From the
A classical model for studying the effects of extracellular
matrix is to culture cells inside a three-dimensional collagen gel.
When surrounded by fibrillar collagen, many cell types decrease the
production of type I collagen, and the expression of interstitial
collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously
induced. To study the role of the collagen-binding integrins
The integrins are a large family of transmembrane proteins,
which form heterodimers and mediate cell-matrix and cell-cell
interactions (Hynes, 1992). Three integrins,
Given the fact that
the role of integrins as signaling receptors has been established,
Transfections were carried out using
Lipofectin reagent (Life Technologies, Inc.) according to the
manufacturer's recommendations. 400 µg/ml G418 (Life
Technologies, Inc.) was added to the culture media of transfected and
control cells. After 2-3 weeks of selection, the control cells
were dead, and G418 resistant clones were isolated and analyzed for
their expression of
In a second set of experiments,
Two integrin-type collagen receptors,
Previous studies with
migrating keratinocytes (Saarialho-Kere et al., 1993; Sudbeck
et al., 1994) and with invasive melanoma cells (Montgomery
et al., 1994) have shown the regulation of MMP-1 after
cell-collagen interactions. Here, we show that the amount of
In addition to MMP-1, other genes are regulated by signals generated
by integrin-type collagen receptors. Our data suggest that
Signal
transduction via integrins has been associated with protein tyrosine
phosphorylation (Schaller and Parsons, 1994). A specific focal adhesion
kinase (pp125
We thank Drs. M. Hemler, A. Weiss, E. Vuorio, P. Fort,
E. Bauer, V. Woods, and D. Carmichael for cDNAs, antibodies, and
vectors, Dr. S. Edwards for critical comments, and M. Potila for
technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1
1 and
2
1 in this process, we used three different
osteogenic cell lines with distinct patterns of putative collagen
receptors: HOS cells, which express only
1
1 integrin, MG-63
cells, which express only
2
1 integrin, and KHOS-240 cells,
which express both. Inside collagen gels,
1(I) collagen mRNA
levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells.
In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells
but not in HOS cells. Transfection of MG-63 cells with
2 integrin
cDNA produced cell clones overexpressing
2
1 integrin.
Transfection of MG-63 cells with
2 integrin cDNA in an antisense
orientation reduced the expression level of
2 integrin. These cell
clones showed induction and reduction of mRNA levels for MMP-1,
respectively. HOS cells normally lacking
2
1 integrin were
forced to express it, and this prevented the down-regulation in the
levels of
1(I) collagen mRNA when cells were grown inside collagen
gels. The data indicate that the level of MMP-1 expression is regulated
by the collagen receptor
2
1 integrin. The down-regulation of
collagen
1(I) is mediated by another receptor. Integrin
2
1 may compete with it and thus be a positive regulator of
collagen synthesis.
1
1,
2
1, and
3
1, have been proposed to be responsible
for anchoring cells to collagenous matrices. The role of
1
1
and
2
1 integrins as the major cellular collagen receptors has
been well documented, whereas in many cell lines
3
1 integrin
is not a collagen receptor. Integrin subunits
1 and
2 contain
an I-domain of about 190 amino acids, which is thought to be essential
for binding to collagen (Kern et al., 1994; Katama et
al., 1994). Both integrins also require the presence of
Mg
for ligand binding (Staatz et al., 1989).
Binding sites for
1
1 and
2
1 integrins in type I and
type IV collagen are in the triple helical area (Gullberg et
al., 1992; Eble et al., 1993). Denatured collagen can be
recognized by cells via RGD-binding integrins, whereas collagen binding
by
1
1 and
2
1 integrins require a native
conformation (Gullberg et al., 1992).
1
1 and
2
1 integrin heterodimers are the major
candidates to mediate the well known cellular responses to
extracellular collagen, i.e. decreased collagen gene
expression and induction of matrix metalloproteinase-1
(MMP-1)
(
)
production (Grinnell, 1994). Cells
surrounded by matrix are also able to reorganize collagen fibrils,
which is seen as the contraction of matrix (Grinnell(1994) and
references therein). Fibroblasts cultured inside floating or anchored
three-dimensional collagen matrices are supposed to mimic dermis or
scars and granulation tissue, respectively (Grinnell, 1994). The
regulation of MMPs by matrix receptors may also have great importance
for cancer cell invasiveness. The role of
2
1 integrin in the
reorganization of collagenous matrix is well documented (Shiro et
al., 1991; Klein et al., 1991a; Riikonen et al.,
1995), whereas the cellular receptors responsible for changes in
collagen and MMP-1 gene expression are not known. Here, we show that
the number of
2
1 integrin heterodimers at the cell surface
can be critical for the expression level of
1(I) collagen and
MMP-1.
Cell Culture
The human osteosarcoma
cell lines MG-63, HOS, and KHOS-240 (HOS cells transformed with Kirsten
murine sarcoma virus) were obtained from American Type Culture
Collection. Cells were maintained in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 10%
fetal calf serum (Life Technologies, Inc.). For collagen gel
experiments, eight volumes of Vitrogen-100 (Celtrix) was neutralized
with two volumes of 1:1 mixture of 10-fold concentrated medium and 0.1
N NaOH before adding detached cells.
Plasmid Constructs and
Transfections
2 integrin cDNA corresponding to
nucleotides 1-4559 in the published sequence (Takada and Hemler,
1989) was kindly provided by Dr. Hemler. It was ligated in both
orientations into the pAWneo2 expression vector (a kind gift from Dr.
Arthur Weiss) (Ohashi et al., 1985), which carries the
neomycin resistance gene.
2 integrin mRNA and protein (Riikonen et
al., 1995).
Northern Blot Hybridizations
Total
cellular RNA was isolated by the guanidium thiocyanate-CsCl method
(Chirgwin et al., 1979). Before isolating total RNA from the
cells inside collagen gels, the gels were briefly treated with 0.5
mg/ml collagenase (type II, Sigma) in phosphate-buffered saline (PBS,
pH 7.4) with 1 mM CaCl. 20 µg of total
cellular RNA was separated in formaldehyde-containing 1% agarose gels,
transferred to nylon membranes (ZETA-probe, Bio-Rad), and hybridized
with
P-labeled (Amersham) cDNAs for human
1(I)
collagen (Mäkelä et al., 1988), human MMP-1
(Goldberg et al., 1986), human
2 integrin (Takada and
Hemler, 1989), human tissue inhibitor of metalloproteinases-1 (TIMP-1)
(Carmichael et al., 1986), and rat glyceraldehyde-3-phosphate
dehydrogenase (a ``housekeeping'' enzyme used as a control)
(Fort et al., 1985) probes. Autoradiograms were quantified
with Microcomputer Imaging Device version M4 (Imaging Research Inc.),
and the resulting measurements were corrected for
glyceraldehyde-3-phosphate dehydrogenase mRNA levels.
Flow Cytometry
Cells were grown to early
confluence and detached with trypsin-EDTA, and trypsin activity was
inhibited by medium supplemented with serum. Cells were washed with PBS
(pH 7.4) and then incubated with PBS containing 10 mg/ml bovine serum
albumin, 1 mg/ml glycine, and 0.02% NaN at 4 °C for 20
min. Cells were collected by centrifugation, exposed to a saturating
concentration of monoclonal antibody against
2 integrin (12F1)
(Pischel et al., 1987) in PBS/bovine serum albumin (1 mg/ml)
containing NaN
at +4 °C for 30 min, and stained
with rabbit anti-mouse IgG coupled to fluorescein (1:20 dilution;
Dacopatts, Denmark) at 4 °C for 30 min. Cells were washed twice
with PBS containing NaN
and suspended in the same buffer.
To measure the amount of
2 integrin on the cell surfaces, the
fluorescent excitation spectra were analyzed by using a FACScan
apparatus (Becton Dickinson). Control samples were prepared by treating
the cells without primary antibodies.
Down-regulation of Collagen
We have taken
advantage of the fact, that the pattern of collagen-binding integrins
varies significantly in different osteogenic cell lines (Takada et
al., 1987; Heino and Massagué, 1989; Dedhar and Saulnier,
1990; Santala et al., 1994). MG-63 cells express mainly
1(I) and Induction
of MMP-1 Gene Expression Correlate with the Presence of
1
1
and
2
1 Integrins, Respectively
3
1 heterodimer and in smaller amounts
2
1 integrin.
Integrin
1
1 is not usually detectable, although its
expression can be induced, e.g. with cytokines (Santala and
Heino, 1991). HOS cells express both
1
1 and
3
1
integrins, whereas
2
1 integrin expression is below the
detection level (Santala et al., 1994). We have also used a
third cell line KHOS-240, a Kirsten sarcoma virus transformed variant
of HOS, which has all three putative integrin-type collagen receptors
(Santala et al., 1994). When cultured inside collagenous
matrices, only KHOS-240 cells showed fibroblast-like alterations in the
expression of MMP-1 and collagen
1(I) (induction of MMP-1 and a
90% decrease in collagen
1(I) mRNA levels; Fig. 1). This
observation is in accordance with the fact that fibroblasts and
KHOS-240 cells have a similar pattern of integrin-type collagen
receptors. Inside collagen gels, HOS cells showed markedly decreased
mRNA levels (80%) for collagen
1(I), whereas no MMP-1 mRNA wes
detected (Fig. 1). In MG-63 cells, there were no alterations in
the mRNA levels of
1(I) collagen. MG-63 cells cultured in
monolayer already expressed some MMP-1, and its expression was
increased when the cells were grown in collagen gels (2-8-fold
range in three measurements; Fig. 2and 3). Thus, in these three
cell lines, collagen
1(I) expression was down-regulated only when
1
1 integrin was present, and the induction of MMP-1
expression was seen only when
2
1 integrin was present.
Figure 1:
Northern blot analysis of 1(I)
collagen and interstitial collagenase (MMP-1) expression in HOS and
KHOS-240 cells grown in monolayer or inside collagen. Cells were grown
for 48 h, total cellular RNA was isolated, and the levels of mRNAs were
analyzed by specific
P-labeled probes and autoradiography.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as
control.
Figure 2:
Northern blot analysis of interstitial
collagenase (MMP-1) and TIMP-1 expression in MG-63 wild type
(wt) and 2 integrin cDNA-transfected cells
(sa2). MG-63 cells were transfected with sense-oriented
2
integrin cDNA in the pAWneo2 expression vector. A, the cells
were grown in monolayer or inside collagen gels before isolating total
RNA. 20 or 30 µg of cellular RNA was separated in 1%
formaldehyde-containing agarose gels, transferred to nylon membranes,
and hybridized with
P-labeled cDNA probes for MMP-1,
TIMP-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
B, autoradiograms were quantified with Microcomputer Imaging
Device, and the values were corrected for glyceraldehyde-3-phosphate
dehydrogenase mRNA levels.
In MG-63 Cells, the Overexpression of
To get direct evidence about the role of
2
1
Integrin Enhances and Its Down-regulation Reduces MMP-1 mRNA
Levels
2
1 integrin in the regulation of MMP-1 expression, we
specifically up- or down-regulated
2 integrin levels in MG-63
cells. We created a permanent MG-63 cell clone constantly
overexpressing
2 integrin by transfecting cells with a cDNA
construct containing the entire coding sequence of
2 integrin
(Riikonen et al., 1995). In this cell clone, the cell surface
level of
2 integrin was 5-fold higher than in wild type cells when
measured by flow cytometry (not shown). Recent studies have shown that
integrins might be sensitive to down-regulation by antisense strategies
(Lallier and Bonner-Fraser, 1993). Instead of using oligonucleotides,
we constructed a plasmid containing the
2 integrin cDNA in
antisense orientation. This construct gave a continuous expression of
an approximately 4.5-kilobase antisense mRNA in two MG-63 cell clones.
In these clones, the expression level of
2 integrin protein was
decreased (to 10% of control; not shown) as were the corresponding mRNA
levels (about 50% of control; Fig. 3). Interestingly, in these
two clones the ratio of antisense mRNA/
2 integrin mRNA were
different (1 and 6), suggesting that they had different copy numbers of
the antisense cDNA. The
2 integrin mRNA level, however, seemed to
be the same even if the amount of antisense mRNA increased
(Fig. 3). The levels of
2 integrin mRNA were strongly
(6-fold) up-regulated when the cells were inside collagen gels
(Fig. 3). Similar phenomena have been previously described with
melanoma cells and fibroblasts (Klein et al., 1991a). In
antisense transfected cell clones grown in collagen gels,
2
integrin mRNA levels were also elevated, although they remained at a
lower level than in control cells (35-50% of controls).
Simultaneously with the increase in
2 integrin mRNA levels, the
amount of antisense mRNA decreased (Fig. 3).
Figure 3:
Northern blot analysis of 1(I)
collagen, interstitial collagenase (MMP-1), and
2 integrin mRNA
expression in MG-63 wild type (wt) and two antisense
2
integrin-transfected cell clones (as#2, as#4).
A, the antisense-oriented
2 integrin cDNA in the pAWneo2
expression vector was transfected into MG-63 cells. The two resulting
clones, in which
2 integrin expression was reduced, were grown in
monolayer or inside collagen gels; total cellular RNA was isolated, and
specific mRNAs were analyzed. Autoradiograms were quantified, and the
MMP-1 mRNA levels in cells grown in monolayer after correction for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels
are shown in B.
In the MG-63 cell
clone overexpressing 2 integrin MMP-1, mRNA levels were 9-fold
higher than in wild type cells when the cells were cultured in
monolayer (Fig. 2). Inside collagen gels, the difference was
6-fold (Fig. 2). In both antisense transfected cell clones, the
basal level of MMP-1 was lower than in control cells (5-50% of
control; Fig. 3). MMP-1 mRNA levels were elevated in antisense
clone cells inside collagen gels, which is in accordance with the fact
that the antisense construct could not entirely block the induction of
2 integrin by collagen. The data indicate that in MG-63 cells,
elevated expression of
2 integrin enhances the expression of
MMP-1, and diminished expression of
2 integrin leads to its
down-regulation.
2 integrin was
overexpressed in HOS cells. The expression of
2 integrin on the
cell surface was confirmed by flow cytometric measurements (not shown).
Inside collagen gels, the level of the 4.5-kilobase plasmid-derived
2 mRNA was surprisingly elevated (Fig. 4), suggesting that
the increased
2 integrin mRNA levels are partially due to
increased stability of the mRNA. In HOS cells overexpressing
2
integrin, MMP-1 was detected in only two out of five experiments (not
shown), suggesting that in HOS cells MMP-1 expression is also regulated
by another factor more strongly than by
2 integrin.
Figure 4:
The levels of 1(I) collagen and
2 integrin mRNAs in HOS cells transfected with sense-oriented
2 integrin cDNA into pAWneo2 expression vector
(p
2AW#7, p
2AW#11). Total cellular RNAs from
cells grown inside collagen gels or in monolayer were isolated, and 20
µg of total cellular RNA was separated in formaldehyde-containing
1% agarose gels, transferred to nylon membranes, and hybridized with
P-labeled
1(I) collagen,
2 integrin, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
probes.
Many cell
types produce, concomitantly with MMP-1, its specific inhibitor, TIMP-1
(see Birkedahl-Hansen et al.(1993)). In 2 integrin
overexpressing MG-63 cells, TIMP-1 mRNA levels were decreased
(Fig. 2). This suggests that
2 integrin elicited
up-regulation of MMP-1 expression may result in increased pericellular
collagenase activity.
In HOS Cells, the Overexpression of
In 2
1
Integrin Prevents the Down-regulation of Collagen
1(I)
2
1 integrin positive wild type MG-63
cells, collagen
1(I) mRNA levels were not down-regulated,
indicating that
2
1 integrin is probably not the receptor
mediating suppression of collagen synthesis by collagenous matrix. The
data suggest that the unidentified receptor responsible for this
phenomenon may not be expressed in MG-63 cells, making
1
1
integrin one of the candidate molecules. Therefore, it was not
surprising that in MG-63 cells up-regulation or down-regulation of
2 integrin did not result in any dramatic changes in collagen mRNA
levels (Fig. 2). In both antisense and sense transfected cell
clones, there was some increase in the basal level of collagen
synthesis that was considered to be nonspecific. In contrast to MG-63
cells, HOS cells responded to collagenous matrix by diminished collagen
synthesis (Fig. 1). In HOS cells overexpressing
2 integrin,
the levels of collagen
1(I) mRNA elevated in response to collagen
matrix (Fig. 4). This elevation was seen in all experiments, but
was not always as prominent as in Fig. 4.
1
1 and
2
1 heterodimers, mediate the interaction of cells with
collagenous matrices. To investigate the role of
2
1 integrin
in the regulation of MMP-1 and collagen
1(I) expression, we used
cDNA transfections to specifically up- and down-regulate its expression
at the cell surface. The use of anti-integrin antibodies would have
given indirect evidence about the role of integrins. That approach,
however, would have been controversial because anti-integrin antibodies
might either block integrin function or simulate ligand binding and
activate signal transduction. Furthermore, anti-integrin
chain
antibodies alone are usually ineffective and can only be used to
potentiate the effect of anti-
1 antibodies (Klein et al.,
1991a; Riikonen et al., 1995).
2
1 integrin at the cell surface regulates the expression
level of MMP-1 in response to collagen. Under different physiological
conditions, the level of
2
1 integrin expression is not
constant but can be up-regulated by growth factors and malignant
transformation (Heino and Massagué, 1989; Dedhar and Saulnier,
1990; Santala et al., 1994). In melanoma cells, the
2
integrin subunit was originally described as the melanoma progression
antigen (Klein et al., 1991b); also, the ability to contract
collagen gels (an
2
1 integrin-related function) separates
aggressive melanoma clones from less invasive clones (Klein et
al., 1991a). In osteogenic HOS cells, transformation with either a
chemical mutagen,
N-methyl-N`-nitro-N-nitrosoguanidine, or
Kirsten murine sarcoma virus induces
2 integrin expression
(Santala et al., 1994). In rhabdomyosarcoma cells, the forced
expression of
2 integrin leads to an invasive cell phenotype (Chan
et al., 1991). The observation that
2
1
integrin-related signals regulate the expression of MMP-1 reveals a
putative invasion mechanism and explains why
2
1 integrin
seems to be important for cancer cells. Furthermore, this observation
provides the basis for new specific strategies to prevent invasion by
malignant tumors. Previous studies have shown that treatment of
fibroblasts with RGD peptides, anti-
5
1 integrin antibodies,
and fibronectin fragments induces the expression of MMPs (Werb et
al., 1989). Transformation can, however, down-regulate the
expression of
5
1 integrin (Plantefaber and Hynes, 1989) or
inhibit its activity (Akiyama et al., 1990), and therefore,
5
1 integrin is probably not involved in the regulation of
MMP-1 expression in transformed cells. Moreover, the forced
overexpression of
5
1 integrin prevents the malignant behavior
of Chinese hamster ovary cells, suggesting that
5
1 integrin
might function as a tumor suppressor (Giancotti and Ruoslahti, 1990).
2
integrin itself is positively regulated by collagenous matrix, whereas
collagen
1(I) is down-regulated in cells surrounded by collagen.
The suppression in collagen gene expression was seen in
1
1
positive-
2
1 negative HOS cells, suggesting that
1
1
integrin rather than
2
1 integrin mediates the signals
required. In HOS cells the overexpression of
2
1 heterodimer
not only prevented the down-regulation of collagen
1(I) mRNA
levels but even elevated them. The molecular mechanism of this
phenomenon is not clear. Previous studies have suggested that the
regulation of collagen synthesis in cells inside collagenous matrices
is a complex phenomenon involving both transcriptional and
posttranscriptional mechanisms (Eckes et al., 1993). Due to
the fact that in
1
1 negative-
2
1 positive MG-63
cells, the altered expression of
2
1 integrin did not have any
significant effect on collagen gene expression, we suggest that
2
1 integrin is a positive regulator of collagen synthesis by
competing with a ``negative regulator'' collagen receptor,
possibly
1
1 integrin. Inhibition of the down-regulation of
collagen synthesis by overexpression of
2 integrin suggests that
the accumulation of collagen in tissues can also be regulated by the
expression pattern of different collagen receptors. In a progressive
fibrotic skin disorder (scleroderma), there are alterations in the
expression of integrin-type collagen receptors (Ivarsson et
al., 1993). The fact that in scleroderma cells the ratio of
1
1 integrin/
2
1 integrin is decreased and that these
cells show reduced response to collagenous matrix (Ivarsson et
al., 1993) is in full accordance with our observations.
) is claimed to be directly linked to
integrins, and it is a strong candidate to be one of the first
components in the cascade (Schaller and Parsons, 1994). Furthermore,
the regulation of MMP-1 by cell-collagen interactions can be prevented
by inhibitors of protein kinase C and protein tyrosine kinases (Sudbeck
et al., 1994). Our observations suggest that different
collagen receptors might generate distinct signals, having opposite
effects on cell behavior (Fig. 5). It is an important challenge
for future research to elucidate the molecular mechanisms giving rise
to the specificity of signals generated by different matrix receptors.
Figure 5:
Model
for the distinct roles of the integrin-type collagen receptors in the
regulation of (1) collagenase (MMP-1) and (2)
collagen 1(I) expression (left). Rightpanel summarizes the main results of this paper supporting the model. It
is not clear whether
2
1 integrin up-regulates
1(I)
collagen mRNA levels directly or whether overexpression of
2
1
integrin competes with another collagen receptor. The
2 integrin
subunit itself is up-regulated in cells exposed to collagen. This
phenomenon might be mediated by
2
1 integrin-related
signals.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.