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Fourth Department of Internal Medicine (M.S., Y.T., S.F., T.F.), University of Tokyo School of Medicine, Tokyo 112, Institute of Molecular and Cellular Biology (M.S., S.K.), University of Tokyo, Tokyo 113, Japan; National Institute for Physiological Sciences (N.U.), Okazaki, Cancer Institute Hospital (K.M.), First Department of Internal Medicine (T.M.), University of Tokushima School of Medicine, Tokushima 770, Japan
Address all correspondence and requests for reprints to: Yasuhiro Takeuchi, M.D., Fourth Department of Internal Medicine, University of Tokyo School of Medicine, 3–28-6 Mejirodai, Bunkyo-ku, Tokyo 112-8688, Japan. E-mail: takeuchi-tky{at}umin.ac.jp
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Introduction |
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Cells prepared from infantile or neonatal bone are able to differentiate into mature osteoblasts that form bone-like nodules in vitro in the presence of serum and ascorbic acid without any other supplements (16, 17). This indicates that eutopic cells in bone have intrinsic mechanisms that promote their differentiation into osteoblasts for bone formation. However, it is yet uncertain what is the mechanism for osteoblastic differentiation and how it works. Impairment of the mechanism could contribute to the pathogenesis of osteoporosis, a principal metabolic disease of losing bone mass, and the way by which it is restored must be useful for the prevention and improvement of bone loss.
The present study was undertaken to explore the mechanisms for osteoblastic differentiation of cells in the osteoblast lineage using nontransformed cell line MC3T3-E1 derived from neonatal mouse calvariae. We present results indicating that endogenously synthesized BMP-2/4 promote osteoblastic differentiation if the cells accumulate collagenous extracellular matrices where BMP-2/4 are stored.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cloning of MC3T3-E1 cells constitutively expressing the
kinase-deficient BMP receptor-IA
Complementary DNA (cDNA) of kinase-deficient BMP receptor type
IA (BMPR-IA) (18) was subcloned into the expression plasmid pcDNA3
(Invitrogen, Carlsbad, CA). This plasmid contains a
3'-nested deletion clone of mouse BMPR-IA, and the cDNA codes for the
extracellular domain and transmembrane domain of the receptor. MC3T3-E1
cells were cultured in MEM supplement with 10% FBS in the presence and
absence of 50 mg/liter L-ascorbic acid. These cells were
transfected at 40–60% confluence in 10-cm plastic dishes with pcDNA3
containing the kinase-deficient BMPR-IA or mock plasmid (pcDNA3 alone)
by calcium phosphate coprecipitation technique. Medium was freshly
changed after 16 h, and cells were maintained for further 48
h to reach confluence. Cells were, then dispersed and cultured in
MEM/10% FBS with G418 (300 µg/ml). Cells expressing mRNA for the
kinase-deficient BMPR-IA were cloned.
Alkaline phosphatase activity in osteoblastic cells
Cells were washed twice with ice-cold PBS and scraped in 10
mM Tris-HCl containing 2 mM MgCl2
and 0.05% Triton X-100, pH 8.2. The cell suspension was sonicated on
ice. Aliquots of supernatants were subjected to protein assay with a
kit from Bio-Rad Laboratories, Inc. (Hercules, CA),
according to Bradford’s method and to ALP activity measurement. In
brief, the assay mixture contained 10 mM p-nitrophenyl
phosphate in 0.1 M sodium carbonate buffer, pH 10,
supplemented with 1 mM MgCl2, followed by an
incubation at 37 C for 30 min. After adding 0.1 M NaOH, the
amount of p-nitrophenol liberated was measured by spectrophotometer
(17).
RNA isolation and RT-PCR
Poly (A)+ RNA was isolated from cells that were
cultured with or without ascorbic acid for 3, 5, and 10 days using the
MicroFastTrack Kit (Invitrogen). For RT-PCR analysis, 100
ng poly (A)+ RNA was treated with DNase I (Gibco BRL, Grand Island, NY) and reverse transcribed using random
9-mer (TAKARA RNA PCR Kit ver.2, TAKARA Co., Ohtsu, Japan). The 5'- and
3'-primers used were as follows: BMP-2, 5'-GACGGACTGCGGTCTCCTAAAG-3'
and 5'-TCTGCAGATGTGAGAAACTCGTCA-3'; BMP-4, 5'-CGCCGTCATTCCGGATTACAT-3'
and 5'-GGCCCAATCTCCACTCCCTT-3'; BMPR-IA,
5'-TAGCACCAGAGGATACCTTGC-3' and 5'-AATGCTTCATCCTGT-TCCAAA-3';
BMPR-IB, 5'-TGCTCTTACGAAGCTCT GGA-3' and 5'-ATCTCTGTCCTTGAGAGGAG-3';
BMPR-II, 5'-CAGAATCAAGGA-ACGGC TATG-3' and
5'-TTGTTTACGGTCTCCTGTCA-3'. Expression of the kinase-deficient BMPR-IA
mRNA was detected by RT-PCR using T7 primer as a 5'-primer and
5'-TGGGAGTGGCACCTTCCA-3', which corresponds to a sequence in the pcDNA3
plasmid as a 3'-primer. PCR conditions are as follows; for BMP-2,
denature 94 C for 1 min, annealing 56 C for 1 min, extension 74 C for 1
min, this process was repeated 40 cycles followed by 74 C for 7 min;
for BMP-4, denature 94 C for 30 sec, annealing 58 C for 30 sec,
extension 74 C for 1 min, this process was repeated 40 cycles followed
by 74 C for 7 min; for BMPR-IA, -IB, and -II, denature 94 C for 30 sec,
annealing 58 C for 30 sec, extension 74 C for 1 min, this process was
repeated 35 cycles followed by 74 C for 7 min; for the kinase-deficient
BMPR-IA, denature 94 C for 1 min, annealing 58 C for 30 sec, extension
74 C for 30 sec, this process was repeated 35 cycles followed by 74 C
for 10 min. Then, PCR products were electrophoresed in 1.0% agarose
gel and visualized by ethidiumbromide staining. As a negative control,
we carried out PCR amplification without RT. No PCR products were
obtained in these experiments for all pairs of primers.
Examination of intracellular distribution of Smad1
Cells expressing mRNA for the kinase-deficient BMPR-IA were
transiently transfected with the expression plasmid of
FLAG-tagged-Smad1 (19) with or without cotransfection of
wild-type BMPR-IA using Cell Electroporator (BRL,
Gaithersburg, MD), and then incubated overnight at 37 C. Cells were
once dispersed by trypsinization, then plated on Lab-Tek chamber slides
(Nunc, Roskilde, Denmark) and allowed to adhere overnight.
Intracellular distribution of Smad1 was examined as described (19). In
brief, cells were washed and incubated for 1.5 h in the medium
with 0.2% FBS in the absence or presence of 400 ng/ml rhBMP-2 before
fixing. Cells on the slides were blocked in 10% goat serum for 1
h at room temperature and then incubated overnight at 4 C in 10% goat
serum containing anti-FLAG M2 mAb (20 µg/ml). After washing four
times, FITC-conjugated goat antimouse IgG polyclonal antibody
(1:200 dilution) in 10% goat serum was added and incubated for 1
h at room temperature followed by washing twice. Intracellular
distribution of FLAG-tagged Smad1 was microscopically examined.
Immunocytochemical analysis of BMP-2 and BMP-4
The cells were fixed with 4% paraformaldehyde in PBS for 5 min.
After washes three times with PBS, cells were immersed in methanol for
1 min. They were treated with 3% hydrogen peroxide for 5 min, and were
washed with PBS. Expression of BMP-2/4 was determined by a mouse mAb
(h3b2/17.8.1) against rhBMP-2 using a LSAB kit (DAKO Corp., Kyoto, Japan). This antibody reacts with both BMP-2 and
BMP-4. Cells were treated with a biotinylated secondary antibody
against mouse IgG, and incubated with horseradish peroxidase-conjugated
streptavidin. Immunoreactivity in cell layers was detected using AEC
substrate-chromogen system (DAKO Corp.).
The localization of BMP-2/4 was determined by an immunogold technique with an electron microscopy using the same mAb. Cells on the six-well culture dishes were fixed in 4% paraformaldehyde/0.5% glutaraldehyde at 4 C for 30 min. Specimens were dehydrated with ethanol and embedded in epoxy resin. Ultrathin sections were made with a diamond knife on Ultracut N (Reichert-Nissei, Tokyo), and were examined with JEM-1200 EX II transmission electron microscope (Nippon Denshi, Tokyo) following the incubation with the primary antibody and the secondary antimouse IgG gold antibody. In brief, the specimen were incubated in PBS/1% BSA to saturate nonspecific binding sites. They were incubated for 1 h in PBS/0.5% BSA containing the anti-BMP-2/4 mAb, and washed with PBS followed by incubating with the antimouse IgG-gold antibody in PBS/0.5% BSA for 1 h.
Mineralized nodules stained by von Kossa’s method
MC3T3-E1 cells expressing mRNA for the kinase-deficient BMPR-IA
or pcDNA3 alone were cultured for 3 weeks in the presence of ascorbic
acid and 5 mM ß- glycerophosphate. Mineralized nodules
were stained with silver nitrate according to von Kossa’s procedure as
described (17).
Preparation of detergent-resistant extracellular matrix layers of
osteoblastic cells
MC3T3-E1 cells were cultured on 6-well plastic dishes in the
presence of ascorbic acid for 10 days. Cells were solubilized with
0.5% sodium deoxycholate in 10 mM Tris-HCl, pH 8.0, at 4 C
for 10 min and washed twice with the same buffer. Solubilized materials
were extensively wiped away by several washes with ice-cold PBS (20).
Extracellular matrix (ECM) components resistant to the detergent
remained on dishes and were stored on ice in 1 ml PBS in the presence
and absence of 10 µg soluble form of BMPR-IA (21) for 1 h
followed by washing 3 times with PBS. Freshly dispersed cells were
inoculated on the coated dishes and cultured in MEM/10% FBS without
ascorbic acid at 37 C in 5% CO2 atmosphere. Cells were
subjected to ALP assay after 24 h.
Statistical analysis
Data were expressed as means ± SE. Statistical
significance was evaluated by ANOVA followed by post hoc method of
Bonferroni. Unadjusted P value less than 0.05 was considered
to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
They remained at the premature stage with low ALP activity in the
absence of ascorbic acid. Detection of transcripts for BMP-2 and BMP-4
by RT-PCR indicated that MC3T3-E1 cells constitutively expressed both
BMPs through the differentiation process (Fig. 1, inset). To
confirm synthesis of BMPs by the cells, proteins of BMP-2/4 were
immunostained with a monoclonal antibody that specifically recognizes
both BMP-2 and BMP-4. Monolayer cells cultured in the presence and
absence of ascorbic acid, were diffusely stained with the anti-BMP
antibody (Fig. 2, A and B).
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). Much less ECM layers were accumulated in the absence of
ascorbic acid, and they did not present significant BMP
immunoreactivity (data not shown). By an electron microscopic
examination, BMP immunoreactivity was distributed along fibrous
structures around cells cultured with ascorbic acid, but was not
present in a pericellular space in the absence of ascorbic acid (Fig. 3). These results indicate that MC3T3-E1
cells constitutively express BMP-2 and BMP-4, and that these BMP
proteins are stored in the ECM produced by the cells.
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). Treatment with soluble BMPR-IA
suppressed ALP activity in cells on plastic dishes in the presence of
ascorbic acid to abrogate the effect of ascorbic acid (0.56 ±
0.06 vs. 0.38 ± 0.01 nmol/min·µg-protein in the
absence and presence of soluble BMPR-IA, respectively,
P < 0.01, n = 3), and only slightly lowered that
in the absence of ascorbic acid (Table 1). Conditioned media from
MC3T3-E1 cells at 10 days of culture in the presence and absence of
ascorbic acid did not stimulate ALP activity (data not shown). These
observations suggest that ECM-associated BMPs were responsible for the
stimulation of ALP activity in MC3T3-E1 cells. Although several other
growth factors than BMPs were possibly accumulated in the ECM, such
growth factors, for example TGF-ß1 (20), activin, insulin-like growth
factor I (IGF-I), epidermal growth factor, basic fibroblast
growth factor, failed to stimulate ALP activity in MC3T3-E1 cells (data
not shown).
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). Thus, the ECM containing BMPs could
induce the differentiation not only of cells in the osteoblast lineage
but of pluripotent mesenchymal cells into osteoblasts.
Expression of kinase-deficient BMP receptor type IA eradicates
response of osteoblastic cells to BMP-2
There are two forms of receptors for BMP-2/4. Those are
hetero-oligomers of BMPR-II with either BMPR-IA or BMPR-IB
serine/threonine kinases (23). According to RT-PCR method, MC3T3-E1
cells constitutively expressed both BMPR-IA and BMPR-II receptors at
any differentiation stages in the presence and absence of ascorbic acid
(Fig. 4). BMPR-IB was not detected in the
cells. Treatment of these cells with rhBMP-2 significantly stimulated
ALP activity in a dose-dependent fashion when cultured in the presence
of ascorbic acid (Fig. 5A) and in the
absence of ascorbic acid to a less extent (data not shown).
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BMPR-IA) to obliterate BMP-2/4
signals. When expressed in Xenopus oocytes, this BMPR-IA
blocks BMP signaling as a dominant negative (DN) receptor (18).
Mock-transfected cells were selected in the presence of G418 as
controls. Seven independent clones expressing BMPR-IA mRNA (DN
clones) normally attached, spreaded and proliferated on culture dishes.
Because characteristics of all clones examined were essentially the
same in terms of their responsiveness to BMP-2 and changes in ALP
activity with time, results obtained from representative clones (DN
clones 1 and 2) were presented. To confirm the expression of BMPR-IA
mRNA, transcribed pcDNA3 fragment containing BMPR-IA sequence in DN
clones 1 and 2 was amplified by a RT-PCR method (Fig. 5B). In control
cells, PCR product corresponding to pcDNA3 vector was obtained. In
contrast, longer PCR product containing BMPR-IA insert was observed
in DN clones. After binding to the receptor, BMP-2/4 activate a
signaling molecule Smad1 that is translocated from cytoplasm to nucleus
upon its phosphorylation by the receptor serine/threonine kinase (19).
We examined the nuclear translocation of FLAG-tagged Smad1 transiently
expressed in DN clone cells. Transfected Smad1 diffusely distributed in
cytoplasmic compartments in DN clone cells either in the presence or
absence of rhBMP-2 (Fig. 6, A and C).
Nuclear translocation of Smad1 upon the stimulation with 400 ng/ml
rhBMP-2 was only observed in DN cells overexpressing wild-type BMPR-IA
expression plasmid (Fig. 6, B and D). These results indicate that DN
clones are refractory to BMP-2 at their receptor level, and that this
refractoriness can be restored by overexpressing the wild-type
BMPR-IA.
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). The effect of
rhBMP-2 was only slight but dose dependent up to 400 ng/ml in DN
clones, although it was saturated at 200–300 ng/ml in control cells.
In addition, ALP activity without rhBMP-2 was significantly lower in DN
clones than the control (Fig. 5A). The effect of rhBMP-2 on the
expression of mRNA for osteocalcin, a late osteoblastic differentiation
marker, in DN clones was also negligible, and its basal level was less
in DN clones than that in control cells (Fig. 5C). Thus, DN clones were
unresponsive to the stimulatory effect of BMP-2 on the osteoblastic
differentiation by the expression of BMPR-IA.
Signaling from BMP receptors are required for osteoblastic
differentiation
Increasing ALP activity during the long-term culture in the
presence of ascorbic acid was observed in control MC3T3-E1 cells but
not in DN clones (Fig. 7A). ALP
activities in DN clones after 10 days of culture were similar to those
in control cells in the absence of ascorbic acid. Cultures for 3 weeks
in the presence of ascorbic acid and 5 mM ß-
glycerophosphate induced to form mineralized nodules in the control
cells, but there were no nodules in cultures of DN clones (Fig. 7B).
mRNA expression of BMP-2 and BMP-4 was observed both in DN clones and
in control cells (Fig. 7C). These observations indicate that BMPR-IA
and its downstream signals are essentially required for differentiation
of osteoblastic cells.
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). ECM layers of DN clones stimulated
ALP activity in control cells (Table 2) but not in DN clone cells (data
not shown), indicating that ECM layers of DN clones were potent enough
to promote osteoblastic differentiation. Results shown here confirm
that DN clones fail to express osteoblastic phenotypes not due to less
competent extracellular milieu or lack of expression of BMP-2/4 but due
to inert receptors for BMP-2/4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 3), and because soluble BMPR-IA completely
blocked the stimulatory effect of the ECM (Table 1). Furthermore, high
ALP activity was induced in nonosteoblastic mesenchymal cells, when
they were maintained on the ECM. Thus, one can assume that the ECM
containing BMPs is effective to induce osteoblastic differentiation. We
did not exclude possibility that other members of BMP family, such as
BMP-6, BMP-7 and GDF-5, known to stimulate osteoblastic
differentiation, might also be accumulated in the ECM of MC3T3-E1
cells, and that they were involved in the stimulatory effect. There are several possibilities that explain the reason why the ECM containing BMPs is competent enough for the differentiation. The first is that the detergent-resistant ECM contains several growth factors, such as TGF-ßs and IGFs (26). Growth factors other than BMPs are also involved in the bone formation. TGF-ß is synthesized by osteoblastic cells (27) and is accumulated in bone matrix, and so is IGF-I (28). TGF-ß strongly stimulates the synthesis and the accumulation of bone matrix proteins by osteoblasts (5, 29). IGF-I enhances the proliferation of cells in the osteoblast lineage (30). Both of them stimulate bone formation (31, 32), and decreases in these factors in bone matrices are proposed to be associated with loss of bone mass. TGF-ß content in bone is less in ovariectomized rats (33). It is also reported that TGF-ß and IGF-I accumulated in human bone matrices decrease with advancing age (34). However, no growth factors but BMPs are able to promote osteoblastic differentiation of nonosteogenic cells. TGF-ß has been shown to suppress the expression of osteoblastic phenotypes and the formation of mineralized nodules in osteoblastic cell cultures (16). Thus, BMPs could be principal factors in the ECM of MC3T3-E1 cells that direct cells in the osteoblast lineage toward mature osteoblasts, and TGF-ß and IGFs may exert their effects on bone formation in a different way from BMPs.
The second one is that ECM proteins, such as collagen and fibronectin,
are also involved in the stimulation of osteoblastic differentiation.
We have reported that an interaction between type I collagen and
2ß1 integrin on osteoblastic cells is required for the promotion
of their differentiation (20). Their interaction activates focal
adhesion kinase (FAK) followed by an activation of its downstream
pathways including MAP kinase, and this FAK-MAP kinase cascade is
involved in the differentiation process (35). Because rhBMP-2 do not
directly enhance the activation of FAK or MAP kinase (unpublished
observations), and because endogenous BMPs are shown to be involved in
the differentiation, it is suggested that BMP signals stimulate
osteoblastic differentiation in conjunction with the integrin-FAK-MAP
kinase pathway activated by ECM proteins. Indeed, it is reported that
Ras, Raf, and AP-1, which are closely associated with the
integrin-FAK-MAP kinase pathway, are involved in BMP-4 signaling during
Xenopus embryonic development (36). The third possibility is
that immobilized BMPs in the ECM may be protected from proteolytic
degradation and ready to access to their receptors on the cell-surface
when cells attach to ECM components. Taken together, results in the
present study suggest that BMP-2/4 accumulated in ECM layers are highly
competent to stimulate osteoblastic differentiation. Thus, endogenous
BMPs are particularly important for the development of osteoblastic
phenotypes if they are accumulated in ECM layers. Although BMPs have
been reported to be bound to type IV collagen and other matrix
components (37), the mechanism whereby they were accumulated in the ECM
of osteoblastic MC3T3-E1 cells remains to be elucidated.
There has been no direct evidence indicating that BMPs initiate and
support osteoblastic differentiation during modeling and remodeling
process of bone. To clarify if BMPs are indispensable for the
differentiation of osteoblasts, we cloned MC3T3-E1 cells stably
expressing a dominant negative receptor for BMP-2/4 (BMPR-IA) (18).
Although there is another type I receptor for BMP-2/4, BMPR-IB,
MC3T3-E1 cells express only BMPR-IA. Thus, BMPR-IA is able to
eradicate effects of BMP-2/4 in these cells. BMPR-IA expressing
cells (DN clones) we selected were refractory to BMP-2 at the step
before the nuclear translocation of Smad1, an immediate signal
transducer of BMPR-I, and failed to express osteoblastic phenotypes
with culture periods of time. Because expressions of BMP-2/4 were
observed in DN clones and the capability of their ECM layers to
stimulate ALP activity in control cells were not less than that of
control cells, signals generated by BMPR-IA were essentially required
for the spontaneous osteoblastic differentiation of MC3T3-E1 cells.
MC3T3-E1 cells do not differentiate in the absence of ascorbic acid because matrix type I collagen does not sufficiently accumulate in extracellular spaces (20, 22). BMP-2/4 and their receptors were all expressed in these cells even in the absence of ascorbic acid. This and results described above suggest that endogenous BMP-2/4 exert their effect if cells accumulate collagen-rich ECM layers. This hypothesis is further corroborated by the fact that ALP activity in control cells increased even in the absence of ascorbic acid on either ECM layer deposited by DN clones or control MC3T3-E1 cells. Therefore, both appropriate ECM layers and BMP actions are required for the osteoblastic differentiation.
A previous report showing that BMP-4 is transiently but highly expressed at fracture sites during the healing process (15) supports our notion that endogenous BMPs are important for the osteoblastic differentiation that promotes bone formation. In another aspect of pathophysiology, augmented BMP actions might be involved in the pathogenesis of some diseases. It is suggested that the expression of BMP receptors is involved in the development of ossification of paravertebral ligaments (38). Osteosarcoma cells sometimes strongly express members of BMP family (39), which may constitutively activate the signals out of physiological regulations and may be involved in their oncogenesis. Results presented here demonstrate that the regulatory mechanism of BMP-2/4 actions in osteoblastic cells as well as that of the ECM production is one of principal issues to be elucidated in the control of bone formation for better understanding of pathogenesis of osteoporosis and for development of its therapeutic procedures.
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Acknowledgments |
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Footnotes |
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Received September 1, 1998.
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2ß1 integrin
interaction is mediated by focal adhesion kinase and its downstream
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