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RESEARCH REPORT |
vß3 Integrin Expression in Human Odontoblasts and Co-localization with Osteoadherin
Laboratory of Development of Dental Tissues, EA 1892, IFR 62, Faculty of Odontology, Lyon 1 University, G. Paradin Str., 69372 Lyon Cedex 08, France;
* corresponding author, farges{at}laennec.univ-lyon1.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 integrin, a putative receptor for osteoadherin. We showed that vß3 integrin expression was restricted to odontoblasts, blood vessels, and small rounded cells in sound and carious pulp. Odontoblast staining intensity increased from the apical to the cusp region. Osteoadherin staining was strong in the whole odontoblast layer (with a slight decrease in the cusp region) and in predentin. Odontoblasts differentiating in vitro were stained with the anti-vß3 integrin antibody, first at the level of intercellular contacts, then throughout the cell membrane. These results suggest that the vß3 integrin could play a role in interodontoblast adhesion and odontoblast binding to the surrounding predentin/dentin/pulp matrix, possibly through osteoadherin.
KEY WORDS: tooth pulp • dentin extracellular matrix • cell adhesion • intercellular contacts • blood vessel
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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and ß subunits show a wide tissue distribution and play key roles in developmental, physiological, and pathological processes. The ligand specificity relies on the combination of both /ß subunits (18 different integrins, 8 different ß integrins). Ligand binding to the receptor extracellular domain induces a conformational change that is propagated to the cytoplasmic domain and initiates downstream signaling events related to cytoskeletal re-arrangements. As a consequence, integrins are involved in many fundamental cellular functions, including proliferation, adhesion, motility, differentiation, survival, and apoptosis (Hynes, 2002). In vivo studies conducted with v- and ß3-null mice supported the implication of the vß3 integrin in placenta development, secondary palate formation, blood vessel resistance to leakage, and bone resorption (Bader et al., 1998; Hodivala-Dilke et al., 1999; McHugh et al., 2000). In vitro studies showed that the vß3 integrin could also be involved in wound repair in connective tissues (Gailit et al., 1997).
The vß3 integrin was first characterized as the vitronectin receptor, but it exhibits a wide spectrum of extracellular ligands, including fibronectin, fibrinogen, von Willebrand factor, thrombospondin, osteopontin, tenascin, bone sialoprotein, plasminogen activator inhibitor-1, prothrombin, neurite cell adhesion molecule L1, metalloproteinase 2, ADAM-15 and -23 disintegrins (Plow et al., 2000), and TGF-ß1 and -ß3 latency-associated proteins (Ludbrook et al., 2003). The vß3 integrin was also proposed to be a membrane receptor for osteoadherin, a small leucine-rich proteoglycan synthesized by bovine osteoblasts (Wendel et al., 1998). We previously showed that osteoadherin was expressed by mouse osteoblasts, odontoblasts, and ameloblasts (Buchaille et al., 2000) and by human odontoblasts (Lucchini et al., 2002). Osteoadherin was recently localized extracellularly in alveolar bone, predentin, and enamel matrices of rat and mouse teeth (Couble et al., 2004). Since odontoblasts and osteoblasts share common protein expression profiles, we hypothesized that the vß3 integrin could mediate odontoblast adhesion to osteoadherin. The aims of this study were to characterize vß3 integrin expression in the human dental pulp by RT-PCR and in situ hybridization, and then to compare vß3 integrin and osteoadherin protein distribution in vivo and in odontoblasts differentiating in vitro.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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RT-PCR
Total RNA was extracted from pulp samples as described by Buchaille et al.(2000) with use of the RNeasy Mini Kit and protocol (Qiagen, Chatsworth, CA, USA). A 200-ng quantity of total RNA was reverse-transcribed and amplified by polymerase chain-reaction with the Titan One Tube RT-PCR System (Roche Diagnostics, Mannheim, Germany). Primers were for the v integrin subunit (forward, ACTGGGAGCACAAGGAGAACC; reverse, CCG CTTAGTGATGAGATGGTC) and for the ß3 integrin subunit (forward, CCTACATGACCGAAAATACCT; reverse, AATCCCTCCCCACAAATACTG). Thirty cycles of PCR amplification were performed with an annealing temperature of 53°C. PCR products (expected fragment sizes: v integrin subunit, 305 bp; ß3 integrin subunit, 517 bp) were analyzed by 2% agarose gel electrophoresis with the NuSieve 3:1 (FMC Bioproducts, Rockland, ME, USA), the bands being visualized with ethidium bromide.
In situ Hybridization
Probe labeling and in situ hybridization were performed as previously described (Bleicher et al., 1999). v and ß3 integrin PCR products were electro-eluted from a 2% agarose gel, and 25 ng of these DNA templates were subjected to asymmetric PCR in 10 mM Tris-HCl (pH 8.3), 50 mM KCL, 1.5 mM MgCl2, 0.2 mM (dATP, dGTP, dTTP), 1.7 µM dCTP, 50 mM (33P)-dCTP (2500 Ci/mmol), 2 units of TaqDNA polymerase, and 20 pmol antisense primer (10 cycles; annealing temperature, 50°C). The reaction was stopped by 0.2 mM ethylene diamine tetraacetic acid. The sense primer was used to synthesize the control (sense) probe under the same conditions. Pulp frozen sections were pre-hybridized for 2 hrs at 37°C by incubation in 50% de-ionized formamide, 10 mM NaPO4 (pH 7.4), 2 x SSC, 5 mM EDTA, 2.5 x Denhardt’s solution, 250 µg/mL denatured herring sperm DNA, and 500 µg/mL yeast tRNAs. Slides were rapidly rinsed in 2 x SSC, dehydrated in ethanol, and air-dried. Hybridization was performed overnight at 37°C, with the radioactive probe diluted in 50 µL of the pre-hybridization solution without tRNAs but with 0.04 g/mL dextran sulfate (final concentration of the probe: 40-80.106 cpm/mL). Slides were then washed in 2 x SSC for 30 min, 1 x SSC for 1 hr, and 0.5 x SSC for 1 hr. After the slides were dehydrated and air-dried, they were dipped in LM-1 emulsion for autoradiography (Amersham Biosciences, Freiburg, Germany). Emulsions were developed in Kodak D-19 developer and fixed in 30% sodium thiosulfate. Slides were slightly counterstained with Masson’s Hemalun.
Cell Culture
Thirty human odontoblast cell cultures were performed [as described by Couble et al.(2000)] from 5 sound non-erupted third molars. The pulp tissue was separated from the dentin/enamel mineralized complex, and its apical end was removed to prevent periodontal cell contamination. Pulp explants (about 2 mm3) were placed in Permanox Lab-Tek chamber slides (Nunc, Naperville, IL, USA), then cultures were performed in Eagle’s basal medium (Invitrogen, Grand Island, NY, USA) supplemented with 15% fetal calf serum (Eurobio, Les Ulis, France), 50 µg/mL acid ascorbic, 10 mM ß-glycerophosphate (Sigma, St. Louis, MO, USA), and 100 IU/mL penicillin-50 mg/mL streptomycin (Invitrogen). Cells were grown at 37°C in a humidified atmosphere of 5% CO2 in air for 4 wks, then prepared for immunohistochemistry.
Immunohistochemistry
Immunoperoxidase histochemistry was performed by routine procedures as previously described (Farges et al., 2003; Couble et al., 2004). Sections and cultures were incubated with anti-vß3 integrin monoclonal antibody (1:100) (clone LM609, Chemicon International, Temecula, CA, USA) or anti-osteoadherin polyclonal antibody (1:500). Osteoadherin antiserum was prepared as described (Couble et al., 2004). It was raised by immunization of a rabbit against 3 synthetic peptides highly conserved among rat, mouse, and human osteoadherin (ETIQLKTQVFRPYQD, residues 371–385; YNSHYYEMQEWQDTI, residues 412–425; CQYEAYRWDDDYDQE, residues 20–34), respectively. Peptide synthesis and the immunization procedure were performed by CovalAb-Lyon (France). Peptides were conjugated to keyhole limpet hemocyanin (KLH), mixed with an equal volume of Freund’s complete adjuvant, and injected into multiple subcutaneous sites in the rabbit. Animals were boosted 3 wks later with the peptide-conjugate in Freund’s incomplete adjuvant until a sufficient antibody titer was obtained, and assessed by ELISA titration. For antiserum purification, peptides were coupled to sepharose gel and antibodies affinity-purified by standard methods.
Antibody detection was performed with the use of a Vectastain Elite ABC kit (Vector Labs, Burlingame, CA, USA) according to the protocol of the manufacturer, peroxidase being localized with diaminobenzidine.
Western Blotting
Proteins were classically extracted from odontoblast cell cultures by means of Laemmli’s buffer and osteoadherin identified by Western blotting. Anti-osteoadherin polyclonal antibody was diluted in PBS-2% BSA at a concentration of 1:4000, and staining was detected with horseradish-peroxidase-conjugated goat anti-rabbit IgG (DAKO, Glostrup, Denmark) (dilution 1:2000). Immunoreactivity was visualized by means of a chemiluminescence ECL system (Amersham Pharmacia Biotech, Buckinghamshire, England). Control was accomplished by omission of the primary antibody.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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v and ß3 integrin subunits (Fig. 1a
). Transcripts were detected by in situ hybridization in mature odontoblasts (Figs. 1b, 1c) and in cells surrounding blood vessels (Figs. 1d, 1e), but not in pulp core cells. No signal was present on sections hybridized with the sense probe (Fig. 1f).
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vß3 integrin showed an intense staining of the cell membrane in mature odontoblasts of the pulp horn, including processes in dentin tubules (Fig. 2a). Some blood vessels were also heavily stained, whereas others were not (Fig. 2b). Isolated cells in the blood vessel wall were also positive (Fig. 2b). On the same tooth section, vß3 integrin staining decreased progressively in the apical direction and became moderate in cervical odontoblasts (Fig. 2c). In newly differentiated odontoblasts of the forming root, the staining was weak and less intense than in blood vessels (Fig. 2d). In teeth with mild caries (Fig. 2e), odontoblasts producing reactionary dentin (situated in the black box on Fig.2e) were stained with the anti-vß3 integrin antibody (Fig. 2f). Blood vessels and small rounded cells present in the pulp parenchyma were also stained (Fig. 2g). Osteoadherin was immunodetected in pulp horn odontoblasts, including processes entering dentin tubules (Fig. 2h). Newly differentiated odontoblasts and predentin in the forming root were heavily stained (Fig. 2i). No obvious difference in osteoadherin staining intensity was observed between odontoblasts under healthy and those under carious dentin (data not shown).
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vß3 integrin antibody and moderate staining of the cytoplasm (Fig. 3a). Cell body and process were clearly delineated in some cells with typical odontoblast morphology (Fig. 3b). At the culture periphery, where newly polarized cells aligned to constitute a typical odontoblast layer, vß3 staining was detected on the membrane of odontoblast cell bodies at the level of contact zones and intercellular junctions (Fig. 3c). Osteoadherin was identified by immunoblot analysis in extracts of odontoblast cell cultures. Under reducing conditions, a single band of 110 kDa was stained with the anti-osteoadherin antibody (Fig. 3d). No immunoreactive band was observed when the anti-osteoadherin antiserum was omitted. By immunohistochemistry, osteoadherin staining was detected intracellularly close to the odontoblast nucleus in vitro, but not in the extracellular compartment (Fig. 3e).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 integrin, in the cell membrane of human odontoblasts in vivo by in situ hybridization and immunohistochemistry. vß3 integrin staining was also observed in blood vessel cells, thus constituting a good positive control. Indeed, endothelial and vascular smooth-muscle cells were previously shown to express vß3 integrin (Eliceiri and Cheresh, 2001; Moiseeva, 2001). vß3 integrin gene expression was previously detected in vitro in human dental pulp stem cells (Shi et al., 2001). In vivo, v, ß1, ß3, and ß5 integrin subunits were detected in mesenchymal papilla cells during early murine tooth germ development, but their presence in differentiated odontoblasts was not reported (Ruch et al., 1995; Salmivirta et al., 1996). In our study, both the odontoblast cell body and process were immunostained. With regard to these localizations, different roles for the vß3 integrin might be suggested. At the basal pole, this receptor could mediate odontoblast adhesion to the sub-odontoblastic pulpal extracellular matrix. vß3 integrin detection on the lateral surfaces of odontoblasts at the level of cell-cell contact suggests that this receptor could be a crucial element for the formation of interodontoblast adhesion sites and odontoblast layer organization. Further, as odontoblasts move centripetally throughout the life of the tooth toward the pulp core, following dentin matrix deposition, the presence of the vß3 integrin on odontoblast lateral sides could help to maintain the structure and to prevent the disruption of the odontoblast layer. Integrin activation would thus contribute by interaction with the cytoskeleton to the continuous re-organization of actin microfilaments that accompanies odontoblast process elongation and the cell body moving toward the pulp core. Apically, the vß3 integrin might be involved in the determination of the position and in the stabilization of the odontoblast process by anchoring to predentin and intratubular matrix, thus sustaining the odontoblast phenotype. The absence of process staining in odontoblasts newly differentiated in vitro seems to preclude a direct role for vß3 integrin in odontoblast process formation and/or initial elongation. In the context of a dental pulp subjected to a carious aggression, a possible role for vß3 integrin would be the maintenance of the organization and of the cohesion of the odontoblast layer to prevent micro-organisms and/or exogenous molecules from diffusing through dentinal tubules to reach the sub-odontoblast peripheral pulp. Further, by maintaining a constant link with the extracellular matrix and with neighboring cells, the vß3 integrin could provide a critical survival signal and prevent the odontoblast anoikis—apoptotic death due to disruption of interactions between cells—that is classically observed under caries lesions (Magloire et al., 1992).
The vß3 integrin was also detected in isolated small rounded cells present in the blood vessel wall, as well as in the pulp paremchyma under caries lesions. The localization and the typical morphology of these cells suggest that they could be immune cells migrating from the blood circulation to the pulpal connective tissue. Indeed, the vß3 integrin was shown to be involved in the migration of monocytes and lymphocytes through the vascular endothelium (Weerasinghe et al., 1998).
Osteoadherin was proposed to be a potential ligand for vß3 integrin in bovine osteoblasts (Wendel et al., 1998). The vß3 integrin could mediate human odontoblast adhesion to osteoadherin, because odontoblasts co-produced both proteins. However, staining patterns for vß3 integrin and osteoadherin were not totally superimposable: Osteoadherin staining was strong at the onset of predentin synthesis in newly differentiated odontoblasts, whereas vß3 integrin staining was weak in these cells and progressively increased during odontoblast maturation. It is thus possible that the vß3 integrin could bind to other odontoblast-derived molecules present in the human odontoblast layer and/or predentin, such as tenascin and fibronectin (Lukinmaa et al., 1991), bone sialoprotein (Bègue-Kirn et al., 1994), and osteopontin (Bronckers et al., 1989), which are all known to bind the vß3 integrin. Other odontoblast-derived RGD-containing proteins, such as dentin sialoprotein and dentin matrix protein-1, are also candidates for binding to odontoblast integrins. Thus, considering the great number of potential ligands, it cannot be excluded that the vß3 integrin ligand may change in relation to the odontoblast differentiation status and the local matrix environment. Finally, the absence of difference in osteoadherin staining intensity under healthy and carious dentin might indicate that osteoadherin expression is not highly affected in odontoblasts re-activated by carious aggression.
In conclusion, our results suggest that the vß3 integrin could play a role in interodontoblast adhesion and odontoblast binding to the surrounding predentin/dentin/pulp matrix, possibly through osteoadherin. This receptor may thus be of great interest for the development of therapeutic strategies, including biomolecules capable of maintaining odontoblast shape/function and spatial organization in pathological conditions.
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ACKNOWLEDGMENTS |
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Received October 16, 2003; Last revision April 20, 2004; Accepted May 6, 2004
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