From the School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom and the ¶ Department of Rheumatology, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne NE2 4HH, United Kingdom
Received for publication, December 4, 2002, and in revised form, January 13, 2003
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ABSTRACT |
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The balance between matrix
metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors
of metalloproteinases (TIMPs), is pivotal in the remodeling of
extracellular matrix. TGF- Timely breakdown and remodeling of the extracellular matrix
(ECM)1 is an essential
process in development, morphogenesis, and reproduction. ECM
degradation is also associated with a variety of physiological and
pathological processes such as joint destruction in the arthritides, wound healing, tumor metastasis, angiogenesis, and fibrosis (1). Pivotal to the turnover of ECM is the matrix metalloproteinase (MMP)
family of enzymes; these enzymes have the capability, between them, of
degrading the majority of the proteins that make up the ECM (2). The
tissue inhibitors of metalloproteinases (TIMPs) protect ECM integrity
by inhibiting MMPs (3). MMP-1, interstitial collagenase, is one of a
subfamily of MMPs that can specifically degrade the collagen triple
helix; hence, MMP-1 plays a central role in pathologies where collagen
turnover is aberrant (4). As well as inhibiting most of the active
MMPs, TIMP-1 is also reported to have diverse effects on cell growth
and apoptosis (5).
Transforming growth factor- TGF- The proximal promoters of both MMP-1 and TIMP-1
genes contain an AP1 site, which, in each case, has been the focus for
research on their regulation. In the murine Timp-1 gene, the
AP1 site is located at Upstream of the AP1 site in the MMP-1 promoter is a putative
TGF- We postulated that the ability of TGF- Here, we investigate the mechanism(s) by which TGF- Cell Culture--
Murine C3H10T1/2 fibroblasts, Swiss 3T3 cells,
and human skin fibroblasts were routinely cultured in minimal essential
medium with Earle's salts and L-glutamine (2 mM) (Invitrogen) containing 10% fetal bovine serum
(Invitrogen), 1% nonessential amino acids, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 20 units/ml nystatin. Serum-free conditions
used minimal essential medium containing 0.1% bovine serum albumin
with the above antibiotics. AP1 knockout cells (c-Jun Reporter Constructs and Expression Plasmids--
Constructs
using Timp-1 promoter driving luciferase expression were in
pGL2-basic (Promega); point mutations altered the wild-type AP1 site
(5'-TGAGTAA-3') to the MMP-1 consensus AP1 site
(5'-TGAGTCA-3') or a nonfunctional mutant AP1 site (5'-GGAGTGA-3').
Constructs using the MMP-1 promoter were in pGL3-basic
(Promega) and were a kind gift from Prof. C. E. Brinckerhoff
(Dartmouth Medical School) (33).
The AP1 expression plasmids in pCMV were described by Harrison
et al. (34) and were a kind gift of Dr. P. R. Dobner (University of Massachusetts). Smad expression plasmids in pCMV
were described by Wicks et al. (18) and were from
the laboratory of Dr. A. Chantry (University of East Anglia).
All mutagenesis was performed using the QuikChange method (Stratagene).
All mutations were verified by sequencing.
p3TP-Lux is an artificial promoter consisting of the plasminogen
activator inhibitor-1 TGF- Transient Transfection--
Cells were seeded in six-well plates
at a density of 8850 cells/cm2 and grown overnight in
medium containing 10% fetal calf serum at 37 °C in a 5%
CO2 atmosphere. Cells were transfected overnight in
serum-containing medium with 1 µg/well reporter plasmid using FuGene
6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. The following day, cells were washed in Hanks' balanced salts solution and incubated in serum-free medium overnight. Cells were
then stimulated with phorbol 12-myristate 13-acetate (PMA; 10
For co-transfection with expression constructs, an additional Nuclear Extracts--
Confluent cells at a density of ~2 × 107/150-mm dish were washed and incubated in medium
containing 0.1% bovine serum albumin overnight. Cells were then
treated with TGF- Probe Labeling--
Oligonucleotides for electrophoretic
mobility shift assay (EMSA) were synthesized by MWG-Biotech.
Timp-1 AP1 site, 5'-AGCTTGGATGAGTAATGCG-3'; MMP-1
AP1 site, 5'-AGCTAGCATGAGTCAGACA-3'; mutant AP1 site
5'-AGCTTGGAGGAGTGAGCGG-3'; S4BE,
5'-GATCTCGAGAGCCAGACAAAAAGCCAGACATTTAGCCAGACAC-3'; S4BEmut, 5'-GATCTCGAGAGCTACACA AAAAGCTACACATTTAGCTACACAC-3'.
Double-stranded probes were labeled with
[ EMSA--
Nuclear extracts (~2 µg), 1 µg of poly(dI-dC),
and radiolabeled probe (~30,000 cpm) were incubated in 1× binding
buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM dithiothreitol, 5 mM MgCl2, and
5% glycerol) with or without competitor DNA for 20 min at room
temperature in a total volume of 10 µl. For antibody supershift analyses, 2 µg of the appropriate antibody (anti-c-Fos, sc-52-Gx; anti-c-Jun, sc-45-Gx; anti-FosB, sc-48-Gx; anti-Fra1, sc183x; anti-Fra2, sc-604x; anti-JunB, sc-46-Gx; anti-JunD, sc-74-Gx; anti-Smad4, sc-7966x (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
and anti-Smad2/3, S66220 (Transduction Laboratories, Lexington, KY))
was incubated with nuclear extract for 20 min at room temperature prior
to the binding reaction. It should be noted that in EMSA, antibodies
may either further retard the migration of a protein-DNA complex
(supershift) or block binding of the protein to the DNA (inhibition),
depending on epitope recognized and binding affinity (36). Samples were
separated on a 5% polyacrylamide gel in 0.5× TBE (45 mM
Tris-HCl, 45 mM boric acid, 1 mM EDTA). Gels
were prerun at 10 mA for 1 h at 4 °C and run at 4 mA for 3-6 h
at 4 °C. Gels were dried and autoradiographed.
Reverse Transcription-PCR--
RNA was isolated from monolayer
cultures using Trizol (Invitrogen). Quantitative reverse
transcription-PCR was performed using the Applied Biosystems ABI Prism
7700 sequence detection system (TaqMan®) as described (37).
Deletion Analysis of the Timp-1 Promoter--
Prior to studying
the response of the Timp-1 promoter to TGF-
A Protein Binding to the Timp-1 AP1 Site--
EMSA and supershift
analysis was used to probe protein binding to the Timp-1 AP1
site under PMA and TGF-
When these experiments were repeated using an oligonucleotide
containing the MMP-1 consensus AP1 sequence, identical
results were obtained (data not shown).
Overexpression of AP1 Factors Transactivates the Timp-1
Promoter--
In order to assess the role of differing Fos and Jun
family members in activating transcription from the Timp-1
promoter, co-transfection experiments were performed. The
Substitution of the wild-type AP1 (5'-TGAGTAA-3') site in the Expression of Timp-1 in AP1 Knockout Cells--
In order to verify
the data gained from overexpression of AP1 factors, a Timp-1
promoter construct was transiently transfected into cell lines deleted
for one of c-fos, c-jun, junD, or
fra-1, and cells were stimulated with either PMA or TGF-
The data show that c-Fos, c-Jun, and JunD are each necessary, and Fra-1
is not necessary for TGF-
EMSA analysis on overlapping oligonucleotides across the TGF-
A shorter MMP-1 promoter construct, Point Mutations in Overexpression of Smads 2, 3, and 4 Does Not Potentiate TGF-
Conversely, co-expression of Smad 7 with the Timp-1 Expression Is Induced by TGF- A Smad-binding Oligonucleotide Blocks TGF- Smad Binding to the Timp-1 or MMP-1 AP1 Motifs--
Using the S4BE
oligonucleotide in EMSA with nuclear extracts from C3H10T1/2 cells,
five DNA-protein complexes were apparent (Fig.
8). Supershift analysis with antibodies
against Smad 2/3 or Smad 4 demonstrated that band A, the slowest
migrating complex, contained all of these Smads. Indeed, competition
with a 100-fold excess of cold S4BE shows that only band A is competed
(data not shown). Mutation of the three 5'-CAGA-3' sequences in S4BE
(to 5'-TACA-3') leaves only band D intact (data not shown).
EMSA using oligonucleotides containing the Timp-1 or
MMP-1 AP1 site and nuclear extracts from C3H10T1/2 or human
skin fibroblasts shows an identical pattern of binding on each
oligonucleotide (as shown in Fig. 2). No supershift band could be seen
in the additional presence of the anti-Smad 4 antibody (data not
shown). However, using the labeled S4BE as probe, the MMP-1
AP1 oligonucleotide competes band A (the Smad-containing complex) at a
100-fold excess, whereas the Timp-1 AP1 oligonucleotide does
not (Fig. 9). Intriguingly, the
Timp-1 AP1 oligonucleotide competes band E, but because
self-competition with cold S4BE shows this band as nonspecific, this
finding is difficult to interpret (Fig. 9).
TGF- The promoter requirements for TGF- A recent report by Verrecchia et al. (31) states that the
Timp-1 gene is Smad-dependent, in
disagreement with the majority of data in the current study. The
following lines of evidence are presented: (i) TGF- The dependence of Timp-1 gene expression on AP1 factors has
been described in other systems and with other inducing agents (e.g. Botelho et al. (41) ascribe the induction
of Timp-1 by oncostatin M to an induction of c-Fos and a
change in the major AP1-binding complex from c-Jun/c-Fos to JunD/c-Fos
in HepG2 cells; Smart et al. (42) demonstrate that JunD,
Fra2, and FosB associate with the TIMP-1 AP1 site during
hepatic stellate cell activation, of which JunD is functionally the
most important. The current study indicates that c-Fos, Fra2, FosB,
c-Jun, JunD, and JunB are all present in TGF- The AP1 motif in the Timp-1 promoter differs at a single
base pair from the MMP-1 consensus (5'-TGAGTAA-3' compared
with 5'-TGAGTCA-3'), with the only known consequence being binding of
an unknown single-stranded DNA-binding protein to the former but not
the latter (23). However, substitution of the wild-type site for the
consensus in a Timp-1 promoter construct does not alter the
response to TGF- TGF- Deletion from a The importance of the AP1 motif in the TGF- The interaction of Smads with AP1 factors has recently been reported by
several groups (27-29). Using EMSA, no supershift was detected using
anti-Smad2/3 or anti-Smad4 antibodies on an AP1 shift; the nuclear
extracts used show Smad binding to a canonical Smad-binding
oligonucleotide (S4BE) using the same methodology. Intriguingly,
however, the MMP-1 AP1-containing oligonucleotide (but not
the Timp-1 equivalent) does compete for binding of the Smad-containing complex to the S4BE. The functional relevance of this
is shown in co-transfection studies, where either Smad 7 or the
wild-type S4BE oligonucleotide relieve the TGF- In agreement with this, a recent report by Yuan and Varga (30) shows
the Smad dependence of TGF- TGF- In conclusion, the ability of TGF- has profound effects on
extracellular matrix homeostasis, in part via its ability to alter this
balance at the level of gene expression. The intracellular signaling
pathways by which TGF-
mediates its actions include the Smad
pathway, specific to the TGF-
superfamily, but also, for example,
mitogen-activated protein kinase pathways; furthermore, cross-talk
between the Smads and other signaling pathways modifies the TGF-
response. The reciprocal effect of TGF-
on the expression of
Timp-1 and MMP-1 supports its role in matrix
anabolism, yet the mechanisms by which TGF-
induces Timp-1 and represses induced MMP-1 have
remained opaque. Here, we (i) investigate the mechanism(s) by which
TGF-
1 induces expression of the Timp-1 gene and (ii)
compare this with TGF-
1 repression of phorbol ester-induced
MMP-1 expression. We report that the promoter-proximal
activator protein 1 (AP1) site is essential for the response of both
Timp-1 and MMP-1 to TGF-
(induction and
repression, respectively). c-Fos, JunD, and c-Jun are essential for the
induction of Timp-1 gene expression by TGF-
1, but these AP1 factors transactivate equally well from both Timp-1 and
MMP-1 AP1 sites. Smad-containing complexes do not interact
with the Timp-1 AP1 site, and overexpression of Smads does
not substitute or potentiate the induction of the gene by TGF-
1;
furthermore, Timp-1 is still induced by TGF-
1 in Smad
knockout cell lines, although to varying extents. In contrast, Smads do
interact with the MMP-1 AP1 site and mediate repression of
induced MMP-1 gene expression by TGF-
1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
) is a multifunctional growth
factor controlling cell growth and differentiation and has marked
effects on ECM homeostasis (6). This includes the induction of ECM gene
expression and generally suppression of MMPs and induction of TIMPs to
give a "synthetic" phenotype (7). Hence, TGF-
is associated, for
example, with fibrosis in a number of diseases (8). TGF-
has
previously been shown to repress the expression of MMP-1,
induced by a variety of stimuli, in a number of cell types (9, 10).
Conversely, TGF-
induces Timp-1 gene expression, often in
synergy with other growth factors and cytokines (11, 12).
signals via transmembrane receptors to intracellular mediators
of the Smad family. Smad 2 and Smad 3 are receptor-specific Smads that
are phosphorylated on serine residues by the type I TGF-
receptor.
Upon phosphorylation, these Smads form heteromeric complexes with a
common mediator Smad 4 and can then be translocated to the nucleus,
where they regulate gene expression either directly or in association
with a number of co-activators and co-repressors. An inhibitory Smad 7 blocks this cascade to prevent TGF-
-mediated alterations in gene
expression (13). TGF-
can also signal through other pathways
(e.g. mitogen-activated protein kinase pathways), although
the mechanisms for activation of these pathways appear diverse (14,
15). Cross-talk between the Smad signaling cascade and other pathways
also adds complexity to the system (16-18).
59 bp and has been shown to be important in
both basal and inducible Timp-1 gene expression; mutation of
this site in either the mouse or human gene results in greater than
90% reduction in expression of promoter-reporter constructs in
transient transfection studies (19, 20). In the human MMP-1
gene, the AP1 site is located at
72 bp and in both human and rabbit
genes is critical for robust expression from promoter-reporter
constructs (21, 22). For each gene, with some dependence on promoter
length used, induction of the gene by phorbol ester is still apparent when the promoter-proximal AP1 site is mutated, although with much
reduced basal expression
(22).2 Interestingly, the
Timp-1 AP1 site is noncanonical (5'-TGAGTAA-3'), and this is
conserved across all species sequenced, whereas the MMP-1
AP1 site is the consensus sequence (5'-TGAGTCA-3'). The Timp-1 AP1 site has recently been reported to bind a
distinct nuclear factor compared with the consensus; this factor, ssT1, is a single-stranded DNA-binding protein of unknown identity or function (23).
-inhibitory element (TIE;
245 bp, 5'-GAATTGGAGA-3'), first described in the rat MMP-3 promoter to mediate repression of
epidermal growth factor-stimulated expression of this gene by
TGF-
(24). Nuclear proteins from TGF-
-treated fibroblasts,
including c-Fos, were shown to bind to this sequence from the MMP-3
promoter. A recent study using the rabbit MMP-1 promoter
transfected into rabbit synovial fibroblasts demonstrated that the
MMP-1 TIE functions as a constitutive repressor of and an
antagonist of phorbol-ester-induced MMP-1 gene expression
(9).
to induce Timp-1
expression but suppress induced MMP-1 expression could be
mediated at several possible levels (e.g. (i) differences in
the AP1 motif, (ii) the presence of the TIE in the MMP-1
gene, and (iii) binding of Smad complexes to either gene). TGF-
regulation of many genes is dependent on AP1 motifs in their promoters
(e.g. clusterin (25), PAI-1, and type I collagen (26)).
Evidence for the first and third possibilities together comes from
Zhang et al. (27), who reported that Smad 3 interacts
directly with the MMP-1 AP1 motif to activate transcription
in response to TGF-
and that Smad 3 and Smad 4 can activate
TGF-
-inducible transcription from this site in the absence of c-Jun
and c-Fos. Smad 3 and c-Jun can bind to the MMP-1 AP1 site
simultaneously, but footprinting suggested that Smad binding was at the
3' end of the AP1 motif (5'-GTCAGCC-3'), which is not identical in the
Timp-1 gene (5'-GTAATGC-3'). Indeed, the authors suggest
that different AP1 sites could have differing affinity for Smad 3, dependent on a few nucleotides flanking the site. Similarly, Smads are
reported to bind directly to Jun family members (28), and Smad 3 is
reported to potentiate the induction of gene expression by Jun family
members in AP1-dependent promoters via protein-protein
interaction (29). Yuan and Varga (30) document that TGF-
repression
of IL-1-induced MMP-1 expression is Smad-mediated, although
the cis-acting sequences through which this effect is mediated are not
localized. Timp-1 has been reported as a Smad-responsive
gene in dermal fibroblasts (31) using microarray and transient
transfection technology.
1 induces
expression of the Timp-1 gene and compare this to the
TGF-
1 repression of PMA-induced MMP-1 expression. We
report that the promoter proximal AP1 site is essential for the
response of both Timp-1 and MMP-1 to TGF-
(induction and repression, respectively). c-Fos, JunD, and c-Jun are
essential for the induction of Timp-1 gene expression by
TGF-
1, but these AP1 factors transactivate equally well from both
the Timp-1 and MMP-1 AP1 sites. Smad-containing complexes do not interact with the Timp-1 AP1 site, and
overexpression of Smads does not substitute or potentiate the induction
of the gene by TGF-
1; furthermore, Timp-1 is still
induced by TGF-
1 in Smad 2, Smad 3, or Smad 4 knockout cell lines,
although to varying extents. In contrast, Smads do interact with the
MMP-1 AP1 site and mediate the repression of induced
MMP-1 gene expression by TGF-
1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
, c-Fos
/
,
and Fra1
/
, a kind gift from Professor E. Wagner (University of
Vienna) and Dr. P. Angel (University of Heidelberg); Jun
D
/
(32), a kind gift from Professor M. Yaniv and Dr. J. Weitzman (Pasteur Institute, Paris, France)) and Smad knockout cells
(Smad 2
/
and Smad 3
/
, a kind gift from Dr. E. Böttinger
(NCI, National Institutes of Health, Bethesda, MD); Smad 4
/
, a kind
gift from Professor T. Mak (University of Toronto)) were cultured in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
with 1% nonessential amino acids, 100 IU/ml penicillin, 100 µg/ml
streptomycin, and 20 units/ml nystatin. Serum-free conditions used
Dulbecco's modified Eagle's medium containing 0.5% bovine serum
albumin with the above antibiotics.
-responsive promoter and three repeats of
the MMP-1 AP1 site (35).
7 M; Sigma) or TGF-
1 (2 ng/ml; R&D
Systems) or both together for varying times as shown, prior to harvest.
Harvest and assay were according to the manufacturer's instructions
(luciferase activity; Promega).
1
µg/well expression vector(s) was included, keeping the total DNA to 2 µg/well using empty vector. For co-transfection with oligonucleotides, an additional 1 µg/well of oligonucleotide
(wild-type or mutant control) was included in the transfection.
1 (2 ng/ml), PMA (10
7 M),
or both together for 3 h. Cells were scraped into ice-cold phosphate-buffered saline, pelleted at 500 × g,
and resuspended in 1 ml of phosphate-buffered saline, 0.1% Nonidet
P-40 for ~30 s. After centrifugation at 13,000 × g
for 10 s, pellets were rinsed twice with phosphate-buffered
saline, 0.1% Nonidet P-40 and then resuspended in 3 volumes of high
salt buffer (25 mM HEPES, pH 7.8, 500 mM KCl,
0.5 mM MgSO4, 1 mM dithiothreitol)
containing 1× Complete protease inhibitors (Roche Molecular
Biochemicals). Samples were incubated on ice for 20 min with occasional
vortex and then centrifuged at 13,000 × g for 2 min at
4 °C. Supernatant was then divided into aliquots, frozen on dry ice,
and stored at
80 °C. Protein concentration in the nuclear extract
was determined by Bradford assay (Bio-Rad) and was typically 2-5 µg
of protein/µl.
-32P]dCTP using Klenow fill-in, whereas
single-stranded probes were labeled with [
-32P]ATP
using T4 polynucleotide kinase. Labeling reactions were followed by
phenol/chloroform extraction and purification through a Sephadex G50
spin column.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1, the
transient transfection protocol was optimized to enable us to assay the
response of the promoter-reporter constructs at early time points.
Previous studies at the level of steady-state mRNA for
Timp-1 have suggested that there is an early primary response to TGF-
1, followed by a later secondary response, which may
be mediated by or in conjunction with TGF-
1-induced autocrine factors (12). Using luciferase as a reporter, transgene expression was
robust at a 6-h time point, and this was therefore used throughout the
studies of the Timp-1 promoter.
925/+47 Timp-1 promoter construct reiterates the
response of the endogenous Timp-1 gene (12) to PMA and
TGF-
1, demonstrating a significant induction by each factor alone
and an augmented response to both factors together (Fig.
1). Deletion from the 5' end of this
construct demonstrated that, whereas upstream sequences may impact on
the level of induction (or indeed on basal expression), this pattern of
expression is maintained in a
62/+47 Timp-1 promoter construct; however, induction is lost in a
50/+47 construct in which
an AP1 site at
59/
53 is absent. This construct loses TGF-
1 inducibility and the synergism between TGF-
1 and PMA, but it maintains a low level of PMA inducibility (~1.5-fold). This
demonstrates that the
59/
53 AP1 site is critical for TGF-
1
induction of the Timp-1 gene. This is confirmed by an
inactivating point mutation in this AP1 site in the context of the
223/+47 construct, whereby PMA and TGF-
1 induction are markedly
reduced compared with wild-type (and the synergism between the two is
lost) although not completely abolished. It should also be noted that
the pattern of response of the Timp-1 promoter to TGF-
1
and PMA is replicated in other cell lines (e.g. Swiss
3T3).
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Fig. 1.
Mutational analysis of the Timp-1
promoter in response to TGF-
induction. A, a series of Timp-1
promoter constructs in pGL2-basic were transiently transfected into
C3H10T1/2 murine fibroblasts. Cells were serum-starved for 24 h
and then stimulated with TGF-
(2 ng/ml), PMA (10
7
M), or both together. Cell lysates were harvested at
t = 6 h and assayed for luciferase activity. Data
are expressed as -fold induction above the control in each case, mean
and S.E. (n = 3). B, a functionally inactive
mutation in the
59/
53 AP1 site was made in the context of
223/+47
Timp-1 and compared with wild-type under identical assay
conditions as A.
1 stimulation (Fig. 2). At this 3-h time point, the AP1 site
is bound in unstimulated nuclear extracts; upon TGF-
1 or PMA
stimulation, binding to this site increases, although the mobility of
the complex remains unaltered. Treatment with both factors together
increases binding further. Specificity of binding was ascertained by
competition with cold self and mutant oligonucleotides (data not
shown). Supershift/antibody blocking analysis using antibodies against
members of the Jun and Fos family was performed to examine the
components of the AP1-binding complex under each condition. Nuclear
extracts from cells induced with PMA or PMA plus TGF-
1 appear to
contain all of the Fos and Jun family members assayed. Stimulation with
TGF-
1 alone gave a similar pattern of response except for the
absence of a supershift with the anti-Fra1 antibody; this indicates
that TGF-
1 alone does not induce Fra1 binding to this
oligonucleotide.
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Fig. 2.
Electrophoretic mobility shift assay on an
AP1 oligonucleotide. C3H10T1/2 murine fibroblasts were
serum-starved for 24 h and then stimulated with TGF- (2 ng/ml),
PMA (10
7 M), or both together. Nuclear
extracts were harvested at t = 3 h. EMSA was
performed on the Timp-1 AP1 site oligonucleotide in the
absence or presence of antibodies to members of the Fos and Jun
families (2 µg per binding reaction). Lane 1, control;
lane 2, TGF-
; lane 3, PMA; lane 4,
TGF-
+ PMA.
95/+47
Timp-1 luciferase construct was transiently transfected into
C3H10T1/2 cells with combinations of expression constructs for c-Fos,
Fra-1, Fra-2, FosB, c-Jun, JunD, and JunB; empty vector was used a
control (Fig. 3). Expression and function
of these factors was assessed by EMSA (data not shown). Alone, none of
the Jun family members could transactivate the Timp-1
promoter, whereas c-Fos and, to a lesser extent, FosB, do
transactivate, presumably in combination with endogenous Jun factors
expressed in these cells. The combination of c-Fos with c-Jun or JunD
gives the most potent induction of the Timp-1 promoter
construct, ~4-fold above c-Fos alone and 8-fold above control.
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Fig. 3.
Co-transfection of 95/+47 Timp-1
reporter with Fos and Jun family expression constructs.
C3H10T1/2 cells were co-transfected with 1 µg of
95/+47
Timp-1 in pGL2-basic and 0.5 µg of Fos and Jun family
expression vectors or empty vector to total 2 µg of DNA per plate.
Cells were then serum-starved for 24 h prior to harvest and
luciferase assay. Data is expressed as -fold induction above the
control (empty vector only), mean and S.E. (n = 3).
95/+47
for the MMP-1 consensus AP1 site (5'-TGAGTCA-3')
(i.e. an A to C transversion) did not alter the pattern of
response to these AP1 family members (data not shown). Furthermore,
using a
125/+60 MMP-1 luciferase construct, transiently
transfected into C3H10T1/2 cells or Swiss 3T3 cells (in which
PMA-induced MMP-1 expression either is not or is repressed
by TGF-
1, respectively; see below), with AP1 factors, gave the same
pattern of response (data not shown).
1
(Fig. 4). In these experiments, a
925/+47 Timp-1 luciferase plasmid was used, since many of
the AP1 knockout cell lines transfect poorly, and this construct gives
a higher level of expression. As a control vector, p3TP-Lux was used;
this is an artificial construct consisting of three copies of the
MMP-1 AP1 site and one copy of the TGF-
1-responsive PAI-1
promoter. p3TP-Lux is known to be both AP1- and Smad-responsive. All
constructs were co-transfected with pRSVCAT and CAT expression was used
to normalize the data for transfection efficiency. It should be noted
that the +/+ cells in the c-Fos and c-Jun experiments are Swiss 3T3
cells, since cells from wild-type littermates of the knockouts were not
available. Conversely, +/+ cells in the JunD and Fra-1 experiments were
from wild-type littermates. Variability in response of these
"wild-type" cells to TGF-
1 and PMA is apparent, and this
reinforces the need for a PMA- and TGF-
1-inducible control plasmid
such as 3TP-Lux.
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Fig. 4.
Transient transfection of p3TP-Lux and
925/+47 Timp-1 into AP1-deficient cell lines.
Plasmids 3TP-Lux or
925/+47 Timp-1 in pGL2-basic were
transiently transfected into cells deficient in individual Fos or Jun
family members or wild-type cells as shown. Cells were serum-starved
for 24 h and then stimulated with TGF-
(2 ng/ml) or PMA
(10
7 M). Cell lysates were harvested at
t = 6 h and assayed for luciferase activity. Data
are expressed as -fold induction above the control in each case, mean
and S.E. (n = 3).
1-induction of the Timp-1 gene,
although PMA still induces expression in the absence of c-Fos.
Interestingly, c-Jun is also necessary for induction of 3TP-Lux by
either TGF-
1 or PMA, and JunD is necessary for induction by PMA.
These data underscore the importance of c-Fos, c-Jun, and JunD as AP1
factors that induce Timp-1 expression (as seen in
overexpression studies above) and place them in the pathway by which
TGF-
1 induces Timp-1 gene expression.
62/+47
region shows no obvious alterations in protein binding after TGF-
1
or PMA stimulation other than on the AP1 site above (data not shown).
1 Represses PMA-induced Expression from the MMP-1
Promoter--
It has been previously reported that TGF-
1 represses
the expression of MMP-1 when induced by a variety of factors
(e.g. PMA, interleukin-1, and tumor necrosis factor-
) (9,
10). In order to reiterate this in our model system, a construct
containing
517/+60 of the human MMP-1 promoter in a
luciferase vector was transiently transfected into both murine Swiss
3T3 cells and primary human skin fibroblasts. In both cases, PMA
potently induces expression from this construct, and TGF-
1 represses
this induction although, at the doses used, not back to control levels
(see Fig. 5, A and B). TGF-
1 alone does not significantly repress basal
expression of MMP-1. These data agree with data published in
other cell lines, and we have confirmed that the same response is seen
with either 8- or 24-h stimulation (data not shown). It should be noted
that TGF-
1 does not repress PMA-induced expression from the
MMP-1 promoter in C3H10T1/2 cells.
View larger version (21K):
[in a new window]
Fig. 5.
Mutational analysis of the MMP-1
promoter in response to PMA and TGF- .
A, a series of MMP-1 promoter constructs in
pGL3-basic were transiently transfected into human skin fibroblasts.
Cells were serum-starved for 24 h and then stimulated with TGF-
(2 ng/ml), PMA (10
7 M), or both together.
Cell lysates were harvested at t = 8 h and assayed
for luciferase activity. Data are expressed as -fold induction above
the control in each case, mean and S.E. (n = 3).
B, functionally inactive mutations in either the
72 AP1
site, the
245 TIE, or both were made in the context of
517/+60
MMP-1 and compared with wild type under identical assay
conditions as A.
153/+60, shows an
identical pattern of response to
517/+60. This suggests that the
putative TIE at
245 is not involved in the repression of
PMA-induced MMP-1 expression. A construct of
80/+60,
containing the proximal AP1 site at
72, gives very low levels of
expression, but the TGF-
1 repression of PMA-induced expression is
still apparent; therefore, elements within the
80/+60 region must be
responsible for this effect of TGF-
1 (Fig. 5A).
517/+60 Confirm the Role of the AP1
Site in TGF-
1-mediated Repression of MMP-1--
Since previous data
suggest that in MMP-1 promoter constructs extending further
5' than
321 (18), the AP1 site at
72 may contribute less to
expression of the transgene, functionally inactivating point mutations
in both TIE and AP1 motifs were made in the context of
517/+60. Fig.
5B shows that mutation of the TIE does not prevent TGF-
1
repression of PMA-induced expression. However, the inactivating mutation in the AP1 motif, in the presence or absence of the TIE mutation, actually leads to a further induction of PMA-induced gene
expression by TGF-
1. Interestingly, the TIE mutation increases absolute levels of expression, whereas the AP1 mutation decreases absolute levels. Exchanging the consensus AP1 sequence in
517/+60 for
the Timp-1 AP1 sequence does not alter the pattern of
expression; moreover, transient transfection of a
95/+47
Timp-1 construct containing the MMP-1 AP1 site
into C3H10T1/2 or Swiss 3T3 cells did not alter the pattern of TGF-
1
and PMA induction seen in the same construct containing the wild-type
Timp-1 AP1 motif (data not shown).
1
Induction of Timp-1 Promoter--
In order to probe the role of the
Smad signaling pathway in the response of the Timp-1 gene to
TGF-
1, expression vectors for Smads 2, 3, 4, and 7 were
co-transfected into C3H10T1/2 with either the
95/+47
Timp-1 promoter construct or the Smad-responsive 3TP-Lux,
using empty vector as a control. Cells were then stimulated for 6 h with TGF-
1. Fig. 6A shows
that the 3TP-Lux construct behaves in a Smad-responsive manner as
expected; TGF-
1 induces expression of luciferase, and this is
further induced by the addition of either Smad 2, 3, or 4 alone.
Combinations of these Smads yield even higher levels of
expression. The Smad dependence of this response is underlined by
co-transfection of the inhibitory Smad 7, which potently blocks
TGF-
1 induction of 3TP-Lux. In comparison with this, TGF-
1
induction of the Timp-1 promoter is not potentiated by Smad
2, 3, or 4 alone, with Smads 3 and 4 acting in a repressive fashion;
combinations of Smads 2 and either 3 or 4 have no effect, whereas Smads
3 and 4 or Smads 2, 3, and 4 potently repress TGF-
1 stimulation of
the Timp-1 construct (Fig. 5B). Furthermore, Smad 7 does not repress TGF-
1-stimulated Timp-1 expression.
Together, these data suggest that the response of the Timp-1
gene to TGF-
1 is not Smad-dependent.
View larger version (21K):
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Fig. 6.
Co-transfection of p3TP-Lux, 95/+47
Timp-1, or
517/+60 MMP-1 reporter
with Smad family expression constructs. A, and
B, C3H10T1/2 cells were co-transfected with 1 µg of
95/+47 Timp-1 in pGL2-basic or p3TP-Lux in combination
with 0.33 µg of Smad family expression vectors or empty vector to
total 2 µg of DNA/plate. Cells were serum-starved for 24 h and
then stimulated with TGF-
(2 ng/ml). Cell lysates were harvested at
t = 6 h and assayed for luciferase activity. Data
are expressed as -fold induction above the control (empty vector only),
mean and S.E. (n = 3). C, human skin
fibroblasts were co-transfected with 1 µg of
517/+60
MMP-1 in pGL3-basic or 3TP-Lux in combination with 1 µg of
Smad7 expression construct or empty vector. Cells were serum-starved
for 24 h and then stimulated with TGF-
(2 ng/ml), PMA
(10
7 M), or both together. Cell lysates were
harvested at t = 8 h and assayed for luciferase
activity. Data are expressed as -fold induction above the control
(empty vector only), mean and S.E. (n = 3).
517/+60 MMP-1
construct blocks the TGF-
1-mediated repression of PMA-induced luciferase expression from the MMP-1 promoter (Fig.
6C). This suggests that the effect of TGF-
1 on the
MMP-1 gene is Smad-dependent.
1 in Smad Knockout Cell
Lines--
In our hands, Smad knockout cell lines and their wild-type
partner lines proved difficult to transfect reproducibly. Hence, Smad
2, Smad 3, or Smad 4 knockout cells and their wild-type partners were
stimulated with TGF-
1, PMA, or both together, and expression of the
endogenous Timp-1 gene was assessed by quantitative reverse transcription-PCR using the Taqman system. All three knockout cell
lines retain some TGF-
1 inducibility, although this is at a reduced
level compared with wild types (data not shown). All three knockout
cell lines also retain the synergism in Timp-1 induction
with PMA and TGF-
1 together. Interpretation of these data is clouded
by the fact that wild-type cell lines show wide variation in their
response to TGF-
1 in both these experiments and, for example, in
Fig. 4.
1-mediated Repression
of the MMP-1 Promoter but Not Induction of the Timp-1 Promoter--
In
order to reinforce the overexpression data above, cells were
co-transfected with the
517/+60 MMP-1 promoter or
95/+47 Timp-1 promoter construct with either the Smad-binding
oligonucleotide, S4BE, containing three Smad-binding sites, or a mutant
oligonucleotide S4BEmut, where the Smad-binding sites were functionally
mutated. Binding of Smads to S4BE and the absence of binding to the
S4BEmut were demonstrated using EMSA (see Fig. 8; data not shown). Fig. 7 shows that wild-type oligonucleotide
does not alter the pattern of TGF-
1 or PMA induction of the
Timp-1 promoter. Conversely, co-transfection of S4BE blocks
TGF-
1-mediated repression of PMA-induced MMP-1
expression, whereas the S4BEmut has no effect. Again, this suggests
that whereas Smads do mediate TGF-
1 repression of the MMP-1 gene, they are not involved in TGF-
1 induction of
the Timp-1 gene.
View larger version (24K):
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Fig. 7.
Co-transfection of 95/+47 Timp-1
or
517/+60 MMP-1 reporter with Smad-binding
oligonucleotides. A, C3H10T1/2 cells were
co-transfected with 1 µg of
95/+47 Timp-1 in pGL2-basic
in combination with 1 µg of the Smad-binding oligonucleotide S4BE.
Cells were serum-starved for 24 h and then stimulated with TGF-
(2 ng/ml), PMA (10
7 M), or both together.
Cell lysates were harvested at t = 6 h and assayed
for luciferase activity. Data are expressed as -fold induction above
the control (empty vector only), mean and S.E. (n = 3).
B, human skin fibroblasts were co-transfected with 1 µg of
517/+60 MMP-1 in pGL3-basic in combination with 1 µg of
either S4BE or the mutant oligonucleotide, which does not bind Smads
(mS4BE). Cells were serum-starved for 24 h and then stimulated
with TGF-
(2 ng/ml), PMA (10
7 M), or both
together. Cell lysates were harvested at t = 8 h
and assayed for luciferase activity. Data are expressed as -fold
induction above the control (empty vector only), mean and S.E.
(n = 3).
View larger version (94K):
[in a new window]
Fig. 8.
Electrophoretic mobility shift assay on the
S4BE oligonucleotide. C3H10T1/2 murine fibroblasts were
serum-starved for 24 h and then stimulated with TGF- (2 ng/ml),
or TGF-
and PMA (10
7 M) together. Nuclear
extracts were harvested at t = 3 h. EMSA was
performed on the S4BE oligonucleotide in the absence or presence of
antibodies to Smad2/3 or Smad4 (2 µg per binding reaction).
T, TGF-
; TP, TGF-
+ PMA. Shift
(A-E) and supershift complexes are marked.
View larger version (44K):
[in a new window]
Fig. 9.
Electrophoretic mobility shift assay on the
S4BE oligonucleotide: competition with MMP-1 or
Timp-1 AP1 oligonucleotides. C3H10T1/2 murine
fibroblasts were serum-starved for 24 h and then stimulated with
TGF- (2 ng/ml). Nuclear extracts were harvested at t = 3 h. EMSA was performed on the S4BE oligonucleotide in the
absence or presence of a 100× excess of cold S4BE, Timp-1
AP1, or MMP-1 AP1 oligonucleotide. Shifted complexes are
labeled A-E as in Fig. 8.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
has profound effects on extracellular matrix homeostasis,
in part via its ability to alter the balance between proteinases and
their inhibitors at the level of gene expression (7, 8). The
intracellular signaling pathways by which TGF-
mediates its actions
are diverse. The Smad pathway, specific to the TGF-
family, is
probably of prime importance, but many reports implicate other pathways
(e.g. mitogen-activated protein kinase pathways (14, 15));
furthermore, cross-talk between the Smads and other signaling pathways
modifies the TGF-
response (16-18). The reciprocal effect of
TGF-
on the expression of TIMP-1 and MMP-1
initially described by Edwards et al. (11) supports its role
in matrix anabolism. The mechanisms by which TGF-
induces
Timp-1 yet represses induced MMP-1 have remained
opaque; hence, the current study sought to address this, with a focus
on Timp-1 gene expression.
-induction of the
Timp-1 gene were ascertained using deletion mutants,
demonstrating that the proximal (
59/
53) AP1 site plays a major
role. Internal substitutions across
50 to +47, leaving the AP1 site
intact in the context of
223/+47 Timp-1, failed to
demonstrate further elements necessary for TGF-
induction (data not
shown). This was reinforced by EMSA on overlapping oligonucleotides
across
50 to +47, which showed no altered protein-DNA interactions.
EMSA on an oligonucleotide containing the AP1 sequence demonstrated an
increase in binding upon either TGF-
or PMA stimulation with the
only significant difference between the two being the induction of
Fra-1-containing complexes by PMA but not TGF-
. Analysis of the
contribution of Fos and Jun family members using both overexpression
and cell lines from AP1 knockout mice revealed a requirement for c-Fos, c-Jun, and JunD in TGF-
induction of Timp-1, whereas
c-Fos was not essential for PMA induction of the gene. Smad
co-expression experiments show that the Timp-1 gene is not
Smad-responsive; nor is TGF-
-induction of the gene blocked by the
inhibitory Smad 7. These facets are shown very clearly in the control
plasmid 3TP-Lux. Finally, co-transfection of a Smad-binding
oligonucleotide has no effect on TGF-
induction of the
Timp-1 gene. From all of these data, it can be concluded
that the induction of Timp-1 gene expression by TGF-
1 is
AP1- but not Smad-dependent. This firm conclusion must be
tempered by the data coming from Smad knockout cells, where induction
of Timp-1 is still evident, but its magnitude is reduced,
compared with wild-type cells. However, it should be reiterated that
the magnitude of response to TGF-
1 varies among wild-type cell lines
and also that the absence of Smads may have secondary consequences to
cellular function that are separate from direct effects on
Timp-1 expression.
induces a
greater than 2-fold induction of the gene within 30 min as assessed by
cDNA microarray analysis, and this is not blocked by the protein
synthesis inhibitor cycloheximide or the c-Jun N-terminal kinase
inhibitor curcumin; (ii) a Timp-1 promoter construct driving
CAT expression is induced by TGF-
at a 24-h time point, and this is
blocked by dominant negative Smad 3 or by Smad 7; and (iii)
co-expression of Smad 3 with the Timp-1 promoter construct
mimics the effect of TGF-
, and no promoter transactivation is seen
in Smad 3
/
cells. There are many differences in these data
compared with the current study. (i) The microarray data in
Verrecchia et al. (31) shows the TGF-
-induction of Timp-1 gene expression maximal at 30-60 min, remaining at
this level at 4 h. In our hands, in both murine fibroblasts and
human fibroblasts (dermal or lung), TGF-
induction of
Timp-1 becomes maximal at 12-24 h, as assessed by Northern
blot (12).2 (ii) The use of cycloheximide as a protein
synthesis inhibitor in such studies is problematic, since it has
been shown to augment the induction of immediate early genes such as
c-fos and c-jun by growth factors (38) via the
p38 mitogen-activated protein kinase pathway (39); indeed, Verrecchia
et al. (31) state that cycloheximide treatment causes "a
broad increase in gene expression ... " Using emetine as a
protein synthesis inhibitor, the induction of Timp-1 by
TGF-
is dependent on new protein synthesis (data not
shown).2 (iii) Inhibitors of the mitogen-activated protein
kinase pathways such as curcumin (c-Jun N-terminal kinase), U0126
(extracellular signal-regulated kinase), or SB202190 (p38) all block
the TGF-
induction of Timp-1 to varying extents in the
C3H10T1/2 cells used in the current study (data not shown). (iv) The
promoter studies in Verrecchia et al. (31) use an
undisclosed length of murine promoter driving CAT expression in human
cells measured at a 24-h time point with no serum starvation prior to
TGF-
treatment (Timp-1 is serum-responsive (40)); the
studies above use constructs driving luciferase expression measured at
a 6-h time point to avoid potential secondary responses of the cells to
TGF-
-induced growth factors. (v) Smad 3
/
cells, in our hands,
were difficult to transfect; however, as discussed above, the
endogenous Timp-1 gene is these cells is still induced by
TGF-
, albeit at a reduced level.
-induced cell nuclear
extracts and can bind the Timp-1 AP1 sequence on EMSA
(presuming the specificity of antibodies used in supershift
experiments). An overall increase in AP1 binding is observed upon
TGF-
treatment compared with control. Whereas c-Fos, c-Jun, and JunD
were shown to be necessary for Timp-1 induction using
knockout cell lines, JunB and Fra2 have not been assessed in this
manner, since cells were not available. It is possible that members of
the cAMP-response element-binding protein family of transcription
factors, which can heterodimerize with AP1 factors, are also important
in the response of Timp-1 to TGF-
; indeed, ATF2 is a
target of TGF-
signaling via both the Smad pathway and TAK1/p38
(43).
; this suggests either that flanking sequence around
the AP1 site is important, as suggested by Zhang et al.
(27), and/or that interactions with other transcription factors binding
at distal sites are involved.
represses PMA-induced MMP-1 expression in human skin
fibroblasts. Our data on Timp-1 expression were gained using
the murine system, allowing us to use a well characterized cell line (C3H10T1/2) for TGF-
response, maintaining parity with our earlier studies and enabling us to exploit cell lines from knockout animals; however, the endogenous MMP-1 gene in mice is controlled
very differently from that in humans (44). Hence, experiments using MMP-1 promoter constructs were performed both in human skin
fibroblasts and in murine Swiss 3T3 cells, where the same pattern of
response was observed; TGF-
is unable to repress PMA-induced
expression from MMP-1 promoter constructs in C3H10T1/2 cells.
517/+60 MMP-1 promoter construct
demonstrates that TGF-
repression is maintained, even down to
80/+60; this suggests that the putative TIE at
245 is not necessary
for this response. This is reinforced by a point mutation in
517/+60, where mutation at the TIE has no functional consequence, but mutation in the AP1 site at
72 abrogates TGF-
-mediated repression. A previous report (9) using the rabbit MMP-1 promoter
conflicts with this, demonstrating that AP1 mutation has no effect,
whereas TIE mutation abrogates TGF-
repression. In agreement, both
groups demonstrate that AP1 mutation severely reduces the level of
transgene expression seen and that TIE mutation increases basal
expression. Potentially, this is a species difference (human
versus rabbit) or a cell type difference (dermal fibroblasts
versus synovial fibroblasts); the MMP-1 AP1 motif
binds at least c-Fos, JunD, and Fra2 from PMA-stimulated rabbit
synovial fibroblasts (22).
response of the
MMP-1 gene was demonstrated by Mauviel et al.
(45), whence TGF-
repressed MMP-1 expression in dermal
fibroblasts via JunB-containing AP1 complexes, whereas TGF-
induced
MMP-1 expression in epidermal keratinocytes via
c-Jun-containing AP1 complexes.
repression of
PMA-induced MMP-1 expression. Hence, our data suggest that the TGF-
-mediated repression of PMA-induced MMP-1
expression is Smad-dependent and mediated through the
promoter-proximal AP1 site.
repression of interleukin-1-induced MMP-1 expression. In this study, Smad3 overexpression mimics
the TGF-
-mediated repression of MMP-1, although this
appears to be mediated via competition between NF-
B and the
coactivator p300, using a cis-acting element distinct from the AP1
site; this difference is presumably due to the use of IL-1 to stimulate
MMP-1 compared with PMA used in the current study. The
current study suggests that the MMP-1 AP1 sequence competes
for Smad binding, and potentially, competition between Smads and AP1
for binding to this site could underlie TGF-
-mediated repression.
However, no evidence for this mode of action comes from the EMSA
studies above, and the increase in AP1 binding upon TGF-
treatment
suggests otherwise.
induces expression of the MMP-13 gene, and this is
at least partly via the AP1 site (5'-TGACTCA-3') (46) and an increase in c-Fos, c-Jun, and JunD binding. Furthermore, the MMP-13
gene cannot be induced by either epidermal growth factor or
platelet-derived growth factor in c-fos-deficient cells
(47). The group of Kahari (48, 49) has demonstrated that TGF-
induction of MMP-13 is dependent on both the p38
mitogen-activated protein kinase pathway and the Smad pathway.
to induce Timp-1 and
repress induced MMP-1 expression is dependent on a
promoter-proximal AP1 motif in each case. Contrary to the repression of
MMP-1, which is Smad-dependent, induction of
Timp-1 does not involve the "classical" Smad pathway;
however, data from Smad-deficient cell lines suggest some role for
Smads that may involve indirect effects on the repertoire of
transcription factors expressed by such cell lines. Future work will
focus on dissecting the signaling pathways that link TGF-
to the
Timp-1 gene.
![]() |
FOOTNOTES |
---|
* This work was supported by the Biotechnology and Biological Sciences Research Council UK.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.
Present address: Dept. of Growth and Development, University of
California, San Francisco, CA 94143.
§ Supported by the Arthritis Research Campaign UK.
Supported by the Norfolk and Norwich Big C Appeal.
** To whom correspondence should be addressed: School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom. Tel.: 44-1603-592760; Fax: 44-1603-592250; E-mail: i.clark@uea.ac.uk.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M212334200
2 M.-C. Hall, D. A. Young, J. G. Waters, A. D. Rowan, A. Chantry, D. R. Edwards, and I. M. Clark, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ECM, extracellular
matrix;
AP1, activator protein 1;
CAT, chloramphenicol
acetyltransferase;
EMSA, electrophoretic mobility shift assay;
MMP, matrix metalloproteinase;
PMA, phorbol 12-myristate 13-acetate;
TGF-, transforming growth factor-
;
TIE, TGF-
inhibitory
element;
TIMP, tissue inhibitor of metalloproteinases
(Timp-1, mouse gene;
TIMP-1, human gene).
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