Author: 〇傅 堯1、宮崎 佳奈子1、吉崎 恵悟1、千葉 雄太2、鮒田 啓太1、田 甜1、湯田 智美1、水田 敢士1、福本 敏2、高橋 一郎1
Affiliation: 1九大 院歯 矯正、2九大 院歯 小児口腔
Abstract:
Purpose: Lipid rafts are composed of glycosylphosphatidylinositol (GPI) anchors and transmembrane proteins, and are localized in the cell membrane. It is known that the lipid raft plays important roles as an organizing center for signal transduction and are essential for organogenesis. However, the function of lipid rafts in tooth development is unclear yet. In this study, we analyzed the expression pattern and the function of GPI-anchored protein LY6/PLAUR domain 1 (Lypd1) as the lipid raft component in tooth development.Materials & Methods: We performed single-cell RNA-seq (scRNA-seq) analysis using embryonic day (E)16 tooth germ. qRT-PCR was conducted using organs from E16 ICR mice and molars from each stage of tooth development. In addition, immunohistochemistry (IHC) and in situ hybridization (ISH) were performed using E15 molar and postnatal day (P)1 incisor to confirm the localization of Lypd1. A lipid raft inhibitor Methyl-&beta-cyclodextrin (M&betaCD) is applied in tooth germ organ culture system. Furthermore, Lypd1 siRNA were transfected into mouse dental mesenchymal cell line (mDP) and its differentiation was analyzed.Results & Conclusion: We analyzed scRNA-seq using mouse embryonic tooth germs and found that Lypd1 was specifically detected in the pre-odontoblast cluster. Lypd1 has been reported to play an important role in tumorigenesis, anxiety control and angiogenesis as a GPI anchor, while the mechanisms of functions in tooth development was not understood. qRT-PCR results showed that Lypd1 was highly expressed in the brain and teeth, especially in mesenchymal cells in teeth germ. The expression of Lypd1 is increased during differentiation stages of odontoblasts. IHC and ISH revealed that Lypd1 was localized in the pre-odontoblasts and in the odontoblasts.To clarify the role of Lypd1 on tooth germ formation, we conducted organ culture method. The expression of dentin sialophosphoprotein (DSPP) and dentin matrix acidic phosphoprotein 1 (DMP1) were downregulated in Lypd1 siRNA transfected to tooth germs. Similarly, M&betaCD inhibited tooth germ formation in dose-dependent manner. We also confirmed that DSPP and DMP1 were downregulated in mDP cells transfected with Lypd1 siRNA. In conclusion, Lypd1 is specifically expressed in pre-odontoblasts and regulates odontoblast differentiation. Lipid rafts may play important roles in tooth germ formation.
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