From the Department of Pathology, University of Alabama School of Medicine, Birmingham, Alabama 35294 and the § Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78284
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ABSTRACT |
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Bone morphogenetic proteins (BMP) transduce their
signals into the cell through a family of mediator proteins known as
Smads. Upon phosphorylation by the BMP receptors, Smad1 interacts with Smad4 and translocates into the nucleus where the complex recruits DNA-binding protein(s) to activate specific gene transcription. However, the DNA-binding protein(s) involved in BMP signaling has not
been identified. Using a yeast two-hybrid approach, we found that Smad1
interacts with Hoxc-8, a homeodomain transcription factor. The
interaction between Smad1 and Hoxc-8 was confirmed by a "pull-down"
assay and a co-immunoprecipitation experiment in COS-1 cells.
Interestingly, purified Smad1 inhibited Hoxc-8 binding to the
osteopontin Hoxc-8 site in a concentration-dependent manner. Transient transfection studies showed that native osteopontin promoter activity was elevated upon BMP stimulation. Consistent with
the gel shift assay, overexpression of Hoxc-8 abolished the BMP
stimulation. When a wild type or mutant Hoxc-8 binding element was
linked to an SV40 promoter-driven reporter gene, the wild type but not
the mutant Hoxc-8 binding site responded to BMP stimulation. Again,
overexpression of Hoxc-8 suppressed the BMP-induced activity of the
wild type reporter construct. Our findings suggest that Smad1
interaction with Hoxc-8 dislodges Hoxc-8 from its DNA binding element,
resulting in the induction of gene expression.
Transforming growth factor- It has been suggested that homeobox genes play a role in downstream
events in BMP signaling (13, 14). In vertebrates, there are 39 Hox transcription factor genes organized into four separated
chromosome clusters, which play critical roles in the patterning of
vertebrate embryonic development (15). These 39 genes are subdivided
into 13 paralogous groups on the basis of duplication of an ancestral
homeobox cluster during evolution, sequence similarity, and position
within the cluster (16). Each paralog group has been demonstrated to be
responsible for the morphogenesis of a particular embryonic domain or
structure (15).
Hoxc-8, as one of the three members of paralog VIII, is
predominantly expressed at a high level in the limbs, backbone, and spinal cord in early mouse embryos (17, 18). Null mutant mice showed
that Hoxc-8 is expressed in the neuron, chondrocyte, fetal liver, and
adult bone marrow (19, 20). Bending and fusion of the ribs, anterior
transformation of the vertebrae, and abnormal patterns of ossification
in the sternum were observed in adult Hoxc-8 null mice (20). Studies
published recently demonstrated that tissue-specific overexpression of
a Hoxc-8 transgene inhibits chondrocyte maturation and stimulates
chondrocyte proliferation (21). The other two members in the Hox VIII
group are Hoxb-8 and Hoxd-8. Hoxb-8 has been shown to activate the
Sonic hedgehog gene, an essential mediator in forelimb development (22,
23), whereas generalized expression of Hoxd-8 modifies
Drosophila anterior head segments (24).
Despite the fact that homeobox genes are DNA-binding proteins, little
has been learned about their natural DNA response elements and role in
transcription (25). In the current study, we report that Smad1
interacts with Hoxc-8, and this interaction specifically activates the
osteopontin gene transcription in response to BMP stimulation. Our data
suggest that Hoxc-8 functions as a transcription repressor and that the
interaction of Smad1 with Hoxc-8 dislodges Hoxc-8 binding from its
element resulting in initiation of gene transcription.
Two-hybrid Library Screening--
A full-length Smad1 coding
sequence from pBluescript-Smad1(9) was cloned into
SalI/PstI sites of pGBT9
(CLONTECH) to generate the pGBT9/Smad1 bait
plasmid. The human U2 OS osteoblast-like pACT2 cDNA library was
screened according to the manufacturer's instruction
(CLONTECH). To confirm the interaction between
Hoxc-8 and Smad1, a full-length mouse Hoxc-8 cDNA (18) was
subcloned into pACT2 vector (CLONTECH) between the
XhoI and EcoRI sites. The pACT/Hoxc-8 was
co-transformed with pBGT9/Smad1 into Y190, and the colonies were
assayed for the production of Expression and Purification of Glutathione S-transferase (GST)
Fusion Proteins--
GST fusion constructs of GST-Smad1 and -Smad3
were generated by restriction digest of pGBT-Smad1
(SalI/HindIII) and pCMV5-Smad3 (9)
(BamHI/SalI) and subsequently inserted into the
SalI/HindIII and BamHI/SalI
sites of the pGEX-KG vector, respectively. GST-Smad2 and -Smad4 were
digested with EcoRI/SalI from pCMV5-Smad2 and pCMV5-Smad4 (9) and inserted into the EcoRI/SalI
sites of the pGEX-5X-2 and pGEX-5X-1 vector (Amersham Pharmacia
Biotech), respectively. The GST-Hoxc-8 and GST-Hoxa-9 (26) were
amplified by polymerase chain reaction using high fidelity
Pfu-Turbo DNA polymerase (Stratagene) and cloned in the
BamHI/EcoRI and BamHI/XbaI
sites of the pGEX-KG vector, respectively. The GST-Msx-1 and -Msx-2
expression plasmids (27) were provided by Dr. C. Abate-Shen (Center for
Advanced Biotechnology and Medicine, Piscataway, NJ). The GST
constructs described above were transformed into BL21. The expression
and purification of the fusion proteins were performed as described (28).
GST Pull-down Assay--
[35S]Methionine-labeled
Hoxc-8 protein was synthesized using the TNT-coupled transcription and
translation system (Promega) with linearized pBluescript-Hoxc-8 plasmid
according to the manufacturer's instruction. The production of labeled
protein was confirmed by SDS-polyacrylamide gel electrophoresis. An
equivalent amount (1 µg) of purified GST or GST-Smad1 fusion protein
was preincubated with 35S-labeled Hoxc-8 protein (5 µl)
for 30 min on ice. Following the addition of GST-agarose, the samples
were incubated for another 30 min at 4 °C. The agarose beads were
washed four times in a phosphate-buffered saline, 0.1% Triton X-100
solution, and bound proteins were eluted by boiling in 2× SDS buffer
for 5 min before loading onto 10% SDS-polyacrylamide gel electrophoresis.
Immunoprecipitation and Western Blot--
HA-tagged Hoxc-8 was
subcloned from pACT2/Hoxc-8 into a mammalian expression vector
pcDNA3 (Invitrogen) at BglII/BamHI and XhoI. Expression vectors for FLAG-tagged Smad1 and Smad4
were provided by Dr. Rik Derynck (University of California, San
Francisco, CA). Expression plasmids for constitutively active BMP type
IA (ALK3) and IB receptors (ALK6) (7) were provided by Dr. Jeffrey L. Wrana (Hospital for Sick Children, Toronto, Canada). COS-1 cells were
transfected with expression constructs as indicated in Fig.
2B using Tfx-50 according to the manufacturer's
description (Promega). Cells were lysed 48 h
post-transfection, and lysates were immunoprecipitated with anti-HA
antiserum (Babco) and immunoblotted with anti-FLAG M2 (Eastman Kodak)
as described (7).
Gel Shift Assay--
Gel shift assays were performed as
described previously (29). In brief, DNA fragments OPN-1, OPN-2, and
OPN-3 were generated by polymerase chain reaction using primers
specific for the osteopontin promoter. The double-stranded oligomers
were created by annealing the pairs of synthetic
oliogonucleotides (only top strands are shown) as follows:
5'-CATGACCCCAATTAGTCCTGGCAGCA-3' (probe-M); 5'-CCTTTCCTTATGGATCCCTG-3'
(OPN-4); 5'-GGTAGTTAATGACATCGTTCATCAG-3'(OPN-5); 5'-GGTAGTGCCGGACATCGTTCATCAG-3'(mOPN-5); and
5'-GACATCGTTCATCAGTAATGCTTTG-3' (OPN-6). Mutated nucleotides in
mOPN-5 are bolded. These DNA fragments were radiolabeled by
T4 polynucleotide kinase and
[ Transfection--
The osteopontin promoter from region Yeast Two-hybrid Library Screening--
To investigate the
transcription mechanism in BMP-induced gene activation, we used a yeast
two-hybrid system to identify transcription factors that interact with
Smad1. An intact Smad1 cDNA fused with the Gal4 DNA-binding domain
was used as a bait plasmid to screen a human U-2 OS osteoblast-like
cell cDNA library constructed in the pACT2 plasmid. After two
rounds of screening, we obtained 25 positive clones. DNA sequence
analysis identified one clone as Hoxc-8 and two clones as Smad4.
Because our objective is to identify downstream transcription factors
in the BMP signaling pathway and Hoxc-8 is a homeodomain DNA-binding
protein, we chose the Hoxc-8 cDNA clone for further study. Cloning
of Smad4 provided a positive control for the two-hybrid library
screening because the interaction between Smad1 and Smad4 is known. The
other 22 clones were not characterized.
The initial Hoxc-8 cDNA clone (Fig.
1B, clone 19) encodes amino
acids 68-237 of a 242-amino acid Hoxc-8 protein. Fig. 1A
shows the growth properties of the two-hybrid clones, suggesting that there is an interaction between Smad1 and Hoxc-8 in vivo.
The yeast bearing both Smad1 and Hoxc-8 plasmids grew on medium
deficient in Trp, Leu, and His. The interaction between Hoxc-8 and
Smad1 was further confirmed with a Smad1 Interacts with Hoxc-8 in Vitro and in COS-1 Cells--
The
interaction between Smad1 and Hoxc-8 was examined in an in
vitro pull-down experiment using
[35S]methionine-labeled Hoxc-8 and purified GST-Smad1 or
GST alone. As shown in Fig.
2A, Hoxc-8 was precipitated
with the purified GST-Smad1 fusion protein (lane 3) but not
with GST alone (lane2), demonstrating a direct interaction between the
two proteins in vitro.
BMP-2 stimulates phosphorylation of Smad1, and phosphorylated Smad1 in
turn binds to Smad4 and takes the complex into the nucleus. It is of
interest whether Smad1, Smad4, or the complex of Smad1 and Smad4 also
interacts with Hoxc-8 in cells. COS-1 cells were transiently
co-transfected with expression plasmids for FLAG-Smad1, FLAG-Smad4,
HA-Hoxc-8, and/or constitutively active BMP type IA receptor ALK3
(Q233D). The cell lysates were immunoprecipitated with anti-HA antibody
and immunoblotted with anti-FLAG antibody. Fig. 2B
demonstrates that Smad1 (lane 3), Smad4 (lane 5)
or both (lane 7) were co-immunoprecipitated with HA-Hoxc-8
in cells. Co-transfection of ALK3 (Q233D) enhanced the interaction of
Smad1 (lane 4) or Smad4 (lane 6) with Hoxc-8.
However, ALK3 (Q233D) did not significantly enhance the interaction of
Smad1 and Smad4 complex with Hoxc-8 (lane 8). These results
show both Smad1 and Smad4 interact with Hoxc-8 in COS-1 cells with or
without BMP stimulation, indicating that the phosphorylation of Smad1
is not required for its interaction with Hoxc-8. If this is the case,
the BMP-dependent regulation of the interaction is inherent
in the intracellular localization of the proteins. Hox proteins are
homeodomain transcription factors localized in the nucleus (30),
whereas both Smad1 and Smad4 are cytoplasmic (5). It is likely that the
interaction occurs only when Smad1 or the complex translocates to
nucleus upon its phosphorylation induced by BMP receptors.
Osteopontin Promoter Contains a Hoxc-8 Binding Element--
To
examine the effect of the interaction between Hoxc-8 and Smad1 on
Hoxc-8 DNA binding activity, we turned our attention to BMP-2 inducible
genes. Putative Hox binding sites that have served as markers for
osteogenic differentiation were found in four BMP-2 inducible genes,
including bone sialoprotein, osteopontin, osteonectin, and osteocalcin
(3, 31). These genes have served as markers for osteoblast
differentiation. The osteopontin promoter was examined for this purpose
because its mRNA expression is rapidly induced in response to BMP-2
treatment in C3H10T1/2 mesenchymal cell (3). Five putative Hox binding
sites with a core sequence of Tt/aAT (32) were identified within the
first 382 base pairs of the 5'-flanking region in the osteopontin gene
(Fig. 3A). When a 212-base
pair DNA fragment from
The specificity of the Hoxc-8 binding to the DNA was determined by a
gel shift competition assay. Unlabeled Hoxc-8 DNA binding element
inhibited the shifted band in a concentration dependent manner (Fig.
3D, lanes 3-5) in which a 100-fold excess of the specific cold probe eliminated the Hoxc-8 binding, whereas a 100-fold excess of the Msx-2 DNA binding element (33) did not (Fig.
3D). Msx-2 is a homeodomain-containing protein, but it does
not belong to the HOX family. The Msx-2 DNA binding element was
identified from the osteocalcin promoter, and its flanking regions of
the core sequence is different from Hoxc-8 binding site.
There are three TAAT and two TTAT putative Hox sites identified from
the osteopontin promoter. Hoxc-8 binds to only one of the TAAT core
sequences ( Smad1 Inhibits Binding of Hox Proteins to DNA--
Purified
GST-Smad1 was examined for the effect of its interaction with Hoxc-8 on
Hoxc-8 DNA binding activity. When GST-Hoxc-8 protein and its DNA
binding element (OPN-5) were incubated with increasing amounts of
GST-Smad1 protein, the binding of Hoxc-8 to the DNA probe was inhibited
in a concentrationdependent manner (Fig.
4A, lanes 5-7).
The same amount of GST protein did not have an effect on Hoxc-8 binding
activity (Fig. 4B, lane 4). These results suggest
that the interaction of Smad1 with Hoxc-8 dislodges Hoxc-8 from its
response element.
Because the signaling networks of the TGF-
To estimate the relative strength of the interactions between the Smads
and homeodomain proteins, the same amounts of Hoxc-8 and Hoxa-9 or
Msx-1 and Msx-2 proteins were used in each of the gel shift assays with
a fixed amount of different Smad proteins (Fig. 4, B and
C). Smad1 and Smad4 inhibited both Hoxc-8 and Hoxa-9 binding, and the inhibition was enhanced when both Smad proteins were
added together (Fig. 4B, lanes 7 and
14). In contrast, neither Smad2 nor Smad3 interacted with
these two Hox proteins. Fig. 4C showed that neither of the
Msx proteins interacted with any of the four Smad proteins. GST did not
affect Hox or Msx protein binding (Fig. 4B, lanes
4 and 11, and Fig. 4C, lanes 4 and 10). The homeodomain, a well conserved DNA binding
motif, is the region highly conserved between Hoxc-8 and Hoxa-9,
suggesting that Smad1 interacts with other Hox proteins involved in BMP signaling.
Hox Binding Site Mediates BMP-induced Transcription--
To
examine whether the Hoxc-8 binding site functions as a BMP response
element, we cloned a 266-base pair osteopontin promoter fragment
containing the Hoxc-8 binding site into the pGL3-basic luciferase
reporter vector to generate an OPN-266 reporter plasmid (Fig.
5A). Transfection of the
OPN-266 construct in C3H10T1/2 mesenchymal cells showed that the
reporter activity was stimulated moderately when Smad1 or Smad4
expression plasmids were co-transfected. The luciferase activity was
significantly enhanced when the OPN-266 reporter construct was
co-transfected with ALK3 (Q233D), Smad1, and Smad4 expression plasmids.
Furthermore, the ALK3 (Q233D)-induced transcriptional activity was
completely abolished when Hoxc-8 was overexpressed (Fig.
5B).
To further define the transcription activity of the Hoxc-8 binding
site, we linked a shorter osteopontin promoter fragment containing the
Hoxc-8 binding site to a luciferase reporter vector under the control
of the SV40 promoter (Hox-pGL3, Fig. 5A). When the Hox-pGL3
construct was co-transfected in C3H10T1/2 cells with ALK3 (Q233D) or
ALK6 (Q203D), luciferase reporter activity was induced more than 13- and 11-fold, respectively. Most importantly, overexpression of Hoxc-8
suppressed the ALK3 (Q233D)-induced or ALK6 (Q203D)-induced reporter
activity (Fig. 5C). These results suggest that the Hox
binding site mediates BMP signaling and that Hoxc-8 functions as a
transcription repressor. In comparison with the osteopontin native
promoter, the Hox-pGL3 construct does not require overexpression of
Smad1 and -4 in responding to BMP stimulation. This is an SV40
promoter-driven construct with a much shorter osteopontin promoter
fragment, which does not contain many other transcription elements like
the native osteopontin promoter construct.
To validate whether the Hoxc-8 site mediates BMP signaling, we mutated
the core nucleotides of the Hoxc-8 binding site from TAAT to GCCG to
create mHox-pGL3 (Fig. 5A). Transfection of the mutant
construct, mHox-pGL3, completely abolished the ALK3 (Q233D)-induced or
ALK6 (Q203D)-induced reporter activity and eliminated Hoxc-8 inhibition
in C3H10T1/2 cells (Fig. 5D). These results confirm that the
osteopontin Hox binding site is a BMP response element.
Several Smad downstream transcription factors have been characterized
in the TGF-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(TGF-
)1-related molecules,
or BMPs, regulate embryonic development, vertebral patterning, and mesenchymal cell differentiation (1, 2). BMP-2 and -4 have been
identified as bone inductive growth factors and are important signaling
molecules during the development of the skeleton in vertebrates (1, 3,
4). Signal transduction in the TGF-
superfamily requires the
interaction of two types of serine/threonine transmembrane receptor
kinases (5). The signaling is mediated by direct phosphorylation of
Smad proteins. Specifically, Smad2 and Smad3 are phosphorylated by
TGF-
and activin receptors (5, 6), whereas phosphorylation of Smad1
is induced by BMPs (7, 8). Upon phosphorylation, these Smads interact
with a common partner, Smad4, which then translocates to the nucleus
where the complexes recruit DNA-binding protein(s) to activate specific gene transcription (5, 7, 9). The downstream DNA-binding proteins in
the TGF-
signaling pathway, such as Fast-1, Fast-2, and TFE3, have
been reported (10-12). However, little is known about the downstream
DNA-binding protein(s) beyond Smad1 in the BMP signal transduction machinery.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-galactosidase using both filter lift
and liquid assays.
-32P]ATP.
266 to
1 relative to the transcription start site was amplified by
polymerase chain reaction from CH10T1/2 cell genomic DNA and cloned
into SmaI and XhoI sites of the pGL3-basic vector
(Promega) to generate a luciferase reporter construct (OPN-266).
Hox-pGL3 reporter bearing the Hoxc-8 binding site (
290 to
166) was
constructed using the same strategy but was put into the pGL3-control
vector (Promega). The Hox recognition core TAAT was replaced with GCCG
in Hox-pGL3 by polymerase chain reaction to create mutant Hox-pGL3
(mHox-pGL3). C3H10T1/2 cells (2.5 × 105 cells/60-mm
dish) were transfected using Tfx-50 with 0.5 µg of luciferase
reporter plasmid (OPN-266, Hox-pGL3, or mHox-pGL3) and different
expression plasmids as indicated. Total DNA was kept constant by the
addition of pSV-
-galactosidase plasmid. Luciferase activities were
assayed 48 h post-transfection using the dual luciferase assay kit
(Promega) according to the manufacturer's direction. Values were
normalized with the Renilla luciferase activity expressed
from pRL-SV40 reporter plasmid. Luciferase values shown in the figures
are representative of transfection experiments performed in triplicate
in at least three independent experiments.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-galactosidase filter lift assay (data not shown) and quantified by a liquid
-galactosidase assay (Fig. 1B). When the full length of Hoxc-8 fused with the
Gal4 transcriptional activation domain was tested in the two-hybrid system, it showed an interaction similar to clone 19. The assays of
both the empty prey vector (pACT2) with Smad1 in the bait plasmid and
the empty bait vector (pGBT9) with full-length Hoxc-8 in the prey
vector showed very little activity. Compared with the interaction between Smad1 and Smad4, the interaction of Smad1 with Hoxc-8 is weaker
in the yeast two-hybrid
-galactosidase assay (Fig. 1B).
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Fig. 1.
Specific interaction of Smad1 with Hoxc-8 in
a yeast two-hybrid system. A, growth properties of
two-hybrid clones. The interaction was assayed in a yeast strain, which
requires His, Leu, and Trp to grow. pGBT9-Smad1 and pACT2-Hoxc-8
plasmids carry Trp and Leu as their selective markers, respectively.
The interaction between Smad1 and Hoxc-8 enables the yeast to
synthesize His. Only clones bearing both pGBT9-Smad1 and pACT2-Hoxc-8
plasmids grew in medium lacking His, Leu, and Trp. All assays were done
in medium containing 45 mM 3-aminotriazole
(3-AT), which inhibits growth because of nonspecific
interaction. B, -galactosidase liquid assay for
two-hybrid interaction.
-Galactosidase activities for yeast bearing
plasmids as indicated were plotted.
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Fig. 2.
Interaction of Smad1 with Hoxc-8 in
vitro and in vivo. A, specific
interaction of Smad1 with Hoxc-8 in vitro. Hoxc-8 protein
was labeled with [35S]methionine by in vitro
translation and incubated with purified GST-Smad1 or GST-protein.
Samples were subsequently incubated with GST-Sepharose, washed, eluted
in SDS buffer, and separated on 10% SDS-polyacrylamide gel
electrophoresis gel. B, the interaction of both Smad1 and 4 with Hoxc-8 in vivo. FLAG-tagged Smad1 and -4 and HA-tagged
Hoxc-8 were co-transfected with or without ALK3 (Q233D). Cell lysates
were immunoprecipitated by anti-HA antibody, and the resulting
complexes were analyzed by Western blotting with anti-FLAG antibody.
The expression levels of Smad1 and -4 were shown by Western blot with
anti-FLAG antibody (middle panel) and of Hoxc-8 with anti-HA
antibody (bottom panel).
382 to
170 (OPN-1 in Fig. 3A)
containing all five putative Hox sites was used for a gel shift assay
with purified GST-Hoxc-8 protein, one shifted band (Fig. 3B,
lane 3) was observed. This band was not present in
lane 1, containing probe only, or in lane 2,
containing probe with GST (Fig. 3B). This result indicates
that there is only one Hoxc-8 binding site in this osteopontin promoter
fragment. Further gel shift assays with shorter probes (OPN-2 and OPN-3
in Fig. 3A) indicated that OPN-2 contains this Hoxc-8
binding element (Fig. 3B, lane 6). When three
single putative Hox binding probes (OPN-4, -5, and -6, Fig.
3A) were used, Hoxc-8 only bound to OPN-5, located at
206
to
180 (Fig. 3C, lane 8). Neither GST alone nor
GST-Smad1 fusion protein could bind to any of the probes used in this
series of gel shift assays (Fig. 3, B, lanes 2,
5, and 8, and C, lanes 2,
3, 6, 7, 10, and 11). When the TAAT core sequence
of Hoxc-8 binding site in OPN-5 was mutated to GCCG (mOPN-5), Hoxc-8
binding was abolished (Fig. 3C, lane 15).
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Fig. 3.
Characterization of a Hoxc-8 DNA-binding site
from the osteopontin promoter. A, DNA fragments of
osteopontin promoter used for gel shift assays in panels B,
C, and D. Nucleotides are numbered
relative to the transcription start site. Filled boxes
indicate putative Hox binding sites. The striped box
represents a putative Hox binding site containing the mutated core
sequence. B, OPN-2 DNA fragment contains a Hoxc-8 binding
site. A gel shift assay was performed using different
32P-labeled DNA fragments as indicated. C, OPN-5
is the Hoxc-8 binding element. A gel shift assay was performed using
shorter DNA fragments as marked. The Hoxc-8 binding element is located
from 206 to
180. D, Hoxc-8 specifically binds to OPN-5.
A gel shift assay was performed using OPN-5 alone (lane 1)
or with GST-Hoxc-8 (lanes 2-8). Lanes
3-5 and 6-8 contained 5-, 25-, and 100-fold molar excess of unlabeled OPN-5 and MSX-2 DNA binding
element (Probe-M), respectively.
206 to
180), suggesting that the flanking regions are
also important for Hoxc-8 binding. The Hoxc-8 binding site, including
its flanking regions, is highly conserved in chicken, mouse, pig, and
human. The other four putative Hox sites may be involved in other
homeodomain protein binding or may not be authentic Hox binding sites.
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Fig. 4.
Smad1 inhibits binding of Hox proteins to
DNA. A, Smad1 inhibits the binding of Hoxc-8 to OPN-5
in a concentration-dependent manner. Gel shift assays were
performed using OPN-5 alone (lane 1) or with 1.5 µg of GST
(lane 2), 1.5 µg of GST-Smad1 (lane 3), or 0.2 µg of GST-Hoxc-8 protein (lanes 4-7) and
different amounts of GST-Smad1 (1.5, 3, and 4.5 µg for lanes
5-7, respectively). B, Hox proteins interact
with Smad1 and -4 but not Smad2 and -3. Hoxa-9 and Hoxc-8 GST fusion
proteins (0.2 µg) were tested for their ability to interact with
Smad1, -2, -3, and -4 or GST (3 µg) in a gel shift assay.
C, Smads do not inhibit binding of Msx-1 and Msx-2
homeodomains containing proteins to their cognate DNA element. Purified
GST-Msx-1 or -Msx-2 (0.5 µg) was incubated together with probe-M and
different Smads (3 µg).
superfamily are very
complex, it is important to understand the specificity of the
interaction between Hox and Smad proteins. Hoxa-9 was chosen as a well
characterized homeobox DNA-binding protein (34, 35) to examine its
interaction with different Smad proteins. Two other homeodomain
proteins, Msx-1 and Msx-2, were also used for gel shift assays for the
same purpose. Msx-1 and Msx-2, found at different loci than the Hox gene clusters, are involved in development
of teeth. The expression of both genes is coordinately regulated by
BMP-2 and BMP-4 (36-38).
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Fig. 5.
BMP-induced osteopontin gene transcription is
mediated by Hoxc-8 binding site. A, schematic
description of the constructs used in the transfection assays:
OPN-266 is the native osteopontin construct;
Hox-pGL3 contains the osteopontin Hox binding site linked to
SV40 promoter; mHox-pGL3 contains the mutated osteopontin
Hox binding site. B, BMP activates the osteopontin promoter. The
OPN-266 plasmid was co-transfected in C3H10T1/2 mesenchymal cells with
Hoxc-8, Smad1, or Smad4 plasmids alone or in a combination of all three
in the presence or absence of ALK3 plasmid. C, the
osteopontin Hox binding site mediates BMP-induced transcription.
Hox-pGL3 construct was co-transfected with ALK6 or ALK3 in C3H10T1/2
mesenchymal cells. D, mutation of Hox binding site abolishes
BMP stimulation. Hox-pGL3 construct or mHox-pGL3 pGL3 control plasmid
was co-transfected with ALK6, ALK3, or Hoxc-8 plasmids in C3H10T1/2
mesenchymal cells. Cell lysates in B, C, and
D were assayed for luciferase activity normalized to
Renilla luciferase levels 48 h after transfection.
Experiments were repeated twice in triplicates.
pathway. Here, we first show that Hoxc-8 interacts with
Smad1 as a downstream DNA-binding protein in the BMP pathway. Our data
demonstrate that Hoxc-8 binds to the osteopontin promoter and represses
the gene transcription. BMP stimulation activates gene transcription by
derepressing the Hoxc-8 protein through the interaction of Smad1 with
the Hoxc-8 protein. The direct interaction between Smad1 and Hox
protein(s) suggests their functional relationship and the mechanisms in
BMP-induced skeleton development.
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ACKNOWLEDGEMENTS |
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We are grateful to R. Wuthier, V. Darley-Usmar, and H. Jo for discussions and comments on the manuscript. We thank J. Warna for kindly providing the constitutively active type IB (ALK6) and type IA (ALK3) BMP receptor expression vectors, R. Derynck for the human Smad1, -2, -3, and -4 cDNA clones, H. Le Mouellic for Hoxc-8 cDNA, C. Largman for Hoxa-9 cDNA, and C. Abate-Shen for GST-MSX1 and -MSX2 expression vectors.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK53757 (to X. C.) and a grant from the Center for Bone Metabolic Disease, University of Alabama at Birmingham (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.
These authors contributed equally to this work.
¶ To whom correspondence should be addressed: 1670 University Blvd., VH G002, Birmingham, AL 35294-0019. Tel.: 205-934-0162; Fax: 205-934-1775; E-mail: Cao{at}path.uab.edu.
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ABBREVIATIONS |
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The abbreviations used are:
TGF-, transforming growth factor-
;
BMP, bone morphogenetic protein;
ALK3, constitutively active BMP type IA receptor (Q233D);
ALK6, constitutively active BMP type IB receptor (Q203D);
GST, glutathione
S-transferase;
Hox, homeobox gene;
HA, hemagglutinin;
OPN, osteopontin;
m, mutant (e.g. mOPN-5).
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REFERENCES |
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