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RESEARCH REPORT |
1 Department of SAU & FAL, University of Bologna, Italy;
2 Institute of Histology and Laboratory Analysis, and
4 Institute of Morphological Sciences, University ‘Carlo Bo’ of Urbino, Italy;
3 Department of Oral Biology, School of Dentistry, Medical College of Georgia, Augusta, GA, USA; and
5 Unit of Dental Sciences and Biomaterials, Department of Biomedicine, University of Trieste, Via Stuparich, 1, I-34129 Trieste, Italy
* corresponding author, lbreschi{at}units.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: dentin • MMP-2 • MMP-9 • Western blotting • zymography • matrix metalloproteinases • gelatinases
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Matrix metalloproteinases (MMPs) are Ca/Zn-dependent endopeptidases involved in extracellular matrix degradation (Visse and Nagase, 2003), in physiologic tissue remodeling, and in tumor growth and invasion (Egeblad and Werb, 2002). These proteinases belong to the wider metzincin group, which in turn constitutes the matrixin peptidases of the metallo -endopeptidase families. They share a conserved structural topology, which consists of a catalytic domain containing the zinc binding site, a prodomain that constitutes the N-terminus of the secreted enzyme (maintaining it in its latent zymogen form until its removal or disruption), and an active site that specifically binds to selective substrates. Further specific regions, such as hinge, hemopexin, and transmembrane domains, additionally characterize several MMPs (Visse and Nagase, 2003). Matrix metalloproteinases MMP-2 and -9 are characterized by the cysteine-rich repeats within the catalytic domain that resemble the collagen-binding type II repeats of fibronectin, necessary for the binding and cleaving activities of these peculiar MMPs (Visse and Nagase, 2003).
Among the different roles attributed to MMPs in the oral environment, their activity has been revealed in various developmental events involving dental tissues (Fukae et al., 1991; Llano et al., 1997; Hall et al., 1999; Caron et al., 2001), pathological processes such as periodontal disease (Ingman et al., 1996), caries (Tjäderhane et al., 1998), and dental pulp inflammation (de Souza et al., 2000; Wahlgren et al., 2002). Recently, MMP-2 was isolated from mature human mineralized dentin matrix (Martin-De Las Heras et al., 2000) and zymographically identified in demineralized dentin (van Strijp et al., 2003). Although MMP-2, -20, and possibly -8 have been identified in normal dentin matrix, MMP-9 has not yet been identified. The purpose of this study was to test the null hypothesis that normal dentin matrix contains no MMP-9.
Accordingly, we attempted to measure the presence and concentrations of all molecular forms of both MMP-2 and MMP-9 in demineralized dentin. We measured enzyme concentrations by immunoassay, form distribution by gelatin zymography, and immunological characterization by Western blotting.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Dentin powder was then equally divided and demineralized (at 4°C for 24 hrs while being stirred) in one of the following water solutions: (1) 0.87 M acetic acid, pH = 2.3; (2) 0.26 M citric acid, pH = 2.3; (3) 0.5 M EDTA, pH = 6.4; or (4) 0.5 M EGTA, pH = 6.4.
Enzyme Extraction of Demineralized Dentin Specimens and Sample Conditioning
The demineralized dentin powder was suspended in extraction buffer (50 mM Tris-HCl, pH 6.0, containing 5 mM CaCl2, 100 mM NaCl, 0.1% Triton X-100, 0.1% NONIDET P-40, 0.1 mM ZnCl2, 0.02% NaN3) and EDTA-free protease inhibitor cocktail (Sigma Chemical, St. Louis, MO, USA). The samples were ultrasonically treated at 40 W (Sonicator Ultrasonic Liquid Processor Model W-385, Heat Systems-Ultrasonic Inc., Farmingdale, NY, USA) output for 3 bursts of 10 sec each at 4°C. The vials were centrifuged at 18,000 rpm for 30 min at 4°C, and the supernatants were collected. All the proteins present in the supernatants were precipitated at 4°C by the addition of powdered ammonium sulphate (w/v) to achieve a final concentration of 85%, pH 7.0. The precipitate was collected by centrifugation at 24,000 rpm for 30 min at 4°C, redissolved in a 10-fold dilution in extraction buffer, dialyzed through a 30-kDa membrane against extraction buffer overnight, and stored at 4°C until analyzed.
Matrix Metalloproteinase and Protein Content Determinations
Total protein concentration in the demineralized dentin extract was measured by the bicinchoninic acid assay (Pierce, Rockford, IL, USA). Total MMP-2 and -9 activity was determined in dentin extracts with the use of immunoassay kits (BiotrakTM MMP systems, Amersham-Pharmacia, Milan, Italy). The specific immunoassay recognizes both proforms and active forms, and shows cross-reactivity for MMP-TIMP complexes, but does not react with other MMPs (Mannello and Sebastiani, 2003). Detection limits were estimated as 0.5 and 0.25 µg/L for MMP-2 and -9, respectively. The activity of gelatinolytic MMP forms was expressed as ng MMP/mg protein. To exclude "matrix" artefacts caused by interfering substances (e.g., proteins of dentin organic matrix, residual ammonium sulphate, phosphate, and hydroxyapatite crystals), we serially diluted the specimens and re-analyzed them for response linearity. For the analytical recovery study, dentin extracts were spiked with 3 concentrations of human plasma purified pro-MMP-2 and -9, according to the manufacturer’s instructions in ELISA kits; dentin extracts from the same specimen were left untreated. The concentrations of MMPs (5, 10, and 20 ng/mg protein, and 3, 6, and 12 ng/mg protein, for MMP-9 and MMP-2, respectively) in spiked and untreated samples were then measured as previously described; the recovery is defined as the percent difference in MMP levels in spiked and unspiked samples (Mannello and Sebastiani, 2003).
Gelatin Zymography
Dentin proteins were subjected to electrophoresis under non-reducing conditions on 7.5% SDS-polyacrylamide gels copolymerized with 2 g/L gelatin from porcine skin (Sigma Chemical), as previously described (Mannello et al., 2003). After electrophoresis, gels were washed in 2.5% Triton-X 100 with agitation and then incubated for at least 24 hrs at 37°C in enzyme incubation buffer (50 mM Tris-HCl, pH 7.5, containing 5 mM CaCl2, 100 mM NaCl, 0.01% Triton X-100, 0.1 mM ZnCl2, 0.2% Brij non-ionic detergent, and 0.002% NaN3). Negative control zymograms were incubated in the presence of 5 mM EDTA and 2 mM 1,10-phenanthroline for specific inhibition studies. Activation of gelatinase proforms was achieved with 2 mM p-aminophenylmercuric acetate (APMA) at 37°C for 1 hr (Mannello et al., 2003). Zymographic gels were stained in 0.2% Coomassie Brilliant Blue R-250 and de-stained. Wet gelatine zymograms were scanned at 600 nm by means of a densitometer equipped with an image analyzer (Cybernetics, Yorktown, VA, USA).
Immunoblotting
To confirm the immunological identity of enzymatic activities with known MMP-2 and MMP-9 gelatinase forms, we performed Western blots as previously described (Mannello and Sebastiani, 2003), using anti-human MMP-2 and MMP-9 monoclonal antibodies (Calbiochem, Milan, Italy).
Statistical Analysis
Mean ± standard error values of independent experiments (16) were calculated. Statistical analyses were performed by Student’s t test and one-way ANOVA, followed by Student-Newman-Keuls’ multiple comparison at p = 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Detection of Matrix Metalloproteinase Activity
Ammonium sulphate precipitation of extracted proteins after sonication provided significantly higher concentrations of MMP enzymes compared with untreated samples (17-fold increase; p < 0.01, Table 1
). Gelatinolytic enzymes were found in all dentin extracts, with significant differences among demineralization treatments. The highest MMP activities were found after citric acid treatment (Table 1). Both acetic acid and EDTA treatments extracted much lower levels of active MMPs as did citric acid. The smallest amount of total extractable active MMPs was detectable in dentin extracts after EGTA demineralization, even after ammonium sulphate concentration, which allowed for recovery of about 40% of total MMPs. Acetic acid demineralization provided an MMP yield of 60% (Table 1) compared with citric acid treatment, considered as 100%.
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). In the higher MMP recovery condition (citric acid demineralizing treatment and ammonium sulphate precipitation), we found 15.9 and 8.4 ng/mg proteins for MMP-2 and -9, respectively. MMP concentrations were significantly higher in citric and acetic acid extracts than those found in other demineralizing conditions (p < 0.05, Table 2).
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, A, lane stds) and immunologically identified to be MMP-2 and -9. When dentin extracts were analyzed by zymography, multiple forms of gelatinolytic enzymes were detected (Fig., A), with the 72-kDa activity identified as proMMP-2, the predominant/constitutive gelatinase form. In addition, other gelatinolytic bands were detected having apparent Mr of 92 and 220 kDa. No gelatinolytic band was present at lower molecular weights, suggesting the absence of autolytic degradation processes of dentin MMPs. In both EDTA and EGTA treatments, only a faint gelatinolytic 72-kDa band and negligible amounts of other gelatinolytic activities were detected. Between citric and acetic acid demineralizing treatments, quantitative but no qualitative differences were detected: In both acidic treatments, gelatinolytic bands (at 225, 92, and 72 kDa) were clearly demonstrated. Interestingly, in these acidic extracts, MMP-9 was present in both latent and activated forms (92 and 86 kDa), whereas both gelatinolytic bands at 225 and 72 kDa were found to be pro-enzymes, known proforms that may be time-dependently activated to lower Mr active forms after incubation in 2 mM APMA (Fig., B).
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, C).
Western blotting analysis (Fig., D) of human whole blood (used as Mr standard) identified MMP-2 (72-kDa) and the 3 forms of MMP-9 (92, 130, and 225 kDa), demonstrating the complete specific immunolabeling of all human gelatinase forms. Using monoclonal anti-human MMP-2 and -9 antibodies, we recognized the constitutive proMMP-2 (72 kDa) and only 2 proMMP-9 forms (weakly detected at 225 and 92 kDa, Fig., D) in dentin extracts. Densitometrically, MMP-2 concentrations appeared to have at least two-fold higher concentrations than MMP-9 (p < 0.01). Moreover, immunoreactivity was not detected against any lower-Mr MMP proteins, either because these did not show appreciable gelatinolytic activity, or because of the loss of the epitope recognized by monoclonal antibodies.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Citric acid demineralization, sonication, and ammonium sulphate protein precipitation allowed us to zymographically characterize proMMP-2 migrating at 72 kDa, the zymogen APMA-activable form of MMP-2. No activated form of MMP-2 (66 kDa) was zymographically and immunologically detectable without the activation procedure.
For the first time, the presence of several forms of both zymogen and activated MMP-9 has been shown in dentin extracts. In fact, zymogen proMMP-9 (92 kDa), activated MMP-9 (86 kDa), and high-molecular-weight enzyme polymer (225 kDa, probably disulfide-bonded dimers of MMP-9) were separated enzymatically and immunologically detected. All of these gelatinolytic enzymes were activated by APMA treatment (generating MMP-activated species). The specific Ca and Zn requirements for their full proteolytic activity (evaluated by EDTA, EGTA, and 1,10-phenanthroline inhibition studies) demonstrate them to be MMPs with gelatinolytic activity, excluding other MMPs, such as stromelysins or collagenases, that show overall a low specific activity for gelatin (Van den Steen et al., 2002; Visse and Nagase, 2003).
The binding properties of both MMP-2 and -9 to native and temperature-denatured collagen are mainly due to the fibronectin type II repeats (Visse and Nagase, 2003). The results suggest that our extraction technique disrupted gelatinase-collagen binding and released soluble gelatinase activity.
The presence of host-derived MMP-2 and -9 in mature dentin may contribute to degradation of the organic matrix of teeth (Fukae et al., 1991; Tjäderhane et al., 1998) during dental caries progression (Dayan et al., 1983; Sulkala et al., 2001), and throughout resin-infiltrated dentin interfaces (Nishitani et al., 2006). This may cause auto-degradation of collagen matrices by slow release or activation of host-derived proteinases. Since incompletely infiltrated collagen fibrils in acid-etched dentin are prone to proteolytic degradation (Pashley et al., 2004; Hebling et al., 2005), our demonstration of MMP-2 and -9 forms in sound dentin under acidic conditions further validates the self-destruction processes.
Caries progression (Sulkala et al., 2001; Chaussain-Miller et al., 2006) or other destructive processes may be correlated to impaired inhibition of MMPs by dentin TIMPs (Ishiguro et al., 1994), thus increasing proteolytic degradation pathways. Moreover, the presence of the membrane type MT1-MMP enzyme, the main activator of MMP-2, suggests an additional possible in situ MMP-2 activation that, balanced by TIMP-inhibitory effects, may lead to bio-molecular switches for dentin matrix destruction (Caron et al., 1998; Palosaari et al., 2002).
Since adverse effects of MMPs in mature human dentin have been identified, further studies are needed to identify effective, non-toxic inhibitors of these MMPs as potential therapeutic agents that can be incorporated into restorative materials.
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ACKNOWLEDGMENTS |
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Received January 10, 2006; Last revision November 19, 2006; Accepted December 12, 2006
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REFERENCES |
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