mTORC1 promotes mineralization via p53 pathway

FASEB J. 2021 Feb;35(2):e21325. doi: 10.1096/fj.202002016R.

Abstract

The objectives of our study were to investigate the roles of mTORC1 in odontoblast proliferation and mineralization and to determine the mechanism by which mTORC1 regulates odontoblast mineralization. In vitro, MDPC23 cells were treated with rapamycin (10 nmol/L) and transfected with a lentivirus for short hairpin (shRNA)-mediated silencing of the tuberous sclerosis complex (shTSC1) to inhibit and activate mTORC1, respectively. CCK8 assays, flow cytometry, Alizarin red S staining, ALP staining, qRT-PCR, and western blot analysis were performed. TSC1-conditional knockout (DMP1-Cre+ ; TSC1f/f , hereafter CKO) mice and littermate control (DMP1-Cre- ; TSC1f/f , hereafter WT) mice were generated. H&E staining, immunofluorescence, and micro-CT analysis were performed. Transcriptome sequencing analysis was used to screen the mechanism of this process. mTORC1 inactivation decreased the cell proliferation. The qRT-PCR and western blot results showed that mineralization-related genes and proteins were downregulated in mTORC1-inactivated cells. Moreover, mTORC1 overactivation promoted cell proliferation and mineralization-related gene and protein expression. In vivo, the micro-CT results showed that DV/TV and dentin thickness were higher in CKO mice than in controls and H&E staining showed the same results. Mineralization-related proteins expression was upregulated. Transcriptome sequencing analysis revealed that p53 pathway-associated genes were differentially expressed in TSC1-deficient cells. By inhibiting p53 alone or both mTORC1 and p53 with rapamycin and a p53 inhibitor, we elucidated that p53 acts downstream of mTORC1 and that mTORC1 thereby promotes odontoblast mineralization. Taken together, our findings demonstrate that the role of mTORC1 in odontoblast proliferation and mineralization, and confirm that mTORC1 upregulates odontoblast mineralization via the p53 pathway.

Keywords: cell signaling; dentinogenesis; mTORC1; molecular biology; odontoblast; tooth development.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cell Proliferation
  • Dentin / cytology
  • Dentin / metabolism
  • Mechanistic Target of Rapamycin Complex 1 / metabolism*
  • Mice
  • Odontoblasts / metabolism*
  • Odontoblasts / physiology
  • Tooth Calcification*
  • Transcriptome
  • Tuberous Sclerosis Complex 1 Protein / genetics
  • Tuberous Sclerosis Complex 1 Protein / metabolism*
  • Tumor Suppressor Protein p53 / metabolism*

Substances

  • Tsc1 protein, mouse
  • Tuberous Sclerosis Complex 1 Protein
  • Tumor Suppressor Protein p53
  • Mechanistic Target of Rapamycin Complex 1