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Abstract
This study was aimed to elucidate the biological function of OD314 (Apin protein), which is related toameloblast differentiation and amelogenesis. Apin protein, calcifying epithelial odontogenic (pindborg) tumors (CEOTs)-associated amyloid, were isolated from CEOTs, and has similar nucleotide sequences to OD314. We examined expression of the OD314 mRNA using in-situ hybridization during tooth development in mice. Expression of OD314 and several enamel matrix proteins were examined in the cultured ameloblast cell line up to 28 days by reverse transcription-polymerase chain reaction (RT-PCR) amplification. After inactivation and over-expression of the OD314 gene in ameloblast cell lines using U6 vector-driven RNA interference and CMV-OD314 construct, RT-PCR were performed to evaluate the effect of the OD314 during amelogenesis.
The results were as follows:
1. In in-situ hybridization, OD314 mRNAs were more strongly expressed in ameloblast than odontoblast.
2. When ameloblast cells were cultured in the differentiation and mineralization medium for 28 days, the tuftelin mRNA expression was maintained from the beginning to day 14, and then gradually decreased to day 28. The expressions of amelogenin and enamelin were gradually decreased according to the ameloblast differentiation.
3. Inactivation of OD314 by U6-OD314 siRNA construct down-regulated the expression of OD314, MMP-20, and tuftelin, whereas over-expression of OD314 by CMV-OD314 construct up-regulated the expression of OD314 and MMP-20 without change in tuftelin.
These results suggest that OD314 is considered as an ameloblast-enriched gene and may play the important roles in ameloblast differentiation and mineralization.
Figures and Tables
Figure 1
Maxillary molar of 1-week-old mouse hybridized with antisense cRNA probes of OD314. Intense signal for OD314 mRNA is detected in ameloblast (arrow).
Figure 3
RT-PCR amplification of enamel matrix proteins in the cultured ameloblast cell line up to 28 days.
Figure 4
RT-PCR amplification of OD314 in ameloblast cell line after over-expression with CMV-OD314 plasmid and inactivation with U6-OD314 siRNA.
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