Tài liệu Báo cáo khoa học: Collagen I regulates matrix metalloproteinase-2 activation in osteosarcoma cells independent of S100A4 pdf

Collagen I regulates matrix metalloproteinase-2 activation
in osteosarcoma cells independent of S100A4
Renate Elenjord
1
, Jasmine B. Allen
2
, Harald T. Johansen
3
, Hanne Kildalsen
1
, Gunbjørg Svineng
2
,
Gunhild M. Mælandsmo
1,4
, Thrina Loennechen
1
and Jan-Olof Winberg
2
1 Department of Pharmacy, University of Tromsø, Norway
2 Department of Medical Biochemistry, University of Tromsø, Norway
3 School of Pharmacy, University of Oslo, Norway
4 Department of Tumor Biology, The Norwegian Radium Hospital, Oslo, Norway
Introduction
The extracellular matrix (ECM) is an intricate network
of macromolecules composed of a wide variety of
locally secreted proteins and polysaccharides which are
closely associated with the surface of the cell that pro-
duced them. The ECM can be found in different
forms, from the hard compositions of bone to the soft
structures of connective tissue. Collagens are the main

components of ECM, and type I collagen is the most
abundant form in bone and connective tissue [1].
Controlled turnover of ECM is critical for a wide vari-
ety of normal physiological processes, such as wound
healing and embryogenesis. The matrix metalloprotein-
ases (MMPs) are considered to be the major enzymes
involved in ECM remodelling, and dysregulated
MMPs have been implicated in several diseases such as
arthritis, cancer and cardiovascular disease [2]. The
family of MMPs consists of over 20 secreted and mem-
brane-bound enzymes which are involved in degrada-
Keywords
collagen I; extracellular matrix; inhibitors of
matrix metalloproteinases; matrix
metalloproteinases; S100A4
Correspondence
J O. Winberg, Department of Medical
Biochemistry, Institute of Medical Biology,
University of Tromsø, 9037 Tromsø, Norway
Fax: +47 776 45350
Tel: +47 776 45488
E-mail:
(Received 9 December 2008, revised 6 July
2009, accepted 20 July 2009)
doi:10.1111/j.1742-4658.2009.07223.x
This work investigates the effect of cell–collagen I interactions on the syn-
thesis and activation of MMP-2, as well as synthesis of MT1-MMP and
TIMP-1, by using an in vitro model with 3D fibrillar and 2D monomeric
collagen. In order to reveal whether the metastasis-associated protein
S100A4 can influence the cell’s response to the two forms of collagen, oste-

osarcoma cell lines with high and low endogenous levels of S100A4 were
used. Attachment of osteosarcoma cells to 3D fibrillar and 2D monomeric
collagen resulted in opposite effects on MMP-2 activation. Attachment to
3D fibrillar collagen decreased activation of proMMP-2, with a corre-
sponding reduction in MT1-MMP. By contrast, attachment to monomeric
collagen increased the amount of fully active MMP-2. This was caused by
a reduction in TIMP-1 levels when cells were attached to monomeric 2D
collagen. The effect of collagen on proMMP-2 activation was independent
of endogenous S100A4 levels, whereas synthesis of TIMP-1 was dependent
on S100A4. When cells were attached to monomeric collagen, cells with a
high level of S100A4 showed a greater reduction in the synthesis of TIMP-
1 than did those with a low level of S100A4. Taken together, this study
shows that synthesis and activation of MMP-2 is affected by interactions
between osteosarcoma cells and collagen I in both fibrillar and monomeric
form.
Abbreviations
APMA, p-aminophenylmercuric acid; ECM, extracellular matrix; MMPs, matrix metalloproteinases; MT-MMPs, membrane type matrix
metalloproteinases; TIMPs, tissue inhibitors of MMPs.
FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS 5275
tion and limited proteolysis of extracellular matrix.
Most MMPs are secreted as inactive proenzymes and
latency is maintained by an interaction between a
cystein residue in the prodomain and Zn
2+
in the
active site of the catalytic domain. Two major types of
endogenous inhibitors regulate the activity of MMPs;
a
2
-macroglobulin and four tissue inhibitors of metallo-

proteinases, TIMP-1 to TIMP-4 [2]. In order to iden-
tify potential drug targets, it is important to know the
role of individual MMPs, their expression pattern and
activation mechanisms. The best-described activation
mechanism for MMP-2 is the two-step cell-surface
activation in which proMMP-2 is activated in a trimo-
lecular complex including membrane type 1 MMP
(MT1-MMP) and the inhibitor TIMP-2, giving an
MMP-2 intermediate that is autocatalysed to fully
active MMP-2 [3]. Other activators of MMP-2 can
directly activate proMMP-2 to fully active MMP-2, for
example, the lysosomal cysteine proteinase legumain,
which is known to activate proMMP-2 by cleaving the
Asn80–Tyr81 (i.e. the autocatalytic site) and Asn82–
Phe83 bonds [4], and MT6-MMP, which can cleave
proMMP-2 at the Asn80–Tyr81 bond [5]. Some activa-
tors are suggested primarily to take part in the second
activation step, in which the MMP-2 intermediate is
converted to the fully active MMP-2. Among these are
TIMP-2 [6] and integrins such as aVb3 [7]. However,
there is some controversy regarding the role of the
aVb3 integrin, because it is also reported to suppress
collagen I-induced activation of proMMP-2 [8].
Another MMP inhibitor, reversion-inducing cysteine-
rich protein with kazal motifs (RECK) is a cell mem-
brane associated inhibitor of MMP-2 that is able to
inhibit the second activation step of MMP-2 [9].
The small calcium-binding protein S100A4 has been
shown to regulate expression of MMPs and their
inhibitors in several cell lines [10]. The protein itself

has no known enzymatic activity, but binds to distinct
intracellular target proteins and regulates specific func-
tions involved in tumour progression such as cell
motility, proliferation and apoptosis [11]. Although
S100A4 is strongly associated with the stimulation of
invasion and metastasis, the actual mechanism for the
metastasis-promoting function of S100A4 is not com-
pletely understood. The protein seems to have several
functions, both intracellularly and extracellularly. By
reducing the S100A4 level in a human osteosarcoma
cell line, and implementing these in mice, the capacity
to metastasize has been shown to decrease [12]. Culti-
vation of the same cell lines on plastic also showed
decreased expression and activation of MMP-2 [13,14].
Previously, we have shown that a reduced endoge-
nous level of S100A4 in human osteosarcoma cell lines
resulted in a reduced in vitro and in vivo invasive and
metastatic capacity [12,13]. Furthermore, we also
showed that the reduction in the endogenous level of
S100A4 in these cell lines resulted in altered levels of
MMP-2, MT1-MMP, TIMP-1 and TIMP-2, as well as
active MMP-2 [13,14]. Therefore, these cell lines were
used in this study to investigate the extent to which
synthesis and activation of proMMP-2, as well as syn-
thesis of MT1-MMP, TIMP-1 and TIMP-2, are
affected by the interaction of the cell with various bio-
logical forms of collagen I. A fibrillar 3D lattice and a
2D layer of monomeric collagen I will, to a certain
extent, mimic the natural environment of osteosarcoma
cells and were used in this study as an in vitro model

to study effects of cell–collagen I interactions.
Results
Cell morphology and actin cytoskeleton structure
As observed by light microscopy (data not shown),
pHb-1 and II-11b cells attached to plastic or mono-
meric 2D collagen were spread in a confluent cell layer
and hence showed maximum cell–cell contact. For cells
seeded on a fibrillar 3D collagen matrix, the cells were
rounded up and seemed to have a more spherical
shape; hence they were separated from most adjacent
cells, but were still attached to the surface.
Confocal microscopy revealed no differences in actin
cytoskeleton organization for cells attached to the dif-
ferent surfaces. In addition, whether the fibrillar 3D col-
lagen gel was attached to or released from plastic, or
whether the cells were attached on the top of or inside
the fibrillar 3D matrix did not influence the organization
of the actin cytoskeleton (data not shown).
Cell viability
As shown in Fig. 1, during 48 h incubation in serum-
free medium only minor changes in the number of via-
ble cells were observed for both cell lines attached to
plastic. However, when the cells were attached to
monomeric 2D collagen, the number of viable cells
increased, whereas for cells attached to fibrillar colla-
gen a small reduction in viable cells was observed.
S100A4 expression is not affected by the cells
binding to collagen I
In order to confirm the difference in S100A4 levels
between the two cell lines, western blot analyses were

performed on cell lysates from cells attached to plastic.
To ensure equal loading, the total amount of cellular
Collagen I-modulated MMP expression R. Elenjord et al.
5276 FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS
protein in the two cell lines was determined as
described in Materials and methods, and 81 lg of pro-
tein was added to each well. The amount of S100A4 in
the II-11b cells was 1.2% of that in the pHb-1 cells
after 2 min exposure of the film and 12% after 5 min
exposure (Fig. 2A).
The level of S100A4 protein expression did not
change when cells were attached to monomeric 2D or
fibrillar 3D collagen surfaces. pHb-1 cells maintained
high S100A4 expression, whereas the II-11b cells main-
tained low S100A4 expression (Fig. 2B). As shown in
Fig. 2B, equal amounts of protein were loaded based
on equal amounts of actin.
Binding of cells to fibrillar 3D collagen I results in
decreased proMMP-2 activation and reduced
MT1-MMP expression
A decrease in the total amount of MMP-2 (72, 64 and
62 kDa bands), as well as in the active MMP-2 forms
(64 and 62 kDa bands), was observed for both cell
lines attached to fibrillar 3D collagen compared with
cells attached to plastic (Fig. 3A). Because gelatin
zymography is a semiquantitative method, the amount
of proMMP-2 was also determined by ELISA. As
shown in Fig. 3B, a large decrease in proMMP-2 was
observed for cells attached to fibrillar collagen. Reduc-
tion in proMMP-2 activation occurred independent of

whether the fibrillar 3D collagen was attached to or
released from plastic, or whether the cells were
attached on top of or inside the fibrillar 3D matrix
(data not shown). Some of the synthesized MMP-2
was adsorbed to the fibrillar collagen, and the ratio of
active to total MMP-2 was the same as that detected
in the conditioned medium (data not shown). Thus,
adsorption may in part explain the reduction in the
total amount of MMP-2 in the medium, but it does
0
100
200
Relative cell
viability (%)
pH
β
–1 II-11b
pp
2D 2D3D 3D
3 h
48 h
*
*
Fig. 1. Cell viability. Relative cell viability (mean ± SEM) for pHb-1
and II-11b cells attached to plastic (P), monomeric 2D collagen I
(2D) and fibrillar 3D collagen I (3D). *P < 0.05 for 48 h compared
with 3 h (n = 7).
pHβ II-11b
A
S100A4

Actin
B
Time (min)
2 5
pHβ pHβ II-11b
II-11b
S100A4
P
2D
3D
P 2D 3D St
20
40
M
r
(kDa)
Fig. 2. Expression of S100A4 in pHb-1 and II-11b cells. (A) Deter-
mination of S100A4 by western blotting of cell lysates from pHb-1
and II-11b cells attached to plastic, using a total protein concentra-
tion of 81 lgÆmL
)1
, and the blot was exposed to the film for 2 or
5 min. (B) Western blot of cell lysates from pHb-1 and II-11b cells
attached to plastic (P), monomeric 2D collagen I (2D) and fibrillar
3D collagen I (3D). Actin was used as loading control. St: molecular
mass standard.
pHβ-1
II-11b
P 2D 3D P 2D 3D
3

2
1
0
Act/Tot MMP-2 (rel)
M
r
(kDa)
72
62
*
*
64
0
0.5
1.0
ProMMP-2 (rel)
P
2D 3D
P 2D 3D
pHβ-1
II-11b
B
*
*
*
1.5
A
Fig. 3. Expression of MMP-2 in serum-free media from pHb-1 and
II-11b cells. (A) Gelatin zymography of harvested media from pHb-1
and II-11b cells attached to plastic (P), monomeric 2D collagen I

(2D) or fibrillar 3D collagen I (3D). Typical zymograms showing
proMMP-2 (72 kDa), intermediate MMP-2 (64 kDa) and fully acti-
vated MMP-2 (62 kDa). Box-plots illustrate the ratio of activated to
total MMP-2. Open boxes denote pHb-1 cells while filled boxes
denote II-11b cells. Lines inside the boxes indicate median values,
and dotted lines illustrate mean values (n = 12). (B) Harvested
media from pHb-1 and II-11b cells attached to plastic (P), mono-
meric 2D collagen I (2D), or fibrillar 3D collagen I (3D) were analy-
sed for proMMP-2 expression by ELISA. Relative values (± SD) are
adjusted for cell viability. Open bars denote pHb-1 cells and filled
bars denote II-11b cells (n = 3). *P < 0.05 compared with cells
attached to plastic.
R. Elenjord et al. Collagen I-modulated MMP expression
FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS 5277
not explain the reduction in active forms. Because both
cell lines showed reduction in active forms, S100A4
did not influence this alteration in activation.
Western blots of cell lysates showed reduced expres-
sion of MT1-MMP for both cell lines when attached
to fibrillar 3D collagen compared with cells attached
to plastic (Fig. 4A). The level was reduced to 34% and
33% in pHb-1 and II-11b cells, respectively.
Cell binding to monomeric 2D collagen I
increased the amount of fully activated MMP-2
For cells attached to both plastic and monomeric 2D
collagen, pHb-1 cells produced more of the activated
forms of MMP-2 than did II-11b cells (Fig. 3A). For
both cell lines, attachment to 2D monomeric collagen
increased the amount of fully activated MMP-2
(62 kDa) and decreased the amount of the intermediate

activated form (64 kDa) compared with cells attached
to plastic (Figs 3A and 5). Although the amount of
activated forms varied between experiments, the
64 kDa intermediate form was always weaker when
cells were attached to monomeric 2D collagen than
when cells were attached to plastic (Fig. 5). However,
the ratio of activated forms to total MMP-2 was
approximately the same for cells attached to monomeric
collagen and to plastic (Fig. 3A). As shown by ELISA
in Fig. 3B, a decrease in proMMP-2 was observed when
cells were attached to momomeric collagen.
Western blots of cell lysates showed only small
changes in MT1-MMP expression for both cell lines
(+3% for pHb-1 and )10% for II-11b) when attached
to monomeric 2D collagen compared with cells
attached to plastic (Fig. 4A). Neither of the cell lines
showed any difference in TIMP-2 expression when
comparing cells attached to plastic and to monomeric
collagen (Fig. 4B). This indicates that TIMP-2 was not
involved in the observed difference in activation.
Cell binding to monomeric 2D collagen I results
in an S100A4-dependent decrease in TIMP-1
expression
Approximately twice as much TIMP-1 was secreted
into the medium from pHb-1 cells compared with
II-11b cells when attached to plastic (5.0 versus 3.1
lgÆmL
)1
Æ10
6

cells
)1
) (Fig. 6). The difference was sus-
tained when cells were attached to fibrillar 3D colla-
gen. However, when cells were attached to monomeric
2D collagen, pHb-1 cells produced significantly less
TIMP-1 (2.8 lgÆmL
)1
Æ10
6
cells
)1
), whereas the level
pH β -1
II-11b
MT1
Actin
P2D
3D
P2D
3D
St
40
50
50
60
M
r
(kDa)
Actin

B
II-11b pHβ-1
TIMP-2
20
40
P 2D P 2D St
M
r
(kDa)
A
Fig. 4. The expression of MT1-MMP and TIMP-2 in pHb-1 and II-
11b cells. Determination of MT1-MMP (A) and TIMP-2 (B) by wes-
tern blot of cell lysates from pHb-1 and II-11b cells. (A) Cells were
attached to plastic (P), monomeric 2D collagen I (2D) and fibrillar
3D collagen I (3D). Quantification of two blots gave mean values
for pHb-1 cells: P = 100%, 2D = 103%, 3D = 34% and for II-11B
cells: P = 100%, 2D = 90%, 3D = 33%. (B) Cells were attached to
plastic (P) and monomeric 2D collagen I (2D). Actin was used as
loading control. St: molecular mass standard.
pHβ-1
P 2D
II-11b
P 2D
72
62
64
72
62
64
M

r
(kDa)
Fig. 5. The effect of monomeric 2D collagen I on MMP-2 activa-
tion. Gelatin zymography of harvested media from pHb-1 and II-11b
cells attached to plastic (P) and monomeric 2D collagen I (2D). Typi-
cal zymograms showing proMMP-2 (72 kDa), intermediate MMP-2
(64 kDa) and fully activated MMP-2 (62 kDa).
0
2
4
6
TIMP-1
(µg·mL
–1
·10
6
cells
–1
)
pHβ-1 II-11b
P
2D
3
D
P
2D
3
D
*
*

Fig. 6. The effect of collagen I on TIMP-1 synthesis from pHb-1
and II-11b cells. The cells were either attached to plastic (P), mono-
meric 2D collagen I (2D) or on the top of fibrillar 3D collagen gel I
(3D). Harvested media were analysed for TIMP-1 expression by
ELISA. Open bars denote pHb-1 cells and filled denote for II-11b
cells. Mean values ± SEM are adjusted for cell viability (n = 6).
*P < 0.05 compared with cells attached to plastic.
Collagen I-modulated MMP expression R. Elenjord et al.
5278 FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS
was reduced to 2.3 lgÆmL
)1
Æ10
6
cells
)1
for the II-11b
cell line (Fig. 6).
TIMP-1 prevents the second step in the
MT1-MMP-induced activation of proMMP-2
Previously, it was shown that TIMP-1 prevents the
p-aminophenylmercuric acid (APMA)-induced auto-
activation of proMMP-2 and other MMPs [15–17]. In
order to test whether TIMP-1 inhibits the second step
in the MT1-MMP-induced activation of proMMP-2,
commercial recombinant proMMP-2 was activated for
24 h at 37 °C, either with membranes isolated from
colchicines-stimulated pHb-1 cells containing a high
level of MT1-MMP [18] or with a commercial recom-
binant soluble form of MT1-MMP containing only the
catalytic domain. As shown in Fig. 7, both the mem-

brane fraction and the recombinant soluble MT1-
MMP activated proMMP-2. As expected, TIMP-1 did
not inhibit the first step where MT1-MMP cleaves the
72 kDa proMMP-2 form into the 64 kDa intermediate.
However, TIMP-1 inhibited the second step that is an
autoactivation of the intermediate 64 kDa form to the
fully active 62 kDa enzyme. Further, autoactivation of
the active 62 kDa form to a C-terminally truncated
45 kDa form was also inhibited by TIMP-1.
Investigation of mechanisms that may explain
the increased activation of MMP-2 when cells are
attached to monomeric collagen I
To investigate whether TIMP-1 affected MMP-2 acti-
vation, recombinant TIMP-1 was added to cells
attached to monomeric 2D collagen. As shown in
Fig. 8A, the intermediate 64 kDa band is stronger in
the presence of TIMP-1 than in the absence of the
inhibitor. In order to determine whether other previ-
ously described mechanisms are also involved, several
experiments were performed. First, we studied whether
an interaction between either the pro (72 kDa) or
intermediate (64 kDa) MMP-2 and the underlying col-
lagen layer caused the formation of a fully activated
62 kDa form of the enzyme. Harvested media from
cells attached to plastic were incubated for 24 h at
37 °C in culture wells with or without monomeric col-
lagen. Neither of these two conditions altered the rela-
tive amount of the activated forms, indicating that
binding of the pro (72 kDa) or intermediate (64 kDa)
forms of MMP-2 to collagen did not result in

enhanced autoactivation (data not shown). Second, we
wanted to investigate whether the cysteine proteinase
legumain is involved in the activation. The presence of
this proteinase was shown in both cell lines, but incu-
bation on monomeric 2D collagen did not change its
level (data not shown). Third, treatment of cells
attached to plastic or monomeric 2D collagen with the
cysteine proteinase inhibitors egg white cystatin, E-64
or E-64d showed no effect on the synthesis and activa-
tion of proMMP-2 (Fig. 8B). Fourth, in order to
determine whether the observed activation took place
intracellularly or extracellularly, surface proteins of
cells attached to plastic and 2D collagen were labelled
with biotin and removed as described in Materials and
methods. The unlabelled intracellular fraction of pro-
teins were analysed by gelatin zymography. No active
MMP-2 was detected, demonstrating that the activa-
tion occurred outside the cell (data not shown). Fifth,
24 h 37 °C
0
0
0
0
rMT1-MMP
0
0.5
0.5
0
1.0
1.0

2.0
2.0
Membr.
[TIMP-1]
[MMP-2]
72
62
64
Fig. 7. Activation of proMMP-2 by isolated cell membranes and
MT1-MMP in the presence of TIMP-1. Human recombinant proM-
MP-2 (3 lgÆmL
)1
;42nM) was incubated for 24 h at 37 °C with
membranes isolated from colchicine treated pHb-1 cells or recombi-
nant human MT1-MMP catalytic domain in the presence of increas-
ing concentrations of TIMP-1 as described in Materials and
methods and analysed by gelatin zymography. As controls, the
proMMP-2 alone was either dirctly added to loading buffer without
incubation or after 24 h incubation at 37 °C.
TIMP-1 (ng·well
–1
)
0
0
75
P 2D
A

C
E64

E64d
Cys
2D
B
72
62
64
72
62
64
Fig. 8. The effect of added inhibitors on MMP-2 activation. Typical
gelatin zymograms showing (A) the effect of TIMP-1 on the activa-
tion of MMP-2 using pHb-1 cells attached to monomeric 2D colla-
gen (2D) compared with pHb-1 cells attached to plastic (P), (B) the
effect of cystein proteinase inhibitors E-64, E-64d and cystatin
(Cys) on the activation of MMP-2 using pHb-1 cells attached to
monomeric 2D collagen (2D). C, control without added inhibitors.
R. Elenjord et al. Collagen I-modulated MMP expression
FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS 5279
we investigated whether new activators on cell mem-
branes from cells attached to monomeric 2D collagen
could be responsible for the increased activation of
proMMP-2. Isolated cell membranes from cells
attached to plastic and monomeric 2D collagen did
not show any difference in their capacity to activate
proMMP-2 at pH 8.0 or at pH 5.8, indicating no new
activators at the cell membrane of cells attached to the
monomeric collagen.
Discussion
The two osteosarcoma cell lines used in this study have

characteristics suggesting they are of osteoblastic origin
[19,20]. In bone, active osteoblasts are embedded in a
3D ECM, whereas fully mature osteoblasts flatten out
and line quiescent bone surfaces. Collagen I is the
main component of ECM, and interactions between
the osteosarcoma cell lines and the 3D fibrillar colla-
gen I network or the 2D layer of monomeric collagen
I will, to a certain extent, mimic the in vivo situation
for these cells. Hence, this model can reveal to what
extent cell–collagen I interactions will affect synthesis
and activation of MMPs and their inhibitors. Further,
this model will discover whether the endogenous level
of the metastasis-associated protein S100A4 is of
importance for how the cells respond to interactions
with these two forms of collagen I.
Included among cells that have been shown to
increase the activation of proMMP-2 when they inter-
act with fibrillar 3D collagen I are various human
tumour cell lines [21,22], human skin fibroblasts
[21,23–27], human umbilical vein and neonatal foreskin
endothelial cells [28], human fetal lung fibroblasts [29],
human hepatic stellate cells [30], rat capillary endothe-
lial cells [31] and rat cardiac fibroblasts [32]. In most
cases, the increased activation of proMMP-2 is shown
to be associated with an increase in MT1-MMP. Fur-
thermore, cells have a changed morphology when
attached to fibrillar 3D collagen compared with the
same cells attached to a 2D surface such as monomeric
collagen or plastic [21–23,29–31,33]. This was also
shown for the osteosarcoma cells in our study where

the cells appeared more rounded in shape. In human
skin fibroblasts there is an increase in proMMP-2 acti-
vation that is independent of the lattice contraction
[23]. It has also been shown that cells grown on fibril-
lar 3D collagen I attached to a plastic surface contain
actin stress fibres, whereas stress fibres are missing
when collagen is released from the surface. Only under
conditions where the cells lack stress fibres, do fibro-
blasts produce increased amounts of activated MMP-2
[29]. A lack of stress fibres is also necessary for the
increase in the activation of proMMP-2 in smooth
muscle endothelial cells [34]. In several of the studies
referred to above, it has also been shown that treat-
ment of cells attached to a planar substrate (such as
monomeric collagen or plastic) with compounds that
dissolve actin stress fibres (cytochalasin D, vascular
endothelial growth factor), results in increased activa-
tion of proMMP-2. However, treating cells with com-
pounds that dissolve the tubulin network (colchicine,
nocodazole) did not induce proMMP-2 activation. In
contrast to this, we have previously shown that colchi-
cine-induced rearrangements of the microtubule
network in osteosarcoma cell lines increase activation
of proMMP-2 along with an increased level of
MT1-MMP [18]. This study shows that the interaction
between osteosarcoma cells and fibrillar 3D collagen
reduces the activation of proMMP-2 because of a
decrease in MT1-MMP. The reduction was indepen-
dent of whether the 3D collagen lattice was attached
to plastic or not. In contrast to the cell lines discussed

above, the actin cytoskeleton in the osteosarcoma cells
was not affected by the surface the cells were attached
to. Hence, the reduction in MT1-MMP and active
forms of MMP-2 could not be attributed to changes in
the actin cytoskeleton. This adds to previous investiga-
tions showing that these osteosarcoma cell lines
respond differently to various stimuli compared with
other cells.
We also show that the osteosarcoma cells produce an
increased amount of fully active MMP-2 when bound
to 2D monomeric collagen (Figs 3A and 5) which is
another example of a different characteristic trait of
these cells compared with fibroblasts and endothelial
cells. No drastic change in the amount of MT1-MMP
was observed in osteosarcoma cells attached to mono-
meric collagen compared with plastic. Although
MT1-MMP here may participate in the activation of
proMMP-2, it cannot account for the increased amount
of fully activated enzyme. MT1-MMP induces the con-
version of proMMP-2 to the intermediate 64 kDa form
by cleaving the Asn37–Leu38 bond [35,36], whereas the
64 kDa intermediate is further processed to the fully
activated 62 kDa species in an autoactivation step. In
this study, various experiments were performed to
determine whether one of the following mechanisms
was responsible for the increase in fully activated
MMP-2 when cells were attached to monomeric colla-
gen: (a) increased expression of an activator enzyme
that cleaves proMMP-2 in or near the autocatalytic site
(Asn80–Tyr81), (b) increased expression of a factor

that stimulates the second step of the MT1-MMP
induced activation, or (c) reduced expression of an
inhibitor of the second step of the activation.
Collagen I-modulated MMP expression R. Elenjord et al.
5280 FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS
The two osteosarcoma cell lines used in this study
produce legumain, a potential intracellular activator of
proMMP-2. However, because we were not able to
detect any intracellular fully activated MMP-2, nor
could we inhibit MMP-2 activation with E-64, we can
rule out legumain or other lysosomal cysteine protein-
ases as activators when the cells are attached to mono-
meric 2D collagen. Furthermore, there was no
difference in activation of exogenously added recombi-
nant proMMP-2 between isolated cell membranes from
cells attached to plastic and monomeric 2D collagen,
irrespective of the pH used. This result excludes the
existence of some enzyme in membranes from cells
attached to monomeric collagen that cleaves proMMP-
2 either in or close to the autoactivation site. Nor
could the activation be explained by autoactivation
caused by a direct binding of either the 72 kDa pro-
form or the 64 kDa intermediate to the cell membrane.
Lafleur et al. [6] have shown that TIMP-2, in addition
to being involved in the first step in the MT1-MMP
activation of MMP-2, can also take part in the second
autoactivation step and function as an activator by
promoting conversion of the 64 kDa intermediate to
the fully active 62 kDa form. In this study, we did not
find any difference in the levels of TIMP-2 when cells

were attached to plastic compared with monomeric 2D
collagen (Fig. 4B), hence TIMP-2 is not the cause of
the observed difference in activation level of MMP-2.
Our results rule out the two first alternatives, (a) and
(b), as an explanation for the increased activation
when cells are attached to monomeric 2D collagen.
However, alternative (c), reduced expression of an
inhibitor of the second step of the activation, seemed
to be an explanation. We have shown that TIMP-1 is
a regulator of the second step in the activation of
proMMP-2 using both recombinant MT1-MMP and
isolated cell membranes rich in MT1-MMP (Fig. 7).
This is consistent with previous observations that
TIMP-1 inhibits autoactivation of several MMPs such
as: the Ca
2+
-induced intramolecular autoactivation of
proMMP-9 covalently linked to the core protein of a
chondroitin sulfate proteoglycan [37]; the APMA-
induced autoactivation of MMP-9 to the 80 kDa inac-
tive intermediate and the 68 kDa active species, where
TIMP-1 prevented the formation of the latter species
[15,38,39]; APMA-induced autoactivation of proMMP-
2 [15]; APMA-induced autoactivation of proMMP-3
and N-terminally truncated proMMP-3 [15,40]; and
APMA-induced autoactivation of proMMP-1 and
proMMP-8 [16,17]. At the cellular level, we have
shown that exogenously added TIMP-1 increased the
intermediate 64 kDa form of MMP-2 (Fig. 8A), indi-
cating that the decreased level of TIMP-1 is the main

cause of increased activation of MMP-2 when cells
were attached to monomeric 2D collagen. Altogether,
our results show that endogenously produced TIMP-1
can act as a modulator of the MT1-MMP-induced
activation of proMMP-2.
The interaction between cells and fibrillar or mono-
meric collagen, respectively, showed opposite effects
on proMMP-2 activation. This effect was independent
of the endogenous level of S100A4 in the two cell lines.
By contrast, the expression of TIMP-1 was dependent
on the cell endogenous level of S100A4. When cells
were attached to plastic and fibrillar 3D collagen,
those with a high endogenous level of S100A4 pro-
duced approximately twice as much TIMP-1 as those
with a reduced S100A4 level. However, when cells were
attached to monomeric 2D collagen, the production of
TIMP-1 from cells with a high level of S100A4 was
reduced to approximately the same amount as from
cells with a low S100A4 level. This suggests that the
interaction between cells and monomeric 2D collagen
causes a block in the S100A4-induced pathway that
upregulates TIMP-1 expression. Taken together, our
results show that osteosarcoma cells interact with two
types of collagen I found in vivo, and the form of the
collagen determines the cells synthesis and activation
of MMP-2 as well as the synthesis of MT1-MMP and
TIMP-1.
Previously, it has been shown that the reduced level
of S100A4 in the II-11b cells compared with pHb-1
cells resulted in a large reduction of in vivo and in vitro

invasive capacity, as well as in vitro motility [12,13].
The reduction in S100A4 also resulted in a decrease in
the expression of MT1-MMP, TIMP-1 and MMP-2 at
both the mRNA and protein levels, in addition to a
decreased amount of activated MMP-2 [13,14]. MMPs
and TIMPs are associated with cell invasion and
metastasis, although their role is dual [41–43]. Both
MMPs and TIMPs, as well as the in vivo substrates of
a given MMP, can prevent or facilitate the invasion
and metastasis process, depending on the time and
localization of their expression. The N-terminal part of
TIMPs is involved in binding to the active site of
MMPs and hence prevents their action, whereas the
C-terminal part can bind to proteins in the cell mem-
brane and modulate cell growth and viability indepen-
dent of MMPs [44,45]. One of the aims of current
research on MMPs and TIMPs in cancer is to establish
the localization and timeframe for their expression, as
well as the identification of the in vivo substrates of
individual MMPs. It is thus important to discover how
each ECM component in the microenvironment of a
given cancer type affects expression and activation of
MMPs and TIMPs. Our investigation shows that two
R. Elenjord et al. Collagen I-modulated MMP expression
FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS 5281
forms of the main ECM component in the microenvi-
ronment of osteosarcoma cells differently affect their
expression of MMPs and TIMPs as well as their acti-
vation of MMP-2. In addition, it adds to the earlier
investigations by showing that the structure of the sur-

rounding matrix components will be of importance for
the effect of S100A4 on MMP and TIMP regulation
and thereby on the cell’s possibility to promote inva-
sion and metastasis.
Materials and methods
Materials
DMEM containing Hams F12 medium, penicillin, strepto-
mycin, geneticin disulfate salt (G418), gelatin 300 Bloom
type A (from porcine skin), egg white cystatin (C-0408),
BSA, Igepal CA-630, bactrerial collagenase and Sigma
serum replacement 1 were all from Sigma-Aldrich (St.
Louis, MO, USA). Fetal bovine serum was from Biochrom
AG (Berlin, Germany), l-glutamine was from Gibco BRL
Life Technologies (Paisley, UK), nonessential amino acids
(100·) were from PAA Laboratories GmbH (Pasching,
Austria), sterile rat-tail collagen I was from Roche Diag-
nostics GmbH (Basel, Switzerland), poly(vinylidene difluo-
ride) membranes were from Millipore (Bedford, MA,
USA), Alexa Fluor 568-labelled phalloidin (A12380), Magic
Mark Western Standard and 4–12% NuPAGE Bis ⁄ Tris
gels were from Invitrogen Life Technologies (Carlsbad, CA,
USA), Biotrac TIMP-1 and MMP-2 ELISA were from
Amersham Biosciences (Little Chalfont, UK) and E-64
(N-1645) and E-64d (N-1650) were from Bachem (Buben-
dorf, Switzerland). Western Blotting Luminol Reagent was
from Santa Cruz Biotechnology (Santa Cruz, CA, USA),
EZ-Link Sulfo-NHS-LC-LC biotin, streptavidin agarose
resins and halt protease inhibitor cocktail were from Pierce
Biotechnology (Rockford, IL, USA). The following anti-
bodies were used; GAPDH rabbit mAb and pan-actin

rabbit polyclonal from Cell Signaling Technology (Danvers,
MA, USA), MT6-MMP mouse mAb from R&D systems
(Minneapolis, MN, USA), TIMP-2 and MT1-MMP rabbit
polyclonal from Panomics (Redwood City, CA, USA),
S100A4 rabbit polyclonal from Abcam (Cambridge, UK)
and RECK mouse mAb from BD Biosciences (San Jose,
CA, USA). Anti- mouse and anti-rabbit IgG horseradish
peroxidase-linked antibodies were from Cell Signalling
Technology. TIMP-1 was from Oncogene Research Prod-
ucts (Boston, MA, USA), purified human recombinant
proMMP-2 and MT1-MMP (catalytic domain) were from
Calbiochem (San Diego, CA, USA). Solution cell prolifera-
tion assay (Cell Titer 96AQueous One) was from Promega
(Madison, WI, USA). Paraformaldehyde was purchased
from Merck (Darmstadt, Germany) and Triton X-100 from
BDH Biochemicals Ltd. (Poole, UK).
Cell cultures
The highly metastatic osteosarcoma cell line, OHS, was
established from a bone tumour biopsy from a patient trea-
ted at the Norwegian Radium Hospital [46]. The OHS cell
line was transfected with a vector encoding a S100A4-spe-
cific ribozyme or, as a control, with the vector alone [12].
The ribozyme-transfected clone was designated II-11b, and
the control cell clone transfected with the vector alone was
designated pHb-1. The II-11b cell line had a reduced level
of S100A4 and a decreased metastatic capacity, whereas the
pHb-1 cell line maintained the S100A4 expression level and
metastatic properties of the parental OHS cell line [12].
Transfectants were subcultivated in a 1 : 1 mixture of
DMEM and Hams F12 medium (basal medium) containing

10% fetal bovine serum, 400 lgÆmL
)1
geneticin, 2.0 nm
l-glutamine, nonessential amino acids (100· dilution), peni-
cillin (100 IUÆmL
)1
) and streptomycin (100 lgÆmL
)1
). The
cells were kept in a humidified 5% CO
2
atmosphere at
37 °C.
Preparation of wells for cell experiments
Cells (6.0 · 10
4
) were, in addition to plastic, attached to
monomeric 2D collagen I and on top and inside fibrillar
lattices of 3D collagen I, all in 0.33 cm
2
wells. Ten microli-
tres of 0.16 mgÆmL
)1
collagen I in 0.2% acetic acid was
spread into the wells to prepare monomeric collagen I. The
wells were dried for 2 h, followed by washing in serum-free
medium (culture medium in which fetal bovine serum was
replaced by 2% serum replacement) and left with 50 lL
serum-free medium for 20 min. 3D fibrillar collagen I gels
were prepared by adding 50 lL neutralized collagen I solu-

tion (7 : 1 : 1 : 1 of each 3 mgÆmL
)1
collagen I in 0.2% ace-
tic acid, 10· serum-free medium, 1.0 m Hepes, pH 7.3, and
0.33 m NaOH, respectively) to the wells. After 2 h of poly-
merization at 37 °C, wells were equilibrated with serum-free
medium for 20 min and the medium was removed before
cells were added in a new aliquot of medium. For cell
attachment inside 3D collagen I gels, cells were mixed with
50 lL neutralized collagen I solution and left for 2 h while
polymerization took place.
Cell viability assay
To determine the viability of the cells, trypsinized cells were
suspended in serum-containing medium. In order to remove
serum, cells were washed three times with serum-free med-
ium prior to seeding on the different plate surfaces as
described above. A 100-lL cell suspension was added to
plastic and monomeric 2D collagen, whereas 50 lL serum-
free medium with or without cells was added to the 3D
collagen gels. After 3 and 48 h incubation in 5% CO
2
at
37 °C, cell viability reagent was added to the wells and the
Collagen I-modulated MMP expression R. Elenjord et al.
5282 FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS
absorbance at 490 nm was determined according to the
instructions of the manufacturer’s protocol. Prior addition
of cell viability reagent to wells containing fibrillar 3D col-
lagen, bacterial collagenase (15 lL of 18.6 mgÆmL
)1

colla-
genase in 0.1 m Hepes pH 7,5 containing 0.9% NaCl) was
added to dissolve the polymerized gel and after  25 min
at 37 °C, EDTA (10 mm) was used to stop the collagenase
activity. For each surface cells were attached to, standard
curves were made to assure a linear response between cell
number and absorbance.
Phalloidin staining of actin filaments
Cells were cultured on glass covers slides, on monomeric
2D collagen I, on top of or inside fibrillar 3D collagen I for
24 h in serum-free media. Cells were fixed in a final concen-
tration of 4% paraformaldehyde for 10 min on ice, washed
with 1· NaCl ⁄ P
i
and permeabilized by incubation with
0.1% Triton X-100 in 1· NaCl ⁄ P
i
for 10 min on ice. After
washing with 1· NaCl ⁄ P
i
, cells were incubated with 1%
BSA in 1· NaCl ⁄ P
i
for 30 min at room temperature before
labelling with 1 unit of Alexa Fluor 568-conjugated phalloi-
din in 1% BSA for 30 min at room temperature. Cells were
then washed five times with 1· NaCl ⁄ P
i
before images were
obtained using a 40· water objective on a LSM 510 META

confocal laser-scanning inverted microscope (Carl Zeiss
International, Go
¨
ttingen, Germany).
Production of conditioned media for MMP and
TIMP determination
To determine the secretion of MMP-2 and TIMP-1 into the
media, trypsinized cells were suspended in serum-containing
medium. In order to remove serum, cells were washed three
times with serum-free medium prior to seeding on the dif-
ferent plate surfaces, as described above. A 100 lL cell sus-
pension was added to plastic and monomeric 2D collagen,
whereas 50 lL serum-free medium with or without cells
was added to the 3D collagen gels. After 48 h incubation in
5% CO
2
at 37 °C, the conditioned media and 3D gels were
harvested. Prior to freezing, the harvested media was centri-
fuged and taken to 10 mm CaCl
2
, 0.1 m Hepes, pH 7.3. To
test whether TIMP-1 or legumain and other lysosomal cys-
teine proteinases were involved in proMMP-2 activation,
cells were attached to plastic and 2D collagen I surfaces
with or without inhibitors (25–300 ngÆwell
)1
of TIMP-1,
10 lm E-64, 10 lm E-64d or 1 lm egg white cystatin).
Isolation of cell membranes
Cells (1.4 · 10

7
) were seeded in serum-free media on 66 cm
2
Petri dishes, uncoated or coated with 2D monomeric colla-
gen I (2 mL 0.16 mgÆmL
)1
in 0.2% acetic acid), and kept
the conditions described above. Plasma membranes were
prepared as previously described [18,47]. Production and
purification of plasma membranes from pHb-1 cells
attached to plastic and treated with colchicine under serum
free conditions were performed as previously described [18].
Isolation of cell lysates
To compare the level of S100A4 in the two cell lines culti-
vated on a plastic surface, confluent cells in a 75 cm
2
were
washed in NaCl ⁄ P
i
, and released from the plastic with a
rubber scraper, suspended in NaCl/P
i
and pelleted at
4000 g. Cells were then sonicated and the lysate centrifuged
at 4000 g for 5 min at 4 °C in order to remove cell debris.
The amount of cellular protein was detected by the Brad-
ford method (Bio-Rad, Hercules, CA, USA), using BSA as
a standard.
Activation of MMP-2 by cell membranes
Membrane-mediated activation of human MMP-2 was per-

formed by incubating the proenzyme (3 lgÆmL
)1
;42nm)
with membrane protein (500 lgÆmL
)1
) from cells attached
to plastic or 2D collagen I in 50 mm Tris ⁄ HCl, pH 8.0,
5mm CaCl
2
, 0,005% Brij 35 or 39.5 mm citric acid, pH
5,8, 121 mm Na
2
HPO
4
, 0.8% NaCl, 0.005% Brij 35 at
37 °C. Aliquots were withdrawn after 0, 6, 12 and 24 h and
analysed by gelatin zymography. Activation experiments
using cell membranes from colchicine stimulated pHb-1
cells were performed as described previously [14,18], with
and without recombinant TIMP-1 present.
Activation of MMP-2 by recombinant MT1-MMP
Activation of MMP-2 by MT1-MMP was performed by
incubating human recombinant MMP-2 (3 lgÆmL
)1
;42nm)
with human recombinant MT1-MMP catalytic domain
(4 lgÆmL
)1
; 200 nm) in the absence and presence of recom-
binant TIMP-1 (0.588–2.352 lgÆmL

)1
; 21–84 nm) for 24 h
at 35 °Cin50mm Tris ⁄ HCl, pH 8.0, 5 mm CaCl
2
, 0.005%
Brij 35. Aliquots of MMP-2 corresponding to 400 pgÆwell
)1
were applied to gelatin zymography.
Biotinylation of cell-surface proteins
Cells (2.0 · 10
6
) were seeded in serum-free media in six-well
plates either uncoated or coated with monomeric 2D colla-
gen (290 lL, 0.16 mgÆmL
)1
collagen I in 0.2% acetic acid),
and kept under the conditions described above. Condi-
tioned media were removed and wells were washed with
cold NaCl ⁄ P
i
(pH 8). To release cells, NaCl ⁄ P
i
was added
and cells were incubated at 37 °C. Cells were suspended at
a concentration of 25 · 10
6
cellsÆmL
)1
in NaCl ⁄ P
i

and
16 lLof10mm biotinreagent solution was added. The
reaction mixture was incubated at room temperature for
R. Elenjord et al. Collagen I-modulated MMP expression
FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS 5283
30 min. Cells were washed with NaCl ⁄ P
i
containing
100 mm glycine to remove excess biotin reagent and there-
after lysed (in 50 mm Tris ⁄ HCl pH 7.4, 150 mm NaCl,
1mm CaCl
2
,1mm MgCl
2
, 0.5% Igepal, 1% inhibitor coc-
tail). To remove biotinylated surface proteins, cell lysate
was added to a streptavidin agarose resin and incubated for
30 min. After centrifugation, only unlabelled intracellular
proteins were found in the supernatant.
Gelatin zymography
Conditioned medium was mixed with loading buffer
(333 mm Tris ⁄ HCl, pH 6.8, 11% SDS, 0.03% bromophenol
blue and 50% glycerol) and loaded onto a 10% gelatin gel.
To determine the amount of gelatinase in the harvested 3D
collagen I gels, an equal volume of gel and loading buffer
were mixed and left for 30 min at room temperature prior
to centrifugation (4000 g for 5 min at 4 °C) and the extract
was applied to the zymography gel. To determine whether
gelatinases bind to monomeric 2D collagen, 10% dimethyl
sulfoxide in serum-free media was added to wells after

removal of conditioned medium, and the extract was added
loading buffer and applied to the gelatin gel. Gelatin
zymography gels were run, washed and stained as described
previously [14]. Gelatinolytic activity was evident as trans-
parent zones in the blue gels. The area of the cleared zones
was analysed using the genetools program from SynGene
(Cambridge, UK).
Western blot analysis
After removal of conditioned media, cells were lysed in gel
loading buffer containing 0.1 m dithiothreitol, and boiled
for 5 min. Samples were electrophoresed on acrylamide gra-
dient gels (4–12%) and proteins were transferred to a
poly(vinylidene difluoride) membrane by electroblotting.
After blocking nonspecific binding sites with non-fat milk
(5% solution), blots were incubated for 1 h at room tem-
perature with primary antibodies against S100A4, MT1-
MMP or TIMP-2. After washing, the blots were incubated
for 1 h at room temperature with horseradish peroxidase-
conjugated secondary antibodies diluted in blocking solu-
tion, and developed using a western blotting luminol
reagent. The membranes were washed, blocked and rep-
robed for the detection of actin or GAPDH. The amount
of protein in the detected spots were analysed on either the
gelbase ⁄ gelblotÔ pro program from Ultra Violet Prod-
ucts (Cambridge, UK) or the genetools program.
ELISA
The levels of TIMP-1 and MMP-2 were determined from
serum-free conditioned media, according to manufacturer’s
instructions. The TIMP-1 assay recognizes total human
TIMP-1, i.e. free TIMP-1 and TIMP-1 bound to MMPs.

The MMP-2 assay recognizes proMMP-2 and proMMP-2
bound to TIMP-2, but not the active form of MMP-2.
Legumain activity
Legumain was measured by recording the cleavage of the
substrate Z-Ala-Ala-Asn-NHMec (Department of Biochem-
istry, University of Cambridge, UK), as previously
described [48,49]. Twenty microlitres of cell lysate were
added to black 96-well microtiter plates (Costar). After the
addition of 100 lL buffer and 50 lL substrate solution
(10 lm final concentration), a kinetic measurement based
on increase in fluorescence over 10 min was performed.
Temperature was kept at 30 °C and all measurements were
performed in triplicate.
Statistics
Statistical analyses were performed using the student t-test
for independent analysis. Data are presented as
mean ± SD (gelatin zymography, western blotting and
ELISA data). A P-value < 0.05 was considered significant.
Analyses were based on three or more independent cell cul-
ture experiments. Conditioned medium from each experi-
ment was run in duplicate on gelatin zymography, ELISA
and western blots.
Acknowledgements
This work was supported in part by grants from The
Norwegian Cancer Society and the Erna and Olav
Aakre Foundation for Cancer Research. We are grate-
ful to Dr Peter McCourt for linguistic revision of the
manuscript.
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