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

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.