Identification of a Cytokine-induced Repressor of Interleukin-1 Stimulated Expression of Stromelysin 1 (MMP-3)*

Ruth Carter BorghaeiDagger , Christine Sullivan, and Eugene Mochan

From the Department of Biochemistry/Molecular Biology, Philadelphia College of Osteopathic Medicine, Philadelphia, Pennsylvania 19131

    ABSTRACT
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Abstract
Introduction
References

Stromelysin 1 (MMP-3) is a matrix metalloproteinase with broad substrate specificity that has been linked to joint and tissue destruction associated with chronic inflammatory diseases such as rheumatoid arthritis and periodontitis. Transcription of the stromelysin gene is induced by inflammatory cytokines such as interleukin 1 (IL-1) and tumor necrosis factor as well as a number of other cytokines and mitogens, but the exact mechanisms involved in its regulation are not fully understood. 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 electrophoretic mobility shift assay for IL-1-induced DNA-binding complexes in human synovial and gingival fibroblasts. Here we report the identification of such a complex binding to the region -1614 to -1595 (5'-G(T)TTTTTCCCCCCATCAAAG-3') termed the stromelysin IL-1 responsive element site. Binding to this site is also induced by tumor necrosis factor but not by platelet-derived growth factor or interleukin 4. UV cross-linking demonstrates that there are at least two DNA-binding proteins involved, of approximately 48 and 52 kDa. Transient transfection experiments in human foreskin fibroblasts demonstrate that proteins binding to this site act as a repressor of IL-1-induced expression of the stromelysin gene.

    INTRODUCTION
Top
Abstract
Introduction
References

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-1beta /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-1beta (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 [gamma -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-1beta /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 Delta 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 beta -galactosidase gene under control of the SV40 promoter (SVbeta 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-1beta /ml where appropriate. Cells were harvested 24 h later, and luciferase and beta -galactosidase activity were determined using reagents and protocols from Promega and CLONTECH, respectively. Luciferase values were normalized to levels of beta -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-1beta /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.


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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).


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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.


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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 alpha  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.


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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.


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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-1beta (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.


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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 (Delta 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 SVbeta gal as a control for transfection efficiency. Interestingly, the Delta 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.


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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.


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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 SVbeta -gal along with a stromelysin luciferase reporter construct. pGLStro contains a 2-kilobase fragment of the wild-type stromelysin promoter; pGLDelta 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 beta -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.

    ACKNOWLEDGEMENTS

We thank Elizabeth Pease and Ruth Thornton for helpful discussions and P. Lyle Rawlings Jr., Jennifer Ross, Bill Laidlaw, and Billie Johnson for technical assistance.

    FOOTNOTES

* This work was supported by NIDR, National Institutes of Health Grants RO3 DE11577-01 and R29 DE12096-02 (to R. C. B.).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.

Dagger To whom correspondence should be addressed. Tel.: 215-871-6454; Fax: 215-871-6865; E-mail: ruthb{at}pcom.edu.

The abbreviations used are: IL, interleukin; EMSA, electrophoretic mobility shift assay; HFF, human foreskin fibroblasts; PCR, polymerase chain reaction; SIRE, stromelysin IL-1 responsive element.
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