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KDM1A regulated the osteo/dentinogenic differentiation process of the stem cells of the apical papilla via binding with PLOD2.

ABSTRACT: OBJECTIVES:Dental tissue-derived mesenchymal stem cells (MSCs)-mediated pulp-dentin regeneration is considered a potential approach for the regeneration of damaged teeth. Enhancing MSC-mediated pulp-dentin regeneration is based on an understanding of the molecular mechanisms underlying directed cell differentiation process. Histone demethylation enzyme, lysine demethylase 1A (KDM1A) can regulate the differentiation of some MSCs, but its role in dental tissue-derived MSCs is unclear. MATERIAL AND METHODS:We obtained SCAPs from immature teeth. Alkaline phosphatase (ALP) activity assay, Alizarin red staining, quantitative calcium analysis, osteogenesis-related genes expression and in vivo transplantation experiment were used to explore the osteo/dentinogenic differentiation. Co-immunoprecipitation (Co-IP) assay was used to investigate the binding protein. RESULTS:Knock-down of KDM1A reduced ALP activity and mineralization, promoted the expressions of osteo/dentinogenic differentiation markers DSPP, DMP1, BSP and key transcript factors, RUNX2, OSX, DLX2 in SCAPs, and also enhanced the osteo/dentinogenesis in vivo. In addition, KDM1A could associate with PLOD2 to form protein complex. And knock-down of PLOD2 inhibited ALP activity and mineralization, and promoted the expressions of DSPP, DMP1, BSP, RUNX2, OSX and DLX2 in SCAPs. CONCLUSIONS:KDM1A might have different role in different stages of osteo/dentinogenic differentiation process by binding partner with PLOD2, and finally resulted in the inhibited function for the osteo/dentinogenesis in SCAPs. Our studies provided a further understanding of the regulatory mechanisms of dynamic osteo/dentinogenic differentiation process in dental tissue MSCs.

SUBMITTER: Wang L

PROVIDER: S-EPMC6528894 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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KDM1A regulated the osteo/dentinogenic differentiation process of the stem cells of the apical papilla via binding with PLOD2.

Wang Lijun L Yang Haoqing H Lin Xiao X Cao Yangyang Y Gao Peipei P Zheng Ying Y Fan Zhipeng Z

Cell proliferation 20180415 4

<h4>Objectives</h4>Dental tissue-derived mesenchymal stem cells (MSCs)-mediated pulp-dentin regeneration is considered a potential approach for the regeneration of damaged teeth. Enhancing MSC-mediated pulp-dentin regeneration is based on an understanding of the molecular mechanisms underlying directed cell differentiation process. Histone demethylation enzyme, lysine demethylase 1A (KDM1A) can regulate the differentiation of some MSCs, but its role in dental tissue-derived MSCs is unclear.<h4>M ...[more]

PMID: 29656462

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Project description:Stem cells from apical papilla (SCAP) possess clear osteo‑/odontogenic differentiation capabilities, and are regarded as the major cellular source for root dentin development. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) serve pivotal roles in the modulation of tooth development and dentin formation. However, the synergistic effects of BMP2 and VEGF on osteo‑/odontogenic differentiation of SCAP remain unclear. The current study aimed to investigate the proliferative and osteo‑/odontogenic differentiating capabilities of BMP2 and VEGF gene-co-transfected SCAP (SCAP-BMP2-VEGF) in vitro. The basic characteristics of the isolated SCAP were identified by the induction of multipotent differentiation and by flow cytometry. Lentiviral vector‑mediated gene transfection was conducted with SCAP in order to construct blank vector‑transfected SCAP (SCAP-green fluorescent protein), BMP2 gene-transfected SCAP (SCAP-BMP2), VEGF gene‑transfected SCAP (SCAP‑VEGF) and SCAP-BMP2-VEGF. The Cell Counting Kit 8 assay was used to analyze the proliferative capacities of the four groups of cells. The expression of osteo-/odontogenic genes and proteins in the cells were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. The mineralized nodules formed by the four group cells were visualized by alkaline phosphatase (ALP) staining. Among the four groups of cells, SCAP‑VEGF was demonstrated to exhibit increased proliferation, and SCAP‑BMP2‑VEGF exhibited reduced proliferation during eight days observation. SCAP‑BMP2‑VEGF exhibited significantly increased expression levels of ALP, osteocalcin, dentin sialophosphoprotein, dentin matrix acidic phosphoprotein gene 1 and dentin sialoprotein than the other three groups at the majority of the time points. Furthermore, the SCAP‑BMP2‑VEGF group exhibited a significantly greater number of ALP‑positive mineralized nodules than the other groups following 16 days culture in vitro. In conclusion, lentiviral vector-mediated BMP2 and VEGF gene co-transfection significantly activated the osteo‑/odontogenic differentiation of human SCAP.

Project description:BackgroundStem cells from apical papilla (SCAP) are a subpopulation of mesenchymal stem cells (MSCs) isolated from the apical papilla of the developing tooth root apex of human teeth. Because of their osteogenic/dentinogenic capacity, SCAP are considered as a source for bone and dentin regeneration. However, little is understood about the molecular mechanism of osteogenic/dentinogenic differentiation of SCAP. Phosphoinositide 3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) signal pathway participates in regulating the differentiation of various cell types, such as MSCs. In this study, we examined the role of the PI3K-AKT-mTOR signal pathway in the osteogenic/dentinogenic differentiation of SCAP. Moreover, we challenge to fabricate scaffold-free SCAP-based spheroidal calcified constructs.MethodsSCAP were pretreated with or without small interfering RNA for AKT (AKT siRNA), PI3K inhibitor LY294402, and mTOR inhibitor rapamycin and were cultured under osteogenic/dentinogenic differentiation to examine in vitro and in vivo calcified tissue formation. Moreover, SCAP-based cell aggregates were pretreated with or without LY294402 and rapamycin. The cell aggregates were cultured under osteogenic/dentinogenic condition and were analyzed the calcification of the aggregates.ResultsPretreatment with AKT siRNA, LY294402, and rapamycin enhances the in vitro and in vivo calcified tissue-forming capacity of SCAP. SCAP were fabricated as scaffold-free spheroids and were induced into forming calcified 3D constructs. The calcified density of the spheroidal constructs was enhanced when the spheroids were pretreated with LY294402 and rapamycin.ConclusionsOur findings indicate that the suppression of PI3K-AKT-mTOR signal pathway plays a role in not only enhancing the in vivo and in vitro osteogenic/dentinogenic differentiation of SCAP, but also promoting the calcification of scaffold-free SCAP-based calcified constructs. These findings suggest that a suppressive regulation of PI3K-AKT-mTOR signal pathway is a novel approach for SCAP-based bone and dentin regeneration.

Dataset Information

KDM1A regulated the osteo/dentinogenic differentiation process of the stem cells of the apical papilla via binding with PLOD2.

Publications

KDM1A regulated the osteo/dentinogenic differentiation process of the stem cells of the apical papilla via binding with PLOD2.

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