From the Institute of Molecular and Cellular
Biosciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo
113-0034, Japan, § CREST, Japan Science and Technology,
4-1-8 Honcho, Kawaguchi, Saitama 332, Japan, and the ¶ Department
of Biochemistry, The Cancer Institute, Tokyo, Japanese Foundation for
Cancer Research, and Research for the Future Program, Japan Society for
the Promotion of Science, 1-37-1 Kami-Ikebukuro, Toshima-ku,
Tokyo 170-8455, Japan
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ABSTRACT |
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Several lines of experiments demonstrated the
interplay between the transforming growth factor- Cell growth, differentiation, and function are tightly regulated
by orchestrated functions of extracellular signals, i.e. growth regulatory factors, such as transforming growth factor- Extensive studies have been directed toward the interplay between the
two factors, TGF- Plasmid Construction--
N-terminally FLAG or HA tagged mouse
Smad3, mouse Smad6, and human Smad7 were inserted between the
EcoRI and XhoI sites of the mammalian expression
vector pcDNA3 (Invitrogen) (FLAG-Smad3, -6, and -7 and HA-Smad6 and
-7, respectively). The original construction of constitutively active
TGF- Transfection and CAT Assay--
COS-1 cells were maintained in
Dulbecco's modified Eagle's medium without phenol red, supplemented
with 5% dextran-coated charcoal-stripped fetal bovine serum. The cells
were transfected at 40-50% confluence in 10-cm Petri dishes with a
total of 20 µg of indicated plasmids using calcium phosphate. All
assays were performed in the presence of 3 µg of pCH110 (Pharmacia)
with a Mammalian Two- and Three-hybrid Assays--
COS-1 cells were
transfected at 70-80% confluence in 6-well dishes by using Lipofectin
(Life Technologies, Inc.) following the manufacturer's instructions. 1 µg of 17m8-luc vector was cotransfected with 100 ng of GAL4-VDR or
GAL4-RXRa, 10 ng of pSG5-VDR, 100 ng of FLAG-Smad6 or Smad7, and 100 ng
of VP-Smad3. In all assays, 200 ng of pRL-tk vector (Promega) was
transfected for the internal control. 6-8 h after transfection, cells
were washed with fresh medium, ligand was added to the medium, and
cells were incubated for an additional 24-36 h. Cell extract
preparations and dual luciferase assays were performed following the
manufacturer's protocols (Promega).
GST Pull-down Assay--
Glutathione S-transferase
(GST)-fused proteins were expressed in Escherichia coli and
purified using glutathione-Sepharose 4B beads (Pharmacia). The beads
were incubated with [35S]methionine-labeled proteins.
Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis
as described (28).
Coimmunoprecipitation and Western Blotting--
COS-1 cells were
transfected with the indicated plasmids, lysed in TNE buffer (10 mM Tris-HCl, pH 7.8, 1% Nonidet P-40, 0.15 M
NaCl, 1 mM EDTA), and immunoprecipitated with anti-FLAG M2
monoclonal antibody (IBI; Eastman Kodak), and interacting proteins were
separated by 8% SDS-polyacrylamide gel electrophoresis, transferred
onto polyvinylidene difluoride membranes (Bio-Rad), and detected with anti-VDR antibody and anti-rabbit IgG conjugated with alkaline phosphatase (Promega) (28).
Smad7, but Not Smad6, Abrogates the Smad3-mediated Potentiation of
VDR Function--
In an attempt to clarify the roles of Smad proteins
for VDR, we found that Smad3 acts as a coactivator of VDR (28). We
extended our studies to another class of Smad proteins, inhibitory Smad proteins, Smad6 and Smad7, in the ligand-induced transactivation function of VDR. To circumvent the confounding actions of DNA binding,
heterodimerization, and endogenous VDR induction, we chose the GAL4
reporter assay system using VDR-AF2 fused to GAL4-DNA-binding domain
and 17mx2G CAT reporter (29). As expected, Smad3 strongly increased the
reporter activity elicited by VDR-AF2 in a ligand-dependent manner, confirming that Smad3 is a bona fide coactivator of VDR-AF2. Neither overexpressed Smad7 nor Smad6 altered the activative function of VDR-AF2 itself (Fig. 1A).
Smad7, however, abrogated the Smad3-mediated potentiation of VDR-AF2
function and the enhanced VDR-AF2 function by constitutively active
TGF-
To verify the suppressive effect of Smad7, we further investigated the
effects of Smad6 and Smad7 on full-length VDR by a CAT assay using CAT
reporters containing various vitamin D response elements (VDREs).
First, we used synthetic VDRE (DR 3). As with VDR-AF2, we found that
full-length VDR transactivation activated by Smad3 or constitutively
active TGF- Smad7 Inhibits the Formation of the VDR-Smad3 Complex--
Because
some coactivators of VDR directly interact with VDR, we thought that a
possible molecular mechanism underlying this inhibitory effect could be
competition between Smad3 and inhibitory Smad proteins for binding to
VDR. Alternatively, inhibitory Smad proteins could affect the VDR-Smad3
complex formation indirectly. Because we have previously shown that
Smad3 binds to VDR in in vivo and in vitro assays
(28), we performed the GST pull-down assay using Smad6 or Smad7 to test
the former possibility. In vitro translated Smad3 bound VDR
weakly but significantly, as described previously (28). However, under
the same conditions, we could detect no interaction between VDR and
Smad6 or Smad7 (data not shown). We then determined whether the
interaction between Smad3 and VDR is affected in the presence of these
inhibitory Smad proteins. First we employed a mammalian two-hybrid
assay system. When Smad7 was cotransfected, the interaction between Smad3 and VDR was clearly inhibited. However, Smad6 did not affect this
interaction (Fig. 2A). Next,
to determine whether Smad3 also binds to the functional VDR unit,
VDR-RXR heterodimer, we employed a mammalian three-hybrid assay system.
As described previously, Smad3 bound VDR-RXR heterodimer in a
ligand-dependent manner. Similar to the result of the
mammalian two-hybrid assay, this interaction was strongly inhibited by
Smad7 but not by Smad6 (Fig. 2B).
To further confirm the observations that Smad3-VDR complex formation is
abolished in the presence of Smad7, we examined the effects of these
inhibitory Smad proteins on the interaction of Smad3 with VDR using a
coimmunoprecipitation assay. Coimmunoprecipitations were performed
using antibody against the N-terminal FLAG tag of Smad3 and followed by
Western blotting using anti-VDR antibody. Smad3 weakly bound VDR by
itself. However, in the presence of SRC-1, Smad3 efficiently bound to
the VDR-SRC-1 complex as described previously (28). The presence of
Smad7 caused almost complete abolishment of this VDR-Smad3 complex
formation. However, Smad6 had little effect on this interaction (Fig.
2C). These results are consistent with the results of the
mammalian two-hybrid and three-hybrid assays.
In all assays we employed, Smad6 had little effect on the
Smad3-activated VDR transactivation or the formation of the VDR-Smad3 complex. These results are in accordance with the previous reports demonstrating that Smad3 phosphorylation is not affected by Smad6 (19),
which is different from Smad7 (20, 21).
We demonstrated here that Smad7, as well as Smad3, is strongly involved
in the interplay between the TGF-
Considering the complex modulations of nuclear receptor transactivation
functions by TGF- (TGF-
) and
vitamin D signaling pathways. Recently, we found that Smad3, a
downstream component of the TGF-
signaling pathway, potentiates
ligand-induced transactivation of vitamin D receptor (VDR) as a
coactivator of VDR (Yanagisawa, J., Yanagi, Y., Masuhiro, Y., Suzawa,
M., Watanabe, M., Kashiwagi, K., Toriyabe, T., Kawabata, M., Miyazono,
K., and Kato, S. (1999) Science 283, 1317-1321). Here, we investigated the roles of inhibitory Smads, Smad6
and Smad7, which are negative regulators of the TGF-
/bone morphogenetic protein signaling pathway, on the Smad3-mediated potentiation of VDR function. We found that Smad7, but not Smad6, abrogates the Smad3-mediated VDR potentiation. Interaction studies in vivo and in vitro showed that Smad7
inhibited the formation of the VDR-Smad3 complex, whereas Smad6 had no
effect. Taken together, our results strongly suggest that the interplay
between the TGF-
and vitamin D signaling pathways is, at least in
part, mediated by the two classes of Smad proteins, which modulate VDR
transactivation function both positively and negatively.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(TGF-
)1 and bone
morphogenetic proteins (BMPs), and lipophilic hormones, such as vitamin
D and retinoic acids. Lipophilic hormones transcriptionally control
gene expression by binding to cognate nuclear receptors (1), which act
as ligand-inducible transcription factors with transcriptional
coactivators such as the SRC-1/TIF2 family and CBP/p300 (2-5). The
cell membrane receptors for TGF-
/BMP are activated by ligand binding
and phosphorylate and activate certain members of the Smad protein
family (Smad1-Smad8) as intracellular signal transducers (6, 7). The
signals for TGF-
are mediated by Smad2 and Smad3 (8, 9), whereas
Smad1, Smad5, and Smad8 are specific signal transducers for the BMP
signals (10, 11). In addition to these pathway-restricted Smads, Smad4,
a common partner Smad, is required for the functional
heterooligomerization with the pathway-restricted Smads (12, 13).
These complexes translocate into the nucleus, where they activate
transcription as coactivators and/or DNA-binding transcription factors
(14-18). In contrast to these positive transducers of TGF-
/BMP
signalings, inhibitory Smad proteins (Smad6 and Smad7) have been
identified (19-22). These Smad proteins directly bind to the
TGF-
/BMP type I receptors, consequently interfering with the
phosphorylation of the pathway-restricted Smads and thereby preventing
TGF-
/BMP signalings. As the gene expressions of Smad6 and Smad7 are
up-regulated upon activation of their own signaling pathways by
TGF-
/BMP, they are considered as negative feedback regulators of the
TGF-
/BMP signaling pathways (19-24).
and vitamin D (25-27). However, little is known
about the molecular mechanism underlying the complicated interplay. In
our recent work, we demonstrated that Smad3, a downstream component of
TGF-
signaling pathway, acts as a coactivator of vitamin D receptor
(VDR) and positively regulates the vitamin D signaling pathway (28). In
view of the complex interplay between vitamin D and TGF-
signalings,
it is likely that other downstream components of the TGF-
signaling
pathway also modulate the transactivation function of VDR. Here, we
show that Smad7 abrogates the Smad3-mediated potentiation of VDR
function, demonstrating another aspect of the molecular mechanism of
the interplay between TGF-
and vitamin D signaling. Thus, our
observations indicate that Smad proteins act as positive and negative
modulators for the potentiation of VDR function by TGF-
signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
type I receptor was as described (T
RI(TD)) (13). Full-length
human SRC-1a was inserted between the XhoI and
XbaI sites of pcDNA3 (pcDNA3-SRC-1) (28).
Full-length rat VDR was inserted between the EcoRI and
BamHI sites of the mammalian expression vector pSG5
(pSG5-VDR) (28). DEF domains of rat VDR and mouse retinoid X receptor
(RXR
) were inserted between the EcoRI and
BamHI sites of the pM vector (CLONTECH) (GAL4-VDR-AF2 (25) and GAL4-RXR-AF2). Full-length mouse Smad3 was
inserted between the EcoRI and SalI sites of the
pVP vector (CLONTECH) (VP-Smad3). Synthetic
oligonucleotides containing eight tandem copies of the 17-mer of
GAL4-DNA-binding site followed by the adenovirus E1A TATA sequence were
inserted between the HindIII and ClaI sites of
pGL3-basic vector (Promega) (17m8-luc).
-galactosidase expression vector as an internal control.
Cognate ligands were added to the medium 1 h after transfection
and at each change of medium. After 24 h of incubation with the
calcium phosphate-precipitated DNA, the cells were washed with fresh
medium and incubated for an additional 24 h. Cell extracts were
prepared by freezing and thawing and assayed for CAT after
normalization for
-galactosidase activity as described elsewhere
(28, 29).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
type I receptor, whereas Smad6 showed no effect. Under these
conditions, we confirmed that approximately equivalent levels of Smad6
and Smad7 were expressed in the cells as detected by Western blot
analysis using antibody directed against FLAG epitope (data not
shown).
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Fig. 1.
Smad7, but not Smad6, abrogates the
Smad3-mediated potentiation of VDR function. A, COS-1
cells were transfected with 3 µg of CAT reporter bearing the
GAL4-binding element (17mx2G-CAT), 5 µg of GAL4-VDR-AF2 vector, along
with 5 µg of T RI(TD), Smad3, Smad6, and Smad7 in the presence (+)
or absence (
) of 10
8 M
1,25(OH)2D3. B and C,
COS-1 cells were transfected with 3 µg of CAT reporter bearing
various vitamin D response elements (B, DR3; C,
upper panel, mouse osteopontin (O.P.) VDRE;
C, lower panel, rat osteocalcin (O.C.)
VDRE), 1 µg of full-length VDR expression vector, along with 5 µg
of T
RI(TD), Smad3, Smad6, Smad7 (shown as +), or increasing amounts
(1, 3, and 5 µg for B; 1 and 5 µg for C) of
Smad6 or Smad7 in the presence (+) or absence (
) of 10
8
M 1,25(OH)2D3. The graphs show the
fold change in CAT activity relative to those in the presence of
1,25(OH)2D3 and in the absence of exogenous
Smad proteins or T
RI(TD). The average of at least three independent
experiments are shown; error bars indicate standard
deviation.
type I receptor was remarkably abrogated when Smad7 was
co-transfected. This suppression was dependent on the dose of the
transfected Smad7 (Fig. 1B). At the maximum dose we
employed, Smad7 suppressed the enhancement of VDR function by Smad3
nearly to the control level (i.e. the level in the absence
of Smad3). In contrast, Smad6 did not alter Smad3-activated responses.
Similar suppressive effects of Smad7 were also observed with mouse
osteopontin VDRE and rat osteocalcin VDRE (30), suggesting that this
suppressive effect of Smad7 on Smad3-activated VDR transactivation does
not depend on the promoter context but is derived from the decreased
transactivation function of VDR.
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Fig. 2.
Smad7 abolishes the VDR-Smad3 complex
formation. A and B, effects of Smad6 and
Smad7 on the interaction of Smad3 with VDR were examined by a mammalian
two-hybrid system (A) and a mammalian three-hybrid system
(B). COS-1 cells were transfected with various combinations
(see "Experimental Procedures" for details) of the indicated
vectors in the presence (+) or absence ( ) of 10
8
M 1,25(OH)2D3. The
graphs show the fold change in luciferase activity relative
to those in the presence of 1,25(OH)2D3 and in
the absence of VP-Smad3, FLAG-Smad6, FLAG-Smad7, and pSG5-VDR. The
average of at least three independent experiments are shown;
error bars indicate standard deviation. C,
effects of Smad6 and Smad7 on VDR-Smad3 complex formation were analyzed
by coimmunoprecipitation (IP) using anti-FLAG antibody
followed by immunoblotting (WB) using anti-VDR antibody.
COS-1 cells were transfected with 10 µg of pSG5-VDR, 5 µg of
pcDNA3-SRC-1, 5 µg of FLAG-Smad3, and HA-Smad6 or HA-Smad7
(upper panel). The expression of proteins in extracts of
transfected cells were determined by direct Western blot analysis using
the epitope tags of each protein (lower panel).
Coimmunoprecipitation and immunoblotting were performed as described
elsewhere (28).
and vitamin D signaling pathways
by modulating VDR transactivation. We propose the model that TGF-
signaling regulates the vitamin D signaling pathway positively by the
nuclear accumulation of Smad3, and this effect is opposed by Smad7,
which presumably inhibits the nuclear accumulation of Smad3. Because
Smad7 was shown to localize also in the nucleus (31), it is possible
that Smad7 abrogates the Smad3-mediated potentiation of VDR function in
the nucleus through unknown molecular mechanism. Our results
demonstrate that relative expression levels of Smad7 to Smad3 determine
the extent of the potentiation of VDR function by TGF-
signaling.
Because TGF-
/BMP treatments are reported to alter the gene
expression of Smad3 (32) and Smad7 (23) in a cell-specific manner,
inconsistent previous studies about the effects of TGF-
on vitamin D
signaling may be due to the differences in the expression levels of the
Smad3 and Smad7 proteins in the tested cells.
/BMP signaling, together with the possibility that
there are more unknown Smad proteins, such Smad proteins could be
involved in the cross-talk between the signaling pathways mediated by
nuclear receptors and by the TGF-
/BMP cell membrane receptors.
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ACKNOWLEDGEMENTS |
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We thank Chugai Pharmaceuticals for 1,25(OH)2D3 and S. Hanazawa and A. Takeshita for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid for priority areas from the Ministry of Education, Science, Sports and Culture of Japan (to S. K.).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: Inst. of Molecular
and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku,
Tokyo 113-0034. Tel.: 81-3-5802-8632; Fax: 81-3-5684-8342; E-mail:
uskato{at}hongo.ecc.u-tokyo.ac.jp.
1
The abbreviations used are; TGF-,
transforming growth factor-
; VDR, vitamin D receptor; VDRE, vitamin
D response element; SRC-1, steroid hormone receptor coactivator 1; BMP,
bone morphogenetic protein; 1,25(OH)2D3,
1
,25-dihydroxyvitamin D3; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; HA,
hemagglutinin; T
RI(TD), TGF-
type I receptor; RXR, retinoid X receptor.
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