From the Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294
Received for publication, July 6, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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Smad2 and Smad3 are downstream transforming
growth factor- Transforming growth factor- Signal transduction by members of TGF- In a previous study (40), we have demonstrated an interaction
between Smad1 and Hoxc-8 in the BMP signaling pathway. The interaction
between Smad1 and Hoxc-8 breaks the equilibrium of Hoxc-8 binding to
its DNA binding site and results in the transcriptional activation of
OPN in response to BMP2 stimulation (40). Here we show evidence that
the DNA-binding protein Hoxa-9 interacts with Smad4, but not with Smad3
(which binds to OPN promoter), and the interaction between Smad4 and
Hoxa-9 results in the transcriptional activation of OPN in response to
TGF- Cell Lines--
Mink lung epithelial cell line Mv1Lu (ATCC,
Manassas, VA) was maintained in minimal essential medium
containing 10% fetal bovine serum and nonessential amino acids. COS-1
(ATCC) cells were maintained in Dulbecco's modified Eagle's medium
supplied with 10% fetal bovine serum.
Plasmid Constructs--
OPN promoter luciferase reporter
constructs Hox-pGL3 and mHox-pGL3, bacteria expression vectors for
GST-Smad2, GST-Smad-3, GST-Smad-4, GST-Hoxa-9, as well as
mammalian expression vectors Smad3-FLAG and Smad4-FLAG were described
previously (40). mSBE-pGL3, which bears four nucleotide alterations at
the Smad binding site in the OPN promoter Expression and Purification of GST Fusion Proteins--
BL21
bacterial cells were transformed with the GST fusion constructs
described above, and the fusion proteins were purified by
glutathione-agarose 4B (Sigma) following induction with
isopropyl- Electrophoretic Mobility Shift Assays--
A 50-bp SBE (Smad
binding element) corresponding to nucleotides Immunoprecipitation and Western Blotting--
COS-1 cells were
transfected with expression vectors as indicated in Fig. 6 using Tfx-50
according to manufacturer's description (Promega). Twenty-four hours
post-transfection, the cells were switched to Dulbecco's modified
Eagle's medium containing 0.2% fetal bovine serum for 12 h and
then treated with 10 ng/ml TGF- Transfection and Luciferase Assays--
Mv1Lu cells were
transiently transfected with the constructs indicated in Fig. 1 using a
mixture of cationic and neutral lipids (Tfx-50, Promega) as described
previously (41). When increasing amounts of expression vectors were
transfected, total DNA was kept constant by addition of PcDNA3
(Invitrogen, Carlsbad, CA). An internal control plasmid pRL-SV40 was
cotransfected to monitor the transfection efficiency. Luciferase
activities were assayed 48 h post-transfection with separate
substrates to detect the luciferase (firefly) in the
promoter-luciferase reporter plasmid and to the second luciferase
(Renilla), encoded by the pRL-SV40 vector (dual luciferase assay kit,
Promega) according to the manufacturer's directions. Values were
normalized to the renilla luciferase activity expressed from the
pRL-SV40 reporter plasmid. Luciferase values shown in the figures are
representative of transfection experiments performed in triplicate in
at least three independent experiments.
TGF- Hoxa-9 Inhibits OPN Gene Transcription--
Previous studies
showed that Hoxc-8 inhibits OPN gene transcription in BMP signaling
pathway through a functional Hox binding site adjacent to the SBE (40).
Therefore, we examined whether this Hox binding element is also
involved in TGF- Smad3 Binds to the OPN Promoter--
We then investigated if the
direct binding of Smad2 or Smad3 to OPN promoter is required. Gel shift
assays were performed using affinity-purified GST-Smad2 or GST-Smad3
fusion protein and a 50-bp DNA fragment containing SBE (
Studies have shown that the MH1 domain, but not the full length of
Smad3, is able to bind DNA (41, 42). Under our experimental conditions,
however, full-length GST-Smad3 was found to bind to OPN promoter
effectively (Fig. 3). To confirm that the full length of mammalian
cell-expressed Smad3 also bound to the SBE, COS-1 cells were
transfected with Smad3-FLAG expression vector. The transfected cells
were treated with or without TGF- Smad4 Interacts with Hoxa-9 in Cells--
Previously we have shown
that Smad1 interacts with Hoxc-8 in response to BMP stimulation (40).
To test whether TGF-
In summary, we have located a 50-bp DNA fragment (
Our previous studies have shown that Hoxc-8 is the downstream
transcription factor of the BMP signaling pathway and that BMP-2 activates OPN gene transcription through an interaction between Smad1
and Hoxc-8. The results of this study show that Hox binding element is
also involved in the TGF- (TGF-
) signaling molecules. Upon phosphorylation
by its type I receptor, Smad2 or Smad3 forms a complex with Smad4 and
translocates to the nucleus where the complex activates target gene
transcription. In the present study, we report that Smad3 binds
directly to the osteopontin (OPN) promoter and that Smad4 interacts
with the Hox protein and displaces it from its cognate DNA binding site
in response to TGF-
stimulation. In gel shift assays, the
glutathione S-transferase-Smad3 fusion protein was
found to bind to a 50-base pair DNA element (
179 to
229) from the
OPN promoter. Also, we found that both Hoxc-8 and Hoxa-9 bound to a Hox
binding site adjacent to Smad3 binding sequence. Interestingly, Smad4,
the common partner for both bone morphogenic protein and TGF-
signaling pathways, inhibited the binding of Hox protein to DNA.
FLAG-tagged Smad4 coimmunoprecipitated with HA-tagged Hoxa-9 from
cotransfected COS-1 cells, demonstrating an interaction between Smad4
and Hoxa-9. Transfection studies showed that Hoxa-9 is a strong
transcriptional repressor; it suppresses the transcription of the
luciferase reporter gene driven by a 124-base pair OPN promoter
fragment containing both Smad3 and Hox binding sites. Taken together,
these data demonstrate a unique TGF-
-induced transcription
mechanism. Smad3 and Smad4 exhibit different functions in activation of
OPN transcription. Smad3 binds directly to the OPN promoter as a
sequence-specific activator, and Smad4 displaces the transcription
repressor, Hoxa-9, by formation of Smad4/Hox complex as part of the
transcription mechanism in response to TGF-
stimulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(TGF-
)1 superfamily growth
factors regulate a diverse range of cellular functions, including proliferation, differentiation, extracellular matrix secretion, and
cell adhesion. TGF-
is also one of the most abundant of the known
growth factors stored within the bone matrix. When bone is resorbed
during remodeling, TGF-
is released and stimulates the proliferation
of precursor cells of osteoblast lineage, which induces new bone
formation (1, 2). TGF-
decreases bone resorbtion by inhibiting both
proliferation and differentiation of osteoclast precursors (3, 4) and
by inducing apoptosis of osteoclasts (5). Osteopontin (OPN), the major
noncollagenous bone matrix protein, is a secreted,
arginine-glycine-aspartate (RGD)-containing phosphorylated glycoprotein
(6, 7). OPN expression is rapidly induced by both bone morphogenic
protein (BMP) and TGF-
(8, 9). It is produced by osteoblasts (10), as well as osteoclasts (10, 11). OPN regulates the adhesion, attachment, and spreading of osteoclasts to the bone surface during bone resorbtion (12). Most recently, OPN has been demonstrated to
regulate the bone remodeling in response to mechanical stress (13), a
novel mechanism by which osteoclast function is related to mechanical
loading of the bone tissue.
superfamily is mediated by
two types of transmembrane receptors (14). Smads are the direct
substrates for kinase receptors. Upon phosphorylation, Smads
translocate to the nucleus and recruit DNA-binding protein(s) to
regulate gene expression. Smads are functionally classified into three
groups: receptor-regulated Smads (R-Smads) including Smad1, -5, and -8 for BMP (15-19) and Smad2 and -3 for TGF-
/activin signaling pathway
(20-23); co-Smad Smad4, which hetero-oligomerizes with R-Smads
(24-29); and anti-Smads, Smad6 and -7, which block signals from being
transduced into the nucleus via a mechanism of associating with type I
kinase receptors (30-33) or competing with Smad4 for pathway-specific
Smads (34). The activated type I receptors phosphorylate specific
R-Smads. Upon dissociation of the phosphorylated R-Smad from type I
receptor, it interacts with Smad4 and moves to the nucleus as a
heteromeric complex. Once in the nucleus, Smads can be recruited to
many DNA-binding proteins such as c-Jun/c-Fos, Fast-1, Fast-2, vitamin
D receptor, glucocorticoid receptor, and Hoxc-8 to regulate
transcriptional responses (24, 35, 37). It is not clear,
however, whether the Smad4-R-Smads complex still remains
associated after translocating to the nucleus (38, 39).
stimulation. Also, unlike most DNA-binding proteins interacting
with Smads that are transcriptional activators, Hoxa-9 functions as a
strong transcriptional repressor, similar to Hoxc-8.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
168 to
229 region, was
generated by standard polymerase chain reaction cloning technique using
primers
5'-TATAACGCGTCTAAATGCCATGGATAAATGAAAAGG-3' (upstream) and
5'-TATACTCGAGTACACAAAGCATTACTGA-3'(downstream) and
inserted in between MluI and XhoI sites of the
pGL3-control vector (Promega). An MluI and an
XhoI site were added to the up- and downstream primers
(bold), respectively. Mutated nucleotides are shown underlined.
HA-Hoxa-9 expression vector was constructed in a similar strategy using
a full-length Hoxa-9 cDNA vector (kindly provided by C. Largman) as
a template. The primer sequences for up- and downstream, respectively,
are: 5'-TATAGGATCCATGGCCACCACCGGGGCCCTGGGCAA-3' and
5'-TATATCTAGACGGACAGTCCTTTCTTTTTCTTGTCT-3'. A
BamHI and an XbaI restriction site (bold) was
added to the upstream and downstream primers, respectively. The
polymerase chain reaction product was cut with XbaI and
inserted between the EcoRI (blunted with S1 nuclease) and
XbaI sites of the PcDNA3-HA-Hoxc-8 (40).
-D-thiogalactopyranoside as described
previously (41).
179 to
229, and
a 25-bp Smad binding element (used in Fig. 4) corresponding to
nucleotides
203 to
228 of the OPN promoter region were
generated by annealing pairs of oligonucleotides (sense strands
are shown).
5'-CTAAATGCAGTCTATAAATGAAAAGGGTAGTTAATGACATCGTTCATCAG-3' for SBE (underlined sequences were changed to ACCCTT and GCGC, respectively, in mutant probe m-SBE), and
5'-CTAAATGCAGTCTATAAATGAAAAG-3' for Smad binding element. The probe
OPN5 used in Fig. 5 was described previously (40). These DNA
fragments were end-labeled with [
-32P]ATP using T4
polynucleotide kinase. The subsequent experiment was performed as
described previously (41). For supershift assays, anti-FLAG M2
antibody (Eastman Kodak Co.) was added to the binding reaction and
incubated for an additional 10 min at room temperature.
1 (R & D Systems, Minneapolis, MN)
for 30 min where appropriate. The cell lysates were immunoprecipitated
with anti-HA polyclonal antiserum (Babco, Berkeley, CA) and
immunoblotted with anti-FLAG M2 monoclonal antibody (Kodak) as
described previously (41). For the gel shift assay (see Fig. 4), the
cells were treated with 10 ng/ml TGF-
1 for 12 h before cells
were lysed.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Activates OPN Gene Transcription--
To investigate the
molecular basis of TGF-
-induced OPN expression, the effect of
TGF-
on OPN promoter activity was tested. Hox-pGL3 (Fig.
1A), a previously
characterized OPN promoter-luciferase reporter (40), was used. Hox-pGL3
contains a 124-bp OPN promoter fragment (nucleotides
166 to
290)
inserted upstream of SV40 promoter in the pGL3-control vector. This
124-bp fragment contains a SBE (nucleotides
217 to
222) and a well
characterized Hox binding site (TAAT, nucleotides
195 to
198)
separated by 20 base pairs. Mv1Lu cells, a TGF-
-responsive cell
line, were transiently transfected with Hox-pGL3 reporter construct.
The transfected cells were then incubated in the presence or absence of
5 ng/ml TGF-
for 48 h before the luciferase activity was
assayed. As shown in Fig. 1B, treatment of transfected cells
with TGF-
stimulated OPN promoter-reporter activity more than
5-fold. To verify whether SBE is involved in the TGF-
-induced
transcriptional activity, a shorter, mutant version of OPN
promoter-luciferase reporter was constructed (nucleotides
168 to
229) in pGL3-control vector (Fig. 1A). This 61-bp OPN
promoter reporter construct (mSBE-pGL3) bears four nucleotide
alterations in the Smad binding sequence (CAGTCT to CCATGG).
Transfection study showed that mutation of the Smad binding site
greatly reduced the basal and TGF-
-induced reporter activity. These
results suggested that the TGF-
-induced OPN gene transcription is
mediated via the SBE and that the direct binding of Smad3 to OPN
promoter might be required for its gene activation.
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Fig. 1.
TGF- activates OPN
gene transcription. A, diagram of OPN promoter-reporter
constructs showing wild-type (Hox-pGL3) and mutated Smad binding site
(mSBE-pGL3). B and C, Hox-pGL3, mSBE-pGL3
(B), or mHox-pGL3 (C) was transfected into Mv1Lu
cells alone or cotransfected with a Hoxa-9 expression vector
(HA-Hoxa-9). The cells were then treated with or without 5 ng/ml of
TGF-
for 48 h, and the luciferase activities were measured.
Luciferase activities are normalized to pRL-SV40 and are presented as
relative light units of mean ± S.D. of triplicates.
signaling. A Hoxa-9 expression vector (HA-Hoxa-9)
was cotransfected with Hox-pGL3, mSBE-pGL3, or mHox-pGL3 reporter
construct. As shown in Fig. 1B, coexpression of a small
amount of Hoxa-9 (50 ng of HA-Hoxa-9 plasmid DNA) significantly
inhibited the promoter reporter activities of both wild-type (Hox-pGL3)
and SBE mutated (mSBE-pGL3) constructs in the presence or absence of
TGF-
treatment. However, Hoxa-9 was unable to inhibit
TGF-
-induced reporter activity when hox binding site was mutated
(mHox-pGL3, Fig. 1C). These results indicate that Hoxa-9
negatively regulates TGF-
-induced OPN gene transcription via this
hox site. Binding of Hoxa-9 to this Hox binding element was examined in
gel shift assays. As shown in Fig. 2,
GST-Hoxa-9 bound to this DNA element (lane 3). The
specificity of the binding was demonstrated by competition shift assays
using unlabeled specific probe (lanes 4-6), as well as
nonspecific probe (lanes 7-9). The GST itself did not bind
to this probe (lane 2). Lane 1 is the probe with
no added protein. These results suggest that the inhibitory effect of
Hoxa-9 on the OPN promoter is mediated via the Hox binding site.
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Fig. 2.
Smad4 inhibits Hoxa-9 binding to SBE.
Gel mobility shift assay showing an interaction between GST-Smad4 and
GST-Hoxa-9. Labeled SBE was used as a probe and incubated with 0.2 µg
of GST-Hoxa-9 (lanes 3-9 and 11-13).
Competition assay using unlabeled SBE (lanes 4-6) and probe
8 (lanes 7-9) as specific and nonspecific probe,
respectively. Lane 10 contained 2 µg of GST-Smad4; in
addition to GST-Hoxa-9, lanes 11-13 also contained 2, 4, and 8 µg, respectively, of GST-Smad4. Lane 1 contained no
protein; lane 2 contained 2 µg of GST protein.
179 to
229). Fig. 3A shows that
GST-Smad3 effectively bound to SBE (lane 3), while GST-Smad2
bound weakly (data not shown). Thus, we focused attention on Smad3. The
specificity of Smad3 binding was shown in a competition shift assay.
Unlabeled SBE dose-dependently competed for Smad3 binding
(lanes 4-6), and a 100-fold excess of it completely
competed off Smad3 binding (lane 6). In contrast, an equal
molar amount of a nonspecific probe did not show such an effect
(lanes 7-9). GST-Smad4, the common partner for all
pathway-specific Smads, did not bind to SBE (lane 10), nor
did it affect Smad3 binding activity (lanes 11-13). To
validate the DNA sequence in the SBE that confers the binding of Smad3,
the core sequences CAGTCT for SBE and TAAT for Hox protein binding
sites were mutated to ACCCTT and GCCG, respectively. As shown in Fig.
3B, mutations of SBE abolished binding of Smad3 (Fig.
3B, compare lane 7 to wild-type probe, lane
3).
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Fig. 3.
Smad3 specifically binds to OPN
promoter. Gel mobility shift assay using GST-Smad3 fusion protein
and a labeled 49-bp DNA fragment covering the nucleotide 180 to
229
region of the OPN promoter sequence (SBE) as a probe. SBE contains a
Smad3 and a Hox binding site. A, Smad3 specifically binds to
SBE. Lane 1 contained no protein; lane 2 contained 2 µg of GST protein; lanes 3-9 and
11-13 contained 2 µg of GST-Smad3. For competition
assays, a 25-, 50-, and 100-fold excess of unlabeled specific, SBE
(lanes 4-6), and nonspecific probe, probe 8 (lanes
7-9), respectively, were added. Lane 10 contained 2 µg of GST-Smad4; lanes 11-13 contained 2, 4, and 8 µg,
respectively, of GST-Smad4 in addition to 2 µg of GST-Smad3.
B, mutations of Smad and Hox binding sites in SBE abolish
both Smad3 and Hoxa-9 binding activities. To confirm the authenticity
of Smad3 and Hoxa-9 binding sites, the CAGTCT and TAAT in wild-type SBE
were mutated to ACCCTT and GCGC (m-SBE), respectively, and the mobility
shift assay was performed. Lanes 1-4, wild-type SBE;
lanes 5-8, m-SBE. Lanes 1 and 5 contained no protein; lanes 2 and 6 contained 2 µg of GST; lanes 3 and 7 contained 2 µg of
GST-Smad3; and lanes 4 and 8 contained 0.5 µg
of GST-Hoxa-9.
(10 ng/ml) for 12 h before
lysates were prepared, then gel shift assays were performed using the
SBE probe. Western blot demonstrated that Smad3 was only expressed in
the cells transfected with the Smad expression plasmid (Fig.
4B). As shown in Fig.
4A, a shifted band is seen from both TGF-
-treated and
untreated lysates of Smad3-FLAG transfected cells (lanes 4 and 5). In contrast, the lysates from control cells did not
yield such a band (lanes 2 and 3). To verify the
presence of FLAG-tagged Smad3 in this new complex, anti-FLAG M2
antibody was added to the binding reaction. The addition of antibodies
disrupted the newly formed DNA-protein complex (lanes 8 and
9), indicating that the complex contained FLAG-tagged Smad3. Lane 1 contained no protein, demonstrating the quality of
the probe.
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Fig. 4.
Overexpressed Smad3 binds to DNA.
A, COS-1 cells were transfected with Smad3-FLAG expression
vector and treated with or without 10 ng/ml of TGF- for 12 h
before cell lysates were prepared. Gel shift assay was performed using
a 25-bp DNA fragment corresponding to nucleotide
203 to
228 of the
OPN promoter region and 8 µg of total lysate protein. Lane
1, free probe; lanes 2 and 3, lysate from
untransfected cells; lanes 4 and 5, lysate from
Smad3-FLAG-transfected cells. Note: cells for lanes 3 and
5 were treated with TGF-
. Lanes 6-7 and
8-9 are same as lanes 2-3 and 4-5,
respectively, except that anti-FLAG M2 antibodies were added to the
binding reactions for lanes 6-9. B, Western blot
showing the levels of Smad3-FLAG expression in COS-1 cells. Equal
amounts of cell lysates, corresponding to samples of lanes
2-5 in (A), were separated on SDS-polyacrylamide gel
electrophoresis, electrotransferred onto polyvinylidene
difluoride membrane, and immunoblotted with anti-FLAG M2
antibody.
regulatory Smads interact with Hoxa-9 in a way
similar to the interaction between Smad1 and Hoxc-8 in regulation of
gene expression, GST-Smad2, -Smad3, or -Smad4 was coincubated with
GST-Hoxa-9 in the binding reaction for gel shift assays. Neither
GST-Smad2 nor GST-Smad3 interacted with GST-Hoxa-9 (Fig.
5, lanes 4 and 5).
GST-Smad4, which did not bind to SBE, inhibited Hoxa-9 binding (Fig. 5,
lane 6; Fig. 2, lanes 10-13). Addition of Smad2
or Smad3 did not enhance the inhibitory effect of Smad4 on Hoxa-9
binding (Fig. 5, lanes 7 and 8). Furthermore, the
interaction of TGF-
regulatory Smads with Hoxa-9 was examined in
mammalian cells. The carboxyl-terminally FLAG-tagged Smad3/Smad4
(Smad3/Smad4-FLAG) and amino-terminally HA-tagged Hoxa-9 (HA-Hoxa-9)
were coexpressed in COS-1 cells in the presence or absence of TGF-
(10 ng/ml) stimulation. Whole cell extract immunoprecipitations were
performed using anti-HA antibodies followed by Western blot with
anti-FLAG M2 antibodies. In the presence of coexpressed Hoxa-9, Smad4
was coimmunoprecipitated (Fig. 6,
lanes 5-8, top). In contrast, Smad3 did not
interact with Hoxa-9 (lanes 3 and 4), which is
consistent with gel shift assay results (Fig. 5). TGF-
stimulation
neither enhanced the interaction between Smad4 and Hoxa-9 (compare
lane 5 with lane 6), nor did it induce the
formation of Hoxa-9-Smad3/4 complex (compare lane 7 with
lane 8). These results are also in agreement with the
observations in the gel shift assays that Smad4, but not Smad3 or
Smad2, interacts with Hoxa-9 (Fig. 5). Since Smad3 and Smad4 are known
to form a heteromeric complex and translocate to the nucleus in
response to TGF-
stimulation (36, 42-43), it seems that both
Smad3 and Hoxa-9 would compete for binding to Smad4. However, in the
presence of Hoxa-9, the association of Smad3 with Smad4 was not seen
(Fig. 6, lanes 7 and 8). Apparently, Smad4
prefers to interact with Hoxa-9. This could be due to a higher affinity
between Smad4 and Hoxa-9 or another specific mechanism. Nevertheless,
release of Smad3 as a result of Smad4/Hoxa-9 interaction allows Smad3
to bind OPN promoter as a part of the transcription mechanism.
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Fig. 5.
Smad4, but not Smad2 or Smad3, inhibits
Hoxa-9 binding to DNA. Gel mobility shift assays showing a
specific interaction between GST-Smad4 and GST-Hoxa-9. Labeled OPN5
probe was incubated with 0.5 µg of GST-Hoxa-9 (lanes 3-8)
and 2 µg each of GST-Smad2 (lane 4), GST-Smad3 (lane
5), GST-Smad4 (lane 6), or GST-Smad2 plus GST-Smad4
(lane 7), GST-Smad3 plus GST-Smad4 (lane
8). Lane 1 contained no protein; lane
2 contained 2 µg of GST protein.
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Fig. 6.
Smad4 interacts with Hoxa-9 in cells.
Coimmunoprecipitation assay showing an in vivo interaction
between Smad4 and Hoxa-9. FLAG-tagged Smad3/Smad4 (Smad/Smad4-FLAG) and
HA-tagged Hoxa-9 (HA-Hoxa-9) were coexpressed in COS-1 cells with or
without TGF- stimulation. Whole cell extracts immunoprecipitations
were performed using anti-HA antibodies followed by Western blotting
utilizing anti-FLAG M2 antibodies (top panel). Lanes
1 and 2, lysates from PcDNA3 transfected cells;
lanes 3 and 4, Smad3-FLAG coexpressed with
HA-Hoxa-9; lanes 5 and 6, Smad4-FLAG coexpressed
with HA-Hoxa-9; lanes 7 and 8, Smad3/4-FLAG
coexpressed with HA-Hoxa-9. In lanes 2, 4,
6, and 8, cells were treated with 10 ng/ml of
TGF-
for 30 min before lysates were prepared. Lanes 9 and
10 (Western), whole cell lysates from Smad3-FLAG and
Smad4-FLAG, respectively, transfected cells showing the molecular sizes
of both proteins. The bottom two panels are Western blots
showing the expression levels of Smad3/4-FLAG and HA-Hoxa-9.
179 to
229) in
the OPN promoter to be responsible for mediating TGF-
-induced OPN
gene transcription. It is interesting to note that this 50-bp fragment
contains a functional Smad3 and a Hox binding sites. Mutations of these
two sites abolished both Smad3 and Hoxa-9 binding activities (Fig.
3B). Disruption of the Smad binding site reduced basal
promoter activity significantly, but did not abolish the TGF-
-induced OPN gene transcription (Fig. 1). This is likely due to
the interactions of endogenous Smad4 with Hox proteins, since the
endogenous Smad2 and -3 will associate with Smad4 and take it into the
nucleus upon TGF-
stimulation where Smad4, in turn, interacts with
and dislodges Hox proteins from their cognate binding sites. As shown
in our previous studies, mutation of the Hox binding site in Hox-pGL3
reporter construct elevates the basal promoter activity and
de-represses the inhibitory effects of Hox proteins on this
promoter-luciferase reporter (40).
-induced OPN gene transcription via an
interaction between Hoxa-9 and Smad4 and that Smad3 directly binds to
the OPN promoter in response to TGF-
. It is well established that
phosphorylated Smad3 associates with Smad4 and translocates to nucleus
as a complex. Smad4 is not required for nuclear accumulation but for
the formation of functional complexes. However, it is not clear whether
Smad4 and Smad3 still remain as complex in participating transcription
after translocation to nucleus. Our data indicate that Smad4 and Smad3
can function separately in activation of OPN gene transcription. While
Smad4 displaces transcriptional repressor, Hoxa-9, from its binding
site, Smad3 directly binds to OPN promoter as a sequence specific activator.
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ACKNOWLEDGEMENTS |
---|
We are grateful to C. Largman for kindly providing the Hoxa-9 cDNA clone and to Rik Derynck for the Smad2, -3, and -4 expression vectors.
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FOOTNOTES |
---|
* This work was supported in part by an Arthritis Foundation Investigator Grant (to X. S.), National Institutes of Health Grant DK53757 (to X. C.), and Department of Army Grant DAMD17-00-1-0066 (to X. C.).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: University of Alabama
at Birmingham, 1670 University Blvd., Volker Hall/G002, Birmingham, AL
35294. Tel.: 205-934-0162; Fax: 205-934-1775; E-mail: cao@path.uab.edu.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M005955200
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
OPN, osteopontin;
SBE, Smad binding
element;
bp, base pair;
GST, glutathione S-transferase;
HA, hemagglutinin;
BMP, bone morphogenic protein.
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REFERENCES |
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