From the Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain
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ABSTRACT |
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Collagenase-3 (MMP-13) is a matrix
metalloproteinase (MMP) originally identified in breast carcinomas
which is also produced at significant levels during fetal ossification
and in arthritic processes. In this work, we have found that
transforming growth factor 1 (TGF-
1), a growth factor widely
assumed to be inhibitory for MMPs, strongly induces collagenase-3
expression in human KMST fibroblasts. In contrast, this growth factor
down-regulated the expression in these cells of collagenase-1 (MMP-1),
an enzyme highly related to collagenase-3 in terms of structure and
enzymatic properties. The positive effect of TGF-
1 on collagenase-3
expression was dose- and time-dependent, but independent of
the effects of this growth factor on cell proliferation rate. Analysis
of the signal transduction mechanisms underlying the up-regulating
effect of TGF-
1 on collagenase-3 expression demonstrated that this
growth factor acts through a signaling pathway involving protein kinase C and tyrosine kinase activities. Functional analysis of the
collagenase-3 gene promoter region revealed that the inductive effect
of TGF-
1 is partially mediated by an AP-1 site. Comparative analysis
with the promoter region of the collagenase-1 gene which contains an AP-1 site at equivalent position, confirmed that TGF-
1 did not have
any effect on CAT activity levels of this promoter. Finally, by using
electrophoretic mobility shift assays and antibody supershift analysis,
we propose that c-Fos, c-Jun, and JunD may play major roles in the
collagenase-3 activation by TGF-
1 in human fibroblasts.
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INTRODUCTION |
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The matrix metalloproteinases (MMPs)1 or matrixins form a family of structurally related metalloendopeptidases that are collectively capable of degrading the different macromolecular components of the extracellular matrix. These enzymes play a major role in normal tissue remodeling processes such as embryonic development, bone growth, and resorption, ovulation, uterine involution, and wound healing (1-4). In addition, abnormal expression of these proteinases may contribute to a variety of pathological processes such as rheumatoid arthritis (5), atherosclerosis (6), pulmonary emphysema (7), and tumor invasion and metastasis (8). At present, the family of human MMPs is composed of 16 members that, according to structural and functional considerations, can be classified into four different families: collagenases, gelatinases, stromelysins, and membrane-type MMPs (1-4), although there are some enzymes like macrophage metalloelastase (9), stromelysin-3 (10), MMP-19 (11), and enamelysin (12) that do not belong to these groupings. Recently, we have cloned from a breast carcinoma a novel MMP that has been called collagenase-3 (MMP-13) (13, 14), since it represents the third member of the collagenase subfamily of human MMPs, the others being fibroblast and neutrophil collagenases. Biochemical characterization of recombinant human collagenase-3 has revealed that it degrades very efficiently the native helix of fibrillar collagens with preferential activity on type II collagen (15, 16). In contrast, fibroblast collagenase (MMP-1 or collagenase-1) is more active against type III collagen (17) and neutrophil collagenase (MMP-8 or collagenase-2) preferentially cleaves type I collagen (18). Consequently, the three human collagenases characterized to date show distinct substrate specificities strongly suggesting that they have evolved as specialized enzymes to degrade tissues with different collagen composition. In addition to its degrading activity on fibrillar collagens, further analysis of the substrate specificity of collagenase-3 has revealed that this enzyme may also act as a potent gelatinase thus contributing to further degrade the initial cleavage products of collagenolysis to small fragments suitable for subsequent metabolism (15). Furthermore, very recent studies have shown that collagenase-3 is also able to degrade the large cartilage proteoglycan aggrecan and other components of the extracellular matrix and basement membranes, including the large tenascin isoform, fibronectin, and type IV collagen (15, 18, 19).
Analysis of the expression of collagenase-3 in human tissues has
revealed that in addition to its presence in breast carcinomas, this
enzyme is produced during fetal ossification (20, 21), and in
degenerative joint diseases including osteoarthritis and rheumatoid
arthritis (21-27). At present, very little information is available on
the mechanisms controlling collagenase-3 expression in both normal and
pathological conditions. Thus, although several groups have reported
that human collagenase-3 gene expression can be induced by IL-1 and
tumor necrosis factor-
in chondrocytes from normal and
osteoarthritic cartilage (22-24), much less is known on the mechanisms
modulating its expression in tumor processes. In this regard, and based
on in situ hybridization experiments and on co-cultures of
fibroblast and epithelial breast cancer cells, we have recently
proposed that collagenase-3 is predominantly expressed within
fibroblasts adjacent to the invasive tumor cells in response to
diffusible factors released from the breast cancer cells (28). A
preliminary search of putative factors with ability to induce
collagenase-3 expression in human fibroblasts revealed that only IL-1
and TPA were able to up-regulate the expression of this gene in KMST
fibroblastic cells. By contrast, a series of cytokines and growth
factors factors including aFGF, bFGF, platelet-derived growth factor,
epidermal growth factor, tumor necrosis factor-
, and TGF-
,
previously found to play important roles in up-regulating expression of
other MMPs, did not show any effect on collagenase-3 expression by
human fibroblasts. Since these findings strongly suggested that the
mechanisms regulating collagenase-3 expression could be distinct to
those operating in the control of other MMPs, we have extended our
preliminary search for factors that could act as mediators of
collagenase-3 expression in breast carcinomas. In this work, we show
that TGF-
, a growth factor widely assumed to be inhibitory for MMPs
like collagenase-1, strongly up-regulates collagenase-3 expression in
KMST fibroblasts. We also analyze the mechanisms mediating this
induction with the finding that this growth factor acts through a
signaling pathway involving tyrosine kinase and protein kinase C
activities. Finally, we perform a functional characterization of the
promoter region of the collagenase-3 gene looking for the putative
elements with ability to mediate the induction of this gene by
TGF-
.
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EXPERIMENTAL PROCEDURES |
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Materials--
All media and supplements for cell culture were
obtained from Sigma except for fetal calf serum, which was from
Boehringer Mannheim (Mannheim, Germany). TGF-1, IL-1
, IL-1
,
aFGF, bFGF, platelet-derived growth factor-BB,
12-O-tetradecanoylphorbol-13-acetate (TPA), cycloheximide,
staurosporine, genistein, herbimycin A, calphostin C, and indomethacin
were from Sigma. H-89 was from Calbiochem. Restriction endonucleases
and other reagents used for molecular cloning were from Boehringer
Mannheim. Double-stranded DNA probes were radiolabeled with
[
-32P]dCTP (3000 Ci/mmol) from Amersham International
(Buckinghamshire, United Kingdom) using a commercial random-priming kit
from Amersham. Antibodies against members of the Fos and Jun family of
transcription factors were from Santa Cruz Biotechnology (Santa Cruz,
CA).
Cell Culture and Cell Growth Estimation--
Human KMST-6 cells
immortalized by -ray irradiation of KMS-6 embryonic fibroblasts were
kindly provided by Dr. M. Namba (Hyougo Medical College, Japan). NRK
and Mv1Lu cells were kindly provided by Dr. F. Ventura (Universidad
Autónoma, Barcelona, Spain). Cells were routinely maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin in a
humidified atmosphere of 5% CO2. Cells were subcultured
weekly by incubation at 37 °C for 2 min with 0.0125% trypsin in
0.02% EDTA, followed by addition of complete medium, washing and
resuspension in fresh medium. For most experiments, approximately
5 × 105 cells/well were plated out in 100-mm dishes
and transferred to serum-free Dulbecco's modified Eagle's medium for
24 h and then exposed to the different growth factors, cytokines,
and tumor promoters at the concentrations and for the times indicated.
All inhibitors or antagonist compounds were added 1 h before
treatment with the different stimulating factors. To test the effect of TGF-
1 on cell number, KMST, NRK, and Mv1Lu cells were plated in
24-well plates and allowed to adhere to substrate for 24 h in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
Afterward, the serum concentration was reduced to 1%, and TGF-
1 was
added at different concentrations. Cells were incubated for 7 days in
the presence of TGF-
1 with medium changes every 2 days. At the end
of the incubation period, the total number of cells was estimated by
the fluorometric protein assay essentially as described by Skehan
et al. (29).
Isolation of RNA and Northern Blot Analysis-- Total RNA from the cells was isolated by the guanidinium isothiocyanate procedure according to Chomczynski and Sacchi (30), separated by electrophoresis in 1% agarose-formaldehyde gels and blotted onto Hybond N nylon filters (Amersham Int.). Filters containing 20 µg of total RNA were prehybridized at 42 °C for 3 h in 50% formamide, 5 × SSPE (1 × = 150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4), 10 × Denhardt's, 2% SDS, and 100 µg/ml denatured herring sperm DNA and then hybridized with radiolabeled collagenase-3 full-length cDNA for 20 h under the same conditions. Filters were washed with 0.1 × SSC, 0.1% SDS for 2 h at 50 °C and exposed to autoradiography. RNA integrity and equal loading was assessed by hybridization with an actin probe or with a probe specific for human glyceraldehyde-3-phosphate dehydrogenase. Densitometry of the x-ray films was carried out with the BioImage software (Millipore Corp., Bedford, MA).
Western Immunoblot Analysis--
Conditioned media were obtained
after incubation of KMST cells in serum-free Dulbecco's modified
Eagle's medium for 48 h or supplemented with TGF-1, filtered,
and concentrated 25-fold in a Centricon filter with a molecular mass
cut-off of 10 kDa. Proteins from conditioned media were separated by
polyacrylamide gel electrophoresis under denaturing and reducing
conditions and transferred to nitrocellulose membranes (Amersham Int.).
After blocking with a 5% nonfat milk solution, the membranes were
incubated with a 1:5000 dilution of rabbit polyclonal antisera raised
against collagenase-3 (13) and then with a goat anti-rabbit IgG
antisera conjugated to horseradish peroxidase. Finally, the membranes
were washed and developed with a horseradish peroxidase
chemiluminescence detection reagent (ECL system, Amersham Int.).
Stable Transfection of Human Collagenase-3 cDNA in KMST Fibroblasts-- The full-length cDNA insert of human collagenase-3 was excised from plasmid pNot3a (13), filled-in with Klenow fragment, and ligated into the BamHI site of the eukaryotic expression vector pCMV, also blunt-ended by treatment with Klenow. The resulting plasmid, pCMV-col3+, containing the collagenase-3 cDNA downstream of the constitutive CMV promoter, was transfected in KMST cells using the LipofectAMINETM reagent (Life Technologies, Inc.) according to the manufacturer's instructions. A parallel transfection was performed with the unmodified pCMV vector as a control. Transfectants were selected with 500 mg/ml G418 (Life Technologies, Inc.). Individual antibiotic-resistant clones were isolated and expanded. A high-expression clonal cell line, called KMST-col3.3, was identified by Western blot analysis of the conditioned culture media and selected for further experiments. Control clones transfected with the empty vector were randomly chosen.
Construction of Chloramphenicol Acetyltransferase Fusion
Plasmids--
All plasmid constructs were prepared using standard
methods (31). The promoterless basic plasmid pCAT-Basic (Promega Corp., Madison, WI) was used for cloning the different promoter fragments obtained from the human collagenase-3 gene upstream of the bacterial CAT gene. The different collagenase-3 promoter constructs (called, 1004 CAT,
402 CAT, and
56 CAT) were generated by polymerase chain
reaction amplification with specific oligonucleotides or by
endonuclease restriction. All constructs were verified by extensive restriction mapping and DNA sequencing. The
1004 CAT,
402 CAT,
CAT, and
56 mut CAT constructs were polymerase chain
reaction-generated by using the following 5' primers corresponding to
nucleotides
1004 to
984 (5'-CTGCAGCCCTAGTTTTCTTGG-3'), nucleotides
402 to
383 (5'-TCTAGAATCAGTACTAAGTT-3'), nucleotides
56 to
38
(5'-AAGTGATGACTCACCATTG-3'), and nucleotides
56 to
38 with two
mutations in the AP-1 site (5'-AAGTGATTTCTCACCATTG-3'). In
all cases the same 3' primer was used, 5'-GGTCTAGATTGAATGGTGATGCCTGG-3'
(nucleotides +10 to +27). The construct containing the AP-1 site of the
collagenase-1 gene was kindly provided by Dr. P. Angel (University of
Heidelberg, Germany). All recombinant plasmids used for transfection
assays were purified by the QIAGEN plasmid kit (QIAGEN Inc.,
Chatsworth, CA).
DNA Transfections and Chloramphenicol Acetyltransferase
Assays--
For each transfection experiment, cells were seeded at
2 × 105 cells/30-mm dish and transfected 18 h
later with 1.5 µg of the indicated reporter plasmid DNA and 0.5 µg
of pRSVgal (Promega Corp.) using the LipofectAMINETM
Reagent and following the manufacturer's indications. 16 h
following the start of transfection, serum-free DNA-containing medium
was replaced by fresh growth medium without serum and the indicated concentration of hormones to be tested. Transfected cells were harvested in phosphate-buffered saline after 24 h of hormone
exposure for chloramphenicol acetyltransferase (CAT) assays. Cell
extracts of transfected cells were prepared by three cycles of
freeze-thaw and finally resuspended in 100 µl of buffer A (15 mM Tris-HCl, pH 8.0, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 1 mM
dithiothreitol).
-Galactosidase activity was assayed according to
Sambrook et al. (31) in 30 µl of the cell extracts. CAT
activity was assayed essentially according to the method of Gorman
et al. (32). 50 µl of extract were incubated in 60 µl of
buffer A containing 0.2 µCi of [14C]chloramphenicol and
2 mM acetylcoenzyme A for 4 h at 37 °C. One ml of
ethyl acetate was added, mixed by vigorous vortexing, and centrifuged
for 5 min. The supernatant was evaporated under vacuum and reaction
products were redissolved in 20 µl of ethyl acetate, which were
applied to the origin of a silica gel TLC plate. Plates were run in
chloroform/methanol (95/5, v/v), dried, and exposed for autoradiography
on Hyperfilm-MP (Amersham) for 16 h. For quantitation,
radioactivity of the TLC plates was measured by using the InstantImager
electronic autoradiography system (Packard Instrument Co., Meriden,
CT). Stimulation of CAT activity was expressed as fold increase over
activity of non-induced transfected cells and was based on at least
three independent experiments.
Electrophoretic Mobility Shift DNA Binding Assay--
Nuclear
extracts from KMST cells untreated or treated with 10 ng/ml TGF-1
for 2 h were prepared as described by Schreiber et al.
(33). DNA probes corresponding to the AP-1 element identified at
position
50 to
44 of the collagenase-3 promoter region were synthesized as two complementary oligonucleotides,
5'-AAGTGATGACTCACCATTG-3' and 5'-CAATGGTGAGTCATCACTT-3'.
Oligonucleotides were annealed, labeled with [
-32P]ATP
by T4 polynucleotide kinase, and further purified by Sephadex G-25
column chromatography (Pharmacia Biotech Inc.). Nuclear extracts (2 µg) were preincubated at room temperature for 15 min with 2 µg of
poly(dI-dC) in 25 mM Tris-HCl, pH 8, 60 mM KCl,
5 mM MgCl2, 1 mM EDTA, 10%
glycerol, and 0.1 mM dithiothreitol in 20 µl. The 30-min
binding reaction was initiated by the addition of 2 µl (0.1 pmol) of
32P-labeled probe (5 × 106 cpm/pmol). The
amount of unlabeled competitor DNA added is indicated in the figure
legends. Supershift reactions were carried out by adding 2-4 µg of
the corresponding antibodies to the binding reactions, 30 min after
addition of the labeled probes. The whole mixture was incubated for
2 h at 4 °C prior to electrophoresis. Samples were
electrophoresed on prerun 4% polyacrylamide gels containing 2.5%
glycerol in 50 mM Tris, 380 mM glycine, 2 mM EDTA buffer at 200 V for 4 h. Gels were dried and
subjected to autoradiography.
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RESULTS |
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Collagenase-3 Expression Is Induced by TGF- in Human
Fibroblasts--
To study the putative effect of TGF-
on the
expression of collagenase-3 in human fibroblasts, KMST cells were
treated with 5 ng/ml TGF-
1 for 24 h and total cellular RNAs
were purified and analyzed by Northern blot using a specific
collagenase-3 cDNA probe (13). As shown in Fig.
1, TGF-
1 induced the accumulation of
two mRNA transcripts of 3.0 and 2.5 kilobases, corresponding to the
two major collagenase-3 transcripts identified in breast carcinomas
(13) and articular cartilage (22). These two transcripts are the result
of utilization of different polyadenylation sites present in the
3'-noncoding sequence of the human collagenase-3 gene, as demonstrated
by using a probe specific for the 3'-end of the collagenase-3 cDNA
(13). Densitometric analysis of the x-ray films revealed that the
observed stimulatory action of TGF-
1 on collagenase-3 expression was
about 2- and 4-fold lower than the respective effects of IL-1 and TPA,
which had been previously characterized as collagenase-3 inducers in
KMST cells (Fig. 1).
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TGF-mediated Induction of Collagenase-3 Expression in Human
Fibroblasts Is Dependent upon Protein Kinase C and Tyrosine Kinase
Activities--
To study the molecular mechanisms and signal
transduction pathways underlying the up-regulating effect of TGF-
1
on collagenase-3 expression, we first performed cell culture
experiments in the presence of the protein synthesis inhibitor
cycloheximide. As shown in Fig.
4A, incubation of KMST cells
with cycloheximide (10 µg/ml, added 1 h before TGF-
1
treatment) blocked the effect of this growth factor on collagenase-3
mRNA levels. We therefore conclude that de novo protein
synthesis is required for the induction of collagenase-3 mRNA by
TGF-
1. We next evaluated the possibility that protein kinase
C-mediated signaling pathways could be involved in the TGF-
1 induced
production of collagenase-3 in human fibroblasts. To this purpose, KMST
cells were incubated with TGF-
1 in the presence or absence of PKC
inhibitors and the levels of collagenase-3 were examined by Northern
blot. As shown in Fig. 4B, staurosporine and calphostin C
are able to completely inhibit the up-regulating effect of TGF-
1 on
collagenase-3 expression, indicating the involvement of a protein
kinase C in this process. To determine whether a tyrosine kinase is
also involved in the collagenase-3 induction by TGF-
1, we treated
KMST cells with 10 ng/ml TGF-
1 for 24 h in the presence or
absence of genistein or herbimycin A, which inhibit tyrosine kinases by
different mechanisms, and the levels of collagenase-3 mRNA were
analyzed by Northern blot. As illustrated in Fig. 4B, both
tyrosine kinase inhibitors were able to completely block the induction
of collagenase-3 expression elicited by TGF-
1. By contrast,
incubation of fibroblasts with indomethacin, which inhibits
prostaglandin synthesis, did not affect TGF-
1 mediated induction of
collagenase-3 (Fig. 4A). To rule out a possible nonspecific effect of these inhibitors on general transcriptional activity, we
developed a KMST-derived cell line, KMST-col3.3, which stably expresses
the collagenase-3 gene under the control of the constitutive CMV
promoter (see "Experimental Procedures"). As can be seen in Fig.
4C, treatment of these cells with cycloheximide,
staurosporine, calphostin C, genistein, or herbimicyn A at
concentrations that completely abrogate the TGF-
1-induced
collagenase-3 expression in the parental KMST cells, failed to produce
any significant reduction on the constitutive expression of the
collagenase-3 mRNA by these transfected cells. Interestingly,
treatment of KMST-col3.3 cells with cycloheximide resulted in a
significant increase of the collagenase-3 mRNA levels, which
reinforces the proposed specificity of these agents on the regulation
of the endogenous collagenase-3 gene. Taken together, these results
indicate that the positive effect of TGF-
1 on collagenase-3
expression in human fibroblasts is exerted through a signal
transduction pathway involving PKC and tyrosine kinase activities.
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TGF- Mediated Induction of Collagenase-3 Expression in Human
Fibroblasts Is Independent of Cell Proliferation Rate--
It is well
established that TGF-
exerts both positive and negative effects on
cell growth, the final effect depending on a number of factors
including cell type (34). Thus, it is widely assumed that TGF-
induces growth of mesenchymal cells and inhibits proliferation of
epithelial cells. From these considerations, we next investigated if
the effect of TGF-
1 on collagenase-3 expression was correlated with
a stimulation of cell proliferation in human fibroblasts. For this
purpose, KMST cells were incubated for 7 days in the presence of
different concentrations (0.1, 1, and 10 ng/ml) of TGF-
1 and the
cell growth was examined by the fluorimetric protein assay described by
Skehan et al. (29). As shown in Fig.
5A, treatment of KMST cells in
exponential growth phase with TGF-
1 only resulted in a very slight
increase in cell number. As a positive control of TGF-
induced cell
proliferation we used NRK rat kidney fibroblasts which have been
described to be sensitive to cell growth stimulation mediated by this
factor (35), whereas Mv1Lu epithelial cells were used as control of cells inhibited in their proliferation by TGF-
(36). These results
indicate that collagenase-3 induction by TGF-
1 is independent of the
effects of this factor on cell proliferation rate. To provide additional support to this observation, we also examined the possible occurrence of variations in the expression levels of different cyclin-dependent kinase inhibitors in KMST cells treated
with TGF-
. These inhibitors play essential roles in the regulation of the cell cycle and have been demonstrated to mediate the growth inhibitory signals of TGF-
in different cell types (36-39). In fact, TGF-
regulates the expression of at least three members of the
cyclin-dependent kinase inhibitor family: p27kip1,
p21WAF1, and p15INK4B. However, Northern blot analysis
of total RNA from TGF-
1-treated KMST cells did not reveal any
variations on p27kip1, p21WAF1, and p15INK4B
mRNA levels when compared with untreated cells (data not shown). These results provide additional evidence that the TGF-
1 mediated induction of collagenase-3 expression in human fibroblasts is independent of its effects on cell cycle progression.
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Induction of Collagenase-3 Expression by TGF- in Human
Fibroblasts Is Partially Mediated by an AP-1 Site--
To further
analyze the molecular mechanisms involved in the TGF-
1 mediated
regulation of collagenase-3 expression by human fibroblasts, we
undertook a functional analysis of the promoter region of this gene,
looking for the putative response elements that could mediate the above
observed up-regulatory effect. To this purpose, we first prepared a
series of DNA constructs containing various lengths of a 1-kilobase
fragment of the 5'-flanking region of the collagenase-3 gene (40),
cloned in front of the CAT reporter gene. Three different collagenase-3
promoter constructs linked to the CAT reporter gene were used:
56 CAT
containing an AP-1 consensus sequence (TGACTCA);
402 CAT including
the AP-site and an additional PEA-3 site assumed to be important for
MMP responsiveness to growth factors and tumor promoters; and
1004
CAT containing both sites and 596 base pairs of additional upstream
sequence (Fig. 6A). These
three constructs were transiently transfected into KMST cells and
tested for inducibility by TGF-
1. As can be seen in Fig.
6A, those cells transfected with the
56 CAT construct showed significant levels of CAT activity (2.2-fold over basal levels)
after TGF-
1 treatment. This increase in CAT activity was similar to
that obtained after treatment of
56 CAT transfected cells with TPA.
Furthermore, simultaneous treatment of these cells with TGF-
1 and
TPA led to a maximal inducibility of 4.3-fold, confirming the above
observation of synergistic effect between these two factors in the
induction of collagenase-3 expression. Addition of sequences located
further upstream of the AP-1 site in the collagenase-3 promoter
(constructs
1004 CAT and
402 CAT), abolished the stimulatory effect
of both TGF-
1 and TPA on CAT activity, suggesting the presence of
putative inhibitory elements in this 5'-flanking region of the
collagenase-3 gene (Fig. 6A). To perform a comparative
analysis between collagenase-3 and collagenase-1 promoters, similar
experiments were then carried out with a construct of the collagenase-1
promoter containing its corresponding AP-1 site located at equivalent
position to that of collagenase-3 (Fig. 7). In agreement with previous studies
(41), CAT levels in cells transfected with this construct were strongly
induced by TPA. However, treatment with TGF-
1 did not have any
effect on CAT activity levels of the collagenase-1 promoter construct
(Fig. 6A), confirming that despite its close similarity in
many structural and functional aspects, collagenase-1 and collagenase-3
may respond in a completely opposite way to identical treatments with
factors like TGF-
1.
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DISCUSSION |
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In this work we have shown that expression of human collagenase-3,
a matrix metalloproteinase produced by breast carcinomas and arthritic
cartilage, is induced by TGF-1 in cultured human fibroblasts. This
up-regulatory effect of TGF-
1 on the production of a potent
proteolytic enzyme like collagenase-3 is in marked contrast with the
widely assumed role of this growth factor as an inducer of anabolic
responses in mesenchymal cells. In fact, TGF-
has been implicated in
the induction of connective tissue formation by stimulating the
synthesis of several extracellular matrix components such as
fibronectin, thrombospondin, and types I and III collagen (35, 48-52).
In addition, TGF-
suppresses the overall degrading activity on these
matrix components through a concerted dual action involving the
reduction in the production of a wide diversity of proteolytic enzymes
including MMPs (53, 54) and plasminogen activators (55), as well as the
concomitant increase in the synthesis of their respective inhibitors
(52, 56-58). The ability of TGF-
family members to induce
collagenase-3 expression is not exclusive of fibroblasts, since a
recent report has shown that TGF-
2 up-regulates its expression in
transformed keratinocytes (59). By contrast, another recent report has
shown that TGF-
1 inhibits collagenase-3 expression in osteoblast
cultures (60), suggesting that the effects of these growth factors are markedly dependent of the cell type. Nevertheless, the ability of
TGF-
to stimulate the production of a proteolytic enzyme by human
fibroblastic cells is not unprecedented, since Overall et al. (58, 61) have reported that levels of gelatinase A (MMP-2) are
increased in gingival fibroblasts, although the magnitude of this
up-regulating effect is lesser than that observed in the present work
for collagenase-3. The possibility that collagenase-3 and gelatinase A
are coordinately regulated in fibroblasts is of interest in light of
recent findings demonstrating that both enzymes form part of an
activation cascade which can generate the extracellular collagenolytic
activity requested for the connective tissue degradation occurring in
both normal and pathological conditions (62). In this context, it is
specially noteworthy that collagenase-1 (MMP-1), despite sharing with
collagenase-3 its unique ability to initiate degradation of the native
helix of fibrillar collagens, shows a completely opposite response to
TGF-
1 treatment. In fact, and as a consequence of the structural
complexity of the extracellular matrix and basement membranes which
must be proteolytically degraded by MMPs, these enzymes are often
coregulated by the same cells in response to the same stimuli. In the
case of collagenase-1 and collagenase-3 production by human
fibroblasts, this statement seems to be true for cytokines like IL-1
and IL-1
and for tumor promoters like TPA, all of them displaying a
marked up-regulatory effect on both MMP genes (Fig. 1). However, their
divergent responses to TGF-
1 clearly indicate that in addition to
common regulatory elements, these genes have distinct transcriptional
elements that determine their specific expression patterns. Functional
studies of the promoter region of the collagenase-3 gene performed in this work have shown that the AP-1 site present in its 5'-flanking region is responsible, at least in part, for the TGF-
1 mediated induction of this gene. AP-1 sites have also been reported to mediate
the response to this growth factor of other genes such as type-1
plasminogen activator inhibitor (63), osteocalcin (64), retinoic acid
and retinoid X receptors (65), as well as the autoinduction of the
TGF-
1 gene itself (66). Nevertheless, the extent of stimulation of
collagenase-3 gene expression by TGF-
1, as detected by Northern
blot, was higher than the induction of promoter activity observed in
transient cell transfection experiments with AP-1 containing
constructs. Similar observations have been previously reported during
the functional analysis of AP-1 sites present in other MMP genes (67,
68). Therefore, it seems likely that this AP-1 site present in the
collagenase-3 gene is necessary but not sufficient for mediating its
response to TGF-
1 in human fibroblasts. The participation of
additional elements which could be located further upstream in the
5'-flanking region of the collagenase-3 gene could contribute to
explain the differences observed between data derived from Northern
blot analysis and those from determination of relative CAT activity of
reporter gene constructs. In addition, it is remarkable that other MMP
genes, including collagenase-1, which are down-regulated by TGF-
,
also contain AP-1 consensus sequences at approximately the same
position as that present in the collagenase-3 gene (Fig. 7), thus
supporting the idea that sequences other than AP-1 influence the
responsiveness of the different MMP genes to this growth factor.
Finally, it is also likely that TGF-
1, in addition to activating the
collagenase-3 promoter, may increase the expression of this gene by
post-transcriptional mechanisms such as stabilization of the
corresponding mRNAs, which have been previously described to
operate in the case of the collagenase-1 gene (69).
In an attempt to determine the molecular basis of the somewhat
paradoxical effect of TGF-1 on collagenase-3 expression in human
fibroblasts, we have further investigated the mechanistic aspects
underlying this up-regulatory effect. In this work, and by using a
series of specific inhibitors for different signaling pathways, we have
found that the TGF-
1 action on collagenase-3 is mediated by PKC and
tyrosine kinase signal transduction pathways. Since TGF-
receptors
are Ser/Thr kinases, it is tempting to speculate that the activity
affected by PKC inhibitors could be that intrinsic to the TGF-
receptors themselves. However, the observation that TGF
-RI and -RII
autophosphorylation is not affected by PKC inhibitors (70) suggests
that additional kinase activities acting downstream from the TGF-
receptor are involved in mediating the induction of collagenase-3 by
TGF-
1. In addition, we have tried to correlate the TGF-
1 positive
effect on collagenase-3 expression with some of the pleiotropic effects
elicited by this multifunctional growth factor. It is well known that
TGF-
displays a wide variety of actions even in the same cell type,
depending on a number of factors including the specific target,
conditions of cell culture, and presence of other growth regulators.
Consistent with this, the observation that collagenase-3 induction by
TGF-
1 in fibroblasts is accompanied by a weak stimulation of cell
growth, together with data showing that in HaCaT keratinocytes TGF-
mediated up-regulation of this enzyme is accompanied by a potent
inhibition of cell growth,2
strongly suggest that induction of collagenase-3 by this growth factor
is independent of its effects on cell proliferation.
Finally, in this work we have examined the possibility that cell
specific induction of distinct members of the Fos/Jun family of
transcription factors is responsible for the divergent regulation of
collagenase-1 and collagenase-3 genes by TGF-1 in human fibroblasts. Electrophoretic mobility shift assays and antibody supershift analysis
revealed that c-Fos, c-Jun, and JunD are found in complexes formed with
nuclear extracts prepared from KMST cells treated with TGF-
1.
Analysis of the pattern of expression of these Fos/Jun proto-oncogenes
in KMST cells treated with TGF-
1 confirmed that collagenase-3
induction is preceded by an increase in levels of expression of c-Fos
and JunD. According to these results, it is conceivable that the
induction of high levels of c-Fos and JunD favors the formation of
specific complexes which bind and transactivate the collagenase-3
promoter, thus resulting in the observed up-regulation of this gene.
Nevertheless, the participation of c-Jun in the process, after
post-transcriptional mechanisms of regulation induced by TGF-
such
as phosphorylation mediated by c-Jun N-terminal kinase (47), cannot be
ruled out. In this regard, it is well known that the preferential
binding to AP-1 sites exhibited by different Fos/Jun proteins is
dependent upon specific flanking and core nucleotide sequences, thus
allowing fine regulation of expression of the diverse AP-1 driven
genes. c-Jun and c-Fos have been proposed to be fundamental
intermediates for collagenase-1 and stromelysin-1 gene activation by
tumor necrosis factor-
in fibroblasts, and by TGF-
in
keratinocytes, whereas inhibitory effects have been reported to be
mediated by transient elevation of JunB (42, 45). However, in KMST
cells, TGF-
had little if any effect on the expression of c-Jun or
JunB, thus indicating that at least in these cells, other mechanisms
should be involved in TGF-
1 elicited down-regulation of
collagenase-1.
In summary, we have provided evidence that TGF- dissociates
production of collagenase-1 and collagenase-3 by human fibroblasts. The
opposite response of both enzymes to TGF-
1 in KMST cells makes them
an appropriate model system for studying the molecular mechanisms
controlling the expression of these two highly related enzymes in terms
of structure and enzymatic properties, but displaying marked
differences in their pattern of tissue expression. Further studies will
be also required to evaluate the putative role of TGF-
1 as an
in vivo inducer of human collagenase-3 in those conditions in which this enzyme has been found at high levels, including breast
carcinomas and arthritic processes.
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ACKNOWLEDGEMENTS |
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We thank Drs. G. Velasco, A. Fueyo, and A. M. Pendás for helpful comments, Drs. P. Angel, M. Serrano, and F. Ventura for kindly providing plasmids and cells, and S. Alvarez for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by Grants SAF94-0892 and SAF97-0258 from the Comisión Interministerial de Ciencia y Tecnología, EU-BIOMED II (BMH4-CT96-0017), and Glaxo-Wellcome, Spain.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.
Recipient of a fellowship from the Fundación para la
Investigación Científica y Técnica (FICYT),
Asturias, Spain.
§ Recipient of a fellowship from the Ayuntamiento de Oviedo, Asturias, Spain.
¶ To whom correspondence should be addressed: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain. Tel.: 34-85-104201; Fax: 34-85-103564 or 34-85-232255; E-mail: clo{at}dwarf1.quimica.uniovi.es.
1
The abbreviations used are: MMP, matrix
metalloproteinase; IL-1, interleukin 1
; TPA,
12-O-tetradecanoylphorbol-13-acetate; aFGF, acidic
fibroblast growth factor; bFGF, basic fibroblast growth factor; TGF,
transforming growth factor; CAT, chloramphenicol acetyltransferase;
PKC, protein kinase C.
2 J. A. Uría and C. López-Otín, unpublished results.
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REFERENCES |
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