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Vol. 10, Issue 11, 3801-3813, November 1999
and
*Department of Biochemistry, The Cancer Institute of the Japanese
Foundation for Cancer Research and Research for the Future Program,
Japan Society for the Promotion of Science, Tokyo 170-8455, Japan;
Department of Biomaterials Science and
Second Department of Oral Surgery, Faculty of Dentistry,
Tokyo Medical and Dental University, Tokyo 113-8549, Japan; and
§Creative BioMolecules, Hopkinton, Massachusetts 01748
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Bone morphogenetic proteins (BMPs) are multifunctional regulators
of cell growth, differentiation, and apoptosis (Reddi, 1994
; Hogan,
1996). BMPs were originally isolated as proteins that induce bone and
cartilage formation in vivo (Wozney et al., 1988), but it is
now known that BMPs play critical roles in morphogenesis during
development in vertebrates and invertebrates. BMPs have been shown to
induce differentiation of mesenchymal cells into osteoblast and
chondroblast lineages and to inhibit their differentiation into
myocytes in vitro (Katagiri et al., 1994; Luyten et
al., 1994; Shukunami et al., 1996). More than a dozen
BMP proteins have been identified in mammals, which can be
subclassified into several groups depending on their structures. BMP-2
and BMP-4 are highly similar to each other and most similar to
Decapentaplegic in Drosophila. BMP-5, BMP-6,
osteogenic protein (OP)-1 (also called BMP-7), and OP-2/BMP-8 are
structurally similar to each other. Growth-differentiation factor
(GDF)-5 (also termed cartilage-derived morphogenetic protein-1),
GDF-6/cartilage-derived morphogenetic protein-2, and GDF-7 form another
group (Kawabata et al., 1998a). In contrast to BMP-2, BMP-4,
BMP-6, and OP-1/BMP-7, which induce bone and cartilage formation in
vivo (Wozney et al., 1988; Sampath et al., 1992;
Gitelman et al., 1994), GDF-5, GDF-6, and GDF-7 more
efficiently induce cartilage and tendon-like structures in vivo
(Hötten et al., 1996; Wolfman et al.,
1997).
BMPs belong to the transforming growth factor (TGF)-
superfamily,
which includes TGF-s, activins, and Müllerian-inhibiting substance. Members of the TGF- superfamily exert their effects via
binding to two types of serine/threonine kinase receptors, both of
which are essential for signal transduction (Kawabata et
al., 1998a; Massagué, 1998). The type II receptors are
constitutively active kinases, which transphosphorylate type I
receptors upon ligand binding. The type I receptors activate
intracellular substrates such as Smad proteins and thus determine the
specificity of intracellular signals. Seven different type I receptors
have been isolated in mammals, which were originally termed activin
receptor-like kinase (ALK)-1-ALK7 (ten Dijke et al.,
1994a,b). BMP type IA receptor (BMPR-IA or ALK-3) and BMPR-IB (ALK-6)
are structurally highly similar to each other and specifically bind
BMPs together with type II receptors. ALK-2 has been shown to bind
activin, but recent data revealed that it is a type I receptor for
certain BMPs, e.g., OP-1/BMP-7 (ten Dijke et al., 1994b;
Macías-Silva et al., 1998). ALK-1 is structurally
highly similar to ALK-2, but its physiological ligand is still unknown.
ALK-5 and ALK-4 are type I receptors for TGF- (TR-I) and activin
(ActR-IB), respectively. ALK-7 is structurally similar to ALK-4 and
ALK-5, but its ligand has not been determined yet.
Type I receptors function as downstream components of type II
receptors. Mutation of Thr-204 in TR-I to acidic amino acids such as
aspartic acid [TR-I(TD)] leads to constitutive activation of the
type I receptor kinase (Wieser et al., 1995). Thus,
TR-I(TD) induces signals in the absence of ligands or type II
receptors. Similarly, mutations of corresponding threonine or glutamine
residues in the other type I receptors to aspartic acid or glutamic
acid lead to constitutive activation of the type I receptor kinases. The specificity of the intracellular signals by type I receptors is
determined by a specific region in the serine/threonine kinase domain,
termed the L45 loop (Feng and Derynck, 1997). Thus, the structures of
the L45 loop of BMPR-IA/ALK-3 and BMPR-IB/ALK-6 (BMPR-I group) are
identical to each other, and they may transduce similar signals in
cells. Similarly, the L45 loops of TR-I/ALK-5, ActR-IB/ALK-4, and
ALK-7 (TR-I group) are identical to each other, and they activate
similar substrates (Y.G. Chen et al., 1998; Persson et
al., 1998). The L45 loops of ALK-1 and ALK-2 (ALK-1 group) are
most divergent from the other type I receptors, but they activate
substrates similar to that of the type I receptors of the BMPR-I group
(Armes and Smith, 1997; Armes et al., 1999; Chen and
Massagué, 1999).
Signals from the serine/threonine kinase receptors may be transduced by
various proteins. Among them, the best-studied molecules are proteins
of the Smad family (Heldin et al., 1997; Attisano and Wrana,
1998; Derynck et al., 1998; Massagué, 1998). Eight different Smad proteins have been identified in mammals, and these proteins are classified into three subgroups, i.e., receptor-regulated Smads (R-Smads), common partner Smads (Co-Smads), and inhibitory Smads.
R-Smads are directly activated by type I receptors, form complexes with
Co-Smads, and translocate into the nucleus. The Smad heteromers bind to
DNA directly and indirectly via other DNA-binding proteins and thus
regulate the transcription of target genes. Smad1, Smad5, and Smad8 are
activated by BMPs, whereas Smad2 and Smad3 are activated by TGF- and
activin. Smad4 functions as a Co-Smad. Smad6 and Smad7 are structurally
distantly related to the other Smads and act as inhibitory Smads.
BMP type I receptors have been shown to exhibit various biological
effects, including osteoblast differentiation (Akiyama et
al., 1997; Namiki et al., 1997; D. Chen et
al., 1998). However, biological effects of the type I receptors of
the ALK-1 group have not been determined. Smad1 and Smad5 have also
been shown to elicit various effects of BMPs, e.g., ventral mesoderm
formation in Xenopus embryos (Graff et al., 1996;
Thomsen, 1996; Suzuki et al., 1997). They were also shown to
induce differentiation of C2C12 cells into osteoblast cells
(Yamamoto et al., 1997), although this differentiation was
not efficient compared with that stimulated by ligands or
constitutively active BMP receptors. It is not known whether Smads are
sufficient for the induction of osteoblast differentiation or whether
other signaling molecules activated by BMP receptors are required for
this induction. Smad6 and Smad7 were shown to inhibit the
transcriptional activity induced by TGF- (Hayashi et al.,
1997; Imamura et al., 1997; Nakao et al., 1997),
but recent data revealed that Smad7 is more potent than Smad6 in
inhibiting TGF--induced growth inhibitory activity (Itoh et
al., 1998). Inhibitory Smads were also shown to inhibit the
effects of BMPs as well as those of activins in Xenopus
embryos (Bhushan et al., 1998; Hata et al., 1998;
Nakayama et al., 1998a,b); however, the effects of
inhibitory Smads on osteoblast and chondroblast differentiation have
not been determined. An adenovirus-based vector system allows high
efficiencies of transfection of DNAs in many cell types. In this study,
we examined the effects of various type I receptors and Smads in the
regulation of differentiation into osteoblast and chondroblast cells
using an adenovirus vector system.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Constructions of Recombinant Adenoviruses
Recombinant adenoviruses were constructed as described
previously (Saito et al., 1985; Miyake et al.,
1996; Saitoh et al., 1998). Briefly, various hemagglutinin
(HA)-tagged type I receptor, FLAG-tagged Smad, and
-galactosidase cDNAs were subcloned into the SwaI site of
the pAxCAwt cassette cosmid. Each cosmid carrying the expression unit
and adenovirus DNA-terminal protein complex were cotransfected into E1
transcomplemental cell line 293. The recombinant adenoviruses generated
by homologous recombination were isolated, and the insertion of Smad or
type I receptor cDNAs was confirmed by digestion using restriction
endonucleases. High-titered stocks of recombinant viruses were grown in
293 cells and purified. Infection of recombinant adenoviruses was
performed at a multiplicity of infection (m.o.i.) of <8 × 102 pfu/cell.
Cell Culture
The mouse muscle myoblast cell line C2C12 was obtained from
American Type Culture Collection (Rockville, MD). The cells were maintained in DMEM containing 15% fetal bovine serum (FBS) and 100 U/ml penicillin. The mouse clonal teratocarcinoma cell line ATDC5 was obtained from Riken Cell Bank (Saitama, Japan).
The cells were grown in a medium consisting of a 1:1 mixture of DMEM and Ham's F-12 medium containing 5% FBS, 10 µg/ml bovine insulin, 10 µg/ml transferrin, 3 × 10
8 M sodium
selenite (Boehringer Mannheim, Indianapolis, IN), and antibiotics as
described (Shukunami et al., 1996).
Immunoblotting
Cells infected with adenoviruses were washed with
phosphate-buffered saline (PBS) and solubilized in a buffer containing
20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Lysates were cleared by centrifugation and subjected to immunoprecipitation by anti-FLAG antibody or directly subjected to SDS-gel electrophoresis (Kawabata et al., 1998b; Yagi et al., 1999). Proteins were
then electrotransferred to polyvinylidene difluoride membranes,
immunoblotted with anti-FLAG M2 antibody (Sigma, St. Louis,
MO), anti-HA 3F10 antibody (Boehringer Mannheim), or anti-phosphoserine
antibody (Zymed Labs, San Francisco, CA), and visualized using an
enhanced chemiluminescence detection system (Pharmacia, Piscataway, NJ).
Assay for Alkaline Phosphatase Activity
Histochemical analysis of alkaline phosphatase activity was
performed as described (Katagiri et al., 1994). Briefly,
cells were fixed for 10 min with 3.7% formaldehyde at room
temperature. After washing with PBS, the cells were incubated for 20 min with a mixture of 0.1 mg/ml naphthol AS-MX phosphate (Sigma), 0.5% N,N-dimethylformamide, 2 mM
MgCl2, and 0.6 mg/ml fast blue BB salt (Sigma) in
0.1 M Tris-HCl, pH 8.5, at room temperature, followed by histochemical
analysis using phase-contrast microscopy. For quantitative analysis of
alkaline phosphatase activity, cells were washed and extracted with a
lysis buffer as described (Asahina et al., 1993; Nishitoh
et al., 1996). Alkaline phosphatase activity was determined
using p-nitrophenyl phosphate (Sigma) as a substrate.
Measurements of Chondrogenic Differentiation
Chondrogenic differentiation of ATDC5 cells was determined by
staining of sulfated glycosaminoglycans of the cells with Alcian Blue
as described (Asahina et al., 1996). Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and stained with 0.5%
Alcian Blue 8GX (Wako, Osaka, Japan) in 0.1N HCl overnight. After washing with distilled water, the cells were examined by histochemical analysis. Synthesis of sulfated glycosaminoglycans was
measured by incorporation of [35S]sulfate as
described (Lietman et al., 1997). Briefly, cells were
labeled with
Na235SO4
(10 µCi; Pharmacia) for 4 h. The radioactive medium was removed, and cells were washed with ice-cold buffer (10 mM EDTA and 0.1 M sodium
phosphate, pH 6.5) and digested in 12-well plates with 200 µl of
proteinase K (1 mg/ml) for 24 h at 37°C. One hundred microliters
of the samples were subjected to chromatography on Microspin G-25
columns (Pharmacia) in 4 M guanidine hydrochloride to remove
unincorporated 35SO4.
Radioactivity in the macromolecular fraction was determined by a
scintillation counter.
Immunofluorescence and Confocal Microscopy
Cells were grown in LAB-TEK chambers (Nalge, Rochester, NY), infected with adenoviruses carrying different Smad and receptor cDNAs, washed with PBS, and fixed with acetone. Cells were then incubated with 10% normal goat serum, washed with PBS, and incubated with an anti-Smad5 antiserum (provided by P. ten Dijke and C.-H. Heldin). The cells were then washed again with PBS, followed by incubation with fluorescein isothiocyanate-conjugated antibody against mouse immunoglobulin (Cappel, West Chester, PA). After a final wash, cells were covered with glycerine and examined by confocal laser-scanning microscopy (Olympus, Lake Success, NY).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Differentiation Induction of C2C12 Cells by Type I Receptors
To obtain a high-transfection efficiency, we infected DNAs in
C2C12 cells using a recombinant adenovirus system. More than 80% of
the cells were infected as determined by staining of the cells for
-galactosidase. Similar results were obtained for ATDC5 cells.
C2C12 undifferentiated mesenchymal cells differentiate into
osteoblast-like cells after treatment with BMP-2 and OP-1/BMP-7 (Katagiri et al., 1994; Takeda et al., 1998).
Osteoblast differentiation by constitutively active forms of type I
receptors was examined by the induction of alkaline phosphatase
activity by staining the cells 4 d after infection. Similar
expression levels of HA-tagged type I receptors were observed when
determined by immunoblotting using anti-HA antibody
(see Figure 1C). Histochemical analysis revealed that the cells infected with adenoviruses carrying
BMPR-IA/ALK-3(QD) and BMPR-IB/ALK-6(QD) were positively stained (Figure
1A). In addition, constitutively active forms of type I receptors of
the ALK-1 group (ALK-1 and ALK-2), but not those of the TR-I group (TR-I/ALK-5 and ActR-IB/ALK-4), induced alkaline phosphatase activity (Figure 1A). Quantitative analysis of alkaline phosphatase activity also revealed that the constitutively active ALK-2 induced alkaline phosphatase activity (Figure 1B). Differences in the alkaline
phosphatase activity were caused by the difference in the numbers of
the alkaline phosphatase-positive cells in certain experiments (see
below). Therefore, further studies on osteoblast differentiation were
performed by histochemical analysis.
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Activation of Smad1 and Smad5 by type I receptors was examined by
immunoblotting using anti-phosphoserine antibody.
Constitutively active forms of ALK-1, ALK-2, BMPR-IA/ALK-3, and
BMPR-IB/ALK-6 induced the phosphorylation of Smad1 and Smad5 (Figure
1C). In contrast, neither ActR-IB/ALK-4 nor TR-I/ALK-5 efficiently
phosphorylated Smad1 or Smad5. Thus, the ability of type I receptors to
induce osteoblast differentiation of C2C12 cells was correlated with their ability to activate Smad1 and Smad5.
Induction of Alkaline Phosphatase Activity in C2C12 Cells by Smad1 and Smad5
Osteoblast differentiation by Smads was then examined by
induction of alkaline phosphatase activity by staining the cells infected with adenoviruses carrying Smad cDNAs. Expression of FLAG-tagged Smads was confirmed by anti-FLAG
immunoblotting (Figure 2,
A and C). Small fractions of the cells expressing Smad1 or Smad5 were
positively stained at an m.o.i. of >450 (Figure 2B). In contrast,
neither Smad2 nor Smad3, which are activated by TGF- and activin
pathways, induced alkaline phosphatase activity even at an m.o.i. of
600 (Figure 2D), although Smad proteins were expressed in the infected
cells. Smad4, the common partner Smad in mammals, did not induce
alkaline phosphatase activity when infected alone (Figure 2D); however,
coinfection of Smad4 potentiated the effect of Smad1 and Smad5 (Figure
2B). Cells positively stained for alkaline phosphatase were only
sparsely observed in the presence of Smad1 or Smad5 and Smad4, whereas
most of the cells infected with the adenoviruses containing type I
receptors were positively stained (see Figure 1A). Interestingly, Smad4
weakly induced alkaline phosphatase activity in the C2C12 cells
infected with the Smad3 adenovirus but not in the cells infected with
the Smad2 adenovirus (Figure 2D), suggesting that Smad3 weakly, but
significantly, activates the transcription of the alkaline phosphatase
gene in the presence of Smad4.
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BMP-6 is structurally most similar to OP-1/BMP-7. BMP-6 (200 ng/ml) induced the differentiation of C2C12 cells into osteoblast cells
(Figure 3A). When the C2C12 cells were
infected with Smad1 or Smad5 and treated with 200 ng/ml BMP-6,
induction of alkaline phosphatase activity was dramatically enhanced,
and most of the infected cells were positively stained for alkaline
phosphatase activity (Figure 3A).
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To study the mechanism of efficient induction of alkaline
phosphatase activity of R-Smads by BMP-6, we examined subcellular localization of Smad5 by indirect immunofluorescence staining of cells
using an anti-Smad5 antiserum (Figure 3B). Smad5 was mainly observed in
the cytoplasm in the absence of BMP-6, whereas addition of BMP-6
induced the nuclear accumulation of Smad5. Constitutively active forms
of ALK-1, ALK-2, BMPR-IA/ALK-3, and BMPR-IB/ALK-6 also induced the
nuclear translocation of Smad5 (our unpublished results; see Figure
4). Overexpression of Smad5 does not lead to its nuclear accumulation, although small fractions of Smad5 may
spontaneously translocate into the nucleus. Acceleration of Smad5-induced alkaline phosphatase induction by BMP-6 may thus be
induced by nuclear translocation of Smad5. Similar results were
obtained for Smad1.
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Inhibitory Smads Block the Differentiation of C2C12 Cells into Osteoblasts Induced by BMPs
Next, the effects of inhibitory Smads on differentiation of osteoblasts were determined. C2C12 cells were infected with the adenoviruses with Smad6 or Smad7 together with Smad5 and treated with BMP-6. Alkaline phosphatase activity was induced by BMP-6 and Smad5 but was inhibited by Smad6 or Smad7 (Figure 4A). Coinfection of Smad6 or Smad7 adenoviruses in the cells expressing BMPR-IA/ALK-3(QD) or BMPR-IB/ALK-6(QD) also prevented differentiation into osteoblast cells (Figure 4, B and C). Similar data were obtained when adenovirus carrying ALK-2(QD) was used (our unpublished results).
Smad5 was observed in the nucleus after stimulation of BMPR-IA/ALK-3(QD) or BMPR-IB/ALK-6(QD), when the cells were stained with anti-Smad5 antibody. However, coinfection of the Smad6 or Smad7 adenovirus clearly blocked the nuclear translocation of Smad5, and the Smad5 protein was observed predominantly in the cytoplasm (Figure 4, D and E). These findings indicate that Smad6 and Smad7 prevent the nuclear translocation of Smad5 and thus inhibit the differentiation of C2C12 cells into osteoblast-like cells.
Chondrogenic Differentiation of ATDC5 Cells by Type I Receptors
We next examined the effects of type I receptors on
differentiation into chondrogenic cells of a mouse teratocarcinoma cell line, ATDC5. Comparable expression levels of the type I receptors were
obtained when the immunoblotting was performed using
anti-HA antibody (Figure 5A). By 8 d
after infection, a certain number of the cells spontaneously
differentiated into chondrogenic cells, and formation of Alcian
Blue-positive cartilage nodules was observed, probably because of
endogenously produced BMPs (Figure 5B) (Shukunami et al.,
1996, 1998). The constitutively active type I receptors of the BMPR-I
group (BMPR-IA/ALK-3 and BMPR-IB/ALK-6) increased cartilage nodules,
whereas those of the ALK-1 group (ALK-1 and ALK-2) did not.
Chondrogenic differentiation was also examined by the
in corporation of [35S]sulfate
into glycosaminoglycans 10 d after adenovirus infection. In
agreement with the results obtained by Alcian Blue staining, BMPR-IA/ALK-3(QD) and BMPR-IB/ALK-6(QD), but not the other type I
receptors, induced incorporation of
[35S]sulfate (Figure 5C).
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Because chondrogenic differentiation is spontaneously induced in ATDC5
cells, we tested the effects of kinase-inactive forms of type I
receptors, which act as dominant-negative receptors (Imamura et
al., 1997). BMPR-IA/ALK-3(KR) and BMPR-IB/ALK-6(KR) prevented the
cartilage nodule formation of ATDC5 cells (Figure 6). In addition, ALK-1(KR) and ALK-2(KR),
but not TR-I/ALK-5(KR) or ActR-IB/ALK-4(KR), blocked chondrogenic
differentiation. Thus, the constitutively active type I receptors of
the ALK-1 group failed to induce chondrogenic differentiation, whereas
the kinase-inactive receptors of the ALK-1 group blocked this
differentiation.
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Effects of R-Smads and Inhibitory Smads on Chondrogenic Differentiation
To determine whether Smads are involved in chondrogenic
differentiation of ATDC5 cells, adenoviruses carrying Smad1-Smad7 were
infected into these cells. None of the Smads efficiently induced Alcian
Blue-positive cartilage nodule formation in the absence (Figure
7A) or presence (our unpublished results)
of BMPR-IB/ALK-6(QD). In the presence of BMPR-IB/ALK-6(KR),
BMP-specific R-Smad did not induce cartilage nodule formation in the
presence or absence of Smad4 (our unpublished results). However, Smad6
and Smad7 strongly inhibited the formation of cartilage nodules,
indicating that R-Smads and Co-Smads do not induce chondrogenic
differentiation but that inhibitory Smads can block it. Chondrogenic
differentiation was also studied by the incorporation of
[35S]sulfate (Figure 7B). In agreement with the
results of histochemical analysis, Smad6 and Smad7 inhibited the
chondrogenic differentiation of ATDC5 cells.
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DISCUSSION |
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|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Induction of Osteoblast Differentiation by Type I Receptors of BMPR-I and ALK-1 Groups
BMP-2, BMP-4, BMP-6, and OP-1/BMP-7 have been shown to induce
osteoblast and chondroblast differentiation both in vitro and in vivo
(reviewed in Kawabata et al., 1998a). In addition, GDF-5 and
its related proteins play important roles in chondrocyte
differentiation (Hötten et al., 1996; Wolfman et
al., 1997; Francis-West et al., 1999; Tsumaki et
al., 1999). BMPR-IA/ALK-3 and BMPR-IB/ALK-6 are type I receptors
that specifically bind BMPs. GDF-5 was shown to bind predominantly to
BMPR-IB/ALK-6, compared with other type I receptors (Nishitoh et
al., 1996). In contrast, ALK-2 was shown to be a type I receptor
for OP-1/BMP-7, BMP-6, and possibly other BMPs (ten Dijke et
al., 1994b; Macías-Silva et al., 1998; Ebisawa et al., 1999) (our unpublished results). Thus, the type I
receptors of the BMPR-I group as well as certain members of the ALK-1
group act as type I receptors for BMPs. A difference in the biological effects of BMPR-IA/ALK-3 and BMPR-IB/ALK-6 has been reported (Zou et al., 1997; D. Chen et al., 1998); D. Chen
et al. showed that BMPR-IA/ALK-3 is important for adipocyte
differentiation, whereas BMPR-IB/ALK-6 is critical for osteoblast
differentiation and apoptosis (D. Chen et al., 1998). No
functional difference was found between the two receptors in our
assays, probably because of the difference in cell types used in the
present study.
The specificity of the intracellular signals is determined by the L45
loops of type I receptors, which are composed of nine amino acid
residues (Feng and Derynck, 1997). The L45 loop of type I receptors
interacts with specific sequences in the C-terminal Mad homology
2 domain of R-Smads, including the L3 loop and 
-helix 1 (Lo et
al., 1998; Y.G. Chen et al., 1998; Chen and
Massagué, 1999). The L45 loops of BMPR-IA/ALK-3 and BMPR-IB/ALK-6
are identical to each other, and those of the TR-I group are also
identical to each other. However, the L45 loops of BMPR-IA and BMPR-IB
and those of the TR-I group differ at four amino acid residues; this appears to be most important for the specific interaction with different R-Smads. Thus, the BMPR-I group activates Smad1, Smad5, and
presumably Smad8, whereas the TR-I group phosphorylates Smad2 and
Smad3. Although the L45 loop of the ALK-1 group diverges from those of
the other type I receptors, it was shown to interact with Smad1 and
Smad5, similar to that of the BMPR-I group (Chen and Massagué,
1999) (our unpublished results). Thus, the ability of type I receptors
to induce alkaline phosphatase activity correlated with their ability
to activate Smad1 and Smad5.
Smad1 and Smad5 Induce Alkaline Phosphatase Activity in C2C12 Cells
An important question with regard to signal transduction by serine/threonine kinase receptors is whether Smads alone are sufficient to induce osteoblast differentiation. As shown in Figure 2, Smad1 and Smad5 could induce osteoblast differentiation, which was enhanced by the presence of Co-Smad, Smad4. However, the differentiation-inducing effects of Smad1 and Smad5 in the presence of Smad4 were less potent than were those of the constitutively active type I receptors. When the cells were stimulated with BMP-6 at a concentration that did not fully induce osteoblast differentiation in culture, Smad1 and Smad5 dramatically induced alkaline phosphatase activity. This may have been because R-Smads efficiently translocated into the nucleus upon ligand stimulation; R-Smads were otherwise predominantly localized in the cytoplasm. Nuclear localization of R-Smads is thus an important event in their biological activity, because they act as transcription factors together with other DNA-binding proteins. Our present findings suggest that Smads are major signaling molecules for the differentiation of osteoblasts, but it is still possible that other signaling pathways independent of that of Smads, which act cooperatively with Smad pathways, may be required for efficient osteoblast differentiation induction.
Inhibitory Smads Inhibit Osteoblast Differentiation
Smad6 and Smad7 have been shown to inhibit the effects of R-Smads
by competing for binding to activated type I receptors (Hayashi et al., 1997; Imamura et al., 1997; Nakao
et al., 1997). It was also shown that Smad6 inhibits the
activity of Smad1 by competing for complex formation with Smad4 (Hata
et al., 1998). Inhibition of BMP signals by inhibitory Smads
has been reported using Xenopus assays (Bhushan et
al., 1998; Hata et al., 1998; Nakayama et
al., 1998a,b), but their effects on osteoblast differentiation
have not been reported. In the present study, we showed that both Smad6 and Smad7 inhibited the osteoblast differentiation induced by ligand or
receptor stimulation. In the presence of inhibitory Smads, Smad5
(Figure 4, D and E) and Smad1 (our unpublished results) were detected
in the cytoplasm, supporting the notion that inhibitory Smads exhibit
their effects by inhibiting the activation of R-Smads. These findings
again indicate that Smad pathways are essential for the induction of
osteoblast differentiation.
We have shown previously that Smad6 inhibits the activation of R-Smad
by BMPR-IB/ALK-6 but not efficiently that by BMPR-IA/ALK-3 (Imamura
et al., 1997). However, using constitutively active forms of
type I receptors in the adenovirus vector, which allowed us to obtain
sufficient protein expression levels, we found that both Smad6 and
Smad7 inhibit the activation of Smad1 and Smad5 by BMPR-IA/ALK-3 and
BMPR-IB/ALK-6, as well as that by ALK-2 (Figure 4; our unpublished results).
Induction of Chondrogenesis by Different Type I Receptors
Formation of cartilaginous bone rudiments is a critical step in
the initiation of endochondral bone formation. BMPs, including BMP-2,
BMP-4, and OP-1/BMP-7, have been shown to regulate the growth and
maturation of chondrocytes in vitro (Luyten et al., 1994;
Rosen et al., 1994; Asahina et al., 1996;
Shukunami et al., 1998). Stimulation of chondrogenesis by
BMPR-IA/ALK-3 and BMPR-IB/ALK-6 was also demonstrated in vivo (Zou
et al., 1997). We therefore studied the chondrogenic
differentiation induced by type I receptors using ATDC5 cells. The
receptors of the BMPR-I group, but not those of the ALK-1 group,
induced chondrogenic differentiation (Figure 5). The lack of ability of
ALK-1 and ALK-2 to induce differentiation of ATDC5 cells suggests that
Smads may not be sufficient for chondrogenic differentiation. In
agreement with this notion, none of the Smads efficiently induced
cartilage formation in vitro (Figure 7).
Prevention of Chondrogenic Differentiation by Kinase-inactive Type I Receptors and Inhibitory Smads
ATDC5 cells spontaneously form cartilage nodules in the absence of
exogenously added BMPs, possibly because of BMPs endogenously produced
by these cells. Spontaneous cartilage nodule formation was blocked by
the kinase-inactive forms of type I receptors of the BMPR-I group as
well as those of the ALK-1 group (Figure 6). Moreover, inhibitory
Smads, Smad6 and Smad7, efficiently blocked ATDC5 differentiation.
These findings suggest that the type I receptors of the BMPR-I group
activate the Smad pathway as well as Smad-independent signaling
pathways and that the latter may not be efficiently activated by ALK-1
or ALK-2. Smad-dependent and -independent pathways may be required to
act in concert for chondrogenic differentiation. Type I receptors have
been shown to activate various Smad-independent signaling pathways,
including ERK, JNK, and p38 MAP kinase pathways (Hartsough and Mulder,
1995; Atfi et al., 1997; Hannigan et al.,
1998; Liberati et al., 1999). Production of fibronectin has
been shown recently to be induced by the JNK pathway (Hocevar et
al., 1999). It may thus be important to determine the
Smad-independent signaling pathways that are involved in chondrogenic differentiation.
Our present data revealed that osteoblast differentiation of C2C12 cells is induced by the type I receptors of the BMPR-I and ALK-1 groups, the signaling from which appears to be mainly transmitted by the Smad pathways. In contrast, chondrogenic differentiation of ATDC5 cells is induced by BMPR-IA/ALK-3 and BMPR-IB/ALK-6, and Smads may be required, but not sufficient, for this differentiation. Further studies will be needed to identify the signaling pathways responsible for these biological effects and to elucidate whether there are cooperative or antagonistic effects between Smad-dependent and -independent signaling pathways.
| |
ACKNOWLEDGMENTS |
|---|
We thank Drs. Chisa Shukunami and Yuji Hiraki for valuable comments, Peter ten Dijke and Carl-Henrik Heldin for the Smad5 antibody, and Yasufumi Yuuki, Yuri Inada, and Aki Hanyu for technical help. This study was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan and by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency.
| |
FOOTNOTES |
|---|
Corresponding author. E-mail address:
fjios2{at}dent.tmd.ac.jp.
| |
ABBREVIATIONS |
|---|
Abbreviations used:
ActR, activin receptor;
ALK, activin
receptor-like kinase;
BMP, bone morphogenetic protein;
BMPR, BMP
receptor;
Co-Smad, common partner Smad;
FBS, fetal bovine serum;
GDF, growth-differentiation factor;
HA, hemagglutinin;
m.o.i., multiplicity
of infection;
OP, osteogenic protein;
PBS, phosphate-buffered saline;
R-Smad, receptor-regulated Smad;
TGF-, transforming growth
factor-;
TR, TGF- receptor.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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N. Dumont, A. V. Bakin, and C. L. Arteaga Autocrine Transforming Growth Factor-beta Signaling Mediates Smad-independent Motility in Human Cancer Cells J. Biol. Chem., January 24, 2003; 278(5): 3275 - 3285. [Abstract] [Full Text] [PDF]
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A. Ishisaki, H. Hayashi, A.-J. Li, and T. Imamura Human Umbilical Vein Endothelium-derived Cells Retain Potential to Differentiate into Smooth Muscle-like Cells J. Biol. Chem., January 3, 2003; 278(2): 1303 - 1309. [Abstract] [Full Text] [PDF]
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 S. Lamouille, C. Mallet, J.-J. Feige, and S. Bailly Activin receptor-like kinase 1 is implicated in the maturation phase of angiogenesis Blood, December 15, 2002; 100(13): 4495 - 4501. [Abstract] [Full Text] [PDF]
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M. Shen, E. Yoshida, W. Yan, T. Kawamoto, K. Suardita, Y. Koyano, K. Fujimoto, M. Noshiro, and Y. Kato Basic Helix-loop-helix Protein DEC1 Promotes Chondrocyte Differentiation at the Early and Terminal Stages J. Biol. Chem., December 13, 2002; 277(51): 50112 - 50120. [Abstract] [Full Text] [PDF]
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M. Tomita, M. I. Reinhold, J. D. Molkentin, and M. C. Naski Calcineurin and NFAT4 Induce Chondrogenesis J. Biol. Chem., October 25, 2002; 277(44): 42214 - 42218. [Abstract] [Full Text] [PDF]
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 J. Skillington, L. Choy, and R. Derynck Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes J. Cell Biol., October 14, 2002; 159(1): 135 - 146. [Abstract] [Full Text] [PDF]
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U. Valcourt, J. Gouttenoire, A. Moustakas, D. Herbage, and F. Mallein-Gerin Functions of Transforming Growth Factor-beta Family Type I Receptors and Smad Proteins in the Hypertrophic Maturation and Osteoblastic Differentiation of Chondrocytes J. Biol. Chem., September 6, 2002; 277(37): 33545 - 33558. [Abstract] [Full Text] [PDF]
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A. Nishihara, T. Watabe, T. Imamura, and K. Miyazono Functional Heterogeneity of Bone Morphogenetic Protein Receptor-II Mutants Found in Patients with Primary Pulmonary Hypertension Mol. Biol. Cell, September 1, 2002; 13(9): 3055 - 3063. [Abstract] [Full Text] [PDF]
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C.-F. Lai and S.-L. Cheng Signal Transductions Induced by Bone Morphogenetic Protein-2 and Transforming Growth Factor-beta in Normal Human Osteoblastic Cells J. Biol. Chem., May 3, 2002; 277(18): 15514 - 15522. [Abstract] [Full Text] [PDF]
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B.-C. Kim, M. Mamura, K. S. Choi, B. Calabretta, and S.-J. Kim Transforming Growth Factor {beta}1 Induces Apoptosis through Cleavage of BAD in a Smad3-Dependent Mechanism in FaO Hepatoma Cells Mol. Cell. Biol., March 1, 2002; 22(5): 1369 - 1378. [Abstract] [Full Text] [PDF]
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A. Hoffmann, S. Czichos, C. Kaps, D. Bachner, H. Mayer, Y. Zilberman, G. Turgeman, G. Pelled, G. Gross, and D. Gazit The T-box transcription factor Brachyury mediates cartilage development in mesenchymal stem cell line C3H10T1/2 J. Cell Sci., February 15, 2002; 115(4): 769 - 781. [Abstract] [Full Text] [PDF]
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S. Bai and X. Cao A Nuclear Antagonistic Mechanism of Inhibitory Smads in Transforming Growth Factor-beta Signaling J. Biol. Chem., February 1, 2002; 277(6): 4176 - 4182. [Abstract] [Full Text] [PDF]
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W. Samuel, C. N. Nagineni, R. K. Kutty, W. T. Parks, J. S. Gordon, S. M. Prouty, J. J. Hooks, and B. Wiggert Transforming Growth Factor-beta Regulates Stearoyl Coenzyme A Desaturase Expression through a Smad Signaling Pathway J. Biol. Chem., January 4, 2002; 277(1): 59 - 66. [Abstract] [Full Text]
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 J. Theilhaber, T. Connolly, S. Roman-Roman, S. Bushnell, A. Jackson, K. Call, T. Garcia, and R. Baron Finding Genes in the C2C12 Osteogenic Pathway by k-Nearest-Neighbor Classification of Expression Data Genome Res., January 1, 2002; 12(1): 165 - 176. [Abstract] [Full Text] [PDF]
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A. Hanyu, Y. Ishidou, T. Ebisawa, T. Shimanuki, T. Imamura, and K. Miyazono The N domain of Smad7 is essential for specific inhibition of transforming growth factor-{beta} signaling J. Cell Biol., December 10, 2001; 155(6): 1017 - 1028. [Abstract] [Full Text] [PDF]
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P. Tylzanowski, K. Verschueren, D. Huylebroeck, and F. P. Luyten Smad-interacting Protein 1 Is a Repressor of Liver/Bone/Kidney Alkaline Phosphatase Transcription in Bone Morphogenetic Protein-induced Osteogenic Differentiation of C2C12 Cells J. Biol. Chem., October 19, 2001; 276(43): 40001 - 40007. [Abstract] [Full Text] [PDF]
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H Aoki, M Fujii, T Imamura, K Yagi, K Takehara, M Kato, and K Miyazono Synergistic effects of different bone morphogenetic protein type I receptors on alkaline phosphatase induction J. Cell Sci., January 4, 2001; 114(8): 1483 - 1489. [Abstract] [PDF]
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 F. De Luca, K. M. Barnes, J. A. Uyeda, S. De-Levi, V. Abad, T. Palese, V. Mericq, and J. Baron Regulation of Growth Plate Chondrogenesis by Bone Morphogenetic Protein-2 Endocrinology, January 1, 2001; 142(1): 430 - 436. [Abstract] [Full Text] [PDF]
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K.-S. Lee, H.-J. Kim, Q.-L. Li, X.-Z. Chi, C. Ueta, T. Komori, J. M. Wozney, E.-G. Kim, J.-Y. Choi, H.-M. Ryoo, et al. Runx2 Is a Common Target of Transforming Growth Factor beta 1 and Bone Morphogenetic Protein 2, and Cooperation between Runx2 and Smad5 Induces Osteoblast-Specific Gene Expression in the Pluripotent Mesenchymal Precursor Cell Line C2C12 Mol. Cell. Biol., December 1, 2000; 20(23): 8783 - 8792. [Abstract] [Full Text]
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Y.-W. Zhang, N. Yasui, K. Ito, G. Huang, M. Fujii, J.-i. Hanai, H. Nogami, T. Ochi, K. Miyazono, and Y. Ito A RUNX2/PEBP2alpha A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia PNAS, August 23, 2000; (2000) 180309597. [Abstract] [Full Text]
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A. Yamaguchi, T. Komori, and T. Suda Regulation of Osteoblast Differentiation Mediated by Bone Morphogenetic Proteins, Hedgehogs, and Cbfa1 Endocr. Rev., August 1, 2000; 21(4): 393 - 411. [Abstract] [Full Text] [PDF]
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W. Ishida, T. Hamamoto, K. Kusanagi, K. Yagi, M. Kawabata, K. Takehara, T. K. Sampath, M. Kato, and K. Miyazono Smad6 Is a Smad1/5-induced Smad Inhibitor. CHARACTERIZATION OF BONE MORPHOGENETIC PROTEIN-RESPONSIVE ELEMENT IN THE MOUSE Smad6 PROMOTER J. Biol. Chem., February 25, 2000; 275(9): 6075 - 6079. [Abstract] [Full Text] [PDF]
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H. Watanabe, M. P. de Caestecker, and Y. Yamada Transcriptional Cross-talk between Smad, ERK1/2, and p38 Mitogen-activated Protein Kinase Pathways Regulates Transforming Growth Factor-beta -induced Aggrecan Gene Expression in Chondrogenic ATDC5 Cells J. Biol. Chem., April 20, 2001; 276(17): 14466 - 14473. [Abstract] [Full Text] [PDF]
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S. Nishimori, Y. Tanaka, T. Chiba, M. Fujii, T. Imamura, K. Miyazono, T. Ogasawara, H. Kawaguchi, T. Igarashi, T. Fujita, et al. Smad-mediated Transcription Is Required for Transforming Growth Factor-beta 1-induced p57Kip2 Proteolysis in Osteoblastic Cells J. Biol. Chem., March 30, 2001; 276(14): 10700 - 10705. [Abstract] [Full Text] [PDF]
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M. Izumi, Y. Fujio, K. Kunisada, S. Negoro, E. Tone, M. Funamoto, T. Osugi, Y. Oshima, Y. Nakaoka, T. Kishimoto, et al. Bone Morphogenetic Protein-2 Inhibits Serum Deprivation-induced Apoptosis of Neonatal Cardiac Myocytes through Activation of the Smad1 Pathway J. Biol. Chem., August 10, 2001; 276(33): 31133 - 31141. [Abstract] [Full Text] [PDF]
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E. Piek, W. J. Ju, J. Heyer, D. Escalante-Alcalde, C. L. Stewart, M. Weinstein, C. Deng, R. Kucherlapati, E. P. Bottinger, and A. B. Roberts Functional Characterization of Transforming Growth Factor beta Signaling in Smad2- and Smad3-deficient Fibroblasts J. Biol. Chem., June 1, 2001; 276(23): 19945 - 19953. [Abstract] [Full Text] [PDF]
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K. Pardali, A. Kurisaki, A. Moren, P. ten Dijke, D. Kardassis, and A. Moustakas Role of Smad Proteins and Transcription Factor Sp1 in p21Waf1/Cip1 Regulation by Transforming Growth Factor-beta J. Biol. Chem., September 15, 2000; 275(38): 29244 - 29256. [Abstract] [Full Text] [PDF]
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Y.-W. Zhang, N. Yasui, K. Ito, G. Huang, M. Fujii, J.-i. Hanai, H. Nogami, T. Ochi, K. Miyazono, and Y. Ito A RUNX2/PEBP2alpha A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia PNAS, September 12, 2000; 97(19): 10549 - 10554. [Abstract] [Full Text] [PDF]
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