(Received for publication, June 23, 1995; and in revised form, November 28, 1995)
From the
Although transforming growth factor (TGF)- 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-
2
1
integrin blocking antibody blocked the changes in ALP activity and
TGF-
receptors, and a DGEA peptide that interferes binding of
collagen to
2
1 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-
2
1 integrin antibody. These results
demonstrate that the interaction of cell surface
2
1 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.
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- (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.
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- 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.
Figure 1:
Effect of TGF-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.
Consistent with the previous observations of the inhibitory effect
of TGF- 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.
Figure 2:
Effect of TGF-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).
Figure 3:
Cell surface expression of TGF-
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.
Figure 4:
Expression of mRNAs for alkaline
phosphatase and type II TGF- 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.
Figure 5:
Effects of a collagen synthesis
inhibitor, L-azetidine-2-carboxylic acid, on cell surface
TGF- 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.
Figure 6:
Effects of anti-2
1 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
2
1 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-
2
1
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.
Figure 7:
Effects of ECM produced by MC3T3-E1 cells
after long-term culture on TGF- 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).
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- 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.
A decrease in TGF- 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.
As to the mechanism of the regulation of the
differentiation and the down-regulation of TGF- 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
2
1
integrin(40, 41) . The present results demonstrate
that an anti-
2
1 integrin antibody that specifically blocks
the interaction of
2
1 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
2
1 integrin (Table 2). These
observations are consistent with the assumption that the interaction of
osteoblastic cells with matrix collagen via
2
1 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
2
1 integrin(42) , contact of osteoblastic cells
with native collagen may be required to cause
2
1
integrin-collagen interaction. However, the possibility cannot be ruled
out that some ECM-associated factors may also be involved in the
osteoblastic differentiation.
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- 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
2
1 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-
2
1 integrin
antibody. These results suggest that bone matrix collagen synthesized
under the stimulatory effect of TGF-
interacts with osteoblasts
via
2
1 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.