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Relative Levels of mRNA Encoding Enamel Proteins in Enamel Organ Epithelia and Odontoblasts

¹ Department of Periodontics and Endodontics and
² Department of Biochemistry, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan;

*corresponding author, nagano-takatoshi{at}tsurumi-u.ac.jp

	ABSTRACT

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Amelogenin, enamelin, sheathlin (ameloblastin/ amelin), enamelysin (MMP-20), and KLK4 (EMSP-1) are the major structural proteins and proteinases in developing tooth enamel. Recently, odontoblasts were reported to express amelogenin, the most abundant enamel protein. In this study, we hypothesized that odontoblasts express all enamel proteins and proteases, and we measured their relative mRNA levels in enamel organ epithelia and odontoblasts associated with porcine secretory- and maturation-stage enamel by RT-PCR, using a LightCycler instrument. The results showed that amelogenin mRNA in secretory-stage EOE is 320-fold higher than in odontoblasts beneath secretory-stage enamel, and over 20,000-fold higher than in odontoblasts under maturation-stage enamel. Similar results were obtained for enamelin and sheathlin. Enamelysin mRNA levels were equivalent in these two tissues, while KLK4 mRNA was higher in odontoblasts than in secretory-stage EOE. These results support the conclusion that odontoblasts are involved in the formation of the enamel layer adjacent to enamel-dentin junction.

KEY WORDS: amelogenin • enamelysin • KLK4 • odontoblasts • ameloblasts

	INTRODUCTION

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During amelogenesis, enamel proteins and their cleavage products form the enamel matrix and are involved in controlling crystal growth. Amelogenin is the most abundant enamel protein during the secretory stage of amelogenesis (Termine et al., 1980). Enamelin is the largest glycoprotein secreted into the matrix (Hu et al., 1997b), while sheathlin cleavage products (Fukae and Tanabe, 1987) concentrate in the sheath space between the rod and interrod enamel (Uchida et al., 1995; Hu et al., 1997a). Amelin (Cerny et al., 1996) and ameloblastin (Krebsbach et al., 1996), first cloned from rat-tooth-specific cDNA libraries, are homologs of porcine sheathlin.

With the technique of in situ hybridization, amelogenin mRNA can be clearly detected in pre-ameloblasts, with the maximum signal being observed in secretory-stage ameloblasts (Inage et al., 1996). Similarly, enamelin signal is detected in ameloblasts but not in odontoblasts or dental pulp (CC Hu et al., 2000). Sheathlin signal is detected in pre-odontoblasts, polarizing odontoblasts, and ameloblasts (Bègue-Kirn et al., 1998). Recently, it was proved by reverse-transcription/polymerase chain-reaction (RT-PCR), with a LightCycler instrument, that the amelogenin mRNA is expressed by odontoblasts, even in erupted young first molar in the root-forming stage (Oida et al., 2002).

There are two proteinases—enamelysin (MMP-20) (Bartlett et al., 1996; Fukae et al., 1998) and kallikrein-4 (KLK4), also known as enamel matrix serine proteinase-1 (EMSP-1) (Simmer et al., 1998)—involved in the degradation of amelogenin. Enamelysin participates in the conversion from 25-kDa amelogenin to 20-kDa amelogenin (Fukae and Tanabe, 1998). KLK4 is a proteolytic enzyme that was first detected in porcine enamel (Fukae et al., 1977), and degrades the 20-kDa amelogenin into two fragments, corresponding to the 6-kDa and 13-kDa amelogenins (Shimizu and Fukae, 1983). Enamelysin mRNA has been detected in odontoblasts and ameloblasts of the enamel organ by in situ hybridization (Bègue-Kirn et al., 1998; Fukae et al., 1998; Caterina et al., 1999). KLK4 mRNA signal was detected in the ameloblasts during the early maturation stage by in situ hybridization (JC Hu et al., 2000). Localization of mRNA encoding enamel proteins and proteinases in the developmental tooth germs by in situ hybridization may miss low levels of expression in tissues adjacent to those having high levels of expression, since development times must be reduced to prevent overexposure of the signal in the high-level tissue. In this study, we examined, semi-quantitatively, enamel protein mRNA levels in several cell layers at different developmental stages of porcine permanent tooth germs.

	MATERIALS & METHODS

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All experimental procedures involving the use of animals was reviewed and approved by the Institutional Animal Care Program at the Tsurumi University.

Isolation of Tissues
Fresh permanent tooth germs were dissected from six-month-old porcine mandibles purchased from a local slaughterhouse. We prepared various cell layers according to the schematic drawing shown in Fig. 1. The secretory and maturation enamel organ epithelia (EOE) layers were collected separately from the labial site of the developing incisor enamel (Oida et al., 2002). We also prepared 2 odontoblast cell layers, taking care to avoid contamination by ameloblasts. Following removal of the EOE and dental pulp (odontoblasts remain attached to the hard tissue when the dental papilla is removed), we divided the tooth into 2 pieces at the position of the transition-stage enamel by cutting it with a dental diamond disc attached to a dental engine (Morita Corporation, Tokyo, Japan). Odontoblasts lining dentin (Oida et al., 2002) under the secretory and maturation stages of developmental enamel were collected separately by being washed with a phosphate-buffered solution and being scraped with a micro-spatula; these were designated ’young’ and ’mature’ odontoblasts, respectively. Two additional samples were prepared from the removed dental pulp. The surface-side cells were collected as the pre-odontoblast layer, and the remaining pulp tissue was collected as dental pulp cells.

View larger version (44K): [in this window] [in a new window]   Figure 1. Schematic drawing of ameloblast and odontoblast tissue preparations. SA, secretory ameloblast (enamel organ epithelia; EOE) layer; MA, maturation ameloblast (EOE) layer; YO, young odontoblast layer; MO, mature odontoblast layer.

RNA Preparation
Total RNA of each cell layer sample was obtained with the Stratagene Total RNA Miniprep Kit and protocol (Stratagene, La Jolla, CA, USA).

Reverse-transcription/Polymerase Chain-reaction (RT-PCR)
Single-strand cDNA was prepared from 3 µg of each total RNA sample by means of a Pharmacia Ready-to-go T-primed first kit and protocol (Amersham-Pharmacia Biotech, Piscataway, NJ, USA). PCR conditions (Perkin-Elmer/GeneAmp PCR system 9600) were as follows: PCR began with a 10-minute denaturation at 94°C, followed by 25 cycles with denaturation at 94°C for 30 sec, primer annealing at 55°C for 30 sec, and product extension at 72°C for 30 sec; in the final cycle, the 72°C extension was for 7 min. PCR products were analyzed by 4.5% polyacrylamide gel electrophoresis in TBE buffer (pH 8.0). Gels were stained with ethidium bromide, and the bands were visualized under UV light. Additionally, the PCR products were cloned into pBluescriptIISK(+) (Stratagene, La Jolla, CA, USA), and their nucleotide sequences were determined by cycle sequencing.

Semi-quantitative PCR with the LightCycler Instrument
Specific primers of enamel proteins and proteinases were designed based on porcine and mouse mRNA sequences. The amelogenin sense and anti-sense primers were: 5’ ACCCCTCTGAAGTGGTACCAG 3’ and 5’ TGTTGGGTTGGAGTCATGGAG 3’, which generated a 236-bp amplification product. The enamelin sense and anti-sense primers were: 5’ AAGAACTGCTGGCCTTACTCC 3’ and 5’ TCCCCAAAGAATGTTTGGTTG 3’, which generated a 433-bp amplification product. The sheathlin sense and anti-sense primers were: 5’ CAGAGGTAGCATCTGGGTGTCC 3’ and 5’ CTCTCAGGGCTCTTGGAAACG 3’, which generated a 320-bp amplification product. The enamelysin sense and anti-sense primers were: 5’ ATACGTGCAGCGAATAGATGC 3’ and 5’ CTATTTAGCAACCAATCCAGG 3', which generated a 290-bp amplification product. The KLK4 sense and anti-sense primers were: 5’ ACCATGGGCGAGGACTGCAA 3’ and 5’ GTTAACTGGCCTGGATGGTCG 3’, which generated a 673-bp amplification product. A primer set amplifying glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Clontech, Palo Alto, CA, USA) was used as a control.

The cDNAs generated from each EOE and odontoblast sample were amplified by means of the DNA Master SYBR Green I kit and protocol and a LightCycler instrument (Roche Molecular Biochemicals, Mannheim, Germany). The relative amount of each mRNA was determined at 50% levels of PCR product, and normalized with use of the relative amount of GAPDH mRNA.

	RESULTS

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To examine the relative levels of mRNA encoding enamel proteins and their related proteinases in the various tissues in developing teeth, we prepared several cell layer samples from the soft tissues surrounding the immature permanent tooth germs (Fig. 1). It was shown histologically that the secretory-stage EOE sample contained secretory ameloblasts, from 4 to 6 cell layers of stratum intermedium-like cells, and a thick layer of stellate reticulum. In contrast, the odontoblast samples contained mostly odontoblasts (Oida et al., 2002; data not shown). Fig. 2 shows PCR products of amelogenin, enamelin, sheathlin, enamelysin, and KLK4, after 25 cycles of standard PCR, normalized to GAPDH, and then visualized on polyacrylamide gels stained with ethidium bromide. The mRNAs encoding the structural enamel proteins were found more or less in both the EOE and odontoblast samples. Although some of these mRNAs were not detected by gel electrophoresis, their presence in these tissues was evident when the LightCycler system was used (Fig. 3). However, the PCR products of enamel structural proteins were not detected in the pre-odontoblast cell layers (PO) and dental pulp cells (DP) after as many as 40 cycles of PCR (data not shown). The DSPP PCR product, which was the marker for odontoblasts, was found only in young and mature odontoblast layer samples (data not shown). DNA sequencing of the PCR products from the EOE and odontoblast samples proved that they were the appropriate gene products (data not shown).

View larger version (59K): [in this window] [in a new window]   Figure 2. Detection of mRNAs encoding GAPDH, amelogenin, enamelin, sheathlin, enamelysin, and KLK4 after 25 cycles of RT-PCR. Amelogenin, enamelin, sheathlin, enamelysin, and KLK4 PCR products were amplified from the cDNA template and separated by electrophoresis on a 4.5% polyacrylamide gel. M, molecular-size standard, PhiX174 DNA-Hae III (New England Biolabs); SA, secretory ameloblast layer; MA, maturation ameloblast layer; YO, young odontoblast layer; MO, mature odontoblast layer; PO, pre-odontoblast layer; DP, dental pulp cells; EO, erupted first molar odontblast layer.

View larger version (32K): [in this window] [in a new window]   Figure 3. Quantitative PCR of amelogenin, enamelin, sheathlin, enamelysin, KLK4, and GAPDH mRNAs isolated from the various cell layer samples. SA, secretory ameloblast layer; MA, maturation ameloblast layer; YO, young odontoblast layer; MO, mature odontoblast layer. The most typical pattern was shown. The vertical axis shows relative amounts, and the horizontal axis shows cycles.

View larger version (31K): [in this window] [in a new window]   Figure 4. The relative amounts of amelogenin (A), enamelin (B), sheathlin (C), enamelysin (D), and KLK4 (E) mRNA in the cell layer samples, after normalization with the amounts of the GAPDH mRNA. SA, secretory ameloblast layer; MA, maturation ameloblast layer; YO, young odontoblast layer; MO, mature odontoblast layer. The vertical axis shows relative amounts. The results were expressed as means ± SD of three samples.

	DISCUSSION

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If we assume that ameloblasts contributed 20% of the GAPDH, and that, among the EOE cells, only ameloblasts expressed enamel proteins and proteinases, then we would estimate from our results that the ratios of amelogenin mRNA in secretory-stage ameloblasts relative to odontoblasts lining dentin under the secretory-stage enamel and maturation-stage enamel would be 320:1 and 20,480:1, respectively. It is expected that similar adjustments are needed to estimate enamelin and sheathlin mRNA levels in ameloblasts relative to odontoblasts.

This study demonstrates that mRNAs encoding all enamel proteins and proteinases are expressed by odontoblasts. Previously, we demonstrated that odontoblasts splice amelogenin differently than do ameloblasts, and we used this difference to demonstrate that our dissection technique did not allow odontoblasts to be contaminated with ameloblasts (Oida et al., 2002). In addition, we prepared another odontoblast sample from an erupted young first molar in the root-forming stage. The molar had finished enamel formation, and the ameloblasts disappeared from the tooth. In spite of the absence of ameloblasts, amelogenin (Oida et al., 2002), enamelin, sheathlin, enamelysin, and KLK4 PCR products were detected in this odontoblast sample (Fig. 2). The expression of amelogenins by odontoblasts explains the histochemical detection of amelogenin in the dentin matrix and odontoblastic processes, as was observed previously (Uchida et al., 1991). The function of amelogenin in the dentin matrix is unknown at present.

Enamelysin and KLK4 were expressed by both ameloblasts and odontoblasts. Enamelysin mRNA expression was 16-fold higher in secretory EOE relative to maturation EOE. For KLK4, this trend was reversed: KLK4 mRNA expression in maturation EOE was 32-fold higher than in secretory EOE. In contrast, changes in enamelysin and KLK4 mRNA levels in odontoblasts were fairly constant, both dropping about 25% in older odontoblasts relative to younger odontoblasts. Enamelysin mRNA levels in secretory EOE were about half those of the odontoblasts, suggesting that secretory ameloblasts specifically may express enamelysin mRNA at slightly higher levels than odontoblasts. KLK4 mRNA levels were twice as high in maturation EOE as in odontoblasts, suggesting that maturation ameloblasts specifically may express KLK4 mRNA at levels 10-fold higher than in odontoblasts. In contrast, KLK4 mRNA in secretory-stage EOE was much lower than in younger and older odontoblasts, indicating that KLK4 mRNA is expressed more in odontoblasts than in the secretory ameloblasts, even when one takes into account the number of cells other than ameloblasts in the secretory EOE sample.

We conclude from this study that, in the pig, secretory ameloblasts express mRNAs encoding enamel structural proteins at levels thousands of times higher than do maturation ameloblasts and odontoblasts lining dentin beneath maturing enamel, and hundreds of times higher than do odontoblasts lining dentin beneath secretory-stage enamel. Younger odontoblasts lining dentin closer to the EDJ express mRNAs encoding enamel structural proteins at levels over 30-fold higher than do older odontoblasts forming dentin further from the EDJ. These findings suggest that enamel structural proteins secreted by odontoblasts may play some part in the formation of dentin, particularly dentin forming at the EDJ. Enamelysin and KLK4 are expressed by both ameloblasts and odontoblasts. Recently, KLK4 activity was detected in the highly mineralized enamel at the EDJ during the secretory stage (Fukae et al., 2002), despite the fact that no KLK4 activity was observed in the inner-layer enamel (Fukae et al., 2001), pre-dentin, or dentin (Fukae et al., 2002). KLK4 activity in the deepest inner-layer enamel during the secretory stage is probably secreted through the odontoblast dental tubules at the EDJ, where it degrades organic matrix to be replaced by mineral.

	ACKNOWLEDGMENTS

 
We thank all the members of our labs, especially Dr. T. Tanabe, Dr. Y. Yamakoshi, and Dr. T. Iwata. This study was supported by a grant-in-aid (No. 12671818) for funding High Technology Research Centers from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Received October 15, 2002; Last revision July 9, 2003; Accepted September 12, 2003

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