From the Bone & Mineral Research Program, Garvan
Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia and the ¶ Department of Molecular Genetics & Microbiology, State University of New York,
Stony Brook, New York 11794
Received for publication, November 30, 2000, and in revised form, February 21, 2001
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ABSTRACT |
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Transforming growth factor- Transforming growth factor- Recently it has been shown that Smad proteins also interact with other
nuclear factors such as c-Ski and the Ski-related novel (Sno) protein
and nuclear hormone receptors, including the vitamin D receptor (VDR)
to modulate TGF- As Ski and Sno can modulate TGF- Plasmid Constructs--
SKIP wild-type cDNA was PCR cloned
with the forward primer (5'-GGG AAT TCC CGG GGT CTA GAA CCA CCA TGG CGC
TCA CCA GCT TTT TA-3') and reverse primer (5'-GCG GGA TCC CTA TTC CTT
CCT CCT CTT-3'). The PCR product was ligated into pGEM-T Easy plasmid (Promega, Madison, WI) from which an EcoRI/BamH1
insert was excised and subcloned into a modified GAL4AD pACTII plasmid
(pACTIIb) and the vector pSG5 (Stratagene, La Jolla, CA). The pACTIIb
plasmid was created by replacing the BglII polylinker
fragment of pACTII (CLONTECH, Palo Alto, CA) with
the double-stranded oligonucleotide: 5'-GAT CTG TGA ATT CCC GGG GAT CCG
TCG ACC TA-3'. The GAL4 DBD wild-type Smad-pBridge yeast two hybrid
constructs were made by EcoRI/XhoI digestion of
Smad2-, Smad3-, and Smad4-pcDNA3 plasmids (3) and MH2 (aa 400-425
of hSmad3)-pBridge by PCR and subcloning of Smad cDNAs into the
EcoRI/SalI sites of pBridge
(CLONTECH). The GST-MH2 construct was made by
cloning cDNA of PCR product into the
EcoRI/XhoI sites of modified pGEX-4T2 plasmid
(Amersham Pharmacia Biotech). The SKIP deletion constructs (aa 1-200,
aa 1-333, aa 201-536, and aa 334-536) were made by PCR and cloned into the EcoRI/BglII site of pACTIIb and the
XbaI/BamH1 site of pCGN. The Sno cDNA was
amplified by PCR using the forward primer 5'-GCA ATC TAG AGA AAG CCC
ACA AGC AAA TTT CCC-3' and reverse primer 5'-GCA AGG ATC CCT ATT TTC
CAT TTC CAT TTT TG-3' and the PCR product ligated into the
XbaI/BamHI site of pCGN. The GST-SKIP construct
was made by PCR and cloned into the EcoRI and
SalI sites of pGEX-KG (24). All PCR primer sequences not
listed are available on request. All constructs were sequenced by
automated fluorescent sequencing and confirmed to be in frame and
correct. The wild-type SKIP-pCGN, c-Ski-pMT2, GST-Smad3, Smad2-,
Smad3-, and Smad4-pcDNA3 constructs and the 3TP-Lux reporter have
been described previously (3, 20, 25-27).
Yeast Two-hybrid Analysis--
Yeast transformation was
performed using a lithium acetate transformation kit (BIO101, Vista,
CA). Wild-type SKIP-pACTII plasmid encoding the SKIP-GAL4-AD fusion
protein was transformed into the Y187 yeast strain and Smad2-, Smad3-,
Smad4-, and MH2-pBridge encoding the Smad-GAL4 DBD fusion proteins were
transformed into the opposite yeast mating strain, CG1945. To
co-express the two different fusion proteins, yeast matings were
performed (CLONTECH Yeast Handbook, PT3024-1).
Yeast ligand experiments were performed as described previously (28).
Cell Culture and Reporter Assays--
COS-1 African green monkey
kidney cells were grown in Dulbecco's modified Eagle's medium with
5% fetal calf serum at 37o in 5% CO2. Cells
were plated the day before transfection in 24-well plates at a cell
density of 2 × 104 cells/well. Transfections was
performed with FuGENE-6 transfection reagent (Roche Molecular
Biochemicals) as per the manufacturer's instructions using 1.5 µl of
FuGENE with 0.75 µg of total DNA/well. Transfected cells were left in
FuGENE transfection reagent for 16-20 h, then treated with TGF- Glutathione-Sepharose Binding Assays--
Expression of
appropriately sized GST-SKIP and GST-Smad3 wild-type or mutant fusion
proteins was confirmed by SDS-PAGE. GST-binding assays were performed
in triplicate with equal amounts of 35S-labeled SKIP, VDR,
or luciferase as a negative control (28). Bound proteins were resolved
on 10% SDS-PAGE gels and subjected to autoradiography. In
vitro translation and transcription was performed according to the
manufacturer's instructions (Promega) with
[35S]methionine (Amersham Pharmacia Biotech).
Far Western and Immunoblot Analysis--
Far Western analysis
and preparation of nuclear extracts were essentially as described
previously (5, 28). COS1 nuclear extracts overexpressing Smad3 were run
on 10% SDS-PAGE and electroblotted onto a polyvinylidene difluoride
membrane (Millipore, Bedford, MA). Proteins were denatured with 6 M guanidine hydrochloride and renatured by the stepwise
dilution of guanidine hydrochloride. The Smad3 membrane was then
blocked and hybridized overnight at 4 °C with 20 µg of COS1
nuclear extracts containing HA-SKIP. The filter was rinsed three times
in HYB (20 mM Hepes-KOH, pH 7.4, 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 1% nonfat
milk, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol) and then probed with an anti-HA antibody
(Roche Molecular Biochemicals), which detected HA-SKIP, followed by
probing with a anti-mouse-HRP secondary antibody (Santa Cruz) prior to
ECL chemiluminescent detection (Amersham Pharmacia Biotech) and autoradiography.
Electromobility Shift Assay (EMSA)--
EMSA was performed with
the PE2 probe from the PAI-1 promoter (29), as described previously
(10), using in vitro translated 35S-labeled
Smad3 and Smad4 with COS1 nuclear extracts overexpressing SKIP, Sno, or Ski.
SKIP Augments TGF- Mapping of SKIP-Smad Interaction Domains in Yeast and Mammalian
Cells--
SKIP interaction with Smad proteins was investigated by
yeast two-hybrid interaction analysis. SKIP interacted with both Smad2 and Smad3 (Fig. 1B). Smad4 induced a high level of reporter
activity, which was unaltered by co-expression of SKIP. However, Smad4, as expected, interacted strongly with v-Ski-GAL4-AD in yeast (data not
shown). Domains of SKIP required for Smad interaction were examined
using deletion constructs of SKIP (Fig. 1B). The
C-terminally deleted aa 1-333 and N-terminally deleted aa 201-536
SKIP mutants had interaction with Smad2 comparable to that of wild-type
SKIP. The interaction between these two mutants and Smad3 were about 50% and 25% of wild-type SKIP, respectively. No interaction of the
SKIP N-terminal (aa 1-200) or C-terminal (aa 334-536) domain with
Smad2 or Smad3 was observed. Thus, these results suggest that the
region of SKIP between aa 201 and 333 interacts with Smad2 and Smad3.
The same SKIP deletion constructs were tested with the 3TP-lux reporter
in the COS1 mammalian cell line (Fig. 1C). Co-expression of
wild-type SKIP (aa 1-536) with Smad3 caused a synergistic 3-fold increase in reporter activity above SKIP or Smad3 alone. The N-terminal domain of SKIP (aa 1-200) had no effect on reporter activity, while
the other SKIP constructs had comparable transactivation to that of
wild-type SKIP. Western blot analysis of these deletion constructs
showed comparable expression with wild-type, except for the aa 1-200
construct, which, despite its lack of transactivation, was 2-3 times
more highly expressed (data not shown). Thus, these transfection data
were consistent with the yeast interaction data and suggest that
expression of the aa 201-333 region of SKIP with Smad3 is sufficient
for near maximal transactivation of the 3TP-lux reporter. Surprisingly,
the aa 334-536 SKIP construct was able to activate the 3TP-lux
reporter with Smad3, even though no interaction was observed with Smad3
in yeast. This suggests that an additional C-terminal domain may also
be transcriptionally functional and possibly recruits other
Smad-interacting co-factors present in mammalian cells, but not yeast.
SKIP Interaction with Smad2 and Smad3 in Vitro--
The potential
direct physical interaction between the Smad proteins and SKIP was
explored using a GST "pull-down" assay. GST-SKIP bound both Smad2
and Smad3 (Fig. 2A). In comparison
there was minimal, if any, binding of Smad2 or Smad3 to GST-0 and no
binding of luciferase to GST-SKIP.
To determine which domains of Smad3 may be involved in SKIP
interaction, a GST-Smad3 binding assay was performed with
35S labeled in vitro translated SKIP (Fig.
2B). GST-wild-type Smad3 bound SKIP and the positive control
VDR (9). Deletion of the MH1 domain of Smad3 (aa 199-427) had no
effect on SKIP binding, but, as expected, VDR binding was abolished.
Both SKIP and VDR binding was lost when both the MH1 and MH2 domains of
Smad3 were deleted (GST-Smad3 aa 199-405). However, no binding of SKIP
was observed to a GST-MH2 construct, which expressed only the last 26 aa of hSmad3. This result was further supported by a lack of interaction between SKIP-GAL4-AD and a MH2-GAL4-DBD construct containing the same C-terminal 26 aa of hSmad3 in vivo in
yeast (data not shown). These results indicate that, although deletion of the C-terminal MH2 domain abrogates SKIP binding, the MH2 domain alone is not sufficient for SKIP interaction.
To further support the existence of a direct protein-protein
interaction in vitro, a Far Western assay was performed
using mammalian cell nuclear extracts overexpressing HA-SKIP
(upper panel) or Smad3 (middle
panel) (Fig. 2C). In the Far Western analysis (lower panel), Smad3 detected by Western analysis
co-localized with HA-SKIP detected by using an anti-HA antibody, but
not with the negative empty vector control extracts. These results
together with the GST-binding studies thus strongly support the
existence of a protein-protein interaction in vitro between
SKIP and Smad3.
Ski and Sno Competitively Inhibit SKIP-dependent
Activation--
The Smad3 transcriptional repressors, Ski and its
related protein, Sno, are known to bind to the MH2 domain of Smad3
(26). Since SKIP also interacts with Ski and Sno, we tested whether SKIP modulates Ski/Sno repression of Smad3-dependent transcription. As
shown above, SKIP increased basal and TGF-
As Ski/Sno interact with both Smad3 and SKIP, one alternative
possibility other than a competitive interaction between these proteins
is that they form a ternary complex. To address this question, a gel
shift analysis was performed (Fig. 5). Using
the PE-2 probe from the PAI-1 promoter, which binds a Smad3/4
heterodimer (29) (Fig. 5, lane 2), we showed
that, with addition of increasing amounts of SKIP nuclear extracts,
there was augmentation of binding of a higher molecular weight complex,
which presumably contained SKIP and Smad3/4 (lanes
3-6). The addition of Sno nuclear extracts also led to
increased Smad3/4 binding with a similar mobility shift
(lanes 9 and 10 and lanes
12 and 13). This complex was specific as it was
abrogated by addition of cold probe (lane 11).
However, in the presence of both SKIP and increasing Sno, although we
observed increased intensity of the upper complex, no further
supershift and hence no ternary complex was observed (lanes
7-10). Similar results were obtained using
Ski-overexpressing nuclear extracts (data not shown).
The Ski and Sno oncoproteins have been shown to negatively
modulate TGF- The C-terminal MH2 domain of Smad2 and Smad3 has been reported to be a
key region involved in multiple protein-protein interactions, including
those with the coregulators CBP/p300 and the Smad repressors Ski and
Sno (1). The N-terminal MH1 domain of the Smads confers only low
affinity DNA binding to a consensus SBE (1). Although natural
TGF- Although SKIP was able to interact with Smad2 and Smad3 in yeast, SKIP
co-transfection with Smad3 with or without Smad4, led to the greatest
increases in reporter activity in mammalian cells, presumably because
the 3TP-lux reporter is Smad3-selective (27). Thus, as SKIP interacted
with Smad2 in vivo and in vitro, it is also
possible that SKIP in mammalian cells may be able to modulate TGF- The deletional analysis of SKIP in yeast and mammalian cells suggests
that the aa 201-333 region of SKIP is required for Smad interactions
in vivo. However, some functional differences were observed
between yeast and mammalian cells. Specifically, although the
C-terminal SKIP construct (aa 334-536) did not interact with Smad2 or
Smad3 in yeast, its transactivation activity in mammalian cells was
comparable to wild-type SKIP. A C-terminal transactivation domain of
SKIP that functions in mammalian cells, distinct from the Smad
interaction domain, is consistent with the domain C-terminal to aa 437 of murine SKIP (NcoA-62) being involved in vitamin
D-dependent transactivation (21).
As SKIP interacts with Ski/Sno and Smad3 and, in turn, Ski/Sno interact
with Smad3, to address the possibility that these proteins form a
ternary complex, we performed a gel shift analysis using the PE2 probe
from the PAI-1 promoter, as used in the transient transfections. The
EMSA clearly showed that both SKIP and Ski/Sno alone formed a slightly
higher migrating complex with Smad3/4. However, we did not observe the
formation of a ternary complex in the presence of all three proteins.
Thus, these data are consistent with the transient transfection
results, which suggests competition occurring between SKIP and Ski/Sno
for Smad3 transactivation, but cannot exclude the presence of a ternary
complex forming between these proteins.
In this study SKIP acted as a coactivator of
TGF- In summary, our results support a model in which SKIP positively
modulates TGF- (TGF-
) signaling
requires the action of Smad proteins in association with other
DNA-binding factors and coactivator and corepressor proteins to
modulate target gene transcription. Smad2 and Smad3 both associate with
the c-Ski and Sno oncoproteins to repress transcription of Smad target
genes via recruitment of a nuclear corepressor complex. Ski-interacting protein (SKIP), a nuclear hormone receptor coactivator, was examined as
a possible modulator of transcriptional regulation of the
TGF-
-responsive promoter from the plasminogen activator inhibitor
gene-1. SKIP augmented TGF-
-dependent transactivation in
contrast to Ski/Sno-dependent repression of this reporter.
SKIP interacted with Smad2 and Smad3 proteins in vivo in
yeast and in mammalian cells through a region of SKIP between amino
acids 201-333. In vitro, deletion of the Mad homology
domain 2 (MH2) domain of Smad3 abrogated SKIP binding, like Ski/Sno,
but the MH2 domain of Smad3 alone was not sufficient for
protein-protein interaction. Overexpression of SKIP partially overcame
Ski/Sno-dependent repression, whereas Ski/Sno
overexpression attenuated SKIP augmentation of
TGF-
-dependent transcription. Our results suggest a
potential mechanism for transcriptional control of TGF-
signaling
that involves the opposing and competitive actions of SKIP and Smad
MH2-interacting factors, such as Ski and/or Sno. Thus, SKIP appears to
modulate both TGF-
and nuclear hormone receptor signaling pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(TGF-
)1 superfamily members
are multifunctional cell-cell signaling proteins, which include the TGF-
s, bone-morphogenetic proteins (BMPs), activins, and inhibins, mullerian-inhibiting substance and growth differentiation factors (1).
Members of this superfamily mediate many key cellular events in growth
and development and are evolutionarily conserved from
Drosophila to mammals (2). TGF-
signaling requires the action of a family of DNA-binding proteins called Smads, including TGF-
-specific (Smad2 and Smad3), BMP-specific (Smad1, Smad5 and Smad8), a common Smad4, and anti-Smads (Smad6 and Smad7). TGF-
signals through sequential activation of two cell surface receptor serine-threonine kinases, which phosphorylate Smad2 and/or Smad3. Phosphorylated Smad2 or Smad3, together with Smad4, translocates into
the nucleus where the Smad heterodimer binds Smad-binding elements
(SBEs) in association with other nuclear factors in promoters of target
genes (1, 3, 4).
signaling (5-10). Ski and Sno are involved in
oncogenic transformation and enhancement of muscle differentiation by
blocking TGF-
signaling (11-14). The mechanism of Ski/Sno
repression of TGF-
signaling appears to involve an interaction with
a complex consisting of the nuclear corepressor (N-CoR) and a histone
deacetylase enzyme (15, 16). N-CoR, and its related co-repressor
silencing mediator for retinoic acid and thyroid receptors (SMRT),
interact with a wide variety of other nuclear factors to mediate
transcriptional repression (17-19). Interestingly, the Ski-interacting
protein (SKIP) was initially identified in a two hybrid screen using
v-Ski as a bait and was later independently identified as a VDR- and
CBF1-interacting factor (20-22). Thus, the recent observation that
SKIP modulates CBF1 and Notch-dependent signaling suggests
that SKIP may play a role in the regulation of a number of different
and distinct cellular signaling pathways (23).
-dependent signaling, it
was of interest to determine whether SKIP could also modulate the TGF-
-signaling pathway through interaction with the Smad proteins. In this study, in contrast to Ski- and Sno-mediated repression, SKIP
augmented TGF-
-dependent transcription. A region of
SKIP, aa 201-333, appeared to be required for the Smad interaction. SKIP interacted in vitro and in vivo with Smad3
and partially counteracted Ski- and Sno-dependent
repression, while Ski/Sno attenuated SKIP transactivation of TGF-
signaling. These results suggest that SKIP may play an opposite role to
Ski and Sno in the control of TGF-
-dependent transcription.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Galactosidase activity in protein lysates was measured with the
Tropix Galactolight chemiluminescence assay (PerkinElmer Life Sciences)
using the Berthold LB953 luminometer (Berthold, Bad Wildbad, Germany)
and expressed as relative light units. All results are shown as
mean ± S.E. of at least three different yeast colonies, from at
least two experiments, and corrected for protein concentration (Bio-Rad
protein assay).
(Sigma) or vehicle (4 mM HCl and 1 mg/ml bovine serum
albumin) in 2% charcoal-stripped medium for 16-24 h. The medium was
then removed, and cells were lysed with 2× Promega lysis buffer.
Luciferase assays were performed in triplicate with the firefly
luciferase assay kit (Promega) and measured with a luminometer (Berthold).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-dependent Transcription--
As
Ski and Sno interact directly with Smad proteins (Smad2 and Smad3) to
repress TGF-
-dependent transcription, the effects of
SKIP on the 3TP-lux TGF-
-responsive reporter construct (27) were
tested (Fig. 1). In COS1 cells this reporter
responded to TGF-
with a 4-fold increase in reporter activity,
consistent with these cells expressing endogenous Smad proteins (30).
Smad3 alone, or Smad2 and Smad4 together (but neither alone) augmented both basal (2-fold) and TGF-
responses (10-fold) of this reporter activity. Smad3 co-transfection with Smad4 led to a 6-fold increase in
basal and a 30-fold increase in ligand-dependent reporter
activity. This augmentation was similar to that of SKIP alone on
ligand-dependent reporter activity (Fig. 1). An interaction
between SKIP and Smads was suggested in co-transfection studies, with
the -fold increase of basal activity progressively increasing when SKIP
was co-transfected with Smad2 (8-fold), Smad2 and 4 (20-fold), Smad3
(53-fold), and Smad3 and Smad4 (164-fold). The comparable increases in
TGF-
induced activity were 39-, 116-, 96-, and 323-fold,
respectively. These data are consistent with a functional interaction
primarily occurring between SKIP and Smad3, with or without exogenous
Smad4.
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Fig. 1.
SKIP augments
TGF- -dependent transactivation in
mammalian cells. A, transient transfections of COS1
cells were performed in 24-well plates with the 3TP-lux reporter (250 ng/well) and the following expression plasmids as indicated: SKIP-pCGN
and Smad2-, Smad3-, and Smad4-pcDNA3. The 3TP-Lux reporter contains
three clusters of Smad-binding elements from the PAI-1 promoter and is
predominantly a Smad3-responsive promoter reporter. Cells were treated
with vehicle (
) or TGF-
ligand (
) (1 ng/ml). The total amount
of transfected DNA was kept constant by use of respective empty
vectors. The results are shown as the mean luciferase activity ± S.E. of three independent experiments performed in triplicate.
B, SKIP interacts in vivo with Smad2 and Smad3 in
a yeast two hybrid system through a domain between 201 and 333 aa.
Deletional analysis of SKIP-GAL4-AD as indicated followed by
co-expression with wild-type Smad2-, Smad3-, and Smad4-GAL4-DBD was
performed as described under "Experimental Procedures." Results of
-galactosidase activity are shown as mean ± S.E. from three
independent colonies obtained in at least duplicate experiments and are
corrected for protein levels determined by the Bio-Rad protein assay.
RLUs, relative light units. C, SKIP domain
between aa 201 and 333 enhances TGF-
transactivation in mammalian
cells. Transient transfections of COS1 cells were performed as
described in A with the 3TP-lux reporter and the
Smad3-pcDNA3 expression plasmid (100 ng) and wild-type SKIP-pCGN or
deletion constructs as indicated (50 ng). Cells were treated with
vehicle (
) or TGF-
ligand (
) (1 ng/ml). The results are shown
as the mean luciferase activity ± S.E. of two independent
experiments performed in triplicate.
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Fig. 2.
SKIP interacts with Smad2 and Smad3 in
vitro. A, glutathione-Sepharose beads were
coated with bacterially expressed GST-SKIP wild-type fusion proteins or
GST alone and were incubated with equal amounts of
35S-labeled wild-type Smad3 and luciferase. Bound proteins
were resolved by SDS-PAGE. Input proteins show one-fifth of loaded
lysate. B, Sepharose beads were coated with bacterially
expressed fusion proteins consisting of GST-Smad3 or deletion mutants
of Smad3, including an MH1 and MH2 domain double mutant (aa 199-405)
and a phosphorylated MH1 domain deletion mutant that retains the MH2
(aa 199-425), a mutant containing the MH2 domain consisting of the
last 26 aa of the C terminus of Smad3 (aa 400-425), or GST alone.
Beads were incubated with equal amounts of 35S-labeled
wild-type SKIP, and hVDR (9) and luciferase as positive and negative
controls, respectively. Bound proteins were resolved on SDS-PAGE. Input
proteins show one-fifth of loaded lysate. C, Far Western
analysis. Twenty µg of nuclear extracts containing HA-SKIP
(upper panel) or Smad3 (middle
panel) was detected by Western analysis. COS1 nuclear
extracts were prepared from cells transfected with the empty vector
pcDNA3 or pCGN (indicated by ) or Smad3-pcDNA3 or
HA-SKIP-pCGN expression plasmid (indicated by +). Proteins in the
Smad3-containing membrane were resolved on SDS-PAGE, electroblotted to
a polyvinylidene difluoride membrane, denatured, renatured with serial
dilutions of 6 M guanidine hydrochloride, and probed with
an anti-HA antibody (lower panel). On Far Western
analysis anti-HA antibody detected a band co-migrating with Smad3, but
not the negative control extract.
-dependent
transactivation, particularly in the presence of Smad3 (Fig.
3). Both Ski and Sno attenuated this
SKIP-dependent transactivation by about 80% and 40%,
respectively (Fig. 3). SKIP transactivation, either alone or with
Smad3, was repressed in a dose-dependent manner by
co-transfection with Ski. Sno had a similar but weaker effect (Fig.
4). These data suggest that SKIP and Ski/Sno
may act as counteracting regulators of the TGF-
transcriptional
response.
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Fig. 3.
SKIP augments
TGF- -dependent transcription and
partially counteracts Ski- and Sno-dependent
repression. Transient transfections of COS1 cells were performed
with the 3TP-lux reporter (250 ng), Smad3-pcDNA3 (100 ng), and
increasing amounts of wild-type SKIP-pCGN as indicated, with either
c-Ski-pMT2 (A, 100 ng) or Sno-pCGN (B, 100 ng)
expression plasmids. Cells were treated with vehicle (
) or TGF-
ligand (
) (1 ng/ml). The results are shown as the mean luciferase
activity ± S.E. of triplicate wells relative to
ligand-dependent reporter activity of Smad3 transfection
alone set at 1, and are representative of three independent
experiments.
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Fig. 4.
Ski and Sno attenuates SKIP augmentation of
TGF- -dependent transcription.
Transient transfections of COS1 cells were performed with the 3TP-lux
reporter (250 ng) and Smad3-pcDNA3 (100 ng), wild-type SKIP-pCGN
(50 ng), and increasing amounts of either c-Ski-pMT2 (A) or
Sno-pCGN (B) expression plasmids as indicated. Cells were
treated with vehicle (
) or TGF-
ligand (
) (1 ng/ml). The
results are shown as the mean luciferase activity ± S.E. of
triplicate wells relative to maximal ligand-dependent
reporter activity with SKIP and Smad3 co-transfection set at 10, and
are representative of three independent experiments.
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Fig. 5.
SKIP and Sno independently bind to Smad3/4
DNA complexes. In vitro 35S-labeled Smad3
and Smad4 (2 µl each) with COS1 nuclear extracts of SKIP and Sno were
added, as indicated (µl), with radiolabeled PE2 probe, a fragment of
the PAI-1 promoter and analyzed by gel shift assay. Lane
1 contains untranslated lysate and lane
2 Smad3/4 lysate alone (lower arrow).
The SKIP/Smad3/4 (lanes 3-6) and Sno/Smad3/4
higher molecular weight complexes (lanes 7-10)
are indicated by upper arrow. CP, cold probe
(lane 11).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
signaling through an interaction with a N-CoR
repressor complex (15). As SKIP, a nuclear hormone receptor-interacting cofactor, also associates with both Ski and Sno, this study was undertaken to determine the potential role of SKIP in TGF-
signaling. In these studies SKIP augmented TGF-
dependent
transcription and exhibited a direct interaction with Smad proteins.
This SKIP-Smad interaction was apparent both in vitro and
in vivo, as demonstrated by GST pull-down assays, Far
Western analysis, and yeast two hybrid protein-protein studies. The
region between aa 201 and 333 within SKIP appeared to act as the
Smad-interacting domain, while, SKIP, like Ski and Sno, interacted with
the MH2 domain of Smad3. Moreover, Ski and Sno attenuated SKIP
transactivation, while SKIP partially counteracted Ski- and
Sno-mediated transcriptional repression.
-responsive promoters contain functional clusters of SBEs, other
DNA-binding factors, such as FAST-1, TFE3, and AP-1, as well as non-DNA
binding factors through protein-protein interaction with the C-terminal
MH-2 domain, are involved in determining the specificity and direction
of Smad target gene action (29, 31, 32). As such, SKIP appears to play
a role in augmentation of TGF-
-specific Smad transcriptional
activity via an interaction with the MH2 domain of Smad3. Our data also
suggest, as SKIP was unable to interact with an isolated MH2 domain
(last 26 aa) of Smad3 in GST binding assays and yeast two-hybrid
studies, that other regions within the Smad proteins possibly within
the context of the whole Smad protein may also modulate Smad-SKIP interaction.
signaling through Smad2 in certain situations (30). Furthermore, in the
transient transfections we observed that Smad3 augmented basal reporter
activity, as previously described with this promoter (5), but this
activity was further increased by SKIP. Further studies will be
required to address the specific reasons for this effect of SKIP.
-dependent transcription. SKIP similarly acts as a
coactivator of nuclear hormone receptor-dependent
transcription, but also as a repressor of Notch signaling through its
interaction with SMRT and associated histone deacetylase enzyme
proteins (21, 23). These divergent effects of SKIP may depend on
interaction of SKIP with other, possibly cell-specific nuclear factors.
For example, SKIP converts CBF1 from a transcriptional repressor to
activator through switching its interaction between the corepressor
SMRT and Notch 1C (23). In our study SKIP and Ski/Sno modulated each
other's opposing transcriptional activities, raising the intriguing
possibility that the relative cellular expression of SKIP
versus Ski or Sno may play a regulatory role on
TGF-
-dependent transcription and hence its effects on
cell growth and differentiation. Interestingly, the Smad-interacting
domain of SKIP (aa 201-333) appears to be also involved in interaction
with Ski and Sno.2 These results
and those showing that SKIP, like Ski/Sno, interacted with the MH2
domain of Smad3 suggest that the opposing transcriptional effects of
SKIP and Ski/Sno may involve competition for Smad3 binding between SKIP
and c-Ski/Sno, and/or other Smad3 MH2-interacting factors, such as with
CBP/p300 (7, 33, 34). Thus, the modulatory effects of SKIP through the
MH2 domain potentially increase the complexity and diversity of
Smad-dependent transcriptional effects. Furthermore, as
SKIP and Ski/Sno interact with each other and also with the related
corepressors N-CoR/SMRT, an additional mechanism could involve
SKIP-mediated derepression (1, 15, 20, 23). This may possibly occur via
SKIP sequestration of corepressors such as SMRT or N-CoR from the
Ski/Sno repressor complex, a mechanism similar to that suggested for
Hoxc-8 and Smad1 (35). Whatever the molecular mechanism of SKIP action,
it is nevertheless clear that SKIP plays a role in modulation of this
important cellular and signaling pathway.
-dependent-transcription and potentially
competes with other MH2-interacting factors, such as c-Ski and Sno, to determine the transcriptional outcome of a TGF-
-responsive target gene. This suggests a potential role for SKIP in the regulation of
TGF-
effects on cell growth and differentiation.
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ACKNOWLEDGEMENTS |
---|
We thank Jean Massague, Takeshi Imamura, Kohei Miyazono, and Robert Weinberg for plasmids; Edith Gardiner, Michelle Henderson, Roger Daly, and Nobuhide Nueki for critical reading of the manuscript; and Colette Fong and other members of the Bone & Mineral Research Program for general technical support, advice, and camaraderie.
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FOOTNOTES |
---|
* This work was supported by a National Health and Medical Research Council (NHMRC) grant to the Bone & Mineral Research Program at the Garvan Institute, in part through a NHMRC medical postgraduate scholarship (to G. M. L.), and by National Institutes of Health Public Service Grants CA28146 and CA42573 (to M. J. H).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.
§ To whom correspondence should be addressed: Bone & Mineral Research Program, Garvan Inst. of Medical Research, 384 Victoria St., Darlinghurst, New South Wales 2010, Australia. Tel.: 61-2-9295-8247; Fax: 61-2-9295-8241; E-mail: g.leong@garvan.unsw.edu.au.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M010815200
2 J. Figueroa and M. J. Hayman, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
SKIP, Ski-interacting protein;
N-CoR, nuclear corepressor;
PAI-1, plasminogen activator inhibitor gene-1;
MH1, Mad homology domain 1;
MH2, Mad homology domain 2;
BMP, bone-morphogenetic protein;
SBE, Smad-binding element;
VDR, vitamin D
receptor;
SMRT, silencing mediator for retinoic acid and thyroid
receptors;
GAL4-AD, GAL4 activation domain;
GAL4-DBD, GAL4 DNA-binding
domain;
aa, amino acid(s);
HA, hemagglutinin;
GST, glutathione
S-transferase;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel electrophoresis.
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