The Affiliated Hospital of Stomatology, School of Stomatology, Zhejiang University School of Medicine, and Key Laboratory of Oral Biomedical Research of Zhejiang Province, Hangzhou, China.
Int Endod J. 2021 Apr;54(4):572-584. doi: 10.1111/iej.13447. Epub 2021 Jan 16.
To investigate the potential role of Krüppel-like factor 6 (KLF6) in the odontoblastic differentiation of immortalized dental papilla mesenchymal cells (iMDP-3) cells.
Alizarin Red S (ARS) and Alkaline phosphatase (ALP) staining was used to examine the mineralization effect of iMDP-3 cells after odontoblastic induction. Real-time PCR and Western blotting were employed to analyse dentine sialophosphoprotein (DSPP), dentine matrix protein 1 (DMP1), RUNX family transcription factor 2 (RUNX2), ALP and KLF6 expression during this process. Co-expression of the KLF6 with DMP1, DSPP and RUNX2 was detected by double immunofluorescence staining to explore their local relationship in the cell. To further investigate KLF6 functions, Klf6 gain- and loss-of-function assays followed by ARS and ALP stainings, real-time PCR and Western blotting were performed using Klf6-overexpression plasmids and Klf6 siRNA to investigate whether changes in Klf6 expression affect the odontoblastic differentiation of iMDP-3 cells. Dual-luciferase reporter assays were used to elucidate the mechanistic regulation of Dspp and Dmp1 expression by Klf6. Means were compared using the unpaired t-test and Kruskal-Wallis one-way anova with P < 0.05 and P < 0.01 defined as statistical significance levels.
The expression levels of Klf6 (P < 0.01), Dspp (P < 0.05), Dmp1 (P < 0.01), Runx2 (P < 0.01) and Alp (P < 0.01) were significantly elevated during odontoblastic differentiation of iMDP-3 cells. KLF6 was co-localized with DSPP, DMP1 and RUNX2 in the cytoplasm and nucleus of iMDP-3 cells. Overexpression of Klf6 promoted the odontoblastic differentiation of iMDP-3, whereas the inhibition of Klf6 prevented this procession. Dual-luciferase assays revealed that Klf6 upregulates Dspp and Dmp1 transcription in iMDP-3 cells during odontoblastic differentiation.
Klf6 promoted odontoblastic differentiation by targeting the transcription promoter of Dmp1 and Dspp. This study may offer novel insights into strategies for treating injuries to dental pulp tissue.
探讨 Krüppel 样因子 6（KLF6）在永生化牙髓间充质细胞（iMDP-3）成牙本质分化中的潜在作用。
采用茜素红 S（ARS）和碱性磷酸酶（ALP）染色检测 iMDP-3 细胞成牙本质诱导后的矿化效果。实时 PCR 和 Western blot 分析在此过程中牙本质涎磷蛋白（DSPP）、牙本质基质蛋白 1（DMP1）、RUNX 家族转录因子 2（RUNX2）、ALP 和 KLF6 的表达。通过双免疫荧光染色检测 KLF6 与 DMP1、DSPP 和 RUNX2 的共表达，探讨它们在细胞内的局部关系。为了进一步研究 KLF6 的功能，通过 Klf6 过表达质粒和 Klf6 siRNA 进行 Klf6 增益和缺失功能测定以及 ARS 和 ALP 染色、实时 PCR 和 Western blot，研究 Klf6 表达变化是否影响 iMDP-3 细胞的成牙本质分化。双荧光素酶报告基因实验阐明了 Klf6 对 Dspp 和 Dmp1 表达的调控机制。采用独立样本 t 检验和 Kruskal-Wallis 单向方差分析比较均值，P 值<0.05 和 P 值<0.01 定义为统计学显著性水平。
iMDP-3 细胞成牙本质分化过程中 Klf6（P<0.01）、Dspp（P<0.05）、Dmp1（P<0.01）、Runx2（P<0.01）和 Alp（P<0.01）的表达水平显著升高。KLF6 与 iMDP-3 细胞质和细胞核中的 DSPP、DMP1 和 RUNX2 共定位。Klf6 的过表达促进了 iMDP-3 的成牙本质分化，而 Klf6 的抑制则阻止了这一过程。双荧光素酶实验显示，Klf6 在 iMDP-3 细胞成牙本质分化过程中上调 Dspp 和 Dmp1 的转录。
Klf6 通过靶向 Dmp1 和 Dspp 的转录启动子促进成牙本质分化。本研究为牙髓组织损伤的治疗策略提供了新的思路。
