KLF9 通过负调控 Notch1 介导的信号通路促进牙髓干细胞的成骨分化。

KLF9 Promotes Osteogenic Differentiation of Dental Stem Cells by Negatively Regulating Notch1 Mediated Signaling Pathway.

机构信息

Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University & Department of Endodontic, Affiliated Hospital of Stomatology, Nanjing Medical University, 211166 Nanjing, Jiangsu, China.

Institute of Stomatology, Nanjing Medical University, 211166 Nanjing, Jiangsu, China.

出版信息

Front Biosci (Landmark Ed). 2023 May 6;28(5):85. doi: 10.31083/j.fbl2805085.

DOI:10.31083/j.fbl2805085

PMID:37258472

Abstract

BACKGROUND

Human dental stem cells (DSCs) are excellent sources of cells for treating dental and craniofacial diseases. However, the mechanisms regulating DSC osteogenic differentiation are still unclear. In this study, we aimed to determine the role of Krüppel-like factor 9 (KLF9) in regulating the biological functions of DSCs and explore the underlying molecular mechanisms.

METHODS

Bioinformatic analyses, quantitative real-time polymerase chain reaction (qRT‒PCR) and Western blotting were performed to determine the KLF9 level during osteogenic differentiation of DSCs. The effects of KLF9 depletion or overexpression on DSC osteogenic differentiation were then evaluated. The osteogenic potential and associated mineralized nodule-forming activities of DSCs were monitored via Alizarin red S staining and quantitative analyses of osteogenic markers. The regulatory effect of KLF9 on the Notch1 signaling pathway was analyzed by luciferase reporter assays.

RESULTS

KLF9 mRNA expression was consistently increased during mesenchymal stem cell osteogenic differentiation in multiple public datasets, and our qRT‒PCR and Western blotting data further validated this finding. In addition, KLF9 depletion promoted proliferation and suppressed osteogenic differentiation of DSCs, while enforced expression of KLF9 promoted the DSC osteogenic potential. Mechanistically, KLF9 negatively regulated the Notch1-mediated signaling pathway by directly binding to the Notch1 promoter. More importantly, Notch1 inhibition/overexpression partially rescued the suppressive/enhancing effects of KLF9 depletion/overexpression on the osteogenic differentiation of DSCs, indicating that Notch1 is a functional downstream target of KLF9.

CONCLUSIONS

In summary, our results strongly demonstrate that KLF9 is a crucial transcription factor that controls the osteogenic differentiation of DSCs by negatively regulating the Notch1 signaling pathway.

摘要

背景

人类牙髓干细胞（DSC）是治疗口腔颌面部疾病的理想细胞来源。然而，调控 DSC 成骨分化的机制仍不清楚。本研究旨在探讨 Krüppel 样因子 9（KLF9）在调控 DSC 生物学功能中的作用及其潜在的分子机制。

方法

通过生物信息学分析、定量实时聚合酶链反应（qRT-PCR）和 Western blot 检测 DSC 成骨分化过程中 KLF9 的表达水平。然后，通过敲低或过表达 KLF9 评估其对 DSC 成骨分化的影响。通过茜素红 S 染色和定量分析成骨标志物监测 DSC 的成骨潜能和相关矿化结节形成活性。通过荧光素酶报告基因检测分析 KLF9 对 Notch1 信号通路的调控作用。

结果

多个公共数据集的研究结果显示，KLF9mRNA 在间充质干细胞成骨分化过程中持续上调，qRT-PCR 和 Western blot 数据进一步验证了这一发现。此外，敲低 KLF9 促进了 DSC 的增殖并抑制了其成骨分化，而过表达 KLF9 则增强了 DSC 的成骨潜能。机制上，KLF9 通过直接结合 Notch1 启动子负调控 Notch1 介导的信号通路。更为重要的是，Notch1 抑制/过表达部分挽救了 KLF9 敲低/过表达对 DSC 成骨分化的抑制/增强作用，表明 Notch1 是 KLF9 的功能性下游靶标。