Induction of Collagenase-3 (MMP-13) Expression in Human Skin
Fibroblasts by Three-dimensional Collagen Is Mediated by p38
Mitogen-activated Protein Kinase*
Laura
Ravanti
§,
Jyrki
Heino§¶,
Carlos
López-Otín
, and
Veli-Matti
Kähäri
§**
From the
Department of Dermatology, Turku University
Central Hospital and the § Department of Medical
Biochemistry, and MediCity Research Laboratory, University of Turku,
FIN-20520 Turku, Finland, the ¶ Department of Biological and
Environmental Science, University of Jyväskylä, FIN-40351
Jyväskylä, Finland, and the
Departamento
Bioquímica y Biología Molecular, Universidad de Oviedo,
33006 Oviedo, Spain
 |
ABSTRACT |
Collagenase-3 (matrix
metalloproteinase-13, MMP-13) is a recently identified human MMP with
an exceptionally wide substrate specificity and restricted
tissue-specific expression. Here we show that MMP-13 expression is
induced in normal human skin fibroblasts cultured within
three-dimensional collagen gel resulting in production and proteolytic
activation of MMP-13. Induction of MMP-13 mRNAs by collagen gel was
potently inhibited by blocking antibodies against
1 and
2 integrin subunits and
augmented by activating antibody against
1 integrin
subunit, indicating that both
1
1 and
2
1 integrins mediate the MMP-13-inducing
cellular signal generated by three-dimensional collagen.
Collagen-related induction of MMP-13 expression was dependent on
tyrosine kinase activity, as it was abolished by treatment of
fibroblasts with tyrosine kinase inhibitors genistein and herbimycin A. Contact of fibroblasts to three-dimensional collagen resulted in
simultaneous activation of mitogen-activated protein kinases (MAPKs) in
three distinct subgroups: extracellular signal-regulated kinase (ERK)1
and ERK2, Jun N-terminal kinase/stress-activated protein kinase, and
p38. Induction of MMP-13 expression was inhibited by treatment of
fibroblasts with a specific p38 inhibitor, SB 203580, whereas blocking
the ERK1,2 pathway (Raf/MEK1,2/ERK1,2) by PD 98059, a selective
inhibitor of MEK1,2 activation potently augmented MMP-13 expression.
Furthermore, specific activation of ERK1,2 pathway by
12-O-tetradecanoylphorbol-13-acetate markedly suppressed
MMP-13 expression in dermal fibroblasts in collagen gel. These results
show that collagen-dependent induction of MMP-13 in dermal
fibroblasts requires p38 activity, and is inhibited by activation of
ERK1,2. Therefore, the balance between the activity of ERK1,2 and p38
MAPK pathways appears to be crucial in regulation of MMP-13 expression
in dermal fibroblasts, suggesting that p38 MAPK may serve as a target
for selective inhibition of collagen degradation, e.g. in
chronic dermal ulcers.
 |
INTRODUCTION |
Controlled degradation of extracellular matrix
(ECM)1 is essential in
physiologic situations involving connective tissue remodeling, such as
tissue morphogenesis, angiogenesis, and tissue repair. On the other
hand, excessive breakdown of connective tissue plays an important role
in the pathogenesis of, e.g., rheumatoid arthritis, osteoarthritis, atherosclerosis, periodontitis, autoimmune blistering disorders of skin, and dermal photoaging, as well as in invasion and
metastasis of tumor cells (see Refs. 1 and 2). Matrix metalloproteinases (MMPs) are a family of structurally related zinc-dependent endopeptidases collectively capable of
degrading essentially all ECM components, and they apparently play an
important role in ECM remodeling in the physiologic and pathologic
situations mentioned above. At present, human MMP gene family contains
16 members, which can be divided into subgroups of collagenases, gelatinases, stromelysins, membrane-type MMPs, and novel MMPs based on
their structure and substrate specificity (1, 2).
Members of the collagenase subgroup of MMPs, i.e.
collagenase-1 (MMP-1), collagenase-2 (MMP-8), and collagenase-3
(MMP-13), are the principal neutral proteinases capable of degrading
native fibrillar collagens in the extracellular space. They all cleave type I, II, and III collagens at a specific site generating 3/4 N-terminal and 1/4 C-terminal fragments, which rapidly denature in
physiologic temperature and become susceptible to degradation by other
MMPs, e.g. gelatinases (see Refs. 1 and 2). In addition,
MMP-13 recognizes another cleavage site in N-terminal non-helical
telopeptide of type I collagen (3). MMP-13 cleaves fibrillar collagens
with preference to type II collagen over type I and III collagens, and
displays over 40-fold stronger gelatinase activity than MMP-1 and MMP-8
(4-6). In addition, MMP-13 degrades type IV, X, and XIV collagens,
tenascin, fibronectin, and aggrecan core protein (7). Apparently due to
its ability to degrade an exceptionally wide range of ECM components,
the physiologic expression of MMP-13 is limited to situations in which
rapid and effective remodeling of collagenous ECM is required,
i.e. fetal bone development and bone remodeling in adulthood
(8, 9). On the other hand, MMP-13 is implicated in excessive
degradation of collagenous ECM in osteoarthritic cartilage (6, 10),
rheumatoid synovium (9, 11, 12), chronic cutaneous ulcers (13), intestinal ulcerations (14), and periodontitis (15), as well as in
malignant tumors, i.e. breast carcinomas (4, 16, 17), squamous cell carcinomas (SCCs) of the head and neck (18), cutaneous basal cell carcinomas (19), and chondrosarcomas (20). In cell culture
conditions, the expression of MMP-13 is also restricted as compared
with MMP-1 and has so far been reported in human fetal and
osteoarthritic chondrocytes (6, 8), and in transformed keratinocytes
and fibroblasts, osteosarcoma, and chondrosarcoma cells (17,
20-22).
Controlled degradation of fibrillar collagens of type I and III
apparently plays an important role in ECM remodeling in cutaneous wound
repair. However, the expression of MMP-13 and MMP-1 in human cutaneous
wounds is clearly different, as MMP-1 is expressed in migrating
keratinocytes (23, 24) and in dermal fibroblasts in acute and chronic
wounds (13, 25). In contrast, our recent observations show that MMP-13
is not expressed by keratinocytes in wound repair, although it is
expressed in dermal fibroblasts in chronic cutaneous ulcers, but not
during acute wound repair (13). We also noted that normal human skin
fibroblasts express MMP-13 mRNAs when cultured in three-dimensional
collagen gel, but not when grown in monolayer (13). In this study, we
show that collagen-specific induction of MMP-13 expression by dermal fibroblasts is mediated via
1
1 and
2
1 integrins and requires tyrosine kinase
activity. We also show that the contact of dermal fibroblasts with
three-dimensional collagen coordinately activates three distinct
classes of mitogen-activated protein kinases (MAPKs), i.e.
ERK1,2, JNK, and p38. The activity of p38 MAPK is essential for MMP-13
expression by fibroblasts in collagen gel, whereas specific inhibition
of ERK1,2 pathway potently augments induction of MMP-13 expression.
These results provide evidence that activity of p38 MAPK pathway plays
an essential role in the activation of MMP-13 expression in fibroblasts
and it may serve as a target for selective inhibition of collagen
degradation, e.g. in chronic dermal ulcers.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Human recombinant tumor necrosis factor-
(TNF-
) and 12-O-tetradecanoylphorbol-13-acetate (TPA)
were obtained from Sigma. C2-ceramide, genistein,
herbimycin A, Ro-31-8220, bisindolylmaleimide, SB 203580, and PD 98059 were obtained from Calbiochem (San Diego, CA). Interleukin-1
(IL-1
) was obtained from Boehringer Mannheim (Mannheim, Germany).
Bovine TGF-
1 was kindly provided by Dr. David R. Olsen (Celtrix Co.,
Santa Clara, CA). Activating antibody against
1 integrin
subunit (mAb13; Ref. 26) was kindly provided by Dr. Kenneth Yamada
(NIDR, Bethesda, MD). Blocking antibody against
1
integrin subunit (SR-84; Ref. 27) was a kind gift from Dr. Wolfgang
Rettig (Boehringer Ingelheim, Germany) and a blocking antibody against
2 integrin subunit (MCA743) was obtained from Serotech
(Oxford, United Kingdom).
Cell Cultures--
Normal human skin fibroblast cultures were
established from a punch biopsy obtained from a healthy male volunteer
(age 27 years). Fibroblast cultures were maintained in Dulbecco's
modified Eagle's medium (DMEM; Flow Laboratories, Irvine, United
Kingdom) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 IU/ml penicillin G, and 100 µg/ml streptomycin.
Collagen Gels--
Collagen gels were prepared from bovine
dermal collagen, which contains 95% type I collagen and 5% type III
collagen (Cellon, Strassen, France). Briefly, eight volumes of Cellon
were mixed with one volume of 10× concentrated DMEM and one volume of
10× concentrated NaOH (0.05 M) in Hepes buffer (0.2 M) and kept on ice. Cells were trypsinized, resuspended in
DMEM supplemented with 1% FCS, mixed into neutralized Cellon solution,
transferred into six-well plates and incubated at 37 °C for 2 h
for collagen polymerization. Thereafter, DMEM supplemented with 1% or
10% FCS, depending on the experimental conditions, was added, the gels were detached from the sides of the wells and the incubations continued
for different periods of time. As controls, fibroblasts were plated on
plastic as monolayer and cultured under similar conditions as the
corresponding fibroblasts in collagen gel. In the experiments involving
cytokines, growth factors, C2-ceramide, or TPA, fibroblasts
were first incubated for 24 h as monolayer or inside collagen
gels, after which the modulators were added and the incubations
continued for an additional 24 h. In experiments involving
tyrosine kinase inhibitors, PKC inhibitors, MAPK inhibitors, or
functional antibodies against different collagen-binding integrin subunits, these were added to the cell suspension prior to mixing it
with neutralized collagen solution or plating the cells on plastic, and
the incubations were continued for 48 h. In experiments involving
two-dimensional collagen, cell culture dishes were coated with rat tail
type I collagen (5 µg/cm2) (Sigma).
Northern Blot Hybridizations--
Fibroblasts were released from
collagen gels by brief treatment with 0.5 mg/ml collagenase (type II,
Sigma) in phosphate-buffered saline (pH 7.4) with 1 mM
CaCl2. Total cellular RNA was isolated from cells using the
single-step method (28). Aliquots of total RNA (5-20 µg) were
fractionated on 0.8% agarose gel containing 2.2 M
formaldehyde, transferred to Zetaprobe filter (Bio-Rad) by vacuum
transfer (VacuGene XL; LKB, Bromma, Sweden), and immobilized by heating
at 80 °C for 30 min. The filters were prehybridized for 2 h and
subsequently hybridized for 20 h with cDNAs labeled with
[
-32P]dCTP using random priming. For detection of
MMP-13 mRNAs, we used three cDNA fragments covering altogether
1637 bp of human MMP-13 cDNA (21) and for collagenase-1 (MMP-1)
mRNA, we used a 2.0-kb human cDNA (29). In addition, a 1.5-kb
human stromelysin-1 (MMP-3) cDNA (30), a 2.7-kb human gelatinase-A
(MMP-2) cDNA (31), a 0.7-kb human MT1-MMP (MMP-14) cDNA (32), a
0.7-kb human pro-
1(I) collagen cDNA (33), and a
1.3-kb rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA
(34) were used. The [32P]cDNA/mRNA hybrids were
visualized with autoradiography, quantified with densitometry, and
corrected for the levels of GAPDH mRNA for each sample.
RT-PCR--
The levels of MMP-13 mRNAs were also determined
by reverse-transcription PCR (RT-PCR) using the Gene Amp RNA PCR kit
(Perkin-Elmer/Roche, Branchburg, NJ). The RT reactions were performed
with random hexamer primers using 1 µg of total RNA from fibroblasts
cultured on plastic or in collagen gel for 48 h as a template.
Subsequently, a 300-bp fragment of human MMP-13 cDNA (nucleotides
534-833) (4) was amplified by PCR with 40 cycles of denaturation
(95 °C, 1.5 min), annealing (65 °C 1.5 min), and extension
(72 °C, 2 min) using sense oligonucleotide
(5'-CATTTGATGGGCCCTCTGGCCTGC-3') and antisense oligonucleotide
(5'-GTTTAGGGTTGGGGTCTTCATCTC-3'), and the PCR products were
electrophoretically fractionated on 2% agarose gel. Total RNA (1 µg)
from HaCaT keratinocytes treated with TGF-
1 (5 ng/ml) for 24 h
was used as a positive control (21).
Assay of MMP-13 Production--
Human skin fibroblasts were
seeded inside collagen gels or on plastic and maintained in DMEM
supplemented with 1% FCS for 48 h. Equal aliquots of the
conditioned media were concentrated and analyzed for the amount of
MMP-13 by Western blotting, as described previously (21) using a rabbit
antiserum against human recombinant MMP-13 (4) in dilution 1:1000
followed by enhanced chemiluminescence detection of bound primary
antibodies (Amersham Corp., United Kingdom).
Assay of MAPK Activation--
The activation of ERK1,2,
JNK/SAPK, and p38 MAPK was determined by Western blotting using
antibodies specific for phosphorylated, activated forms of the
corresponding MAPKs (New England Biolabs, Beverly, MA). Fibroblasts
were seeded in collagen gels in DMEM with 0.5% FCS, released from gels
at various time points, as described above, and lysed in 100 µl of
Laemmli sample buffer. The samples were then sonicated, fractionated by
10% SDS-PAGE, and transferred to Hybond ECL membrane (Amersham Corp.).
Western blotting was performed as described previously (35), with
phosphospecific antibodies for ERK1,2, JNK, and p38 in dilution 1:1000.
Specific binding of antibodies was detected with peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence (ECL)
detection system (Amersham).
 |
RESULTS |
Collagenase-3 (MMP-13) Expression in Dermal Fibroblasts Is Induced
by Contact to Three-dimensional Collagen--
We have recently noted
that MMP-13 is expressed by fibroblasts in chronic human cutaneous
ulcers in vivo but not in normally healing acute dermal
wounds (13). In addition, we observed that MMP-13 mRNAs are
expressed by human skin fibroblasts cultured in type I collagen gel but
not by fibroblasts in monolayer cultures (13). In the present study, we
have elucidated the signaling mechanisms mediating the
collagen-dependent induction of MMP-13 expression in human
skin fibroblasts. Initially, we cultured fibroblasts for 48 h on
plastic, on type I collagen-coated dishes, or in collagen gels and
determined the expression of MMP-13 mRNAs by Northern blot
hybridizations. Marked expression of two MMP-13 transcripts of 2.0 and
2.5 kb was noted in dermal fibroblasts cultured inside collagen,
whereas no MMP-13 mRNA could be detected in fibroblasts maintained
in monolayer cultures on plastic or on two-dimensional collagen (Fig.
1A). In comparison, MMP-1
mRNA abundance was slightly enhanced by contact to 2-dimensional
collagen and markedly up-regulated (36.5-fold) in cells maintained
inside collagen (Fig. 1A). Examination of the time
dependence of MMP-13 induction showed marked expression of MMP-13
mRNAs in dermal fibroblasts 24 h after seeding the cells in
collagen gel, and the induction was still detected after 72 h
(data not shown).

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Fig. 1.
Collagenase-3 (MMP-13) expression in dermal
fibroblasts is induced by three-dimensional collagen.
A, confluent human skin fibroblasts were cultured in
monolayer (Plastic), on type I collagen-coated dishes
(2-d), or within collagen gel (3-d) in DMEM with
10% FCS for 48 h. MMP-13, MMP-1, and GAPDH mRNA levels were
determined by Northern blot hybridizations of total RNA (10 µg).
B, total RNA (1 µg) from human skin fibroblasts cultured
in monolayer (Plastic) or in collagen gel
(Collagen) for 48 h was used as a template to amplify a
300-bp fragment of MMP-13 cDNA by RT-PCR with specific
oligonucleotide primers, as described under "Experimental
Procedures." In parallel RT-PCR reactions, total RNA (1 µg) from
HaCaT keratinocytes treated with TGF- (5 ng/ml) for 24 h was
used as a positive control. Aliquots of RT-PCR reactions were
fractionated on 2% agarose gel and DNA fragments visualized by
ethidium bromide. The sizes of molecular mass markers (in bp) are shown
on the left. C, human skin fibroblasts were
cultured in monolayer (Plastic) or in collagen gel
(Collagen) for 48 h in DMEM supplemented with 1% FCS.
Levels of MMP-13 in aliquots of concentrated (plastic, 7.8-fold;
collagen, 4.3-fold) conditioned media corresponding to equal number of
cells were assayed by Western blot analysis using an MMP-13 antibody.
In parallel, conditioned medium of HaCaT cells treated with TNF- (20 ng/ml) for 48 h was analyzed. The migration positions of molecular
size markers (Mr × 10 3) are shown
on the left.
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To confirm the observation that expression of MMP-13 is specifically
induced in human skin fibroblasts inside collagen we determined the
levels of MMP-13 mRNA by RT-PCR. As shown in Fig. 1B, a
specific 300-bp fragment of MMP-13 cDNA was amplified when RNA from
fibroblasts cultured in collagen gel for 48 h was used as a
template. The same 300-bp MMP-13 cDNA fragment was amplified in
RT-PCR reactions using RNA from TGF-
-treated HaCaT keratinocytes known to express high levels of MMP-13 mRNAs (21) (Fig.
1B). In contrast, a very low level of the same MMP-13
cDNA fragment was amplified by PCR using RNA from fibroblasts
cultured on plastic indicating that under these conditions normal human
skin fibroblasts express very low levels of MMP-13 mRNA (Fig.
1B).
Next, we assayed MMP-13 production by human skin fibroblasts cultured
in collagen gel or on plastic for 48 h by Western blot analysis.
No detectable amounts of immunoreactive MMP-13 were produced by
fibroblasts cultured in monolayer on plastic, supporting the
observations above that in dermal fibroblasts in monolayer cultures the
expression of MMP-13 mRNAs is very low. In contrast, a specific
band with an apparent molecular mass of 52 kDa was detected in
conditioned media of fibroblasts cultured in collagen gel (Fig.
1C). Interestingly, this immunoreactive MMP-13 was clearly smaller in size than the 62-kDa latent MMP-13 produced by
TNF-
-treated HaCaT keratinocytes, indicating that it had been
proteolytically processed to active form (Fig. 1C). No
latent MMP-13 was detected in the conditioned media, indicating that
all MMP-13 produced by fibroblasts in collagen gel was activated.
Expression of MMP-13 by Dermal Fibroblasts in Collagen Gel Is
Down-regulated by TGF-
but Not Altered by IL-1
or
TNF-
--
In chronic dermal ulcers, fibroblasts are exposed to
cytokines and growth factors, which have the ability to regulate the expression of several MMPs including MMP-1 (see Refs. 1 and 2). To
examine the regulation of MMP-13 expression by cytokines and growth
factors, dermal fibroblasts were cultured in collagen gel for 24 h
to allow induction of MMP-13 expression, and were subsequently treated
with IL-1
, TNF-
, or TGF-
1 for another 24 h.
Interestingly, treatment of fibroblasts in collagen gels with IL-1
(5 units/ml) or TNF-
(20 ng/ml) did not markedly alter MMP-13
mRNA levels, whereas treatment with TGF-
1 (5 ng/ml) decreased MMP-13 mRNA levels (by 60%), as compared with the levels of
untreated cells in collagen gel (Fig. 2,
A and B). No MMP-13 mRNAs were detected in
fibroblasts cultured in monolayer after a 24-h treatment with TNF-
,
IL-1
, or TGF-
1 (Fig. 2A). In comparison, MMP-1
mRNA levels were enhanced up to 1.8-fold by IL-1
and up to
3.2-fold by TNF-
, whereas stromelysin-1 (MMP-3) mRNA abundance
was up-regulated 6.1-fold by both IL-1
and TNF-
in fibroblasts
inside collagen gel (Fig. 2, A and B).
Interestingly, TGF-
1 had no marked effect on the expression of MMP-1
mRNA by fibroblasts inside collagen gels and it even slightly
enhanced the abundance of MMP-3 mRNA (Fig. 2, A and
B).

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Fig. 2.
Expression of MMP-13 by dermal fibroblasts
cultured in collagen gel is down-regulated by TGF- , but not altered
by IL-1 or TNF- . A, human skin fibroblasts were
cultured in monolayer (Plastic) or within collagen gel
(Collagen) for 24 h and then treated with IL-1 (5 units/ml), TNF- (20 ng/ml), or TGF- (5 ng/ml) for another 24 h. MMP-13, MMP-1, MMP-2, and MT1-MMP (MMP-14) mRNA levels were
determined by Northern blot hybridizations of total RNA (12 µg).
B, densitometric quantitation of MMP-13, MMP-1, MMP-2,
MMP-3, and MT1-MMP (MMP-14) mRNA levels of dermal fibroblasts
cultured in collagen gel and treated with IL-1 , TNF- , or TGF- ,
as indicated in A. The values for each MMP are shown
relative to untreated control cells in collagen gel (1.00).
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In addition to MMP-13 and MMP-1, the mRNA levels of MMP-2 (72-kDa
gelatinase), MMP-3, and MT1-MMP (MMP-14) were clearly up-regulated in
fibroblasts cultured inside collagen gels, as compared with cells
cultured in monolayer (Fig. 2A). In accordance with our previous observations (32), the levels of MT1-MMP mRNA in
fibroblasts cultured on plastic were not markedly altered by any of the
treatments although a slight up-regulation by IL-1
and TNF-
was
detected in fibroblasts cultured inside collagen gel (Fig. 2,
A and B).
Induction of MMP-13 Expression in Dermal Fibroblasts by
Three-dimensional Collagen Is Mediated by
1
1 and
2
1
Integrins--
Up-regulation of MMP-1 expression in human osteosarcoma
cells and dermal fibroblasts in collagen gel, as well as the induction of MMP-1 expression in epidermal keratinocytes cultured on
two-dimensional collagen is mediated via collagen receptor
2
1 integrin (36-38), whereas
collagen-specific inhibition of type I collagen gene expression in
fibroblasts and osteosarcoma cells is mediated via
1
1 integrin (36, 37, 39). To investigate
the roles of these two distinct collagen receptors in the
collagen-dependent induction of MMP-13 expression, we added
blocking antibodies against
1 or
2
integrin subunits to fibroblasts prior to seeding them inside collagen gels. Interestingly, addition of anti-
1 integrin
antibody potently reduced MMP-13 mRNA levels (by 52%) of the
levels in control cells treated with rat IgG (Fig.
3A). Previous observations
show that most antibodies against integrin
subunits do not alone
completely inhibit the signaling function of the corresponding receptor
(37). Here, addition of the anti-
1 integrin antibody had
no marked effect on enhancement of MMP-1 mRNA abundance, whereas
marked (80%) down-regulation of pro-
1(I) collagen
mRNA levels in fibroblasts in collagen gel was entirely abrogated
by anti-
1 integrin antibody, indicating that in the
concentration used (1 µg/ml) this antibody entirely blocks the
signaling function of
1
1 integrin (Fig. 3A). In comparison, addition of the anti-
2
integrin antibody resulted in dose-dependent inhibition of
the collagen-induced MMP-13 expression, the maximal inhibition (90%)
noted with the concentration 5 µg/ml (Fig. 3B). Together,
these observations show that the induction of MMP-13 expression in
fibroblasts by three-dimensional collagen involves activation of
signaling via both
1
1 and
2
1 integrins. However,
2
1 integrin appears to play a more
important role, as inhibiting signaling via
2
1 integrin results in nearly complete
inhibition of collagen-elicited induction of MMP-13 expression by
fibroblasts.

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Fig. 3.
Induction of MMP-13 expression by
fibroblasts in collagen gel is mediated by
1 1 and 2 1
integrins. A and B, human skin fibroblasts
were cultured in monolayer (Plastic) or within collagen gel
(Collagen), as indicated, for 48 h and total RNA was
extracted. Prior to seeding in collagen gel, rat IgG (IgG; 2 µg/ml), or blocking antibodies against A, 1
(anti- 1; SR-84, 1 µg/ml), or B,
2 (anti- 2; MCA743, 1 or 5 µg/ml) integrin subunits were added to cell suspension. Ctl, no
antibodies added. Aliquots of total RNA (7 or 15 µg) were analyzed
for expression of MMP-13, MMP-1, pro- 1(I) collagen, and
GAPDH mRNAs by Northern blot hybridizations. A and
B, lower panels, densitometric
quantitation of MMP-13 ( ), MMP-1 ( ), and pro- 1(I)
collagen ( ) mRNA levels in dermal fibroblasts cultured in
monolayer on plastic (pl) or in collagen gel
(Collagen) treated with rat IgG, anti- 1
(a- 1), or anti- 2
(a- 2) integrin antibodies, as indicated. The
values for each mRNA are shown relative to the levels in untreated
control cells in collagen gel (ctl) (MMP-13), or on plastic
(pl) (1.00) (pro- 1(I) collagen) after
correction for GAPDH mRNA levels. C, before plating
dermal fibroblasts in monolayer (Plastic) or in collagen gel
(Collagen), rat IgG (IgG; 2 µg/ml), activating antibody
against 1 integrin subunit (mAb13; 1 µg/ml), or the
Fab fragments of mAb13 (Fab13; 1 µg/ml), or a combination of mAb13
and anti-rat IgG (anti-rat) (1 µg/ml each) were added to cell
suspension. Cells were cultured for 48 h, and aliquots of total
RNA (7 µg) were analyzed for expression of MMP-13, MMP-1, and GAPDH
mRNAs by Northern blot hybridizations.
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To further elucidate the role of the
1 integrins in
collagen-dependent induction of fibroblast MMP-13
expression, we used a monoclonal antibody (mAb13) against
1 integrin subunit, shown to activate
1
integrin-mediated signaling in keratinocytes resulting in enhanced
92-kDa gelatinase (MMP-9) expression (40). Interestingly, addition of
mAb13 to fibroblasts before seeding them in collagen gel potently
(4.7-fold) augmented the induction of MMP-13 mRNA levels, as
compared with cells treated with rat IgG (Fig. 3C). In
contrast, treatment with mAb13 did not induce MMP-13 expression by
fibroblasts plated on plastic, and it had no effect on MMP-1 mRNA
levels in fibroblasts cultured on plastic or inside collagen (Fig.
3C). In accordance with a previous study (37), mAb13 also inhibited contraction of collagen gel by fibroblasts (data not shown).
These observations show that induction of MMP-13 expression in dermal
fibroblasts is mediated via activation of
1 integrin subunit, but only when cells are in contact with three-dimensional collagen.
Triggering the signaling via
1 integrin subunit by a
multivalent ligand or by an activating antibody may require integrin clustering, which results in subsequent activation of downstream signaling pathways (41, 42). To examine the role of
1
integrin clustering in the induction of MMP-13 expression, we utilized Fab fragments of mAb13, which bind to
1 integrins and
activate their signaling without inducing their clustering.
Interestingly, treatment of fibroblasts with Fab13 prior to seeding in
collagen gel enhanced MMP-13 mRNA expression even more potently
(8.3-fold) than the intact mAb13, indicating that
1
integrin-mediated induction of MMP-13 expression does not require
clustering of
1 integrins (Fig. 3C). However,
treatment of fibroblasts plated in monolayer on plastic with Fab13 did
not induce the expression of MMP-13 mRNA (Fig. 3C).
Furthermore, addition of a secondary antibody (anti-rat IgG) together
with mAb13 to further increase
1 integrin clustering did
not induce the expression of MMP-13 mRNA in fibroblasts in
monolayer, indicating that clustering and activation of
1 integrins is not alone sufficient to induce expression
of MMP-13 in fibroblasts in monolayer culture (Fig. 3C).
Induction of MMP-13 Expression in Fibroblasts Cultured in Collagen
Gel Is Dependent on Tyrosine Kinase Activity--
One of the initial
events following the ECM ligand binding and activation of
1 integrins is the association of the cytoplasmic domain
of
1 integrin with focal adhesion kinase (FAK), a
cytosolic non-receptor tyrosine kinase, leading to the phosphorylation
of FAK (41, 42). Accordingly, tyrosine phosphorylation has been shown
to be involved in the signal transduction triggered by contact of
fibroblasts to three-dimensional collagen (43) and it has been shown
that contraction of collagen gel by fibroblasts requires protein-tyrosine phosphorylation (44). To elucidate the signaling mechanisms mediating the collagen-dependent induction of
MMP-13 expression, we first added two different tyrosine kinase
inhibitors to fibroblasts before seeding them in collagen gel.
Interestingly, induction of MMP-13 mRNA expression in fibroblasts
within collagen gel was entirely inhibited by genistein (50 µM) and herbimycin A (1 µM), indicating
requirement for tyrosine kinase activity (Fig.
4). In accordance with previous
observations (37) genistein and herbimycin A also potently inhibited
the induction of MMP-1 gene expression by three-dimensional collagen
(Fig. 4). In contrast, MMP-13 induction was not altered by two specific
PKC inhibitors, bisindolylmaleimide (5 µM) and Ro-31-8220
(1 µM), indicating that PKC activity is not required for
induction of MMP-13 expression in collagen gel (data not shown).

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Fig. 4.
Induction of fibroblast collagenase-3
(MMP-13) expression in collagen gel is dependent on tyrosine kinase
activity. Normal human skin fibroblasts were cultured in monolayer
(Plastic) or within type I collagen gel
(Collagen) and incubated for 48 h without
(Ctl) or with tyrosine kinase inhibitors genistein
(Genist., 50 µM), or herbimycin A
(Herb. A, 1 µM), added to cell
suspension before seeding in collagen gel. Total RNA (10 µg) was
analyzed for levels of MMP-13, MMP-1, and GAPDH mRNA by Northern
blot hybridizations.
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Activation of ERK1,2, JNK/SAPK, and p38 in Dermal Fibroblasts by
Collagen Gel--
Activation of tyrosine kinase-dependent
signaling by
1 integrin stimulation can result in
subsequent activation of Ras, which in turn can activate downstream
signaling cascades including MAPKs (42, 45). In this context, we first
examined the activation of ERK1,2 by Western blot analysis of cellular
proteins from dermal fibroblasts at various time points after seeding
them in collagen gel using an antibody against the active,
phosphorylated forms of ERK1 and ERK2 (p44 and p42 MAPK, respectively).
The levels of activated ERK2 were 2-fold increased 2 h after
seeding the cells inside collagen and a marked activation (5-fold) was
detected at 3- and 6-h time points (Fig.
5, A and B). The
activated form of ERK1 was also detected at 3- and 6-h time points,
although at a markedly lower level, as compared with activated ERK2
(Fig. 5, A and B). The activation of ERK2 and
ERK1 occurs rapidly after the formation of collagen gel, which takes
place approximately 2 h after neutralizing the collagen solution
and seeding the cells inside it.

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Fig. 5.
Activation of ERK1,2, JNK/SAPK, and p38 in
dermal fibroblasts in collagen gel. A, human skin
fibroblasts were seeded in collagen gel and incubated in DMEM
supplemented with 0.5% FCS for different periods of time, as
indicated. The levels of activated ERK1 and ERK2 (ERK1-P,
ERK2-P), JNK (JNK1-P), and p38 (p38-P)
were determined by Western blot analysis using phosphospecific
antibodies for the corresponding MAPKs. The migration positions of the
molecular size markers (Mr × 10 3)
are shown on the left. B, the levels of activated
ERK1 and ERK2 (ERK1,2, ), JNK/SAPK ( ), and p38 ( ) were
quantitated by scanning densitometry and are shown relative to the
levels at 1 h of incubation (1.00).
|
|
In addition to ERK1,2 pathway, Ras can activate two additional
subgroups of MAPKs, i.e. Jun N-terminal
kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK,
e.g. via small GTPases Rac and Rho (see 42, 45). In the next
set of experiments, we examined the activation of JNK and p38 utilizing
antibodies specific for phosphorylated JNK or p38, respectively.
Contact of dermal fibroblasts to three-dimensional collagen resulted in
a marked activation (9-fold) of JNK1 (p46 SAPK), noted 3 h after
seeding the cells inside collagen and the activation gradually
decreased by 12 and 24 h of incubation (Fig. 5, A and
B). A lower abundance activated JNK with molecular mass of
48 kDa was also detected at 3- and 6-h time points, possibly
representing an alternatively spliced form of JNK1 (Fig.
5A).
A marked (4.5-fold) activation of p38 MAPK was also detected in human
skin fibroblasts, as early as 2 h after seeding the cells inside
collagen with the maximal activation (7-fold) noted at 3 h,
followed by a gradual decrease in the cellular levels of the activated
p38 by 12 h (Fig. 5, A and B). The
activation of p38 MAPK was quite persistent, as elevated (3.5-fold)
levels of activated p38 were still detected after 24 h of
incubation (Fig. 5, A and B). Together, these
results show that contact of fibroblasts to three-dimensional collagen
results in coordinate activation of MAPKs in three distinct subgroups,
i.e. ERK1 and ERK2, JNK1, and p38 MAPK, all capable of
activating the expression of c-Jun and c-Fos, components of the
classical AP-1 dimer (45).
Collagen-dependent Induction of MMP-13 Expression in
Dermal Fibroblasts Requires p38 Activity and Is Inhibited by Activation
of ERK1,2--
To elucidate the roles of ERK1,2 and p38 MAPKs in
mediating the collagen-dependent induction of MMP-13
expression in dermal fibroblasts, we used selective inhibitors and
activators of these MAPKs. First, to block the ERK1,2 pathway
(Raf/MEK1,2/ERK1,2), we added PD 98059, a specific inhibitor of the
activation of MEK1 and MEK2 (46, 47), to fibroblasts immediately before
seeding them inside collagen. Surprisingly, PD 98059 (40 µM) potently (7.3-fold) augmented the induction of MMP-13
mRNA as compared with untreated fibroblasts in collagen gel (Fig.
6A). In contrast, addition of
selective p38 inhibitor SB 203580 (10 µM) (48, 49) to
fibroblasts before seeding them inside collagen entirely abrogated the
induction of MMP-13 mRNA expression by collagen (Fig.
6A). In the same cells, collagen-dependent
induction of MMP-1 mRNA levels was slightly (by 40%) reduced by
treatment of cells with PD 98059 and somewhat more potently (by 65%)
by SB 203580 (Fig. 6A). These results show that the activity
of p38 is essential for MMP-13 expression in collagen-embedded dermal
fibroblasts, whereas induction of MMP-1 is only partially inhibited by
blocking p38 or ERK1,2 pathways.

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Fig. 6.
Induction of collagenase-3 (MMP-13) mRNAs
in dermal fibroblasts in collagen gel is dependent on the activity of
p38 MAPK and inhibited by ERK1,2 activity. A, normal
human skin fibroblasts were seeded in monolayer (Plastic) or
within type I collagen gel (Collagen), as indicated, and
incubated for 48 h with or without PD 98059 (40 µM),
a specific inhibitor of ERK1,2 kinases MEK1,2, or with SB 203580 (10 µM), a selective inhibitor of p38
MAPK, both added to cell suspension prior to seeding in collagen gel.
Total RNA was extracted and 20-µg aliquots analyzed for levels of
MMP-13, MMP-1, and GAPDH mRNA by Northern blot hybridizations.
B and C, human skin fibroblasts were cultured in
monolayer (Plastic) or within type I collagen gel
(Collagen) for 24 h and incubated for another 24 h
without ( ) or with (+) TPA (60 ng/ml) (B), or without ( )
or with (+) C2-ceramide (C2-cer; 50 µM) for another 24 h (C). Total RNA was
extracted and 10-µg aliquots analyzed for expression of MMP-13,
MMP-1, and GAPDH mRNAs by Northern blot hybridizations.
A-C, lower panels, densitometric
quantitations of MMP-13 ( )and MMP-1 ( ) mRNA levels corrected
for GAPDH mRNA levels are shown relative to the levels of untreated
control cells in collagen gel (1.00).
|
|
Augmentation of MMP-13 expression by dermal fibroblasts inside collagen
obtained by blocking the ERK1,2 pathway by PD 98059 (Fig.
6A) provides evidence that ERK1,2 MAPKs play an inhibitory role in MMP-13 expression. To further examine this possibility, we
cultured dermal fibroblasts inside collagen gel for 24 h to allow
induction of MMP-13 expression and subsequently treated them for
24 h with phorbol ester TPA, which selectively activates ERK1,2,
but not p38 in dermal fibroblasts (50) (data not shown). As shown in
Fig. 6B, treatment with TPA markedly (76%) reduced the
levels of MMP-13 mRNAs in fibroblasts, providing further evidence for the inhibitory role of ERK1,2 pathway in the regulation of MMP-13
expression. In comparison, the abundance of MMP-1 mRNA in
fibroblasts in collagen gel was not markedly altered by treatment with
TPA, indicating differential regulation of MMP-13 and MMP-1 expression
by ERK1,2 MAPKs (Fig. 6B).
To further investigate the role of distinct MAPK pathways in the
regulation of MMP-13 expression, dermal fibroblasts cultured for
24 h in collagen gel were incubated for another 24 h in the presence of C2-ceramide (50 µM), which
coordinately activates ERK1,2, JNK/SAPK, and p38 in these cells (35).
Interestingly, treatment with C2-ceramide further enhanced
(2-fold) the levels of MMP-13 mRNAs in fibroblasts cultured in
collagen gel, whereas MMP-1 mRNAs were only slightly (1.2-fold)
increased (Fig. 6C). In accordance with our previous
observations (35) C2-ceramide potently enhanced the levels
of MMP-1 mRNA in dermal fibroblasts cultured on plastic, but did
not induce their MMP-13 mRNA expression (Fig. 6C).
 |
DISCUSSION |
Culturing fibroblasts in collagen gel is a well established model
for studying the formation of ECM in vitro under conditions resembling three-dimensional collagen matrix surrounding fibroblasts in vivo (see 51). The floating, contracting collagen gel is
suggested to mimic the cellular environment in dermal layer of normal
skin or scar, and the anchored gel simulates granulation tissue (51). In the present study we show for the first time that the expression of
collagenase-3 (MMP-13) in normal human skin fibroblasts is specifically
induced by three-dimensional type I collagen via both
1
1 and
2
1
integrins and that this requires tyrosine kinase activity. We also show
that contact of fibroblasts to three-dimensional collagen results in
coordinate activation of three distinct subgroups of MAPKs,
i.e. ERK1,2, JNK/SAPK, and p38, and that
collagen-dependent induction of MMP-13 expression requires
p38 MAPK activity and is in turn potently augmented by blocking the
ERK1,2 MAPK pathway (Fig. 7).

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Fig. 7.
Signaling pathways mediating regulation of
MMP-13 expression in dermal fibroblasts by three-dimensional type I
collagen. Contact of dermal fibroblasts to type I collagen via
1 1 and 2 1
integrin collagen receptors results in activation of ERK1,2 and p38
MAPKs. Induction of MMP-13 expression by three-dimensional collagen is
inhibited by tyrosine kinase inhibitors (genistein, herbimycin A).
Activity of p38 is also required for MMP-13 expression, whereas
blocking the ERK1,2 pathway augments induction of MMP-13 expression. In
addition, activation of ERK1,2 cascade by phorbol ester (TPA) inhibits
MMP-13 expression indicating that ERK1,2 MAPKs mediate inhibitory
signals on MMP-13 expression. MEK, MAP/ERK kinase;
MKK, MAPK kinase; MAPKKK, MAPKK kinase; SB
203580, a specific inhibitor of p38 MAPK; PD 98059, a
specific inhibitor of MEK1,2.
|
|
Contact of human skin fibroblasts with three-dimensional collagenous
matrix has profound effects on their morphology, growth factor
response, and matrix gene expression (51). In fibroblasts, collagen
receptors
1
1 and
2
1 integrins play an important role in
regulating ECM remodeling, as shown by observations that
1
1 integrin mediates signals responsible
for down-regulation of type I collagen gene expression and
2
1 integrin activates signaling pathways
mediating induction of MMP-1 expression (36, 37, 39). The results of
the present study show that collagen-dependent induction of
MMP-13 expression in dermal fibroblasts is partially prevented by
blocking antibody against
1 integrin subunit and nearly
completely abrogated by anti-
2 integrin antibody,
indicating that collagen-dependent activation of MMP-13
expression involves activation of signaling pathways via both
1
1 and
2
1
integrins. Thus, in contrast to regulation of MMP-1 and type I collagen
expression mediated by specific integrin
subunits, our results show
that common
1 subunit is more important in regulation of
MMP-13 expression. This notion is further supported by our finding that
induction of fibroblast MMP-13 expression by three-dimensional collagen is potently augmented by an activating antibody against
1 integrin (mAb13) and by Fab fragments of the same
antibody (Fab13), indicating that collagen receptor-mediated induction
of MMP-13 expression involves direct activation of signaling via
1 integrin subunit and does not require clustering of
1 integrins. However, induction of MMP-13 expression by
both collagen and anti-
1 integrin antibody was dependent
on the presence of three-dimensional collagen matrix, indicating that
activation of
1 integrin subunit alone is not sufficient
for triggering signaling pathways mediating MMP-13 induction. It is
apparent that ligand binding sites of
1
1
and
2
1 integrins are fully occupied when
fibroblasts are in three-dimensional type I collagen recognized by
specific I domains of
1 and
2 integrin
subunits (52, 53). In contrast, mAb13 binds to the
1
integrin subunit and may cause conformation changes resulting in
further stimulation of signaling via
1 integrin.
Previous observations have shown that seeding fibroblasts in collagen
gel results in tyrosine phosphorylation of a 125-kDa protein
corresponding to FAK (43) and that down-regulation of TGF-
receptor
by collagen contact is dependent on the activity of FAK (54). In
addition, activation of fibroblast and keratinocyte MMP-1 expression by
three- and two-dimensional collagen, respectively, is dependent on
tyrosine kinase activity (37, 55). Our observations show that induction
of MMP-13 expression by collagen gel is also dependent on tyrosine
kinase activity, indicating that tyrosine kinases play an essential
role in signaling pathways regulating the collagenolytic capacity of
fibroblasts. It has also been shown that contact of dermal fibroblasts
to three-dimensional collagen results in activation of an atypical PKC,
PKC-
, the activity of which is required for induction of MMP-1 in
this model (56). However, in our study, induction of MMP-13 expression
in fibroblasts in collagen gel was not altered by specific PKC
inhibitors bisindolylmaleimide and Ro-31-8220, indicating that PKC
activity is not essential for the activation of MMP-13 expression.
Our results show that contact of fibroblasts to three-dimensional
collagen results in rapid and coordinate activation of three distinct
subgroups of MAPKs, i.e. ERK1,2, JNK/SAPK, and p38. A recent
study also showed that contact of fibroblasts to two- and three-dimensional collagen results in tyrosine phosphorylation of ERK1
and ERK2 (57). In addition, in osteoblasts contact to two-dimensional
collagen activates ERK1,2 (52). In the present study we show for the
first time that contact of fibroblasts to three-dimensional collagen
also results in activation of JNK/SAPK and p38 MAPK, generally
activated by cellular stress and cytokines (45). Interestingly,
induction of MMP-13 expression by collagen gel is entirely inhibited by
a selective p38 inhibitor SB 203580, indicating that p38 activity is
essential for induction of MMP-13 expression. In contrast, expression
of MMP-13 was potently augmented by blocking the ERK1,2 pathway by PD
98059, a selective inhibitor of MEK1,2 activation. Furthermore,
activation of ERK1,2 pathway by phorbol ester TPA potently inhibited
MMP-13 expression in fibroblasts in collagen gel. Together these
results provide evidence that, although ERK1 and ERK2 are activated by
contact of fibroblasts to three-dimensional collagen, they mediate an
inhibitory rather than activating signal on MMP-13 expression. It is
therefore likely that the balance between the activity of ERK1,2 and
p38 MAPK pathways is crucial in regulation of fibroblast MMP-13
expression in dermal fibroblasts (Fig. 7).
The expression of MMP-13 in fibroblasts in monolayer cultures is not
inducible by TNF-
, IL-1
, TPA, or C2-ceramide, all of which are capable of stimulating expression of MMP-1 under similar conditions (see Refs. 1 and 2). Furthermore, once turned on by contact
to three-dimensional collagen matrix, the expression of MMP-13 remains
relatively unresponsive to modulation, as it is not stimulated by
TNF-
and IL-1
, which enhance the expression of MMP-1 and MMP-3 by
dermal fibroblasts in collagen gel. In contrast, the expression of
MMP-13 in fibroblasts within collagen gel was somewhat inhibited by
TGF-
. The different modulation of fibroblast MMP-13 and MMP-1
expression by TNF-
and IL-1 may be one explanation for distinct
localization of fibroblasts expressing MMP-13 and MMP-1 in chronic
dermal ulcers (13). Three-dimensional collagen contact also induces
fibroblast expression of stromelysin-1 (MMP-3), 72-kDa gelatinase
(MMP-2), and MT1-MMP (MMP-14), all capable of activating latent MMP-13
(5, 7). It is therefore not surprising that all MMP-13 detected in the
culture media of dermal fibroblasts in collagen gel is in active form.
Thus, contact to three-dimensional collagen matrix most likely plays an
important role in stimulating the proteolytic capacity of fibroblasts
in ECM remodeling during normal wound repair and in excessive
degradation of ECM in chronic ulcers (13, 14, 25). In analogy, the
expression of MMP-2 and MT1-MMP in endothelial cells is also induced by
three-dimensional collagen and this may play an important role in
angiogenesis during wound repair (58). Similarly, we have recently
observed that expression of MMP-13 by periodontal ligament cells is
induced by contact to two-dimensional collagen, which may be an
important factor in inducing MMP-13 expression by these cells during
chronic inflammation of oral mucosa in vivo (15).
The expression of MMP-13 in vivo and in cultured cells is
clearly more restricted than the expression of most other MMPs (see Refs. 1, 2, and 22). In addition to chronic dermal and intestinal
ulcers, and periodontal inflammation (13-15), the only situations in
which the expression of MMP-13 has been detected in fibroblasts
in vivo are SCCs of the head and neck, and breast carcinomas
(17, 18). The expression of MMP-13 has been recently documented in
monolayer cultures of human immortalized fibroblasts of embryonal
origin (KMST cells), in which its expression is stimulated by IL-1
and TGF-
(17, 59). Together these observations are in accordance
with a recent study, in which expression of MMP-13 mRNA was
detected in several transformed human fibroblast lines, whereas the
expression in normal human skin fibroblasts was low (22). As it has
been shown that stromal fibroblasts derived from SCCs can also modulate
the phenotype of tumor cells (60), it is possible that the stromal
fibroblasts in SCCs and breast carcinomas expressing MMP-13 represent a
dedifferentiated fibroblast phenotype, in which MMP-13 expression is
regulated differently as compared with normal human skin fibroblasts.
This notion is supported by our observations that the expression of
MMP-13 in normal dermal fibroblasts is inhibited by TGF-
and not
altered by IL-1
, whereas both clearly enhance MMP-13 expression in
KMST fibroblasts (17, 59).
The ability to degrade type I collagen is essential for migration of
epidermal keratinocytes (38). However, human epidermal keratinocytes do
not express MMP-13 under any conditions in culture or in
vivo (13, 21), indicating that MMP-1 is the collagenase of choice
for restricted cleavage of type I collagen required for keratinocyte
migration. In contrast, the ability of MMP-13 to degrade several ECM
components is obviously beneficial for invading transformed
keratinocytes, e.g. SCC cells (18). It is possible that
degradation of ECM components in chronic dermal ulcers by MMP-13 alters
the cell-matrix interactions of both epidermal keratinocytes and
fibroblasts and may affect their migration capacity (61). In this
context, identification of p38 MAPK as an essential signaling pathway
in the induction of MMP-13 in normal human skin fibroblasts suggest p38
MAPK as a target for selective inhibition of degradation of collagenous
ECM in chronic dermal ulcers.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the expert
technical assistance of Ulla Paasio and Marita Potila. We also thank
Drs. E. Bauer, M. Kurkinen, G. Goldberg, and P. Fort for plasmids.
 |
FOOTNOTES |
*
This work was supported by grants from the Academy of
Finland, the Sigrid Jusélius Foundation, the Cancer Research
Foundation of Finland, Turku University Central Hospital, and the Turku
University Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: MediCity Research
Laboratory, University of Turku, Tykistökatu 6A, FIN-20520 Turku,
Finland. Tel.: 358-2-3337025; Fax: 358-2-3337000; E-mail: veli-matti.kahari{at}utu.fi.
The abbreviations used are:
ECM, extracellular
matrix; MMP, matrix metalloproteinase; TNF-
, tumor necrosis
factor-
; IL-1, interleukin-1; TGF-
, transforming growth
factor-
; MAPK, mitogen-activated protein kinase; ERK, extracellular
signal-regulated kinase; JNK/SAPK, Jun N-terminal
kinase/stress-activated protein kinase; MEK, MAPK/ERK kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; PKC, protein kinase C; SCC, squamous cell carcinoma; FAK, focal adhesion kinase; RT, reverse
transcription; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mAb, monoclonal antibody; bp, base pair(s); kb, kilobase pair(s); DMEM, Dulbecco's modified Eagle's
medium; FCS, fetal calf serum.
 |
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