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(Received for publication, June 23,
1995; and in revised form, November 28, 1995) From the
Although transforming growth factor (TGF)- Cells of osteoblast lineage exert various functions to maintain
bone formation. After bone resorption, osteoblast precursors migrate
and proliferate at the site of bone formation. They, then, synthesize
type I collagen and other matrix proteins. Onto the newly formed
unmineralized matrices, hydroxyapatite crystals are accumulated, and
mineralization of bone is completed(1) . In order to form
lamellar bones that maintain structural integrity and physical
strength, it appears to be of critical importance for osteoblastic
cells to maintain sequentially ordered development of these multiple
functions. Using cultured osteoblastic cells obtained from bone or
of clonal origin, it has become clear that these cells express various
functional properties in a differentiation-dependent manner from the
early proliferation phase, via the matrix formation phase with active
matrix protein synthesis, to the mineralization
phase(1, 2, 3) . Various hormones and
cytokines are shown to affect the differentiation process and
functional properties of these cells. However, the mechanism that
controls the differentiation of osteoblastic cells is as yet unclear. Transforming growth factor- Osteoblast-like MC3T3-E1
cells in long-term culture exhibit various phenotypes in a sequential
manner, and, after the avid production of matrix proteins, develop the
capacity to induce mineralization(15, 16) . However,
when MC3T3-E1 cells were cultured in the absence of ascorbic acid,
these cells cannot differentiate to induce mineralization(16) .
Because ascorbic acid is essential for collagen synthesis as a cofactor
for prolyl-lysyl hydroxylase, the inhibition of differentiation by the
removal of ascorbic acid may be due to a suppression of the synthesis
of collagen. In addition, the expression of ALP is reported to be
dependent upon the production of type I collagen in osteoblastic cell
lines(3, 17) . From these previous observations,
there is a possibility that the actions of TGF-
Figure 1:
Effect of TGF-
Consistent with the previous observations of the inhibitory effect
of TGF-
Figure 2:
Effect of TGF-
Figure 3:
Cell surface expression of TGF-
Figure 4:
Expression of mRNAs for alkaline
phosphatase and type II TGF-
Figure 5:
Effects of a collagen synthesis
inhibitor, L-azetidine-2-carboxylic acid, on cell surface
TGF-
Figure 6:
Effects of anti-
Figure 7:
Effects of ECM produced by MC3T3-E1 cells
after long-term culture on TGF-
Production of type I collagen is one of the early events
associated with osteoblastic differentiation. Following the synthesis
of type I collagen, sequential expression of ALP and osteocalcin,
markers of osteoblastic differentiation, is
observed(31, 32, 33) . The present studies
demonstrate that the differentiation of osteoblastic MC3T3-E1 cells, as
indicated by the elevation of ALP activity, is markedly suppressed when
collagen synthesis is inhibited by either eliminating ascorbic acid or
adding a collagen synthesis inhibitor. Thus, the synthesis and the
accumulation of matrix collagen appear to be involved in the
osteoblastic differentiation of these cells. These results are
consistent with the previous observations that the production of
collagen is required for the development of ALP activity in
osteoblastic cells(3, 17) . TGF- A decrease in TGF- As to the mechanism of the regulation of the
differentiation and the down-regulation of TGF- In conclusion, the present studies
demonstrate that the synthesis and the accumulation of matrix collagen
are involved in the differentiation of osteoblastic cells, and that
osteoblastic differentiation is associated with a reduction in
TGF-
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3938-3944
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Receptors Are Regulated by Interaction with Matrix Collagen in Murine
Osteoblastic Cells (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
enhances bone
formation, it inhibits the differentiation of osteoblasts. To clarify
the regulatory mechanism of osteoblastic differentiation and TGF-
actions, the relationship among differentiation, TGF- actions, and
matrix protein synthesis was examined using murine osteoblast-like
MC3T3-E1 cells. Alkaline phosphatase (ALP) activity continued to
increase during long-term cultures, and the increase was closely
associated with a reduction in cell surface TGF- receptors
competent to bind TGF-. Both the stimulation of proteoglycan
synthesis and the inhibition of ALP activity by TGF- were also
suppressed. Collagen synthesis inhibitors and an anti-
21
integrin blocking antibody blocked the changes in ALP activity and
TGF- receptors, and a DGEA peptide that interferes binding of
collagen to 21 integrin also blocked the increase in ALP
activity. Furthermore, when MC3T3-E1 cells were cultured on
extracellular matrix layers obtained from these cells, all the
differentiation-associated changes could be observed without collagen
production, and the extracellular matrix-induced differentiation was
also blocked by an anti-21 integrin antibody. These results
demonstrate that the interaction of cell surface 21 integrin
with matrix collagen synthesized by osteoblasts themselves is involved
in the osteoblastic differentiation and the reduction in cell surface
receptors and actions of TGF-. It is suggested that matrix
collagen synthesized under the stimulation by TGF- plays an
important role in the regulation of osteoblastic differentiation and
TGF- actions by differentiation-associated down-regulation of
TGF- receptors.
(TGF-) (
)is stored
in bone matrix as a latent form(4) , is thought to be released
and activated during osteoclastic bone resorption(5) , and to
play an important role in the regulation of bone
metabolism(6) . TGF- is one of the most potent stimulators
of the production of type I collagen in various types of
cells(7, 8) , and the most pronounced effect of
TGF- in bone is the stimulation of matrix protein synthesis
including type I collagen, proteoglycans, and
fibronectin(9, 10) . Thus, TGF- stimulates bone
formation when given to bone in
vivo(11, 12) . In contrast, TGF- inhibits
the differentiation of many types of mesenchymal cells(13) ,
and is also shown to prevent the development of
differentiation-associated phenotypes such as the expression of
alkaline phosphatase (ALP) (9) and osteocalcin (14) in
cells of osteoblast lineage. Therefore, the actions of TGF- have
to be suppressed when osteoblastic cells differentiate from matrix
formation phase into mineralization phase. on collagen and
other matrix protein synthesis and on the differentiation of these
cells may be mutually regulated, and that the interaction of
osteoblastic cells with matrix proteins including collagen may play an
important role in the regulation of osteoblastic differentiation and
TGF- actions. The present studies are undertaken to clarify the
relationship among the differentiation, TGF- actions, and matrix
collagen synthesis in osteoblasts using MC3T3-E1 cells in culture.
Materials
[I]TGF-
1
and [P]dCTP were purchased from Amersham Japan
and [
S]sulfuric acid from DuPont NEN. Human
recombinant TGF-
1 was obtained from R & D systems
(Minneapolis, MN); hamster monoclonal antibody against mouse
21 integrin (clone HM2) and against mouse 51
integrin (clone HM5-1) from Sumitomo Denko Co. (Osaka,
Japan); hamster non-immune IgG from Cedarlane Laboratories Ltd.
(Ontario, Canada); disuccinimidyl suberate from Pierce; ascorbic acid
and L-azetidine-2-carboxylic acid from Wako Pure Chemicals
(Osaka, Japan); L-proline and sodium deoxycholate from Sigma;
and Sephadex G-50 and Q-Sepharose were from Pharmacia Biotech Inc.
(Tokyo, Japan). Human type II TGF- receptor cDNA and rat ALP cDNA
were generous gifts from Dr. K. Miyazono (Ludwig Institute for Cancer
Research, Uppsala, Sweden) and Dr. G. A. Rodan (Merk, Sharp and Dohme,
West Point, PA), respectively. DGEA and KDGE peptides were synthesized
by Drs. M. Hane and N. Tamura of Mitsubishi Chemical Co. (Tokyo,
Japan). MC3T3-E1 cells were provided by Dr. H. Kodama (Ohu Dental
College, Japan).Cell Culture
MC3T3-E1 cells were cultured in
minimum essential medium supplemented with 10% fetal bovine serum,
penicillin, and streptomycin and with or without 50 mg/liter L-ascorbic acid. The medium did not contain phenol red, and
was changed twice a week. Experiments were performed in the presence of
10% fetal bovine serum unless otherwise indicated. TGF-1 of 1
µM was dissolved in 17.5 mM acetic acid as a
stock solution. Aliquots of 5 mg/ml ascorbic acid, 0.3 ML-azetidine-2-carboxylic acid, 0.5 M cis-4-hydroxyproline, 0.5 M 3,4-dehydroproline, and 0.3 ML-proline dissolved in distilled water were added
to the culture medium as indicated.Alkaline Phosphatase Activity in Osteoblastic
Cell
Cells were washed twice with ice-cold phosphate-buffered
saline (PBS) and scraped in 10 mM Tris-HCl containing 2 mM MgCl
and 0.05% Triton X-100, pH 8.2. The cell
suspension was homogenized using Pellet Pestle (Kontes, Vineland, NJ)
on ice following two cycles of freeze and thaw. Aliquots of
supernatants were subject to protein assay with a Bio-Rad kit according
to Bradford's method and to ALP activity
measurement(15) . In brief, the assay mixture contained 10
mMp-nitrophenyl phosphate in 0.1 M sodium
carbonate buffer, pH 10, supplemented with 1 mM MgCl, 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.Affinity Cross-linking Analyses of Cell Surface TGF-
Osteoblastic cells in 10-cm culture dishes were washed
twice with ice-cold PBS. Binding of 100 pM
ReceptorsI-TGF-
1 (total radioactivity of approximately
200,000 cpm) to osteoblastic cells was performed in a buffer containing
128 mM NaCl, 5 mM KCl, 5 mM MgSO
, 1.3 mM CaCl, and 25 mM Hepes, pH 7.4, containing 0.3% bovine serum albumin at 4 °C
for 4 h as reported previously(18) . After washing the cells
with ice-cold PBS, bound I-TGF-
1 was chemically
cross-linked in 2 ml of PBS containing 0.3 mM disuccinimidyl
suberate at 4 °C for 20 min(19) . Chemical reaction was
terminated by adding 200 µl of quenching buffer (100 mM Tris-HCl, pH 7.5, 200 mM glycine, and 20 mM EDTA), and cells were kept at room temperature for 1 min. After
washing twice with PBS, cells were scraped into a tube on ice
containing 1 ml of PBS with 1 mM EDTA and a protease inhibitor
mixture of 1 mM phenylmethylsulfonyl fluoride, 10 mMN-ethylmaleimide, and 10 µg/ml pepstatin A. Cells
were then collected by centrifugation, and solubilized in 40-100
µl of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, 0.5%
Nonidet P-40, 1 mM EDTA, and protease inhibitors) for 40 min
on ice. Lysates were centrifuged and supernatants were subject to
SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel
followed by autoradiography. Loading samples were adjusted to contain
the same amount of protein. Some experiments were performed at least
three times, and representative results from reproducible experiments
are shown in the figures.Northern Blot Analysis
Ten µg of total RNA
from each osteoblastic cell culture was loaded on each lane of an
agarose gel. Human type II TGF- receptor cDNA (20) and rat
ALP cDNA (21) were used as probes. Probes were radiolabeled
with [P]dCTP using the Megaprime DNA labeling
system (Amersham) according to the manufacturer's instructions.
Assays for Proteoglycan Synthesis
Synthesis of
proteoglycans by osteoblastic cells was determined as described
previously(22, 23) . Cells were labeled with 100
µCi/ml [S]sulfate for 1 h at 37 °C after
treatment with various concentrations of TGF-
1. Cell layer and
media were simultaneously extracted with 4 M guanidine HCl, pH
6.0, containing 0.5% Triton X-100, 50 mM sodium acetate, 50
mM EDTA, and protease inhibitors (4 M guanidine HCl
buffer). Unincorporated isotopes were removed by Sephadex G-50 column
chromatography eluted with 7 M urea buffer containing 0.25 M NaCl, 50 mM sodium acetate, and 0.5% Triton X-100,
pH 6.0. Void fractions containing proteoglycans were collected and
applied onto freshly prepared Q-Sepharose column (bed volume of 100
µl). The column was washed with 1 ml of 7 M urea buffer
containing 0.3 M NaCl, and then with the same buffer
containing 0.5% CHAPS. Proteoglycans bound to Q-Sepharose were eluted
with 4 M guanidine HCl buffer containing 0.5% CHAPS.
Radioactivity in each eluent was counted.Coating of Plastic Culture Plates with Extracellular
Matrix Produced by Osteoblastic Cells
MC3T3-E1 cells were
cultured on 6-well plastic dishes (Falcon) in the presence and absence
of ascorbic acid for 10-14 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(24) . Solubilized
materials were extensively wiped away by several washes with ice-cold
PBS. Extracellular matrix (ECM) components resistant to the detergent
remained on dishes and were stored in 1 ml of PBS on ice. Freshly
dispersed cells were inoculated on the coated dishes and cultured at 37
°C in 5% CO atmosphere. Cells were subject to ALP assay
after 7 days.Statistical Analysis
Data were expressed as means
± S.E. and analyzed by an independent t test.
Impaired Osteoblastic Differentiation in the Absence of
Collagen Production
Cellular ALP activity in MC3T3-E1 cells
continues to increase during long-term culture in the presence of
ascorbic acid as reported previously(15) , while it is markedly
suppressed in the absence of ascorbic acid(17) . Ascorbic acid
is essential for the synthesis of collagen, and when a collagen
synthesis inhibitor, L-azetidine-2-carboxylic acid, was
included, the increase in ALP activity after 7 days of culture in the
presence of ascorbic acid (1.68 ± 0.22 and 0.23 ± 0.01
nmol/min/µg of protein in the presence and absence of ascorbic
acid, respectively, p < 0.01) was almost completely
eliminated (0.26 ± 0.01 nmol/min/µg of protein, p < 0.01 versus the group with ascorbic acid alone). L-Azetidine-2-carboxylic acid is a proline analog that
replaces prolyl residues in collagen molecules and inhibits prolyl
hydroxylation(25) . By an addition of a 10 times higher
concentration of L-proline (3 mM) together with 0.3
mML-azetidine-2-carboxylic acid, the inhibitory
effect of L-azetidine-2-carboxylic acid on the increase in ALP
activity was reversed (1.68 ± 0.32 nmol/min/µg of protein).
Thus, osteoblastic differentiation of MC3T3-E1 cells requires the
presence of ascorbic acid, and the effect of ascorbic acid is mediated
via the production of collagen in these cells.Effect of TGF-
As reported previously(26) ,
treatment with TGF-1 on Proteoglycan Synthesis and ALP
Activity in MC3T3-E1 Cells1 for 24 h stimulated proteoglycan synthesis in
a dose-dependent manner after 4 days of culture when MC3T3-E1 cells
reached confluence, and 100 pM TGF-1 stimulated
proteoglycan synthesis to 340% of the control (Fig. 1). The
stimulation of proteoglycan synthesis by 100 pM TGF-1
decreased with the progression of culture period, and was reduced to as
low as 141% of the control at 14 days of culture. In contrast, when
ascorbic acid was removed from the culture medium, stimulation of
proteoglycan synthesis by 100 pM TGF-1 was not reduced
until day 7, and decreased only slightly at day 14 (Fig. 1).
1 on proteoglycan
synthesis during long-term cultures of MC3T3-E1 cells in the presence
and absence of ascorbic acid. MC3T3-E1 cells were cultured in the
presence (shaded columns) and absence (open columns)
of 50 mg/liter ascorbic acid until 14 days. Cells were treated with
various concentrations of TGF-1 for 24 h before experiments. Cells
were washed with PBS and kept in the medium without any supplements for
1 h at 37 °C. Cells were metabolically radiolabeled with 100
µCi/ml [S]sulfate for 1 h at 37 °C.
Labeled proteoglycans were extracted and isolated as described under
``Materials and Methods.'' The amounts of labeled
proteoglycans in each group were calculated as percentages of the
control without ascorbic acid or TGF-
1. Data are expressed as
means ± S.E., n = 3.
on the expression of ALP(9) , treatment with 100
pM TGF-1 inhibited ALP activity in MC3T3-E1 cells until 7
days of culture in the presence of ascorbic acid (Fig. 2).
However, the inhibitory effect of TGF-1 on ALP activity
disappeared with the increase in ALP activity after 14 days of culture
in the presence of ascorbic acid (Fig. 2). Thus, both the
stimulation of matrix protein synthesis and the suppression of
differentiation-associated phenotypes by TGF- were lost during the
differentiation process of MC3T3-E1 cells when ascorbic acid was
present and collagen production was maintained.
1 on alkaline
phosphatase activity during long-term cultures of MC3T3-E1 cells in the
presence of ascorbic acid. MC3T3-E1 cells were cultured in the presence
of 50 mg/liter ascorbic acid until 14 days. At the indicated periods of
culture, cells were treated with 100 pM TGF-1.
Twenty-four hours later, cells were washed twice with ice-cold PBS and
were harvested into 50 mM Tris-HCl, 2 mM MgCl, 0.05% Triton X-100, pH 8.2. Cells were
homogenized on ice and supernatants were collected by centrifugation.
Alkaline phosphatase activity and protein content of the supernatants
were assayed. Data are expressed as means ± S.E., n = 3. *, significantly different from the control without
TGF-1 (p < 0.01).
Cell Surface Expression of TGF-
In an effort to clarify the mechanism whereby
TGF- Receptors in
MC3T3-E1 Cells actions are reduced after long-term cultures of MC3T3-E1
cells with ascorbic acid, changes in TGF- binding to its receptors
were examined. As shown in Fig. 3, affinity cross-link studies
using I-TGF-
1 revealed that types I and II
signal-transducing receptors and beta-glycan were abundantly present in
MC3T3-E1 cells at 4 days of culture. The presence of the type II
receptor in MC3T3-E1 cells was confirmed by an immunoblot analysis (27) . A 72-kDa band bearing I-TGF-
1
corresponded to the type I receptor because of its molecular size and
disappearance after pretreatment of cells with dithiothreitol (28) (data not shown). In the presence of ascorbic acid, the
expression of types I and II receptors competent to bind TGF-1 was
reduced after 7 days, and was almost undetectable after 14 days of
culture (Fig. 3). In the absence of ascorbic acid, the abundance
of TGF- receptors competent to bind ligands at 7 days of culture
was similar to that at 4 days, and, although it declined thereafter,
the abundance of receptors after 14 days of culture was still higher
than that in the presence of ascorbic acid at day 7 (Fig. 3).
Thus, the changes in the expression of the receptors competent to bind
TGF- were closely correlated with the changes in the actions of
TGF-1 after long-term cultures of MC3T3-E1 cells.
receptors during long-term cultures of MC3T3-E1 cells in the presence
and absence of ascorbic acid. TGF- receptors on the cell surface
of MC3T3-E1 cells in the presence (lanes 2, 4, and 6)
and absence (lanes 1, 3, and 5) of 50 mg/liter
ascorbic acid were analyzed by an affinity cross-linking method using I-TGF-
1. Cells were cultured for 4 days (lanes 1 and 2), 7 days (lanes 3 and 4), and 14
days (lanes 5 and 6). Molecular weight markers are
indicated in the right margin.
Changes in mRNA Expression for ALP and Type II TGF-
In parallel with the increase in ALP activity after
long-term cultures of MC3T3-E1 cells with ascorbic acid, Northern blot
analysis revealed that ALP mRNA markedly increased after 14 days of
culture (Fig. 4). In contrast, the type II TGF-
Receptor receptor
mRNA level was not obviously affected after 14 days of culture (Fig. 4).
receptor in MC3T3-E1 cells during
long-term cultures. MC3T3-E1 cells were cultured in the presence of 50
mg/liter ascorbic acid until 14 days. Total RNA was extracted at 4 and
14 days of culture. Northern blot analyses were performed using cDNAs
for rat ALP and human type II TGF- receptor as probes. Ten µg
of total RNA was electrophoresed in 1% agarose gel and was transferred
onto a nylon membrane followed by hybridization with the radiolabeled
cDNA probes. mRNA expression of -actin was indicated as an
internal reference of a housekeeping gene.
Effects of Collagen Synthesis Inhibitors on Cell Surface
TGF-
In order to find out if the effect of
ascorbic acid on the abundance of TGF- Receptors receptors was also mediated
by its effect on collagen synthesis, the influence of collagen
synthesis inhibitors on TGF-1 binding to its receptors was
examined. When MC3T3-E1 cells were cultured in the presence of ascorbic
acid and treated with 0.3 mML-azetidine-2-carboxylic
acid, the reduction in the binding of TGF-1 to its receptors after
7 days of culture was blocked, whereas a simultaneous addition of 3
mML-proline eliminated the effect of 0.3 mML-azetidine-2-carboxylic acid (Fig. 5). Similar
results could be obtained when MC3T3-E1 cells were treated with other
inhibitors of collagen synthesis, cis-4-hydroxyproline and
3,4-dehydroproline, in the presence of ascorbic acid (data not shown).
None of these collagen synthesis inhibitors affected the binding of
TGF-1 to its receptors in the absence of ascorbic acid. These
results demonstrate that the synthesis of collagen is required for the
down-regulation of TGF- receptors competent to bind ligands as
well as for the increase in ALP activity after long-term cultures of
MC3T3-E1 cells.
receptors in MC3T3-E1 cells. MC3T3-E1 cells were cultured in
the absence of ascorbic acid for 4 days. Cells were then treated with (lanes 2, 4, and 5) or without (lanes 1, 3, and 6) 0.3 mML-azetidine-2-carboxylic
acid in the presence (lanes 3-6) or absence (lanes 1 and 2) of 50 mg/liter ascorbic acid for 3 days. Ten times
higher concentration of L-proline (3 mM) was added
with 0.3 mML-azetidine-2-carboxylic acid and
ascorbic acid in lane 5. Cell surface TGF- receptors were
demonstrated by a procedure described in the legend to Fig. 3.
Molecular weight markers are indicated in the right margin of the
figure.
Effect of Anti-
Type I collagen interacts with various types of cells
via the specific binding of DGEA motif on 21 Integrin Blocking Antibody
and DGEA Peptide on the Differentiation and TGF-
Receptors1 chain to cell surface
21 integrin(29, 30) . In order to clarify if
the effect of ascorbic acid on the differentiation of MC3T3-E1 cells
was in fact mediated via the interaction of collagen with 21
integrin, the influence of anti-21 integrin blocking antibody
and DGEA peptide on ALP activity was examined. As shown in Table 1, 10 mg/liter anti-21 integrin blocking antibody
significantly inhibited the effect of ascorbic acid on ALP activity.
The same concentration of an anti-51 integrin antibody that
interferes with the binding of 51 integrin to fibronectin did
not affect ALP activity. The addition of 5 mM DGEA peptide
also blocked the increase in ALP activity by ascorbic acid, but a
related peptide, KDGE, without binding affinity for 21
integrin had no effect (Table 1). Furthermore, the decrease in
TGF-1 binding to its receptors induced by ascorbic acid was also
blocked by the anti-21 integrin antibody (Fig. 6). The
antibody had no effect on TGF- receptors in the absence of
ascorbic acid. The addition of neither anti-21 integrin
blocking antibody nor DGEA peptide visibly disrupted the collagen
matrix organization or dislodged osteoblastic cells from culture
dishes.
21 integrin
blocking antibody on TGF- receptors in MC3T3-E1 cells. MC3T3-E1
cells were cultured in the absence of ascorbic acid. After 3 days of
culture, cells were treated with 50 mg/liter ascorbic acid, and 10
mg/liter monoclonal anti-mouse 21 integrin antibody was added
to cultures. TGF- receptors on the cell surface were determined as
described in the legend to Fig. 3. Cell surface TGF-
receptors in MC3T3-E1 cells cultured with (lanes 4-6) or
without (lanes 1-3) ascorbic acid. Anti-21
integrin antibody (lane 3 and 6) or non-immune IgG (lanes 2 and 5) was added along with ascorbic acid.
Migration positions of type I and type II TGF- receptors and
beta-glycan are indicated. Molecular weight markers are indicated in
the right margin.
Effect of Extracellular Matrix on the Differentiation and
TGF-
If a direct interaction of extracellular
collagen with Receptors21 integrin is involved in the differentiation
and the reduction in cell surface TGF- receptors of osteoblastic
cells, contact of MC3T3-E1 cells with collagen fibrils should cause
similar changes even when collagen synthesis is inhibited. In order to
test this possibility, ECM layer was first produced by culturing
MC3T3-E1 cells for 10 days in the presence of ascorbic acid, and then
cells were removed by a treatment with nonionic detergent. On these ECM
layers, MC3T3-E1 cells were cultured with or without ascorbic acid. As
shown in Table 2, when cells were cultured on ECM layers, ALP
activity was elevated even in the absence of ascorbic acid to a similar
level to that in cells cultured in the presence of ascorbic acid
without ECM layers. Although ECM layers contained a small amount of
ALP, ALP activities associated with ECM layers were less than 0.1
nmol/min/µg of protein, and could not account for the increase
observed in MC3T3-E1 cell cultures with ECM. The addition of ascorbic
acid to cultures on ECM layers did not further increase ALP activity.
The stimulation of proteoglycan synthesis in response to TGF-1 was
also inhibited when cells were cultured on the ECM (Table 2).
Furthermore, the addition of 10 mg/liter anti-21 integrin
blocking antibody at the seeding of cells onto the ECM layer inhibited
the increase in ALP activity, and partially restored the responsiveness
in proteoglycan synthesis to TGF-1 (Table 2). The attachment
of cells to ECM layers was not apparently inhibited by the antibody. In
parallel with these changes, TGF- receptors competent to bind
ligands decreased without ascorbic acid if cells were cultured on the
ECM (Fig. 7). The ECM layer without MC3T3-E1 cells did not
appreciably bind TGF-1, indicating that the reduction in the
binding of labeled TGF-1 to its receptors was not due to
sequestration of labeled TGF-1 by binding to the ECM layers (Fig. 7, lane 4).
receptors in MC3T3-E1 cells. Cell
surface TGF- receptors were demonstrated by a procedure described
in the legend to Fig. 3using I-TGF-
1 after 7
days of culture on plastic plates without (lanes 1 and 2) and with ECM layer in the absence (lanes 1 and 3) and presence of ascorbic acid (lane 2). A control
sample was prepared from ECM-coated plates without cells (lane
4).
is thought to
play an important role in bone formation(6) . The most
pronounced effect of TGF- in osteoblasts is the stimulation of the
synthesis of bone matrix proteins including type I collagen,
proteoglycans, and fibronectin. In contrast, it strongly inhibits the
expression of differentiation-associated phenotypes of osteoblasts such
as ALP and osteocalcin(9, 14, 33) .
Therefore, if TGF- actions persist, osteoblasts continue to
accumulate matrix proteins but cannot further differentiate to
mineralize the extracellular matrix, and bone formation cannot be
promoted. However, when applied in vivo, TGF- markedly
stimulates bone formation(11, 12) . In the present
studies, the development of ALP activity is associated with a
suppression of the actions of TGF-1 on both the stimulation of
proteoglycan synthesis and the inhibition of ALP activity ( Fig. 1and 2). The changes in these actions of TGF-1 are
closely correlated with a reduction in TGF-1 binding to its
receptors (Fig. 3). In addition, when collagen synthesis is
inhibited, all the differentiation-associated changes described above
are blocked. From these results, it is plausible to assume that the
accumulation of matrix collagen enhances the differentiation of
osteoblastic cells and suppresses the actions of TGF- by reducing
the receptors competent to bind TGF-, and that the change in the
responsiveness to TGF- allows these cells to escape from the
inhibitory effect of TGF- on osteoblastic differentiation and to
further differentiate into cells with mineralization capacity. Thus, it
is suggested that type I collagen in bone matrix plays an important
role in the regulation of osteoblastic differentiation, and that bone
matrix collagen synthesized under the stimulatory effect of TGF-
regulates the actions of TGF- by differentiation-associated
down-regulation of its receptors. receptors
has also been reported during retinoic acid-induced osteoblastic
differentiation of pluripotent mesenchymal cells(34) . In
addition, myogenic differentiation of myoblasts to myotubes is reported
to be inhibited by TGF-(35, 36) , and myocytes
lose TGF- receptors after the terminal
differentiation(37) . Stimulation of monocytes by cytokines (38) and suspension of lipocytes obtained from rat liver (39) have also been shown to result in down-regulation of
TGF- receptors on the cell surface. Taken together with our
results, these observations suggest that a decrease in TGF-
receptors is one of the mechanisms by which cells escape from the
control of TGF-. The decrease in TGF- receptors competent to
bind ligands does not appear to be caused by a reduction in the
synthesis of receptor proteins, because the abundance of the transcript
for type II TGF- receptor is not reduced to a similar extent (Fig. 4)(39) . Because matrix proteins can bind
TGF-, and because matrix layers surrounding these cells can hinder
TGF- receptors from binding to TGF-, the reduction in
TGF- binding to its receptors may be caused by a reduction in the
accessibility of labeled TGF-1 to its receptors. However, labeled
TGF-1 does not appreciably bind to ECM layers without MC3T3-E1
cells (Fig. 7). In addition, when cells were preincubated at 4
°C for 16 h before the TGF-1 binding studies, a procedure
known to inhibit endocytosis, ascorbic acid-induced down-regulation of
TGF-1 binding to its receptors was inhibited. ()These
observations are consistent with the assumption that the decrease in
the binding of TGF- to its cell surface receptors is not due to a
reduction in the synthesis of receptors, or entrapment of TGF- or
sequestration of TGF- receptors by collagen matrix, but may be due
to a change in the intracellular trafficking of receptor proteins.
However, further investigation is required on the precise mechanism
whereby TGF- binding to its receptors is reduced with osteoblastic
differentiation. receptors by
matrix collagen, there are possibilities that the synthesis of collagen per se or the interaction of osteoblastic cells with the
accumulated collagen affects the differentiation of osteoblastic cells.
Matrix collagen interacts with various cells through a specific binding
of DGEA motif to cell surface 21
integrin(40, 41) . The present results demonstrate
that an anti-21 integrin antibody that specifically blocks
the interaction of 21 integrin with its ligands inhibits not
only the differentiation but also the decrease in TGF-1 binding to
its receptors of MC3T3-E1 cells (Table 1, Fig. 6). The
differentiation of these cells is also blocked when a peptide
containing DGEA motif is added to the culture (Table 1).
Furthermore, when these cells are cultured on dishes coated with ECM
produced by MC3T3-E1 cells themselves, these cells are able to
differentiate in association with the decrease in the TGF- action
through the reduction in TGF- binding to its receptors even in the
absence of ascorbic acid (Table 2, Fig. 7). The
ECM-induced differentiation of these cells along with the decrease in
the responsiveness to TGF- are also inhibited by a blocking
antibody against 21 integrin (Table 2). These
observations are consistent with the assumption that the interaction of
osteoblastic cells with matrix collagen via 21 integrin plays
an important role in the differentiation and associated down-regulation
of TGF- receptors and actions in these cells. Interestingly, when
these cells were cultured on dishes coated with denatured type I
collagen, none of these changes could be observed. Because
maintenance of the conformation of collagen fibrils is shown to be
important for the specific binding of DGEA motif on collagen molecule
to 21 integrin(42) , contact of osteoblastic cells
with native collagen may be required to cause 21
integrin-collagen interaction. However, the possibility cannot be ruled
out that some ECM-associated factors may also be involved in the
osteoblastic differentiation. binding to its receptors and a suppression of the actions of
TGF- on both the stimulation of matrix protein synthesis and the
inhibition of differentiation. Furthermore, the
differentiation-associated changes are suppressed by a blocking
antibody against 21 integrin and a DGEA peptide that
interfere with the binding of cells to type I collagen. The
osteoblastic differentiation is also promoted by culturing cells on
ECM, which is again inhibited by an anti-21 integrin
antibody. These results suggest that bone matrix collagen synthesized
under the stimulatory effect of TGF- interacts with osteoblasts
via 21 integrin and plays an important role in the regulation
of osteoblastic differentiation and TGF- actions by
differentiation-associated down-regulation of TGF- receptors.
Recent studies demonstrate that the interaction of integrins with
matrix proteins provokes various changes in cellular proliferation,
differentiation, and functions via the activation of intracellular
signal transduction pathways. These include non-receptor tyrosine
kinase cascades such as Src and focal adhesion kinase, and the
Ras-mitogen-activated protein kinase pathway (43) . The present
observations warrant further investigation into the mechanism of the
control of osteoblastic differentiation and TGF- receptors by the
interaction between matrix collagen and cell surface integrin.
),
transforming growth factor-; ALP, alkaline phosphatase; PBS,
phosphate-buffered saline; ECM, extracellular matrix; CHAPS,
3-(cyclohexylamino)propanesulfonic acid.)
We thank Prof. Toshiro Fujita for encouragement during
the preparation of the manuscript, and Tomoko Kikuchi for excellent
technical assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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