(Received for publication, May 16, 1994; and in revised form, October 27, 1994)
From the
The contraction of floating collagen gels is suggested to mimic
the reorganization of collagenous matrix during development and tissue
healing. Here, we have studied two osteogenic cell lines, namely MG-63
and HOS, and a chemically transformed subclone of HOS cells, HOS-MNNG.
Transforming growth factor- (TGF-
), a putative regulator of
bone fracture healing, increased collagen gel contraction by MG-63 and
HOS-MNNG, but not by HOS cells. Our data show that TGF-
-induced
fibronectin synthesis is not sufficient for the process. Instead,
anti-
1 integrin antibodies could prevent the contraction. There
are three different integrin heterodimers that are known to mediate the
cell-collagen interaction, namely
1
1,
2
1, and
3
1. In MG-63 cells TGF-
increased the expression of
2
1 integrin and decreased the expression of
3
1
integrin, whereas
1
1 integrin is not expressed. HOS cells had
no
2
1 integrin, neither did TGF-
induce its expression.
However, HOS-MNNG cells expressed more
2
1 integrin when
treated with TGF-
. Thus, we suggest that the mechanism of the
enhanced collagen gel contraction by TGF-
is the increased
expression of
2
1 integrin heterodimer. To further test this
hypothesis, we expressed a full-length
2 integrin cDNA in HOS
cells and in MG-63 cells. We obtained HOS cell clones that expressed
2
1 heterodimer, and the ability of these cells to contract
collagen gels was greatly enhanced. Furthermore, the contraction by
MG-63 cells transfected with
2 integrin cDNA was enhanced, and the
contraction by cells transfected with antisense oriented
2
integrin cDNA was decreased. Thus, both in MG-63 and HOS cells the
increased
2 integrin expression alone was sufficient for the
enhanced contraction of collagen gels. Furthermore, the amount of
2 integrin is critical for the process, and its decrease leads to
diminished ability to contract gels.
Hydrated collagen lattices were first described as a method to
mimic soft-tissue matrices (Elsdale and Bard, 1972). Watery milieu
within fibrous collagen net has since been used as a substrate for
growth and differentation for many types of cells. When fibroblasts are
embedded in collagen they are able to reduce the areas of the collagen
gels and form a tissue-like structure. The ultrastructure of
fibroblasts during contraction differ from the same cells plated on a
plastic surface. Differences in the prominence of cell coat, changes of
the prevalence of cell processes and the state of aggregation of their
associated microfilamentous material are seen (Bellows et al.,
1981). Collagen gels can be used to study the contraction of either
floating matrix or anchored matrix (Grinnell, 1994). In the two
variations of in vitro collagen matrix reorganization model,
the morphology and the behavior of fibroblasts differ. The first one is
suggested to mimic dermis or scars and the latter granulation tissue
(Grinnell, 1994). The exact molecular mechanism of collagen gel
contraction is unknown. Previous studies have suggested an essential
role for cell surface collagen receptors (Gullberg et al.,
1990; Shiro et al., 1991; Klein et al., 1991). Also
cellular fibronectin (FN) ()has been suggested to be
required in the collagen matrix contraction by fibroblasts (Asaga et al., 1991).
Transforming growth factor- (TGF-
)
has been discovered to enhance collagen gel contraction by skin
fibroblasts (Montesano and Orci, 1988). Later, a similar effect has
been described with other growth factors and cytokines (Clark et
al., 1989; Gullberg et al., 1990). Some others, like
interferon-
, can inhibit the process (Dans and Isseroff, 1994).
TGF-
is a multifunctional regulator of cells which acts in the
process of wound healing (for review, see
Massagué, 1990). Many cell types can in vitro and in vivo produce TGF-
, and it has been found
abundant in platelets and in bone. It stimulates the accumulation of
extracellular matrix (Ignotz and Massagué, 1986)
and regulates cell adhesion apparatus (Ignotz and
Massagué, 1987). TGF-
is supposed to have a
central role in tissue healing. The fact that it is present in large
amounts in bone and that it increases bone formation in vivo in test animals (Noda and Camilliere, 1989; Joyce et al.,
1990) indicates the possible role of TGF-
in the healing of bone
fractures. Here, we have shown that TGF-
can increase collagen gel
contraction by osteogenic cells. Since TGF-
is known to regulate
the integrin type collagen receptors in different cell lines (Heino and
Massagué, 1989; Heino et al., 1989) and
also to increase production of cellular FN (Ignotz and
Massagué, 1986; Roberts et al., 1987),
these are the candidate mechanisms to be responsible for the
phenomenon. Integrins are a family of cell surface receptors consisting
of two subunits,
and
(for review, see Hynes, 1992;
Ruoslahti, 1991). Three different integrin heterodimers are known to
mediate cell-type I collagen interaction, namely
1
1,
2
1, and
3
1. In addition to
1,
2, and
3 subunits,
1 subunit can form heterodimers with
4-
9 subunits and
v subunit. Several of these
1
integrins recognize FN as their ligand, namely
3
1,
4
1,
5
1, and
v
1. Laminin is the ligand for
1
1,
2
1,
3
1,
6
1, and
7
1 integrin heterodimers.
We have determined the effects
of TGF- on collagen gel contraction by osteogenic cell lines, in
which the pattern of collagen-binding integrins differs from each
other, and identified the molecular mechanism by which TGF-
enhances collagen gel contraction. Our data suggest that up-regulation
of
2
1 integrin is required and alone sufficient to increase
contraction.
The same 4.5-kb cDNA was
linked to pAW vector also in antisense orientation. Cells were
transfected and selected as above. Among the first 28 cell clones
analyzed, two showed markedly reduced expression of 2 integrin and
the presence of antisense
2 integrin mRNA.
Figure 1:
The effects of TGF- and IL-1
on collagen gel contraction by MG-63 (A), HOS (B),
and HOS-MNNG (B) cells. Collagen gels were prepared by using
Vitrogen-100 collagen. 50,000 cells were added in neutralized collagen
and poured into 24-well plates. Gels were let to polymerize at 37
°C for 45 min after which the edges of the gels were gently removed
from the sides of the wells, and DMEM supplemented with 10% FCS was
added on them. Concentrations of TGF-
and IL-1
used were 200
pM and 10 units/ml, respectively. After 2-4 days of
contraction, the surface areas of the collagen gels were measured. Mean
± S.D. of four parallel measurements is
shown.
Figure 2:
The
effects of 10 µg/ml plasma fibronectin (pFN) and cellular
fibronectin (cFN) on collagen gel contraction by MG-63 cells.
Cells were added together with pFN or cFN into neutralized Vitrogen-100
collagen solution and were then plated into 24-well culture plates.
Collagen polymerization was initiated by incubating the plates at 37
°C for 45 min. After the polymerization was complete, DMEM
supplemented with 10% FCS was added on gels, and the sides of the gels
were gently removed from the sides of the wells. MG-63 cells treated
with 200 pM TGF- were used as a contraction control.
After 4 days the areas of the gels were measured. Mean ± S.D. of
four parallel measurements is shown.
Figure 3:
The blocking of TGF--induced collagen
gel contraction by MG-63 cells with functional antibodies against
different collagen-binding integrin subunits. Antibodies were added
into neutralized Vitrogen-100 collagen together with the cells. After
incubation at 37 °C for 45 min, the edges of the gels were detached
from the sides of the wells, and DMEM supplemented with 10% FCS and 200
pM TGF-
was added on them. The areas of the gels were
measured after 4 days of contraction. Antibodies against
1,
1,
2, and
3 subunits were used. Mean ± S.D. of
four parallel measurements is shown.
Figure 4:
Effect of TGF- on
2 integrin
subunit biosynthesis. Confluent cultures of MG-63 and HOS-MNNG cells
were incubated in serum-free medium with TGF-
for 12 h. Cells were
transferred to methionine-free medium containing 50 µCi/ml
[
S]methionine and 200 pM TGF-
. An
equal amount of radioactivity from the cell lysates was precipitated
with anti-
2 subunit antibody. The immunoprecipitants were analyzed
by electrophoresis followed by
fluorography.
Figure 5:
Integrin expression in and collagen gel
contraction by HOS cells. A, immunoprecipitation of
collagen-binding integrins from HOS cells. Cells were labeled with
[S]methionine in methionine-free medium for 24
h. An equal amount of radioactivity from the cell lysates was incubated
with antibodies against
1,
2, and
3 integrin subunits.
Immunoprecipitants were analyzed by SDS-PAGE under nonreducing
conditions followed by fluorography. B, blocking of the
collagen gel contraction of HOS cells by functional antibody against
1 integrin subunit. 10,000 cells were added together with mAb13
into neutralized Vitrogen-100 collagen solution. Gels were let to
polymerize in 96-well plates before detaching the gels from the sides
of the wells and adding DMEM supplemented with 10% FCS on them. The
areas of the gels were measured after 6 days of incubation. The figure
shows four parallel control and anti-
1 antibody-containing
wells.
Chemical transformation of HOS cells turns on the
expression of 2 integrin subunit (Santala et al., 1994).
Here, TGF-
could slightly (about 2.5-fold) increase the expression
of
2 integrin in HOS-MNNG cells (Fig. 4). TGF-
also
enhanced the contraction by these cells (Fig. 1). The data
suggest that the increased expression of
2
1 integrin
correlates with the enhanced collagen gel contraction by TGF-
.
Figure 6:
Integrin expression in pAW2
transfected HOS cells. A, immunoprecipitation of
2
integrin in HOS cells transfected with full-length
2 integrin
cDNA. Human full-length
2 integrin cDNA was linked into pAWneo2
expression vector, which carries the neomycin resistance gene. HOS
cells were transfected by using Lipofectin reagent, and stable cell
clones were selected by using G418. G418-resistant clones were tested
for their expression of
2 integrin protein in immunoprecipitation
assays (in the figure clone 16 is shown). Control cells
(marked as C) were transfected with pAWneo2 expression vector
only. Wild type HOS cells are marked as Wt. Cells were
metabolically labeled with [
S]methionine, and an
equal amount of radioactivity from each cell lysate was precipitated
with
2 integrin antibody. Immunoprecipitants were analyzed by gel
electrophoresis and fluorography. B, Northern blot
hybridization of total RNA from
2 integrin-transfected HOS cells (clones 11 and 16) and MG-63 wild type cells (MG). cDNA probe specific to
2 integrin was
used.
Figure 7:
Adhesion to different substrates and
collagen gel contraction by pAW2 transfected HOS cells. A, adhesion of HOS cells expressing
2 integrin (clones 16 and 7) to type I collagen (T I
COL), laminin (LM), type IV collagen (T IV COL),
and cellular fibronectin (cFN). 96-well immunoplates were
coated with different matrix molecules, and bovine serum albumin (BSA) was used to measure the nonspecific binding. Residual
protein absorption sites were blocked with 1% bovine serum albumin.
10,000 cells were let to adhere for 45 min after which the adherent
cells were fixed with paraformaldehyde, stained with crystal violet,
and air-dried. The cell bound stain was dissolved in acetic acid and
measured spectrophotometrically at 600 nm. B, collagen gel
contraction by HOS cells expressing
2 integrin (clone
16). HOS cells transfected with pAWneo2 vector and wild type HOS
cells were used as a control for a cell clone to be tested. 50,000
cells were added in neutralized type I collagen solution and
immediately transferred into 24-well plates. Collagen gel
polymerization was initiated by incubating the plates at 37 °C for
45 min. DMEM supplemented with 10% FCS was added on gels, and the edges
of the gels were gently removed from the sides of the wells. After 2
days of incubation at 37 °C, the areas of the gels were measured.
Two parallel experiments are shown.
Figure 8:
Effects of the expression of sense and
antisense 2 integrin mRNA in MG-63-cells. Immunoprecipitation of
MG-63 cells transfected with sense (A, clone 4) or
antisense (B, clones 1-4)-oriented
2
integrin cDNA. MG-63 cells were transfected with pAWneo2 expression
vector containing the
2 integrin cDNA. Transfections were done by
using Lipofectin reagent, and G418-resistant cell clones were tested
for their expression of
2 integrin by immunoprecipitation.
Immunoprecipitants were analyzed by SDS-PAGE followed by fluorography.
Contraction of collagen gels by MG-63 cells transfected with sense (C, clone 4) or antisense (D, clones 1 and 4)-oriented
2 integrin cDNA. MG-63 cells
transfected with pAWneo2 vector only (pAW1) were used as
control cells for sense-transfection clones, and wild type MG-63 cells
were used as controls for antisense-transfected cell clones. 50,000
cells were added into neutralized Vitrogen solution and poured into
24-well plates. Collagen polymerization was initiated by incubating the
plates at 37 °C. After polymerization was complete DMEM
supplemented with 10% FCS was added on gels. After 4 days the areas of
the collagen gels were measured. Mean ± S.D. of four parallel
measurements is shown.
TGFs- are a family of growth and differentiation
factors. Among other biological functions, TGFs-
are supposed to
be essential for wound healing and tissue repair processes
(Massagué, 1990). In general, they increase the
accumulation of connective tissue macromolecules and angiogenesis
(Massagué, 1990). Both phenomena are important in
the formation of scars. A major source of TGF-
1, and also
TGF-
2, is bone matrix. In the healing of bone fractures, recently
discovered members of TGF-
superfamily called bone morphogenetic
proteins (BMP 2-7) are suggested to be involved in the initiation
of the healing process (Reddi, 1992). TGF-
is more probably
involved in the other stages of the healing process, e.g. in
the synthesis of new matrix (Reddi, 1992). The important role of
extracellular matrix and growth factors for bone forming cells has also
been emphasized in the recent review by Robey et al.(1993).
We have tested two osteogenic cell lines, MG-63 and HOS, for their
ability to contract collagen gels and studied the effect of TGF-
on the process. Both cell lines could contract collagen gels, and
anti-
1 integrin antibodies could inhibit this contraction. We and
others (Takada et al., 1987; Heino and
Massagué, 1989; Dedhar and Saulnier, 1990;
Santala et al., 1994) have previously described the
1
integrin pattern in both cell lines. There are three putative
integrin-type collagen receptors, namely
1
1,
2
1,
and
3
1. In MG-63 cells
2
1 and
3
1 are
expressed, whereas no
1
1 is present. HOS cells express
distinct integrin pattern:
1
1 and
3
1 are present,
but
2
1 is missing. Both MG-63 cells and HOS cells express
5
1 fibronectin receptor, whereas
6
1 laminin
receptor is expressed only in HOS cells (Santala et al.,
1994). Previous reports have suggested the involvement of
1
integrins in the collagen gel contraction, especially by skin
fibroblasts (Klein et al., 1991; Shiro et al., 1991).
Here, we show that osteogenic cells can still induce contraction, even
if the
1
1 or
2
1 heterodimers are missing. Thus, the
data suggest that these heterodimers can replace each other or that
they both can be replaced by a third
1 integrin containing
heterodimer. TGF-
could increase collagen gel contraction by MG-63
cells but not by HOS cells. In MG-63 cells TGF-
strongly induces
the synthesis of
2 integrin and concomitantly decreases the
expression of
3 subunit, whereas it cannot turn on the expression
of
1 subunit (Heino and Massagué, 1989).
This suggests that the effect of TGF-
is due to increased
expression of
2 integrin. Here, the increased contraction could be
inhibited totally by anti-
1 integrin antibody and partially by
anti-
2 integrin antibody, whereas anti-
1 integrin antibody
had no effect. Previous studies have shown that anti-
integrin
antibodies can not alone inhibit collagen gel contraction by
fibroblasts, but only enhance the effect of anti-
1 antibody (Klein et al., 1991). Interestingly, anti-
3 antibody constantly,
in three out of three experiments, stimulated contraction. The
mechanism of this phenomenon stays unknown. However, we have recently
shown that in keratinocytes
3
1 heterodimer is connected to a
signal transduction pathway regulating the expression of gelatinases
(Larjava et al., 1993a). The role of
2 subunit was also
suggested by the fact that in HOS cells TGF-
cannot induce its
expression (Santala et al., 1994). We have previously shown
that transformation of HOS cells with MNNG induces the cells to express
2 integrin subunit (Santala et al., 1994). Here, we show
that TGF-
increases both
2 integrin expression and collagen
gel contraction by HOS-MNNG cells.
In addition to altered integrin
expression, TGF- induces several other changes in cell metabolism
that could have explained the increased collagen gel contraction. Here,
we have excluded the involvement of TGF-
-induced increase in
fibronectin synthesis. More importantly, we have shown that increased
expression of
2 integrin subunit alone is sufficient to increase
contraction by both MG-63 and HOS cells. This was done by expressing
2 integrin cDNA in these cells. We have previously shown that both
cell lines express a large intracellular pool of excess precursor
1 integrin subunit (Heino and Massagué,
1989; Santala et al., 1994). Therefore, it is possible to get
functional integrin heterodimers by forced expression of an
subunit. Furthermore, transfection of MG-63 cells with
antisense-oriented
2 integrin cDNA generated cell clones with
reduced ability to contract gels.
Cell behavior can be regulated by
both growth factors and extracellular matrix. The fact that a growth
factor, TGF-, can regulate the synthesis of matrix molecules and
their cellular receptors connects the two mechanisms together.
Previously, some of the effects of TGF-
on cell growth and
differentiation have been partially explained by the altered structure
of extracellular matrix in TGF-
-treated cell cultures (Heino and
Massagué, 1990; Nugent and Newman, 1989). The
data presented here show that TGF-
regulates the cell behavior
also by altering their integrin pattern. We and others have shown, that
in addition to TGF-
also other cytokines, including interleukin-1,
tumor necrosis factor-
, and interferon-
, can regulate
integrin expression in numerous tissue-cultured cell types (Santala and
Heino, 1991; Defilippi et al., 1991). In in vivo conditions, including wound healing (Larjava et al.,
1993b) and chronic inflammation (Nikkari et al., 1993), where
cytokines are present in large amounts, it is possible to detect
dramatic changes in integrin expression. The role of osteoblast
integrins in the healing of bone fractures is unknown. Recent studies
have shown, that osteoblastic cells in bone and in culture express
several integrin heterodimers. However,
2
1 is expressed only
in small quantities, if at all (Hughes et al., 1993; Brighton
and Albelda, 1992; Clover et al., 1992; Saito et al.,
1994). Our data suggest that TGF-
might be involved in the
reorganization of collagenous matrix also by osteogenic cells; however,
only in the case that these cells already express
2
1
integrin. We have previously suggested the presence of an inhibitory
element, other than DNA methylation, in HOS cells, which prevents the
expression of
2 integrin gene (Santala et al., 1994).
Transformation of cells with both MNNG and Kirsten murine sarcoma virus
can induce the expression of
2 integrin in HOS cells (Santala et al., 1994), but we have not yet found a physiological
inducer.
To conclude, we have shown that TGF- induces collagen
gel contraction by osteogenic cells. The process is due to increased
expression of
2
1 integrin-type collagen receptor. In cells
which do not express
2 subunit, TGF-
can not turn on its gene
expression or induce collagen gel contraction. The data show that
TGF-
can regulate cellular functions by altering integrin pattern.
Furthermore, we propose that TGF-
might be one of the factors
involved in reorganization of bone matrix during the healing of
fractures.