KLF6介导的谷氨酰胺代谢调控牙髓干细胞的成牙分化和基质矿化。

KLF6-mediated glutamine metabolism governs odontogenic differentiation and matrix mineralization of dental pulp stem cells.

作者信息

Wu Wenzhi, Xu Zekai, Zhang Yulian, Zhang Xiatong, Huang Xiaoyuan, Xie Zhijian, Chen Zhuo

机构信息

Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Engineering Research Center of Oral Biomaterials and Devices of Zhejiang Province, Hangzhou, 310000, China.

出版信息

Stem Cell Res Ther. 2025 Apr 15;16(1):179. doi: 10.1186/s13287-025-04308-3.

DOI:10.1186/s13287-025-04308-3

PMID:40234972

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12001474/

Abstract

BACKGROUND

When a tooth suffers severe injuries, dental pulp stem cells migrate and differentiate into odontoblast-like cells to form restorative dentin. Kruppel-like factor 6 (KLF6) activates the odontoblast differentiation of dental papilla cells during tooth development. However, the mechanisms by which KLF6 regulates the function of odontoblast-like cells differentiated from human dental pulp stem cells (hDPSCs) remain unknown.

METHODS

KLF6 was over-expressed or silenced by lentivirus transfection. Transcriptome sequencing and metabolomics were performed to reveal main changes in KLF6 high expressed hDPSCs. Mitochondrial morphology was observed by confocal microscope and cryo-transmission electron microscopy. Metabolic assays and metabolic flux were used to determine changes in cellular metabolic characteristics. Glutamine, glutamate, α-KG, and citrate concentrations were detected in cultured cells. Citrate and Ca concentration were detected in ECM. Adeno-associated virus were used to silence KLF6 in mice. A mouse dental injury model was established to investigate the role of KLF6 and glutamine metabolism in dentin repair in vivo.

RESULTS

RNA sequencing and metabolomics showed a remarkable influence on glutamine metabolism, mitochondrial respiration, and the TCA cycle by KLF6 overexpression. Metabolic assays and mitochondrial morphology observation found KLF6 promoted glutamine metabolism and mitochondrial function, and glutamine metabolism and mitochondrial respiration are enhanced during odontogenic differentiation of hDPSCs. Deprivation of glutamine inhibited mineralization of hDPSCs and restrained deposition of citrate and Ca in ECM. Increased glutamine entry into the tricarboxylic acid (TCA) cycle was both observed in differentiated hDPSCs and KLF6 overexpressed hDPSCs. ChIP-qPCR experiments revealed that KLF6 can directly bind to the promoter sequences of GLS1 and GDH. Supplementation of α-KG rescued suppression of odontogenic differentiation and mineralization induced by KLF6 knockdown. Inhibition of glutamine metabolism and knockdown of KLF6 attenuated tertiary dentin formation in vivo.

CONCLUSIONS

Our study shows that KLF6 mediates biomineralization in the newly generated functional odontoblast-like cells differentiated from hDPSCs by altering cell metabolism preferences. KLF6 facilitated glutamine influx into the TCA cycle, leading to increased deposition of citrate in the ECM.These findings may inspire the development of novel strategies for reparative dentin formation.

摘要

背景

当牙齿遭受严重损伤时，牙髓干细胞会迁移并分化为成牙本质细胞样细胞，以形成修复性牙本质。Kruppel样因子6（KLF6）在牙齿发育过程中激活牙乳头细胞的成牙本质细胞分化。然而，KLF6调节人牙髓干细胞（hDPSCs）分化而来的成牙本质细胞样细胞功能的机制尚不清楚。

方法

通过慢病毒转染过表达或沉默KLF6。进行转录组测序和代谢组学分析，以揭示KLF6高表达的hDPSCs中的主要变化。通过共聚焦显微镜和冷冻透射电子显微镜观察线粒体形态。使用代谢分析和代谢通量来确定细胞代谢特征的变化。检测培养细胞中谷氨酰胺、谷氨酸、α-酮戊二酸和柠檬酸的浓度。检测细胞外基质（ECM）中柠檬酸和钙的浓度。使用腺相关病毒在小鼠中沉默KLF6。建立小鼠牙齿损伤模型，以研究KLF6和谷氨酰胺代谢在体内牙本质修复中的作用。

结果

RNA测序和代谢组学分析表明，KLF6过表达对谷氨酰胺代谢、线粒体呼吸和三羧酸循环有显著影响。代谢分析和线粒体形态观察发现，KLF6促进谷氨酰胺代谢和线粒体功能，并且在hDPSCs的成牙本质分化过程中谷氨酰胺代谢和线粒体呼吸增强。剥夺谷氨酰胺会抑制hDPSCs的矿化，并抑制ECM中柠檬酸和钙的沉积。在分化的hDPSCs和KLF6过表达的hDPSCs中均观察到进入三羧酸（TCA）循环的谷氨酰胺增加。染色质免疫沉淀-定量聚合酶链反应（ChIP-qPCR）实验表明，KLF6可以直接结合到GLS1和GDH的启动子序列上。补充α-酮戊二酸可挽救KLF6敲低诱导的成牙本质分化和矿化的抑制。抑制谷氨酰胺代谢和敲低KLF6可减弱体内第三期牙本质的形成。