Expression and function of OD314, Apin protein during ameloblast differentiation and amelogenesis / 대한치과보존학회지

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.

Role of OD314 During Odontoblast Differentiation / 체질인류학회지

Odontoblasts are responsible for the formation and maintenance of dentin which is a mineralized part in dentin-pulp complex of tooth. OD314 was obtained by subtractive hybridization between odontoblasts and osteoblast/dental papilla cells, and differentiatially expressed in the odontoblasts but not in osteoblasts and dental papilla cells. In this study, to better understand the biological function of new odontoblast-enriched gene, OD314, we examined expression of OD314 in cultured MDPC-23 cells and intracellular localization of OD314 protein. We also evaluate the effect of OD314 over-expression and inactivation on the cells by northern analysis. When MDPC-23 cells are cultured in the differentiation and mineralization medium for 28 days, OD314 mRNA expression was gradually increased from the beginning to day 21 and remained relatively high on day 28. Immunofluorescent staining of cultured MDPC-23 revealed localization of OD314 on the cytoplasm, especially near the nuclear membrane. However, a small amount of fluorescence was also observed in the nucleus. Inactivation of OD314 by RNA interference up-regulated the expression of DSPP, whereas over-expression of OD314 by CMV-OD314 plasmid down-regulated the expression of ON. These results suggest that OD314, a odontoblat-enriched gene, may play important roles in the odontoblast differentiation and dentin mineralization.

Expression of OD314 during ameloblast differentiation and maturation / 대한치과보존학회지

Ameloblasts are responsible for the formation and maintenance of enamel which is an epithelially derived protective covering for teeth. Ameloblast differentiation is controlled by sequential epithelial-mesenchymal interactions. However, little is known about the differentiation and maturation mechanisms. OD314 was firstly identified from odontoblasts by subtraction between odontoblast/pulp cells and osteoblast/dental papilla cells, even though OD314 protein was also expressed in ameloblast during tooth formation. In this study, to better understand the biological function of OD314 during amelogenesis, we examined expression of the OD314 mRNA and protein in various stages of ameloblast differentiation using in-situ hybridization and immunohistochemistry. The results were as follows : 1. The ameloblast showed 4 main morphological and functional stages referred to as the presecretory, secretory, smooth-ended, and ruffle-ended. 2. OD314 mRNA was expressed in secretory ameloblast and increased according to the maturation of the cells. 3. OD314 protein was not expressed in presecretory ameloblast but expressed in secretory ameloblast and maturative ameloblast. OD314 protein was distributed in entire cytoplasm of secretory ameloblast. However, OD314 was localized at the proxiamal and distal portion of the cytoplasm of smooth-ended and ruffle-ended ameloblast. These results suggest that OD314 may play important roles in the ameloblast differentiation and maturation.

Inactivation of the OD314 Gene by RNA Interference in Preodontoblast Cell Lines / 체질인류학회지

Tooth development depends on reciprocal interactions between oral epithelium and ectomesenchyme. The ectomesenchyme-derived odontoblasts secrete several collagenous and non-collagenous proteins to form a unique extracellular matrix. OD314 was obtained by subtractive hybridization between odontoblasts and osteoblast/pulp cells, and differentially or predominantly expressed in odontoblasts of rat incisors compared to osteoblasts and pulp cells. However, little is known about the function of OD314 in odontoblast differentiation. In this study, to better understand the function of OD314, we inactivated the OD314 gene in mouse MDPC23 cells using U6 promoter-driven siRNA method. After the characterization of mineralized nodule formation and molecular expression of MDPC23 cell, Inactivation effects of the OD314 were evaluated by RT-PCR and western blot. 1. Mineralized nodule formation was observed after 14 days of culture in MDPC23 cells. 2. The OD314, OC and DSPP mRNA were highly expressed throughout the cultures, while the expression of ON decreased as the MDPC23 cells differentiated. 3. The OD314 protein was weakly expressed at 7 days of culture, but increased gradually as MDPC23 cells reached mineralization stage. 4. The inactivation of OD314 by RNA interference downregulated the expressions of OD314, DSPP, OC, and ON mRNA and OD314 protein in MDPC23 cells. These results suggest that OD314 may play a important role in mineralization process of odontoblast and also regulate odontoblast-related genes such as OC, ON, and DSPP.

Expression and functional characterization of odontoblast-derived gene: OD314 / 대한치과보존학회지

Odontoblasts are responsible for the formation and maintenance of dentin. They are known to synthesize unique gene products including dentin sialophosphoprotein (DSPP). Another unique genes of the cells remain unclear. OD314 was isolated from the odontoblasts/pulp cells of rats and partially characterized as an odontoblast-enriched gene (Dey et al., 2001). This study aimed to elucidate the biological function of OD314, relating to odontoblast differentiation and dentinogenesis. After determining the open reading frame (ORF) of OD314 by transient transfection analysis using green fluorescent protein (GFP) expression vector, mRNA in-situ hybridization, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and western analysis were performed. The results were as follows: 1. In in-situ hybridization, OD314 mRNAs were expressed in odontoblasts of developing coronal and root pulp. 2. OD314 was a novel protein encoding 154 amino acids, and the protein was mainly expressed in cytoplasm by transient transfection analysis. 3. Mineralized nodules were associated with multilayer cell nodules in the culture of human dental pulp cells and first detected from day 21 using alizarin-red S staining. 4. In RT-PCR analysis, OD314, osteocalcin (OC) and DSPP strongly expressed throughout 28 days of culture. Whereas, osteonectin (ON) mRNA expression stayed low up to day 14, and then gradually decreased from day 21. 5. Western blots showed an approximately 17 kDa band. OD314 protein was expressed from the start of culture and then increased greatly from day 21. In conclusion, OD314 is considered as an odontoblast-enriched gene and may play important roles in odontoblast differentiation and dentin mineralization.