Immunolocalization of Enamelysin (Matrix Metalloproteinase-20) in the Forming Rat Incisor
Matrices Extracellulaires et Biominéralisation, EA 2496, Faculté de Chirurgie Dentaire, Université René Descartes Paris V, Montrouge, France (KBB,DS,MG); Department of Clinical Dental Sciences, University of Liverpool, Liverpool, United Kingdom (RH); and U 532 INSERM, Hôpital Saint-Louis, Paris, France (SM)
Correspondence to: Michel Goldberg, Faculté de Chirurgie Dentaire, Université Paris V, 1, rue Maurice Arnoux, 92120 Montrouge, France. E-mail: mgoldod{at}aol.com
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Summary |
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(J Histochem Cytochem 52:437445, 2004)
Key Words: matrix metalloproteinase-20 TIMP-2 stratum intermedium ameloblast odontoblast enamel amelogenin dentin
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Introduction |
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Amelogenin is the most abundant protein in the forming enamel and is hydrolyzed within hours after its secretion to the matrix (Smith et al. 1989). The cleavage of amelogenin is highly regulated during development and is believed to produce biologically active fragments that may have different functions from those of the intact molecule. The degradation of amelogenin at early stages of enamel formation implies that amelogenin-degrading enzymes are particularly important in the initial development of enamel. Enamelysin, or MMP-20, is an MMP with a specificity for amelogenin and is considered a predominant enzyme for the processing of enamel matrix. It was shown to have an Mr of about 54.1 kD in humans (Llano et al. 1997
) and 51.9 kD in swine (Bartlett et al. 1996
), whereas the lower molecular weight active species (43, 38, 33, 25, and 21 kD) described by Li et al. (1999)
and MoradianOldak et al. (2001)
probably represent autoproteolytic products. MMP-20 has the same domain structure as most other MMPs including a signal peptide, a propeptide involved in maintaining enzyme latency, a catalytic domain with a Zn-binding site, and a hinge region that links the catalytic domain to the C-terminal hemopexin-like domain (Bartlett and Simmer 1999
). It has recently been shown that MMP-20 can be activated by MT1MMP in human odontoblasts and pulp cells (Palosaari et al. 2002
). MT1MMP is considered the physiological activator of the gelatinase MMP-2 on the cell membrane in a mechanism that requires a complex of TIMP-2 with the pro-MMP-2. The implication of TIMP-2 in pro-MMP-20 activation remains to be clarified.
Cloned from porcine enamel organ by Bartlett et al. (1996), MMP-20 was believed to be exclusively expressed in teeth, but its presence in human tongue carcinoma cells (Vaananen et al. 2001
) and in cultured granulosa cells in follicles of porcine ovaries (Kimura et al. 2001
) has recently been described. In the developing teeth, MMP-20 is expressed primarily during the secretory to late transition stages of amelogenesis but not in enamel maturation. In situ hybridization (ISH) and immunohistochemical (IHC) investigations revealed the presence of MMP-20 in both ameloblasts and in odontoblasts and in pulp cells (BegueKirn et al. 1998
; Fukae et al. 1998
; Caterina et al. 2000
).
In this study we used the continuously growing rat incisor to study the expression pattern of MMP-20 and its presumed substrate, amelogenin, during different stages in the formation of mineralized rat dental tissues.
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Materials and Methods |
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Zymography and Immunoblotting Analyses
Segments of the forming part of the rat mandibular incisor, as defined by Leblond and Warshawsky (1979), were homogenized in an ice-cold buffer (50 mM Tris-HCl, pH 7.5, containing 5 mM CaCl2, 0.9% NaCl, and 0.2% Triton X-100 with or without a cocktail of protease inhibitors (Set V, EDTA-free; Calbiochem, La Jolla, CA) using a potter homogenizer (two incisors in 500 µl buffer). The resulting homogenate was then briefly sonicated on ice (three times for 5 sec), cleared by centrifugation at 10,000 x g for 15 min at 4C, and analyzed for the presence of MMP-20 by zymography and immunoblotting. MMP-20 was activated by incubating lysates with 1 mM p-aminophenylmercuric acetate (APMA) for 30 min at 37C.
Casein zymography was performed using 10% SDS-polyacrylamide gels co-polymerized with 1 mg/ml casein, Extracts (5 µg) were mixed with Laemmli sample buffer without reducing agents and without heating and subjected to SDS-PAGE. After electrophoresis, gels were incubated (30 min at 22C) in renaturing solution (2.5% Triton X-100), rinsed briefly in distilled water, and incubated (24 hr at 37C) in developing buffer (50 mM Tris-HCl, pH 7.5, containing 5 mM CaCl2 and 0.02% NaN3). The gels were then stained with 0.1% Coomassie Blue R-250 in a solution of 20% isopropanol and 10% acetic acid and destained in 20% isopropanol and 10% acetic acid to reveal gelatinolytic activity.
For immunoblotting analysis, 20 µg of protein lysate was subjected to Laemmli 12% SDS-PAGE under reducing conditions, followed by transfer to a nitrocellulose membrane. The membrane was blocked with 5% non-fat dry milk in TBS (100 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.1% Tween-20 (T-TBS) and was then incubated for 2 hr at room temperature (RT) with either MMP-20 antibody at 1:1000 dilution, anti-TIMP-1 at 1:2000 dilution, or TIMP-2 at 1:2000 dilution in T-TBS. The membrane was washed and incubated with a 1:1000 dilution of an IgGperoxidase conjugate for 2 ht at RT. Immunodetection of the antigen was performed using the Renaissance Western Blot Chemiluminescence Reagent Plus Kit from NEN Life Science (Boston, MA) according to the manufacturer's instructions.
Light and Immunoelectron Microscopy
The study design was approved by the Ethics Committee of the Faculty. Five SpragueDawley male rats (100 g body weight) were anesthetized with an IP injection of chloral hydrate (400 mg/ kg), then perfused intracardially with a solution containing 4% paraformaldehyde, buffered with 0.1 M sodium cacodylate, pH 7.27.4, for 24 hr at 4C. After several rinses in cacodylate buffer, the teeth were dissected out from the mandibles, dehydrated in graded ethanol, and without demineralization embedded in Paraplast. Five- to 7-µm-thick sections were treated in methanol to inhibit endogenous peroxidases. Sections were saturated at 4C overnight with 1% PBSBSA and incubated in the primary antibody at 1:100 dilution for 2 hr at RT. The optimal working dilutions in terms of detection and acceptable background labeling were determined after testing a series of antibody concentrations between 1:50 to 1:200. A swine anti-rabbit IgG conjugated with peroxidase (DAKO; Glostrup, Denmark) in 1% PBSBSA was used as a secondary antibody at 1:100 dilution, and the conjugates were revealed by using 30 mg DAB:100 ml plus H2O2 (30 vol) in PBS (40 µl/100 ml). Controls were performed by omitting or substituting the primary antiserum with non-immune serum.
Electron Immunogold Labeling
Five SpragueDawley male rats (100 g body weight) were anesthetized with an injection of chloral hydrate, then perfused intracardially with a solution containing 1% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.27.4. After 10 min the two mandibular incisors were dissected out. The segments were prepared from the forming part of the incisor (the first 6 mm). The undemineralized mandibular incisors were sliced transversely with a razor blade into three equal segments about 2 mm thick. The segments were immersed in the fixative solution for 1 hr at 4C, then immersed overnight in 0.2 M cacodylate buffer. After dehydration in graded ethanols, the segments were embedded in Lowicryl K4M and polymerized at -20C with UV light.
Sections were incubated in PBS5% BSA at RT for 90 min and then incubated with the primary antibodies (1:150 dilution) for 2 hr at RT. After three rinses with PBS1% BSA, sections were incubated with a 1:100 dilution of the secondary antibody, a goat anti-rabbit IgG coupled to 15-nm colloidal gold (AuroProbe GAR IgG-G15; Amersham, Poole, UK) for 90 min at RT. Sections were rinsed in PBS and stained with aqueous uranyl acetate for 5 min and then with lead citrate for 1 min. They were examined with a JEOL 100B electron microscope operating at 80 kV. Controls were performed by omitting or substituting the primary antiserum with non-immune serum.
Quantitative Evaluation
The presence and distribution of MMP-20 and the intensity of its staining were evaluated by electron microscopy on two distinct presecretory and secretory areas as described (Goldberg et al. 2003). On electron micrographs enlarged to a final magnification of x54,000, goldantibody conjugates were scored on 40 to 90 squares per area (2 cm x 2 cm) and were expressed as the number of gold particles per µm2 (grain density) followed by the standard deviation. The standard deviation mean was also calculated. Grain density was calculated in the stratum intermedium, in the basal mitochondrion-rich area, and in the distal supranuclear area of presecretory and secretory ameloblasts. In the presecretory area, calculation was further carried out in the non-mineralized mantle dentin and in polarizing odontoblasts. In the secretory area, immunogold complexes were scored again in the stratum intermedium and in secretory ameloblast cell bodies and Tomes' processes. Gold particles were scored also in the outer interrod and rod enamel growth sites. Calculations were carried out in the older inner enamel near the dentino-enamel junction (DEJ), mantle dentin, circumpulpal dentin, and metadentin (the 0.52.5-µm-thick border at which dentin mineralization is initiated) (Goldberg and Septier 1996
), predentin, odontoblast cell bodies and processes, and finally in pulp cells. Background labeling was scored in the lumen of vessels and in lateral parts of the section where no biological material was present. Statistical comparison between two mean values was carried out using Student's t-test, and only p
0.05 was considered significant.
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Results |
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Electron Immunogold Staining
Here we compared the distribution patterns and staining intensities of MMP-20 at two stages of enamel formation, an early stage characterized by a basement membrane separating the presecretory ameloblasts from the non-mineralized dentin and a later stage of development in which translucent enamel was partially formed, although not fully matured.
At the early stage, the most striking observation was the high labeling intensity in the stratum intermedium (Figure 3A). In the presecretory ameloblasts and odontoblasts, staining was mainly cytosolic and nuclear, although a few vesicles were also stained. No labeling was detected in the non-mineralized dentin, basement membrane, matrix vesicles, or with collagen fibrils (not shown).
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Discussion |
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The significance of the localization of amelogenin or MMP-20 in odontoblasts is not clear. Although it was postulated that low molecular weight amelogenin degradation products diffuse through the basement membrane separating preameloblasts and preodontoblasts to become trapped in the forming dentin (Sawada and Nanci 1995), the localization of amelogenin and MMP-20 to the same sites argues for local synthesis of both enzyme and substrate. This would be in accord with the localization of MMP-20 RNA in the odontoblasts by in situ hybridization (Caterina et al. 2000
). The local degradation of amelogenin by MMP-20 would produce low molecular mass peptides that have been suggested to serve as signaling molecules. Indeed, two gene splice products of 59-kD amelogenin [A+4] and [A-4], synthesized by odontoblasts, were shown to interact with immature mesenchymal cells to initiate a change in the cell phenotype and the maturation pathway (Nebgen et al. 1999
; Veis et al. 2000
). However, in view of our results showing the localization of both MMP-20 and amelogenin at the same sites, it appears likely that the amelogenin peptides extracted from dentin by Nebgen et al. may also represent degradation products. Indeed, the amelogenin present in the tooth was suggested to be a complex mixture of gene isoforms and degradation products (Simmer 1995
).
When we examine the staining of MMP-20 within the cell, the decreased density of goldantibody complexes observed from the cell bodies to the processes favors the possibility of active secretion. This may be the case for the forming enamel, in which staining was strong in the outer forming enamel and gradually diminished near the DEJ, but not for the matrix secreted by odontoblasts, because dentin and predentin were unstained. In addition, examination of ultrathin sections revealed that the gold-antibody complexes were located mostly in the cytosol and the nucleus, with a few gold particles seen in the secretory vesicles. This is not a unique phenomenon. We have previously shown that MMP-3 is mostly associated with cytoskeletal structures or/and small structures that may be storage sites for MPs in the cytosol (Hall et al. 1999).
A striking observation by both light and electron microscopy is the strong concentration of MMP-20 in the SI, particularly in the earlier stages of enamel development during which staining appears even stronger than in the presecretory ameloblasts. We have no explanation for this high SI labeling and why relatively weaker staining was observed by IHC. It is unlikely that the MMP-20 is synthesized locally only to be transferred to ameloblasts or to the mineralization front. It is possible, however, that MMP-20 at this location contributes to the polarized organization of the enamel organ by the cleavage of laterally secreted enamel proteins that might otherwise accumulate in intercellular spaces (Ruch et al. 1975; Matsuo et al. 1988
,1990
).
The molecular forms of MMP-20 in the rat incisor were also investigated by casein zymography and immunoblotting. Zymographic analysis of extracts prepared in the presence of protease inhibitors revealed the 46- and 41-kD bands corresponding to the calculated active form and the 57-kD band to the proform of MMP-20, as was previously shown for human and bovine MMP-20 (Bartlett et al. 1996; Llano et al. 1997
). We noted, however, that when different extracts were compared, although these three bands were consistently present the relative intensity of the latent vs active bands varied quite significantly. This may be due to the fact that the forming part of the rat incisor represents multiple stages of tooth formation expressing different stages of MMP-20 activation. Interestingly, when extraction of the tooth protein was performed in the absence of proteinase inhibitors, the main lysis band was observed at 21 kD, indicating degradation of the proteinase during extraction. This may suggest that the smaller molecular weight species previously reported (MoradianOldak et al. 2001
) are not physiological but are processed during the extraction procedure, although it is not excluded that they can be, under other circumstances, also produced naturally.
The higher molecular weight 78-kD band revealed by both immunoblotting and zymograms was also stained with the TIMP-2 antibody but not with TIMP-1, and its molecular weight, as well as its resistance to proteolysis, may indicate a complex proMMP-20/TIMP-2. Such MMP/TIMP complexes that do not dissociate under SDS-PAGE conditions have been previously described (Kolkenbrock et al. 1995; Kossakowska et al. 1998
). It might resemble the specific interaction of the proMMP-2 with TIMP-2 via the C-terminal end of proMMP-2, an interaction that is a prerequisite for the activation of MMP-2 by MT1MMP (Strongin et al. 1995
). It is interesting, in this respect, that proMMP-20, like proMMP-2, is also activated by MT1MMP (Palosaari et al. 2002
). However, further work will be necessary to determine the exact nature of the interaction of TIMP-2 with MMP -20.
Although the significance of MMP-20 and amelogenin in odontoblasts, but not in dentin, remains to be determined, its transient expression during cell maturation, as well as its intracellular location not linked to the classical secretion pathway, may reinforce the additional role previously proposed for MMP-20 in signaling and therefore in the crucial event of epithelomesenchymal interaction.
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Footnotes |
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Literature Cited |
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Bartlett JD, Simmer JP (1999) Proteinases in developing dental enamel. Crit Rev Oral Biol Med 10:425441[Abstract]
Bartlett JD, Simmer JP, Xue J, Margolis HC, Moreno EC (1996) Molecular cloning and mRNA tissue distribution of a novel matrix metalloproteinase isolated from porcine enamel organ. Gene 183:123128[Medline]
BegueKirn C, Krebsbach PH, Bartlett JD, Butler WT (1998) Dentin sialoprotein, dentin phosphoprotein, enamelysin and ameloblastin: tooth-specific molecules that are distinctively expressed during murine dental differentiation. Eur J Oral Sci 106:963970[Medline]
Caterina J, Shi J, Sun X, Qian Q, Yamada S, Liu Y, Krakora S, et al. (2000) Cloning, characterization, and expression analysis of mouse enamelysin. J Dent Res 79:16971703[Abstract]
Fukae M, Tanabe T, Murakami C, Dohi N, Uchida T, Shimizu M (1996) Primary structure of the porcine 89-kDa enamelin. Adv Dent Res 10:111118[Medline]
Fukae M, Tanabe T, Uchida T, Lee SK, Ryu OH, Murakami C, Wakida K, et al. (1998) Enamelysin (matrix metalloproteinase-20): localization in the developing tooth and effects of pH and calcium on amelogenin hydrolysis. J Dent Res 77:15801588[Abstract]
Goldberg M, Septier D (1996) A comparative study of the transition between predentin and dentin using various preparative procedures in the rat. Europ J Oral Sciences 104:269277
Goldberg M, Septier D, Bourd K, Hall R, George A, Goldberg H, Menashi S (2003) Immunohistochemical localization of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the forming rat incisor. Connect Tissue Res 44:143153[Medline]
Hall R, Septier D, Embery G, Goldberg M (1999) Stromelysin-1 (MMP-3) in forming enamel and predentine in rat incisor-coordinated distribution with proteoglycans suggests a functional role. Histochem J 31:761770[Medline]
Hoyhtya M, Fridman R, Komarek D, Porter-Jordan K, Stetler-Stevenson WG, Liotta LA, Liang CM (1994) Immunohistochemical localization of matrix metalloproteinase 2 and its specific inhibitor TIMP-2 in neoplastic tissues with monoclonal antibodies. Int J Cancer 56:500505[Medline]
Kimura A, Kihara T, Ohkura R, Ogiwara K, Takahashi T (2001) Localization of bradykinin B(2) receptor in the follicles of porcine ovary and increased expression of matrix metalloproteinase-3 and -20 in cultured granulosa cells by bradykinin treatment. Biol Reprod 65:14621470
Kolkenbrock H, Orgel D, HeckerKia A, Zimmermann J, Ulbrich N (1995) Generation and activity of the ternary gelatinase B/TIMP-1/LMW-stromelysin-1 complex. Biol Chem Hoppe Seyler 376: 495500[Medline]
Kossakowska AE, Edwards DR, Lee SS, Urbanski LS, Stabbler AL, Zhang CL, Phillips BW, et al. (1998) Altered balance between matrix metalloproteinases and their inhibitors in experimental biliary fibrosis. Am J Pathol 153:18951902
Leblond CP, Warshawsky H (1979) Dynamics of enamel formation in the rat incisor tooth. J Dent Res 58:950975[Medline]
Li W, Machule D, Gao C, DenBesten PK (1999) Activation of recombinant bovine matrix metalloproteinase-20 and its hydrolysis of two amelogenin oligopeptides. Eur J Oral Sci 107:352359[Medline]
Llano E, Pendas AM, Knauper V, Sorsa T, Salo T, Salido E, Murphy G, et al. (1997) Identification and structural and functional characterization of human enamelysin (MMP-20). Biochemistry 36:1510115108[Medline]
Matsuo S, Ichikawa H, Wakisaka S, Akai M (1990) Influence of colchicine on the addition of a sugar to the enamel protein in secretory ameloblasts of cultured germs of rat molar tooth by 3H-galactose radioautography. Cell Tissue Res 260:521528[Medline]
Matsuo S, Takano Y, Wakisaka S, Ichikawa H, Nishikawa S, Akai M (1988) Effect of colchicine on the transport of precursor enamel protein in secretory ameloblasts studied by 3H-proline radioautography in vitro. Anat Rec 221:812822[Medline]
MoradianOldak J, Jimenez I, Maltby D, Fincham AG (2001) Controlled proteolysis of amelogenins reveals exposure of both carboxy- and amino-terminal regions. Biopolymers 58:606616[Medline]
Nebgen DR, Inoue H, Sabsay B, Wei K, Ho CS, Veis A (1999) Identification of the chondrogenic-inducing activity from bovine dentin (bCIA) as a low-molecular-mass amelogenin polypeptide. J Dent Res 78:14841494[Abstract]
Overall CM, Limeback H (1988) Identification and characterization of enamel proteinases isolated from developing enamel. Amelogeninolytic serine proteinases are associated with enamel maturation in pig. Biochem J 256:965972[Medline]
Palosaari H, Ding Y, Larmas M, Sorsa T, Bartlett JD, Salo T, Tjaderhane L (2002) Regulation and interactions of MT1-MMP and MMP-20 in human odontoblasts and pulp tissue in vitro. J Dent Res 81:354359
Robinson C, Brookes SJ, Shore RC, Kirkham J (1998) The developing enamel matrix: nature and function. Eur J Oral Sci 106 (suppl 1):282291[Medline]
Robinson C, Kirkham J, Brookes SJ, Bonass WA, Shore RC (1995) The chemistry of enamel development. Int J Dev Biol 39:145152[Medline]
Ruch JV, KarcherDjuricic V, Staubli A, Fabre M (1975) Effects of cytochalasin B and colchicine on dental cytodifferentiation in vitro. Arch Anat Microsc Morphol Exp 64:113134[Medline]
Sawada T, Nanci A (1995) Spatial distribution of enamel proteins and fibronectin at early stages of rat incisor tooth formation. Arch Oral Biol 40:10291038[Medline]
Simmer JP (1995) Alternative splicing of amelogenins. Connect Tissue Res 32:131136[Medline]
Smith CE, Pompura JR, Borenstein S, Fazel A, Nanci A (1989) Degradation and loss of matrix proteins from developing enamel. Anat Rec 224:292316[Medline]
Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA, Goldberg GI (1995) Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J Biol Chem 270:53315338
Tanabe T, Fukae M, Shimizu M (1996) Possible actions of metalloproteinases found in porcine enamel in an early secretory stage. Adv Dent Res 10:170172[Medline]
Vaananen A, Srinivas R, Parikka M, Palosaari H, Bartlett JD, Iwata K, Grenman R, et al. (2001) Expression and regulation of MMP-20 in human tongue carcinoma cells. J Dent Res 80:18841889[Abstract]
Veis A, Tompkins K, Alvares K, Wei K, Wang L, Wang XS, Brownell AG, et al. (2000) Specific amelogenin gene splice products have signaling effects on cells in culture and in implants in vivo. J Biol Chem 275:4126341272