From the Division of Endocrinology and Bone
Metabolism, Department of Medicine, University of Texas Health Science
Center at San Antonio, San Antonio, Texas 78284 and the
§ Department of Biochemistry, Faculty of Dentistry, Osaka
University, 565-0871 Osaka, Japan
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ABSTRACT |
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Since the bone morphogenetic proteins (BMPs) are
members of the transforming growth factor- (TGF-
) superfamily
that induce the differentiation of mesenchymal precursor cells into the
osteogenic cells, we identified the relevant signaling molecules
responsible for mediating BMP-2 effects on mesenchymal precursor cells.
BMP-2 induces osteoblastic differentiation of the pluripotent
mesenchymal cell line C2C12 by increasing alkaline phosphatase activity
and osteocalcin production. As recent studies have demonstrated that cytoplasmic Smad proteins are involved in TGF-
superfamily
signaling, we plan to isolate the relevant Smad family members involved
in osteoblastic differentiation. We identified human Smad5, which is
highly homologous to Smad1. BMP-2 caused serine phosphorylation of
Smad5 as well as Smad1. In contrast, TGF-
failed to cause serine
phosphorylation of Smad1 and Smad5. We found Smad5 is directly activated by BMP type Ia or Ib receptors through physical association with these receptors. Following phosphorylation, Smad5 bound to DPC4,
another Smad family member, and the complex was translocated to the
nucleus. Overexpression of point-mutated Smad5 (G419S) or a C-terminal
deletion mutant DPC4 (DPC4
C) blocked the induction of alkaline
phosphatase activity, osteocalcin production, and Smad5-DPC4 signaling
cascades upon BMP-2 treatment in C2C12 cells. These data suggest that
activation of Smad5 and subsequent Smad5-DPC4 complex formation are key
steps in the BMP signaling pathway, which mediates BMP-2-induced
osteoblastic differentiation of the C2C12 mesenchymal cells.
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INTRODUCTION |
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The bone morphogenic proteins
(BMPs)1 are members of the
transforming growth factor- (TGF-
) superfamily which have been
implicated in embryogenesis, organogenesis, and morphogenesis (1).
Among BMPs, BMP-2 and BMP-4 have been shown to promote the development of bone and cartilage by inducing the differentiation of
undifferentiated mesenchymal cells into the osteoblastic cells or
cartilage cells, respectively (2). BMP-2 has been shown to induce
ectopic bone or cartilage formation when implanted in muscular tissue
in vivo (2, 3) and stimulate osteoblastic differentiation of
the mesenchymal cells in vitro as assessed by the
stimulation of calcification, alkaline phosphatase (ALP) expression, or
osteocalcin production (4, 5). However, the molecular mechanisms
responsible for the effects of BMP on differentiation of these cells
toward bone and cartilage are poorly understood.
BMPs exert their diverse biological effects through two types of
transmembrane receptors, BMP type I (BMPIR) and type II (BMPRII) receptors (6, 7), which possess intrinsic serine/threonine kinase
activity (6, 7). BMPIR is further subclassified into BMP type IaR (also
called ALK3) and IbR (also called ALK6) (6, 7), but their functional
difference in BMP signaling is unknown at the present time. Upon
binding to the type II receptors, BMPs induce heterodimerization
between BMP type I and type II receptors, and transduce signals into
the cytoplasm (6, 7). Recent studies have shown that cytoplasmic
signaling molecules, including Mad (mother against dpp), the
Xenopus homologue of Mad, Xmad1, Xmad2, and several human
homologues of Mad, Smads, play critical roles in TGF- superfamily
signaling (7-9). To date, seven Smad family members that possess
ligand selectivity have been identified (7, 8). For example, Smad1 has
been implicated in BMP responses (10-12), whereas Smad2 (13, 14) and
Smad3 (15, 16) are activated upon treatment with TGF-
. DPC4 (Smad4)
was initially found to be a tumor suppressor in pancreatic cancers
(17). DPC4 is not phosphorylated but forms a complex with Smad1, Smad2,
and Smad3 upon BMP or TGF-
treatment (18, 19). Thus, DPC4 may have
unique roles in TGF-
superfamily signaling. Interestingly, Smad6
(20) and Smad7 (20, 21) are found to possess unique structures compared
with other Smad family members and both Smad6 and Smad7 inhibit TGF-
effects. Despite these data, it is not known whether these Smad family
members are specific for these growth factors, or whether the growth
factors may utilize different Smad family members for different
biological effects. Furthermore, considering the multifunctional
properties of the BMPs, it is possible that there are still
unidentified Smad family members.
In the present study, we first sought Smad family members that might be
involved in BMP-induced osteoblastic differentiation of pluripotent
mesenchymal precursor cells. We isolated several human Smad family
members including Smad5. Because the functional roles of Smad5 in
TGF- superfamily signaling have not been fully characterized as yet,
we focused our efforts on Smad5 and found that Smad5 was directly
activated by BMP type Ia or Ib receptors upon BMP-2 stimulation and
transduced BMP-2 signals to the nucleus by forming a complex with DPC4.
Moreover, we demonstrate that Smad5 and DPC4 play a critical role in
the induction of the osteoblastic differentiation of the pluripotent
mesenchymal cells C2C12 by BMP-2.
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MATERIALS AND METHODS |
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Cells and Antibodies-- 293 cells, L6 cells and C2C12 cells were cultured in DMEM containing 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT). NMuMg cells were purchased from ATCC and cultured in DMEM containing 10% FBS and 10 µg/ml insulin (Sigma). Anti-HA polyclonal antibody, anti-phosphotyrosine monoclonal antibody, and anti-Flag monoclonal antibody were purchased from BAbCO, Transduction Laboratories, and IBI, respectively. Anti-phosphoserine and anti-phosphothreonine polyclonal antibodies were purchased from Zymed Laboratories Inc.
cDNA Cloning--
Human homologues of Mad cDNA were
isolated using the PCR-nested cloning approach. Degenerate
oligonucleotide primers for PCR were designed based on the conserved
region of Drosophila Mad (22, 23) and Caenorhabditis
elegans Sma-2 (24) proteins (forward primer;
5-CA(C/T)AT(A/C/T)GGNAA(A/G)GGNGT-3
encoding HIGKGV; reverse primer;
5
-TG(A/T/G)AT(C/T)TC(A/G/T)ATCCA(A/G)CANGGNGT-3
, encoding
TPCWIEIH). After PCR amplification, predicted size PCR products were
subcloned into TA cloning vector (Invitrogen) and their DNA sequences
were determined by dideoxy DNA sequencing kit (Upstate Biotechnology,
Inc., Lake Placid, NY). The PCR products were released from TA-cloning
vector with EcoRI, and radiolabeled with random primed
labeling kit (Boehringer Mannheim) and [32P]dCTP (NEN
Life Science Products). Human 293 cell cDNA library (gifted by
Joseph Schlessinger and Ivan Dikic) were screened with radiolabeled
probes and positive clones were isolated by two additional round of
screening. The isolated clones were subjected to DNA sequence
analysis.
Constructs and Transfection--
BMP type Ia (BMPIaR), Ib
(BMPIbR), BMP type II (BMPRII), TGF- type I (TGF-
RI), and TGF-
type II (TGF-
RII) receptors, and Flag-Smad1 were used as described
previously (10) (kind gifts of Kohei Miyazono, John Massagué, and
Jeffrey L. Wrana). Constitutively active mutant BMPIaR (233D), BMPIbR
(203D), and TGF-
RI (204D) were point-mutated from glutamine 233, glutamine 203, or threonine 204 into aspartic acid, respectively
(kindly gifted by Jeffrey L. Wrana and John Massague). Smad3, Smad5 or
DPC4 cDNA was subcloned into pcDNA3 (Invitrogen) tagged with a
Flag (10, 11) or HA (18) epitope in the N terminus. The site-directed
mutagenesis of Smad1, Smad3, Smad5, and DPC4 was performed using the
Altered Sites in vitro mutagenesis system (Promega), and the
mutations were confirmed by DNA sequences analysis. Mutant Smad1,
mutant Smad3, and mutant Smad5 were generated by replacing glycine 419, 379, or 419 by serine, respectively. Mutant DPC4 was generated by
introducing stop codon at C-terminal region of DPC4. 293 cells, NMuMg
cells, L6 cells, or C2C12 cells were transfected using LipofectAMINE (Life Technologies, Inc.) or Tfx-50 (Promega) according to the manufacture's protocol.
Immunoprecipitation, Western Blotting, and Metabolic
Labeling--
The cells were serum-starved with DMEM containing 0.2%
FBS for 16 h, and treated with 100 ng/ml BMP-2 or 10 ng/ml TGF-
for 15 min. The cells were washed three times with ice-cold
phosphate-buffered saline buffer (PBS), and solubilized in lysis buffer
(20 mM Hepes (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl2, 10%
glycerol, 1% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin,
1 mM phenylmethylsulfonyl fluoride, 0.2 mM
sodium orthovanadate) (25). The lysates were centrifuged for 15 min at
4 °C, 16,000 × g. The lysates were incubated with
antibodies for 4 h at 4 °C, followed by immunoprecipitation
with protein A-Sepharose (Zymed Laboratories Inc.) or
protein G-agarose (Boehringer Mannheim). Immunoprecipitates were washed
five times with lysis buffer and boiled in SDS sample buffer containing
0.5 M
-mercaptoethanol, and supernatants were recovered
as immunoprecipitate samples. These samples were separated by SDS-PAGE,
transferred to nitrocellulose membranes, immunoblotted with
anti-antibodies. The samples were visualized with horseradish peroxidase coupled to protein A (KPL) or horseradish peroxidase-coupled anti-mouse IgG antibodies (Cappel), and enhanced by ECL detection kits
(Amersham).
Immunofluorescent Staining-- The cells were serum-starved with DMEM containing 0.2% FBS for 16 h, and treated with 100 ng/ml BMP-2 for 40 min. The cells were washed three times with ice-cold PBS and fixed with 3.8% paraformaldehyde-PBS. After 15 min of incubation with 0.1% Triton-PBS, the cells were blocked with 1% bovine serum albumin-PBS, incubated with anti-Flag antibody (1:500) for 2 h, and washed six times with 0.1% Triton-PBS, followed by incubation with fluorescein isothiocyanate-conjugated anti-mouse IgG antibody (Jackson Immunoresearch Laboratories Inc.). The cells were extensively washed with PBS and visualized by fluorescence microscope (Zeiss). The number of cells whose nucleus was stained by anti-Flag antibody was counted per hundred cells at four independent fields.
GST Fusion Proteins and in Vitro Binding Assay-- Smad5 and DPC4 cDNA were subcloned into pGEX-2T (Pharmacia Biotech Inc.) in frame. GST fusion proteins were expressed and purified by glutathione-agarose affinity column (25). Recombinant GST or GST fusion proteins (0.5 µg) immobilized on agarose beads were incubated with the cell lysates for in vitro binding assay (25). After extensively washing the beads, the molecules associated with GST fusion proteins were determined by Western blotting using anti-HA polyclonal or anti-Flag monoclonal antibodies.
In Vitro Kinase Assay for Smad5--
BMPIbR or TGF-RI was
immunoprecipitated from 293 cells overexpressing HA-tagged BMPIbR or
TGF-
RI using anti-HA antibody, and the amount of immunoprecipitated
receptors were examined by Western blotting using anti-HA antibody.
Soluble form of recombinant GST-Smad5 or GST-DPC4 fusion proteins (1 µg) were incubated with immunoprecipitated HA-tagged BMPIbR or
TGF-
RI in the presence of 5 mM MnCl2 and 1 µCi of [
-32P]ATP (NEN Life Science Products) at
28 °C for 20 min (26). The supernatants were collected, boiled in
the presence of SDS-sample buffer at 95 °C for 5 min, and subjected
onto SDS-PAGE, followed by autoradiography.
Determination of Alkaline Phosphatase Activity and
Osteocalcin--
C2C12 cells transfected with pcDNA3, mutant
Flag-Smad1 (G419S), mutant Flag-Smad3 (G379S), mutant Flag-Smad5
(G419S), or mutant Flag-DPC4 (DPC4C) were cultured with 300 ng/ml
BMP-2 for 3 days. Osteocalcin in the culture media was measured by
radioimmunoassay (Biomedical Technologies Inc.) (27). ALP activity was
determined as described previously (27).
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RESULTS |
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Isolation of Human Smad5--
To identify Smad-related genes,
human Smad genes were isolated by cDNA cloning using PCR technique.
We designed de-generated primers based on conserved region of Mad (22,
23) and Sma-2 (24). We isolated several human cDNA clones, which
encode Smad-related genes including Smad3 (15, 16) and DPC4 (Smad4;
Ref. 17) from a human 293 cell cDNA library. One of clones was
approximately 90% homologous to the human Smad1 (10-12) and identical
to dwarfin-C, which has been cloned previously and shown to mediate
TGF- superfamily signaling (28). A partial sequence of this clone
was also reported as JV5-1 (29). According to the nomenclature for
Smad family (30), we named the clone Smad5. Human Smad5 cDNA
encodes 465 amino acids containing highly conserved MH1 and MH2
domains, which are separated by a proline-rich linker domain (Fig.
1). Smad5 is less homologous to other
Smad family members, including Smad2 (13, 14) (55%), Smad3 (15, 16)
(60%), and DPC4 (17) (31%) than Smad1.
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Specificity of Smad5 Responsiveness for BMP-2--
Since Smad5
shows high homology to Smad1 that is phosphorylated by BMP-2 (10, 12),
we examined whether BMP-2 also phosphorylates Smad5. To carry out the
experiments, we tagged Flag-epitope (10, 11) to the N terminus of Smad5
to distinguish from other Smad proteins, and transiently expressed
Flag-tagged Smad5 in 293 cells. As shown Fig.
2A, Smad5 was clearly
phosphorylated by BMP-2 (lane 2). In contrast, TGF- had
no effect on the phosphorylation of Smad5 (Fig. 2A,
lane 6). Western blotting analysis using antibodies to
phosphoserine, phosphothreonine, and phosphotyrosine revealed that the
serine residue(s) of Smad5 was phosphorylated (Fig. 2B, lane 2). Neither tyrosine nor threonine residues were
phosphorylated (Fig. 2B, lanes 6 and
8). Point-mutated Smad5 in which the highly conserved 419 glycine residue was replaced by serine was not phosphorylated by BMP-2
(Fig. 2, lane 4 in A and B), as shown
previously for Smad1 (10). Of note, Yingling et al. (28)
have reported dwarfin-C, which is identical to Smad5, is phosphorylated
by TGF-
, but not BMP-2 in NMuMg cells. Since their results are not
consistent with our present data, we determined Smad5 phosphorylation
in response to TGF-
or BMP-2 in NMuMg cells. As shown in Fig.
2C, BMP-2 caused Smad5 phosphorylation (lane 6),
whereas TGF-
did not cause Smad5 phosphorylation in NMuMg cells
(lane 5). Smad1 showed identical responsiveness to BMP-2
(lanes 2 and 3) to that of Smad5, as shown previously (10-12). On the other hand, we observed that Smad3 was not
phosphorylated by BMP-2, but TGF-
clearly phosphorylated Smad3 in
NMuMg cells (lanes 8 and 9). We further confirmed
the specificity of Smad5 responsiveness to BMP-2 by overexpressing constitutively active BMPIR or TGF-
type I receptors (TGF-
RI). It
has been demonstrated that point mutation of BMPIaR at position 233 (glutamine to aspartic acid), BMPIbR at 203 (glutamine to aspartic
acid), or TGF-
RI at 204 (threonine to aspartic acid) renders these
receptors constitutively active without their corresponding ligands and
type II receptors (10). Smad5 was serine-phosphorylated in the presence
of constitutively active mutant BMPIaR (233D) or BMPIbR (203D) without
BMP-2 stimulation (Fig. 2D, lanes 2 and 6). In contrast, Smad5 phosphorylation did not occur in the
presence of constitutively active mutant TGF-
RI (204D) (Fig.
2D, lane 10). Of note, the mutant Smad5 (G419S)
in which glycine 419 was replaced by serine was not phosphorylated,
even in the presence of constitutively active mutant BMPIaR (233D)
(Fig. 2D, lane 4) or BMPIbR (203D) (Fig.
2D, lane 8). Wild-type BMPIaR or BMPIbR (lanes 1 and 5) failed to serine-phosphorylate
Smad5 in the absence of BMP-2. Moreover, recombinant GST-Smad5 was also
phosphorylated by BMPIRs, but not by TGF-
RI in vitro
(Fig. 2E), whereas GST-DPC4 was not phosphorylated by BMPIbR
or TGF-
RI. These data clearly show that Smad5 is a downstream
signaling molecule specific for BMP in two different cell models.
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Association of Smad5 with BMPIR-- To further determine the interaction of Smad5 with BMPIR, we next explored whether Smad5 physically associates with BMPIRs. We performed co-immunoprecipitation experiments in 293 cells that were co-transfected with HA-BMPIbR and Flag-Smad5. As shown in Fig. 3A, Smad5 was co-immunoprecipitated with BMPIbR in a BMP-2-dependent manner (lanes 1 and 2). Consistent with this result obtained in living cells, GST-Smad5 associated with BMP-2-stimulated BMPIaR (Fig. 3B, lanes 1 and 2) or BMPIbR (Fig. 3, lanes 3 and 4 in B and C). The difference in the position of the bands of BMPIaR and BMPIbR observed in Fig. 3B is due to the difference of molecular size of BMPIaR and BMPIbR. GST alone (Fig. 3C, lanes 1 and 2) or GST-DPC4 (lanes 5 and 6) was unable to bind to BMPIR. We also found that GST-Smad5 was phosphorylated by BMPIRs in vitro (Fig. 2E). These findings suggest that activated BMPIRs directly phosphorylate Smad5 through physical association. Interestingly, the mutant Smad5 (G419S) that is not phosphorylated by BMP-2 was also able to bind to activated BMPIbR (Fig. 3A, lane 4). The result suggests that the mutant Smad5 (G419S) may compete with the intact Smad5 for the binding to BMPIRs regardless the state of phosphorylation. We also found that GST-Smad5 lacking the MH2 domain was unable to bind to activated BMPIbR (Fig. 3D, lanes 5 and 6), whereas MH1 domain-deleted GST-Smad5 still retained binding capacity (Fig. 3D, lanes 3 and 4). These data indicate that the MH2 domain is responsible for the physical association of Smad5 to BMPIbR.
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Association of Activated Smad5 with DPC4--
Smad1 that has high
homology with Smad5 is shown to form a heterocomplex with DPC4 (18,
19). To unravel further downstream of Smad5, we determined the
interaction of Smad5 with DPC4 by co-immunoprecipitation and an
in vitro binding assay using GST-DPC4 (25). As shown in Fig.
4A, Smad5 associated with DPC4
in a BMP-2-dependent manner (lane 2). In
contrast, the mutant Smad5 (G419S) that is not phosphorylated by BMP-2
failed to bind to DPC4 (Fig. 4A, lane 4). Of
note, BMP-2-activated Smad5 failed to associate with the C-terminal
deletion mutant DPC4 (DPC4C) (Fig. 4A, lane
6). Consistent with these results obtained in
co-immunoprecipitation experiments, BMP-2-activated Smad5 also
associated with GST-DPC4 (Fig. 4B, lane 2), but
not with GST-DPC4
C (lane 6) in vitro.
Furthermore, mutant Smad5 (G419S) was not able to bind to GST-DPC4
(lane 4). These results demonstrate that the phosphorylated
Smad5 forms heterocomplex with DPC4 and suggest that the
phosphorylation of Smad5 and the presence of the C terminus of DPC4 are
required for heterocomplex formation between Smad5 and DPC4. Since DPC4 did not physically associate with BMPIRs (Fig. 3C,
lane 6), DPC4 most likely serves as a downstream molecule of
Smad5 in the BMP-2 signaling pathway.
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Translocation of Smad5 and DPC4 into Nucleus by BMP-2--
We next
examined whether Smad5-DPC4 complex translocates into the nucleus, as
shown in the case of Smad1 and Smad2 (11, 31). Immunofluorescent
staining demonstrated Flag-Smad5 and Flag-DPC4 clearly translocated and
accumulated in the nucleus as early as 40 min after BMP-2 treatment
(Fig. 5, A, B,
E, and F). The mutant Smad5 (G419S) or mutant
DPC4 (DPC4C) did not show nuclear translocation by BMP-2 treatment
(Fig. 5, C, D, G, and H).
These results suggest that phosphorylated Smad5, following complex
formation with DPC4, translocates to the nucleus and might function as
a transcription-regulating factor, as is the case of Smad2 (31).
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Role of Smad5 and DPC4 in Osteoblastic Differentiation of C2C12 Cells-- Since BMP-2 plays an important role in osteogenesis by regulating the differentiation of the undifferentiated mesenchymal cells in vivo (2, 3, 32) and in vitro (4, 5, 27), we next explored the biological role of Smad5 in a pluripotent mesenchymal cell line C2C12, which shows osteoblastic differentiation in the presence of BMP-2 (27). BMP-2 induced serine phosphorylation of Flag-Smad5 that was transfected in C2C12 cells (Fig. 6A). In conjunction with this, BMP-2 also induced a marked increase in ALP activity (Fig. 6B) and production of osteocalcin (Fig. 6C) in C2C12 cells as reported previously (27). ALP and osteocalcin are widely recognized phenotypic markers of cells of osteoblast lineage (27). Importantly, overexpression of the mutant Smad5 (G419S) in C2C12 cells strongly inhibited BMP-2-induced ALP activity (Fig. 6B) and osteocalcin production (Fig. 6C). In addition, co-transfection of the mutant Flag-Smad5 (G419S) with wild-type Flag-Smad5 into C2C12 cells abolished BMP-2-induced serine phosphorylation of wild-type Flag-Smad5, showing dominant negative effects of the mutant Smad5 (G419S) on Smad5 activation (Fig. 6D). This dominant-negative effect is probably due to a competition for the binding to BMPIR between wild type and mutant Smad5 (Fig. 3A). The data demonstrate an inhibition of Smad5 phosphorylation by dominant-negative mutant Smad5 is associated with an inhibition of BMP-2-induced osteoblastic differentiation of C2C12 cells and suggest that the phosphorylation of Smad5 is necessary for the osteoblastic differentiation of C2C12 cells induced by BMP-2.
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DISCUSSION |
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The bone- and cartilage-inducing effects of the BMPs have been
studied extensively (2-5, 27, 32). Recent studies have markedly
increased our understanding of BMP signal transduction pathways at
molecular levels through identification of BMP-activated cytoplasmic
signaling molecules including Smads and DPC4 (7, 8). Nevertheless, the
precise role of these signaling molecules in the bone-inducing effects
of BMPs is not defined to date. In this study, we isolated human Smad5
and found that Smad5 was involved in BMP-2 signaling cascades, which
mediate the bone-inducing effects of BMP-2. Smad5 was directly
serine-phosphorylated by BMPIR through a physical interaction. The
activated Smad5 subsequently formed a complex with DPC4, and this
complex was then translocated to the nucleus. Overexpression of mutant
Smad5 (G419S), which inhibits the phosphorylation of intact Smad5,
blocked the BMP-2-induced osteoblastic differentiation of C2C12 cells.
Furthermore, suppression of the complex formation of the Smad5 with
DPC4 by overexpressing DPC4C also blocked BMP-2-induced osteoblastic
differentiation of C2C12 cells. Thus, interruption of BMP-2 signaling
cascades by inhibiting Smad5 activation or interfering with the
association between Smad5 and DPC4 abolished the osteogenic effects of
BMP-2 on C2C12 cells. These results strongly suggest that both
activation of Smad5 and following heterocomplex formation with DPC4 are
critical to the BMP-2 signaling, which mediates the induction of the
osteoblastic differentiation of the pluripotent mesenchymal cells
C2C12. In contrast, TGF-
did not induce the phosphorylation of Smad5
in living cells and in vitro, constitutively active
TGF-
RI failed to phosphorylate Smad5, Smad5 was unable to bind to
the activated TGF-
RI, and a previous study has shown that TGF-
fails to promote osteoblastic differentiation of C2C12 cells (27).
Collectively, these results suggest that Smad5, in addition to Smad1,
is an intracellular molecule specifically involved in the BMP
signaling. Thus, Smad5 is a new cytoplasmic signaling molecule of human
Smad family members that mediates the osteogenic effects of BMP-2.
In conflict with our data, an earlier study has reported that
mouse homologues for Smad5 and Smad1, dwarfin-C and dwarfin-A, respectively, are phosphorylated by TGF-, but not by BMP-2 (28). To
examine whether this apparent discrepancy between that study and ours
is due to a difference in experimental models, we performed identical
experiments to those described here in NMuMg cells that were used in
the previous study. In our hands, Smad5 was selectively activated by
BMP-2 and TGF-
did not activate Smad5 in NMuMg cells. It is possible
that Smad1 and Smad5 antibodies used in the previous study might
recognize other Smads including Smad2 and Smad3 that are responsive to
TGF-
(13-16), since homology between these Smad members is high (7,
8). Indeed, the authors raised the same possibility in the report.
Furthermore, we experienced that the antibody we generated against
GST-Smad5 recognized other Smad molecules including Smad1, Smad3, and
DPC4.2 However, it still
remains possible that endogenous Smad5 behaves in different manners
from that of transfected exogenous Smad5 or Smad1. Resolution of this
issue awaits generation of specific antibodies for Smad5.
Finally, our results suggest that there are no distinctive functional differences between Smad1 and Smad5 in BMP-2 signal transduction and BMP-2-induced osteoblastic differentiation in C2C12 cells. Dominant-negative Smad1, like dominant-negative Smad5, inhibited BMP-2-induced C2C12 differentiation into osteoblasts. Dominant-negative Smad5 interfered with Smad1 phosphorylation by BMP-2 stimulation. Whether Smad5 characterized herein has specific roles that are distinguishable from those of Smad1 in BMP-2 signaling that mediates the biological effects of BMP-2 is an important issue. However this is beyond the scope of the present study. Antibodies that specifically recognize Smad5 and development of additional experimental models to C2C12 cells may clarify this point.
In conclusion, we demonstrate an important role of Smad5 and DPC4 in the BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal stem cell C2C12.
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ACKNOWLEDGEMENTS |
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We thank Anthony J. Celeste and Vicki
Rosen (Genetic Institute) for BMP-2, Liliana Attisano and Jeffrey L. Wrana (The Hospital for Sick Children) for the BMP receptor and
Flag-Smad1 constructs, Joan Massagué (Sloan-Kettering Cancer
Center) and Kohei Miyazono (Cancer Institute) for TGF- receptor
constructs, and Joseph Schlessinger and Ivan Dikic (New York
University) for human 293 cell cDNA library, respectively.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant RO1-DK45229-05.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: Dept. of Medicine, Div. of Endocrinology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284-7877. Tel.: 210-567-4900; Fax: 210-567-6693; E-mail: yoneda{at}uthscsa.edu.
1
The abbreviations used are: BMP, bone
morphogenetic protein; TGF-, transforming growth factor-
; BMPIR,
BMP type I receptor; BMPIaR, BMP type Ia receptor; BMPIbR, BMP type Ib
receptor; BMPRII, BMP type II receptor; TGF-
RI, TGF-
type I
receptor; TGF-
RII, TGF-
type II receptor; Mad, mother against
dpp; Smad1 G419S, point mutant Smad1 (glycine 419 replaced by serine);
Smad5 G419S, point mutant Smad5 (glycine 419 replaced by serine);
DPC4
C, C-terminal deletion mutant of DPC4; Smad3 G379S, point mutant
Smad3 (glycine 379 replaced by serine); ALP, alkaline phosphatase;
DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum;
PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PAGE,
polyacrylamide gel electrophoresis; GST, glutathione
S-transferase.
2 R. Nishimura, Y. Kato, D. Chen, S. E. Harris, G. R. Mundy, and T. Yoneda, unpublished observation.
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REFERENCES |
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