RESUMO
The formation of dentin and enamel is initiated by the differentiation of odontogenic precursor cells into odontoblasts and ameloblasts, respectively. This study was performed to identify new molecules involved in the differentiation of odontogenic cells. The genes expressed differentially between the root stage (after the differentiation of odontogenic cells and dental hard-tissue formation) and the cap stage (before the differentiation of odontogenic cells and dental hard-tissue formation) were searched using differential display PCR. For the first time, synaptic vesicle protein (SV) 2b, an important transmembrane transporter of Ca(2+) -stimulated vesicle exocytosis, was identified as a differentially expressed molecule. Real-time PCR and western blotting revealed an increase in the transcriptional and translational levels of SV2b during or after the differentiation of odontogenic cells. Immunofluorescence revealed this molecule to be localized in not only fully differentiated odontoblasts but also in pre-odontoblasts before dentin matrix secretion. The expression pattern of the SV2a isoform was similar to that of the SV2b isoform, whereas the SV2c isoform showed a contrasting pattern of expression. After treatment with alendronate, an inhibitor of protein isoprenylation for the transport of secretory vesicles, the expression of SV2a and SV2b decreased, whereas that of SV2c increased. These results suggest that the SV2 isoforms are functional molecules of (pre)odontoblasts which may be involved in vesicle transport.
Assuntos
Diferenciação Celular/genética , Exocitose/genética , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Odontoblastos/metabolismo , Odontogênese/genética , Vesículas Sinápticas/metabolismo , Germe de Dente/citologia , Alendronato/metabolismo , Animais , Regulação da Expressão Gênica , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Odontoblastos/citologia , Odontogênese/fisiologia , Isoformas de Proteínas/genética , Isoformas de RNA/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Germe de Dente/metabolismoRESUMO
A complex and intricate cascade of gene expression is essential for late stage tooth development. This study was performed to detect molecules involved in dental hard tissue formation and tooth eruption by comparing gene expression in cap stage molar germs (before eruptive movement and dental hard tissue formation) with that in root formation stage molar germs (after eruptive movement and dental hard tissue formation). DD-PCR revealed that cyclophilin A (Cyp-A), a potent chemoattractant for monocytes as well as a ligand for extracellular matrix metalloproteinase inducer (EMMPRIN) was expressed differentially in the two stages molar germs. The levels of Cyp-A and EMMPRIN mRNA were significantly higher at the root formation stage than at the cap and crown stages of the molar germs. Immunofluorescence showed that Cyp-A and EMMPRIN were expressed strongly in the follicular cells overlaying the occlusal region of the molar germs at the root formation stage. In contrast, their immunoreactivity was weak in the follicular tissues and was not region-specific in molar germs at the cap stage. In addition, the MCP-1 and CSF-1 mRNA levels increased in parallel to that of Cyp-A mRNA and the increased number of osteoclasts at the occlusal region. Immunoreactivity against Cyp-A and EMMPRIN was also observed in the fully differentiated ameloblasts and odontoblasts. This study suggests that Cyp-A and EMMPRIN play roles in the maturation of dental hard tissue and the formation of an eruption pathway.