RESUMO
Runx2, of the Runx family, is an essential transcription factor that controls bone and tooth development by regulating osteoblast and odontoblast differentiation. However, the function of Runx2 in late stage odontoblast differentiation is not clear. We studied the function of Runx2 in dentinogenesis by generating transgenic mice expressing Runx2 specifically in odontoblasts. We observed dentin formation in postnatal day 3 (P3), P7 and P28 mice and measured the expression levels of Runx2 and matrix proteins in dentin. The odontoblasts in transgenic mice (Tg) lost their tall columnar shape and polarization and dentinal tubules were absent. The dental pulp chamber was dramatically enlarged and the dentin in Tg mice was thinner. Osteoblast-like cells were seen instead of normal odontoblasts and were embedded in a bone-like matrix, indicating that dentin formation was replaced with bone. Predentin was disorganized possessing lacunae that contained odontoblasts. The mandibular molars of Tg mice showed noticeable defects by Micro-CT. Using quantitative real-time PCR, the expression of dentin matrix proteins, particularly dentin sialophosphoprotein (DSPP), was found to be upregulated in 3-day-old Tg mice and downregulated at 1 month of age. These findings indicate that Runx2 inhibited odontoblast terminal differentiation and induced transdifferentiation of odontoblasts to osteoblasts at the late cell differentiation stage. Therefore, Runx2 should be inhibited in odontoblasts to encourage normal cell maturation, differentiation and dentinogenesis.
Assuntos
Diferenciação Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Dentina/crescimento & desenvolvimento , Dentinogênese/genética , Odontoblastos/citologia , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Camundongos , Camundongos Transgênicos , Dente Molar/crescimento & desenvolvimento , Dente Molar/metabolismo , Dente Molar/ultraestruturaRESUMO
Amelogenin (AMG) is a highly conserved protein secreted by ameloblasts. Some research indicates that AMG might induce the differentiation and maturation of odontoblasts. The aim of this study was to clarify the function of AMG during the differentiation of odontoblast-like MDPC-23 cells. The results revealed that the alkaline phosphatase activity and the number of mineralized nodules were significantly enhanced in AMG-overexpressing MDPC-23 cells during the mineralization process. Tissue-specific markers such as dentin matrix protein 1 and dentin sialophosphoprotein also elevated significantly, indicating the cell differentiation and maturation process. Furthermore, AMG could upregulate the phosphorylation levels of ERK1/2 and p38 MAPK. However, JNK, another MAPK pathway molecule, didn't change the activity at all. And the differentiation induced by AMG was abrogated when the MDPC-23 cells were treated with U0126 and SB203580, the inhibitors of ERK1/2 and p38, respectively. Taken together, our present results showed that AMG could promote the differentiation of odontoblast-like MDPC-23 cells via ERK1/2 and p38 MAPK pathways.
Assuntos
Amelogenina/metabolismo , Diferenciação Celular , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Odontoblastos/citologia , Proteínas Recombinantes/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adenoviridae , Fosfatase Alcalina/metabolismo , Amelogenina/genética , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Linhagem Celular , Clonagem Molecular , Ensaios Enzimáticos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Expressão Gênica , Vetores Genéticos , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismoRESUMO
OBJECTIVE: Notch signalling controls cell fate decisions in adult and embryonic tissues. The Notch ligand Delta1 is known to influence proliferation and differentiation of many kinds of tissue specific stem cells. In the present study, we investigated the role of Delta1 in the regulation of dental pulp stem cells (DPSCs) in vitro. METHODS: DPSCs were isolated from impacted third molars. Expression of human Notch1, 2 and Delta1 in DPSCs were detected by immunochemistry. Delta1 overexpressed DPSCs were constructed by a retroviral method. Delta1 transduced DPSCs proliferation changes were examined by means of colony-forming assay, BrdU incorporation assay and cell cycle analysis. Delta1 transduced DPSCs were cultured in differentiation-inductive medium. The nodule formation and DSPP expression were evaluated. RESULTS: It was shown that the Notch receptors and Delta1 ligand were expressed throughout the proliferation and differentiation process of cultured dental pulp stem cells. Furthermore, it was found in our study that Delta1 could significantly enhance the proliferation of DPSCs and permit DPSCs differentiating into odontoblast-like cells in differentiation-inductive environments. CONCLUSIONS: Our findings verified that Notch-Delta1 signalling was expressed in human DPSCs in vitro and appeared to play pivotal role in DPSCs proliferation enhancement and differentiation regulation, thereby consistent with the hypothesis that the Notch pathway controls stem cell fate during pulp regeneration.