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

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-

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

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

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

(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

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

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
*
*

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

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

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

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

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

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-

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

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

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

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

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

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.
References
1 Gelse K, Poschl E & Aigner T (2003) Collagens – struc-

MT1-MMP-mediated MMP-2 activation is repressed by
alphaVbeta3 integrin in human breast cancer cells.
Matrix Biol 26, 291–305.
9 Oh J, Takahashi R, Kondo S, Mizoguchi A, Adachi E,
Sasahara RM, Nishimura S, Imamura Y, Kitayama H,
Alexander DB et al. (2001) The membrane-anchored
MMP inhibitor RECK is a key regulator of extracellu-
lar matrix integrity and angiogenesis. Cell 107, 789–800.
10 Elenjord R, Ljones H, Sundkvist E, Loennechen T &
Winberg JO (2008) Dysregulation of matrix metallopro-
teinases and their tissue inhibitors by S100A4. Connect
Tissue Res 49 , 185–188.
11 Helfman DM, Kim EJ, Lukanidin E & Grigorian M
(2005) The metastasis associated protein S100A4: role
in tumour progression and metastasis. Br J Cancer 92 ,
1955–1958.
12 Maelandsmo GM, Hovig E, Skrede M, Engebraaten O,
Florenes VA, Myklebost O, Grigorian M, Lukanidin E,
Scanlon KJ & Fodstad O (1996) Reversal of the in vivo
metastatic phenotype of human tumor cells by an anti-
CAPL (mts1) ribozyme. Cancer Res 56, 5490–5498.
13 Bjornland K, Winberg JO, Odegaard OT, Hovig E,
Loennechen T, Aasen AO, Fodstad O & Maelandsmo
GM (1999) S100A4 involvement in metastasis: deregula-
tion of matrix metalloproteinases and tissue inhibitors
of matrix metalloproteinases in osteosarcoma cells
transfected with an anti-S100A4 ribozyme. Cancer Res
59, 4702–4708.
14 Mathisen B, Lindstad RI, Hansen J, El-Gewely SA,
Maelandsmo GM, Hovig E, Fodstad O, Loennechen T

regulated by parathyroid hormone and bone morphoge-
netic protein-2 in osteoblastic cells. Biochem Biophys
Res Commun
324, 218–223.
21 Azzam HS & Thompson EW (1992) Collagen-induced
activation of the M(r) 72,000 type IV collagenase in
normal and malignant human fibroblastoid cells. Cancer
Res 52, 4540–4544.
22 Takino T, Miyamori H, Watanabe Y, Yoshioka K,
Seiki M & Sato H (2004) Membrane type 1 matrix
metalloproteinase regulates collagen-dependent mito-
gen-activated protein ⁄ extracellular signal-related kinase
activation and cell migration. Cancer Res 64, 1044–
1049.
23 Seltzer JL, Lee AY, Akers KT, Sudbeck B, Southon
EA, Wayner EA & Eisen AZ (1994) Activation of
72-kDa type IV collagenase ⁄ gelatinase by normal fibro-
blasts in collagen lattices is mediated by integrin recep-
tors but is not related to lattice contraction. Exp Cell
Res 213, 365–374.
24 Lee AY, Akers KT, Collier M, Li L, Eisen AZ & Selt-
zer JL (1997) Intracellular activation of gelatinase A
(72-kDa type IV collagenase) by normal fibroblasts.
Proc Natl Acad Sci USA 94, 4424–4429.
25 Zigrino P, Drescher C & Mauch C (2001) Collagen-
induced proMMP-2 activation by MT1-MMP in human
dermal fibroblasts and the possible role of alpha2beta1
integrins. Eur J Cell Biol 80, 68–77.
26 Ruangpanit N, Chan D, Holmbeck K, Birkedal-Hansen
H, Polarek J, Yang C, Bateman JF & Thompson EW

MMP-2 activation coincides with up-regulation of
membrane type 1-matrix metalloproteinase and TIMP-2
in cardiac fibroblasts. J Biol Chem 278, 46699–46708.
33 Sato K, Hattori S, Irie S & Kawashima S (2003) Spike
formation by fibroblasts adhering to fibrillar collagen I
gel. Cell Struct Funct 28 , 229–241.
34 Ispanovic E & Haas TL (2006) JNK and PI3K differen-
tially regulate MMP-2 and MT1-MMP mRNA and
protein in response to actin cytoskeleton reorganization
in endothelial cells. Am J Physiol Cell Physiol 291,
C579–C588.
35 Strongin AY, Collier I, Bannikov G, Marmer BL,
Grant GA & Goldberg GI (1995) Mechanism of cell
surface activation of 72-kDa type IV collagenase. Isola-
tion of the activated form of the membrane metallopro-
tease. J Biol Chem 270, 5331–5338.
36 Will H, Atkinson SJ, Butler GS, Smith B & Murphy G
(1996) The soluble catalytic domain of membrane type
1 matrix metalloproteinase cleaves the propeptide of
progelatinase A and initiates autoproteolytic activation.
Regulation by TIMP-2 and TIMP-3. J Biol Chem 271,
17119–17123.
37 Winberg JO, Berg E, Kolset SO & Uhlin-Hansen L
(2003) Calcium-induced activation and truncation of
promatrix metalloproteinase-9 linked to the core protein
of chondroitin sulfate proteoglycans. Eur J Biochem
270, 3996–4007.
38 Morodomi T, Ogata Y, Sasaguri Y, Morimatsu M &
Nagase H (1992) Purification and characterization of
matrix metalloproteinase 9 from U937 monocytic

pendent biological activities. Sci Signal 1, re6.
46 Fodstad O, Brogger A, Bruland O, Solheim OP,
Nesland JM & Pihl A (1986) Characteristics of a cell
line established from a patient with multiple osteosar-
coma, appearing 13 years after treatment for bilateral
retinoblastoma. Int J Cancer 38, 33–40.
47 Kurschat P, Zigrino P, Nischt R, Breitkopf K, Steurer
P, Klein CE, Krieg T & Mauch C (1999) Tissue inhibi-
tor of matrix metalloproteinase-2 regulates matrix
metalloproteinase-2 activation by modulation of mem-
brane-type 1 matrix metalloproteinase activity in high
and low invasive melanoma cell lines. J Biol Chem 274,
21056–21062.
48 Chen JM, Dando PM, Rawlings ND, Brown MA,
Young NE, Stevens RA, Hewitt E, Watts C & Barrett
AJ (1997) Cloning, isolation, and characterization of
mammalian legumain, an asparaginyl endopeptidase.
J Biol Chem 272, 8090–8098.
49 Johansen HT, Knight CG & Barrett AJ (1999) Colori-
metric and fluorimetric microplate assays for legumain
and a staining reaction for detection of the enzyme after
electrophoresis. Anal Biochem 273, 278–283.
Collagen I-modulated MMP expression R. Elenjord et al.
5286 FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS