 |
INTRODUCTION |
Stromelysin 1 (MMP-3) is a metalloproteinase capable of degrading
proteoglycans, fibronectin, laminin, and type IV collagen (1) and
activating procollagenase (2, 3). It is produced, along with
interstitial collagenase (MMP-1), by fibroblasts in response to
increased levels of cytokines (e.g. interleukin 1 (IL-1)1 and tumor necrosis
factor) in inflammatory diseases such as rheumatoid arthritis and
periodontitis (4, 5) and has been linked to joint and soft tissue
destruction associated with those diseases. Although the induction of
stromelysin by IL-1 occurs primarily at the transcriptional level (6,
7), the precise mechanisms involved are not yet fully understood.
Transcription factors AP-1 and PEA3/ets are believed to be involved in
the transcriptional regulation of stromelysin by a number of stimuli
(8-10). However, although AP-1 activity is necessary for basal
transcription, it is not sufficient for IL-1 induction of the gene in
normal fibroblasts (11-14), and 5' deletion studies suggest that other
factors further upstream are involved in determining the magnitude of
the induction (7, 15). In addition, Borden et al. (16) have
published a revised sequence of the stromelysin promoter region based
on 12 independently isolated genomic clones. Their work indicates that
the clone used in previous studies contained a 1-kilobase deletion and
an inversion at the 5' end. Thus, previous studies would have missed
any regulatory elements in the deleted portion and may have over- or
underestimated the importance of other elements by studying them in an
incorrect context.
Although 5' deletion analysis has been used successfully in the past,
there is some concern that such studies might fail to identify elements
and factors whose roles are more subtle and that transcription elements
are best studied in larger constructs that more closely resemble the
natural context (17, 18). To identify transcription factors and
cis elements potentially involved in the IL-1 induction of
stromelysin, the human stromelysin 5'-flanking region was screened by
EMSA for previously unidentified IL-1-induced DNA-binding complexes in
human fibroblasts. Here we report the identification of such a complex
and present evidence that it is a repressor of IL-1-induced expression
of the stromelysin gene.
 |
MATERIALS AND METHODS |
Cell Culture--
Human synovial fibroblasts are obtained from
the synovia of patients with osteoarthritis undergoing reconstructive
and restorative surgery, and gingival fibroblasts are obtained from
patients undergoing periodontal surgery or surgical removal of third
molars. Tissue samples are processed by enzymatic dispersion to produce
primary cultures as described previously (16, 19, 20). These cultures are maintained in Eagle's minimal essential medium supplemented with
10% fetal bovine serum and antibiotic/antimycotic (penicillin, streptomycin, amphotericin) (Life Technologies, Inc.). Cells in passages 5-8 were used in experiments. Cultures were serum-deprived for 16 h in serum-free Eagle's minimum essential medium
supplemented with 10% insulin, transferrin, and sodium selenite
(Sigma) prior to stimulation with 100 ng of IL-1
/ml (a generous gift
of Robert Newton, DuPont-Merck Pharmaceutical Co.). Human foreskin
fibroblasts (HFF) (ATCC) were maintained and treated in the same manner
described above. Cells in passages 5-10 were used in transfection experiments.
EMSA--
Nuclear extracts were isolated from synovial,
gingival, or human foreskin fibroblast cultures at various times after
stimulation with IL-1
(100 ng/ml), as well as from control cultures,
according to the method of Schreiber et al. (21). DNA
fragments used as probes for promoter dissection were generated by PCR
according to standard protocols (primer 1, 5'-CACTGCCACCACTCTGTTCTC-3'; primer 2, 5'-TTCTATGGTTCTCCATTCCTT-3'). This DNA fragment as well as
the complementary oligodeoxynucleotide pairs (4, 5'-ACAAGACATGGTTTTTTCCCCCCATCAAAG-3'; 4B (SIRE),
5'-GTTTTTTCCCCCCATCAAAG-3') were end-labeled using polynucleotide kinase and [
-32P]dATP. Binding
reactions contained 5 or 10 µg of protein, 20 mM
Hepes-OH, pH 7, 50 mM NaCl, 0.2 M EDTA, 5%
glycerol, 4 µg of poly(dI-dC), and a 10,000 cpm probe. Samples were
electrophoresed on native 5% polyacrylamide gel electrophoresis in
0.5× TBE (44.5 mM Tris-Cl, 50 mM boric acid, 3 mM EDTA).
UV Cross-linking--
An oligonucleotide probe corresponding to
the SIRE binding site was labeled with bromodeoxyuridine and
32P. Binding reactions containing 40 µg of gingival
nuclear extract, were isolated 1 h after stimulation with 100 ng
of IL-1
/ml in the presence or absence of 100 µg/ml cycloheximide
and were exposed to UV light (Macrovue Transilluminator, LKB) for 15 min before being electrophoresed in 10% SDS-polyacrylamide gel
electrophoresis beside prestained molecular mass markers.
Site-directed Mutagenesis and Transient Transfection--
A
fragment of human stromelysin 5'-flanking region corresponding to the
sequences published by Borden et al. (16) was a gift from
Dinesh Tewari, Temple University, Philadelphia. A 2050-base pair
fragment was subcloned first into pBluescript SK
, then into the
pGL3Basic luciferase reporter vector (KpnI/AvaI)
(Promega). The
Sac construct was made by excising the
KpnI/SacI fragment containing the binding site of
interest filling in the ends and re-ligating. The binding site of
interest was altered by site-directed mutagenesis using the four primer
PCR method (22) and mutant oligonucleotides (primer 1, 5'-GGTACAAGGTACCATTGA-3'; primer 2, 5'-CTTTGATGGTGAGAAAAAA-3'; primer 3, 5'-GTTTTTTCTCACCATCAAAG-3'; primer 4, 5'-CACAGGTGATAGCCACTTG-3'). The resulting fragment was digested with
KpnI and SacI and used to replace the wild-type KpnI/SacI fragment in pGLStro. Each of these
stromelysin constructs was transiently transfected into HFF cells along
with a
-galactosidase gene under control of the SV40 promoter
(SV
Gal) (Promega) as a control for transfection efficiency.
Transfection was accomplished in triplicate samples using 20 µg of
Lipofectin (Life Technologies, Inc.) in serum-free Eagle's minimum
essential medium. Approximately 16 h after transfection, the
transfection medium was removed and replaced with serum-free Eagle's
minimum essential medium supplemented with 10% insulin, transferrin,
and sodium selenite, along with 100 ng of IL-1
/ml where appropriate.
Cells were harvested 24 h later, and luciferase and
-galactosidase activity were determined using reagents and protocols
from Promega and CLONTECH, respectively. Luciferase
values were normalized to levels of
-galactosidase. Data shown are
from three independent experiments done in triplicate.
 |
RESULTS |
Identification and Localization of IL-1-induced DNA Binding
Activity--
A 2-kilobase fragment of the human stromelysin
5'-flanking region was subcloned into pBluescript SK
(Stratagene).
Because the proximal promoter has already been well studied, the region upstream of
480 starting from the 5' end at
2000 was screened for
IL-1-induced DNA-binding proteins. 100-base pair fragments were
generated by PCR and screened by EMSA using nuclear extracts isolated
from synovial and gingival fibroblast cultures treated with 100 ng of
IL-1
/ml. One of these fragments,
1693 to
1575, was selected for
further study because of the strength and reproducibility of the
IL-1-induced binding. Two DNA-binding complexes were observed, which
were both increased by treatment with IL-1. The more slowly migrating
complex 1 was absent in the control, induced within 30 min, and
maintained for at least 12 h. The smaller complex 2 was often
present in the control but further increased at 3, 6, and 12 h
after the addition of IL-1 (Fig. 1). The
same binding pattern was observed with multiple sets of nuclear extract
isolated from both synovial and gingival fibroblasts, although the
presence of complex 2 in control samples was somewhat variable.

View larger version (107K):
[in this window]
[in a new window]
|
Fig. 1.
IL-1 induced binding to the region 1693 to
1575 of stromelysin 1. The region from 1693 to 1575 of the
stromelysin 5'-flanking region was amplified by PCR and end-labeled
with 32P. This probe was then incubated with nuclear
extracts (10 µg) isolated from synovial fibroblast cultures at the
indicated times after addition of IL-1 (100 ng/ml). Similar results
were obtained with extracts isolated from three different individuals
and also with extracts isolated from gingival fibroblasts from three
different individuals. P, probe alone, no extract;
C, control extract, no IL-1.
|
|
To determine where on the 100-base pair fragment the IL-1-induced
binding occurred, overlapping sets of oligodeoxynucleotides were
synthesized, as shown in Fig.
2A, and used as probes in
additional EMSA experiments. Only fragment 4 (
1624 to
1595) showed
a pattern of IL-1-induced binding similar to that observed with the
larger, PCR-generated probe. IL-1-induced binding to this fragment was specific as demonstrated by the fact that it could be easily competed by the autologous fragment 4 but was not competed with even 100-fold excess of fragment 5 (Fig. 2B). Fragment 4 was further
divided into smaller fragments, 4A and 4B. Again, the IL-1-induced
pattern of binding originally observed with the 100-base pair fragment was seen only with fragment 4B (
1614 to
1595) (Fig.
3).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2.
Identification of SIRE at 1614 to
1595. A, schematic for isolation of SIRE. Overlapping
sets of oligodeoxynucleotide probes were made encompassing the region
1693 to 1575. The pattern of IL-1-induced binding to probe 4 and
finally to 4B was identical to that seen with the larger probe.
B, 10 µg of nuclear extract isolated from synovial
fibroblasts treated for 6 h with IL-1 were incubated with
32P-labeled probe 4. Binding was competed with indicated
molar excess of the unlabeled probe 4 but not by a 100-fold excess of
probe 5. Identical results were obtained with extracts isolated from
gingival fibroblasts. P, probe alone, no extract.
|
|

View larger version (89K):
[in this window]
[in a new window]
|
Fig. 3.
Mutation of two nucleotides in probe 4B
abolishes DNA binding. The same nuclear extract (5 µg) isolated
from human gingival fibroblasts at the indicated times after
stimulation with IL-1 was incubated with both wild-type
(5'-GTTTTTTCCCCCCATCAAAG-3') and mutant
(5'-GTTTTTTCTCACCATCAAAG-3')
32P-labeled oligonucleotide probes as shown. P,
probe alone, no extract; C, control extract, no IL-1.
|
|
Mutation of Two Nucleotides Abolishes Binding--
Analysis of the
sequence comprising fragment 4B revealed an unusual sequence containing
5 or 6 Ts (our clone has 5 Ts, whereas the published sequence (7, 16)
has 6) followed by 6 Cs. To determine whether or not the Cs were
involved in the IL-1-induced binding, a mutant probe was synthesized in
which two of the Cs were altered. As shown in Fig. 3, this mutation
abrogated the IL-1-induced binding, suggesting that at least one of the
mutated Cs is essential for binding. This binding site was designated SIRE.
Induction of SIRE DNA Binding Activity by Other Cytokines--
As
a first step in further characterization of DNA binding to the SIRE
site, the ability of cytokines other than IL-1 to induce binding was
investigated. As shown in Fig. 4, tumor
necrosis factor
also induces binding to this site, but the pattern
of binding is different from that of IL-1; the smaller complex 2 is
much more prominent than complex 1 at 6 h. Neither
platelet-derived growth factor nor IL-4 was able to induce either
complex. Although IL-4 has been shown to suppress some of the
activities of IL-1 (23-26), IL-4 did not inhibit the IL-1 induction of
binding to the SIRE site when added simultaneously with IL-1.

View larger version (77K):
[in this window]
[in a new window]
|
Fig. 4.
Binding to SIRE is also induced by tumor
necrosis factor but not by platelet-derived growth factor or IL-4.
Nuclear extracts (5 µg) isolated 6 h after stimulation of
gingival fibroblast cultures with the indicated cytokine (10 µg/ml)
were incubated with a 32P-labeled probe containing the
wild-type SIRE region. Similar results were observed with extracts
isolated from synovial fibroblasts. P, probe alone, no
extract; C, control extract, no cytokine; TNF,
tumor necrosis factor; PDGF, platelet-derived growth
factor.
|
|
De Novo Protein Synthesis Is Not Required for IL-1-induced Binding
to the SIRE Site--
To begin to characterize the protein(s) binding
to the SIRE site, gingival fibroblasts were stimulated with IL-1 in the
presence and absence of cycloheximide prior to isolation of nuclear
extracts. Results of EMSA analysis, shown in Fig.
5, show a slight increase in the
intensity of the larger complex 1 at 1, 3, and 6 h. The intensity
of the smaller complex 2 was unaffected at 1 h but decreased at 3 and 6 h. Interestingly, there appeared to be a slight increase in
the mobility of complex 2 in extracts exposed to cycloheximide. These
results suggest that de novo protein synthesis is not
required for binding to the SIRE site and that the two complexes are
regulated independently of each other.

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 5.
De novo protein synthesis is not
required for IL-1-induced binding to the SIRE site. 5 µg of
nuclear extract isolated at the indicated hours after stimulation with
IL-1 (100 ng/ml) in the presence or absence of 100 µg/ml
cycloheximide (CHX) were incubated with the
32P-labeled probe containing the SIRE binding site.
P, probe alone, no extract.
|
|
UV Cross-linking of Two Proteins to the SIRE Site--
UV
cross-linking experiments were undertaken to begin to characterize the
protein composition of the IL-1-induced binding to the SIRE site and to
determine whether the altered migration of complex 2 in the presence of
cycloheximide was more likely because of a different protein
composition or potential differences in post-translational processing.
The SIRE probe was tagged with bromodeoxyuridine and labeled with
32P before incubation with nuclear extracts isolated from
gingival fibroblasts induced with IL-1 in the presence and absence of
cycloheximide. Following exposure to UV irradiation, the reactions were
then separated on SDS-polyacrylamide gel electrophoresis beside
molecular mass standards. Results, shown in Fig.
6, show that there are at least two
proteins binding to the SIRE site, of approximately 48 and 52 kDa, and
that the same two proteins are observed in the presence of
cycloheximide.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 6.
UV cross-linking of two proteins to the SIRE
site. 40 µg of nuclear extract isolated from gingival fibroblast
cultures 1 h after IL-1 stimulation in the presence and absence of
cycloheximide (CHX) were incubated in EMSA with a
bromodeoxyuridine-tagged, 32P-labeled probe corresponding
to the SIRE site. The binding reaction was exposed to UV irradiation
for 15 min before electrophoresis on 10% SDS-polyacrylamide gel
electrophoresis along with molecular mass standards.
|
|
Mutation of the SIRE Site Increases IL-1-induced
Transcription--
Transient transfection experiments were conducted
to determine what role, if any, the SIRE site and its binding
protein(s) play in the IL-1 induction of stromelysin gene expression.
The entire 2-kilobase fragment was subcloned into the pGL3Basic
luciferase reporter (Promega) as a wild-type construct, and a deletion
mutant (
Sac) was constructed by deleting stromelysin sequences
upstream of the SacI site at
1477. Because of poor
transfection efficiency in synovial and gingival fibroblasts, transient
transfection experiments were done in HFF cells, which showed an
identical pattern of IL-1-induced binding to the SIRE probe (Fig.
7). The luciferase constructs were
co-transfected along with SV
gal as a control for transfection efficiency. Interestingly, the
Sac construct consistently
showed a higher IL-1 induction than the wild-type construct (Fig.
8), suggesting that the deleted sequences
may contain a repressor element.

View larger version (99K):
[in this window]
[in a new window]
|
Fig. 7.
IL-1 induced binding to the SIRE site in HFF
cells. Five µg of nuclear extract isolated from HFF cells at the
indicated times after stimulation with IL-1 were incubated with the
32P-labeled probe corresponding to the SIRE binding site.
P, probe alone, no extract; C, control extract,
no IL-1.
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 8.
Site-directed mutagenesis of the SIRE site
increases IL-1-induced transcription from the stromelysin
promoter. Subconfluent cultures of human foreskin fibroblasts were
co-transfected with SV -gal along with a stromelysin luciferase
reporter construct. pGLStro contains a 2-kilobase fragment of the
wild-type stromelysin promoter; pGL Sac is a deletion
mutant missing the SIRE region, and pGLmStro is a mutant construct with
the SIRE region altered as shown in Fig. 3. Results are from three
independent experiments performed in triplicate and normalized with
-galactosidase for transfection efficiency and expressed as fold
IL-1 induction. Basal activities were similar for all three
constructs.
|
|
To verify these results and to determine more precisely the role of the
SIRE site in its natural context, the wild-type construct was altered
by site-directed mutagenesis to reflect the sequence of the mutation
already shown to abrogate binding in EMSA. Results, shown in Fig. 8,
were consistent with those obtained with the deletion mutant showing an
IL-1 induction approximately twice that of the wild-type construct.
Results are from three independent experiments performed in triplicate,
and the basal activities of all three constructs were very similar.
 |
DISCUSSION |
The stromelysin 1 gene encodes a metalloproteinase with broad
substrate specificity that plays an important role in tissue remodeling
and wound repair (1, 27-29). However, in chronic inflammatory
conditions such as rheumatoid arthritis and periodontitis, it is found
in abnormally high levels and has therefore been linked to the joint
and tissue destruction associated with these diseases (28, 30). A
better understanding of the mechanisms involved in the regulation of
stromelysin expression by inflammatory cytokines may provide new
insight into potential therapies aimed at limiting the tissue
destruction of chronic inflammation.
The data presented here identify an IL-1-inducible repressor of
stromelysin gene expression. Site-directed mutagenesis of the binding
site resulted in 2-fold greater induction of the mutant construct as
compared with the wild-type construct in HFF cells. UV cross-linking
suggests the presence of at least two proteins, and results of
cycloheximide experiments suggest that the two binding complexes are
regulated independently of each other. The composition of the binding
complexes was not determined (i.e. two monomers
versus homo- or heterodimers); however, the results so far
are consistent with two monomers binding separately.
Although the physiological significance of this repressor is unknown,
it seems likely that repressor binding and/or activity may also be
modified by other cytokines or growth factors in vivo. In
fact, binding to the SIRE site is also enhanced by tumor necrosis factor albeit with a somewhat different pattern of complexes. The
presence of such a repressor element might therefore serve as a
mechanism to "fine tune" the levels of a gene product with a
critical but potentially destructive function in tissue remodeling preventing its overexpression. Interestingly, although IL-4 has been
shown to inhibit the IL-1 induction of stromelysin expression in
synovial fibroblasts (23), it does not appear to do so by inducing
binding to this repressor element.
It is important to note that this binding site has also been identified
as the site of a common genetic polymorphism. Ye et al. (31,
32) have reported that the presence of six Ts at this site rather than
five is associated with more rapid progression of atherosclerosis.
Furthermore, they presented evidence that this element acts as a
repressor element in human fibroblasts, because the transcriptional
activity of a 6T reporter construct was about one-half of a 5T
construct in the presence of serum. They hypothesize that the more
rapid disease progression is because of decreased expression of
stromelysin resulting in decreased degradation of the atherosclerotic plaques.
Our finding that binding to this element is induced by inflammatory
cytokines but not by the anti-inflammatory cytokine IL-4 or the mitogen
platelet-derived growth factor may have interesting implications for a
number of diseases involving both inflammation and tissue remodeling.
For example, it is tempting to speculate that individuals with the 5T
polymorphism might experience more rapid progression and/or increased
tissue destruction in chronic inflammatory diseases such as rheumatoid
arthritis and periodontitis because of increased expression of
stromelysin in response to inflammation. However, because mutation of
this site results in increased IL-1-induced transcriptional activity
compared with the 5T wild type, the site is likely to be active as a
repressor element even in individuals with the 5T polymorphism. Further study of this element and the proteins binding to it is necessary to
determine the in vivo significance of both the repressor and the polymorphism, as well as the feasibility of modifying disease progression by overexpression of these proteins.