Characterization of osteoblastic differentiation of stromal
cell line ST2 that is induced by ascorbic acid
Eri
Otsuka1,
Akira
Yamaguchi2,
Shigehisa
Hirose3, and
Hiromi
Hagiwara1
1 Research Center for
Experimental Biology and
3 Department of Biological
Sciences, Tokyo Institute of Technology, Yokohama 226-8501; and
2 Department of Oral
Pathology, Nagasaki University School of Dentistry, Nagasaki
852-8588, Japan
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ABSTRACT |
The stromal cell line ST2, derived from mouse bone marrow,
differentiated into osteoblast-like cells in response to ascorbic acid.
Ascorbic acid induced alkaline phosphatase (ALPase) activity, the
expression of mRNAs for proteins that are markers of osteoblastic differentiation, the deposition of calcium, and the formation of
mineralized nodules by ST2 cells. We investigated the mechanism whereby
ascorbic acid induced the differentiation of ST2 cells. Inhibitors of
the formation of collagen triple helices completely blocked the effects
of ascorbic acid on ST2 cells, an indication that matrix formation by
type I collagen is essential for the induction of osteoblastic
differentiation of ST2 cells by ascorbic acid. We furthermore examined
the effects of bone morphogenetic proteins (BMPs) on the
differentiation of ST2 cells induced by ascorbic acid. Ascorbic acid
had no effect on the expression of mRNAs for BMP-4 and the BMP
receptors. However, a soluble form of BMP receptor IA
inhibited the induction of ALPase activity by ascorbic acid. These
results suggest that ascorbic acid might promote the differentiation of
ST2 cells into osteoblast-like cells by inducing the formation of a
matrix of type I collagen, with subsequent activation of the signaling
pathways that involve BMPs.
ST2 cell; osteoblast; type I collagen; alkaline phosphatase; bone
morphogenetic protein
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INTRODUCTION |
ST2 CELLS, a clone of stromal cells, were
isolated from the bone marrow of BC8 mice and have characteristics
typical of preadipocytes (13). They do not exhibit an osteoblastic
phenotype in standard cultures. ST2 cells are generally maintained in
RPMI 1640 medium, which does not include ascorbic acid. However, when
we subcultured ST2 cells in RPMI 1640 medium that had been supplemented
with ascorbic acid, they developed an osteoblastic phenotype.
Ascorbic acid is necessary for the expression of osteoblastic markers
and for mineralization in a variety of osteoblast culture systems that
include primary cultures of fetal rat calvarial cells (1), chick
preosteoblasts (7), and osteoblast-like cell lines (5, 6, 17, 18). In
particular, MC3T3-E1 cells, derived from mouse calvaria, have been used
to study the role of ascorbic acid in osteoblast differentiation (5, 6,
17-19). The addition of ascorbic acid to cultures of clonal
preosteoblastic MC3T3-E1 cells stimulates the deposition of a
collagenous extracellular matrix, followed by the induction of specific
genes associated with the osteoblastic phenotype, such as genes for
alkaline phosphatase (ALPase) (5, 6) and osteocalcin (5, 6, 22).
ST2 cells also differentiate into osteoblast-like cells when bone
morphogenetic proteins (BMPs) are added to the culture medium (12, 23).
These proteins belong to the transforming growth factor-
superfamily, and they induce the development
and differentiation of bone cells and the formation of bone (9, 10, 21,
24). In mesenchymal cells, BMPs are thought to induce stem cell
precursors to commit to the osteoblast lineage and to express the
osteoblast phenotype. However, there is little evidence for
interactions between the effects of ascorbic acid and those of BMPs.
In the present study, we found that ST2 cells that had been cultured
with ascorbic acid exhibited characteristics typical of osteoblasts,
with induction of the expression of mRNAs for marker proteins of
osteoblastic differentiation and the formation of mineralized nodules.
We attempted to characterize the mechanism whereby ascorbic acid
induces the osteoblastic differentiation of ST2 cells by using the
induction of ALPase activity as an index. The effects of ascorbic acid
were inhibited by inhibitors of the formation of triple helices of type
I collagen. Furthermore, we obtained evidence to suggest that the
ascorbic acid-induced differentiation of ST2 cells might
involve BMPs and their receptors.
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MATERIALS AND METHODS |
Materials.
L-Ascorbic acid was obtained
from Wako Pure Chemical Industries, Osaka, Japan.
3,4-Dehydro-L-proline (DHP) and
cis-4-hydroxy-L-proline (CHP) were purchased from Sigma (St. Louis, MO). Recombinant human BMP-2 produced in Chinese hamster ovary cells was provided by Yamanouchi Pharmaceutical (Tsukuba, Japan). Soluble BMP receptor IA
(sBMPR-IA) was kindly provided by Drs. N. Ueno and S. Iemura (National
Institute for Basic Biology, Okazaki, Japan).
32P-labeled nucleotides were
obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). RPMI
1640 medium, fetal bovine serum, and a penicillin-streptomycin
antibiotic mixture were obtained from Life Technologies (Grand Island, NY).
Cell culture.
ST2 cells were obtained from the RIKEN Cell Bank (Tsukuba, Japan).
Cells were maintained in 55-cm2
dishes in RPMI 1640 medium supplemented with 10% fetal bovine serum,
50 U/ml penicillin, and 50 µg/ml streptomycin in a humidified atmosphere of 5% CO2 in air at
37°C. After reaching 70% confluence, the cells were detached by
treatment with 0.05% trypsin, replated in
55-cm2 dishes, 6-well plates (9.4 cm2/well), and 12-well plates (3.8 cm2/well) at a density of 1 × 104
cells/cm2 and grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 U/ml penicillin,
50 µg/ml streptomycin, 5 mM
-glycerophosphate, and various
concentrations of ascorbic acid. During subculture, the medium was
replaced every 3 days, and fresh ascorbic acid was added to the medium
just before use.
Northern blot analysis.
RNA was extracted from ST2 cells by the acid
guanidinium-phenol-chloroform method (2). Total RNA (20 µg) was
subjected to electrophoresis on a 1% agarose gel that contained 2.2 M
formaldehyde and was then transferred to a MagnaGraph nylon membrane
(Micron Separations, Westborough, MA). After the membrane was baked,
the RNA on the membrane was allowed to hybridize overnight with cDNA for ALPase, osteocalcin, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at 42°C in 50% formamide that contained 6× SSPE
(1× SSPE is 0.15 M NaCl, 15 mM
NaH2PO4,
pH 7.0, and 1 mM EDTA), 2× Denhardt's solution [BSA,
polyvinylpyrrolidone, and Ficoll (0.1% each)], 1% SDS, and 100 µg/ml herring sperm DNA. Each cDNA probe was radiolabeled with a
Ready-to-Go kit (Amersham Pharmacia Biotech, Uppsala, Sweden). The
membrane was washed twice in 1× SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0) that contained 0.1% SDS at room temperature for 5 min
each and twice in 1× SSC that contained 0.1% SDS at 55°C for
1 h each, and then it was exposed to an imaging plate for 4 h. The
plate was analyzed with a Bioimage analyzer (BAS 2000; Fuji Film,
Tokyo, Japan).
Staining of ALPase and von Kossa staining.
ST2 cells were subcultured in RPMI 1640 medium that contained 10%
fetal bovine serum, 5 mM
-glycerophosphate, and various concentrations of ascorbic acid. The cells were fixed with 10% formalin for 30 min and washed three times with 10 mM
Tris · HCl, pH 7.2. Fixed cells were subjected to
staining for ALPase and von Kossa staining. ALPase was stained with
naphthol AS-MX phosphate and fast blue BB salt (Sigma). The von Kossa
staining for calcium was performed as follows. Fixed cells were
incubated with 5% silver nitrate for 5 min in daylight, washed twice
with H2O, and then treated with
5% sodium thiosulfate. Mineralized nodules were counted under a BH
microscope (Olympus, Tokyo, Japan).
Assay of ALPase activity.
Cells were subcultured in RPMI 1640 medium that contained 10% fetal
bovine serum, 5 mM
-glycerophosphate, and various concentrations of
ascorbic acid. The cells were washed twice with 10 mM
Tris · HCl, pH 7.2, and sonicated in 1 ml of 50 mM
Tris · HCl, pH 7.2, that contained 0.1% Triton X-100
and 2 mM MgCl2 for 15 s with a
sonicator (ultrasonic disrupter UD-201; Tomy, Tokyo, Japan). The ALPase
activity of the sonicate was determined by an established technique
with p-nitrophenyl phosphate as the
substrate (8). Concentrations of protein were determined with
bicinchoninic acid protein assay reagent (Pierce Chemical, Rockford,
IL), with BSA as the standard.
Quantitation of calcium.
Mineralized nodules from a 21-cm2
dish were washed twice with PBS and incubated overnight in 2 ml of 2 N
HCl with gentle shaking. The calcium ions in the sample were
quantitated by the o-cresolphthalein Complexone method with a Calcium C kit (Wako Pure Chemical
Industries) (8).
RT-PCR.
We detected mRNAs for BMPs and BMP receptors (BMPRs) in ST2 cells by
RT-PCR. Total RNA (5 µg) isolated from ST2 cells was reverse
transcribed by Moloney murine leukemia virus RT (200 U; Superscript;
Life Technologies), with random primers (50 ng) and a 20-µl reaction
mixture. The cDNA was amplified during 30 cycles of PCR in 20 µl of
Taq DNA polymerase mixture (Takara,
Tokyo, Japan) that contained 250 nM sense primer
5'-CGGGAACAGATACAGGAAGC-3' and antisense primer
5'-GCTGTTTGTGTTTGGCTTGA-3' for mouse BMP-2 (403 bp), 250 nM
sense primer 5'-ACTCACCTCCACCAGACACG-3' and antisense primer 5'-CCTCTACCACCATCTCCTGA-3' for mouse BMP-4 (493 bp),
250 nM sense primer 5'-AGAAGAAGGTTGGCTGGAAT-3' and
antisense primer 5'-ACAGTAGTTGGCAGCGTAGC-3' for mouse BMP-6
(421 bp), 250 nM sense primer 5'-GGCTTCTCCTACCCCTACAA-3'
and antisense primer 5'-GTGGTTGCTGGTGGCTGTGA-3' for mouse
BMP-7 (399 bp), 250 nM sense primer
5'-TAGCACCAGAGGATACCTTGC-3' and antisense primer
5'-AATGCTTCATCCTGTTCCAAA-3' for mouse BMPR-IA (448 bp), 250 nM sense primer 5'-TGGGCTGGAGCAGGACGAGAC-3' and antisense primer 5'-TCCACGCCACTTTCCCATCCA-3' for mouse
BMPR-IB (193 bp), or 250 nM sense primer
5'-AATCAAGAACGGCTGTGTGCA-3' and antisense primer
5'-CATGCTGTGAAGACCCTGTTT-3' for mouse BMPR-II (473 bp). The
reaction cycle consisted of 94°C for 30 s, 58°C for 30 s, and
72°C for 30 s. mRNA from mouse embryos (day
14), which express mRNAs for all BMPs and their
receptors, was used as a positive control. Products of PCR were
subjected to electrophoresis on a 2% agarose gel and visualized by
staining with ethidium bromide. DNA markers (molecular weight marker V;
Boehringer Mannheim, Tokyo, Japan) were used as size markers.
Statistical analysis.
Data are expressed as means ± SE of results from three or four wells
for each experiment. Statistical significance was determined by the
unpaired Student t-test.
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RESULTS |
Stimulation of ALPase activity in ST2 cells by ascorbic acid.
After 18 days in control culture in RPMI 1640 medium, ST2 cells had
very low levels of ALPase activity (Fig.
1A).
However, ALPase activity increased markedly when the cells were
subcultured in RPMI 1640 medium with 50 µg/ml ascorbic acid (Fig. 1,
B and D).
-Glycerophosphate (5 mM)
was not involved in the activation of ALPase in ST2 cells (Fig. 1,
C and
D), even though
-glycerophosphate has been reported to increase the expression of ALPase in osteoblastic cells (11). As shown in Fig. 2, the
induction of ALPase activity by ascorbic acid in ST2 cells was time and
dose dependent. ALPase activity was markedly induced by ascorbic acid
at 10 days after the latter had been added to the culture medium, and
the ALPase activity (52.3 ± 1.9 µmol · mg
protein
1 · 30 min
1) in ST2 cells that
had been treated with 50 µg/ml ascorbic acid for 18 days was 86-fold
higher than the control value (0.61 ± 0.01 µmol · mg
protein
1 · 30 min
1).

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Fig. 1.
Stimulation of alkaline phosphatase (ALPase) activity in ST2 cells that
had been cultured with ascorbic acid. ST2 cells in 6-well plates (9 cm2/well) were cultured for 18 days with RPMI 1640 that contained 10% fetal bovine serum plus 5 mM
-glycerophosphate and/or 50 µg/ml ascorbic acid.
A: control;
B: 50 µg/ml ascorbic acid added;
C: 5 mM -glycerophosphate added;
D: both ascorbic acid and
-glycerophosphate added. Cells were stained for ALPase activity as
described in MATERIALS AND METHODS.
Results are representative of experiments performed 5 times with
similar results.
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Fig. 2.
Time- and dose-dependent stimulation of ALPase activity in ST2 cells by
ascorbic acid. ST2 cells in 6-well plates (9 cm2/well) were cultured for the
indicated periods of time with RPMI 1640 that contained 10% fetal
bovine serum and ascorbic acid at indicated concentrations. ALPase
activity was determined as described in MATERIALS AND
METHODS. Data are means ± SE of results from 4 different wells and are representative of results from 4 different
experiments. * P < 0.0001 vs.
control.
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Northern blot analysis revealed that ascorbic acid increased the
steady-state levels of mRNAs for proteins that are markers of
osteoblastic differentiation. As shown in Fig.
3, the addition to the culture medium of 50 µg/ml ascorbic acid increased the expression of mRNA for ALPase 1.6-, 6.9-, and 4.7-fold, and that of mRNA for osteocalcin 1.4-, 5.8-, and
30.6-fold, at days
5, 10, and
20, respectively.

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Fig. 3.
Expression of mRNAs for ALPase and osteocalcin in ST2 cells cultured
with ascorbic acid. Total RNA was isolated from ST2 cells after they
had been cultured with 50 µg/ml ascorbic acid for 5, 10, and 20 days.
Then, 20 µg of total RNA were subjected to electrophoresis in a 1%
agarose gel, and bands of RNA were allowed to hybridize with
32P-labeled cDNAs for ALPase and
for osteocalcin as described in MATERIALS AND
METHODS. Expression of mRNA for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as an
internal reference because gene for GAPDH is a housekeeping gene and
fold stimulation reflects normalization to GAPDH. Data are
representative of results from 5 different experiments.
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Induction of the deposition of calcium and the formation of
mineralized nodules by ascorbic acid.
We measured the deposition of calcium by ST2 cells that had been
treated for 24 days with ascorbic acid at various concentrations in
21-cm2 dishes (Fig.
4). Ascorbic acid enhanced the accumulation
of calcium by ST2 cells in a dose-dependent manner at levels above 25 µg/ml. The maximal accumulation, at 75 µg/ml ascorbic acid, was
18.9 ± 3.2 µg calcium/well, and this level was nine times the
control level (2.1 ± 1.6 µg calcium/well).

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Fig. 4.
Deposition of calcium by ST2 cells in response to ascorbic acid. ST2
cells in 21-cm2 dishes were
cultured for 24 days with RPMI 1640 that contained 10% fetal bovine
serum, 5 mM -glycerophosphate, and ascorbic acid at indicated
concentrations. Quantitative analysis of calcium ions derived from
hydroxyapatite was performed as described in
MATERIALS AND METHODS. Data are means ± SE of results from 3 different wells and are representative of
results from 3 different experiments.
* P < 0.05 vs. control;
** P < 0.005 vs. control.
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We next examined whether ascorbic acid could induce the formation of
mineralized nodules. As shown in Fig. 5,
mineralized nodules developed in cultures supplemented with ascorbic
acid for 24 days. In control cultures, no nodules were formed during the same 24-day period. Ascorbic acid at 50 µg/ml induced the formation of 21.0 ± 1.5 (n = 3)
mineralized nodules per 55-cm2
dish. We confirmed that nodules formed by ST2 cells contained type I
collagen by immunostaining with a specific antibody against type I
collagen (results not shown).

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Fig. 5.
Phase-contrast photomicrograph of mineralized nodules in a culture of
ST2 cells that had been supplemented with ascorbic acid. ST2 cells in a
55-cm2 dish were cultured for 24 days with RPMI 1640 that contained 10% fetal bovine serum, 5 mM
-glycerophosphate, and 50 µg/ml ascorbic acid. Mineralized nodules
were subjected to von Kossa staining as described in
MATERIALS AND METHODS. Bar, 40 µm.
Data are representative of results from 3 different experiments.
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Effects of type I collagen on the differentiation of ST2 cells.
Ascorbic acid has been shown to induce the formation of collagen triple
helices via the hydroxylation and processing of procollagen during the
differentiation in vitro of cells to osteoblasts (4). Therefore, we
examined the effects of two inhibitors of the formation of such triple
helices, DHP and CHP, on the ALPase activity of ST2 cells that had been
treated with 50 µg/ml ascorbic acid. As shown in Fig.
6A, DHP
and CHP at 0.5 mM reduced ALPase activity by 80 and 40%, respectively,
compared with the control level on day
18. DHP inhibited ALPase activity more effectively than
did CHP. DHP at 1 mM completely inhibited the ALPase activity of ST2 cells (Fig. 6B).


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Fig. 6.
Inhibition of ALPase activity induced by ascorbic acid by inhibitors of
formation of collagen matrix. A: ST2
cells in 6-well plates (9 cm2/well) were cultured for 10 or
18 days with RPMI 1640 that contained 10% fetal bovine serum and 5 mM
-glycerophosphate, as well as 50 µg/ml ascorbic acid, 0.5 mM
3,4-dehydro-L-proline (DHP),
and/or 0.5 mM
cis-4-hydroxy-L-proline
(CHP), as indicated. ALPase activity was determined as described in
MATERIALS AND METHODS.
B: dose-dependent inhibition of ALPase
activity by DHP on day 18. ,
Presence of ascorbic acid; , absence of ascorbic acid. Data are
means ± SE of results from 3 or 4 different wells and are
representative of results from 2 different experiments.
* P < 0.05 vs. control;
** P < 0.005 vs. control;
# P < 0.001 vs. control.
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Type I collagen interacts with various types of cells through the
binding of the DGEA motif to
2
1-integrin
(17, 18) and facilitates differentiation (17). We examined the effect of a DGEA peptide on the induction of ALPase activity by ascorbic acid.
As shown in Fig. 7, the addition of peptide
at 5 mM blocked ~60% of the ALPase activity induced by ascorbic
acid.

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Fig. 7.
Suppression by DGEA peptide of the induction of ALPase activity by
ascorbic acid. ST2 cells in 24-well plates (2 cm2/well) were cultured with RPMI
1640 that contained 10% fetal bovine serum and 50 µg/ml ascorbic
acid or 5 mM DGEA peptide, as indicated. DGEA peptide was added to the
culture medium of ST2 cells on day 3 of culture, and ALPase activity was measured on day
9. Data are means ± SE of results from 3 different wells. * P < 0.001 vs. results obtained in absence of DGEA and ascorbic acid.
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Identification of the BMPs and corresponding BMPRs in ST2
cells and the role of BMPs in the differentiation induced by ascorbic
acid.
We performed RT-PCR with specific primers for BMP and BMPR mRNAs to
identify BMP systems in ST2 cells. Our results showed that the mRNAs
for BMP-4 and for BMPR-IA and -II were present in ST2 cells during
culture for 20 days (Fig. 8). During this period, ascorbic acid (50 µg/ml) had no effect on the expression of
mRNAs for BMP-4 and BMPR-IA and -II. Exogenous BMP-2 dose dependently enhanced ALPase activity in ST2 cells, in the absence of ascorbic acid
(Fig. 9). In the presence of 50 µg/ml
ascorbic acid, the effects of BMP-2 were enhanced still further and
were synergistic. These results indicated that ascorbic acid did not
affect the expression of BMPs and their receptors but acted
to increase the efficiency of the BMP system. Figure
10 shows the dose-dependent inhibition by
a soluble form of BMPR-IA (sBMPR-IA) on the ALPase activity that was
induced by ascorbic acid in ST2 cells. sBMPR-IA is an extracellular
domain of the type IA receptor for BMP-2 and BMP-4 and
binds to BMP-4 (12). ST2 cells had low ALPase activity in the absence
of ascorbic acid (control culture). However, when the cells were
cultured in the presence of ascorbic acid (50 µg/ml), the level of
ALPase activity increased to four times that in the control culture.
sBMPR-IA at 1 µg/ml is enough to inhibit BMP-4 secreted from ST2
cells and inhibited 40% of the ascorbic acid-induced ALPase activity.

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Fig. 8.
Detection of subtypes of bone morphogenetic protein (BMP) and BMP
receptor (BMPR) in ST2 cells and effects of ascorbic acid on expression
of BMPs and BMPRs. Total RNA was isolated from ST2 cells after they had
been cultured with 50 µg/ml ascorbic acid for 5, 10, and 20 days, and
products of RT-PCR amplification were analyzed. Reaction cycle and
sequences of specific primers are described in
MATERIALS AND METHODS. RNA from mouse
embryos on day 14 was used as the
standard sample.
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Fig. 9.
Stimulation of the expression of ALPase by BMP-2 and/or ascorbic acid
in ST2 cells. ST2 cells in 12-well plates (3.8 cm2/well) were cultured for 6 days
with RPMI 1640 that contained 10% fetal bovine serum and BMP-2 at
various concentrations in absence and presence of 50 µg/ml ascorbic
acid. ALPase activity was determined as described in
MATERIALS AND METHODS. Data are means ± SE of results from 3 different wells.
* P < 0.005 vs. control;
** P < 0.0001 vs.control.
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Fig. 10.
Inhibitory effects of soluble BMPR-IA (sBMPR-IA) on ALPase
activity in ST2 cells cultured with ascorbic acid. ST2 cells in 24-well
plates (2 cm2/well) were cultured
in absence and presence of 50 µg/ml ascorbic acid. sBMPR-IA at
indicated concentrations was included in culture medium of ST2 cells
for 9 days, and ALPase activity was determined as described in
MATERIALS AND METHODS. Data are
means ± SE of results from 3 different wells.
* P < 0.05 vs. control;
** P < 0.01 vs. control.
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DISCUSSION |
Osteoblasts, chondrocytes, adipocytes, myoblasts, and fibroblasts that
form connective-tissue cells are thought to arise from a common
population of mesenchymal stem cells whose progeny become committed to
specific lineages in response to growth factors, hormones, and other
signals. Such signals induce the tissue-specific transcription that is
necessary for the expression of each differentiated phenotype. In
normal cultures, stromal ST2 cells have characteristics of
preadipocytes and none of the features typical of the osteoblastic phenotype (23). In this study, we found that ascorbic acid induced the
osteoblastic differentiation of ST2 cells. To our knowledge, this is the first report of the ascorbic acid-induced osteoblastic differentiation of stromal ST2 cells. We also demonstrated that the
formation of a collagen-containing extracellular matrix in response to
ascorbic acid was essential for expression of the osteoblastic
phenotype of ST2 cells. Furthermore, we showed that signaling pathways
involving BMP might participate in the differentiation of ST2 cells
that is induced by ascorbic acid.
Ascorbic acid stimulated the expression of genes for proteins
associated with the osteoblast phenotype, such as ALPase and osteocalcin, and it increased ALPase activity and the deposition of
calcium by ST2 cells. These events are the hallmarks of cells that are
committed to the osteoblast lineage. We showed that ascorbic acid has
the potential to induce these events in ST2 cells at concentrations
from 5 to 25 µg/ml for the activation of ALPase and from 25 to 75 µg/ml for the deposition of calcium. The normal concentration of
ascorbic acid in human plasma ranges from 5 to 10 µg/ml. Our results
suggest that the effect of ascorbic acid on ST2 cells might be
physiological and that ascorbic acid might be necessary for the
differentiation of stromal cells into osteoblast-like cells.
Ascorbic acid has been shown to stimulate the formation of a collagen
matrix at multiple levels that include gene transcription, hydroxylation, and the processing of procollagen. Type I collagen in
the extracellular matrix mediates the differentiation in vitro of
preosteoblast MC3T3-E1 cells (5, 6, 17, 18) and of osteoprogenitor
ROB-C26 cells (16). Franceschi and Iyer (5) and Franceschi et al. (6)
showed that ascorbic acid increases the levels of mRNAs for ALPase and
osteocalcin through the production of collagen in MC3T3-E1 cells.
Takeuchi et al. (17, 18) also demonstrated that collagen formed in
response to ascorbic acid causes the differentiation of MC3T3-E1 cells
and downregulation of the expression of the receptor for transforming
growth factor-
in MC3T3-E1 cells. We showed, in this study, that
inhibitors of the formation of a collagen matrix completely blocked the
induction of ALPase activity by ascorbic acid (Fig. 6). These
observations strongly suggested that type I collagen itself and/or the
type I collagen-containing extracellular matrix formed in response to
ascorbic acid might be essential for the osteoblastic differentiation of ST2 cells. Therefore, we attempted to identify the signaling pathways that are operative after the formation of the type I collagen-containing extracellular matrix that occurs in response to
ascorbic acid.
The interaction of integrins with matrix proteins has various effects
on the proliferation and differentiation of cells via the activation of
a variety of signal transduction pathways (3).The
2
1-integrin
serves as a collagen-specific receptor, and matrix collagen interacts
with a variety of cells through the specific binding of the DGEA motif
to
2
1-integrin
on cells (17). It has been reported that the interaction between
collagen and integrin induces the differentiation of preosteoblastic
MC3T3-E1 cells that are committed to the osteoblastic phenotype (18).
We also examined the effect of a DGEA peptide on the induction of
ALPase activity by ascorbic acid. The addition of the peptide at 5 mM blocked ~60% of the ALPase activity induced by ascorbic acid. Our
results suggest that the matrix of type I collagen formed in response
to ascorbic acid directly provokes osteoblastic differentiation of ST2
cells. Takeuchi et al. (18) reported that, after the attachment of
MC3T3-E1 cells to collagen, integrin activates the focal adhesion
kinase and the mitogen-activated protein kinase pathway.
We next tried to determine whether pathways involving BMPs are
operative in the ascorbic acid-induced osteoblastic differentiation of
ST2 cells.
BMPs have been reported to induce the differentiation of mesenchymal
cells into osteoblastic cells (10, 15, 23, 24). Clonal preosteoblast
MC3T3-E1 cells express the osteoblastic phenotype, for example, they
have elevated ALPase activity when they respond to BMPs that they
produce themselves (12). By contrast, ST2 cells express the
osteoblastic phenotype when BMPs are added to the culture medium (12,
23, 25). BMPs stimulate the expression of mRNAs for proteins that are
markers of osteoblastic differentiation, such as ALPase and
osteocalcin, in ST2 cells. Moreover, ST2 cells generated mineralized
bone after transplantation with BMP into the peritoneal cavities of
athymic mice (23). These results indicate that the signal transduction
pathways distal to BMPRs are operative in ST2 cells. However, the
mechanism of induction of differentiation of ST2 cells by BMPs has not
been fully clarified. We examined whether BMPs might be involved in the
osteoblastic differentiation of ST2 cells that is induced by ascorbic
acid. We first identified the subtypes of BMP and BMPR in ST2 cells because their expression differs from cell to cell. ST2 cells expressed
mRNAs for BMP-4 and for BMPR-IA and -II (Fig. 8). Ascorbic acid had
little effect on the levels of mRNAs for BMP-4 and the receptors during
the differentiation of ST2 cells. However, as shown in Fig. 9, ALPase
activity was synergistically elevated in ST2 cells when ascorbic acid
was added with BMP-2 to the culture medium. These results suggest that
ascorbic acid can potentiate the actions of BMPs on ALPase activity. To
investigate whether BMPs might actually be involved in the ascorbic
acid-induced differentiation of ST2 cells, we added sBMPR-IA, the
extracellular domain of the type I receptor for BMP-2 and -4 (12), with
ascorbic acid to the culture medium. The added soluble BMPR is
considered to bind BMP-4 that is secreted by ST2 cells into the culture
medium. sBMPR-IA caused the dose-dependent inhibition of the induction
of ALPase activity by ascorbic acid (Fig. 10). Although sBMPR-IA at 1 µg/ml was enough to completely inhibit the ALPase activity induced by exogenous BMP-4 (100 ng/ml) in ST2 cells (12), it inhibited only 40%
of the ALPase activity induced by ascorbic acid. Our results suggest
that part of the action of ascorbic acid might be due to BMP-4 produced
by ST2 cells, even though ascorbic acid acts predominantly through the
collagen-integrin pathway for the differentiation of ST2 cells. BMPs
are stored in the extracellular matrix (19). Therefore, it is possible
that BMP-4 might function more effectively when stored in the
collagen-containing extracellular matrix that forms in response to
ascorbic acid. The facts that the collagen matrix formed in response to
ascorbic acid was essential for the differentiation of ST2 cells (Fig.
6B) and that ascorbic acid accelerated the effects of BMP (Fig. 9)
strongly support this hypothesis.
In this report, we have demonstrated that ascorbic acid induces the
osteoblastic differentiation of ST2 cells. This process requires the
formation of a collagen-containing extracellular matrix in response to
ascorbic acid. Furthermore, we obtained evidence that suggests that the
ascorbic acid-induced differentiation of ST2 cells might be due to the
BMP system in addition to the interaction between collagen and
2
1-integrin.
Commercial medium may or may not contain ascorbic acid.
Similar situations might occur with other components and with other
types of cells, and therefore such possibilities should be kept in mind
when cultured cell systems are used. ST2 cells retain the potential to
support osteoclast differentiation (20). A very recent study showed that ascorbic acid is necessary for osteoclast differentiation in
cocultures of bone marrow cells and ST2 cells (14). Our present results
suggest that ascorbic acid might induce ST2 cells at a particular stage
of differentiation to support osteoclast differentiation.
 |
ACKNOWLEDGEMENTS |
We thank Drs. N. Ueno and S. Iemura (National Institute for Basic
Biology, Okazaki, Japan) for the generous gift of the sBMPR-IA used in
this study and A. Inoue (Tokyo Institute of Technology, Yokohama,
Japan) for helpful discussions.
 |
FOOTNOTES |
This work was supported by Grants-in-Aid for Scientific Research from
the Ministry of Education, Science, Sports and Culture of Japan and by
grants from the Smoking Research Foundation and Ground Research
Announcement for Space Utilization promoted by NASDA and the Japan
Space Forum.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Hagiwara,
Research Center for Experimental Biology, Tokyo Institute of
Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
(E-mail: hhagiwar{at}bio.titech.ac.jp).
Received 21 September 1998; accepted in final form 2 April 1999.
 |
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