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Amelogenin Gene Expression in Porcine Odontoblasts
S. Oida1,*,
T. Nagano2,
Y. Yamakoshi1,
H. Ando1,
M. Yamada3 and
M. Fukae1
1 Department of Biochemistry, School of Dental Medicine, Tsurumi University, 2- 1-3 Tsurumi, Tsurumi-ku, Yokohama, 230-8501, Japan;
2 Department of Periodontology, School of Dental Medicine, Tsurumi University, 2- 1-3 Tsurumi, Tsurumi-ku, Yokohama, 230-8501, Japan; and
3 Department of Physical Therapy, School of Health Science, Niigata University of Health and Welfare, 3198 Shimami-cho, Niigata 950-3198, Japan;
Correspondence: *corresponding author, oida-s{at}tsurumi-u.ac.jp
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ABSTRACT
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Amelogenin is the major organic component in the enamel matrix of developing teeth and plays an important role in enamel biomineralization. Amelogenin has been reported to be a specific secretory product of ameloblasts. In this study, we examined amelogenin gene expression in various cell layers prepared from a porcine permanent tooth germ using reverse transcription-polymerase chain-reaction (RT-PCR). Amelogenin amplification products were detected only in the secretory ameloblast layer after 20 cycles of PCR. After 30 cycles of PCR, amelogenin amplification products were detected in secretory and maturation-stage ameloblasts and in odontoblasts. The relative levels of amelogenin gene expression in secretory and maturation-stage ameloblasts and odontoblasts were determined. Secretory ameloblasts expressed over 1000 times the level of amelogenin mRNA found in odontoblasts. Amelogenin gene expression in odontoblasts was confirmed in an erupted porcine permanent first molar, which has no ameloblasts. Amelogenin PCR amplification products were identified from 4 different alternatively spliced transcripts in the ameloblast samples, and the same spliced forms were detected in the odontoblast samples.
Key Words: amelogenin • ameloblast • odontoblast • RT-PCR • enamel
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INTRODUCTION
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Amelogenin is the most abundant and best-characterized enamel matrix protein. The enamel layer of developing teeth contains a complex mixture of amelogenin peptides. This complexity is the combined result of alternative RNA splicing and proteolytic processing of secreted proteins. Alternative splicing of porcine amelogenin has been confirmed by analyses of both protein (Yamakoshi et al., 1994) and cDNA (Hu et al., 1996) sequences. The processing of secreted amelogenins is due to the activity of the matrix metalloproteinase enamelysin (MMP20) (Fukae et al., 1998; Ryu et al., 1999).
A significant body of evidence supports the conclusion that amelogenin is expressed only by ameloblasts. The technique of in situ hybridization has been used in several investigations of developing teeth from different organisms, and each came up with the same general conclusions: Amelogenin mRNAs are specifically detected in ameloblasts, but not in odontoblasts or other cells of the dental pulp, or in Hertwig's epithelial root sheath (HERS) (Snead et al., 1988; Luo et al., 1991; Fong et al., 1996; Inage et al., 1996; Wurtz et al., 1996; Karg et al., 1997; Bleicher et al., 1999; Wakida et al., 1999; Hu et al., 2001). Amelogenin protein expression is also well-characterized by immunohistochemistry. In general, strong amelogenin signal is observed in the enamel layer, with some signal evident in the dental pulp, particularly in odontoblasts opposite pre-secretory ameloblasts (Inai et al., 1991; Nakamura et al., 1994; Simmer et al., 1994). Taken together, the in situ hybridization and immunohistochemical studies suggest that amelogenin is expressed solely by ameloblasts, but that ameloblast secretory products diffuse into the dental pulp. Consistent with this interpretation is the finding that defects in the amelogenin gene cause X-linked amelogenesis imperfecta (AI) in man (Lagerström et al., 1990, 1991). Over half a dozen separate mutations have been identified in different kindreds afflicted with X-linked AI, and in all cases the defect appears to be confined to the enamel layer (Lagerström et al., 1991; Aldred et al., 1992; Lench et al., 1994; Lagerström-Fermer et al., 1995; Lench and Winter, 1995; Collier et al., 1997; Hart et al., 2000). These reports suggest that amelogenin plays an important role in enamel formation. Two additional pieces of evidence attest to the highly restricted pattern of amelogenin expression: Only ameloblasts and stratum intermedium displayed positive signal when a β-galactosidase reporter gene was expressed from 3.5 kb of the bovine amelogenin gene (promoter) upstream of intron 1 in transgenic mice (Adeleke-Stainback et al., 1995), and no amelogenin expressed sequence tags (ESTs) from non-dental tissues have been reported to GenBank.
But recently, amelogenin peptides were purified from rat dentin extracellular matrix (Nebgen et al., 1999), and amelogenin cDNAs were cloned from a rat odontoblast-pulp cell cDNA library (Veis et al., 2000). In both cases, however, the sources of the amelogenin protein and mRNA were not rigorously controlled, so that a pulpal origin was only suggested.
To investigate the potential expression of amelogenin by secretory-stage ameloblasts, maturation-stage ameloblasts, odontoblasts, and dental pulp, we have used a reliable surgical method for the isolation of ameloblasts, odontoblasts, and dental pulp from developing pig teeth. The cervical region of the developing crown in the area of the cervical loop, where ameloblasts and odontoblasts are in proximity (and risk being mixed), was excised and discarded. Total RNA was isolated from carefully separated soft tissues of the developing tooth, and quantitative RT-PCR was used for assay of the relative levels of amelogenin expression. These surgical techniques were essential to prevent the masking of low levels of amelogenin expression by nearby cells that express very high levels of amelogenin, which we believe occurs with the in situ hybridization technique.
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MATERIALS & METHODS
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Preparation of Tooth Germ Cells
Fresh, unerupted permanent incisor tooth germs and erupted young permanent first molars were dissected from six-month-old porcine mandibles. The secretory ameloblast layer was dissected from the inner surface of the removed enamel organ, and maturation ameloblasts were prepared from the labial surface of the incisor. After preparation of the ameloblast layers, the remaining cells and cell debris on the enamel surface were cleaned off completely with the use of kimwipe paper (Kimberly-Clark Co., Yokohama, Japan), and the apical portion of the tooth was cut off to avoid contamination by ameloblasts. Tissue remaining in the apical portion, consisting of odontoblasts and dental pulp cells, was carefully removed (Fig. 1A ). The mature odontoblast layer, still embedded in the predentin matrix, was washed with phosphate-buffered solution and collected by being scraped with a micro-spatula. We prepared another odontoblast sample from the erupted young molar in the root formation stage to obtain ameloblast-free odontoblasts. Moreover, we prepared dental pulp cells, oral epithelium cells, muscle cells, and connective tissue cells from porcine mandible.
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Figure 1. (A) Schematic drawing of ameloblast and odontoblast tissue preparations. The secretory ameloblast layer was dissected from the inner surface of the removed enamel organ epithelia (EOE), and the maturation ameloblast layer was prepared from the labial surface of the incisor. (B) The secretory ameloblast sample was prepared from the inner surface of the EOE, which contained secretory ameloblasts (SA), stratum intermedium (SI), and stellate reticulum cells (SR). After removal of all the cells covering the enamel matrix completely, the apical portion of the tooth was cut off, and the dental pulp was removed carefully. Almost all the odontoblasts remained on the surface of predentin matrix following the removal of the pulp tissue. (C) Cells remaining attached to the predentin matrix following removal of the dental pulp. Almost all the cells on predentin matrix are odontoblasts.
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RNA Preparation and PCR Primers
Total RNA was extracted from each tissue sample by the acid guanidinium thiocyanate-phenol-chloroform (AGPC) method (Chomczynski and Sacchi, 1987); cDNA was synthesized with oligo-dT primer from 3 µg of the total RNA with use of the You-prime First-Strand Beads kit and protocol (Amersham-Pharmacia Biotech, Piscataway, NJ, USA). Primer sets for amelogenin [U43405] and mouse dentin sialophosphoprotein (DSPP) [NM010080] were designed with the use of nucleotide sequences in the GenBank database. Two pig amelogenin-specific primer-pairs were used: 5'-ACCCCTCTGAAGTGGTACCAG (exon 5) and 5'-TGTTGGGTTGGAGTCATGGAG (exon 6), which generated a 236-base-pair (bp) amplification product, and 5'-CATCAAGAAATGGGGACCTGG (exon 2) and 5'-TCTGATCTTTTAATCCACTTC (exon 7), which generated multiple amplification products from alternatively spliced amelogenin transcripts. The DSPP primers set was: 5'-ACAGCAGCAACAGCAGTGAC and 5'-CTAATCATCACT GGTTGAGTG, which generated a 176-bp amplification product. A primer set amplifying glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Clontech, Palo Alto, CA, USA) was used as a control. The sense and antisense primers of amelogenin were designed for hybridization to different exons to guard against genomic DNA contamination.
RT-PCR
RT-PCR was carried out by means of a PCR amplification kit (Amersham Pharmacia Biotech). The reactions had a five-minute denaturation at 94°C, followed by 30 cycles each with denaturation at 94°C for 30 sec, primer annealing at 55°C for 30 sec, and product elongation at 74°C for 30 sec. Final elongation was performed at 74°C for 5 min. Polyacrylamide gel electrophoresis (4.5%) and ethidium bromide staining were carried out for the analysis of PCR products. Additionally, the PCR products were cloned into pBluescriptIISK(+) (Stratagene, La Jolla, CA, USA), and their nucleotide sequences were determined by cycle-sequencing.
Quantitative PCR by the LightCycler Instrument
The cDNAs generated from the ameloblast and odontoblast layers were amplified quantitatively by means of the DNA Master SYBR Green I kit and protocol and a LightCycler instrument (Roche Molecular Biochemicals, Mannheim, Germany). The value for each PCR amplification product was normalized with use of the amount of GAPDH product to control for variations in the amounts of tissue in each sample.
Histology
After extraction of dental pulp from a porcine incisor, the tooth with remaining odontoblasts was immersed in 2% paraformaldehyde, 1% glutaraldehyde, and 0.1 M cacodylate buffer (pH 7.4) at 4°C for 3 days. After fixation, the sample was demineralized with 10% EDTA (pH 7.4) at 4°C for about 2 wks and embedded in Epon. Sections were mounted on glass slides and stained with toluidine blue. The sample of secretory ameloblasts was fixed in 10% formalin and embedded in paraffin. Sections were stained with hematoxylin-eosin.
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RESULTS
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Odontoblasts were carefully prepared to avoid contamination by ameloblasts. To confirm the retention of odontoblasts on the predentin matrix following removal of the pulp tissue, we fixed the tooth matrix with retained odontoblasts and observed it by light microscopy. Almost all the odontoblasts remained on the predentin surface, while no contaminating ameloblasts were observed in this section (Fig. 1C ).
To investigate the expression of amelogenin mRNA in porcine tooth germs, we performed RT-PCR using cDNAs prepared from ameloblasts, odontoblasts, and dental pulp cells. The upstream primer, specific for exon 5, and a 3'-primer, specific for exon 6 of the pig amelogenin gene, were used. The PCR products were detected by 4.5% polyacrylamide gel electrophoresis. The amelogenin gene product was found only in the secretory ameloblast layer after 20 cycles of PCR (Fig. 2A). When the PCR was carried out for 30 cycles, amelogenin gene products were found in the secretory and maturation-stage ameloblasts, as well as in the odontoblast layer (Fig. 2B). A little expression was detected in dental pulp cells, but no amelogenin amplification products were detected in oral epithelium cells, connective tissue cells, and the muscle cells after 30 cycles (Fig. 2B), and a trace of amelogenin gene expression was detected only in the oral epithelium sample after 40 cycles of PCR (data not shown). The PCR product of the DSPP gene was found only in the incisor and the molar odontoblast layers (Fig. 2C). The product of GAPDH was detected in all of the samples, at similar levels (Fig. 2D). The specificity of the PCR products was confirmed by nucleotide sequencing.
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Figure 2. RT-PCR amplification products with amelogenin (panels A, B), DSPP (panel C), and GAPDH (panel D) specific primer sets. The amplified products were separated on a 4.5% polyacrylamide gel and stained with ethidium bromide. Lane M: molecular-size standard,  x 174 HaeIII-digested (New England Bio-Lab). Lane 1, oral epithelium; lane 2, muscle; lane 3, connective tissue; lane 4, dental pulp cells; lane 5, secretory ameloblasts from the incisor; lane 6, secretary ameloblasts from the molar; lane 7, maturation ameloblasts from the incisor; lane 8, odontoblasts from the incisor; and lane 9, odontoblasts from the molar.
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The relative amounts of amelogenin mRNA were determined with the use of a LightCycler instrument, with templates isolated from secretory ameloblasts, maturation ameloblasts, and odontoblasts, all from the incisor tooth germ. The results of the quantitative PCR of the amelogenin and the GAPDH are shown in Fig. 3. The relative amounts of amelogenin, after normalization with the amounts of the GAPDH, for maturation-stage ameloblasts, odontoblasts, and secretory-stage ameloblasts were 1:4:4000, respectively. The result of quantitative PCR indicates that a considerable amount of amelogenin mRNA was expressed by odontoblasts, but the quantity was 1/1000 of the amount in secretory ameloblasts.
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Figure 3. Quantitative PCR of amelogenin and GAPDH mRNA isolated from secretory ameloblasts, maturation ameloblasts, and odontoblasts, which permitted calculation of the relative amounts of amelogenin mRNAs from these different tissues.
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To examine the alternative splicing of the amelogenin gene in the ameloblasts and the odontoblasts, we carried out PCR using an upstream primer from exon 2 and downstream primer from exon 7 of the amelogenin gene. Gel electrophoresis showed that the same pattern of amelogenin mRNA was expressed in the odontoblast and ameloblast samples (Fig. 4A). The PCR products were cloned into a plasmid vector, and their nucleotide sequences were determined. RNA messages containing exon 2-3-5-6-7 (P173), exon 2-5-6-7 (P157), exon 2-3-5-6D-7 (6D: deletion in exon 6, P56), and exon 2-3-6D-7 (P41) were confirmed to be in both the odontoblast and the ameloblast layers. The lowest molecular band in Fig. 4A contained the PCR products from exons 2-3-5-6D-7 (P56) and 2-5-6D-7 (P41).
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Figure 4. (Panel A) Analysis of RT-PCR products with amelogenin exon 2 and exon 7-specific primer sets. PCR was carried out for each sample by 25 cycles. The amplified products were analyzed by 4.5% polyacrylamide gel electrophoresis. Lane M: molecular-size standard, x 174 HaeIII digested (New England Bio-Lab). Lane 1, secretory ameloblasts in the molar; lane 2, secretory ameloblasts in the incisor; lane 3, maturation ameloblast in the incisor; lane 4, odontoblasts in the molar; and lane 5, odontoblasts in the incisor. (Panel B) Amelogenin gene expression in erupted first molar odontoblasts. Lane 1: PCR with upstream primer at exon 5 and downstream primer at exon 6; lane 2, PCR with upstream primer at exon 2 and downstream primer at exon 7; and lane 3, PCR with GAPDH-specific primers.
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To confirm amelogenin expression in odontoblasts, we examined another odontoblast sample from the erupted young first molar in the root-forming stage. The odontoblast sample from this developing stage could not be contaminated by ameloblasts or ameloblast RNA, because the tooth had already erupted and had no remaining ameloblasts. PCR was carried out with two sets of amelogenin primers. The amelogenin gene expression was detected by both of the primer sets in this odontoblast sample (Fig. 4B).
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DISCUSSION
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It has previously been reported that odontoblasts remain on the surface of predentin after pulp extraction in human (Gotjamanos and Swedlow, 1974), bovine (Munksgaard and Moe, 1980; Oida et al., 1982), and rat teeth (Munksgaard et al., 1978). We also observed, using light microscopy, the retention of odontoblasts in pig teeth on the surface of predentin following the removal of the dental pulp (Fig. 1C). We obtained highly enriched odontoblasts from porcine teeth on this basis. Secretory ameloblasts, on the other hand, were removed from the developing enamel layer along with the rest of the enamel organ epithelia. Maturation-stage ameloblasts were more strongly adherent to the underlying enamel layer. This enhanced attachment could be mediated by the basal lamina, which is assembled immediately following the transition stage (Takano, 1979).
Analysis of RT-PCR (Fig. 2) and quantitative PCR by LightCycler instrument (Fig. 3) revealed that amelogenin mRNA is expressed in odontoblasts as well as ameloblasts. Analysis of alternative splicing of amelogenin showed that the mRNAs of exon 2-3-5-6-7, exon 2-5-6-7, exon 2-3-5-6D-7 (6D: deletion in exon 6), and exon 2-3-6D-7 were expressed in both the odontoblasts and the ameloblasts (Fig. 4A).
In this study, it was very important to avoid ameloblast and odontoblast cross-contamination while obtaining samples. To confirm the amelogenin expression in odontoblasts, we prepared another odontoblast sample from the erupted young first molar in the root-forming stage. The molar had finished enamel formation, and the ameloblast layer had already disappeared from the tooth. In spite of the absence of ameloblasts, amelogenin PCR products were detected in odontoblast samples (Fig. 4B). Under these conditions, ameloblast contamination could be ruled out.
Quantitative PCR with the LightCycler revealed that odontoblasts expressed considerable amount of amelogenin, but that secretory ameloblasts expressed more than 1000 times as much amelogenin mRNA as the odontoblasts. As can be seen from Fig. 1C, the odontoblast layer was contaminated by very few cells from the adjacent dental pulp. Although we exercised great care in separating secretory ameloblasts from adjoining cells in the enamel organ epithelia, we suspect that there may have been more nearby cells included in our secretory ameloblast preparations than in our odontoblast samples. Therefore, we place 1000 to 1 as the lowest possible ratio of amelogenin expression in secretory ameloblasts relative to odontoblasts, and if adjustments could be made for the inclusion of non-ameloblasts from the enamel organ epithelia (EOE), the ratio might be as high as 1500 to 1. Regardless of the impact of this factor, analysis of our data demonstrates that pig odontoblasts express amelogeninin, but that secretory ameloblasts express amelogenin at a rate over 1000 times that of odontoblasts. This large difference in amelogenin expression between secretory ameloblasts and odontoblasts probably explains why odontoblast expression of amelogenin has not been detected by in situ hybridization. The positive reaction in the odontoblast layer was attributed to background because of the greater than 1000-fold stronger reaction in the adjacent secretory-stage ameloblasts. Recently, Veis et al. (2000) arrived at the same opinion.
We report here that odontoblasts express amelogenin mRNA. Several other enamel proteins, enamelin, sheathlin (ameloblastin and amelin), enamelysin, and EMSP1 are expressed in ameloblasts. We detected the expression of these genes as well as amelogenin in the odontoblast sample (manuscript in preparation). It was considered that enamel proteins secreted by ameloblasts play important roles in mineralization and crystal formation in enamel matrix. Nevertheless, enamel proteins secreted by odontoblasts may be involved in ameloblast differentiation and enamel biomineralization.
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ACKNOWLEDGMENTS
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We thank Dr. J.P. Simmer for critical review of the manuscript. This work was partially supported by grant-in-aid No. 12671818, funding from the Bio-Venture and High Technology Research Centers Project from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
Received for publication June 18, 2001.
Revision received December 17, 2001.
Accepted for publication December 20, 2001.
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Journal of Dental Research, Vol. 81, No. 2,
103-108 (2002)
DOI: 10.1177/154405910208100204
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ABSTRACT
INTRODUCTION