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J Dent Res 86(4):352-356, 2007
© 2007 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Fibrillin-2 Degradation by Matrix Metalloproteinase-2 in Periodontium

E. Tsuruga*, K. Irie, and T. Yajima

Department of Oral Anatomy, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan

* corresponding author, tsuru{at}hoku-iryo-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Elastic system fibers, comprised of microfibrils and tropoelastin, are extracellular components of periodontal tissue. During development, the microfibrils act as a template on which tropoelastin is deposited. However, the process of elastic system fiber remodeling is not fully understood. Therefore, we examined whether matrix metalloproteinases (MMPs) are involved in the remodeling of fibrillins (major components of microfibrils) by human gingival fibroblasts and periodontal ligament (PDL) fibroblasts. Gingival and PDL fibroblasts were cultured for 6 weeks. In some cultures, MMP inhibitor or tissue inhibitor of matrix metalloproteinsase-2 (TIMP-2) was added to the medium for an additional 2 weeks. Active MMP-2 (62 kDa) appeared as cell-membrane-associated or in extracellular matrix only in PDL fibroblast cell layers. The addition of MMP inhibitor or TIMP-2 significantly increased fibrillin-2 accumulation in PDL fibroblast cell layers, and decreased the amount of fibrillin-2 fragments, suggesting that active MMP-2 may degrade fibrillin-2, and that MMPs may play a role in the remodeling of elastic system fibers in PDL.

KEY WORDS: degradation • fibrillin • periodontal ligaments • matrix metalloproteinases • remodeling


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Elastic system fibers are an integral part of the extracellular matrix (ECM), which is distributed in blood vessels, lung, skin, and periodontal tissue (Rosenbloom et al., 1993). These fibers are comprised of two distinct components: microfibrils and tropoelastin. During elastic fiber formation, microfibrils are thought to act as a template on which tropoelastin is deposited (Mecham and Davis, 1994; Kielty and Shuttleworth, 1995).

Fibrillin-1 and –2 are the principal structural components of microfibrils. We have biochemically investigated the metabolism of elastic system fibers by comparing human gingival fibroblasts and human periodontal ligament (PDL) fibroblasts (Tsuruga et al., 2002a,b,c, 2004), and demonstrated that expression of the fibrillin-2 gene is correlated with that of the tropoelastin gene (Tsuruga et al., 2002c), and that fibrillin-2 is required for the deposition of tropoelastin (Tsuruga et al., 2005). However, the mechanism involved in the remodeling of elastic system fibers is still unknown.

Turnover of the ECM, involving production, degradation, and replacement, is a fundamental process of homeostasis. The matrix metalloproteinases (MMPs) are a major group of enzymes responsible for degradation of the ECM. MMPs are secreted as latent forms (proMMPs), and require activation, possibly by cleavage of the N-terminal prodomain. MMP-1, –2, –3, –8, and –9 have been found in human inflammatory periodontal biopsy specimens (Birkedal-Hansen et al., 1993; Reynolds, 1996; Westerlund et al., 1996). MMP-2 knockout mice show no skeletal abnormalities (Itoh et al., 1997). Nevertheless, MMP-2 has been considered a major enzyme involved in the turnover of ECM (Creemers et al., 1998). Using in situ hybridization and immunohistochemistry, Tervahartiala et al.(2000) have demonstrated MMP-2 expression in healthy gingiva. It has been reported that healthy gingiva contains only proMMP-2 (Korostoff et al., 2000). However, there have been no reports of MMP-2 in specimens of human PDL.

In contrast, in vitro studies have shown that gingival and PDL fibroblasts express the MMP-2 gene (Bolcato-Bellemin et al., 2000), and that only proMMP-2, and not active MMP-2, is present in both cell layers and culture media (Choi et al., 2001). In the absence of inflammatory cytokines or periodontal bacteria, active MMP-2 has not been found in gingival and PDL fibroblasts cultured for short periods. Previously, we demonstrated the accumulation of microfibrils (fibrillin-1 and –2) and tropoelastin in cell layers after 6 wks of culture. Therefore, we hypothesized that a culture period of least 6 wks may be required for examination of the remodeling of elastic system fibers.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Culture
The protocol for these experiments was reviewed and approved by the Health Sciences University of Hokkaido Research Ethics Committee, and informed consent was obtained from the tissue donors.

Gingival and PDL fibroblasts were isolated from three different donors and cultured in minimum essential medium (MEM) as described previously (Tsuruga et al., 2002b). After 6 wks, the cultures were supplemented with 0–50 µM hydroxamic acid (MMPI) (INH-3850-PI; Peptides International, Louisville, KY, USA) (Ellerbroek et al., 2001), or 0–100 ng/mL TIMP-2 (Sigma Chemical Co., St. Louis, MO, USA). A stock solution of MMPI was dissolved in dimethyl sulfoxide (DMSO), and subsequently diluted with MEM. Control cultures contained an equivalent concentration of DMSO in MEM, instead of MMPI. The final concentration of DMSO did not exceed 0.2% (v/v).

Biotinylation of Cell Surface and Extracellular Proteins
Gingival and PDL fibroblasts were harvested by means of a cell-scraper, washed with MEM, and then collected by centrifugation at 500 x g for 30 min. These cells were then biotinylated by 40-minute incubation in phosphate-buffered saline (PBS) containing 0.5 mg/mL non-cell-permeable sulfo-NHS-biotin (Pierce Chemical Co., Rockford, IL, USA), with gentle shaking at 4°C. The reaction of sulfo-NHS-biotin was terminated by the addition of 100 mM glycine/PBS and washing with PBS.

Separation of Biotinylated and Non-biotinylated Proteins
After centrifugation, the biotinylated cell samples were solubilized in PBS containing 1% Triton X-100 (PBST), sonicated 6 times at 4°C for 10 sec, and then centrifuged at 40,000 x g for 15 min, and adjusted to a concentration of 500 µg of protein/200 µL. An aliquot (200 µL) of the supernatant was mixed with 30 µL of immobilized avidin-agarose beads (Sigma Chemical Co., St. Louis, MO, USA), which were pre-washed twice with PBST containing 2% ovalbumin. The mixture (200 µL of sample plus 30 µL of avidin beads) was incubated overnight at 4°C on a rotator, then centrifuged at 400 x g for 30 sec. The supernatant (100 µL) was taken as the avidin-unbound "intracellular fraction". The collected beads were washed twice with PBST, twice with 2 M guanidine HCl, and twice again with PBST, with the tube changed for the last spin. The adsorbed proteins were then eluted with 100 µL electrophoresis sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.001% bromophenol blue, 100 mM ß-mercaptoethanol), and taken as the "biotinylated fraction".

Extraction of Microfibrils from Cell Layers
Cell layers consist of cells and ECM. Microfibrils were extracted from gingival and PDL fibroblast cell layers cultured for 6 or 8 wks, as described previously (Tsuruga et al., 2002c), and taken as the "microfibril-containing fraction".

Fibrillin-2 Immunoprecipitation from the Microfibril-containing Fraction
The microfibril-containing fraction was subjected to lysis with RIPA buffer (Sigma, St. Louis, MO, USA), subjected to immunoprecipitation with anti-human fibrillin-2 polyclonal antibody (Elastin Products Co., Owensville, MO, USA), as described previously (Tsuruga et al., 2002a), and taken as the "immunoprecipitated fibrillin-2 fraction".

Western Blot Analyses
Western blot analyses were performed on 10-µg aliquots (i) taken prior to incubation with beads (total cell lysates), (ii) of the intracellular fraction, (iii) of the biotinylated fraction, (iv) of the microfibril-containing fraction, and (v) of the immunoprecipitated fibrillin-2 fraction, as described previously (Tsuruga et al., 2002a). The fractions of (i) were for MMP-2, membrane-type matrix metalloproteinases-1 (MT1-MMP) and TIMP-2 analyses, (ii) and (iii) were for MMP-2 analysis, that of (iv) was for fibrillin-1 and –2 analyses, and that of (v) was for fibrillin-2 analysis. The antibodies used were anti-human monoclonal MMP-2 antibody (Fuji Chemical Ind. Ltd., Toyama, Japan), anti-human fibrillin-1 and –2 polyclonal antibodies (Elastin Products Co., Inc.), and anti-human polyclonal MT1-MMP, TIMP-2, and actin antibodies (Sigma Chemical Co.).

Densitometric analysis of the signals was performed with the NIH Image program (National Institutes of Health, Bethesda, MD, USA). We used this program after finding the linear portion by sequential dilutions of the proteins.

Gelatin Zymography
Gelatinase activities in the conditioned media at 6 wks or in the MMPI-treated cell lysates at 8 wks were determined by SDS-polyacrylamide gel electrophoresis zymography. Cell lysates (10 µg) underwent electrophoresis without reduction on SDS-polyacrylamide gels prepared with 10% acrylamide containing 0.1% gelatin. The SDS was removed by a one-hour incubation in 2.5% Triton X-100, and the gels were then incubated in 30 mM Tris-HCl (pH 7.4), 200 mM NaCl, 5 mM CaCl2, and 1 mM ZnCl2, at 37°C for 24 hrs prior to being stained with Coomassie Blue. Enzyme activity was visualized as zones of gelatin clearance within the gels.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Presence of Active MMP-2 in PDL Fibroblast Cell Layers
We first examined the MMP-2 that had accumulated in the gingival and PDL fibroblast cell layers. Immunoblotting of both gingival and PDL fibroblast cell layers at 6 wks detected the 72-kDa proMMP-2 (Fig. 1AGo). The 62-kDa active MMP-2 was observed only in PDL fibroblasts. In both cell layers, active MT1-MMP and TIMP-2 were present at the same level. Next, we used zymography to detect MMP-2 in the culture media. At 6 wks of culture, 2.5 times more proMMP-2 was detected in PDL fibroblast culture media than in gingival fibroblasts (Fig. 1CGo).


Figure 1
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  		Figure 1. Active MMP-2 is present only in human periodontal ligament (PDL) fibroblast cell layers. (A) Immunoblotting of MMP-2, membrane-type matrix metalloproteinases-1 (MT1-MMP), and tissue inhibitor of matrix metalloproteinsase-2 (TIMP-2). Gingival and PDL fibroblasts were cultured for 6 wks. A 10-µg quantity of cell lysate underwent electrophoresis on 10% polyacrylamide gels and was immunoblotted for MMP-2, MT1-MMP, and TIMP-2 analyses. Equivalent protein loading was confirmed by ß-actin. (B) MMP immunoreactivity in biotinylated and intracellular fractions of PDL fibroblasts. PDL fibroblasts were cultured for 6 wks. Cell-surface and extracellular proteins were then biotinylated with non-cell-permeable biotin, and the biotinylated and intracellular fractions were immunoblotted with MMP-2 (upper panel) and actin (lower panel). (C) MMP-2 in the culture media. Serum-free culture media were collected from gingival and PDL fibroblasts at 6 wks. The harvested media (10 µg) were subjected to gelatin zymography.

 
Lee et al.(1997) have reported that some fibroblasts activate MMP-2 intracellularly. Therefore, to examine whether or not active MMP-2 was present intracellularly, we performed biotinylation experiments. The biotinylation reagent we used was a non-cell-permeable type, which ensured that only the cell membrane and extracellular matrices, and not intracellular proteins, were biotinylated. The intracellular cytoskeletal protein actin was not labeled by the biotinylation reagent, confirming that cell-surface and extracellular proteins were being labeled (Fig. 1BGo). The 62-kDa active form of MMP-2 was present in the biotinylated fraction, and not in the intracellular fraction.

Inhibition of Active MMP-2 in PDL Fibroblast Cell Layers by MMPI or TIMP-2
To block the active MMP-2 present in PDL fibroblast cell layers, we added various concentrations of MMPI (0, 5, 50 µM) or TIMP-2 (0, 50, 100 ng/mL) to the medium during weeks 6 to 8 of incubation. Zymography of PDL fibroblast cell lysates treated with MMPI indicated that a concentration of 5 µM of MMPI, or 50 ng/mL TIMP-2, completely inhibited the formation of the 62-kDa active MMP-2 (Fig. 2Go).


Figure 2
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  		Figure 2. MMPI or TIMP-2 inhibits active MMP-2. Gingival and PDL fibroblasts were cultured for 6 wks, and then the medium was changed to MEM containing DMSO or various concentrations of MMPI (0, 5, 50 µM) for an additional 2 wks. TIMP-2 (0, 50, 100 ng/mL) was also added to the media through the same experimental periods. At 8 wks, the gingival and PDL fibroblast cell layers were harvested, and the cell lysates were analyzed by gelatin zymography.

 
Degradation of Fibrillin-2 by Active MMP-2 in PDL Fibroblast Cell Layers
Previously, we reported that there was no difference in fibrillin-1 accumulation between gingival and PDL fibroblasts, although fibrillin-2 accumulated to a much smaller extent in PDL fibroblasts after 6 wks of culture (Tsuruga et al., 2002c). To investigate whether active MMP-2 in PDL fibroblast cell layers degraded fibrillin-1 and –2, we added 5 µM MMPI or 50 ng/mL TIMP-2 to the medium of PDL fibroblasts from weeks 6 to 8 of incubation (total, 2 wks). Addition of MMPI or TIMP-2 resulted in a significant increase of fibrillin-2 accumulation in PDL fibroblast cell layers (Fig. 3Go, right panel). However, fibrillin-1 accumulation in PDL fibroblast cell layers was unchanged by the addition of MMPI (Fig. 3Go, left panel). As expected, fibrillin-1 and –2 in the gingival fibroblast cell layers showed no change, because of the absence of active MMP-2. Since TIMP-2 is known to be an inhibitor of MMP-2 (Howard et al., 1991), these results suggest that MMP-2 degrades fibrillin-2 in PDL fibroblast cell layers.


Figure 3
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  		Figure 3. MMP-2 degrades fibrillin-2, not fibrillin-1, in PDL fibroblast cell layers. (A) Gingival fibroblasts (G) and PDL fibroblasts (P) were cultured for 6 wks, after which MMPI (5 µM) or TIMP-2 (50 ng/mL) was added to the media for 2 wks. Microfibril-containing fractions (10 µg) were analyzed by Western blotting. (B) Densitometric scanning of the bands was performed with National Institute of Health imaging software, as shown in (A). The values for each six-week culture sample were arbitrarily assigned a value of 100. The results represent the means ± SD (standard deviation) of 3 independent experimental determinations (*p < 0.05).

 
To examine the fragmentation of fibrillin-2 in PDL fibroblast cell layers, we performed fibrillin-2 immunoprecipitation from the microfibril-containing fraction. In control samples at 6 wks, a single 350-kDa protein (fibrillin-2) band was detectable (Fig. 4Go), and from 6 wks to 8 wks, both this band and a 180-kDa band were evident. Treatment with MMPI resulted in a decrease of the 180-kDa band, and a corresponding increase of the 350-kDa band.


Figure 4
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  		Figure 4. MMPs degrade fibrillin-2 and produce a fibrillin-2 fragment in PDL fibroblast cell layers. The microfibril-containing fraction of the PDL fibroblast cell layer was subjected to fibrillin-2 immunoprecipitation. In control samples at 6 wks, a single 350-kDa protein (fibrillin-2) was detectable (arrowhead). From 6 to 8 wks of culture, a 180-kDa band (asterisk), as well as the 350-kDa band, was detectable. Upon treatment with MMPI, the 180-kDa band decreased in parallel with an increase of the 350-kDa band.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
All MMPs are produced as latent forms (proMMPs) requiring activation for catalytic activity, a process that is possibly accomplished by proteolytic removal of the propeptide domain. Under certain in vitro conditions, MMP-2 can be activated by MT1-MMP at the cell membrane (Sato et al., 1994). In the present study, active MMP-2 was detected only in PDL fibroblast cell layers, though active MT1-MMP was detected in both gingival and PDL fibroblast cell layers. TIMP-2 is known to be a MMP-2 inhibitor (Howard et al., 1991). However, it is also known that formation of a ternary complex (proMMP-2/MT1-MMP/TIMP-2) is important for activation of proMMP-2 (Kinoshita et al., 1998). At present, TIMP-2 is known to be a bifunctional molecule; it stimulates MMP-2 activation at low concentration, whereas it inhibits activation at high concentration (Hernandez-Barrantes et al., 2000). TIMP-2 was detected at the same level in both cell layers. In this study, more MMP-2 was secreted into the media of the PDL fibroblasts than that of gingival fibroblasts. Therefore, the higher expression of MMP-2 by the PDL fibroblasts may lead to the presence of active MMP-2 not in gingival fibroblasts but in PDL fibroblast cell layers.

Lee et al.(1997) demonstrated intracellular activation of MMP-2 in normal fibroblasts. Therefore, using Western blotting, we separated the intracellular fraction to examine whether active MMP-2 was present. Active MMP-2 was not detected in the intracellular fraction, and was present only extracellularly in the PDL fibroblast cell layers.

It is known that PDL cells have a high turnover rate, and that the ECM is continuously synthesized, degraded, and replaced (Beertsen et al., 1997). This turnover is achieved mainly by fibroblasts, which are the principle cells of the PDL. We have shown previously that fibrillin-2 is related to the formation of elastic system fibers (Tsuruga et al., 2002a,c, 2005). However, there are currently no reported biochemical data about the remodeling of elastic system fibers in periodontal tissue. Analysis of the present data regarding the degradation of fibrillin-2 suggests that this plays some role in the remodeling process.

In the present study, using MMPI and the MMP-2-specific inhibitor, TIMP-2, we have demonstrated for the first time that some active MMPs that include MMP-2 degrade fibrillin-2 in PDL fibroblast cell layers. MMP-2 is thought to play a role in turnover and remodeling of the ECM (Creemers et al., 1998). Previously, we cultured gingival and PDL fibroblasts for 8 wks under normal conditions, and found that fibrillins and tropoelastin began to accumulate in the cell layers after 6 wks (Tsuruga et al., 2002c). Therefore, it seems reasonable to conclude that active MMP-2 available for remodeling appears in these cells after 6 wks, and analysis of our data supports the hypothesis that a period at least as long as this may be needed to investigate the remodeling of elastic system fibers. This longer period of culture may be useful for examining remodeling of the ECM.

Analysis of our data showed that treatment with MMPI blocked the formation of active MMP-2 at least, leading to the increase of fibrillin-2. To identify the fragment of fibrillin-2, we performed an immunoprecipitation study, because of difficulties in detecting a sufficient amount of fibrillin-2 in PDL fibroblast cell layers. The 180-kDa band in the control culture at 8 wks is likely to be a degraded fragment of fibrillin-2, because it was present between 6 and 8 wks of culture, and it decreased in parallel with an increase of the full-length band (arrowhead) upon treatment with MMPI. In our culture system, this cleaved fragment was not detected in the culture medium (data not shown). Since microfibrils become stable through intermolecular cross-linkages (Kielty et al., 2002), it seems difficult to identify this cleaved fragment released from the cell layers.

Since it is difficult to extract a full-length molecule from assembled fibers, many researchers have used recombinant fibrillin peptide to examine its characteristics. It was demonstrated that recombinant partial peptides of fibrillin-2 are degraded by MMP-2, –12, and –13, whereas those of fibrillin-1 are degraded by MMP-2, –3, –9, –12, –13, and –14 (Ashworth et al., 1999; Hindson et al., 1999). Analysis of the present data showed that only fibrillin-2 was degraded by some MMPs, which included MMP-2. This may be because, at the cellular level, the cleavage site of fibrillin-1 is masked by structural folding or glycoprotein modification. Further study will be necessary to resolve this issue.

The present study has shown that, at the cellular level, activated MMP-2 in PDL fibroblast cell layers may be associated with degradation of fibrillin-2, suggesting that MMPs may play a role in the remodeling of elastic system fibers in the PDL.


   ACKNOWLEDGMENTS
 
This work was supported by a Grant-in-Aid for Scientific Research (No. 17591920) from the Ministry of Education, Science, Sports and Culture of Japan.

Received June 8, 2006; Last revision November 7, 2006; Accepted November 17, 2006


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 MATERIALS & METHODS
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 DISCUSSION
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