the MH2 domain of SMAD3 might form a steric
hindrance to the interaction of SMAD3 with exportin 4, which
blocks the nuclear export of SMAD3. Such an effect mimics a
continuous activation of TGF-b–SMAD3 to exert its
transcriptional activity and other cellular actions (Fig. 9).
Indeed, Chan et al. recently report that TRB3 interacts with
BMPRII and positively regulates BMP and TGF-b signaling.
Because TRB3 is predominantly localized in the nucleus, these
authors surmised that TRB3 might play a direct role in the
transcriptional regulation of BMP and TGF-b target genes in
concert with SMAD proteins in the nucleus (Chan et al., 2007).
SMAD proteins are crucial intracellular mediators of TGF-bsignaling pathways. An alteration in SMAD protein levels can
profoundly affect signaling transduction and cellular functions.
Smurf2 is a HECT class ubiquitin E3 ligase, which can interact
with SMAD1, SMAD2 and SMAD3 (Lin et al., 2000). Several
studies have shown that Smurf2 ubiquitylates and degrades
SMAD1 and SMAD2, but not SMAD3 at steady state (Lin et al.,
2000; Zhang et al., 2001). Recently, Wu et al. reported
phosphorylated SMAD3 is a target for Smurf2 ubiquitylation
(Wu et al., 2008). We found that TRB3 interacts with Smurf2 to
promote degradation of Smurf2. Overexpression of TRB3
increases, whereas silencing of TRB3 decreases the levels of
both SMAD2 and phosphorylated SMAD3. SMAD2 and SMAD3
Fig. 9. Schematic diagram of the TRB3–SMAD3 interaction in the
regulation of TGF-b–SMAD3 signaling. (1) TRB3 keeps SMAD3 in the
nucleus by physically interacting with it; (2) TRB3 forms a complex with
Smurf2 that leads to the degradation of Smurf2, which in turn stabilizes the
targets of Smurf2, such as SMAD2 and phosphorylated SMAD3 to enhance
reciprocally the TGF-b–SMAD3 signaling activity.
TRB3 interacts with SMAD3 3243
Jour
nal o
f Cel
l Sci
ence
are both activated in response to TGF-b1 and act, in cooperation
with SMAD4, as effectors of the TGF-b response (Massague et
al., 2005). Thus, augmentation of TGF-b signaling by TRB3 may
not only rely on the decrease in the expression of Smurf1 (Chan
et al., 2007), but also depend on the TRB3 downregulation of
Smurf2 (Fig. 8) to maintain the stability of both phosphorylated
SMAD3 and SMAD2.
TGF-b1 plays an important role in tumor progression. An
increased level of TGF-b1 has been observed in many human
tumors and enhanced TGF-b1 can promote tumor invasion or
metastasis in the established tumor (Bierie and Moses, 2006). In the
present study, we found that silencing TRB3 inhibits cell migration
and invasion, even in the presence of TGF-b1. Overexpression of
TRB3 augments SMAD3-mediated transcription activity. TGF-b–
SMAD3 signaling in turn upregulates the expression of TRB3, thus
establishing a positive feedback loop that amplifies TGF-b–
SMAD3 signaling. Given that TRB3 is highly expressed in
human tumors (Bowers et al., 2003) and it positively promotes
TGF-b–SMAD3 signaling, as demonstrated by this study, the
expression level of TRB3 may, at least partly, be responsible for the
different roles of TGF-b–SMAD3 signaling in tumor inhibition or
tumor progression at the different stages of tumor development
(Bierie and Moses, 2006).
EMT is crucial in the conversion of early stage tumors into
invasive ones during tumor progression, because it allows tumor
cells to infiltrate the adjacent tissue, gain invasive properties and
ultimately form metastases at distal sites (Lee et al., 2006). TGF-b–
SMAD3 signaling regulates EMT through SMAD3-dependent or -
independent mechanisms (Xu et al., 2009). We found that silencing
TRB3 increases the expression of E-cadherin and decreases the
expression of mesenchymal marker a-SMA, suggesting that TRB3
is involved in maintaining the mesenchymal status of tumor cells.
The ability of TRB3 to maintain the mesenchymal status of cancer
cells could result from TRB3 enhancing SMAD3-mediated
transcription activity. Several developmentally important genes,
including those encoding for Twist-1, Twist-2, Snail, Slug and
Zeb-2 have been found to induce EMT through repression of the
gene encoding E-cadherin, a hallmark of EMT (Peinado et al., 2007).
Indeed, in our study, silencing TRB3 attenuated the expressions of
Twist-1 and Snail that have been identified as promoters of invasion
and metastasis in various types of human cancer (Batlle et al., 2000;
Yang et al., 2004; Yang et al., 2008). Moreover, Snail can form a
Snail–SMAD3 or –SMAD4 transcriptional repressor complex to
promote TGF-b-mediated EMT (Vincent et al., 2009). Thus, TRB3
induces EMT, cell migration and invasion through interacting with
SMAD3 to promote SMAD3-induced transcriptional activity and the
expression of EMT-related genes such as those encoding the
transcription factors Twist-1 and Snail.
In summary, our studies demonstrate that TRB3 is a novel
partner of SMAD3. The interaction of TRB3 and SMAD3 has a
crucial role in the regulation of SMAD3-mediated transcriptional
activity and in the reciprocally positive regulation of TRB3
expression. TRB3 augments TGF-b1–SMAD3 signaling through
two mechanisms: (1) keeping SMAD3 in the nucleus by physical
interaction; and (2) preventing the degradation of SMAD2 and
phosphorylated SMAD3 by promoting the degradation of Smurf2
(Fig. 9). Our studies indicate that TRB3 is important for
maintaining the mesenchymal status of tumor cells and may be
a potential therapeutic target for the treatment of human tumor
metastasis.
Materials and MethodsPlasmids constructionHA-tagged TRB3 and its truncations: DC (amino acids 1–179), DN (amino acids180–358) and KD (amino acids 71–315) were constructed in a pcDNA3.1-HAvector by standard subcloning. The TRB3-shRNA-targeting sequence has beenreported previously (Du et al., 2003). The sequence was subcloned intopSilencerTM3.1-H1 hygro-expressing vector (Ambion, Texas, USA). A controlshRNA oligonucleotide, which does not match any known human coding cDNA,was used as control. pTRB3-luc was generated by ligating the human TRB3promoter region (–1038 to +847) with pGL3-basic. Myc-tagged SMAD3A andSMAD3D mutants were generated by PCR as described previously (Liu et al.,1997). Full-length SMAD3 was subcloned in frame to the pGEX4T-1 to makeGST fusion proteins. Smurf2-DDK/Myc expression plasmid was purchased fromOrigene (Rockville, MD, USA). The ubiquitin gene was synthesized according tothe sequence published on NCBI web site (accession number: M17524) and clonedinto pFLAG-CMVTM-2 expression plasmid. SBE4-luc reporter plasmid wasobtained from Dr Bert Vogelstein of Johns Hopkins University MedicalInstitutions, Baltimore, MD. The 3TP–lux reporter plasmid was obtained fromDr Joan Massague of Sloan-Kettering Institute, New York, NY.
Cells lines and transfectionAll cell lines were obtained from the ATCC. Transient transfection was carried outusing LipofectAMINE2000 (Invitrogen, Carlsbad, CA, USA) according to themanufacturer’s instructions. To generate HepG2 cell populations stably expressingTRB3 shRNA, the control-shRNA and the TRB3-shRNA plasmids were bothtransfected into HepG2 cells with siPORTTM XP-1 tansfection reagent (Ambion)according to the manufacturer’s instructions. After 24 hours of transfection, stabletransfectants were selected in medium containing 200 mg/ml hygromycin(Calbiochem, San Diego, CA, USA) for 7 days. After two or three passages in thepresence of hygromycin, the cultures were used for experiments without cloning.
Immunoprecipitation and western blottingCells were washed three times with phosphate-buffered saline, harvested, and lysed inco-immunoprecipitation (co-IP) buffer that has been described before (Du et al., 2003).Total cell lysate (5 mg protein) was subjected to immunoprecipitation with appropriateantibodies, as indicated, overnight at 4 C with gentle agitation, followed by incubationwith protein A/G Plus–agarose for 2,4 hours at 4 C. The immunocomplex waswashed three times and then mixed with 23SDS sample buffer and boiled for5 minutes. For western blotting, co-precipitates or whole cell extracts were resolved bySDS-PAGE and blotted on PVDF membranes (Millipore, Bedford, MA, USA). Themembranes were immunoblotted with the indicated antibodies and developed using anECL detection system (Amersham Bioscience, Piscataway, NJ, USA).
GST pull-down assayGST fusion constructs of SMAD3 were expressed in Escherichia coli and purifiedusing glutathione–Sepharose 4B beads (Amersham Bioscience). Equal amounts ofGST or GST fusion proteins bound to glutathione–Sepharose beads were incubatedwith lysates from HEK293T cells transiently transfected with TRB3–HA. Beadswere washed three times and interacting proteins were detected by immunoblotting.Expression of GST fusion proteins was confirmed by Coomassie Blue staining.
Luciferase reporter assayHEK293T or HepG2 cells were seeded on 12-well plates at a density of 26105/well, and transfected with the indicated plasmids. TK-Renilla expression plasmidwas used as an internal control. The total amount of plasmid per well wasnormalized by the addition of pcDNA3 empty vector. After 24 hours oftransfection, cells were incubated in the presence (+) or absence (–) of TGF-b1for another 24 hours. Cells were harvested with lysis buffer, and the luciferaseactivity was then measured using the dual luciferase assay kit according to themanufacturer’s instruction (Promega, Madison, Wisconsin, USA).
Immunofluorescence microscopyHepG2 cells were plated on sterile coverlips and transfected with the expressionplasmids, as indicated, for 24 hours. Cells were fixed with 4% paraformaldehydefor 10 minutes at room temperature, permeabilized with 0.5% Triton X-100 for10 minutes, and blocked with 3% BSA for 30 minutes at 37 C. The coverslipswere incubated with primary antibodies at 4 C overnight. After washing threetimes with PBS, cells were stained with Alexa Fluor 594 goat anti-mouse IgG andFITC-conjugated goat anti-rabbit antibodies for 30 minutes at 37 C then washedthree times with PBS and mounted on glass slides. Images were acquired using aconfocal microscope (Leica Microsystems Heidelberg GmbH, TCS SP2).
Cellular fractionationTo prepare the nuclear and cytoplasmic fractions, the cultured cells were lysedusing a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute ofBiotechnology, Suzhou, China) following the manufacturer’s instructions. Briefly,cells was washed with iced PBS and added to buffer A for 15 minutes and then
Journal of Cell Science 124 (19)3244
Jour
nal o
f Cel
l Sci
ence
centrifuged at 12,000 g for 15 minutes at 4 C. The supernatants were collected asthe cytoplasmic fraction. The pellet was washed with PBS and resuspended in anuclear extraction buffer. The mixture was votexed vigorously for 15 seconds andput on ice for 10 minutes. The process was repeated 4 or 5 times, and thencentrifuged again (12,000 g for 15 minutes, at 4 C). The obtained supernatant wascollected as the nuclear fraction. The separated cytoplasmic and nuclear fractionswere subjected to SDS-PAGE and immunoblotting.
Cell migration and invasion assays
Transwell migration assays were performed using Transwell chambers with filtermembranes of 8 mm pore size (Millipore). Chambers were precoated with 10 mg/mlfibronectin on the lower surface as previously described (Katoh et al., 2006). Cellswere starved overnight in assay medium [Dulbecco’s modified Eagle’s medium(DMEM) containing 0.4% FBS] and then seeded into the upper chamber (56104
cells per well in 0.4% FBS in DMEM). After 24 hours, non-migrating cells on theupper side of the filter were removed with a cotton swab. Cells migrated were stainedand counted. Transwell invasion assays were done under the same conditions as thetranswell migration assays, but before invasion assays, the polycarbonate filter wascoated with Matrigel (30 mg/well; BD Bioscience, Bedford, MA, USA).
Semi-quantitative RT-PCR
Total RNA was extracted using TRIzol (Invitrogen) following the manufacturer’sinstructions. Reverse transcription of the total cellular RNA was carried out usingoligo(dT) primers and M-MLV reverse transcriptase (Promega). PCR wasperformed using an Mycycler thermal cycler and analyzed using agarose gels.
Statistical analysis
Data are expressed as mean ¡ standard error of the mean (s.e.m.). Student’s t-testwas used for two-group comparisons. Comparisons between three or more groupswere analyzed by one-way ANOVA followed by Duncan’s test in SPSS 13.0(SPSS Inc.). P,0.05 was considered statistically significant.
AcknowledgementsWe thank Dr Bert Vogelstein for the SBE4–luc reporter plasmid, andDr Joan Massague for the 3TP–lux reporter plasmid. This work wassupported by grants from the National Major Basic ResearchProgram of China (973: #2006CB503808), the National NaturalScience Foundation of China (30472025; 30973557) and Creation ofMajor New Drugs (2009ZX09301-003-13; 2009ZX09301-003-9-1).Dr Zhuowei Hu is also supported by the Cheung-Kong ScholarsProgramme of Ministry of Education and by a Senior OverseasChinese Scholar Fund from the Ministry of Personnel of PRC. DrFang Hua is supported by grants from the Basic Research Program ofthe Institute of Materia Medica (2006QN33; 2010CHX12).
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.082875/-/DC1
ReferencesBatlle, E., Sancho, E., Franci, C., Dominguez, D., Monfar, M., Baulida, J. and
Garcia De Herreros, A. (2000). The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat. Cell Biol. 2, 84-89.
Bezy, O., Vernochet, C., Gesta, S., Farmer, S. R. and Kahn, C. R. (2007). TRB3blocks adipocyte differentiation through the inhibition of C/EBPb transcriptionalactivity. Mol. Cell. Biol. 27, 6818-6831.
Bierie, B. and Moses, H. L. (2006). Tumour microenvironment: TGF-b: the molecularJekyll and Hyde of cancer. Nat. Rev. Cancer 6, 506-520.
Bowers, A. J., Scully, S. and Boylan, J. F. (2003). SKIP3, a novel Drosophila tribblesortholog, is overexpressed in human tumors and is regulated by hypoxia. Oncogene
22, 2823-2835.
Chan, M. C., Nguyen, P. H., Davis, B. N., Ohoka, N., Hayashi, H., Du, K., Lagna, G.
and Hata, A. (2007). A novel regulatory mechanism of the bone morphogeneticprotein (BMP) signaling pathway involving the carboxyl-terminal tail domain ofBMP type II receptor. Mol. Cell. Biol. 27, 5776-5789.
Chen, Y. G., Wang, Z., Ma, J., Zhang, L. and Lu, Z. (2007). Endofin, a FYVE domainprotein, interacts with Smad4 and facilitates transforming growth factor-betasignaling. J. Biol. Chem. 282, 9688-9695.
Conery, A. R., Cao, Y., Thompson, E. A., Townsend, C. M., Jr, Ko, T. C. and Luo,
K. (2004). Akt interacts directly with Smad3 to regulate the sensitivity to TGF-binduced apoptosis. Nat. Cell Biol. 6, 366-372.
Ding, L., Wang, Z., Yan, J., Yang, X., Liu, A., Qiu, W., Zhu, J., Han, J., Zhang, H.,
Lin, J. et al. (2009). Human four-and-a-half LIM family members suppress tumorcell growth through a TGF-b-like signaling pathway. J. Clin. Invest. 119, 349-361.
Du, K., Herzig, S., Kulkarni, R. N. and Montminy, M. (2003). TRB3: a tribbleshomolog that inhibits Akt/PKB activation by insulin in liver. Science 300, 1574-1577.
Feng, X. H., Lin, X., Derynck, R. (2000). Smad2, Smad3 and Smad4 cooperate with
Sp1 to induce p15(Ink4B) transcription in response to TGF-b. EMBO J. 19, 5178-
5193.
Grosshans, J. and Wieschaus, E. (2000). A genetic link between morphogenesis and
cell division during formation of the ventral furrow in Drosophila. Cell 101, 523-531.
Hill, C. S. (2009). Nucleocytoplasmic shuttling of Smad proteins. Cell Res. 19, 36-46.
Jin, G., Yamazaki, Y., Takuwa, M., Takahara, T., Kaneko, K., Kuwata, T., Miyata,
S. and Nakamura, T. (2007). Trib1 and Evi1 cooperate with Hoxa and Meis1 in
myeloid leukemogenesis. Blood 109, 3998-4005.
Kang, Y. and Massague, J. (2004). Epithelial-mesenchymal transitions: twist in
development and metastasis. Cell 118, 277-279.
Katoh, H., Hiramoto, K. and Negishi, M. (2006). Activation of Rac1 by RhoG
regulates cell migration. J. Cell Sci. 119, 56-65.
Keeshan, K., He, Y., Wouters, B. J., Shestova, O., Xu, L., Sai, H., Rodriguez, C. G.,
Maillard, I., Tobias, J. W., Valk, P. et al. (2006). Tribbles homolog 2 inactivates
C/EBPa and causes acute myelogenous leukemia. Cancer Cell 10, 401-411.
Kim, I. Y., Kim, M. M. and Kim, S. J. (2005). Transforming growth factor-beta:
biology and clinical relevance. J. Biochem. Mol. Biol. 38, 1-8.
Kurisaki, A., Kurisaki, K., Kowanetz, M., Sugino, H., Yoneda, Y., Heldin, C. H. and
Moustakas, A. (2006). The mechanism of nuclear export of Smad3 involves exportin
4 and Ran. Mol. Cell. Biol. 26, 1318-1332.
Lee, J. M., Dedhar, S., Kalluri, R. and Thompson, E. W. (2006). The epithelial-
mesenchymal transition: new insights in signaling, development, and disease. J. Cell
Biol. 172, 973-981.
Lin, X., Liang, M. and Feng, X. H. (2000). Smurf2 is a ubiquitin E3 ligase mediating
proteasome-dependent degradation of Smad2 in transforming growth factor-bsignaling. J. Biol. Chem. 275, 36818-36822.
Liu, X., Sun, Y., Constantinescu, S. N., Karam, E., Weinberg, R. A. and Lodish,
H. F. (1997). Transforming growth factor b-induced phosphorylation of Smad3 is
required for growth inhibition and transcriptional induction in epithelial cells. Proc.
Natl. Acad. Sci. USA 94, 10669-10674.
Massague, J. (1998). TGF-b signal transduction. Annu. Rev. Biochem. 67, 753-791.
Massague, J., Seoane, J. and Wotton, D. (2005). Smad transcription factors. Genes
Dev. 19, 2783-2810.
Mata, J., Curado, S., Ephrussi, A. and Rørth, P. (2000). Tribbles coordinates mitosis
and morphogenesis in Drosophila by regulating String/CDC25 proteolysis. Cell 101,
511-522.
Micalizzi, D. S., Christensen, K. L., Jedlicka, P., Coletta, R. D., Baron, A. E.,
Harrell, J. C., Horwitz, K. B., Billheimer, D., Heichman, K. A., Welm, A. L. et al.
(2009). The Six1 homeoprotein induces human mammary carcinoma cells to undergo
epithelial-mesenchymal transition and metastasis in mice through increasing TGF-bsignaling. J. Clin. Invest. 119, 2678-2690.
Moustakas, A., Souchelnytskyi, S. and Heldin, C. H. (2001). Smad regulation in TGF-
b signal transduction. J. Cell Sci. 114, 4359-4369.
Ohoka, N., Yoshii, S., Hattori, T., Onozaki, K. and Hayashi, H. (2005). TRB3, a
novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved
in cell death. EMBO J. 24, 1243-1255.
Peinado, H., Olmeda, D. and Cano, A. (2007). Snail, Zeb and bHLH factors in tumour
progression: an alliance against the epithelial phenotype? Nat. Rev. Cancer 7, 415-
428.
Qi, L., Heredia, J. E., Altarejos, J. Y., Screaton, R., Goebel, N., Niessen, S.,
Macleod, I. X., Liew, C. W., Kulkarni, R. N., Bain, J. et al. (2006). TRB3 links the
E3 ubiquitin ligase COP1 to lipid metabolism. Science 312, 1763-1766.
Seher, T. C. and Leptin, M. (2000). Tribbles, a cell-cycle brake that coordinates
proliferation and morphogenesis during Drosophila gastrulation. Curr. Biol. 10, 623-
629.
Seong, H.-A., Jung, H., Kim, K.-T. and Ha, H. (2007). 3-Phosphoinositide-dependent
PDK1 negatively regulates transforming growth factor-beta-induced signaling in a
kinase-dependent manner through physical interaction with Smad proteins. J. Biol.
Chem. 282, 12272-12289.
Tang, M., Zhong, M., Shang, Y., Lin, H., Deng, J., Jiang, H., Lu, H., Zhang, Y. and
Zhang, W. (2008). Differential regulation of collagen types I and III expression in
cardiac fibroblasts by AGEs through TRB3/MAPK signaling pathway. Cell. Mol. Life
Sci. 65, 2924-2932.
Tian, F., DaCosta Byfield, S., Parks, W. T., Yoo, S., Felici, A., Tang, B., Piek, E.,
Wakefield, L. M. and Roberts, A. B. (2003). Reduction in Smad2/3 signaling
enhances tumorigenesis but suppresses metastasis of breast cancer cell lines. Cancer
Res. 63, 8284-8292.
Vincent, T., Neve, E. P., Johnson, J. R., Kukalev, A., Rojo, F., Albanell, J., Pietras,
K., Virtanen, I., Philipson, L., Leopold, P. L. et al. (2009). A SNAIL1-SMAD3/4
transcriptional repressor complex promotes TGF-beta mediated epithelial-mesench-
ymal transition. Nat. Cell Biol. 11, 943-950.
Wesolowska, A., Kwiatkowska, A., Slomnicki, L., Dembinski, M., Master, A., Sliwa,
M., Franciszkiewicz, K., Chouaib, S. and Kaminska, B. (2007). Microglia-derived
TGF-b as an important regulator of glioblastoma invasion-an inhibition of TGF-b -
dependent effects by shRNA against human TGF-b type II receptor. Oncogene 27,
918-930.
Wu, Q., Kim, K. O., Sampson, E. R., Chen, D., Awad, H., O’Brien, T., Puzas, J. E.,
Drissi, H., Schwarz, E. M., O’Keefe, R. J. et al. (2008). Induction of an
osteoarthritis-like phenotype and degradation of phosphorylated Smad3 by Smurf2 in
transgenic mice. Arthritis Rheum. 58, 3132-3144.
TRB3 interacts with SMAD3 3245
Jour
nal o
f Cel
l Sci
ence
Xu, J., Lv, S., Qin, Y., Shu, F., Xu, Y., Chen, J., Xu, B. E., Sun, X. and Wu, J. (2007).TRB3 interacts with CtIP and is overexpressed in certain cancers. Biochim. Biophys.
Acta 1770, 273-278.Xu, J., Lamouille, S. and Derynck, R. (2009). TGF-b-induced epithelial to
mesenchymal transition. Cell Res. 19, 156-172.Xu, L., Kang, Y., Col, S. and Massague, J. (2002). Smad2 nucleocytoplasmic shuttling
by nucleoporins CAN/Nup214 and Nup153 feeds TGF-b signaling complexes in thecytoplasm and nucleus. Mol. Cell 10, 271-282.
Xue, J. F., Hua, F., Lv, Q., Lin, H., Wang, Z. Y., Yan, J., Liu, J. W., Lv, X. X., Yang,
H. Z. and Hu, Z. W. (2010). DEDD negatively regulates transforming growth factor-b1 signaling by interacting with Smad3. FEBS Lett. 584, 3028-3034.
Yang, J., Mani, S. A., Donaher, J. L., Ramaswamy, S., Itzykson, R. A., Come, C.,
Savagner, P., Gitelman, I., Richardson, A. and Weinberg, R. A. (2004). Twist, a
master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell
117, 927-939.
Yang, M. H., Wu, M. Z., Chiou, S. H., Chen, P. M., Chang, S. Y., Liu, C. J., Teng,
S. C. and Wu, K. J. (2008). Direct regulation of TWIST by HIF-1alpha promotes
metastasis. Nat. Cell Biol. 10, 295-305.
Zhang, Y., Chang, C., Gehling, D. J., Hemmati-Brivanlou, A. and Derynck, R.
(2001). Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin
ligase. Proc. Natl. Acad. Sci. USA 98, 974-979.
Journal of Cell Science 124 (19)3246
Jour
nal o
f Cel
l Sci
ence