J Korean Acad Conserv Dent.  2004 Jul;29(4):399-408. 10.5395/JKACD.2004.29.4.399.

Expression and functional characterization of odontoblast-derived gene: OD314

• Kim DH
• Kim HJ
• Jeong MJ
• Son HH
• Park JC

• KMID: 2175634
• DOI: http://doi.org/10.5395/JKACD.2004.29.4.399

Abstract

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.

Keyword

Odontoblast; OD314; Dentin; Differentiation; Mineralization

MeSH Terms

Amino Acids
Animals
Blotting, Western
Cytoplasm
Dental Pulp
Dentin
Dentinogenesis
Humans
Immunohistochemistry
Odontoblasts
Open Reading Frames
Osteocalcin
Osteonectin
Rats
RNA, Messenger
Transfection
Amino Acids
Osteocalcin
Osteonectin
RNA, Messenger

Figure

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  Figure 1 OD314 protein expression in short-ORF transfected HEK 293T cells (× 400). OD314 protein are expressed in the cytoplasm of the HEK 293T cell.

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  Figure 2 The nucleotide sequence of cDNA and deduced amino acid sequence of OD314. Contains 1197 nucleotides in all. The poly a tail is preceded by the polya (A+) signal AATAAA (italics). The start codon ATG is in bold type and begins from nucleotide 423. The stop codon at nucleotide 884 is also in bold denoting the end of the open reading frame (ORF).

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  Figure 3 In-situ hybridization for OD314 in rat mandibular first molar at postnatal day 7. Arrows indicate the OD314 mRNA in the developing odontoblasts. × 100.

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  Figure 4 Expression of mRNA for OD314 in rat mandibular first molar at postnatal day 7. OD314 transcripts are strongly expressed in the developing odontoblasts (arrows). D; dentin, P; pulp, Alv, alveolar bone. × 100.

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  Figure 5 Negative control of in-situ hybridization. No positive signals were found in this section. × 100.

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  Figure 6 Immunohistochemical localization of OD314 protein in rat mandibular first molar at postnatal day 7. Arrows indicate the OD314 protein in the developing odontoblasts. P, pulp; Alv, alveolar bone. × 100.

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  Figure 7 Immunohistochemical localization of OD314 protein in rat mandibular first molar at postnatal day 40. OD314 proteins are strongly expressed in subodontoblasts (arrows). D, dentin; P, pulp; PDL, periodontal ligament; Alv, alveolar bone. × 100.

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  Figure 8 Human dental pulp cells after 4 days of culture. × 100.

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  Figure 9 Human dental pulp cells after 21 days of culture. Formation of mineralization nodules in human dental pulp cells in vitro after β-glycerophosphate treatment. × 200.

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  Figure 10 Visualization of mineralization nodules by Alizarin-red S stain after 21 days of culture. × 200.

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  Figure 11 RT-PCR amplification of OD314, DSPP ON, and OC transcripts in the culture of human dental pulp cells. Total RNAs were extracted from cultured cells after 0, 4, 7, 14, 21, and 28 days of culture. GAPDH used as a control. Sizes are indicated at right. DSPP, dentin sialophosphpprotein; ON, osteonectin; OC, osteocalcin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Figure 12 Temporal changes in the translational activity of OD314 in the culture of human dental pulp cells. Proteins were extracted from cultured cells after 0, 4, 7, 14, 21, and 28 days and analyzed by immunoblotting with anti-OD314. OD314 was expressed as 17 kDa protein in cultured cells.

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