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