Perlecan, a Basement Membranetype Heparan Sulfate Proteoglycan, in the Enamel Organ : Its Intraepithelial Localization in the Stellate Reticulum
Division of Oral Pathology, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
Correspondence to: Takashi Saku, Division of Oral Pathology, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8126, Japan. E-mail: tsaku{at}dent.niigata-u.ac.jp
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Summary |
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Key Words: tooth germ heparan sulfate proteoglycan intraepithelial stroma stellate reticulum enamel organ perlecan
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Introduction |
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The enamel organ of tooth germs is histologically characterized by the stellate reticulum, which is composed of multiple layers of uniquely differentiated epithelial cells with pronounced intercellular spaces between them. Only a small number of investigators have given attention to this peculiar structure of the stellate reticulum. But the existence of extracellular matrix (ECM) molecules, such as syndecan 1 (Thesleff et al. 1988), fibronectin (Thesleff et al. 1981
), and laminin 5 (Yoshiba et al. 1998
), as well as functional molecules, such as transforming growth factor-ß1 (Cam et al. 1990
), bone morphogenetic protein (BMP)-2, BMP-4 (Vainio et al. 1993
), and fibroblast growth factor (FGF)-2 (Cam et al. 1992
) has been previously demonstrated in the stellate reticulum. However, these investigations had no specific point of view in terms of how ECM molecules function in this particular milieu of extended intercellular space, and any further functions of the enamel organ itself remain unknown. Many other ECM molecules have been shown to play important roles in normal (Lesot et al. 2002
) and abnormal (Yonemochi et al. 1998
,1999
) tooth morphogenesis, although little is known about perlecan in terms of its function or even its distribution in normal tooth germ development. Only cation histochemistry (Goldberg and Septier 1987
) and heparan sulfate lyase histochemistry (Kogaya et al. 1990
) have suggested the presence of HSPG in the tooth germ basement membranes and in the stellate reticulum. Furthermore, little attention has been given to ECM function as a carrier for nutrient transport by diffusion, which is thought to be indispensable in tissues with poor vascularity, such the enamel organ.
The purpose of this study is to determine the mode of perlecan expression during murine molar development, paying special attention to the enamel organ, and to demonstrate the biosynthetic activity of perlecan in tooth germ cells in culture. On the basis of this primary data regarding perlecan localization in the tooth germ, we discuss newly suggested properties of perlecan in odontogenesis.
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Materials and Methods |
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Tissue Preparation
Heads of mice were removed under ether anesthesia and fixed with 100% methanol or 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) (pH 7.4) overnight at 4C and processed for embedding in paraffin. Serial sections (5-µm) cut in the sagittal or frontal planes were stained with hematoxylin-eosin and immunohistochemically with the antibody described below. Another set of sections was used for in situ hybridization.
Antibodies
Mouse perlecan core protein was prepared from the murine Engelbreth-Holm-Swarm tumor, and the antibodies against mouse perlecan were raised in rabbits as described elsewhere (Saku and Furthmayr 1989).
Immunohistochemistry
The Envision+/HRP system (DAKO Japan; Kyoto, Japan) was used for immunohistochemical staining. Before incubation with the primary antibodies, sections were treated with 3 mg/ml bovine testicular hyaluronidase (type I-S, 440 U/mg; Sigma Chemical Co., St Louis, MO) in PBS for 30 min at 37C or with 0.4% pepsin (Sigma) in 0.01 N HCl for 30 min at 37C. The primary antibody was diluted to concentrations of 50 µg/ml. For visualization of reaction products, sections were treated with 3,3'-diaminobenzidine (Dohjin Laboratories; Kumamoto, Japan) in the presence of 0.005% hydrogen peroxide, and the sections were counterstained with hematoxylin. For control experiments, the primary antibodies were replaced with preimmune rabbit IgGs.
Preparation of RNA Probes
RNA probes for the mouse perlecan were prepared by using digoxigenin (DIG) RNA labeling kits (F. Hoffmann-La Roche; Basel, Switzerland) and T7/T3 RNA polymerases (Promega; Madison, WI). Template cDNA (516 bp, corresponding to domain I of mouse perlecan) was subcloned with RT-PCR, and the resultant 516-bp fragment was ligated into the plasmid vector (pBluescript II, Promega). The vector plasmid was linearized with BamHI and HindIII, and the linearized plasmids were used as templates to synthesize DIG-labeled RNA antisense probes by T7 RNA polymerase and sense probes by T3 RNA polymerase (Ikarashi et al. 2004).
In Situ Hybridization
After deparaffinization, sections were washed in three changes of 2x standard saline citrate (SSC) and treated with 5 µg/ml of proteinase K (Sigma) for 20 min at 37C. They were then washed with 0.2% glycine in PBS, fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.5) for 5 min, dehydrated with a series of ethanol (70% to 100%), and air dried. Hybridization was performed for 15 hr at 48C in a moist chamber. The hybridization solution contained 10% dextran sulfate, 1x Denhardt's solution, 100 µg/ml of salmon sperm DNA, 125 µg/ml of yeast tRNA, 3x SSC, 50% formamide, and 500 ng/ml of probes in 10 mM phosphate buffer (pH 7.4). After hybridization, the sections were rinsed in 2x SSC, and then the hybridized probes were detected with DIG detection kits (Roche). The sections were counterstained with methyl green (Ikarashi et al. 2004).
Primary Culture of Enamel Organ and Dental Papilla/Pulp Cells
First molar tooth germs of the mandible from day 1 postnatal mouse pups were dissected in Hanks solution under a stereomicroscope. They were incubated with 0.25% trypsin (Roche) in 1 mM ethylenediaminetetraacetic acid solution for 10 min at 37C. Then, the enamel organ and the dental papilla were separated manually under a stereomicroscope. For immunofluorescence and immunoprecipitation experiments, these isolated tissues were placed in 0.1% collagenase/DMEM solution for 4 hr at 37C to dissociate the enamel pulp cells and the dental papilla cells. The suspended enamel organ cells and the dental papilla/pulp cells were plated into 35-mm plastic dishes in 2 ml of DMEM containing 10% fetal calf serum (FCS; ICN Pharmaceuticals, Costa Mesa, CA), 1% glutamine, 50 µg/ml streptomycin, and 50 IU/ml penicillin. They were incubated at 37C under a humidified 5% CO2/95% air atmosphere. When the cells became confluent, they were split again and used for RT-PCR and immunoprecipitation experiments.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from enamel organ and dental papilla tissues using the ISOGEN system (Nippon Gene Co.; Tokyo, Japan). cDNA was synthesized from the RNA with the SuperScript First-Strand Synthesis System (Invitrogen; Carlsbad, CA). Following the RT, PCR was carried out in an Astec thermal cycler PC-800 (Astec; Fukuoka, Japan). Oligonucleotide primers flanking the exon of domain I of perlecan core protein (nucleotide number 183-550, No. M85289, GeneBank) were synthesized as follows: 5'-CCTGA GGACA TAGAG AC-3' forward and 5'- TCGGA AGGGA ATGCG GA-3' reverse, to generate a 503-bp product. For the competitive PCR, we also synthesized oligonucleotide primers of mouse ß-actin as follows: 5'-TGGAA TCCTG TGGCA TCCAT GAAAC-3' forward and 5'-TAAAA CGCAG CTCAG TAACA GTCCG-3' reverse, to generate a 348-bp product. The thermocycling protocol during 30 amplification cycles was as follows: denaturation at 94C for 1 min, annealing at 55C for 1 min, extension at 72C for 1 min, and termination with a final cycle: annealing at 55C for 1 min and extension at 72C for 7 min. The amplified DNA fragments were analyzed by electrophoresis on 3% agarose gels. The electrophoresis image was analyzed quantitatively: amounts of perlecan relative to ß-actin were determined with NIH Image, an image processing and analyzing program.
Immunoprecipitation
Cell labeling and immunoprecipitation experiments were performed as described elsewhere (Kimura et al. 1999). Briefly, cells were preincubated with methionine-free minimum essential medium (MEM) for 1 hr and then incubated in fresh MEM containing 50 µCi of [35S]-methionine for 3 hr. After removal of the medium, the cell layer was lysed and both the cell lysate and medium were centrifuged at 15,000 x g for 10 min. The resultant supernatants were subjected to immunoprecipitation. The precleared lysates and media were incubated with the antibodies to the perlecan core protein overnight, and immune complexes were isolated with protein A-Sepharose (Amersham-Pharmacia Biotech; Uppsala, Sweden). For antibody control experiments, the antibodies to the perlecan core protein were replaced with preimmune rabbit IgGs. Immunoisolated materials were dissolved in Laemmli's sample buffer, boiled for 5 min, and centrifuged at 10,000 x g for 5 min to remove beads. The supernatants were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was done on a 5% polyacrylamide slab gel with 2.5% 2-ß-mercaptoethanol, according to Laemmli (1970)
. Gels were stained with Coomassie Brilliant Blue and then air dried. The dried gels were fluorographed on X-ray film (Hyperfilm-MPTM; Amersham, Buckinghamshire, UK). Apparent molecular mass was determined by co-electrophoresis of marker proteins.
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Results |
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Dental Lamina Stage (E11.5)
The initial morphological sign of tooth organogenesis was a site-specific thickening of the oral epithelium, which was presumed to be dental epithelial cells (Figure 1A). Perlecan was definitely immunolocalized along the basement membrane of the oral epithelium and faintly and diffusely in the underlying mesenchyme (Figure 1B). mRNA signals for perlecan core protein were detected correspondingly to its immunolocalization in both the oral epithelial cells and the underlining mesenchymal cells (Figure 1C).
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Cap Stage (E15.5)
At the cap stage, the dental epithelial cells became differentiated into the enamel organ, in which the inner and outer enamel epithelia, stratum intermedium, and stellate reticulum were distinguished (Figure 1G). The immunolocalization mode for perlecan in the stellate reticulum was almost the same as that in the bud stage, although the positive area was enlarged more with its growth. On the other hand, the positive staining in mesenchymal cells condensed around the enamel organ was more enhanced (Figure 1H). Although perlecan mRNA signals were found in most of the epithelial cells of the enamel organ, they were more strongly positive in those of the stellate reticulum areas, especially in the secondary enamel knots. In addition, the surrounding mesenchymal cells, including those in the dental papilla area enclosed by the invaginated enamel organ, had definite hybridization signals (Figure 1I).
Bell Stage (E17.5)
During the early bell stage, the characteristic cusp pattern of the enamel organ started to be formed, and mesenchymal areas were condensed and enclosed by the enamel organ as definite forms of the dental papilla (Figure 1J). The intercellular immunolocalization for perlecan within the stellate reticulum was much more enhanced with the development of the enamel organ. At the same time, strongly positive reactions were obtained within the dental papilla and in the surrounding interstitium (Figure 1K). Perlecan mRNA signals were most intensely localized at inner ameloblasts as well as at dental papilla cells facing tall ameloblasts. In addition, signals were also seen in the stellate reticulum and outer ameloblasts as well as in mesenchymal cells in the dental papilla and its surrounding areas (Figure 1L).
Postnatal Differentiation Stage (P1)
The tooth germ began to assume the crown shape of molar teeth. Cusps were clearly recognized, with largely regressed enamel organ spaces where blood vessels entered with extravasation of erythrocytes, and the dental papilla space was instead proportionally enlarged. Alignments of tall columnar ameloblasts and odontoblasts were obvious (Figure 2A). Immunohistochemically, perlecan was localized diffusely in the narrowed stellate reticulum and in the immature dental pulp (Figure 2B). Its mRNA signals were detected in the cells with perlecan immunopositivities in both stellate reticulum and dental pulp (Figure 2C).
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At 4 weeks of age, molar roots were completely formed, with the periodontal ligament in its functional state (Figure 2G). In the dental pulp, the immunolocalization of perlecan was scarcely found in the crown part but focally preserved in the root part. Within the pulp, it was strongly immunolocalized in the predentin. The periodontal ligament, especially at the cervical area, was diffusely immunopositive for perlecan (Figure 2H). Perlecan mRNA signals were demonstrated in odontoblasts, especially those in the root area, and in cementoblastic, fibroblastic, or osteoblastic cells in the periodontal ligament, which was not always synchronous to the immunolocalization (Figure 2I). In the histological experiments described above, neither immunopositivities nor mRNA signals for perlecan core protein were obtained when the antibodies were replaced with preimmune rabbit IgGs or when the antisense probes were replaced with sense probes (not shown).
PCR Confirmation of Perlecan Expression in Tooth Germ Tissues
To analyze the perlecan mRNA expression level in each dental component during the tooth development, enamel organ and dental papilla/pulp tissues at postnatal days 1, 6, 10, and 12 were separated, and total RNA was extracted from each fraction. Total RNA samples, 3 µg each, were reverse-transcribed with oligo-dT primers, and the resultant cDNAs were further amplified for perlecan core protein domain I with ß-actin oligonucleotide primer pairs as an internal control. A 503-bp PCR product for perlecan was obtained from all samples with different band densities, whereas a 348-bp PCR product for ß-actin was almost stable among the samples (Figure 3A). The PCR products for perlecan and ß-actin were determined by densitometry, and the relative values for perlecan expression levels against those of ß-actin in the time course after birth were plotted and are shown in Figure 3B. Perlecan mRNA levels in the enamel organ decreased gradually after birth, which was in accordance with regression of the enamel organ space and the immunohistochemical results (Figure 2E). In contrast, perlecan mRNA expression levels in the dental papilla/pulp increased with aging, which was also similar to histological and immunohistochemical observations (Figure 2I).
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Discussion |
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Perlecan, a basement membranetype HSPG, bears three major HS side chains and 12 probable N-linked carbohydrates on its large core protein of 400 kDa molecular mass (Noonan et al. 1991
). This core protein consists of five distinct domains, and this multidomain structure suggests that it possesses multifunctional properties of the molecule, although its actual functions are still poorly understood. On the other hand, the role of HS chains has been studied extensively, especially in binding with various growth factors by their negative charges (Jiang and Couchman 2003
). Their primary function as a member of glycosaminoglycan chains is thought to be retention of water molecules around the straight chains of disaccharide units (Jackson et al. 1991
). In previous studies, we showed that perlecan was most enhanced in the early phase of granulation tissue formation processes, which showed myxoid appearance, in the oral cavity (Murata et al. 1997
; Yamazaki et al. 2004
) and gastrointestinal tracts (Ohtani et al. 1993
). Similar results were obtained in the myxoid stroma of adenoid cystic carcinoma (Cheng et al. 1992
), pleomorphic adenoma (Saku et al. 1990
), and adenomatoid odontogenic tumor (Murata et al. 2000
). Thus, the myxoid histology resulting from the retention of water via HS chains takes place irrespective of the tissue, epithelial or mesenchymal.
The histological phenotype of the stellate reticulumlike appearance is characteristic of ameloblastomas, whose term has been adopted due to the resemblance of their tumor cell nests to the enamel organ. We have already demonstrated that this characteristic stellate reticulumlike histology of ameloblastoma is caused by perlecan retention in the intercellular space of the tumor cells (Ida-Yonemochi et al. 2002). On the basis of the results, we have predicted that perlecan must be present within the authentic stellate reticulum of the enamel organ. However, there has been no direct evidence on the localization of any concrete proteoglycan species, including perlecan, in the enamel organ, although the presence of HSPG was suggested by cation histochemistry (Goldberg and Septier 1987
) and HS lyase histochemistry (Kogaya et al. 1990
). Thus, in the present study, the existence of perlecan has been confirmed for the first time in the intercellular spaces within the enamel organ, and the nomenclature of ameloblastoma has been confirmed functionally by this aspect of perlecan deposition in the intercellular space of epithelial cells.
The stellate reticulum has been believed to function as a spacer device for mechanical protection for the tooth crown formation as well as for nutritional recruitment from the outlying vascular circulation to the stellate cells (Kallenbach 1980). However, the real function of the enamel organ and the stellate reticulum has been mostly unexplored to date. The present evidence of perlecan deposition in the intercellular space of the stellate reticulum may indicate that it acts as a carrier for transport of nutrients to epithelial cells for enamel formation, because there is no entry of blood vessels into the enamel organ before birth, and because no nutrient supplies can be expected from the dental papilla side because of the presence of dental hard matrices. Because proteoglycans and/or some groups of polyanions have been functionally implicated in the transport and diffusion of calcium ions in the secretory ameloblast layer (Goldberg and Septier 1987
), it is likely that the stellate reticulum also takes part in transport or condensation of calcium ions from the blood circulation to the ameloblastic layers through the plentiful HS of perlecan.
Furthermore, we have demonstrated the presence of HS chains in the cell membrane of the stratum intermedium cells and in the papillary layer of the tooth germs and have concluded that HS chains regulate the transport of minerals through their negatively charged cell membranes and play an important role in cellcell interaction by preserving local growth factors in the tooth germ development (Nakamura et al. 1995). Basic fibroblast growth factor (bFGF), which is known to bind HS chains, has been reported to exist in the enamel organ as well as in the basement membranes (Cam et al. 1992
), and the immunolocalization pattern of bFGF is almost the same as that of perlecan, as shown in the present study. On the other hand, other basement membrane molecules, such as laminins and collagen type IV, have been localized only within the basement membrane zones at the epithelialmesenchymal interface (Thesleff et al. 1981
). Thus, it is highly likely that perlecan participates not only in basement membrane assembly but also in the regulation of the proliferation and differentiation of epithelial cells during tooth morphogenesis.
In the present immunofluorescence study of cells in primary culture, it was obvious that perlecan was deposited on the cell surface, especially along plentiful and long cytoprocesses of stellate-shaped enamel organ cells. The perlecan deposition in thread-like and parallel fashions may represent a trace of focal contacts by which cells are stretched. This further suggests that cytoskeletal networks specific to such cell shapes are affected by perlecan, although there have been no reports on the relationship between perlecan and cytoskeletal fibers.
The mRNA expression levels for perlecan by RT-PCR as well as the protein level of perlecan by immunoprecipitation in enamel organ cells/tissues, supported the immunofluorescence data. Although it was difficult to evaluate quantitatively their modes of biosynthesis, a gradual decrease in perlecan mRNA expression levels by enamel organ cells in the 12-day period after birth was at least confirmed by the RT-PCR result, which should represent the regression of the enamel organ space. On the other hand, the modes of biosynthesis of perlecan in the dental papilla/pulp cells characterized by immunofluorescence, immunoprecipitation, and RT-PCR were distinct from those in enamel organ cells. Their production of perlecan was, rather, increased with time until day 12 after birth, which was a reverse tendency in comparison to that by enamel organ cells, and the mesh-like mode of perlecan deposition in the monolayer culture of dental papilla/pulp cells was not obtained in enamel organ cells. Thus, the present results indicate that perlecan functions differently between the enamel organ and the dental papilla/pulp.
In addition to the enamel organ, the immunolocalization of perlecan was also demonstrated to occur throughout the dental mesenchyme during murine odontogenesis. The results suggest that perlecan is involved in the formation of the dental papilla/pulp and the periodontal ligaments. The perlecan immunolocalization in the predentin and its transcripts in odontoblasts indicate its function in odontoblastic differentiation and dentin matrix maturation. By using immunoelectron microscopy for HSPG, laminin, type IV collagen, and fibronectin in the dental papilla of the Macaca fuscata monkey, Sawada and Inoue (1998) suggested the function of the basement membrane in supporting and positioning of odontoblasts toward their differentiation. Moreover, in the periodontal ligament, perlecan expression in osteoblasts and cementoblasts was also first confirmed in the present study. The modes of perlecan expression seemed to be dependent on the developing stage of the structure and cell types. A more extensive examination for the dental papilla/pulp and periodontal ligament is needed for a more accurate understanding of the whole function of perlecan in dentinogenesis and cementogenesis.
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Acknowledgments |
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Footnotes |
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